ACTUAL AND POTENTIAL NITROGEN FIXATION IN PEA AND FIELD BEAN
ASAFFECTED BY COMBINED NITROGEN
promotor: dr. i r . E.G. Mulder, oud-hoogleraar in de
algemene microbiologie en de microbiologie
van bodem en water
V^n <9rcM
(S-}o
Michiel van Mil
ACTUAL AND POTENTIAL NITROGEN FIXATION IN PEA
AND FIELD BEAN AS AFFECTED BY COMBINED NITROGEN
Proefschrift
terverkrijgingvandegraadvan
doctorindelandbouwwetenschappen,
opgezagvanderectormagnificus,
dr. C.C.Oosterlee,
hoogleraarindeveeteeltwetenschap,
in hetopenbaarteverdedigen
op vrijdag6november1981
desnamiddagstevier uurindeaula
vandeLandbouwhogeschool teWageningen
99' 3
Cf
BIBLIOTHEEK LH.
0 6 NOV.1981
ONTV. TÎJOSCHR. AOM.
This study was carried out at the Laboratory of Microbiology,
Agricultural University,Wageningen,
The Netherlands
These investigations were subsidized by theAgricultural Bureau
oftheNetherlands Nitrogen Fertilizer Industry
^m°\
STELLINGEN
1. De feitelijke stikstofbinding van erwt en tuinboon is lager dan de
potentiële stikstofbinding, hetgeen berust op onvoldoende toevoer
van fotoassimilaten.
Dit proefschrift
2. Het belang van waterstof-oxydatie voor de aktiviteit van nitrogenase
in symbiontische systemen wordt schromelijk overschat.
Evans,H.J. el al, 1981. pp. 84 -96
in Current perspectives in nitrogen
fixation (eds.A.H. Gibson and W.E.
Newton) Canberra
Dit proefschrift
3. Toediening van stikstofmeststof gedurende de periode van peulvulling kan
de zaadopbrengst van vroege erwterassen verhogen.
Sinclair,T.R. and De Wit,C.T. 1976.
Agronomy J. 68 :319 - 324
4. De verbouw van gewassen voor de produktie van ethanol als brandstof is
in Nederland zinloos en in ontwikkelingslanden misdadig.
P. van Zijl, Intermediair 26 juni 1981
5. Teelt van veldbonen voor eiwitrijk ruwvoer,als tussengewas bij de
herinzaai van grasland, kan in Nederland jaarlijks evenveel energie
besparen als een stad van 20.000 inwoners verbruikt.
6. Daar de bouw van rioolwater-zuiveringsinstallaties in Nederland vrijwel
voltooid is,moet de heffing voor huishoudens op basis van de wet
'Verontreiniging Oppervlaktewateren' worden afgeschaft teneinde meer
menskracht vrij te maken voor andere taken in het milieubeheer.
B1ÎM
:•• •.'•'*.
7. Zij die de invoering van referenda bepleiten om aktievoerders de wind
uit de zeilen te nemen, houden er onvoldoende rekening mee dat vaak de
rechtmatigheid van het overheidsbeleid wordt aangevochten en niet
zozeer de steun van de bevolking voor dat beleid.
Couwenberg, S.W., Intermediair 29 mei 1981
8. Bij de recente diskussie over het al dan niet verbieden van politieke
partijen met een racistische inslag zijn de partijen met een theokratische
inslag ten onrechte buiten beschouwing gebleven.
Michiel van Mil
Actual and potential nitrogen fixation in pea and field bean as affected by
combined nitrogen.
Wageningen,6 november 1981
CONTENTS
Voorwoord
6
General Introduction
7
1 Actual andpotential nitrogen fixation inPisum sativum L.
and Vieia fabaL.
2 Actual andpotential nitrogen fixationbypearootnodules
as affectedbycytokininandethylene
3 Alternative,cyanide-resistant,respiration inroot systems
of Pisum sativum L.asaffectedbystrainof Rhizobium
leguminosarum andnitrate supply
MichielvanMil,RiesdeVisserandFrank Houwaard
4 Nitrogen fixationby pea-Rhizobium symbiosis containinga
bacterial uptake hydrogenase
Michiel vanMilandHans Brons
21
35
45
63
5 Nodule formationandnitrogen fixation insymbiosesof
pea {Pisum sativum L.)anddifferent strainsof
Rhizobium leguminosarum
75
6 EffectofN-fertilizersonnitrogen fixationandseed
yieldof peà-Rhizobium symbiosesofdifferent nitrogenfixing capacity
93
General Discussion
115
Summary
123
Samenvatting
125
Curriculum vitae
128
VOORWOORD
Iederonderzoek isafhankelijk van een samenwerking tussen degene die het
uitvoert en veleanderen. Inversterktemate geldt dit vooreenpromotieonderzoek. Het onderwerp is immers al in grote lijnen bepaald,de voornaamste
methoden zijn al in huis en het geld komtook ergens vandaan.Het spreekt dus
eigenlijk vanzelf dat velen mij inwoord en daad geholpen hebben.
Het projekt 'Stikstofbemesting van vlinderbloemige gewassen'waaraan ik
vier jaar heb gewerkt,was een lang gekoesterde wensdroom van prof.Mulder.
Zijn steedsweer nieuwe ideeën en kritisch kommentaar zorgden ervoordatde
vaart erniet uitging.
Hetwelslagen van ditonderzoek isook in belangrijke mate te danken aan
LieTek-An diemij veel suggesties gegeven heeft,waaronder de goedegreep
inde stammenkollektie. Frank Houwaard,ex-lotgenoot in knollenland,heeft
mij van begin tot einde op velemanieren bijgestaan.Vooradviezen en hulp
metmethoden kon ikaltijd terecht bijWim Roelofsen enAntoon Akkermans.
Het ijverige en nauwgezettewerken van Piet Dijksman heeft alle stikstofanalyses van dit proefschrift opgeleverd. De planten werden met toewijding
verzorgd doorde heren 0. van Geffen,E.van Velsen enA. Houwers.Annie
Mol-Rozenboomwiste alleongewenste sporen van mijn arbeid uit,waarbijgeen
overstroming haar te veel was.Demooie plaatjes werden vervaardigd door de
heren J. van Velzen en A.Wessels. De laatste schakel werd gevormd door mw.
C.MölIer-Mol die hetmeestal deerlijk toegetakeldemanuscript op uitmuntende
wijzewist omte toveren tot hetboekje datnu voor u ligt.
Graag wil ik iedereen die in deafgelopen jaren meegewerkt heeft van harte
bedanken.
Tenslotte mag niet onvermeld blijven dat hetonderzoek voor een aanzienlijk
deel gefinancierd werd door het Landbouwkundig Bureau der Nederlandse Stikstofmeststoffen Industrie.
GENERAL INTRODUCTION
Grain legumes and protein
production
Theyieldofgrain legumesinthepast decadeshasnottaken advantageOf
modern agricultural growing practices,breedingandresearch likeother important
crops,for example cereals.Bothindevelopedandindeveloping countries, the
yieldofgrain legumes amountstoless than one-halfofthatofcereals (Fig.1)
(24).Thelowyieldsofgrain legumesascompared with cerealscanbeexplained
by thedifferences inseed composition. Legume seeds contain more lipidsand
protein than cereal seeds,andconsequently requireahigher amountofphotosynthesized carbohydrates fortheir synthesis {e.g. with groundnutsandbarley
0.43and0.75gofseed produced pergofcarbohydrates,respectively)(72).
The ratesofyield increase arealso differentforthetwo groupsofcrops:in
the past decade,yieldsofmaize increasedby15%,whereas thoseofbeans
(Phaseolus spp)rose onlyby3%(92).However,with theeconomically more
attractive soybeansandgroundnuts,yield increases amountedto20and 37%,
respectively, indicating that grain legumesmayrespondtoresearch,breedingand
improved management.
The area used forgrowing grain legumeshas decreasedby21%inthepast
decadeindeveloped countries,whereas indeveloping countries thisarea (excluding thatwith soybeansandgroundnuts)hasrisenatanequal rateasthetotal
arable land (approx. 10%).Thesoybean area indeveloping countries increasedby
55%inthis period (FAO production yearbooks).
The reduced interestingrain legumesindeveloped countries iscoupled with
an increased consumptionofanimal protein,ascanbeseeninTable1fromthe
decreasing ratioofconsumedtoconsumable protein.This ratioisstrongly
dependentonincome per capita,ahigh income leadingtoahigh protein consumptionaswellasasubstitutionofanimalforvegetable protein (29).Indeveloping countries,population isgrowingbut also agricultural exportsare increasingbyallocating more landtocash crops.This tendstoachangeofthe type
1950
1960
1970
1980
Years
Fig. 1a,b. Seed production ofcereals (o, • ) , pulses (excluding soybeans and
peanuts)(A,A)and soybeans (a , • ) in developed countries (o,A , a )
and developing countries (t,A , « ) .Source:FAO Production Yearbooks.
ofcrops grown for local food,the high-yielding cereals replacing the low-yielding legumes (92).The resulting dietmay contain sufficient carbohydrates for
the daily energy requirement,but it istoo low in protein. Ingeneral,the ratio
A of cereals to legumes in the diet of developing countries is9,whereas it
should be 7to 5to prevent malnutrition (10,92).Thus,adecreasing interest
ingrain legumes,occurring in developed countries,isa symptom ofan affluent
society,whereas in developing countries it isa sign of a low standard of
1iving.
The agricultural pattern inThe Netherlands shows thecharacteristics ofan
affluent society. Together with the build-up ofan extensive system of dairy
and meat production, thearea of grain legumes shrank considerably, e.g. that
Table 1.Production andconsumption ofprotein in1965.Source:(29)
Production
Consumpt ion
ofconsumab' e ofconsumable
protein D
protein 1)
Consumed
protein 2)
Ratio of
consumed to
consumable
protein
(kg/capita. year)
NorthAmerica
Western Europe
Developed countries
(total)
LatinAmerica
South-EastAsia
Africa
Developing countries
(total)
Incomeclass ($) 3 '
< 100
100-200
200-400
400-800
800-1600
> 1600
152
49
76
104
81
80
35
33
34
0.34
0.41
0.42
47
24
29
29
43
26
26
29
24
19
22
21
0.57
0.73
0.85
0.72
24
26
32
52
57
106
24
27
32
49
70
98
19
20
22
29
33
35
0.80
0.73
0.69
0.59
0.47
0.35
1) Protein invegetable products,fitforhuman consumption plus proteinin
animal products from vegetable foddernotfitforhuman consumption.
2)From vegetableandanimal origin.
3)Countries classified according toaverage incomepercapita,year in1965
based upon dataof85countries.
of green peas grown fordry-seed production from 30000hain1960to2400hain
1979.Typically,theareaofpeas grown forthecanning industry increased slowly,
from 5000hain1960to5400hain1980,indicating thatalow-yielding crop
grown forluxury consumption islessaffected when income rises.Field beans have
almost disappearedasafood crop,butrecently,growers have resumed interest,
nowasafeed cropforsilage (source:Landbouwcijfers 1980).
Agricultural production intheNetherlands hasgreatly benefittedbythe
increased useofnitrogen fertilizers (97kgNperhain1960,216kgNperha
in 1977,values thatarehigher than thoseofotherdeveloped countries).However,
with increasing energy prices agricultural practices thatuselower levelsof
combined nitrogen than atpresentarelikely tobepropagated. Thismightwell
leadtoarenewed attentiontolegumecrops indeveloped countries.Indeveloping
countries,theuseofgrain legumes infood production will probably also depend
on improved yieldsofthese cropsascompared with cereals.
Nitrogen fertilizer usehasbeenofcrucial importancetotheenhanced
productivity ofcereals.Onereasonfor the fact thatyieldsoflegume crops
lag behind thoseofcerealsisthatingeneral theydonotrespondtoadded
fertilizernitrogen.Combined nitrogen interferes with thebiological nitrogen
fixation processofthe~\equme-Rhizobium symbiosis.Legume crops seemtobe
closetoa 'yield barrier' resulting from the limitsofphotosynthetic capacity
andofthecapacityofnitrogen supplyofthe symbiotic system. Both aspects
will havetobeinvestigated when attempting tosurmount this barrier.Therefore,
in this introductionthe data available from the present literature willbe
briefly summarized with regardtothenitrogenandcarbon limitationstoseed
productionofgrain legumes.
Nitrogen limitation and combined-nitrogen dressings
Ifthegrowthoflegumes,capableoffixing atmospheric nitrogenbymeansof
rootnodules,would alwaysbenitrogen-limited,consistent yield increases would
beobtained bythe supplyofcombined nitrogen.However,thedata available from
literatureonthis topic are often conflicting.
Suboptimum symbioses Dressingsofcombined nitrogen nearly always enhance yield
of legumes growing under circumstances unfavourablefor the establishmentofa
fully effective,nitrogen-fixing symbiosis.
The formationofroot nodulesbyRhizobium strains does not occuratalow
soil pH,thevalueofwhichisdifferentfor each host species (53),while also
the rhizobial strainsmayvary inacid tolerance (39,40).Inthese cases,yield
of legumesisincreased bynitrogen fertilizer dressings (9,18).Also,adeficiencyoftrace elements likeB(50)orMo(52)preventsthe developmentofa
fully effective symbiosis.Theadditionofcombined nitrogencanpartly overcome
the resulting nitrogen limitation togrowth.
The nitrogen-fixing capacityofthe symbiosis might alsobeimpairedby
infestation with plaguesanddiseases.Combined nitrogen was foundtoincrease
seed yieldofpea plantswhentheroot nodules were invadedbySitona larvae
(50),whentheplants were attackedbyFusarium (50)orbyinsects.Lodgingof
the plants cannotbealleviated bycombined nitrogenbutiseven aggravatedat
high dressings(51).
Legume seed yieldscanalsobedecreased ifRhizobium strainsarepresentin
the soil whichdonotestablishafully effective symbiosis withthehost plant.
Rhizobium strains that produce ineffective nodules (without nitrogen fixation)
ormoderately effective nodules (withlowlevelsofnitrogen fixation)
10
considerably reduce theyield of plant nitrogen and seed as compared with
strainswhich produce highly effective nodules with a specific hostplant.
Nitrogen fertilizerdressings may improveyield ofplants infected by Rhizobium
strains of poornitrogen-fixing capacity(27).
Apart from fertilizer dressings,inoculation of the sowing-seed with a
highly effective strain can produce good results iffew indigenous rhizobia
are present in the soil (11,37).However,typical problems may be encountered
with inoculant strains regarding:(a) competition with the indigenous rhizobia
fornodulation sites on the legume roots (42);(b)survival in the soil as
free-living bacteria (11,55);(c)anarrow host-plant range of the inoculant
strain leading toan ineffective symbiosiswhen a different host species of the
samecross-inoculation group isgrown at the inoculated plot(4).
The introduction of new plant varieties toareas outside the region where
they originated may also result in symbioses possessing a poor nitrogen-fixing
capacity with the locally occurring rhizobia (17).Host plants may requirea
specific Rhizobium strain foran effective symbiosis,whereas other Rhizobium
strains produce ineffective nodules (41,42)or inhibit nodule formation by other
strains without producing nodules themselves (43).Nitrogen dressings are likely
to increase yield inall these cases,although no reports are available from
field experiments.
Optimum symbioses Itis hardly surprising thatyield increases are obtained by
the supply of combined nitrogen to suboptimum symbioses.To acertain extent,
it isa consequence of thedefinition of 'suboptimum',although always specific
factors can be earmarked as being responsible for the low nitrogen gain of the
symbiosis.Therefore,amore interesting problem,at least from a theoretical
point of view,concerns the question whether also theyield of afully effective
symbiosis with a high rateof nitrogen fixation would respond tocombined nitrogen.With regard to field experiments many conflicting reports can be found in
the literature,which isnot exhaustively treated here.
No enhancement of seed yield was found in trials with combined nitrogen dressings of pea (21,50);soybean (1,36,81,85,87)and field bean [vicia sp.;
15,28,63).Butyield increases were met in other experiments, e.g. with soybean
(15, 10,36);cowpea (14);pea (15,75);field beans {vicia sp.;65)and beans
{Phaseolus
sp.; 79).
Ingrowth chamber and greenhouse experiments,uninoculated legume plants
supplied with combined nitrogen from sowing consistently show a higher dry matter
11
production than plants inoculated withaneffective Rhizobiwn strainbut
without added fertilizer.This phenomenon hasbeen attributed tohigher energy
costsfor the fixationofatmospheric nitrogen than for assimilationofcombined nitrogen.Itmaybetrueofplants growing with NHt-N,butithasnot
convincingly been established that growth with N0Ö-N would requiremuch less
energy than symbiotic growth with atmospheric N„ (equal costs: 22,49;lower
costsofN0Ô-N assimilation thanofN„ fixation: 45,57,67,71).Theproblem
iscomplicated bythe fact thataconsiderable partofthenitrate takenup
is reducedtoammonia intheleaves,using reducing equivalents derived from
photosynthetic activity,which thus decreasetherespiratory costsofnitrate
reduction (3,84).Onthe other hand,ahigh carbohydrate requirementof
nitrogen-fixing rootsmight provideastronger sinkforphotoassimilates leading
toanincreased photosynthesisesmore carbohydratescanbeexported fromthe
leaves (44, 66).
The growth ratesofnitrogen-fixing plantsareusually lower than thoseof
plants growing with combined nitrogen from sowing. Seedlings grown without
added nitrogenous fertilizer faceaperiod of'nitrogen hunger' beforethe
infection process andnodule formation start,whereas seedlings growing with
combined nitrogen maintainaconstantly high growth rate (19,69).Inoculated
seedlings thatare supplied withalowlevelofcombined nitrogen (not interfering with nodule formation)produceahigher shootmassandleaf areawhich
arecapableofsupportingahigher nitrogen-fixing activity than inoculated
seedlings without added nitrogen (6).This 'starter dose' effectisoften aimed
atinagricultural practice (13,6 4 ) ,butresultscanonlybedetectedifboth
the naturally available nitrogen level ofthesoilandthe residual nitrogen
from the preceding crop are low.
Physiological effect of combined nitrogen on nitrogen fixation
Combined nitrogen exertsadetrimental effectonsymbiotic nitrogen fixation,
aswas already foundinthebeginningofnitrogen fixation research (48,62,64,
86). However,theexactwayinwhich combined nitrogen suppresses boththe
formationofnewnodulesandthe activityofexisting nodules,has evokeda
good deal ofcontroversy.
Nodule formation The processofnodule formation byinvasionofroothairsby
free-living rhizobiahasbeen described indetail (38).During this complicated
process, Rhizobium bacteria enter the root via aninfection thread,inducecell
divisionsintherootcortex,andare normally transformed into large,non-motile,
12
and often branched bacteroids,capable of fixing nitrogen.The inhibition of
the initiation and growth of nodules bycombined nitrogen was shown to bea
local effect ina series of experiments with split-root systems ofwhich one
halfwas kept nitrogen-free and the other halfwas supplied with nitrate(90).
The hypothesis that nitrate inhibition ofnodule formation would be exerted
via the production of nitrite has to bediscarded as this does not explain the
similar effect of ammonium ions (23,47).Also the theory that theC/N ratio in
the plant determined theextent of nodule formation has tobeamplified as it
offers no sufficient physiological interpretation.A relationship between
carbohydrate supply and the effect ofcombined nitrogen on nodule formation is
apparent (61).The restraint of nodule formation by combined nitrogen was
counteracted by theaddition ofcarbohydrate compounds(91).
Plant growth regulators may play an important part innodule formation.The
addition of nitrate to legume seedlings decreases root hair curling and nodule
initiation by lowering the level ofthe auxin indolyl-acetic acid (IAA)(80).
This adverse effectof nitrate could be reversed byexternal supply of IAA
(54,82).Furthermore,cytokinins might be involved in the interaction between
host plant and rhizobial microsymbiont,asfree-living rhizobia were shown to
excrete sufficiently largequantities of cytokinins to induce cell divisions in
a soybean-callus test (59).Phytohormoneactivity has been demonstrated both in
nodule formation with actinomycetal symbioses (88)and inrhizobial symbioses
(26,77,78)although noclear-cut solution was offered to the problem of
external influences {e.g. combined nitrogen)on the process of infection and
nodule growth. However,nodules contain cytokinins and auxins in concentrations
well above those found in theadjacent root tissue (26),similar to root tips
(70). This indicates that further research into the role of phytohormones in
nodule formation is necessary toelucidate the effect of combined nitrogen on
nodule formation.
Nodule activity The decline of nitrogenase activity upon the supply of combined
nitrogen toalready nodulated roots is not due toan accumulation ofamino
acids inthenodules (32)or to adirect inhibition of nitrogenase by added
ammonium ions (30)as bacteroids do not take upammonium ions (33).Inmediumterm experiments with nodulated pea plants the nitrogenase content of the
bacteroids was notaffected by combined nitrogen; nitrogenase could bereactivated by supplying the bacteroids with ATP and reducing equivalents(30).
However,leghemoglobin synthesis was strongly repressed resulting in the
13
colourofthenodules turning from pinktogreen (8).Theadverse effectof
combined nitrogenonnitrogen fixationofnodulatedpeaplants couldbecounteractedbyahigh light intensityorbyadded sucrose(31).
14
Inexperiments with shootsofleguminous plants exposedto C0 ? , addition
ofcombined nitrogen evokedachangeinthetranslocation patternofphotosynthates from the shoottotheroots,resulting inalower proportionof
carbohydrates suppliedtothe nodulesandahighertoother partsofthe root
system (34,74).Thisisconsistent withtheinvolvementofplant growth regulatorsasdiscussed above.Theadditionofcombined nitrogen enhancedthe
cytokinin levelsofroot tips (68,83)andprobably increased photosynthate
supplytothese tips whichwould inturn leadtoalower supplyofcarbohydrates
to thenodules.
Carbohydrate limitation to nitrogen fixation
Asthephotosynthate supplyofthenodules seemstobeofcrucial importance
for the explanationofthe effectofcombined nitrogenonnitrogen fixation,it
isworthwhiletosubjecttherelation between photosynthesisandnitrogen
fixationtoacloser examination.The fact thatoptimum legume-Rhizobium symbiosesdonotrespondtonitrogen fertilizer dressingsinanunequivocalway,
pointstothe possibilityofaphotosynthate-1imited growth during the entire
growth cycleorpartofit.Actually,carbon dioxide enrichmentoftheatmosphere resulted inastrongly increased nitrogen fixationandyield (25, 58).
Furthermore,source-sink manipulations including defoliationandshading
reduced nitrogen fixation (5,12,35,50).Thesameeffectoccurred when light
intensity was low,whereasanincreased light intensity raised nitrogen fixation
(2,7,46)asdidgraftingoftwoshootsonasingle root(76).
However,the'nitrogen-hunger' period during theearly phasesofseedling
growth was prolongedandgrowth was retardedbyahigh light intensity because
thenitrogen deficiency was aggravated (20,56,89).Later on,the plants grown
at high light intensity showedahigherdrymatter productionaswell asa
higher nitrogen fixation than plants grownatalower light intensity(89).
Alsointhegenerative growth phaseoflegumes,anenhancementofphotosynthesis
might eventually resultinalower seedyield.Ahigh rate ofphotosynthesis
would accelerate seed production butalso increase thedemand for nitrogen,
which would leadtoamobilizationofnitrogenous compounds from vegetative
tissuesfortranslocationtotheseeds.Thus,photosynthesis wouldbereduced
soon,inturn lowering nitrogen fixationandendingupin'self-destruction'
ofthe plant(72).
14
Outline of the
investigations
The investigations reported inthis paper are focused onthe question
whether growth andyieldof ~\equme-Rhizobiwn symbiosesaredeterminedby
nitrogenorcarbohydrate limitation,with emphasisontheformer.Thestudy
was carried outwith Rhizobium strains selected fordifferences innitrogenfixing capacity,andwith host speciesandcultivars with different seedproduction ratesandphotosynthetic capacities.
InChapters 1and2,the in vitro nitrogenase activityofisolated bacteroids
was compared with the in vivo nitrogenase activityofnodulated peaandfield
bean plants inordertofind outwhether nitrogen fixation inthese plantswas
limited byanintrinsic factorofthebacteriaorbyexternal factors,suchas
photosynthate supply.Chapter3reportsonthe efficiencyofenergy-yielding
respiratory processesinthe root systeminrelationtothe nitrogen source
(atmosphericorcombined nitrogen). Chapter4dealswithastudyonnitrogen
fixation inasymbiosis containingabacterial uptake hydrogenase,capableof
recirculating thehydrogen produced bynitrogenase concomitantly with nitrogen
fixation.InChapter5,nodule formationandnitrogen-fixing capacityofa
numberofR. leguminosarum strainsinassociation withpeaplantswere described
indetail. Finally,Chapter6containsastudyontheinfluenceofcombined
nitrogenonnitrogen fixationofsymbiosesofpeaplants with the R. leguminosarum strains studied inChapter 5.Atentative modelwaspresented forthe
interactions between nitrogen fixationandphotosynthesis.
REFERENCES
1.Alios,H.F.,andBartholomew,W.V. 1959.Replacementofsymbiotic nitrogen
fixationbyavailable nitrogen. Soil Sei. 87:61-66.
2.Antoniw,L.D.,andSprent,J.I. 1978.Growthandnitrogen fixationof Phaseolus
vulgaris L.attwoirradiances. II.Nitrogen fixation.Ann.Bot.42:399-410.
3. Beevers,L.,andHageman,R.H. 1969.Nitrate reduction inhigherplants.
Ann. Rev. Plant Physiol.20:495-522.
4. Berg,E.H.R.vanden. 1977.The effectivenessofthe symbiosisof Rhizobium
leguminosarum onpeaandbroad bean.PlantandSoil 48:629-639.
5.Bethlenfalvay,G.J.,Abu-Shakra,S.S.,Fishbeck,K.,andPhillips,D.A. 1978.
The effectofsource-sink manipulationsonnitrogen fixation inpeas. Physiol.
Plant.43:31-34.
6. Bethlenfalvay,G.J.,Abu-Shakra-,S.S.,andPhillips,D.A. 1978. Interdependence
of nitrogen nutritionand photosynthesisinPisum sativum L.I.Effectof
combined nitrogenonsymbiotic nitrogen fixationandphotosynthesis. Plant
Physiol.62:127-130.
7. Bethlenfalvay,G.J.,andPhillips,D.A. 1978. Interactions between symbiotic
nitrogen fixation,combined-N applicationandphotosynthesis in Pisum sativum.
Physiol.Plant. 42: 119-123.
15
8.Bisseling,T.,Bos,R.C.van den,and Kammen,A. van. 1978.The effect of
ammonium nitrate on the synthesis of nitrogenase and the concentration of
leghemoglobin in pea root nodules induced by Rhizobium
leguminosarum.
Biochim. Biophys.Acta 539: 1-11.
9. Boskma,K. 1966.Kalktoestand en stikstofbemesting voor conservenerwten op
zand- en veenkoloniale grond. 17pp. Inst. Bodemvruchtbaarheid Haren.
10. Bressani,R.,and Elias,L.G. 1980.Nutritional value of legumecrops for
humans and animals,pp. 135-156 in Advances in legume science (eds.R. Summerfield and A.H.Bunting) Kew Gardens,U.K.
11. Burton,J.C. 1980.New developments in inoculating legumes,pp. 380-405
in Recent advances inbiological nitrogen fixation (ed.N.S.Subba Rao)
Arnold, London.
12. Ching,T.M., Hedtke,S.,Russell,S.A., and Evans,H.J. 1975.Energy state
and dinitrogen fixation in soybean nodules ofdark-grown plants. Plant
Physiol. 55:796-798.
13. Copeland,R., and Pate,J.S. 1970.Nitrogen metabolism of nodulated white
clover in the presence and absence of nitrate nitrogen in White clover
research (ed.J. Lowe),Occ. Symp. British Grassl. Soc.6: 71-77.
14. Dart,P.J., Huxley,P.A., Eaglesham,A.R.J.,Minchin,F.R., Summerfield,
R.J.,and Day,J.M. 1977.Nitrogen nutrition of cowpea {vigna
unguiaulata).
IIEffects of short-term applications of inorganic nitrogen on growth and
yield ofnodulated and non-nodulated plants. Expl.Agric. 13:241-252.
15. Dean,J.R., and Clark,K.W. 1980.Effect of low level nitrogen fertilization
on nodulation,acetylene reduction and dry matter infababeans and three
other legumes.Can.J. Plant Sei.60: 121-130.
.,16. Deibert,E.J., Bijeriego,M.,and Olson,P.A. 1979.Utilization of Nfertilizer by nodulating and non-nodulating soybean isolines.Agron. J.
71: 717-723.
17. Devine,T.E.,and Breithaupt,B.H. 1980.Significance of incompatibility
reactions of Rhizobium japonioum strains with soybean host genotypes. Crop
Sei. 20:269-271.
18. Dilz,K.,and Mulder,E.G. 1962.The effect of soil pH,stable manure and
fertilizer nitrogen on thegrowth of red clover enof red clover associations
with perennial ryegrass.Neth.J.Agr.Sei. 10: 1-22.
19. Duke,H.S.,and Ham,G.E. 1976.The effect of nitrogen addition on N2fixation and on glutamate dehydrogenase and glutamate synthase activities
innodules and roots of soybeans inoculated with various strains of Rhizobium
japonioum. Plant and Cell Physiol. 17:1037-1044.
20. Fred,E.B.,Wilson,P.W., and Wyss,0. 1938.Light intensity and the nitrogen
hunger period in the Manchu soybean. Proc.Nat.Acad. Sei. USA 24:46-52.
21. Geering,J. 1957.Ueber die Düngung der Drescherbsen, insbesondere mit
Stickstoff. Landwirtsch. Jahrbuch der Schweiz 6: 473-503.
22. Gibson,A.H. 1966.The carbohydrate requirements for symbiotic nitrogen
fixation:a 'whole plant' growth analysis approach.Aust.J. Biol. Sei.
19:499-515.
23. Gibson,A.H.,and Pagan,J.D. 1977.Nitrate effects on the nodulation of
legumes inoculated with nitrate-reductase deficient mutants of Rhizobium.
Planta 134: 17-22.
24. Hardy,R.W.F. 1976.Potential impact ofcurrent abiological and biological
research on theproblem of providing fixed nitrogen,pp.693-717 in Nitrogen
fixation,vol.2 (eds.W.E.Newton and C.J. Nyman)Washington State Un.Press.
25. Hardy, R.W.F.and Havelka,U.D. 1976.Photosynthate as amajor factor
limiting nitrogen fixation by field-grown legumes with emphasis on soybeans,
pp. 421-439 in Symbiotic nitrogen fixation in plants (ed. P.S. Nutman),
IBP 7.Cambridge Un.Press,Cambridge.
16
26. Henson, J . E . , and Wheeler, C T . 1976. Hormones in plants bearing nitrogenf i x i n g nodules: the d i s t r i b u t i o n of cytokinins in Vicia faba L. New Phytol.
76: 433-439.
27. H i e l e , F.J.H. van. 1960. Enting van lupinen, erwten en stambonen met
verschillende Rhizobiumstammen; veldproeven. Verslagen Landb. Onderzoekingen
66.19, PUDOC Wageningen.
28. Hill-Cottingham, D.G., and Lloyd-Jones, C.P. 1980. The influence of n i t r a t e
supply on nitrogen f i x a t i o n during growth of the f i e l d bean (vicia faba) in
sand. Physiol. Plant. 48: 116-120.
29. Hoogh, J . de, Keyzer, M.A., Linnemann, H., and Heemst, H.D.J, van.1976.
Food f o r a growing world population. Eur. Rev. Agric. Econ. 3: 459-499.
30. Houwaard, F. 1978. Influence of ammonium chloride on the nitrogenase a c t i v i t y
of nodulated pea p l a n t s . Appl. Envir. M i c r o b i o l . 35: 1061-1065.
31. Houwaard,F.1980.Influenceofammoniumandnitratenitrogenonnitrogenase
activityofpeaplantsasaffectedbylight intensityandsugaraddition.
PlantandSoil 54:271-282.
32. Kamberger,W.1977.Regulationofsymbiotic nitrogen fixationinroot nodules
ofalfalfa (Medicago sativa) infected with Rhizobium meliloti.
Arch.Microbiol. 115:103-108.
33. Laane,C ,Krone,W.,Konings,W.,Haaker,H.,andVeeger,C.1980.Shortterm effectofammonium chlorideonnitrogen fixationby Azotobacter
vinelandii
andbybacteroidsof Rhizobium leguminosarum. Eur.J.Biochem.
103: 39-46.
34. Latimore,M.,Giddens,J.,andAshley,D.A. 1977.Effectofammoniumand
nitrate nitrogen upon photosynthate supplyandnitrogen fixationbysoybeans.
Crop Sei. 17:399-404.
35. Lawn,R.J.,andBrun,W.A. 1974.Symbiotic nitrogen fixation insoybeans.
I Effectofphotosynthetic source-sink manipulations.Crop Sei. 14:11-16.
36. Lawn,R.J.,andBrun,W.A. 1974.Symbiotic nitrogen fixation insoybeans.
IllEffectofsupplemental nitrogenandintervarietal grafting.Crop Sei.
14: 22-25.
37. Leng,E.R.,andWhigham,D.K. 1974.Ferti1izing soybeansintheearly stages
of their introduction. Proc.1st FAO/SIDA Seminaronimprovementand
productionoffield food crops.417-423.
38. Libbenga,K.R.,andBogers,R.J. 1974.Rootnodulemorphogenesis,pp.430-472
in Thebiologyofnitrogen fixation (ed.A.Quispel)North Holland Publ. Co.
Amsterdam.
39. Lie,T.A. 1969.TheeffectoflowpHondifferent phasesofnodule formation
inpeaplants.PlantandSoil 31:391-406.
40. Lie,T.A. 1971.Symbiotic nitrogen fixation under stress conditions.Plant
and Soil spec, vol.117-127.
41. Lie,T.A. 1978.Symbiotic specialization inpeaplants:therequirementof
specific Rhizobium strainsforpeas fromAfghanistan.Ann.Appl. Biol. 88:
462-465.
42. Lie,T.A., HilleRisLambers,D.,andHouwers,A.1976.Symbioticspecialisationinpeaplants:some environmental effectsonnodulationandnitrogen
fixation,pp.319-333 in Symbiotic nitrogen fixationinplants (ed. P.S.
Nutman)IBP7,Cambridge Un.Press,Cambridge.
43. Lie,T.A., Winarno,R.,andTimmermans,P.C.J.M. 1978. Rhizobium strains
isolated fromwildandcultivated legumes:suppressionofnodulationbya
non-nodulating Rhizobium strain,pp.398-401 in Microbial Ecology (eds.
M.W.LoutitandJ.A.R.Miles)Springer Verlag Berlin.
44. Lovell,P.H., Oo,H.T.,andSagar,G.R. 1972.Aninvestigation intotherate
and controlofassimilate movement from leavesinVisum sativum. J. Exp.
Bot.23:255-266.
17
45. Mahon,J.D. 1977.Respiration and theenergy requirement for nitrogen
fixation innodulated pea roots.Plant Physiol.60:817-821.
46. Mahon,J.D. 1977.Rootand nodule respiration inrelation to acetylene
reduction innodulated peas.Plant Physiol.60:812-816.
47. Manhart,J.R., and Wong,P.P. 1980.Nitrate effect on nitrogen fixation
(acetylene reduction),activities of legume root nodules induced by rhizobia
with varied nitrate reductase activities.Plant Physiol.65:502-505.
48. Maze,M. 1898.Les microbes des nodosités des légumineuses.Ann. Inst.Pasteur
12: 1-25 and 128-155.
49. Minchin,F.R., and Pate,J.S. 1973.The carbon balance ofa legume and the
functional economy of its rootnodules.J. Exp.Bot.24:259-271.
50. Mulder,E.G. 1949. Investigation of the nitrogen nutrition of pea plants.
Plant and Soil 1:179-212.
51. Mulder,E.G. 1954.Effect ofmineral nutrition on lodging of cereals.Plant
and Soil 5:246-306.
52. Mulder,E.G.,Bakema,K.,and Veen,W.L.van. 1959.Molybdenum in symbiotic
nitrogen fixation and innitrate assimilation. Plant and Soil 10:319-334.
53. Mulder,E.G.,Lie,T.A., Dilz,K.,and Houwers,A. 1966.Effect ofpH on
symbiotic nitrogen fixation of some leguminous plants.Proc. IXth Int.
Congress for Microbiol.,Moscow,pp.133-149.
54. Munns,D.N. 1968.Effects of nitrate on root hairs and infection. Plantand
Soil 29:33-47.
55. Obaton,M. 1977.Effectiveness,saprophytic and competitive ability: three
properties of Rhizobium essential for increasing theyield of inoculated
legumes,pp. 127-133 in Biological nitrogen fixation infarming systems of
the tropics (eds.A.Ayanaba and P.J. Dart)Wiley,Chichester.
56. Orcutt,F.S.,and Fred,E.B. 1935.Light intensity as an inhibiting factor
in the fixation of atmospheric nitrogen by Manchu soybeans.J. Am. Soc.
Agron. 27:550.558.
57. Pate,J.S.,Layzell,D.B.,and Atkins,C.A. 1979.Economy ofcarbon and
nitrogen inanodulated and non-nodulated (NO,-grown) legume.PlantPhysiol.
J
69: 1083-1088.
58. Phillips,D.A., Newell,K.D.,Hassell,S.A., and Telling,C E . 1976.The
effect ofCO« enrichment on root nodule development and symbiotic N~
reduction in Visum sativum L.Am. J. Bot.63:356-362.
59. Phillips,D.A., and Torrey,J.G. 1972.Studies on cytokinin production by
Rhizobium. Plant Physiol.49:11-15.
60. Rabie,R.K.,and Kumazawa,K. 1979.Effect of nitrate application and shade
treatment on the nitrogen fixation andyield of soybean plant.Soil Sei.
Plant Nutr.24:467-476.
61. Raggio,M., Raggio,N.,and Torrey,J.G. 1965.The interaction of nitrate and
carbohydrates in rhizobial root nodule formation. Plant Physiol.40:601-606.
62. Rautenberg,F.,and Kühn,G. 1864.Vegetationsversuche imSommer 1863.
Landwirtsch.Vers.Stat.6:355-359.
63. Richards,J.E.,and Soper,R.J. 1979.Effect ofN fertilizer on yield,
protein content,and symbiotic N fixation infababeans.Agron.J. 71:807-811.
64. Ritter,G. 1911.Beiträge zur Stickstoffernährung durch Leguminosen. Versuche
mit Lupinen auf schwerem Boden.Zentralblatt für Bakt. II 29:650-668.
65. Roberts,S.,and Weaver,W.H.,1971.Relationship among N-nutrition,growth
andyield of three field bean varieties.Washington Agric. Exp.Sta.Bull.
742: 1-7.
66. Russell,W.J. 1975.Carbon-14-assimilate translocation innodulated and
non-nodulated soybeans.Diss,abstr.B35:4319.
67. Ryle,G.J.A.,Powell,C E . ,and Gordon,A.J. 1979.The respiratory costs
ofnitrogen fixation insoybean,cowpea and whiteclover. IIComparisons
ofthecostofnitrogen fixation and the utilization ofcombined nitrogen.
J. Exp.Bot.30:145-153.
68. Sattelmacher,B.,and Marschner,H. 1978.Nitrogen nutrition and cytokinin
activity in Solanum tuberosum. Physiol.Plant.42:185-189.
69. Schreven,D.A. van. 1959.Effect ofadded sugars and nitrogen onnodulation
of legumes.Plantand Soil 11:93-112.
70. Short,K.C.,and Torrey,J.G. 1972.Cytokinins in seedling rootsofpea.
Plant Physiol.49:155-160.
71. Silsbury,J.H. 1977.Energy requirement for symbiotic nitrogen fixation.
Nature 267:149-150.
72. Sinclair,T.R., and Wit,C T . de.1975.Photosynthate and nitrogen requirements for seed production by various crops.Science 189:565-567.
73. Sinclair,T.R., and Wit,C T . de. 1976.Analysis ofthe carbon and nitrogen
limitations tosoybean yield.Agron.J. 68:319-324.
74. Small,J.G.C, and Leonard,O.A. 1969.Translocation ofC1 -labeled photosynthate innodulated legumes as influenced bynitrate nitrogen.Am.J.Bot.
56:187-194.
75. Sozulski,F.,and Buchan,J.A. 1978.Effects of Rhizobium and nitrogen
fertilizer on nitrogen fixation and growth offield peas.Can.J. Plant Sei.
58:553-556.
76. Streeter,J.G. 1974.Growth oftwo soybean shoots ona single root.J. Exp.
Bot. 25:189-198.
77. Syono,K.,Newcomb,W.,and Torrey,J.G. 1976.Cytokinin production in
relation tothedevelopment ofpea rootnodules.Can.J. Bot.54:2155-2162.
78. Syono,K.,and Torrey,J.G. 1976. Identification ofcytokinins inroot
nodules ofthe garden pea, Visum sativum L. PlantPhysiol.57:602-606.
79. Tanaka,A.,and Fujita,K. 1979.Growth,photosynthesis andyield components
inrelation tograinyield of thefield bean.J. Fac.Agr.Hokkaido59:
145-238.
80. Tanner,J.W., and Anderson,J.C 1964.External effectofcombined nitrogen
on nodulation.Plant Physiol.39:1039-1043.
81. Thornton,G.D. 1956.Theeffectoffertilizer nitrogen on nodulation,growth
and nitrogen contentof several legumes grown insandy soils.Proc. Soil and
Crop Sei. Soc.Florida 16:145-151.
82. Valera,C , andAlexander,M. 1965.Reversal ofnitrate inhibition of
nodulation by indolyl-3-acetic acid.Nature 206;326.
83. Wagner,H.,and Michael,G. 1971.Der Einflusz unterschiedlicher Stickstoffversorgung aufdieCytokininbildung inWurzeln von Sonnenblumenpflanzen.
Biochem. Physiol.Pflanz. 162:147-158.
84. Wallace,W.,and Pate,J.S. 1965.Nitrate reductase inthefield pea {visum
awense L.). Ann.Bot.29:655-671.
85. Weber,C R . 1966.Nodulating and non-nodulating soybean isolines. IAgronomic
and chemical attributes.Agron.J. 58:43-49.
86. Weber,E. 1930.Salpeterdüngung als Beeinträchtigung der Stickstoffsammlung
durch Leguminosen.Zentralblatt für Bakt. II82:353-379.
87. Welch,L.F.,Boone,L.V., Chambliss,C G . , Christiansen,A.T.,Mulvaney,D.L.,
Oldham,M.G.,and Pendleton,J.W. 1973.Soybeanyieldswith direct and
residual nitrogen fertilization.Agron.J.65:547-550.
88. Wheeler,C T . ,Henson,I.E.,and'McLaughlin,M.E.1979.Hormones in plants
bearing actinomycetenodules.Bot.Gaz.140 (suppl):S52-S57.
89. Williams,L.E.,and Phillips,D.A. 1980.Effectof irradianceon development
ofapparentnitrogen fixation and photosynthesis in soybean.PlantPhysiol.
66:968-972.
19
90.Wilson,J.K. 1917.Physiological studies ofthe Bacillus radieicola ofthe
soybean {Soja maxPiper)and ofthefactors influencing nodule production.
N.Y. (Cornell)Agr.Exp.Sta.Bul.386:459-507.
91. Wong,P.P. 1980.Nitrate and carbohydrate effects on nodulationand nitrogen
fixation (acetylene reduction)activity of lentil (Lens esaulenta Moench).
Plant Physiol.66:78-81.
92.Zwartz,J.A.,and Hautvast,J.G.A.J. 1979.Food supplies,nutrition and
plant breeding,pp.1-47 in Plantbreeding perspectives (eds.J. Sneepand
A.J.T.Hendriksen)PUDOC,Wageningen.
20
1 ACTUAL AND POTENTIAL NITROGEN FIXATION IN
PISUM SATIVUM L. AND VICIA FABA L
ABSTRACT
Inthis study,actual nitrogen fixationofpea (Pisum sativum L.) and
field bean {vicia faba L,), measuredasthe in vivo acetylene reductionof
nodulated roots,was compared with potential nitrogen fixation,measuredas
the in vitro acetylene reductionofisolated bacteroidsof Rhizobium
leguminosamm. Actual nitrogen fixationofpeaandfield bean growingin
jarsorinthefield equalled potential nitrogen fixation during vegetative
growth.Inthegenerative phase,however,theactual nitrogen fixationwas
much lower thanthepotential nitrogen fixation.
Infield beans supplied with nitrateatsowing,valuesofactual andpotential
nitrogen fixation weremuch lower thaninthoseofcontrol plants untilpod
formation.Actual nitrogen fixationofnitrate-grown plants increased slowly
duetoagradual formationofnodulesonlateral roots. Specific nitrogenase
activitiesofthese nodules were twiceashighascompared tonodulesonprimary
roots.Frompodformation,potential nitrogen fixationofnitrate-grown plants
became equal tothatofplants grown without added nitrate.Asecond nitrate
dressingatmid-pod fill causesaready dropoftheactual nitrogen fixation.
However,potential nitrogen fixation remained constantfortendays before
collapsing.
During growth ofpeaandfield bean,nodule mass increased whereas
the available nitrogenase was only partly utilized. Therefore,inthelong
run,theamountofnitrogen fixed seemstoberegulated bynodule mass
rather thanbynodule activity.
From the data presented inthis paperitisconcluded thatinpeaand
field bean,vegetative growth isnitrogen-limited,whereas during the generative
stage nitrogen fixation islimitedbyaninadequate carbohydrate supply.
21
INTRODUCTION
The question ifgrowthofleguminous crops isrestrained bycarbonor
nitrogen limitation hassince long attractedtheattentionofmany scientists
(e.g. Wilson,17).Inthepresent paper,attention isfocused onthe
nitrogen-fixing capacityofthe lequme-Rhizobium symbiosis,inparticular
onthequestion whether this capacity isrestricted bythe amountof
nitrogenase intheplantorbyother factors,suchasthe energy supplyof
theenzyme.
Quantitative dataonnitrogen fixationbylegume-ff/ziaoMum symbiosesare
usually derived from momentary acetylene-reducing valuesofthe root system
or from long-term changes innitrogen contentoftheplantmaterial.Inthis
study,the in vivo acetylene reductionbynodulated roots (actual nitrogen
fixation)iscompared with the in vitro acetylene reductionbyisolated
bacteroids (potential nitrogen fixation).Inthelatter caseATPandreducing
power are supplied tobacteroids treated with EDTA-toluene (15)toensure
maximum nitrogenase activity. This in vitro acetylene-reducing techniquewas
used earlier (Houwaard,8,9)toassesstheinfluenceofammoniaonnitrogen
fixation bybacteroidsofRhizobium leguminosarum in symbiosis withpea plants.
Uponthe additionofammoniatopeaplants,the 'in vitro nitrogenase activity
ofthebacteroids remained unchanged whereas the in vivo activity declined,
presumably duetoashortageofphotoassimilatesinthe nodules.
Inthepresent investigation the in vitro method was usedtodetermine
whether duringthegrowth cycleofleguminous plantsthepotential nitrogen
fixation differs from the actual nitrogen fixation. Plants usedforthese
tests were grown under optimum conditions comparabletothoseofplants
cultivated inagricultural practice, i.e. insoilandinopen air.Insuch
plantstheinfluenceofcombined nitrogenonactual andpotential nitrogen
fixation hasbeen studied.
MATERIALSANDMETHODS
Plant material
and growth
Enamelled Mitscherlich jarsof6litercapacity,sterilizedwith alcohol,
were filled withamixture (—)of1partofheat-sterilized sandand2parts
of non-sterile clay.Inpreliminary experimentstheclay soil,obtained from
newly reclaimed polder land,hadbeen showntohaveavery low population
22
of indigenous /?. leguminosarton. Theeffective strains PREandRB1of
R. leguminosarvm were usedfor peasandfield beans,respectively,andthe
ineffective strainP8forboth peasandfield beans.Allstrains were
obtained from the culture collectionofour laboratory.Forinoculation,
50mlofa7-daysoldculture wasmixed with6literofsoil.Abasal dressing
of 540mgofPpO,-and900mgofK„0was applied per pot.
Seedsofpea {Pisum sativum I., cultivar Rondo)andfield bean (Vicia
faba L.,cultivar Minica)were surface-sterilizedbyimmersion inasolution
containing4%ofH?0pandafewdropsofTeepol (detergent)for25min
before sowing.Thesoilwasthen covered with sterilized gravel toprevent
clogging after watering. Plants were keptintheopenairunderwirefor
protection against birds.Ineachjarultimately8peaplants or3field
bean plants were retained.
Inaseparate experiment,field bean seeds were inoculated with R. leguminosarum, strain RB1,andsowninafield plotinrowsof45cm.Abasal
2
dressingof18gofP^Ogand29gofKpOwas applied perm.
Hydrogen production and acetylene reduction by nodulated roots
The shootsofthe plants were detachedandtheroots freed from soil
particlesbygentle shaking.Thewhole root system,orinsome casesthe
primaryandthelateral roots separately,were incubated instoppered
1-literErlenmeyerflasks.After25min,a100-ylgassample was assayed
for hydrogen,usingathermal conductivity detector. Subsequently, 100ml
of acetylene was addedanda100-ylgassample was taken after 15min.
Ethylene production was determined withagas Chromatograph equipped with
a hydrogen flame detector.Ratesofhydrogen productionandacetylene
reduction were linearwith timeupto80min.
Nitrogenase determinations in bacteroids
In vitro nitrogenase activityofbacteroids was determined withthe
EDTA-toluenemethod (EDTA=ethylenediaminetetra-acetic acid)accordingto
van StratenandRoelofsen (15).After completion ofthe in vivo assay, roots
werewashed intapwaterandthenodules carefully collected. These nodules
were squeezed inaBergersen press (2)under argonat0°Cinabuffer
solution containing TRIS (50mM) (TRIS=tris(hydroxymethyl)aminomethane),
MgCl 2 (2.5 mM),4%(^) PVP(PVP=polyvinylpyrrolidone)andNa 2 S 2 0 4 (20 mM).
ThepHwas adjustedto7.2with HCl.Bacteroids were centrifuged for10minat
5500xg under argon,washed withabuffer solution (same buffer withoutPVP),
23
centrifugea again,andresuspended inthebuffer solution withoutPVP(1 ml
of buffer per80mgofnodule fresh weight).
Nitrogenase activityofthe bacteroids was restoredbyregenerationat25°C
for30min.Subsequently,1mlofbuffer containing 1ymoleofEDTAand3drops
of toluene was addedto1mlofbacteroid suspension,andthemixture vigorously
shakenfor1min.After1minofsettlement,a0.5-ml aliquotofthe aqueouslayer
was transferred toa16.5-ml Hungate tube finally containing50ymolesofTrisHC1,15ymolesofMgClp, 18.4ymolesofcreatine phosphate,5.6ymolesof
adenosine-5-triphosphate (disodium salt),30ygofcreatine kinaseand20ymoles
of Na 2 S 2 0.,inavolumeof1.0ml,pH7.2.Thegas phase consistedof10%
acetyleneinargon. Ethylene production wasmeasuredbyflame detection after
10min incubationonashaker bathof25°C,200strokes per min.
RESULTS
Nitrogenase activity duping ontogenesis
Profilesofactual andpotential nitrogen fixationbyfield beansandpeas
growing injars during ontogenesisareshown inFig.1.Infield beans,there
wasapeakinnitrogenase activity coinciding withthestartofthe pod-filling
stage.Nosuchapattern was observed inpeas. Bothinfield beansandinpeas,
potential nitrogen fixation was equal tothe actual fixation duringthe vegetative stage.Starting from flowering inpeasandfrompodformation infield
beans,thepotential nitrogen fixation became significantly higher thanthe
actual fixation inaone-sided t-testatthe0.001 level(14).
Actual nitrogenase activityperplantinfield beanswas about five times
higher thaninpeas.Thedifference was mainlyduetothehigher nodule weight
of the field bean-Rhizobium symbiosis (Fig.2a),resulting from the higher plant
weightoffield beans.Theamountofbacteroid protein infield bean nodules
decreased duringthegrowth season from90to40mgpergofnodule fresh weight
(Fig.2b).Peanodules retainedaconstant level ofabout40mgofbacteroid
protein pergofnodule fresh weight (Fig.2b).
Based uponthe dataofFigs.1,2aand2b,thespecific activityofnitrogenasewas calculated (Fig.2c).Inpeaplants,thedecreasing specific
24
100
120
Days
Fig.1.Actual (•,•)andpotential ( Q ,o)nitrogenase activityofpea( B , 0 )
and field bean (•,o)during ontogenesis.Plotted valuesofactual
nitrogen fixation aremeansof2setsof8plants each (pea)andof
2 setsof3plants each (field bean). Valuesofpotential nitrogenase
activityperplantwerecalculated from nodule fresh weightof3plants
and triplicate nitrogenase determinations inbacteroids obtained from
a known quantityofnodules.Arrow1indicatesthestartofflowering
inpeaandarrow2thestartofpodformation infield bean.
activity was compensatedbyanincreasing nodule mass,resulting inaconstant
levelofin vivo nitrogenase activity duringthegrowth season (Fig. 1). In
contrast,thepeakofnitrogenase activityperplantinfield beanswasdue
toariseofspecific activityaswellastoanincreased nodular mass.
Effect of combined nitrogen
To study theeffectofcombined nitrogenonactualandpotential nitrogen
fixation,field beans growinginjarswere given 1130mgofNasNaNO,both
atsowingandatmid-pod fill (Fig.3 ) .Actualandpotential nitrogen fixation
ofnitrate-grown field bean plantsweremuch lowerthan thoseofplantsgrown
without added nitrate, e.g.,atday621.2and12.4umolesofC^Hg reduced/
plant.h (Figs.3and1,respectively).From thebeginningofpodformationat
25
_2000
Ü 1000-
100
120
Days
Fig.2(a).Nodules,fresh weight,mg/plant.Pea( O ) , field bean (o);
meansofduplicate determinations,
(b). Bacteroidalproteinperunitoffresh weight nodules.Meansof
duplicate determinationsofpea( O )andfield bean(o).
(c). Nitrogenase,specific activities,ymolesC2H4/gofbacteroidal
protein.h;mean valuesoftriplicate determinations,ofpea
{in vivo •,in vitro O )andfieldbean (in vivo •, in vitro o).
For startofflowering (pea)andofpodfill (field bean) see
Fig.1.
day 77,potential nitrogen fixationofnitrate-grown plantsapproached thatof
control plants.Actual nitrogen fixationofnitrate-grown plants roseata
lower rate,sothat thedeviation from the potential nitrogen fixation became
more pronounced thaninthecontrol plants.Forexample,atday89actual
nitrogen fixation amountedto29.2ymolesofC^H« reduced/plant.hascompared
to 36.3ymolesofC 2 H~ reduced/plant.hincontrol plants,whereas potential
nitrogen fixation valueswere 48.8ymolesofC„H 2 reduced/plant.hinboth cases
(Figs.1and 3 ) .
26
100
120
t Days
N
Fig.3.Actual (•)andpotential (o)nitrogenaseactivityoffield beans
supplied with 1130mgNasNaN03atsowingandatmid-pod fill (arrow)
For sample sizesseelegendofFig.1.
The second nitrate dressingatmid-pod fill drastically lowered theactual
nitrogen fixation (Fig.3)butleftthepotential fixation unaffectedfor10
days.After that,analmost complete lossofactivity took placewithin4days.
Location of nitrogenase
activity
Additionofnitrateatsowing delayed nodule formationbyfield beans,as
demonstrated inTable 1.Themorphologyoftheroot systemsoffield bean plants,
which possessawell-developed taproot,enabledustodifferentiate between
nodulesonprimaryandlateral roots.Owingtothe delayinnodule formation,
significantly more nodules were formedonlateral rootsofnitrate-grown plants
as comparedtoplants grown without nitrate(p<0.05).This resulted ina
considerably larger contributionofthe activityofnodulesonlateral roots
to total nitrogenase activityinnitrate-grown plants than found incontrol
plants without added nitrate (p<0.01). Acetylene-reducing activityoflateralroot nodules,when calculated per unitofnodule fresh weight,washigher than
27
Table1.Actual nitrogenase activitiesofnodules from primaryandlateral roots
offield beans,asaffectedbytheadditionofnitrateatsowinga^
Time
(days
after
sowing)
Noduleson
primary roots
Noduleson
lateral roots
Freshwt. Nitrogenase
(mg/plant) activity b)
Freshwt.
Nitrogenase
(mg/plant) activity b)
Without nitrate
62
515
75
881
77
973
89
1570
1380
98
Contributionof
nodulesonlateral
rootstototal
nitrogenase
activity(%)
14.9
18.4
26.5
19.9
17.3
0
36
85
306
139
36.9
35.5
16.5
28.0
10
14
14
12.4
22.2
20.3
17.5
78
222
353
776
995
16.3
12.4
32.1
29.9
22.4
100
49
56
79
91
With nitrate
62
75
77
89
98
0
235
407
295
125
Data represent meansofduplicatemeasurements.Forstatistical inference
seetext.
b)umolesofC„H./gofnodules freshweight.h.
x I2O0
c
' I 1000
r
o. 800
I 600
E
*
400
^r
X
IM
o
200
60
80
100
120
Days
Fig.4.In vivo (•,A)andin vitro (o,A)specific activitiesofnitrogenase
ofbacteroidsfrom primary (•,o)andlateral (A,A)rootnodulesof
field bean plantsgrownwithoutadded nitrate.Values representmeans
ofduplicatedeterminations.
28
Table2.Nitrogenaseactivityinpeaandfieldbeanduringtheentiregrowth
period
Nitrogenase activity
Actual {in vivo)
Potential
in vitro)
Product ion Fixât ion
Production
Fixation
of:
of:
of:
N2
H2
C2H4
(mmoles /
plant)
1
2
of:
N2
N2 C 2 H 4
(mg/
plant)
3
4
(mmoles/
plant)
5
Actuala s%
of poten tial
N„ fixât ion
(mg/
plant)
6
7
8
Experiment I
Pea
Field bean
Field bean
grown with
11
40
18
4 106
15 374
4 167
65 14
235 48
134 35
132
447
324
81
84
51
50
53
41
24
31
11 227
13 291
127 37
174 45
346
416
66
70
37
42
N03
Experiment II
Field bean
Field bean
(jar)
(field)
1)Inair+10%C ? H 2
2)Inair
3)Calculatedfrom1
4)Calculatedfrom1minus2
6)Calculatedfrom5
7)Valuesof3as%ofthoseof6
8)Valuesof4as%ofthoseof6
thatofprimary-rootnodulesinanFtestatthe0.05level (Table1). Whenthe
nitrogenaseactivitieswereexpressedperunitofbacteroid protein,differences
betweennodulesofprimaryandlateral rootsweremoresignificant(p<0.01)
(Fig.4 ) .Specificnitrogenaseactivityofnodulesfromlateral rootswas
often50-100%higherthanthatofprimary-rootnodules.
Actual and potential nitrogen fixation
Baseduponthemomentarydataofnitrogenaseactivity,aspresentedabove,
theamountsofnitrogenfixedduringtheentiregrowthperiodwerecalculated
(Table 2). Undertheconditionsofthein vivo nitrogenaseassaywith 10%of
acetyleneinair,acetylenereduction representstheentirenitrogenase
activity.However,inair,intheabsenceofacetylene,afractionofthe
29
total electron flowthrough nitrogenaseisnotusedinnitrogen fixationbut
gives risetotheproductionofhydrogen.Therefore,inthecalculationsof
nitrogen fixation,ethylene production wascorrectedfor hydrogen evolvedin
air.Astoichiometrical factorof3.0was usedtoconvertacetylene reduction
intonitrogen fixation.Forsimplicity,nitrogen fixation wasassumedto
continueatthesame levelfor24haday.
Actual nitrogen fixation amountedto51-84%ofthe potential nitrogen
fixation inthecases studied.Whenthedistributionofenergy among hydrogen
productionandnitrogen reduction occurring in vivo was taken intoaccount,
theactual nitrogen fixation even declinedto37-53%ofthe potential nitrogen
fixation (Table2 ) .
The dataonnitrogen fixation discussed above,were obtained from plants
growinginjars.Inaseparate experiment field-grown field bean plantswere
testedfor actualandpotential nitrogen fixation (Table 2 ) .Ingeneral,
field-grown plants produced more drymatter (not shown) andpossesseda
higher levelofnitrogen fixation than plants growinginjars.Theratioof
potential toactual nitrogen fixation,however,was equal inbothcases.
DISCUSSION
Actual and -potential nitrogen fixation
The central themeofthis paperisthedistinction between actualand
potential nitrogen fixation,ascalculated from in vivo andin vitro nitrogenaseactivities,respectively.Thelimitationsofthe in vivo acetylene
reduction assayofnodulated roots have been extensively discussedbyvarious
authors (5,'10). Therefore,this discussionisrestrictedtothe in vitro
method.
Amajor disadvantageoftheEDTA-toluene techniqueofassaying in vitro
nitrogenase activityofisolated bacteroidsisitssensitivitytooxygen.
Even tracesofoxygen irreversibly lowernitrogenase activity.Removingthe
plants from thesoil might havedamagedthe nodules.
Explainingthedifference between in vivo andin vitro nitrogenase activity
asaresultofscratchingtothenodulesisunlikely because oxygen probably
decreases both valuesofthesamedegree.
The conditionsoftheEDTA-toluene method havebeen carefully optimized(15),
30
Nevertheless,the questionmayberaised whether the in vitro activity measured
in thiswayreallyisthemaximum activity,orthatmaximum activityiseven
higher,asitwas recently shown that electron donorstonitrogenasemaydiffer
in efficiency (16).However,our in vitro method was adequatetodemonstrate that
starting from flowering,anexcess amountofnitrogenase ispresent,presumably
not utilized innitrogen fixation (Figs.1and 2c).
To date,indirect evidenceforanexcessofnitrogenase inroot nodules
has been derived from Arrhenius plotsofnitrogenase activity versus temperature
(5,6 ) .Our direct nitrogenase determinations corroborate this conclusionfor
plantsinthe generative growth phase only.Theassumption that below 20°C
nitrogenase content was limiting nitrogen fixation (6)wasnotsubstantiated
inthepresent studyinwhich field beans growing injars were compared with
those growing inthe field.Injars,soil temperatures were almost equalto
ambient airtemperature (15-30°C),whileinthe fieldat10cmdepth,soil
temperatures ranged from 13-18°C. However,injar-grownaswellasinfieldgrown plants,potential nitrogen fixation was higher than actual nitrogen
fixation (Table2 ) .
Additionofnitratetofield beans with full-grown nodulesatmid-pod fill
inducedanimmediate drop ofin vivo nitrogenase activity,butthe in vitro
activity remained unchanged,conformabletotheresultsofHouwaard (8)with
added ammonium chloridetopeas. Finally,the in vitro nitrogenase activity
collapsed within4days (Fig. 3 ) ,inaccordance with Bisseling et al. (3).
To their estimates,nitrogenase turn-overinR. leguminosarwn bacteroids
amountsto50%within2days.
Nodule mass and
activity
Drymatter production inpeaplants was about five times lower than that
in field beans (datanotshown). This difference was also observed innodule
fresh weightofthese plantsandinnitrogenase activityperplant (Figs.1,2a).
Additionofnitrateatsowing mainly reduces nodule massandnotnitrogenase
activityofthe nodules (Table 1).Some authors have found thatanenhancementofphotosynthesisbyincreasing light intensityoratmospheric carbon
dioxide concentration initially increasedthenitrogenase activityofthe
nodules.However,after several daysoftreatment,ariseofthe nodular mass
accounted forthemajor partofthe increased nitrogen fixation (1,11,12).
Thus,inthelong run,nitrogen fixation seemstoberegulatedbynodule mass
rather thanbynodule activity. Nodule formationandgrowth even continue
when energy supplytothenodules isinsufficient forafull utilizationof
nitrogenase (Figs. 2a,c).
31
The specific {in vitro) nitrogenase activitiesofnodulesonprimaryand
lateral roots show pronounced differences (Fig.4 ) .VandenBos et al. (4)
demonstrated that the in vitro nitrogenase activityofmature bacteroids
decreased with age.Inthepresent experiment,lateral-root nodulesmayhave
contained young bacteroidsofahigher degreeofnitrogen-fixing activityas
compared t.othebacteroidsofprimary-root nodules,whichwould accountfor
thehigher in vivo andin vitro activityoflateral-rootnodulesascompared
with primary-root nodules.
nitrogen or carbohydrate
limitation
The in vitro methodofdetermining nitrogenase activity inbacteroidsis
based uponanoptimized supplyofATPandreducing equivalents (15).Therefore,
ifthe in vitro activityishigher than the in vivo activity,the difference
maybeinterpreted intermsofanenergy shortageofthenodules.This view
iscorroborated bycomparingthepresent results with literature data.During
thegenerative stageofgrowth,flower primordiaanddeveloping podsactas
carbohydrate sinks,reducingthetranslocationofphotoassimilatestothe
rootnodules (7).Inthe present experiments,thepotential nitrogen fixation
deviated from theactual fixation from the onsetofflowering (pea)orpod
fill (field bean) (arrows1and2,respectively,ofFig. 1).Additionof
nitrate also causesadeclineofphotosynthate translocation tothenodules(13).
Concomitantly,theratioofthe actual tothepotential nitrogen fixationis
lowerinnitrate-grown plants thaninplants growing without combined nitrogen
(Fig.3 ) .
Ifourinterpretationofthe physiological significanceofpotential nitrogen
fixation iscorrect,then leguminous plantsaregrowing under nitrogen-limitation duringthevegetative stage.Duringthegenerative stage nitrogen fixationislimitedbycarbohydrate supply leadingtoexcessofnitrogenasein
thenodules.Duringthegrowth cycleofthe legumestheaverage nitrogenase
utilization amountedto66-81%intheabsenceofcombined nitrogen (Table2 ) .
Therefore,inordertoincreaseyieldsof ~\&q\me-Rhizobium symbioses,efforts
should probablybedirectedtotheutilizationofthe entire potential nitrogen
fixationbytheenhancementofphotosynthesis,rather thanbycreating Rhizobium
strainsofahigher nitrogen-fixing capacity.
32
REFERENCES
1.Antoniw,L.D.,andJ.I.Sprent. 1977.Growthandnitrogen fixationof
Phaseolus vulgaris L.attwoirradiances. II.Nitrogen fixation.
Ann. Bot.42:399-410.
2. Bergersen,F.J. 1966.Some propertiesofnitrogen-fixing breis prepared
from soybean root nodules.Biochim.Biophys.Acta 130:304-312.
3.Bisseling,T.,J.vanStraten,andF.Houwaard. 1980.TurnoverofnitrogenaseandleghemoglobininrootnodulesofVisum sativum. Biochim.
Biophys.Acta 610:360-370.
4. Bos,R.C.vanden,T.Bisseling,andA.vanKammen. 1978.Analysisof
DNA content,nitrogenaseactivityandin vivo protein synthesisof
Rhizobium leguminosarum bacteroidsonsucrose gradients.J.Gen.Microbiol.
109: 131-139.
5.Hardy,R.W.F.,R.D. Holsten,É.K.Jackson,andR.C.Burns.1968. The
acetylene-ethylene assay forN2fixation:laboratoryandfield evaluation.
Plant Physiol.43:1185-1207.
6. Hardy,R.W.F.,andU.D.Havelka. 1976.Photosynthateasamajorfactor
limiting nitrogen fixationbyfield-grown legumeswith emphasisonsoybean,p.421-439. In P.S.Nutman (ed.), Symbiotic nitrogen fixationin
plants,IBP7,Cambridge University Press,Cambridge.
7. Herridge,D.F.,andJ.S.Pate. 1977.Utilizationofnet photosynthatefor
nitrogen fixationandprotein production inanannual legume.PlantPhysiol.
60: 759-764.
8. Houwaard,F.1978.Influenceofammonium chlorideonthenitrogenase activity
ofnodulatedpeaplants {visum'^ativum).
Appl. Env.Microbiol.35:1061-1065.
9.Houwaard,F.1980.Influence of'^ammoniumandnitrate nitrogenonnitrogenase
activityofpeaplantsasaffectedbylight intensityandsugaraddition.
PlantandSoil 54:271-282. ,.:
10.Masterson,C.L.,andP.M. Murphy';1980.Theacetylene reduction technique,
p. 8-33. In N.S.SubbaRao(ed.), Recentadvancesinbiological nitrogen
fixation,E.Arnold London.
11.Phillips,D.A., K.D.Newell,S.A. Hassell,andC E . Felling. W6. The
effectofCO2enrichmentonrootnodule developmentandsymbfptic N2
reduction inVisum sativum L.Amer.J.Bot.63:356-362. /">'
12. Sheikholeslam,S.N., K.A. Fishbeck,andD.A. Phillips. 1980.Fjfectof
irradianceonpartitioningofphotosynthatetopearoot nodules.
Bot. Gaz. 141:48-52. •
13. Small,J.G.C.,andO.A. Leonard. 1969.TranslocationofC -labeled photosynthateinnodulated Tggumesasinfluencedbynitrate nitrogen.Amer.J.
Bot. 56:187-194. * •
14. Steel,R.G.D.,andJ.H. Torrie. 1960.Principlesandproceduresof
statistics,McGraw-Hill New York.
15. Straten,J.van,andW.Roelofsen. 1976.Improved methodforpreparing
anaerobic bacteroid suspensionsofRhizobium leguminosarum for the acetylene
reduction assay.Appl.Env.Microbiol.31:859-863.
16. Veeger,C ,Laane,C ,Scherings,G., Matz,L.,Haaker,H.andZeelandWolbers,L.van. 1980.Membrane energizationandnitrogen fixationin
Azotobacter
vinelandii
and Rhizobium
leguminosarum,
pp 111-137 in Nitrogen
fixation vol.I(eds.W.E.NewtonandW.H.Orme-Johnson)Un.Park Press,
Baltimore.
17. Wilson,P.W. 1940.The biochemistryofsymbiotic nitrogen fixation,Univ.of
Wisconsin Press,Madison.
33
ACTUAL AND POTENTIAL NITROGEN FIXATION BY PEA
ROOT NODULES AS AFFECTED BY CYTOKININ AND
ETHYLENE
ABSTRACT
In this paper,the in vivo nitrogenase activity (actual nitrogen fixation)
ofnodulated pearoots {visum sativum L.)hasbeen compared with the in vitro
nitrogenase activity (potential nitrogen fixation)ofbacteroidsoftwo strains
of Rhizobium leguminosarwn,one withandonewithout hydrogenase.Theactual
nitrogen fixationofnodulesof28daysoldplants,keptinagrowth chamber,
amounted to77-82%ofthepotential nitrogen fixation.Treatmentofthenodules
orthelower leaveswith benzyladenine,asynthetic cytokinin,raisedthe
nitrogenase activityofpeaplantsto100%ofthepotential value.Ethephon,an
ethylene-releasing compound,raisedthe nitrogen fixationofpeaplants only
whenitwas supplied tothenodules.Thebeneficial effectofboth ethephonand
benzyladenineonnitrogen fixation was maximal after2daysanddecreased afterwards.
Additionofnitratetothe nutrient solution (final concentration 10mM)
decreased the actual nitrogen fixationofthe nodulesto55%ofthepotential
nitrogen fixationofthe bacteroids within48hours. Simultaneous additionof
benzyladeninetothenodules counteracted the nitrate effect,raisingtheactual
nitrogen fixation to81%ofthe potential value.
Treatment with benzyladenine raised theenergy supplyto thenodules,as it
was shown intranslocation experiments with assimilated ^CCL.Noquantitative
relationship between energy supplyandnitrogenase activity was found.
INTRODUCTION
Until now,little attentionhasbeen paidtothe question whether allor
only partofthenitrogenase presentinRhizobium bacteroidsofleguminous root
nodulesisactive during growthofthelegume under various environmental conditions.
35
Inthis paper,the in vivo nitrogenase activityofrootnodules (actual
nitrogen fixation)wascompared with the in vitro nitrogenase activity (potential
nitrogen fixation)ofisolated bacteroids,supplied withATPandreducing
equivalents.InapreviousChapter (Ch.1,pages 21-33),thecourseofthenitrogenaseactivity during ontogenesisofpeaandfield beanwas reported. The
results obtained indicated thatinthevegetative stageoflegumes,growingin
theopen air,nitrogenase was fully utilized,butinthegenerative stageactual
nitrogen fixation amountedto51-84%ofthepotential nitrogen fixation.
Energy supplyoftheenzymeisconsideredtobeamajor factor regulating
actual nitrogenase activity (19).Therefore,inthis paperanattemptwasmade
toaltertheratioofactual topotential nitrogen fixationbytreatments that
eitherwould lower photosynthate supplytothenodules (additionofnitrate)or
would enhance photosynthate availabilitytothenodules,suchaslocal applicationofplantgrowth regulators.Theratioofactual topotential nitrogen
fixation might alsobeinfluencedbyamore efficient useoftheenergyavailable,suchasfunctioningofahydrogen uptake systeminthebacteroids.This
system enablesthebacteroidstorecoverpartoftheenergy lostby
hydrogen production concomitantlytonitrogen fixation.Therefore,alsoa
comparison wasmadeofnitrogenase utilizationofpea plants inoculated with
strainsofRhizobium leguminosarum withorwithoutanuptakehydrogenase.
MATERIALSANDMETHODS
Plant
material
Pea seeds {Pisum sativum L.cultivar Rondo)were sterilizedbyimmersionin
a4%solutionofHLOpandafewdropsofTeepol (adetergent).After20min,
seedswere sowninheat-sterilized gravel which was inoculated withastrainof
Rhizobium leguminosarum. Strain S310a,containinganuptake hydrogenase,and
strain PRE,withoutahydrogenase,were obtained from theculturecollectionof
our laboratory.Thegravelwassoaked before sowing withanitrogen-free nutrient
solution containing (mg/1oftapwater)K 2 HP0 4 .3H 2 0,360;KH 2 P0 4 , 120;MgS04.7H20,
250; CaS0 4 .2H 2 0,250;Fe-citrate,30;MnS0 4 .4H 2 0,1;ZnS0 4 .7H 2 0,0.25;CuS04.5H20„
0.25;H 3 B0 3 ,0.25;Na 2 Mo0 4 .2H 2 0,0.05. Plantsweregrowninagrowth chamberat
20°C,witha16hlight-8hdark periodandalight intensity (W/m)of8.8
(blue),7.2(red)and12.0 (far-red).
At21days after sowing,plantswere transferredto300-ml Erlenmeyerflasks
containing 100mlofthe nutrient solution plus75mgofKCl.Each flaskcon-
36
tained4plants, supported byaplugofcotton wool, keeping theroot nodules
abovethenutrient solution.
Treatments
Five days after transferring theplantstoErlenmeyer flasks,theroot nodules
or the firstandsecond leaves (from the bottom)were treated with 1ml/4 plants
ofanaqueous solutionofbenzyladenine (10, 100and1000 ug/ml,pH9with KOH)
or ethephon (2-chloroethylphosphoric acid, 250and1000 ug/ml); both solutions
contained atraceofTeepol. Control plantsofthe benzyladenine experiment
received 1mlofa670 yg/ml solutionofadenine under similar conditions.In
some cases, the nitrogen-free nutrient solution was replaced byasolution
containing KN0, (10mM) instead ofKCl.Nutrient solutions were renewed daily.
Hydrogen production and acetylene reduction
Setsof4intact plants were placed inastoppered 300-ml Erlenmeyer flask
and assayedforhydrogen production after20min,usingagaschromatograph
equipped withathermal conductivity detector. Subsequently,30mlofacetylene
was addedandethylene production determined after 15minbyagaschromatograph
equipped withahydrogen flame detector.Therateofacetylene reductionwas
linear for60min.
Invitro nitrogenase assay of bacteroids
Bacteroids were isolated from nodules after the in vivo assayasdescribedby
van StratenandRoelofsen (16).Isolated bacteroids were treated with ethylenediaminetetraaceticacidandtoluene, supplied with reducing equivalents (Na^SpOJ
andanATP-generating system,andincubatedat24°C underanatmosphereof90%
argonand10%acetylene. Ethylene production was determined after 10min.
RESULTS
Actual nitrogenase activity as affected by ethephon and benzyladenine
Local application ofethephontoroot nodulesofpeaplants markedly affected
nitrogenase activity after2days,asshown inFig.1.When65ygofethephon
was applied per plant, nitrogenase activity was enhanced significantly (Ftest;
p<0.05). However,when nodules were treated with 250ygofethephon perplant,
nitrogenase activity was significantly lowered (p<0.05)and the nodules turned
green. Treatment ofthe first and second leaves (from the bottom)didnotproduce
significant changes innitrogenase activity.
Application ofbenzyladenine significantly raised nitrogenase activity (Ftest;
p<0.05) both when applied tonodulesortothe firstandsecond leaves (Fig.2 ) .
37
150
300
Ethephon,/ng/plant
Fig.1
2.5
25
250
Benzyladenine,A4g / p l a n t
Fig.2
Fig.1.Nitrogenase activityof28daysoldpeaplants inoculated with
R. leguminosarvm PRE.Data representmeansoftriplicate
measurementsofsetsof4plants,48hafter applicationof
ethephontothefirstandsecond leaf from the bottom (0)or
to nodules (o).
Fig.2.Nitrogenase activity
of28daysoldpeaplants inoculated with
Data representmeansoftriplicate
measurementsofsets
of4plants,48hafter applicationof
benzyladeninetothe firstandsecond leaf from the bottom(0),
tonodulesofplants
growingonanitrogen-free nutrient
solution (o)andto
nodulesofplants transferred toanutrient
solution containing 10mmolesofKNO,per1(•). Control sets
were given 150ygof
adenineperplant.Control plants without
adenine (not shown) possessedanequal nitrogenase activityas
comparedtoadenine
treated control plants.
R. leguminosarum PRE
The siteofapplication didnotproduce significant differences.Theeffect of
benzyladenineonnitrogenase activity was dependentonconcentration.
The highest values were reached when250ygofbenzyladenine was usedper plant.
Increasing concentrationsto500ygperplant lowered nitrogenase activity (data
not shown).
Bothinthe experimentsofFigs.1and2,nitrogenase activity was measured
2 days afterthe applicationofethephonandbenzyladenine,respectively.In
preliminary experiments,amaximum response was obtained 2days after treatment
with growth regulators.After4days,noeffectsonnitrogen fixation were
observed incomparison with thecontrol plants.Arepeated applicationof
38
benzyladenine,4days aftertheinitial treatment,again raised nitrogenase
activityofplants growing without combined nitrogen afteranincubation period
of24hours.
Inplants,transferred toanutrient solution containing 10mmolesofKNO,
per 1,nitrogenase activity was significantly reduced (p<0.05)(1.06and 1.42
ymolesofC ^ produced/plant.hbyplants withandwithout nitrate,respectively).
Treatmentofthe nodules with benzyladenine counteracted thenitrate effectby
increasing nitrogenase activity (1.55 ymolesofC 2 H.produced/plant.h with
250yg/plantofbenzyladenine).
Actual and potential
nitrogen
fixation
Thewayinwhich benzyladenineandnitrate treatments affect nitrogenaseactivity was studied withtwo R. leguminosarum strains,S310aandPRE,bycomparisonofthe actual nitrogenase activityofnodulated rootsandthepotential
nitrogenase activityofisolated bacteroids,thelatter being supplied with
reducing equivalents (Na 2 S 2 0jandanATP-generating system.Theratioofthe
actual tothepotential nitrogen fixation wasdistinctly influenced bythetwo
treatments,asshowninTable 1.Untreated peaplants utilized thenitrogenase
Table1.EffectofstrainofB. leguminosarum, benzyladenineandnitrate
treatmentsonactualandpotential nitrogen fixation,photosynthate supply
and relative efficiencyofnitrogen fixationbypeaplants'!)
Treatment
Strain
of
microsymbiont
S310a
PRE
Nitrogenase activity
(ymolesofC2H4
produced/g nodules
fresh wt.h)
Ratioof
actualto
potential
nitrogenase
activity
Actual
Potential
{%)
Control
BA
Control
BA
16.3 d
21.9 a
27.9 b
34.1 a
21.1 a
21.9 0
100
N05
NOJ+BA
19.0 od
33.9 a
35.1a
34.6 a
82
97
55
27.7 b
34.2 a
81
11
Nodule label 2) Relative
(kcpm/plant) efficiency
(*)3>
94 I
125 m
111 lm
155 n
61 k
72 k
95p
95 p
71 q
60 r
68 q
62 r
Values represent meansoftriplicate measurements,2days after treatment
of nodules with 250yg/plantofbenzyladenine (BA)ortransferofplantsto
a nutrient solution containing 10mmolesofKNOo/1. Values followed bythe
same letter arenotsignificantly different inTukey's testatthe5%level.
2)
Forexplanation see text.
^Calculatedas(1-H 2 evolved inair
C 2 H 4 producedair+10%C 2 H 2 '- ldU/o U 4 ;
39
present in the bacteroids foronly 80%.Benzyladenine (250yg per plant) raised
theactual nitrogen fixation to 100%or nearly 100%of the potential values.
Inplants supplied with nitrate,the ratio ofactual topotential nitrogen
fixation dropped to 55%.Simultaneous application of benzyladenine to nodules
of-such plants raised the actual nitrogen fixation aswell as the ratio actualpotçntial nitrogen fixation to values similar to those of control plants without
combined nitrogen (Table1).
Inorder tocheck whether the favourable effect of benzyladenine on nitrogenase activity wascoupled with an increased photosynthate supply of the nod14
ules,a pulse-label experiment with C0owas carried out.Shoots of pea plants
14
were exposed in light to C0 2 for 15min,24hafter the start of the nitrate
and benzyladenine treatments. Roots were carefully excluded from labelling by
sealing themwith plastic foil and beeswax.Plants were killed in liquid nitrogen
24hafter labelling, i.e. 48hafter the start of the treatments,to ascertain
a possible correlation with the nitrogenase data obtained from other sets of
plants.Root nodules were crushed in liquid nitrogen and aliquots counted ina
liquid scintillation counter,using Instagel (Packard Inc.)as a scintillation
liquid.
The results of this experiment show (Table 1)thatwith strain PRE,more
radioactivity was recovered in the nodules than with strain S310a. Benzyladenine
treatment ofnodules gave a pronounced rise of recovered radioactivity with both
S310a and PRE.Benzyladenine (250yg per plant) increased nodule counts permin
significantly in2outof 3treatments.Specific radioactivity ofnodules was
equal to that of shoots,whereas that of roots was 3-5 times lower (data not
shown). Nitrate in the nutrient solution significantly lowered nodule counts per
min ascompared to thecontrol.
The nitrogenase activity of nodules formed with strain S310a,which contains
an uptake hydrogenase,responded to benzyladenine similarly to that of nodules
formed with strain PRE (Table 1).The presence of the hydrogenase in S310a
resulted in ahigher relative efficiency than with strain PRE, i.e. fewer moles
of hydrogen evolved inair permole of ethylene produced under acetylene.With
strain PRE,adecrease of the relative efficiency was observed when thenitrogenase activity was increased by benzyladenine treatment,both in the presence
and absence of nitrate in the nutrient solution.
The ratio ofactual to potential nitrogen fixation with S310a was slightly
below thatwith PRE.The lower actual nitrogen fixation with S310a was due to
a lower potential nitrogenase activity of the bacteroids. Expressed on a protein
40
basis, in vitro nitrogenase activity amounted to365 ymolesofC 2 H- produced/g
of protein.h inS310a bacteroids,ascompared with 732 ymolesofC^H. produced/g
of protein.h inPRE bacteroids.
DISCUSSION
Mode of action
of benzyladenine
and
ethephon
Plant growth regulators belonging tothe cytokinin group,asbenzyladenine,
are knowntoattract assimilates tothe site where the hormone isapplied. They
reduce protein degradation (10),increase ionuptakeandtransport,andinduce
cell division (8,17).
Ethephon,anartificial compound, isdegraded within theplanttoethylene
atpHvalues above 3.5. Ethylene mobilizes storage products,directs themto
the siteoffruit growthandpromotes fruit ripening andsenescence (15).
In this study, benzyladenineandethephon were used inanattempt toalter
photosynthate availability tothe nodules which might influencetheratioof
actual topotential nitrogen fixation. Fairly large quantitiesofbenzyladenine
were usedascompared toliterature dataonleaf application (11).Nodata were
availableonlocal application ofgrowth regulators tonodules.Asnodulesare
considered tobeimpermeable towater, the uptakeofbenzyladenineandethephon
probably hastooccur throughtheroot cortex. With our methodofbrush applicationofanaqueoussolution tothe nodules, inevitably also adjacent root parts
were treated. However, both with leaf and nodule treatments,the quantitiesof
growth regulator actually takenupare unknown.
In line with'literature data (1),the observed effectofethephononnodules
(Fig. 1)might beexplained intermsofmobilizationofassimilates. Consequently,
a low concentration ofethephon would stimulate nitrogenase activity bymobilizing
storage products (e.g. starch).Ahigh concentration ofthis'growth regulator
initially might have enhanced nitrogen fixation, but would lateroncausean
exhaustionofsubstrates andconsequently adecreaseofnitrogen fixation: Treatmentofnodules with benzyladenine may enhancethesink functionofthe nodules
and stimulate nitrogenase activity duetoahigher supplyofphotosynthates
(Fig.2,Table1).
Lower leaves have been showntoactassourceofsubstratestonodules(11,13).
Application ofethephon orbenzyladenine tothese source leaves was aimedatincreasing photosynthate supplyofthe nodules inanindirect way. Ethephonwas
thoughttomobilize storage carbohydrates intheleaves,making them available
41
for translocationtothenodules.Evenifthiswould have happened,noeffectof
itonnitrogenase activity was observed (Fig. 1).Benzyladenine treatmentofthe
source leaves was intendedtoreduce protein degradationandthustoenhance photosynthesisintheleaf.Alternatively,thetranslocation pattern intheplant
couldbechanged,diverting photosynthate transportation from the shoot apexto
the nodules.Ineither way,theobserved riseinnitrogen fixation after leaf
application ofbenzyladenine islikelytobeduetoanincreased supplyof
photosynthatetothe nodules.
Actual and potential nitrogen fixation
Benzyladenine application tonodules increasedtheactual nitrogenase activity
(Table 1).This phenomenon was correlated withanincreaseinenergy supply.
However,noquantitative relationship couldbeestablished betweentheadditional
photosynthates translocated tothe nodulesandtheriseinnitrogenase activity.
The same difficulty was encountered byother authors (7,14);itmightbe
attributed tothesynthesisofstorage products,totheaccumulationofassimilatesintheplant tissueofthenodules (7)ortodifferences inrespiratory
activityofthe nodules,leading toasmaller pooloflabelled compounds.
A considerable gap exists betweenthe occurrenceofmaximum nitrogenase
activity,48hafter treatment with benzyladenine,andthe translocation time
14
of Clabelled photosynthate (3-9 h)(6). Oneexplanationofthis deviation
mightbethe occurrenceofuptake problemsofexternally applied growth regulatorsasdiscussed above.
Onacellular level,benzyladenine isknowntoincrease respiration,proton
extrusionandtransmembrane potential inpeaplants (8,9,10).Ontheother
hand,Laane et al. (5)showed thatinbacteroidsofR. leguminosarum, transmembrane potential wasactingasamajor force regulating nitrogenase activityby
directing theelectron flowtonitrogenase.Combining these results,theeffect
of benzyladenineonnitrogenase activity mightbeexplained byahigher supply
of photosynthates,anenhanced respiration,leadingtoahigher transmembrane
potential and,consequently,ahigher nitrogenase activity.
Combined nitrogen decreasedtheratioofactual topotential nitrogen fixation
(Table 1 ) ,aswasfound earlierinthis laboratory (3;Chapter 1,pp.21-33)
Additionofsucrosetothenutrient solution alleviated theeffectofcombined
nitrogenonnitrogenase activity (4)partlybyovercomingashortageofenergy
inthe nodules butalsobydecreasing nitrogen uptake.Inourexperiment,a
decreaseofnitrate uptake was unlikely becauseingeneral,benzyladenine stimulatesionuptakeandtransport.However,the benzyladenine effectonphotosyn-
42
thate supply (Table1)waslowerinnitrate-grown plantsascompared toplants
growing inanitrogen-free nutrient solution.
The possibilityofincreasing nitrogen fixation bytheuseofcytokininsand
other growth regulators (especially those synthetized inroots),asindicated
bytheresultsofthepresent investigation,might open interesting perspectives
toagriculture iftheshort durationofthehigher activityandtheimpractical
application sitemightbeovercome.Another approach wouldbetoincreasethe
rateofnatural cytokinin synthesisinroot nodules.The main cytokinin activity
ofnodules seemstobelocated inthenodule meristem, Rhizobium-infected cells
possessingalower activity (17).Butalso free-living rhizobia have been shown
tobecapableofsynthetizing cytokinin-like substances insufficient quantity
to induce cell division inasoybean-callus test (12). Whether also bacteroids
produce such substances isunknown.However,bacteroids might influence
photosynthate supplytothenodules either directlybyexcretionofcytokinins,orindirectly bytriggering offsynthesisofcytokinins inthe
plant(18).
REFERENCES
1.Abeles,F.B.,andL.E.Torrence. 1970.Temporal andhormonal controlof
31,3glucanase inPhaseolus vulgaris L.Plant Physiol.45:395-400.
2. Evans,H.J., D.W. Emerich,T.Ruiz-Argiieso,S.L. Albrecht,R.J. Maier,
F. Simps,andS.A. Russell. 1978.Hydrogen metabolism inlegume nodules
and rhizobia: some recent developments,p.287-306. In H.G. Schlegeland
K.Schneider (eds.),Hydrogenases,their catalytic activity, structure
and function,E.Goltze,Göttingen.
3.Houwaard, F.1978.Influenceofammonium chlorideonthenitrogenase
a c t i v i t y of nodulated pea plants {Pisum sativum).
35: 1061-1065.
Appl. Envir. M i c r o b i o l .
4. Houwaard,F.1980. Influenceofammoniumandnitrate nitrogenonnitrogenase
activityofpeaplantsasaffectedbylight intensityandsugar addition.
PlantandSoil 54:271-282.
5.Laane,C ,W.Krone,W.N. Konings,H.Haaker,andC.Veeger. 1979. The
involvementofthe membrane potential innitrogen fixationbybacteroids
of Rhizobium leguminosarum. FEBS Letters 103:328-332.
6. Latimore,M,Jr.,J.Giddens,andD.A. Ashley. 1977.Effectofammonium
and nitrate nitrogen upon photosynthate supplyandnitrogen fixationby
soybeans.Crop Science 17:399-404.
7. Lawrie,A.C.,andC.T.Wheeler. 1973.Thesupplyofphotosynthetic assimilatestonodulesofPisum sativum L.inrelationtothefixationof
nitrogen.NewPhytol. 72:1341-1348.
43
8. Marre, E.,P. Lado,F.,Rasi-Caldogno,and R. Colombo. 1973. Correlation
between cell enlargement in pea internode segments and decrease in the
pH of the medium of incubation. II.Effects of inhibitors of respiration
oxidative phosphorylation and protein synthesis. Plant Sei. Letters
1: 185-192.
9. Marre, E.,P. Lado,A. Ferroni,and A. Ballarin-Denti. 1974. Transmembrane
potential increase induced by auxin, benzyladenine and fusicoccin.
Correlation with proton extrusion and cell enlargement. Plant Sei. Letters
2: 257-265.
10. Mothes,K. 1960.Lieberdas Altern der Blätter und die Möglichkeit ihrer
Wiederverjüngung. Naturwissenschaften 47:337-351.
11. Pate,J.S. 1966. Photosynthetizing leaves and nodulated roots as donors
of carbon to protein of the shoot of field pea (Piswn arvense L . ) .
Ann. Bot. 30:93-109.
12. Phillips, D.A., and J.G. Torrey. 1972.Studies on cytokinin production by
Rhizobium. Plant Physiol. 49: 11-15.
13. Schubert, K.R., and H.J. Evans. 1976.Hydrogen evolution, amajor factor
affecting the efficiency of N fixation in nodulated symbionts. Proc.Natl.
Acad. Sei. U.S.A. 73: 1207-1211.
14. Sheikholeslam,S.N., K.A. Fishbeck, and D.A. Phillips. 1980. Effect of
irradiance on partitioning of photosynthate to pea root nodules. Bot. Gaz.
141: 48-52.
15. Stoddart, J.L., and M.A. Venis. 1980.Molecular and subcellular aspects of
hormone action, p.445-510. In J. Mac Millan (ed.), Hormonal regulation
of development I,Encyclopaedia of plant physiology vol.9, Springer, Berlin.
16. Straten, J. van,and W. Roelofsen. 1976.An improved method for preparing
anaerobic bacteroid suspensions of Rhizobivm leguminosarum for the
acetylene reduction assay. Appl. Envir. Microbiol. 31:859-863.
17. Syono, K., W.Newcomb, and J.G. Torrey. 1976.Cytokinin production in
relation to the development of pea root nodules.Can. J. Bot. 54:2155-2162.
18. Syono, K., and J.G. Torrey. 1976. Identification of cytokinins in root
nodules of the garden pea, Piswn sativum L. Plant Physiol. 57:602-606.
19. Yates,M.G.,and R.R. Eady. 1980.The physiology and regulation of nitrogen
fixation, p.88-120. In N.S.Subba Rao (ed.), Recent advances in biological
nitrogen fixation, E.Arnold, London.
20. Zeroni,M., and M.A. Hall. 1980.Molecular effects of hormone treatment,
p. 511-586. In J. Mac Millan (ed.), Hormonal regulation of development I,
Encyclopaedia of Plant Physiology, vol.9, Springer, Berlin.
44
ALTERNATIVE, CYANIDE-RESISTANT, RESPIRATION IN
ROOT SYSTEMS OFPISUM SATIVUM L. AS AFFECTED
BY STRAIN OFRHIZOBIUM LEGUMINOSARUM AND
NITRATE SUPPLY
ABSTRACT
A study has been made of the a l t e r n a t i v e , cyanide-resistant, respiratory
pathway in non-nodulated pea plants {Pisum sativum L . , c u l t i v a r Rondo) as well
as in pea-Rhï-zobiumsymbioses of d i f f e r e n t n i t r o g e n - f i x i n g capacity. Alternative
respiratory a c t i v i t y was low in root systems of 26-days old non-nodulated or
nodulated plants in the absence of n i t r a t e , regardless of the n i t r o g e n - f i x i n g
capacity of the nodules.
Supply of the plants with n i t r a t e (10 mM) raised the respiration of the root
system due to a l t e r n a t i v e - r e s p i r a t i o n a c t i v i t y . In non-nodulated root systems
respiration rate was doubled, 48 h a f t e r the supply of n i t r a t e , and a l t e r n a t i v e
respiration increased to 60% of the electron flow to oxygen. In nodulated root
systems, the nitrate-induced enhancement of the a l t e r n a t i v e respiration was
inversely related to the n i t r o g e n - f i x i n g capacity.
Carbohydrate levels in i n e f f e c t i v e l y nodulated plants were higher than those
in e f f e c t i v e l y nodulated plants in the absence of n i t r a t e . When n i t r a t e was
s u p p l i e d , carbohydrate levels f e l l concomitantly with the r i s e in a l t e r n a t i v e
r e s p i r a t i o n . In n i t r o g e n - f i x i n g p l a n t s , they remained unchanged upon the addition
of n i t r a t e .
The response of growth rate and the induction of n i t r a t e reductase to n i t r a t e
supply were lower with plants lacking the capacity to f i x nitrogen than in n i t r o gen-fixing piants.Nit r a t e supply reduced nitrogenase a c t i v i t y both in highly and
moderately e f f e c t i v e Rhizobiwn s t r a i n s . Upon n i t r a t e supply, hydrogen production
by nitrogenase decreased more rapidly than t o t a l nitrogenase a c t i v i t y , leading
to an increased r e l a t i v e e f f i c i e n c y . I n h i b i t i o n of the a l t e r n a t i v e r e s p i r a t i o n
of isolated bacteroids led to a sharp decline of nitrogenase a c t i v i t y due to
oxygen i n a c t i v a t i o n .
45
Pea plants with and without nitrogen f i x a t i o n , responded to added n i t r a t e
by increased a l t e r n a t i v e respiration consuming 6-10%and 24-43%, respectively,
of the carbohydrates supplied to the roots by the shoots w i t h i n 48 h. A main
function of a l t e r n a t i v e respiration in pea roots seems to be the degradation of
excess carbohydrates supplied by the shoot following a growth stimulus by the
supply of n i t r a t e . As a l t e r n a t i v e respiration was also found in pea plants with
a high n i t r o g e n - f i x i n g capacity upon the supply with n i t r a t e , i t is concluded
that these plants were growing under nitrogen l i m i t a t i o n .
From respiration measurements of detached nodules i t is concluded that
u t i l i z a t i o n of the a l t e r n a t i v e pathway in these nodules decreases the amount
of ATP generated with 8-21% of the maximum ATP production from glucose. Theref o r e , the a l t e r n a t i v e respiration should be taken into account when calculating
energy requirements of nitrogen f i x a t i o n , based on the differences in r e s p i r ation between n i t r o g e n - f i x i n g and non-nodulated n i t r a t e - u t i l i z i n g plants.
INTRODUCTION
In plantmitochondria,analternative respiratory pathway is knowntofunction
inadditiontothe cytochrome pathway (BahrandBonner, 1973a,b).This alternative pathway isinsensitivetocyanide butcanbeinhibitedbyhydroxamic
acids.The alternative respiratory pathway branches from the cytochrome pathwayatubiquinone (Storey, 1976),which easily transfers electronstothecytochrome pathway,butwhich reduces the first carrierofthealternative pathway
only whenitisinitslargely reduced state (Bahrand Bonner, 1973b). Thealternative pathway isgenerallyassumedtoyieldnoATP during electron transferto
oxygen (Solomos,1977).Inthis aspect,alternative respiratory activitymay
be regardedasa 'wastage'ofenergy.
Inarecent review,Lambers (1980)suggested thatthe alternative routemight
functionasan'overflow mechanism'togetridofexcessive amountsofcarbohydrates,not utilized for growth,maintenanceorformationofstorage products
in plants.AccordingtoDeVisser and Lambers (1979),rootsofnodulated,
nitrogen-fixing peaplants possessavery lowalternative-pathway activity.In
contrast,alternative respiration inrootsofnon-nodulated peaplants grown
on ammoniaornitrate,amountedto25-50%oftotal respiration (Lambers, 1980).
These results show thatnitrogen sourceandnitrogen supply are important factors
indetermining respiratory efficiency inlegumes.
46
In experimentswith nodulated legumes,the quantityofcarbohydrates supplied
totherootswas estimated byPateandco-workerstoamountto55-74%ofthecarbohydrates generated innetphotosynthesis.Nodulesacquired 32%ofthenet photosynthateinVisum sativum (MinchinandPate, 1973). Twelve percentofthenet
photosynthate was usedinnodule respiration formaintenanceandnitrogen fixation;theremainder wouldbeusedforsynthesisandtransportofamino acids.
In spiteofthe large quantitiesofcarbohydrates suppliedtonodulated roots,
a lowalternative respiratory activity was found (DeVisserandLambers, 1979).
Duetothehigh costsofenergy fornitrogen fixationandsynthesisofamino
acidsnoexcessofcarbohydrates isgenerated which wouldberespired viathe
alternative pathway.
In ordertofind outwhetherarelation exists between alternative respiration
and nitrogen fixation,inthepresent paper this typeofrespiration was determined innon-nodulatedandnodulated root systemsofpeaplantswith different
nitrogen-fixing capacityand with different relations between growth, storage
and respiration.Also separated nodulesandbacteroids were investigated.The
influenceofthenitrogen sourceonrespiration,growthandnitrogen fixation
was analyzed inaseriesofexperiments inwhich nitrate was addedtonodulated
and non-nodulated plants.Thedataon'energy loss'bythe alternative respiration were compared with thedataonenergy costsofnitrogen fixationand
hydrogen productionbynitrogenase.
ABBREVIATION: SHAM (salicylhydroxamic acid).
MATERIALS AND METHODS
Plant
growth
Seedsofpeaplants {visum sativum L.cultivar Rondo)were sterilizedby
immersion inanaqueous solutionof4%HpO«andafewdropsofTeepol (detergent).
After20min, seeds were sowninsterilized gravel orperlite,whichwas
inoculated withastrainofRhizobium leguminosarum. Allstrains used inthese
experiments were obtained from theculture collectionofthe Laboratoryof
Microbiology, Wageningen.
The plantswerewatered withasterile nitrogen-free nutrient solution containing(mg/loftapwater) K 2 HP0 4 > 360;KH 2 P0 4 , 120;MgS0 4 .7H 2 0,250;CaS0 4 ,250;
Fe(III)citrate,30;MnS0 4> 4H 2 0,1;ZnS0 4 .7H 2 0,0.25;CuS0 4 .5H 2 0,0.25; H 3 B0 3 ,
0.25;Na2Mo04.2HoO,0.05. Plantswere keptinagrowth chamberatalight/dark
periodof16/8hat20°Candarelative humidityof70%.At21days after sowing,
47
they were transferred to300-ml Erlenmeyer flasks,containing 100mlofnutrient
solution plus KCl (10 mM). Experimental sets consisted of4plants supportedby
a cotton wool plug.Insome experiments,the nutrient solution was replacedby
a solution containing KNO, (10mM) insteadofKCl,6days after transferofthe
plants.
Plant
respiration
Measurements started 5days after transferofthe plantstothe water culture.
Respiration was determined according to^LambersandSteingröver(1978).Root systems
were detached from the shootsandfitted intoa300-ml vessel equipped witha
CIark-typeelectrodeandamagnetic stirrer.The vesselwasfilled withanairsaturated nutrient solution plus10mmolesofKClorKNO,per1,but without
Fe(III) citrate,andsealed withamixtureofequal partsofvaseline and
beeswax. Respiration measurements were performed inawater bathat20°C. Linear
ratesofoxygen consumption were monitored foratleast 10min.Then the solution
was replacedbyanequal one containing inaddition 25mmolesofSHAM per1,and
oxygen consumption measured again for10min.
Respirationofroot nodules,30-50mgoffresh weight,was determined
polarographicallyinaYSImagnetic-stirrer bathat20°C. Nutrient solutions
of the composition asdescribed above were saturated with 0 2 before usein
ordertoimprove linear response.
Baateroid respiration
Nodules were detached from the rootsandsqueezed inaBergersen press
(Bergersen, 1966)inabuffer containing 50mmoles ofTris(hydroxymethyl)-aminomethane,2.5mmoles ofMgCl-^HpO, 300 mmoles ofsucroseand4%byweightof
polyvinylpyrrolidone(PVP),in11ofdemineralized water. ThepHwas adjustedto
7.2 with HCl before use.The nodule brei was centrifuged at5500xgfor10min
and the supernatant (nodule cytosol)used inrespiration measurements. The
bacteroids (pellet)were washed inthesame buffer without PVPandresuspended
toaconcentration of80mgofnodule freshwtpermlofbuffer. Respiration
measurements were carried outinaYSI magnetic-stirrer bathat20°C, using
succinate (10mM)asthe substrate.- Inhibitor constants (apparent Ki)were calculated from the reciprocal ofrespiratory rate against concentration ofSHAM
(0.1-10 mM) (Bahr and Bonner, 1973a).
Hydrogen production and acetylene reduction
Detached rootsof4plants were incubated inastoppered 300-ml Erlenmeyer
flask. After20min,a100pigassample was takenandhydrogen production
determined withagas Chromatograph equipped withathermal conductivity detector
48
Subsequently,30mlofacetylenewasaddedtotheflaskandafter15mina100pi
gas samplewasanalyzed forethylene production byhydrogen-flame detection.
Nitrate reductase
Nitrate reductasewasassayed inthefirst unfolded leaf fromthetopusing
NADHasanelectron donor,asdescribed byStulen (1974). Leaf extracts were
incubatedat27°CinthepresenceofNADHandKNO,. Nitrite productionwas
determined colorimetrically after30min,usingthesulphanilamidemethod (Snell
and Snell, 1949).
Chemical analyses
Ethanol-solublecarbohydrates ('sugars')were determined bytheanthrone
method (TrevelyanandHarrison,1952)inethanol-extracted plantdrymatter.
The residuewasheatedat100°Cfor2hwith0.1NHCl.Thecontentofethanolinsolublecarbohydrates ('starch')wasmeasured inthesupernatant usingthe
anthrone method.
Plant nitrogen wasdetermined bytheKjeldahl method,using CuSO,andSe
ascatalysts.
RESULTS
Alternative respiration and nitrogen fixation
Pea-Rhizobium symbioses with different nitrogen-fixing capacity were obtained
by inoculation ofvisum sativum L.(cultivar Rondo)with different strainsof
Rhizobium leguminosarum. AsshowninTable 1,these symbioses displayed different
properties with regardtonodule formation,hydrogen productionandacetylene
reduction,andgrowth.
Table1.Characteristics of pea-Rhizobium symbioses,28days after sowing.
Values representmeansof7determinationsofsetsof4plants.Valuesin
onecolumn followedbythesame letterarenotsignificantly differentin
Tukey's testatthe0.05level
Strain
of
microsymbiont
C2H4 production
(umoles/g
nod frwt.h)
Nonnodulated
0
a
P8
0
0
S313
S310a
PRE
PF2
11.1 b
14.1 b
28.1 a
25.1 a
H2production
(umoles/g
nod frwt.h)
Relative
efficiency
0 e
0 e
5.5c
1.6 d
10.3 b
14.0 a
—
W
—
50
89
63
44
Nodule
fresh
wt (mg/
plant)
Plant
nitrogen
(mg/
plant)
Shoot
0d
54 0
197 a
56e
107 b
102 b
4.9 0
5.8e
10.6 b
9.8 b
12.2 b
16.1 a
1.5
1.6
1.7
1.9
1.8
2.0
to
root
ratio
49
With strain S310a,theratioofhydrogen evolved inairtoethylene produced
inairwith 10%acetylenewaslowerthan with other strains owingtothepresence
inthis strainofahydrogenase thatenabledtheutilizationofH ? producedby
thenitrogenase system (Table 1).Assuming full utilizationofhydrogen takenup
byhydrogenase-containing rhizobial strains,SchubertandEvans (1976)calculated
relative efficiencies accordingtotheformula (1- 2 r ^ ^ V " ^ i r ).100%.
Thusahigh relative efficiency indicates thatlittle energyislostinhydrogen
productionbynitrogenase activity.
Respiration measurementsofroot systemsofthese symbioses were performedat
26daysafter sowing (Table 2). Root systemswithoutanitrogen-fixing symbiosis
generally showedalower respiration rate than thosewith suchasystem.Asignificant difference between total respirationandcytochrome (SHAM-resistant)
respirationwasfound onlyinthesymbiosis RondoxPF ? ,which also possessed
the highest shoottoroot ratio (Table 1).Withtheother R. leguminosarum strains
no significant differenceswerefound,inspiteofthedistinctly different
nitrogen-fixation parameters.
Table2.Total andSHAM-resistant respirationof26-daysoldpearoot
systems grownwithout nitrate.Values representmeansof3setsof
4 plants.Values followedbythesame letterarenotsignificantly
differentinTukey's testatthe0.05 level
Strain of
microsymbiont
Non-nodulated
P8
S313
S310a
PRE
PF2
Respirât ion
(mg09/gdrywt.h)
Total
SHAM-resistant
(1)
(2)
3.8 d
3.0 ef
4.7 ab
4.8 ab
3.9 cd
5.1 a
3.5 de
2.9ƒ
4.5 ab
4.4 ba
3.9 od
4.4 ba
Ratio
(1:2)
1.1
1.0
1.0
1.1
1.0
1.2
Effect of nitrate supply
Alternative
respiration.
Aspeaplantswithalow nitrogen-fixing capacity were
likelytogrow under nitrogen limitation,aseriesofexperimentswascarried
outinwhich nitrogen limitationwasovercomebytheadditionofnitrate.
Immediately aftertheinitial respiration measurementsatday26,theplants
were transferredtoanutrient solution containing 10mmolesofKN0 3 instead
of 10mmolesofKClper1.
50
As shown inFig. 1a.,alternative respiration was generated within 24hafter
the supply ofnitrate.An analysis ofvariance showed that the ratio of totalto
SHAM-resistant respiration increased more sharply innon-nodulated plants than
innodulated plantswith a lownitrogen-fixing capacity,which intheirturn
produced ahigh ratio ascompared with nodulated plantswith ahigh nitrogenfixing capacity (Ftest,p< 0.01). Thealternativerespiratory activitywas
generated inaddition tothecytochrome (SHAM-resistant)respiration (Fig. 1b)
Fig. 1a.Ratio oftotal to SHAM-resistant respiration ofroot systems ofpea
plants (non-nodulated,V; inoculated with R. legum-inosarum strains
P8,•; S313,A;PRE,D ;and P F 2 , 0 )asaffected bynitrate supply
(10mM) (day 0,26days after sowing). Values aremeans of9determinations of sets of4plants from 3experiments.For statistical
interference seetext.
Fig. 1b.Total (V,0) and SHAM-resistant(•,•)respiration (mg02/gdrywt.h)
of root systems of pea plants (non-nodulated,V,•and inoculated
with strain P F 2 Ô , # )as affected by nitrate supply (day 0,26days
after sowing). Valuesaremeans of3determinations ofsets of4
plants,from oneexperiment out ofthe3experiments ofFig.1a.
51
- 80
S
60
S
.c
•o 4 0
o
o
20
E
2
Days
Fig.2
3
Days
Fig.3
Fig.2.Ethanol-soluble(•, A,+)andethanol-insoluble (o,A , 0 ) carbohydrates
(mgglucoseeq./g drywt)inrootsofpeaplants inoculated with
R. leguminosarum strainsP8(•,o ) ;S313 (A,A)andPF2(•,O ) as
affectedbynitrate supply (10mM)(day0,26days after sowing).
Valuesaremeansoftriplicatedeterminations.
Fig.3.Nitrate-reductase activity (ymolesNO^/gfrwt.h)inthefirst unfolded
leaffromthetopofpeaplants,inoculated with R. leguminosarum
strainsP8(•), S313 (A)andPF 2 ( O ) , asaffectedbynitrate supply
(10mM) (day0,26days after sowing). Values aremeansofduplicate
determinationsof4leaves.
Carbohydrate content. Intheineffective symbiosis (P8),carbohydrate levels
in rootsaswellasinshootswere much higher thanineffective symbioses
(Fig.2,roots only).Ontheadditionofnitrate,however,both ethanol-soluble
and ethanol-insoluble carbohydrates droppedtothelevelsofeffectively nodulated plantswithin48and72h,respectively.Carbohydrate levelsinnitrogenfixing plants remained unaffectedbynitrate supply.
Nitrate reductase. Synthesisofnitrate reductaseinleaveswas inducedby
nitrate (Fig.3 ) .Amore rapid synthesiswas observed inleavesofplantswith
a high nitrogen-fixing capacity. Growth ratesofplantswithout nitrogen fixation
respondedtonitrate2-3days after transfertothenitrate-containing nutrient
solutions andthus lagged behindthesynthesisofnitrate reductase.
52
20
E '0
„80-
» 40
er
2
3
Days
Fig. 4a.Ethylene productionofpeaplants inoculated with R. legwninosarum
strains S313 (A),PRE( D ) ,andPF 2 ( O ) , asaffected bynitrate
supply (10mM) (day0,26days after sowing).
Fig. 4b.Relativeefficiencyofpeaplants inoculated with R. leguminosarum
strains S313 (A)andPRE( D )asaffectedbynitrate supply (10mM)
(day0,26days after sowing).
Nitrogenase activity.
Transferofnodulated peaplantstothenutrient solution
with nitrate (10mM)causedadecreaseofnitrogenase activityinsymbiotic
associations withahighaswell asalownitrogen-fixing capacity (Fig.4a).
Hydrogen production decreased more rapidly than acetylene reduction, resulting
inariseoftherelative efficiency (Fig.4b).
Alternative respiration in baoteroids
Although SHAM-sensitive respirationhasbeen described inmitochondriaof
higher plants (Solomos, 1977),itbecame interestingtocheck whether notonly
the plant partofthenodules butalsothebacteroid fraction contributestothis
typeofrespiration. Respirationofnodule cytosol (nodule brei minus bacteroids)
was sensitivetoSHAM,asshowninTable 3,butSHAM-sensitive respirationwas
also presentinbacteroids supplied with succinate (10mM). Respiration ratesof
53
Table3.Inhibitor constants (apparent Ki)ofKCNandSHAMinR. leguminosarum
bacteroidsandnodule cytosol.Values representmeansof6determinations.
Values followed bythesame letter arenotsignificantly different inTukey's
testatthe 0.05 level
Strainof
microsymbiont
Nodule
cytosol
(mM SHAM)
Bacteroids
(mM SHAM)
Bacteroids
(mM SHAMinthe
presenceof
0.8mMKCN)
Bacteroids
(mM KCN)
P8
S313
PRE
N.D.
N.D.
9.4 ha
27.1 a
25.7 a
15.8 b
4.4 a
N.D.
4.9 a
2.1 q
N.D.
0.2p
N.D.=NotDetermined
bacteroidsofstrain PRE,capableofnitrogen fixation,were higher butK^ values
were lower than thoseofbacteroidsofstrainP8lackingthe capacitytofix
nitrogen.
i
20
l
i
l
I
/
15
-
10
5
0
-
-
•
•
20
t
40
60
80
Û2 i n p u t , / n m o l e s / m i n
Fig.5.Ethylene production (nmoles/mg protein.min)bybacteroidsofR. leguminosarum PREasaffected by02 inputandconcentration ofSHAM (0,0,
12.5mM,Aand25mM,A)For experimental conditionssee text.
54
Nitrogen fixationwas absent under the experimental conditionsofTable3»
duetooxygen inactivation. Therefore,the functionofthe alternative respiration innitrogen fixation was investigated inaseparate experiment,inwhich
respiration ofPREbacteroids was measured under nitrogen-fixing conditions
(Fig. 5).Bacteroids were incubated under varying oxygen tension inthepresence
of reduced myoglobinandbovine serum albumine,using succinate (20mM)as the
substrate,asdescribedbyLaane et al. (1978). With SHAM (12.5 mM), respiration
wasmuch lowerascompared tothe control,resulting inalowermaximumof
nitrogenase activityatalower oxygen input (cf.Fig. 5,Table3 ) .
DISCUSSION
Alternative respiration and nitrate supply
In rootsofpeaplants growing without nitrate,alternative respirationwas
only found inplants inoculated with R. leguminosarum strain PF„,whichhada
high growth rateandahigh nitrogenase activity (Tables 1and2 ) .Although
the symbiosis with PREwasasactiveasthatwith PF„ regarding N„ fixation,
its activity startedanumberofdays later,ascanbeseen from its lower yield
of plant nitrogen. When nitrate was supplied,thegrowth ratesofall plants
were enhanced andalternative respiration was generated (Fig. 1).This happened
in addition tothe cytochrome-1inked respiration (Fig. 1b;see also Bahrand
Bonner, 1973b). Alternative respirationofnitrogen-fixing plantswasstimulated
by nitrate supplytoamuch lower extent than thatofplants without nitrogen
fixation (Fig. 1).Thelatter plants,whichhadbeen growing under nitrogen
limitation,contained high levelsofcarbohydrates that declined (Fig.2)
beforeagrowth response became manifest.With theineffective strain P8, the
ratiooftotal toSHAM-resistant respiration decreased tothe valuesofthe
effective strain after the excessofcarbohydrates hadbeen decreased (Figs 1a
and 2 ) .Therefore, the degradation ofexcess carbohydrates isprobablyafunctionofthe alternative respiratory pathway,assuggested byLambers (1980).
According tohis 'overflow'-hypothesis,alternative respiration isgeneratedif
thereisanimbalance between the carbohydrates supplied bytheshootandthe
carbohydrate requirementsofthe roots for growth,maintenance, storageor
osmoregulation.Asalternative respiration was also generated uponthesupply
of nitratetopeaplants withahigh nitrogenase activity, this would mean
that under theexperimental conditions employed, these plants werenotgrowing
underClimitation butprobably underNlimitation.
55
Alternative respiration and carbohydrate metabolism
Based uponthedataonrespiration,carbohydrate levelsandplant growth,
as presented inthe Results section,thecontributionofthe alternative
respirationtothe carbohydrate metabolismofthepeaplantwas calculated
(Table 4 ) .Tocalculate theamountsofsubstrate usedascarbon skeletonsfor
the synthesisofcell material,acoefficientof1.4gwas usedtoconvertg
ofdry weight produced intogofglucose utilized (PenningdeVries, 1974).
Carbohydrate supplyoftherootbytheshootwas calculated fromthe
substrate usedintherespirationoftherootandinthesynthesisofroot
material. This value therefore doesnotincludethe amountofcarbohydrates
recirculated totheshootinamino acids,which was estimatedat14%oftotal
carbohydrate supplyofthe root in cowpea (HerridgeandPate,1977)and 20%
in pea(MinchinandPate, 1973).Toconverttherespiration data from
oxygen consumed perhinto glucose consumed perday,arespiratory quotient
of1.0was assumedanddiurnal variation was neglected.Inarecent paper,
Lambers et al. (1980)observed diurnal variationsinrootsofLupinus albus L.
bothincarbon dioxide productionandoxygen consumption.Therefore,the
conversion employed inthepresent study mustbeconsidered asarough estimate.
Net photosynthesis was determined from the substrate used inthesynthesisof
shootmaterial andintranslocation tothe root.
Innitrogen-fixing plants,bothnetphotosynthesisandcarbohydrate supply
ofthe rootswere higher thaninplants without nitrogen fixation owingtothe
improved nitrogen supply (Table 4 ) .Although carbohydrate supplyofthe roots
is increasedbyadded nitrate,the distribution pattern intheroot systemis
altered (SmallandLeonard, 1969), resulting inless carbohydrates being
suppliedtothe nodules.This explains themoderate decreaseofnitrogenase
activityduetothesupplyofnitrate (Fig.4 ) .Similarly,Bethlenfalvay et al.
(1978b)found that photosynthesis was enhancedbythe additionofammoniato
non-nodulated peaplants.With different Rhizobium strains,photosynthesiswas
positively correlated with nitrogen fixation (Bethlenfalvay et al. 1978a).
The alternative respirationofthe root system was activeincatabolizing
1-7%ofthecarbohydrate supplyóftherootwhentheplants were growing
without nitrate (Table 4 ) .With nitrate,however,theproportionofthe
alternative respiration incarbohydrate catabolismwas much higher (5-11%in
nitrogen-fixing plantsand15-31%innon-nodulatedorineffectively nodulated
plants). Therefore,thealternative respiration shouldbetaken into account
in calculationsofenergy requirementofnitrogen fixationandnitrate reduction,
based upon respiration measurementsofnitrogen-fixingandnitrate-reducingplants.
56
Table 4. Proportion of the a l t e r n a t i v e respiration in carbohydrate metabolism
of pea plants (non-nodulated or infected by a s t r a i n of R. leguminosarum),
growing without n i t r a t e (day 0, 26 days a f t e r sowing) and 48 h a f t e r the supply
of n i t r a t e (10 mM; day 2 ) . For explanation see t e x t .
Item
Non-nodulated
P8
S313
PRE
1a.Total root
5.0
8.5
5.3 7.0
9.0 9.8
7.8 8.7
respiration
(mg0?/gdry
wt.h)
(cfFig.1b)
1b.Alternative
8
52
4
31
4 20
2
10
respirationas
%oftotal
respiration
(cf Fig. 1a)
2.Shootdrywt
164
179
174 190
260 354
241 298
(mg/plant)
3.Rootdrywt
109
112
108 112
157 176
133 148
(mg/plant)
4.Carbohydrate
15
36
supplyof
rootbyshoot
(mgglucose/
plant.day)
(la+3)
5.Netphoto19
60
synthesis
(mgglucose/
plant.day)
(2 + 4 )
6.Alternative
respirationof
therootsystem
a.as%of
7
31
carbohydrate
supply(1b)
b.as %ofnet
5
19
photosynthesis(1b)
17
23
37
70
42
69
60 130
3
15
3
11
2
8
2
6
43
57
79 113
1
1
5
3
57
Alternative respiration and nitrogen fixation
Nitrogen fixation was calculated from ethylene production underairwith 10%
ofacetylene (representing total nitrogenase activity)minus hydrogen production
inair,representing that partofnitrogenaseactivity notutilized innitrogen
fixation (Table 5). Thecarbohydrate requirementofnitrogen fixationwas
calculated from respiration measurementsofdetached nodules.Theresulting
valuesof3.1-4.2mgofcarbon utilized permgofnitrogen fixed with strain
PREcorrespondtothevaluesof4.1mgC/mgNinpeanodules (MinchinandPate,
1973)and2-5mgC/mgNincowpea nodules (HerridgeandPate,1977).The
largeamountofnodular tissue formedwith strain S313enhancedthe overall
costofnitrogen fixationto6.3-11.1mgC/mgNowingtoahighernodule
respirationatequal amountsofnitrogen fixed.
Energy costsoftotal nitrogenase activitywere expressed intermsofmoles
ofATP derived from nodule respiration permoleofnitrogen fixed (Table5 ) .
Onemoleofglucosewasassumedtoyield36molesofATPwhen respired viathe
cytochrome pathwayascompared with 12moleswhenthealternative pathwaywas
utilized.Thepossible influenceofwound respiration wasneglected,sothat
the reported valuesmustbeconsideredasrough estimates.Thevaluesofmoles
ofATP utilized permoleofnitrogen fixed areconsiderably higher thanthe
valueof28mol ATP/mol NpasreportedbyEvans et al. (1981),asinourcase
also nodulemaintenance respirationisaccounted for.
A calculationoftheamountofATP 'lost'byhydrogen productionsimultaneouslywithnitrogen fixationwas based upon theassumption that productionof1
moleofhydrogen requires7molesofATP (Pate et al., 1981).TheamountofATP
'lost'intheproductionofhydrogenisofthesameorderofmagnitudeas
compared withtheamountofATP lostbythealternative respiration with strain
S313,producingalargeamountofnodular tissue.However,with strainPRE
considerably more ATPwas lostinhydrogen production thanbyuseofthealternative pathway (Table5 ) .
Alternative respiration was found notonlyintheplant partofthenodule
(cytosol),butalsointhebacteroids (Table 3 ) . Theinhibitorconstantsfor
SHAMinbacteroidswerealmost 50-100 times higherascomparedtothose reported
from plantmitochondria (TomlinsonandMoreland,1975;HenryandNyns, 1975).
Althoughtthe alternative oxidaseisless sensitivetoSHAMwhen succinateis
usedassubstrate,permeability difficultiesinaddition mightexplain this
difference. Similartothedata obtained withmitochondria,anincreased
electron fluxvia thealternative pathwayyieldedalowerK-forSHAM {e.g.
presenceorabsenceofKCN;PREincomparisontoP8) (Wedding et al. 1973).
58
Table5.Costsofnitrogenfixationin pea-Rhizobium symbiosesgrowingwithout
nitrate(day0,26daysaftersowing)and48hafterthesupplyofnitrate
(10mM;day 2). Forexplanationseetext.
P8
Item
2
0
1.Production(ymoles/
plant.day)of(cfTable1)
a.ethylene
b.hydrogen
c.nitrogenfixed(1a,b)
2. a. Total respiration of
nodule (mg glucose/
plant.day)
b. A l t e r n a t i v e nodule
respiration as %of
total
c.Totalnodulerespiration
as %ofcarbohydrate
supplyofroots
(Table4;2a)
0
0
0
1.0
1.0
38
6
5.Overallcostsofnitrogen
fixation (molesofATP/
molesN2fixed)
a.actualATPproduction(4a,1c)
b.assumingfullphosphorylationefficiency (noalternativerespiration)(4a,b;1c)
72
36
12
5.3
36
72
145
49
2
0
50
21
10
7.5
19
23
12
11
6.3
3.Overall costsofnitrogen
fixation (mgCrespired/
mgN)(1c,2a)
4.ATP(ymoles/plant.day)
a.productioninnodules(2a,b)
b. 'lost'inalternative
respiration(2b)
c. 'lost'inhydrogen
production(1b)
2
0
0
0
0
52
PRE
S313
11.1
67
39
9
2.6
26
3.1
49
23
9
2.5
11
4.2
927
135
1270
230
424
89
467
37
252
147
273
161
77
89
131
155
46
55
54
58
59
Treatment ofnitrogen-fixing bacteroidswith SHAM,final concentration
12.5mM,significantly reduced nitrogenase activity (Fig.5)by decreasing
respiration.As an inhibition ofalternative respiration does not involvea
decrease inATP production during electron transfer from succinate to oxygen
(Storey and Bahr, 1969),this decreasemight point to arespiratory protection
asa possible function ofthealternative respiration in bacteroids.The
alternative oxidase is known tobe less sensitive tooxygen than cytochrome
oxidase (Solomos, 1977). Inanalogy,Appleby et al. (1976)reported respiratory
protection by an oxygen-insensitive oxidase in R. japonioum bacteroids.Also,
a non-phosphorylating,oxygen-insensitive oxidasewas found in Azotobaoter
vinelandii
(Ackrell and Jones, 1971;Eilermann,1973)and assumed to function
in protecting nitrogenase against high pO-.
REFERENCES
1.Ackrell,B.A.C.and Jones,C.W. 1971.The respiratory system of Azotobaoter
vinelandii
IIOxygen effects.Eur.J. Biochem.20:29-35.
2.Appleby,C A . , Bergersen,F.,MacNicol,P.K., Tunner,G.L., Wittenberg,B.A.,ar
Wittenberg,J.B. 1976.Roleof leghemoglobin in symbiotic nitrogen fixation,
pp274-292 in Proc. 1st Symp.on Nitrogen Fixation (eds.W.E.Newton and
C.J. Nyman)Washington State University Press.
3.Bahr,J.T.and Bonner,W.D. 1973a.Cyanide-insensitive respiration. I.The
steady stateof skunk cabbage spadix and bean hypocotyl mitochondria. J. Biol
Chem. 248:3441-3445.
4. Bahr,J.T.and Bonner,W.D. 1973b.Cyanide-insensitive respiration. II.Contn
ofthealternative pathway. J. Biol. Chem. 248:3446-3450.
5.Bergersen,F.J. 1966.Some properties ofnitrogen-fixing breis prepared from
soybean rootnodules.Biochim. Biophys.Acta 130:304-312.
6. Bethlenfalvay,G.J., Abu-Shakra,S.S.,and Phillips,D.A. 1978a. Interdependem
ofnitrogen nutrition and photosynthesis in Pisum sativum L. IEffect of
combined nitrogen on symbiotic nitrogen fixation and photosynthesis. Plant
Physiol.62:127-130.
7. Bethlenfalvay,G.J.,Abu-Shakra,S.S.,and Phillips,D.A. 1978b. Interdepended
ofnitrogen nutrition and photosynthesis in Pisum sativum L. IIHost plant
response to nitrogen fixation by Rhizobium strains.Plant Physiol.62:131-13
8. Eilermann,L.J.M. 1973.Some aspects ofenergy conservation in Azotobaoter
vinelandii.
Thesis,University of Amsterdam.
9. Evans,H.J., Purohit,K.,Cantrell,M.A.,Eisbrenner,G., Russell, S.A.,
Hanus,F.J., and Lepo,J.E. 1981.Hydrogen losses and hydrogenases in nitroge
fixing organisms,pp84-96 in Current perspectives innitrogen fixation
(eds. A.H.Gibson andW.E.Newton)Elsevier/North Holland,Amsterdam
ISBN 0444802916
10.Henry,M.F.and Nyns,E.J. 1975.Cyanide-insensitive respiration.An
alternatiove mitochondrial pathway. Sub-cell Biochemistry 4: 1-65.
11. Herridge,D.F. and Pate,.J.S. 1977.Utilization of net photosynthate for
nitrogen fixation and protein production in anannual legume.PlantPhysiol.
60: 759-764.
60
12. Laane,C , Haaker,H.,and Veeger,C. 1978. Involvement of thecytoplasmatic
membrane innitrogen fixation by Rhisobium leauminosarum bacteroids.Eur. J.
Biochem.87:147-153.
13. Lambers,H. 1980.The physiological significance of cyanide-resistant
respiration in higher plants.Plant,Cell and Environment 3:293-302.
14. Lambers,H.,Layzell ,0.B.,and Pate,J.S. 1980.Efficiency and regulation of
root respiration in a legume:effects of theN source.Physiol.Plant.50:
319-325.
15. Lambers,H.and Steingröver,E. 1978.Efficiency of root respiration ofa
flood-tolerant and aflood-intolerant Seneoio species as affected by low
oxygen tension. Phys.Plant. 42:197-184.
16. Minchin,F.R.and Pate,J.S. 1973.Thecarbon balance ofa legume and the
functional economy of its roots.J. Exp.Bot.24:259-271.
17. Pate,J.S.,Atkins,CA.,and Rainbird,R.M. 1981.Theoretical and experimental costing of nitrogen fixation and related processes in nodules of
legumes,pp 105-116 i n Current perspectives innitrogen fixation (eds.
A.H.Gibson and W.E. Newton) Elsevier/North Holland Amsterdam ISBN
0444802916.
18. Penning deVries,F.W.T. 1974.Use ofassimilates inhigher plants.
i n Photosynthesis and productivity in different environments (ed.J. Cooper)
Cambridge University Press.
19. Schubert,K.R. and Evans,H.J. 1976.Hydrogen evolution:amajor factor
affecting the efficiency ofnitrogen fixation in nodulated symbionts.
Proc.Nat.Acad. Sei. USA 73: 1207-1211.
..
20. Small,J.G.C.and Leonard,O.A. 1969.Translocation of C-labeledphotosynthate innodulated legumes as influenced by nitrate nitrogen.Amer. J.
Bot. 56:187-194.
21. Snell .F.D.and Snell,C.T. 1949.Nitrites,pp802-807 i n Colorimetric
methods ofanalysis.D. van Nostrans Co.,New York.
22. Solomos,T. 1977.Cyanide-resistant respiration in higher plants.Ann.Rev.
Plant Physiol. 28:279-297.
23. Storey,B.T. 1976.Respiration chain of plantmitochondria. XVIII Point of
interaction of the alternate oxidase with the respiratory chain. Plant
Physiol. 58:521-525.
24. Storey,B.T.and Bahr,J.i. 1969.The respiratory chain of plantmitochondria.'IIOxidative phosphorylation in skunk cabbagemitochondria. Plant
Physiol.44:126-134.
25. Stulen,G. 1974.Interference ofNADHwith the reaction on nitrite in
nitrate reductase estimation.Thesis Groningen.
26.Tomlinson, P.D. and Moreland,D.E. 1975.Cyanide-resistant respiration in
sweet potatomitochondria. Plant Physiol.55:365-369.
27. Visser,R.de,and Lambers,H. 1979.Root respiration of visum sativum L.:
activity of thealternative pathway and relation toN~ fixation. Plant
Physiol, suppl. 63-86.
28. Wedding,R.T.,McCready, C.C.and Harley,J.L. 1973.Inhibition and
stimulation of the respiration of Arum mitochondria by cyanide and its
relation to thecoupling ofoxidation and phosphorylation.New Phytol.
72: 1-13.
61
NITROGEN FIXATION BY A PEA-RH/ZOB/UM SYMBIOSIS
CONTAINING A BACTERIAL UPTAKE HYDROGENASE
ABSTRACT
Pea plants {Pisum sativum cv.Rondo)were sowninsoil withlownumbersof
indigenous rhizobiaandinoculated with Bhizobivan leguminosarum strain S310a
containinganuptake hydrogenase (Hup ) ,orstrain PRE,without hydrogenase
(Hup ).Rootsofpeaplants grownintheopen airand inoculated with S310adid
evolve tracesofhydrogen inair,whereas with strainPRE(Hup")38%ofthe
electron flow through nitrogenase was allocated tohydrogen production during
theentire growth cycleoftheplants.However,total plant nitrogenofthe
symbiosis with strain S310a was considerably lower than thatwithPRE(720and
1380mgNperpot,respectively).This wasduetoalower nitrogenase activity
ofthe nodulesofS310a,toalower bacteroid contentofthese nodulesandtoan
early senescenceofthe nodules incomparison with PRE.Also potential nitrogen
fixation (ascalculated from in vitro nitrogenase activityofisolated bacteroids,
supplied with ATPandreducing equivalents)waslowerwith S310a thanwith PRE.
From the later partofthevegetative phaseofthepeaplants,anexcess amount
of nitrogenasenotutilized innitrogen fixation waspresent both inthe
nodulesofS310aandinthoseofPREwhich amounted to27%inS310a (Hup+) and
19%inPRE(Hup"). The in vitro hydrogenase activityofS310a bacteroids
decreased duringthepod-filling periodofthe plant.
Pea plants inoculated with S310a,cultivated inaqrowth chamber,showedno
positive effectsonnitrogen fixationbyenrichmentofthe root atmosphere
with 1%ofhydrogen,evensowhen theenergy supplyofnitrogenase was minimized
by combination oflowlight intensityandnitrate addition. Nitrogenase
activityofisolated S310a bacteroids kept under aerobic conditions wasnot
enhanced when 10%ofhydrogen was presentasasubstrate.However, hydrogen
oxidation providedarespiratory protectionofnitrogenase against high oxygen
levels.
63
As theenergy gain by theoxidation of hydrogen was estimated at 0.6 -4.7%
of theATP produced inthe nodules by the respiration of carbohydrates, it is
concluded that the occurrence of hydrogenase plays only aminor role in the
energy supply of nitrogenase.
INTRODUCTION
Most Rhizobium species possess anumber of strainswhich contain a hydrogenase
capable of oxidizing all or part of the hydrogen produced by nitrogenase simultaneously with nitrogen fixation {e.g. Schubert and Evans,1976;Evans et al.
1980). The energy loss by thisATP-dependent H„ production might be minimized
by hydrogenase activity,although ithas been assumed that the production of
1mole of H„ requires 7moles of ATP (Pate,Atkins and Rainbird, 1981)but that
theoxidation of 1mole of H„would yield only 2moles ofATP (Dixon,1972)
The greater part of the papers published on the subject deal with thehydrogenase of R. japon-iaum.This hydrogenase can virtually recycle all of theH„
produced by nitrogenase and the presence of the enzyme has been shown to increase
nitrogenase activity of isolated bacteroids supplied with H~ (Ruiz-Argu'eso,
Emerich and Evans, 1979). Dry-matter production aswell as nitrogenase activity
were higher in greenhouse-grown soybean plants inoculated with hydrogenasepositive (Hup )strains than in similar plants inoculated with hydrogenasenegative (Hup )strains (Schubert,Jennings and Evans, 1978). However,when
nitrogenase activity and dry matter-production of aHup'-mutant strainwere compared with the values of the Hup wild-type strain,only minor differences of
weak significance were found (Zablotowicz,Russell and Evans, 1981;Lepo et al.,
1981). Inafield experiment with soybean,the use of aHup strain of
R. japoniaum as inoculant resulted only ina low increase in seed protein but
not in a higher seed yieldthanwas obtained with soybean inoculated with a Hup
strain (Hanus et al., 1981).
With the less-explored Hup strains of R. leguminosarum, the hydrogen uptake
rates usually are not sufficient to recycle all of the hydrogen generated by
nitrogenase activity (Ruiz-Arglieso,Hanusand Evans, 1978;Bethlenfalvay and
Phillips, 1979). The alleged beneficial effects of hydrogen oxidation to energy
supply of nitrogenase start from the assumption that energy supply isthe ratelimiting step of nitrogen fixation. Ina previous Chapter (Ch. 1,pp.21-33)it
64
was shown that nitrogenase content was limiting nitrogen fixation duringthe
main partofvegetative growthofpeaplants withaHup strainof
R. leguminosarum, whereas energy supply was likelytoberate-limiting during
generative growth.Inordertoascertain whether the presenceof hydrogenase
could increase nitrogen fixation inR. leguminosarum, inthepresent paper
both actual andpotential nitrogen fixation valuesarereported inpeaplants
inoculated withHup strain S310a duringthegrowth cycleinopenairinsoil
and under energy-limiting conditions inagrowth chamber.
MATERIALS AND METHODS
Plant material
and growth
Seedsofpeaplants [Pisum sativum L.cv. Rondo)were sowningravelas
described earlier (Chapter 2,p.36),andinoculated with R. leguminosarum
S310a,whichcontains hydrogenase.Theplantswere keptinagrowth chamberat
20°Cand70% relative humidity,witha16hlight/8hdark cycle.At14days
after sowing,when nodule formation hadstarted,plantswere transferred toa
4.2-1 containerwith2.51ofnitrogen-free nutrient solution
andanadditional supplyofKCl,10mmolesper1.Thenodules were keptinthe
gas phase.Tenplants,supported byrubber stoppers,were placed inaplasticlid.
Subsequently,thelidwas closely fittothe container andthenutrient solution
aerated with C0«-freeair containing 1%ofH« (treatment)orwith CO^-freeair
(control).Afterthehydrogenaseandnitrogenase measurementsat21days after
sowing,the nutrient solution was replacedbyasolution containing KNCu
(10mmolesper1)insteadofKCl.Thenitrogen-containing nutrient solutionwas
refreshed everytwodays.
Inaseparate experiment,plantswere grownintheopenairinpotsinsoil
containing lownumbersofindigenous rhizobia,asdescribed before(Chapter1,
pp. 21-33).
Hydrogenase and nitrogenase
measurements
In vivo hydrogen productionandnitrogenase activity innodulated roots were
determinedasdescribed before(Chapter1
).
For the in vitro anaerobic assayofhydrogenaseandnitrogenase,nodules
were picked from the roots after completionofthe in vivo assay.Thenodules
were squeezed inapress under argon,at0°C,inabuffer containing (mmoles/1):
Tris(hydroxymethyl)aminomethane,50;MgCl«,2.5;sucrose,300;1,4dithiotreitol,
5;andpolyvinylpyrrolidone (PVP),4% {-). ThepHwas adjustedto8.0with HCl.
65
Bacteroids were centrifugedfor10minat5500g under argon,washed withabuffer
solution (samebufferwithout PVP)andresuspended inthis buffer solution.
Bacteroids from1gofnodules were transferredtoa16.5mlHungate tubewith
2mlofbuffer containing 10mMofmethylene blue,underanatmosphereof0.6%
H ? inargon.H„consumptionat25°Cwas followed gas-chromatographicallyat
30min intervals after pre-incubation for10min(Roelofsenand Akkermans, 1979).
In vitro nitrogenase was determined inbacteroids treated with EDTAandtoluene,
and supplied withATPandsodium dithioniteasdescribed before (Van Stratenand
Roelofsen, 1976; Chapter 1 ).
RESULTS
Inapreliminary experiment,only2Hup strains were found among47strains
of R. leguminosamm ofthe culture collectionofthis laboratory,showing that
the occurrenceofahydrogenaseisarare featureinR. leguminosamm. The
hydrogenase found infree-living R. leguminosarum S310a showed derepression
characteristics similartothoseofR. japonicum 110when growninamediumas
described byLimandShanmugam (1978). For example,uptake hydrogenase activity
incellsofS310a with succinateandcyclicAMPamounted to1.8mmolesH„/g.h
protein.h,ascompared with2.3mmoles h^/g-hwith cellsofstrain 110withmalatt
and cyclic AMP,reportedbyLimandShanmugam.
Nitrogenase and hydrogenase activities of plants growing in open air
To assesstheinfluenceofanuptake hydrogenaseonnitrogen fixationand
growth,apot experiment was carriedoutinwhichpeaplants were cultivatedin
soil inoculated with R. leguminosamm S310a(Hup)andPRE(Hup"). Duringthe
entire growth period,considerably less nitrogen was fixedbypeaplants grown
in symbiosis with S310a thanbyplants grown with PRE,asevidenced bytotal
plant nitrogen values (720and1380mgNperpotatmaturity, respectively).
Actual nitrogen fixation {in vivo nitrogenase activityofnodulated roots)
and potential nitrogen fixation [in vitro nitrogenase activityofisolated
bacteroids)areshowninFig.1afor the entire growth cycleofthe plants.
With strain S310a,both actual andpotential nitrogen fixation were significantly lower than with strainPRE(p<0.001). During themain partof vegetativegrowth,actualwasequal topotential nitrogen fixation with both strains,
but from61days after sowingthepotential washigher than theactual nitrogen
fixation (p<0.01). Actual andpotential nitrogen fixationperunitofnodule
weight decreased with time (Fig.1a).
66
l~"\-q
o
a.
v
V
V
V
V
V
-0.5
i
40
i
60 t
i
i
80
i
*
100
2-1.,
c
120
Days
Fig. 1a.Actual (•,•) and potential ( o , D ) nitrogenase activity during
ontogenesis of pea plants inoculated with R. leguminosarum strains
S310a (t,o)and PRE (• , D ). Values aremeans of triplicate
determinations (potential nitrogenase activity)orduplicate
determinations of8 plants (actual nitrogenase activity)growing in
pots in theopenair.
Fig. 1b.Bacterial-proteincontent ofnodules from pea plants inoculated with
R. leguminosarum strains S310a (o)orPRE (n ). Values aremeansof
duplicate determinations.
Fig. 1c.Specific nitrogenase activity {in vivo, »,• ; in vitro, o , D )of
pea plants inoculated with R. leguminosarum strainsS310a (•, o)and
PRE (• , D ).Values aremeans ofduplicate determinations.
67
When nitrogen fixation was calculated forthe entire growth season,assuming
a nitrogenase activityof24hper daywithout diurnal variation,andafactor
of3.0toconvert molesofethylene produced intomolesofnitrogen fixed,
actual andpotential nitrogen fixation inpeaplants inoculated with S310a
would amountto2.3and3.1mmoles N 2 per plant,respectively. Thus,withthe
Hup strain S310a 27%ofthe nitrogen-fixing capacity wouldnotbeutilized.
In plants inoculated withtheHup strain PREactual andpotential nitrogen
fixation duringthegrowth period would amountto3.8and4.7mmolesofN„per
plant,respectively,which means that 19%ofthe nitrogen-fixing capacity would
notbeutilized.
The differences innitrogenase activity between S310aandPREcan partlybe
explainedbydifferencesinbacterial-protein contentofthe nodules (Figs. 1b,c),
especially duringthepod-filling period. However,during thevegetative growth
phasethe bacterial protein presentinS310a was clearly lessactiveinnitrogen
fixation thantheproteinofPRE(Fig. 1c).This mightbecausedbyaless
complete transformationofbacteria (rods,nonitrogenase activity) into bacteroids (branched shape,high nitrogenase activity)during nodule development with
strain S310aascompared with strain PRE.Analmost equal nitrogenase activity
per individual bacteroidatagreatly different activityperunitofnodule
weightwas found earlier inthis laboratory with field beans growing with
R. leguminosarum strains PREandPF,,(vandenBerg, 1977).
Hydrogen production inairbynodulated rootswas significantly lowerin
symbiosis with S310a than withPRE(Fig.2a) resulting inapronounced difference
in relative efficiency (SchubertandEvans,1976)duringthegrowth period
(95% inS310aand62%inPRE).Bacteroid hydrogenase activity,asdetermined
with themethylene-blueassay,wasfoundinS310a only (Fig.2b).Similarto
nitrogenase activity, specific hydrogenase activity declined with time.Ifthe
nitrogenaseofstrain S310aisassumedtoproduce hydrogen inthesame proportion
to ethyleneaswas found with strain PRE,thehydrogenase valuesofFig.2bare
toolowtoexplain thealmost absentnethydrogen production inairbynodulated
rootswith S310aupto70days after sowing.
Nitrogenase and hydrogenase activities under reduced light conditions
Inordertobeabletonotice evenaminor positive effectofthe hydrogenase
activityonthe energy supplyofnoduleswith strain S310a,anexperimentwas
carried outwithpeaplants inoculated with this strain,keptinagrowth chamber
under suboptimal conditions.Asalow nitrogenase activity under poor energy
conditions might leadtoahydrogen production which couldbetoolowfor satu-
68
Days
120
Fig.2a.Hydrogen production inair by pea roots nodulated with R. leguminosarum
strains S31;0a(o)and PRE ( D ).Values aremeans of duplicate
determinations in sets of8 plants from pots intheopen air.
Fig. 2b.Specific in vitro hydrogenase activity of pea root nodules formed
with R. leguminosarum strain S310a (o).Values aremeans of duplicate
determinations.
ration of thehydrogenase,treatment groupswere given \%ofH 9 in the root
2
atmosphere.Ata light intensity of90W/m nodifferences were observed in
noduleweight per plant and in vivo and in vitro nitrogenase activity dueto
growth with \%ofH ? during 7days,as compared tocontrol plants grown without
added hydrogen.When nitrate (10mM)was supplied,noduleweight and both in vivo
and in vitro nitrogenase activity decreased equally in hydrogen-treated
plants andin plants without added hydrogen.Therefore,experiments were carried
out inwhich light intensity was reduced to 50W/m and after 7days nitrate
(10mM)was added inorder tominimize energy supply tonitrogenase (Table1).
Under this reduced light intensity,the in vivo nitrogenase activity and nodule
fresh weight amounted to only one-third of thevalues found at high light intensity,butnodifferences inthese parameters were found thatwere associated
with hydrogen enrichment.Also the in vitro hydrogenase activity was equal,but
the in vitro nitrogenase activity wasclearly higherwhen the plants weretreated
with H„;nitrate partly eliminated this effect.
69
Table1.Hydrogenaseandnitrogenase activitiesofpeaplantsasaffected
bythesupplyof \%ofH2totherootatmosphere from14daysaftersowing.
The plantswere inoculatedatsowingwith R. legwninosarum S310aandwere
keptonanN-free nutrient solutioninagrowth chamberat50W/m2.At21
daysafter sowingtheplantswere supplied withasimilarnutrient solution
containing KNO,,10mmoles/1
Item
Days aftersowing
21(-NO3)
28(+N0~)
noH 2
noHp
+H„
+H
2
In vivo CpH.production
(ymoles/plant.h)
1.0+0.1
1.0+0.1
Nodule fresh weight
(mg/plant) 1)
75 ± 8
In vitro hydrogenase
activity (ymolesH„
consumed/gnodules,
fresh wt.h) 2 )
1.2± 0 . 3 0.9±0.1
In vitro nitrogenase
activity (ymoles CpH,
produced/g nodules,
fresh wt.h) 2 ^
5.7± 1 . 3 9.8± 1 . 4
72 ± 6
1.2+0.2
143 ±6
166 +10
0.4±0.2
0.3± 0.0
5.2± 1 . 0
1)
Meansandstandard deviationofduplicate samplesof10plants.
2)
Meansoftriplicatesamples.
70
1.4+
6.5± 0.2
0.3
150r
c 100-
0.6
0.8
p02
Fig.3. EffectofpO„onthenitrogenase activityofisolated bacteroidsof
R. leguminosarum strain S310a,supplied with 10%ofH 2 (o)orwithout
added H(•).
2
Nitrogenase activity of bacteroids supplied with H„
The in vitro nitrogenase assay determines theactivityofthe enzyme under
anaerobic conditions,using ATPandsodium dithioniteassourceofenergyandof
reducing equivalents,respectively. Therefore thecontributionofhydrogen uptake
toenergy supply fornitrogen fixationhadtobemeasured underaerobic conditionswith isolated bacteroids capableofusingthe hydrogen addedassubstrate.Washed bacteroidsofstrain S310a from 630mgofnodular tissuewere
incubated under 10%ofacetyleneand varying pCL,inthe presenceofreduced
myoglobinandofbovine serum albumine (final volumeofthe incubation mixture
2 ml),asdescribedbyLaane,Haakerand Veeger (1978).The effectof10%ofH„
on nitrogenase activityofbacteroids without added carbon substrateisshown
in Fig.3.Thehydrogen uptake systeminS310a bacteroidsdidnotraisenitrogenase activity above the rateofbacteroids dependentonendogenous respiration.
However,theactivityofnitrogenaseinbacteroidsinthepresenceof10%ofH ?
ismaintainedatahigher p0„, showing thatinS310a bacteroidsthehydrogenase
system only offers some respiratory protection.
71
DISCUSSION
Hydrogen uptake and utilization by bacteroids of the Hup strain S310a of
R. leguminosavum did not increase nitrogenase activity but only provided
respiratory protection (Fig.3 ) ,incontrast to the results obtained with
R. japoniaum bacteroids (Ruiz-Argüeso et al., 1979). It isdoubtful,however,
towhat extent a respiratory protection isfunctional inthe legume nodule,
where bacteroids are present incompartments,buffered as to high oxygen level
by leghemoqlobin.
No stimulation of nitrogen-fixing characteristics was found in theexperimentwith nodulated pea plants in hydrogen-enriched environment (Table1 ) ,
except the increased in vitro nitrogenase activity of plants growing at
reduced light intensity. Even in that case,however,the benefit of hydrogen
isdoubtful as the in vitro nitrogenase activity was lower than the in vivo
activity. This has been found earlier with plants growing inwater culture
(Houwaard, 1978). As thenodules produced inwater culture had awhitish
surface due to the formation of lenticel tissue,theoxygen content of this
tissue might have caused a partial inactivation of nitrogenase during preparation of the bacteroids.
The in vitro nitrogenase activity was higher than the in vivo activity
during the generative phase of pea plants growing in pots in the open air
(Fig. 1a),supporting theconcept of potential nitrogen fixation (Chapter 1,pp.
21-34)
The hydrogenase present in strain S310a prevented the generation of
large amounts of hydrogen in air (Fig.2a)butdid not contribute to improved
utilization of the potential nitrogen fixation which amounted to 73%with S310a
(Hup+)and to 81%with PRE (Hup"). Inthe generative phasewith PRE aswell as
with S310a,nitrogen fixation seems limited by photosynthate supply, regardless
of nitrogen-fixing capacity and regardless of the presence of hydrogenase
(Fig. 1a).Both with PREand S310a,the full capacity of the nitrogen-fixing
system was only realized by an enhanced photosynthate supply (Chapter 2,page
39).
Even under energy-limiting conditions the presence of hydrogenase had
no detectable effect on nitrogen fixation.Thus,hydrogen oxidation will
certainly not enhance nitrogenase activity when energy supply isnot the
limiting factor, e.g. during early vegetative growth under optimum conditions
when thecontent of theenzyme is limiting nitrogen fixation.
Ina previous paper ( Chapter 3
)theenergy consumption of the
nodules of the highly effective strain PRE inacetylene reduction was estimated
72
at6.3 moles ofATP permole of C„H 2 reduced,whereas innodules of themoderately effective strain S313 avalue was calculated of 12.9moles ATP consumed
permole ofC ? H 2 reduced. When one of these values isassumed to be valid for
the nodules of themoderately effective strain S310a,and when the oxidation
of 1mole of H„ isassumed toyield 2moles ofATP (Dixon, 1972), thecontribution of hydrogenase to energy generation in the nodule can be calculated.
Assuming that thecosts of acetylene reduction with themoderately effective
strain S310a are equal to thosewith the highly effective strain PRE,hydrogenase activity would produce 1.1 -2.9%of theATP generated in respiration
in the experiment with added hydrogen (Table 1).In the pot experiments with
soil these values amounted to3.3 -9.7%during the period when hydrogenase
activity was detected (Fig.2b).However,acomparison with the intermediate
strain S313 ismore realistic,as nodule weight per plant as well as nodule
activity are similar to those with S310a. Inthat case,the hydrogenase
contributes 0.6 - 1.4%of the ATP generated in respiration during the experiment
in hydrogen-enriched air,whereas in the pot experiment 1.6 -4.7%of theATP
production would beprovided by hydrogenase activity. These benefits of hydrogenase activity are lower than those calculated by Evans et al. (1981)for
R. japoniaum (9.1%ofATP supply to nitrogenase) based on adifferent setof
assumptions.
Whatever the benefits of hydrogenase activity may be,they do not compensate
forother,lessfavourable,characteristics. The shorter span ofnodule activity
with S310a ascompared with strain PRE isone of such characteristics. With
hydrogenase-containing strains of R. leguminosarum, nitrogen fixation and dry
matter production are lower than it is true ofmost of the hydrogenase-less
strains {e.g. Fig. 1,S310a vs PRE;strain 128C53us 175G11,Ruiz-Arglieso
et al., 1978). Inaddition,hydrogenase-positive strains might beweak competitors (e.g. S310a vs PRE,Van Mil,in prep.). Summarizing, itmay be concluded
that the presence of hydrogenase should only play aminor role in selecting a
Rhizobiwn strain for use as an inoculant.
REFERENCES
1.Berg,E.H.R.van den. 1977.The effectiveness of the symbiosis of Rhizobium
leguminosarum on pea and broad bean. Plant and Soil 48:629r639.
2. Bethlenfalvay,G.J., and Phillips,D.A. 1979.Variation in nitrogenase and
hydrogenase activity ofAlaska pea root nodules. Plant Physiology 63:
816-820.
3. Dixon,R.O.D. 1972.Hydrogenase in legume root nodule bacteroids: occurrence
and properties.Archives ofMicrobiology 85:193-201.
73
4. Evans,H.J., Emen'ch,D.W., Lepo,J.E.,Maier,R.J., Carter,K.R., Hanus,F.J
and Russell,S.A. 1980.The role of hydrogenase innodule bacteroids and
free-living rhizobia,pp 55-81 in Nitrogen Fixation,Proceedings of the
Phytochemical Society of Europe Symposium,Sussex 1979 (eds.W.D.P. Stewart
and J.R. Gallon),Academic Press London.
5.Evans,H.J., Purohit,K.,Cantrell,M.A.,Eisbrenner,G.,Russell, S.A.,
Hanus,F.J., and Lepo,J.E. 1981.Hydrogen losses and hydrogenases in
nitrogen-fixing organisms,pp84-96 in Current perspectives in nitrogen
fixation,Proceedings of theFourth International Symposium on Nitrogen
Fixation,Canberra 1980 (eds.A.H.Gibson and W.E. Newton), Elsevier/
North Holland Amsterdam
6. Hanus,F.J.,Albrecht,S.L., Zablotowicz,R.M. ,Emerich,D.W., Russell, S.A.,
and Evans,H.J. 1981.Yield and nitrogen content of soybean seed as
influenced by Rhizobium japoniaum inoculants possessing the hydrogenase
characteristic.Agronomy Journal 73:368-372.
7. Houwaard,F. 1978.Influence ofammonium chloride on thenitrogenase activity
of nodulated pea plants {visum sativum). Applied and Environmental Microbiology 35:1061-1065.
8. Laane,C , Haaker,H.,and Veeger,C. 1978.Involvement of thecytoplasmatic
membrane in nitrogen fixation by Rhizobium leguminosarum bacteroids.
European Journal of Biochemistry 87:147-153.
9. Lepo,J.E.,Hickok,R.E.,Cantrell,M.A.,Russell,S.A., and Evans,H.J. 1981,
1981. Revertible hydrogen uptake-deficient mutants of Rhizobium
japoniaum.
Journal of Bacteriology 146:614-620.
10. Lim,S.T.,and Shanmugam,K.T. 1979.Regulation of hydrogen utilisation in
Rhizobium japoniaum bycyclic AMP.Biochimica et Biophysica Acta584:
479-492.
11. Pate,J.S.,Atkins,C A . , and Rainbird,R.M. 1981.Theoretical and experimental costing ofnitrogen fixation and related processes in nodules of
legumes,pp 105-116 in Current perspectives innitrogen fixation,
Proceedings of the Fourth International Symposium on Nitrogen Fixation,
Canberra 1980 (eds.A.H.Gibson and W.E.Newton). Elsevier/North Holland
Amsterdam.
12. Roelofsen,W.,and Akkermans,A.D.L. 1979.Uptake and evolution ofH„and
reduction ofC ? H„ by root nodules and nodule homogenates of Alnus
glutinosa.
Plant and Soil 52:571-578.
13. Ruiz-Argüeso,T., Emerich,D.W., and Evans,H.J. 1979.Hydrogenase system
in legume nodules:amechanism of providing nitrogenase with energy and
protection from oxygen damage.Biochemical and Biophysical Research
Communications 86:259-264.
14. Ruiz-Argüeso,T.,Hanus,F.J., and Evans,H.J. 1978.Hydrogen production and
uptake by pea nodules asaffected by strains of Rhizobium
leguminosarum.
Archives ofMicrobiology 116:113-118.
15. Schubert,K.R.,and Evans,H.J. 1976.Hydrogen evolution:amajor factor
affecting theefficiency of nitrogen fixation innodulated symbionts.
Proceedings of the National Academy of Sciences U.S.A. 73:1207-1211.
16. Schubert,K.R., Jennings,N.T.,and Evans,H.J. 1978.Hydrogen reactions
of nodulated leguminous plants. II Effects on dry matter accumulation
and nitrogen fixation. Plant Physiology 61:398-401.
17. Straten,J. van,and Roelofsen,W., 1976. Improved method for preparing
anaerobic bacteroid suspensions of Rhizobium leguminosarum for the
acetylene reduction assay. Applied and Environmental Microbiology 31:
859-863.
18. Zablotowicz,R.M., Russell,S.A., and Evans,H.J. 1980.Effect of the
hydrogenase system in Rhizobium japoniaum on the nitrogen fixation and
growth of soybeans at different stages of development.Agronomy Journal
72: 555-559.
74
NODULE FORMATION AND NITROGEN FIXATION IN
SYMBIOSES OF PEA (PISUM SATIVUM L ) AND
DIFFERENT STRAINS OFRHIZOBIUM LEGUMINOSARUM
ABSTRACT
Symbiosesofonepeavariety {Pisum sativum L.,cultivar Rondo) inoculated
with different strainsofRhizobium lëguminosarum showed marked differences
innitrogen-fixing characteristics.Nitrogen fixation,ascalculated from
plant-nitrogen data,washighestwith strain PREwhich producedalownodular
masswithahigh nitrogenase activity.With this strainrednoduleswere
located mainlyontheprimary roots,butlateral-root nodules were steadily
produced.Peaplants inoculated with strains S313andS310a fixed less nitrogen
than those inoculated with strain PRE.Theirnodule formation patternwas
different from thatofPRE,strain S313 producing largeamountsofpink,
whitish nodularmass ontheprimary rootbutvirtuallynonodulesonthe
lateral roots,whereaswith strain S310atheprimary (red)root nodules rapidly
were replacedbylarge amountsofredlateral root nodules.Peaplants inoculatedwiththeineffective strainP8produced small,white nodules predominantly
onthelateral roots.
Nodule numbersontheprimary rootswereequal with all strains,whereas
numbersofnodulesonlateral roots were inversely correlatedtonodule weight
ontheprimary root.From the data presented itisconcluded that nodule formation isconnected with nitrogenase activity,alowactivity being compensated
forbythe productionoflarge nodules (S313)orbytheformationofmanynew
nodules (S310a).
Nitrogenase activityofthe nodulesdeclined with time.Less electrons
were spentinhydrogen productionascompared with acetylene reduction,
resulting inariseofrelative efficiency with proceeding growth period.
The valuesofnitrogen fixation,ascalculated from acetylene-reduction data,
usingaconversion factorof3.0,were lower than thosederived from thedifferences between yieldofplant nitrogenoftheeffective symbioses (with strains
75
PRE,S313andS310a)andthatofthe ineffective symbiosis with strain P8.When
the hydrogen produced inairbyeffectively nodulated rootswas subtracted from
the acetylene-reduction values,nitrogen fixation was even more seriously underestimated,butinaddition severely biased infavourofthe hydrogenasecontaining strain S310a,which producedalowplant-nitrogen yield.
The real valuesoftheconversion factor,asobtainedbydivisionofthe
acetylene-reduction figuresbytheyieldofplantnitrogen derived from nitrogen
fixation,decreased gradually duringthegrowth period.With all strains,
nitrogen fixation was highest duringthepod-filling stage.
Inaseparate experiment3peacultivars,differing inseed-production rates,
were inoculated withasingle R. leguminosarum strain.Nitrogen fixation with
an early variety (Florix)waslower than thatwithanintermediate variety
(Rondo)oralate variety (Mercato).Mercato fixed more nitrogen than Rondo
butitsseed yield was less becauseofapoorer translocationofnitrogento
the seed from vegetative matter.
INTRODUCTION
Inagriculture,yieldsofleguminous crops aredeterminedtoaconsiderable
extentbythequalityofthesymbioses, i.e. bythematchingofthehost cultivar
withtherhizobial strain (5,11).Asplant breeding programmesare usually aimed
at increased yield,andatimproved resistance against pestsanddiseases,ahigh
nitrogen fixation capacity isselected inanindirectway.
A direct approachtoenhance nitrogen fixationistofind new Rhizobium
strains with better nitrogen-fixing characteristics.Inthepresent paper,the
relation between nitrogen fixation,yieldandsymbiotic characteristicshas
been investigated inassociationsofpeaswithanumberofselected strainsof
Rhizobium leguminosarum. Aslowlevelsofnitrogen fixation havetobecompensatedbythe supplyofcombined nitrogen forobtaining satisfactory yields, the
influenceofcombined nitrogenonnodulation,nitrogen fixation andyieldis
described inasecond paper.
MATERIALS AND METHODS
Plant material
and growth
Enamelled Mitscherlich jarsof6-1capacity,sterilized with alcohol,were
filled withamixture(-)of1partofheat-sterilized sandand2partsofnonsterileclay,obtained from newly reclaimed polder land.Forinoculation,50ml
76
ofa7-daysoldcultureofRhizobium leguminosarum was mixed with61ofsoil.
A basal fertilizer giftof3.8mmolesofphosphorusand9.6mmolesofpotassium
was applied per pot.
Seedsofpeaplants {visum sativum L.), cultivars Florix,MercatoandRondo,
were surface-sterilizedbyimmersion inasolution containing4%ofH^O«anda
few dropsofTeepol (detergent) for25min before sowing.Thesoilwasthen
covered with sterilized gravel toprevent clogging afterwatering. Plants were
keptintheopen airunderwireforprotection against birds.Ineachpotfinally
8 plantswere retained.
Hydrogen production
and acetylene
reduction
The shootsoftheplants were detached,theroots freed from soil particles
bygentlyshakingandincubated instoppered 1-1Erlenmeyer flasks.After25min
a 100-ulgassamplewas assayedfor hydrogen production usingagasChromatograph
equipped withathermal conductivity detector. Subsequently, 100mlofacetylene
was addedanda100-ylgassamplewas taken after15min. Ethylene production
was determined withagasChromatograph equipped withahydrogen-flame detector.
Ratesofhydrogen productionandacetylene reduction were linearwith timeup
to80min.
Nitrogen
analyses
of the plant
material
Nitrogen inoven-dried plantmaterial wasdeterminedbytheKjeldahl method,
using CuSO«andseleniumascatalysts.
RESULTS
Inapreliminary experiment,the successofinoculationofthenatural soil
used intheexperiments,which was assumedtocontain lowlevelsofindigenous
rhizobia,was tested with mutant strainsofR. leguminosarum resistant against
streptomycin aswellasacriflavine (Table 1 ) .Seedswere sowninsmall0.5-1
test potsasdescribed intheMethods section,using5mlofa4-daysoldbacterial culture grownonyeast-mannitol brothasaninoculum (approximately5.10
bacteriapergofsoil).Thenodulesofthepeaplants were checked40days after
sowingbyprickinganeedle first intoasurface-sterilized noduleandthen into
yeast-mannitol agar plateswithoutandwith streptomycinandacriflavine. Inoculation with R. leguminosarum strains PRE andS313a provedtobesuccessful,
77
Table 1.Nodule formationonpea {visum sativum cv.Rondo)byindigenous
rhizobiaandbyinoculated strains (antibiotic-resistant),40days after
sowing inclay soil.Values representmeansandstandard deviationsof
duplicate determinations
Strain
P8
Pinknodules
perplant
Antibioticresistant
colonies
(%)
0
2±1
SA
S310a
SA
S313
pRE SA
Uninocul ated
CpHpreduction
(nmoles/pl.h)
80± 50
15±5
34± 9
15±2
88+12
370±170
20±4
78±15
240±120
0
7±2
350± 80
100± 50
SA
whereas strain S310a wasless abletocompetewith indigenous Rhizobium strains
for nodulation sites. Inoculation with thewild-type strainP8gavemany small
white ineffective nodulesonpeaplantsbutafewlarger pinknodules which were
apparently derived from indigenous strains.From these resultsitwas concluded
thatinalarge-scale experiment inoculation shouldbeheavier (5.10 bacteria
pergofsoil)inordertoprevent competition difficulties.Inthelarge-scale
experiment only wild-type strainswere used.
Nodule formation
and nitrogen
fixation
Nodulation brought aboutbythree R. leguminosarum strains showed clearly
different patterns (Plate 1).Theineffective strainP8produced small white
nodulesinagreat numberonthelateral roots.Incontrast,strain S313formed
big,branched noduleswith afaintlypink colourinagreatmass,virtually only
ontheprimary roots.Strain PREoccupiedanintermediate position with mediumsized,rednodules spread over primaryandlateral rootsinamore even way.
Strain S310a (not shown)behaved similarlytoPRE,exceptfor thenodule colour
which changed fromredtogreen more rapidly, indicating early senescence.
Therewasnodifference inthe numberofnodulesonprimary roots producedby
each rhizobia! strain (Fig. 1a).The numbersoflateral-root nodules formed
during growth were significantly different withthefour strains (Ftest,
p<0.01,Fig._1b).The highest numbersoflateral-root noduleswere found with
S310aandP8.
78
Plate 1.Rootsystemsof64-daysold pea plants inoculated with Rhizobium
strains P8 (a),PRE (b),and S313(c).
79
60
40
20 -
0 Hh
_j
i
i
i
i
e
c
300
•o
o
200
100
-
100
120
Days
Fig. 1.Root nodule numbers during ontogenesis of pea plants,cv. Rondo
inoculated with R. legum-inosarum strains P8 (•),S310a (o),S313(A),
and PRE ( O ) .Values represent means ofduplicate samples of 4 plants
from different pots, (a)Nodule numbers on primary roots, (b)Nodule
numbers on lateral roots.
Nodule fresh weight per plant increased gradually until mid-pod fill,as
shown in Fig. 2a.With S313 and S310a nodule weight per plantwas significantly
higher (p< 0.01) than thatwith PRE.From mid-pod fill,noduleweights decrease!
slowly with PRE and rapidly with S313.With strain S310a,aconsiderable proportion of the nodules turned green early.The contribution of primary-root
nodules tototal noduleweight decreased during plant growth,thegreatest decrease being found with S313 (Fig.2b).Primary-root nodules contributed significantly (p<0.01) more to total noduleweight per plant inthe sequence S313>
PRE >S310a,P8.
80
- 1200 -
100
120
Days
Fig. 2.Root nodule fresh weight during ontogenesis of pea plants,cv. Rondo
inoculated with R. leguminosarum strains P8 (•),S31Oa (o), S313(A),
and PRE ( D ) .Values represent means of duplicate samples of 4 plants
from different pots, (a)Total nodule fresh weight per plant,
(b)Weight ratio of primary to lateral rootnodules.
Nitrogenase activity of the nodules formed by PRE,S313and S310awith cv.
Rondo declined during growth,the greater fall being found with PRE (Fig.3a).
Roots nodulated with P8displayed no nitrogenase activity. The quantity of
hydrogen produced inair by nitrogenase decreased more rapidly as compared with
the decrease in ethylene produced under acetylene,resulting ina steady rise of
H„ produced in air
the relative efficiency (1~i j—r- •. ,,.„, r ,,)(see 13;Fig3b).
3
C ? H d proauced inair + 10%C^H,,
81
100
120
Days
Fig. 3.Nitrogenase activity of rootnodules during ontogenesis of pea plants,
cv. Rondo,inoculated with R. leguminosavum strains S310a(o),
S313 (A),and PRE (•). Values aremeans ofduplicate potswith
8 plants each, (a)Ethylene produced underair + 10%acetylene,
expressed pergof nodule fresh weight, (b)Relative efficiency
(see text).
Strain S310a on theother hand displayed a high relative efficiency owing to the
presence ofahydrogenase capable of utilizing part ofthe hydrogen evolved by
nitrogenase (Chapter 4,pp.63-75 ) .Relative efficiency with this strain
initially amounted to 96-99%but decreased tothe values ofthe strains without
a hydrogenase at pod fill (day 78).This occurred togetherwith the formation of
a few large nodules on the lateral roots,which weremorphologically different
from the nodules initially produced with S310a.When these deviating nodules were
assayed separately (day 93)a relative efficiency of48%was found indicating
that they were formed byone of the few rhizobia originally present in the soil.
The R. leguminosarwn strains studied inthisexperimentshowed distinctdifferences in the supply ofnitrogen to the plant (Fig.4c).With the ineffective stra
P8, the plantwas fully dependent on seed and available soil nitrogen,resulting
in avery lowyield ofnitrogen atmaturity (15mg N/plant). The amounts of plant
82
100
120
Doys
Fig.4.Nitrogen fixation during ontogenesis of pea plants,cv.Rondo,inoculated with if. leguminosarum strains P8 (•),S310a (o),S313 (A),and
PRE ( D ) .Values represent means of duplicate samples, (a) Calculated
from ethylene production (under 10%ofacetylene inair)minus hydrogen
production in air. (b)Calculated from ethylene production under 10%
of acetylene in air. (c)Total plant nitrogen as determined by the
Kjeldahl method.
nitrogen contained incv. Rondo differed significantly (p< 0.01) with the
rhizobial strains in the sequence PRE >S313 >S310a >P8.
The fact that pea plants inoculated with strain P8displayed no nitrogenase
activity,enabled us tocalculate theamount of nitrogen fixed by nitrogenfixing symbioses by subtracting the nitrogen yield of the P8-associations from
thevalues of theeffective symbioses.During theentire growth period,nitrogen
fixation of the 3associations tested amounted to 122,96,and 56mg N per plant
with PRE,S313and S310a,respectively (fig.4c).
83
Nitrogen fixation during thegrowth period was also estimated fromacetylenereduction data,assuming an activity of the nitrogen-fixing system during 24h
perday without diurnal variation,and using a factor of3.0 toconvert moles
of ethylene produced under acetylene intomoles of nitrogen fixed (Fig.4b).
These values underestimated nitrogen fixation as compared with the plant nitrogen
data: 71,61,and 52mg N per plantwith PRE,S313and S310a,respectively.This
underestimation was more serious with strains PRE and S313 (37-42%)than with
strain S310a(8%).
Hydrogen production in air by nitrogenase isusually considered tooccur in
competition with nitrogen fixation.Therefore,nitrogen fixation was alsoestimated from ethylene production under air + 10% ofacetylene (representing total
nitrogenase activity)minus hydrogen production inair (Fig.4a).According to
thismethod,nitrogen fixation during the growth period would amount to43,38
and 48mg N per plantwith strains PRE,S313 and S310a,respectively.Thus,the
inclusion of the hydrogen production not only aggravated the underestimation of
nitrogen fixation but even caused amisrepresentation of the nitrogen-fixing
capacity infavour of the hydrogenase-positive strain S310a.
The distribution of the nitrogen-fixing activity over the growth period was
influenced by the rhizobial strain (Table 2 ) .With S310a,nitrogen fixation
declined rapidly during pod formation,whereaswith S313 and PREa considerable
partof the total nitrogen fixation took place during the pod-filling period.
When the conversion factors ofethylene production underacetylene into nitrogen
fixed werecalculated on agrowth season basis,assuming an activity of 24h
perdaywithout diurnal variation,the values deviated considerably from the
theoretical value of 3.0 (Table 2). The ratiowas high during vegetative growth
butdecreased afterwards.When theallocation ofelectrons between nitrogen and
proton reduction was taken into account,the ratio dropped below 3.0 except for
strain S310a.This strain,which contains an uptake hydrogenase,gave riseto
a consistently higher ratio between ethylene produced and nitrogen fixed than
the other strains.However,alsowith S310a this ratio declined during the growth
period.
84
Table 2.Nitrogen fixation during ontogenesisofpea(cv. Rondo)-Rhizobzum
symbioses''
Growth stage
Strainofmicrosymbiont
S310a
Vegetative (1-68days) „%
N2fixed (mmoles/plant)
C
2V N 2 3 )
.,
(C 2 H 4 -H 2 )/N 2
4;
Flowering and pod formation (69-86 days)
N9 fixed (mmoles/plant)2)
S313
PRE
1.00
1.61
1.60
4.33
3.54
4.14
4.25
1.67
1.82
0.64
0.71
1.12
C 2 H 4 /N 2 J ;
4.07
2.87
2.27
(C 2 H 4 -H 2 )/N 2 4 )
3.83
2.37
1.89
0.49
1.24
1.62
Pod-fill andripening (87-114 days)
N 2 fixed (mmoles/plant)2)
C 2 H 4 /N 2
3)
(C 2 H 4 -H 2 )/N 2 4 )
Entire growth period (1-114 days)
N9 fixed (mmoles/plant)2)
3)
C 2 H 4 /N 2 J ;
(C 2 H 4 -H 2 )/N 2
4)
2.86
1.67
1.37
2.31
1.41
1.13
2.11
3.56
4.38
3.90
2.76
2.62
3.68
1.72
1.58
1)
Valuesareaveragesofduplicate samplesof8plants
2)
'Calculated from plant nitrogenoftheeffective symbiosis minus plant nitrogen
oftheineffective symbiosis (RondoxP8)
3)Conversion factorforconverting C H4produced inair+10%ofC H into
2
2 2
Infixed
4)
Conversion factorforconverting C 0 H.produced inair+10%ofC 2 H„,minus
H 2 produced inair,into N 2 fixed/
85
Host influenae
Inaseparate experiment,theinfluenceofthehost plantwas briefly checked
in3peacultivars with different ratesofseed production: Florix (early),
Rondo (intermediate),andMercato (late).Theplantswere inoculated witha
single strainofR. leguminosarum, PF~.Thedistribution ofnitrogen fixation
duringthegrowth period isshown inTable3.Theconsiderably shorter growth
periodofFlorix resulted inaloweryieldoffixed nitrogenandseed yield
(Fig. 5a)ascompared with RondoandMercato.Thelate-flowering cultivar Mercato
apparently hadahighernitrogen-fixing capacity than Rondo,when calculated
from valuesofethylene produced underacetylene,usingaconversion factorof
3.0toconvert molesofethylene produced into nitrogen fixed (Fig.5b).However,
the actual difference inplant nitrogen between MercatoandRondowasnotas
largeassuggestedbytheethylene production data (Fig.5c).With Mercato,seed
yield was lower thanwith Rondo (Fig.5a)butfinal yieldofplant nitrogenwas
higher owingtoahigher nitrogen contentoftheleaves (atmaturity 35.4and
18.6mgNperplantwith MercatoandRondo,respectively).
Table3.Nitrogen fixation duringontogenesisofpeacultivars inoculated
with R. leguminosarum
Strain PFo
Growth stage
Cultivar
Florix
Vegetative (days)
.^
N„ f i x e d (mmoles/plant)
1-47
1.13
Rondo
1-57
1.79
Mercato
1-64
2.00
Flowering and pod formation (days)
N„ fixed (mmoles/plant) 1 )
48-58
0.37
58- 73 65-86
0.70
1.40
P o d - f i l l and ripening (days)
N~ f i x e d (mmoles/plant) 1 )
59-86
0.54
73-100 87-105
1.45
0.67
Entire growth period (days)
N9 fixed (mmoles/plant)1)
1-86
2.04
1-100
3.94
'Calculated from plantnitrogenoftheeffective symbiosis minus plant
nitrogenoftheineffective symbiosis RondoxP8.Values are averages
of duplicate determinationsof8plants
86
1- 105
4.07
90
110
Days
Fig.5.Seed productionandnitrogen fixation during ontogenesisofpea
cultivars (Florix,x;Rondo,^;andMercato,^) inoculated with
R. leguminosarum strain PF£. Values represent meansofduplicate
samples, (a)Seed production, (b)Nitrogen fixationascalculated
from acetylene reduction values.Forexplanation seetext, (c) Plant
nitrogenasdetermined bytheKjeldahl method.
DISCUSSION
Soduîe
formation
The difference innodule formation between legumes infected withaneffective
strainofRhizobium andlegumes infected withanineffective strain was reported
earlyinthestudyofnitrogen fixation (6).However,differences innodule
formation between effective strains have received lessattention (18).With
strain PRE,ahigh nitrogenase activityofthe nodules (Fig. 3a)wasassociated
87
with a relatively lownodular mass,mainly located on the primary root (Fig.2 ) .
With strain S313,the lowernitrogenase activity of the nodules was almost
compensated by the high nodularmass produced (compare Figs 2,3and 4)whereas
with strain S310a the low nitrogenase activity led to aconstant formation of
nodular tissue on lateral roots (Fig.2 ) .However,with S310a this did not result
in anitrogen-fixing capacity as high as that of PRE,as the oldernodules aged
atan early stage.
The nodulation pattern of strain S310a was similar to that of strain P8
(Figs 1,2 ) .Aswith S310a and P8nodule numbers were equal but nodule weights
were different (Figs 1 , 2 ) ,the hypothesis that nodule formation is regulated
by the synthesis ofan unknown inhibiting compound is unlikely,as this hypothesis cannot explain theoccurrence of different noduleweights at equal nodule
numbers (12).An explanation of theobserved differences innodulation patterns
might be offered byassuming differences in synthesis of growth-stimulating
phytohormones,such ascytokinins.A function of phytohormones in nodule
formation as has been proposed byLibbenga (10),has so far not been verified
adequately.Therefore,we restrict to the general view that nitrogenase activity
and nodule formation are related by strategies that are differentwith each
strain.A high nitrogenase activity isassumed to beassociated with a relatively
lowamount ofnodular tissue (PRE)and viae versa (S313),whereas a rapid
senescence iscompensated by the formation of new nodules.These patterns are
of course dependent on the interaction between hostand microsymbiont.A large
supply of photo-assimilates increases nodule mass,aswas discussed earlier in
a comparison of peawith field bean (Chapter 1,page31).
Hydrogen
Hydrogen production inairdecreased relatively to ethylene production
(Fig. 3b)with strains PREand S313 during the first 60days of growth.Subsequently,itdeclined much more sharply than theethylene production,so that
the relative efficiency of the symbioses with these strains rose toapproximately
90%. The relative efficiency with strain S310awas nearly 100%owing to the
presence of hydrogenase which recycled the hydrogen produced by nitrogenase,but
obviously this process did notcompensate for the low nitrogenase activity and
unfavourable nodule formation pattern with this strain (Fig.4 ) .
The observed rise in relative efficiency ofnitrogen-fixing nodules (Fig.3b)
during the generative growth phase of the pea plants coincides with the presumably decreasing supply of photosynthates to the nodules due to the formation
of pods (9).This observation is inagreementwith results reported ina
'production
previous Chapter 1,(pp.21 -34) which showed thatenergy supply ofnitrogenase during the generative phase is insufficient for amaximum nitrogenase
activity, incontrast with the vegetative phase,during which nitrogenase is
entirely utilized. The decrease in hydrogen production during presumably reduced
photosynthate supply of the nodules seems tocontradict evidence from experiments
with isolated nitrogenase (16,7, 17).These experiments generally indicate that
under low energy conditions, e.g. when ATP levels are low (16),orwhen electron
flow through nitrogenase Mo^Fe component is low (7),electrons are allocated
to proton reduction rather than to the fixation of nitrogen.Also more hydrogen
is produced when the ratio of Fecomponent (II)to Mo-Fe component (I)of
nitrogenase is low, i.e. when fewelectrons are transferred by theFe protein
to theMo-Fe protein(17).
Van den Bos et al.(3) have shown that the ratio of Fe protein toMo-Fe
protein increases with the age of bacteroids of R. legwminosarum strain PRE.
Therefore,the increase in relative efficiency during the growth period of
pea plants (Fig. 3; 1,2)might beassociated with the altered ratio ofnitrogenase components rather than tothe inadequate photosynthate supply of the
nodules.
The observation that nitrogenase is not fully utilized during generative
growth of pea plants and field bean plants is likely tobeexplained by the
lower photosynthate supply,as in short-term experiments an excess ofnitrogenase,present in the nodules,can be activated by increasing photosynthate
supply (Chapter 2)
The calculation ofnitrogen fixation from acetylene-reduction figures is
hazardous,as can be seen from Fig.4and Table 2.Even when nitrogenase
activity was assumed tocontinue for 24h per day at its daytime level,the
plant nitrogen yields were underestimated by the use ofaconversion factor
of 3.0 (Fig.4;8 ) .Inany case,hydrogen production should not be taken into
accountwhen comparing strainswith and without hydrogenase,as not only an
underestimation occurs but even amisrepresentation is found in favour ofa
strain with hydrogenase but of low nitrogen-fixing capacity. The data of
Table 2also indicate that aconversion factor of 3.0 underestimates nitrogen
fixation more seriously when applied to plants in the generative growth phase
than to plants in thevegetative phase (15).Inthe latter case nitrogenfixation data are overestimated when derived from acetylene-reduction values.
89
Host
plants
Some desirable featuresofpeavarieties with regard tonitrogen fixation
have been described intheliterature (4).From Fig.5itcanbederived that
theearly variety Florix showed 'self-destructive' traits (14)duetoarapid
seed production,resulting inalowoverall level ofnitrogen fixation.The
late flowering variety Mercato fixed more nitrogen than theintermediate variety
Rondo,especially during theprolonged stagesofvegetative growthandflowering. However,seed production ofMercatowaslower than thatofRondo,stressinç
oncemore that nitrogen-fixing capacity isjustoneofthemain features tobe
reckoned with intheselection ofhost plants.
REFERENCES
1.Bethlenfalvay,G.J.,Abu-Shakra,F.F.,Fishbeck,K.,andPhillips,D.A.1978
The effectofsource-sink manipulations onnitrogen fixation inpeas.
Physiol. Plant 43:31-34.
2. Bethlenfalvay,G.J.,andPhillips,D.A.1977.Ontogenetic interactions
between photosynthesis andsymbiotic nitrogen fixation inlegumes.
Plant Physiol.60:419-421.
3.Bos,R.C.vanden,Bisseling,T.,andKammen,A.van.1978.Analysisof
DNA content,nitrogenase activity and in vivo protein synthesisof Rhizobium
leguminosarum bacteroids onsucrose gradients.J.Gen.Microbiol.109:
131-139.
4.Caldwell,B.E.,andVest,H.G.1977.Genetic aspectsofnodulationand
dinitrogen fixation bylegumes:themacrosymbiont,pp557-576 in Atreatise
on dinitrogen fixation (Section III),(R.W.F.HardyandW.S. Silver,eds.)
Wiley,NewYork.
5. Devine,T.E.andBreithaupt,B.H.1980. Significance ofincompatibility
reactions of Rhizobium japonicum strainswith soybean hostgenotypes.
Crop Sei.20:269-271.
6. Fred,E.B.,Baldwin,I.L.,andMcCoy,E.1932.Root nodule bacteriaand
leguminous plants,Madison.
7.Hageman,R.V.,andBurris,R.H.1980. Electron allocation toalternative
substrates of Azotobaater nitrogenase iscontrolled bytheelectron flux
through nitrogenase. Biochim. Biophys.Acta 591:63-75.
8.Hardy,R.W.F.,Burns,R . C ,andHolsten,R.D.1973.Applications ofthe
acetylene-ethyleneassay formeasurement ofnitrogen fixation. Soil Biol.
Biochem.5:47-81.
9. Lawrie,A.C.,andWheeler,C.T.1974.Theeffects offlowering andfruit
formation onthesupply ofphotosynthetic assimilates tothenodulesj)f
Visum sativum L.inrelation tothefixation ofnitrogen.NewPhyto!.73:
1119-1127.
10. Libbenga,K.R.,andBogers,R.J. 1974.Root nodule morphogenesis,pp430-472
in Thebiology ofnitrogen fixation (A.Quispel,ed.)North-Holland Publ.
Co.,Amsterdam.
11.Lie,T.A.1978. Symbiotic specialization inpeaplants:therequirementof
specific Rhizobium strains forpeas from Afghanistan.Ann.Appl.Bot. 88:
462-456.
12. Nutman,P.S.1952.Studiesonthephysiology ofnodule formation.
III. Experiments ontheexcision ofroot tipsandnodules.Ann.Bot. 16:
80-101.
90
13. Schubert,K.R.,and Evans,H.J. 1976.Hydrogen evolution,amajor factor
affecting theefficiency ofNfixation innodulated symbionts. Proc.Natl.
Acad. Sei. U.S.A. 73:1207-1211.
14. Sinclair,T.R., and DeWit,C.T. 1976.Analysis of thecarbon and nitrogen
limitations to soybeanyield.Agron.J. 68:319-324.
15. Sprent,J.I.,and Bradfort,A.M. 1977.Nitrogen fixation infield beans
{vicia faha) asaffected by population density,shading and its relationship
with soil moisture.J.Agric. Sei.88:303-310.
16. Stiefel,E.J., Burgess,B.K.,Wherland,S.,Newton,W.E.,Corbin,J.L.,and
Watt,G.D. 1980. Azotobaater vinelandii
biochemistry:H2(D2)/N2 relationships
ofnitrogenase and someaspects of ironmetabolism,pp211-222 in Nitrogen
fixation,vol.1(eds.W.E.Newton and W.H.Orme-Johnson)University Park
Press,Baltimore.
17. Yates,M.G.,and Walker,C.C. 1980.Hydrogenase activity and hydrogen
evolution bynitrogenase innitrogen-fixing Azotobaater
ahroooocoum,
pp95-109 in Nitrogen fixation,vol.1(eds.W.E.Newton and W.H.OrmeJohnson)University Park Press,Baltimore.
18.Wright,W.H. 1925.Thenodule bacteria ofsoybeans.II.Nitrogen fixation
experiments. Soil Science 20:131-141.
91
EFFECT OF N-FERTILIZERS ON NITROGEN FIXATION AND
SEED YIELD OF PEA-RH/ZOB/UM SYMBIOSES OF
DIFFERENT NITROGEN-FIXING CAPACITY
ABSTRACT
The supplyofnitratetopea(cultivar Rondo)-Rhisobium symbioses with
different nitrogen-fixing capacity growing innatural soil,lowered nitrogen
fixation more seriouslyinsymbioseswiththe highly effective strainPREthan
with themoderately effective strain S313.Nitrogen fixation with strain S310a
was lowerthan thatwith S313,butwas enhancedbylowlevelsofnitrate supply.
Inpeaplants inoculated with strain PRE,nitrogen fixationwas decreasedby
theadditionofcombined nitrogen irrespectiveofthe timeofdressing during
thegrowth period.
Leafdryweightandseed yieldswere significantly increasedbythe supply
ofnitrate,eveninthe caseofsymbioses withthehighly effective strain PRE.
Seed yield increases owingtothe applicationofnitratewith strains S310a
and S313were higher than thosewith PRE.Peaplants inoculated with S313
produced more seedathigh nitrate levels than plants infected with PRE,
associated withahigher leafmassandnitrogen fixation.Thedifferential
effectsofcombined nitrogenonnitrogen fixationandseed yield
were described inamodel.Accordingtoapath analysis,thefact that nitrogen
fixationofaless effective strainisrelatively unaffected bycombined
nitrogen couldbeattributedtoincreased photosynthesis.Theharvest index
was dependentonthe rateofseed productionbythepeacultivar andwasnot
affectedbythe rhizobial strainorbynitrate supply. Onlyinthecaseofthe
ineffective symbiosis RondoxP8the harvest index increased owingtonitrate
supply.
Nodule numbersandnoduleweight perplant decreased with all strains after
the supplyofnitrate,whereas theweight pernodulewas raised with strains
PREandS310a.With those strains,the fewer nodules also showedahigher
nitrogenase activity pernodule than thoseofthe plantswithout added nitrate.
Inthexylemexudateofplants inoculated with effective strains,asparagine
93
and aspartic acid accounted for 50and 20%of the total arnino-acid nitrogen,
respectively. With the ineffective symbiosis Rondo x P8 lessasparagine and
more aspartic acid was produced, viz. 10and 50%,respectively,of the total
amino-acid nitrogen.Theamino-acid pattern did not varywith nitrate supply.
Neither ethanol-soluble nor insoluble carbohydrate contentof the roots of
an effective symbiosis was influenced by nitrate supply.With an ineffective
strain,however,the ethanol-soluble carbohydrates increased with nitrate
supply up to 800mg N per pot butdecreased thereafter.This phenomenon was
attributed toan increased production of photosynthatewhich could not be
used ingrowth,due to lack of nitrogen.
From thedata presented in this paper it isconcluded that strains whose
nitrogen fixation has a low sensitivity in symbioses to combined nitrogen
offer no favourable prospects for use as an inoculant.
INTRODUCTION
Ina previous paper,a number of pea-Rhizobium symbioses were described with
distinctly different nitrogen-fixing capacities.The results obtained with
those symbioses gave rise to the hypothesis that associations with a poor
nitrogen-fixing capacity,giving plants with a low nitrogen content and a low
seedyield,might respond tocombined nitrogen differently from a symbiosis with
a high nitrogen-fixing capacity.
Combined nitrogen is known toexert adetrimental effect on nodule formation
and on nitrogen fixation,aswas already observed in the beginning of nitrogen
fixation research (17,24). Ithas no direct effect on the nitrogen-fixing
system (11)but reduces the photosynthate supply to the root nodules,leading
toa lower nitrogenase activity (18; Chapter 2,page 39/.The addition of combined nitrogen therefore often leads to a simultaneous decrease in nitrogen
fixation without increasing yield (2,21).However,also beneficial effects of
combined nitrogen on nitrogen fixation have been reported,for example the
addition ofa 'starter dose'which increases nitrogen fixation by enhancing
photosynthesis (4,.12,13, 15). Sincethese effects so far have not been examine
inacoherent study,the investigationsreported in the present paperdeal with
the effect of combined nitrogen on pea-Rhizobium symbioses with high and low
nitrogen-fixing capacities in relation to nitrogen fixation, photosynthetic
capacity and seed yield.
94
MATERIALSANDMETHODS
Plant growth and nitrogen
fertilizer
The various pea-Rhisobium symbioses were growninnon-sterile soil asdescribed
intheprevious paper.Ryegrass,Westerwolds,cv.Tewera (Lolium perenne) was
sowninsoil without gravel cover.
A solutionofNH.NCUorNaNO-,wasgivenatdifferent growth stagesofthe
15
1e;
plant.Inthe NexperimentasolutionofK NO,wassuppliedatsowingandat
15
pod fill (enrichment percentages varying from 1.28-2.79atom %N ) .
Amino acids in xylem
exudate
Potswerewateredandtheplantswere decapitated after2h.Xylem sapwas
collected inglass capillary tubesfor1handstoredat-20°C until analysis.
Proteinwasremoved fromthesamplesbyadditionofsulphosalicylic acid, 2%by
weight,followedbycentrifugation.Thesupernatantwasanalyzed foramino acids
withaBiotronik Amino Acid Analyzer.
Carbohydrates
Ethanol-soluble carbohydrates ('sugars')were determined inethanol extracts
of rootsbytheanthrone method (20).Theresiduewasheatedat100°C for2h
with0.1NHCltohydrolyzetheethanol-insoluble carbohydrates ('starch')which
were subsequently measured inthesupernatantwith theanthrone method.
Nitrogen
determinations
Nitrogen contentofshoot,root,podwall andseedwasdeterminedbythe
Kjeldahl method.A100mg sampleofdryplantmaterialwasdigested for3hat
360°C with3mlofconcentrated H 2 S0.,usingamixed catalystofCuSO»,Seand
K 2 S0». Subsequently,theammonia generated indigestionwassteam-distilled into
10mlofH,B0 3 (40g/1)andtitrated with 0.02 NH 2 S0 4 ,usingamixed indicator
ofmethylredandbromocresolgreen.
15
When samples wereanalyzedfor N,the ammonia generated inthedigestion
was steam-distilled into10ml0.1NHCl.Ammonia wasdetermined ina1-ml
aliquotwith Nessler's reagent.Theremaining distillatewasanalyzed for N
by emission spectrometry,usingtheRittenberg methodoffreeing N~ fromNH-.C1
15
( 1 ) . Enrichment of
15
e
N was calculated by comparison with a standard set supplied
n
by VEB Chemie, B e r l i n , D.D.R. ( 7 ) .
95
RESULTS
Nitrogen
fixation
and combined nitrogen
supply
Thenitrogenase activityofeffectively nodulatedpeaplants (cv.Rondox
PRE)wasreducedbythesupplyofcombined nitrogenatsowing (Fig. 1).Evena
lowdoseofammonium nitrate decreased in vivo nitrogenaseactivityanddelayed
theoccurrenceofthemaximum enzymeactivity.Inplantswithout added nitrogen,
maximum nitrogenase activity was foundatthebeginningofthepod-filling
stage.
The effectonnitrogen fixationofsplit dressingsofcombined nitrogenat
variousmomentsofthegrowth period was studiedinaseparate experimentwith
pea plants inoculated with R. legwninosarum strainPRE(effective)andstrain
P8 (ineffective).Nitrogen fixation was calculatedasthedifferenceintotal
plant nitrogen betweentheeffectiveandineffective symbiosis (Fig.2 ) .
Ö
£
oHH-
t70
90
Days
Fig.1.Profilesofnitrogenase activity inthesymbiosispea(cv.Rondo)x
R. leguminosarum PREduring ontogenesis (1978experiment),as
affectedbythesupplyofNH4NO3atsowing:noadded N,D ;314,•;
and 942mgN/pot,0.Arrow indicatesthebeginningofthepod-filling
stage,65daysafter sowing.Valuesaremeansofduplicate samples
of8plants.
96
750
CT
E
x 500 -
250
/ 2 5 \ N a N 0 3 at s o w i n g ,%
\ 7 5 / N a N 0 3 at p o d - f i l l , %
Fig.2.Yieldsofnitrogen fixed inthesymbiosispea(cv.Rondo)x R. legvminosarvanPRE(1979 experiment)asaffected bytheadditionatvarious
growth stagesofNaNC>3:515,• ;1130mgN/pot,» . N(Kj)analyses
were performedatmaturityoftheplants.Thebroken line indicates
theamountofnitrogen fixedbyplants growing without added NaNC^.
Values represent meansofduplicate determinations.
Obviously,nitrogen fixationisreduced less seriouslybythesupplyofcombined
nitrogen lateinthegrowing season thanbysupplyatanearlierdate.
Seed yieldofeffectively nodulated peaplantswashardly affectedbyvarying
themomentof(split)dressingsofcombined nitrogen duringthegrowth period
(Table 1).Supplyofthenitrogen during floweringandpodfilling tendedto
give higher seedyields.
is —
Experiments with
NO .Theresponsetocombined nitrogenofnitrogen fixation
in symbioses with different nitrogen-fixing capacitieswas studied inan
15
experimentinwhich N-labeled
nitratewasusedinordertodifferentiate
between fixed nitrogenandplant nitrogen derived from combined nitrogen.
Nitrate was giveninsplit dressingsofequal quantitiesatsowingandat
pod fill toprevent damagebyhigh dosesofthefertilizer.
Yieldofplant nitrogenofall symbioseshadincreased substantially bythe
supplyofnitrate.The highestamountofnitratehadbeen takenupbythe
moderately effective symbiosisofcv. Rondowith R. leguminosarum, strain S310a
(column6ofTable2 ) .
97
Table 1.Effectofthemomentofsplit dressings with NH^NOjontheseed yield
ofpeaplants (cv.Rondo) inoculated with R. legvminosanm strain PRE ''
Total amount of
Split dressings ofNH
NH 4 N0- added
during the stage of
(mgN/ pot)
0
314
628
4 N0 3
(mg N/pot)
Seed dry
weight
Sowing
Vegetative
growth
Flowerir g
0
314
0
0
0
314
314
314
0
0
0
0
0
314
0
0
314
0
0
314
314
0
0
0
0
314
0
0
314
0
314
0
314
Pod-filling
0
0
0
0
314
0
0
314
0
314
314
(g/pot)
13.0
15.0
18.1
18.2
17.8
a
a
b
b
b
17.7
19.8
18.1
22.0
19.2
19.3
b
ab
b
a
b
b
1) Valuesaremeansofdeterminations of5pots. Values followed bythesame
letterarenotsignificantly different inTukey's testatthe0.05level.
To estimate theamountofnitrogen fixed bythevarious symbiotic systems,
total plant nitrogen oftheeffective symbiosiswasdiminished with theamount
of plant nitrogen oftheineffective symbiosis dressed with thesame amount
ofcombined nitrogenastheeffective association (column4ofTable 2).As
alternativefortheineffective symbiosis,thenitrogen yield ofryegrass
plantswasemployed forcalculating theyields offixed N (column 5ofTable 2),
A somewhat more accuratewaytoestimate theamountoffixed nitrogenin
symbioses grown innatural soil anddressed with combined nitrogen consistsof
the calculation oftotal plant nitrogen minus (a),thenitrate-derived nitrogen
oftheplantmaterial and(b)theamountofnitrogen taken upfrom soiland
seed.Thenitrate-derived nitrogen inplant material canbedirectly measured
by supplying a labeled nitrogen compound (column6ofTable 2 ) .Available soil
+ seed Nwasderived fromtheineffective symbiosis cv.RondoxP8,dressed
with anequal amountofnitrate (column 7ofTable 2). Theresultsofthese
calculations aregiven incolumns 4,5and8ofTable 2.Comparison ofboth
waysofestimation shows thatatlowlevelsofcombined nitrogen thefirstmentioned method gave somewhat lower values thanthelast-mentioned,whereas
at high levels theresults tended tobetheotherwayround.
98
cu
ro
>
3 -i
O !
S- !
Dl-,
I
c/l CU
CU - Q
4->
O
, Q.
S-
p
CO
4-
4 - ^
o ro
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15
A simplified method of calculating nitrogen fixation from N-fertilizer
trials has been proposed byFried and Middelboe ( 8 ) .According to their method,
the amount of fixed nitrogen (Avalue) is calculatedas
15
/. excess atom % Ninlegume
x ..,M,
(1
3-r
)xtotal N legume
excess atom % Ninreference crop
The calculated Avalues of our experiment correspond closely to the nitrogen
fixation values found bysubtraction of nitrate-derived nitrogen and available
soil nitrogen from total nitrogen ofthe effective symbioses (Table 2 , column 9).
Of the three effective R. leguminosarum
strains tested the highly effective
strain PRE insymbiosis with cv. Rondo fixed the highest amounts of nitrogenin
the absence of added nitrate or with alow amount of thisNcompound. With
high amounts of added nitrate (800, 1200 and 1600 mgNper pot) nitrogen fixation with strain PRE was considerably reduced. With the moderately effective
strain S313 lower amounts o fnitrogen were fixed than with PRE inthe absence
of combined nitrogen, but the reverse was true at high levels of combined
nitrogen. The low nitrogen-fixing capacity with strain S310a was enhanced by3 8 %
by nitrate supply upto 800 mgNper p o t , whereas with higher levels of combined
nitrogen, nitrogen fixation decreased, similar to that of the other strains.
Nitrogen fixation
during the growth period. Thegreaterpartofnitrogen
fixation occurred during the pod-filling period (Table 3)ashas been found
earlier inpea ( 1 9 ) ,soybean (9) and cowpea ( 6 ) .The higher nitrogen fixation
during the pod-filling stage coincided with asomewhat higher efficiencyo f
nitrate uptake than during the period of vegetative growth and flowering.
Nitrogen fixation of pea-ff. leguminosarum
strains with alow or intermediate
nitrogen-fixing capacity (S310a and S 3 1 3 , respectively) responded favourably
to added nitrate during the generative phase of the growth cycle. The yields
of fixed Nuntil maturity wereincreased bythe supply of nitrate uptoa dose
of 800 mg Nper pot at the beginning of the pod-filling period, whereas nitrate
supply atsowing decreased nitrogen fixation with all strains.
100
15
Table 3.UptakeofK NO3and N2fixation bypea plants (cv.Rondo)from sowing
tothebeginning ofpod-fill (1-72days)andfrom pod-fill until maturity
(73-114days). Values representmeans ofduplicate samplesof8plantsand
aregivenasmg N/pot
Strainof
K 1 5 N0 3 supplied 1)
Uptakeof 15N03
microsymbiont
P8
S313
S310a
PRE
N 2 fixation
" ( T ^ ) (73-114)"
0
400
800
1200
1600
0
400
800
1200
1600
0
400
800
1200
1600
0
400
800
1200
1600
"(wFj - (73-114)
108
157
378
524
.
121
218
452
491
106
218
374
504
_
137
241
392
525
240
367
521
567
167
246
531
650
181
278
457
755
112
411
479
568
429
262
164
85
171
278
174
139
196
125
442
436
246
174
102
568
674
746
688
364
314
471
680
417
214
786
682
478
353
275
1)
Splitdressing,50%at sowingand 50%atday73.
101
400
800
1200
1600
KN03,mg N/pot
Fig.3.Seedyieldsatmaturityof pea-Rhizobium symbiosesasaffected bysplit
dressingsofKN0 3 viz. 50%atsowingand50%atpodfill.Values
representmeansofyieldsof4pots.
(a)Peacv.Rondo inoculated with R. leguminosarwn strainsP8 (t),
S310a (o),S313(A),andPRE(O)(1980 experiment).PRE(1978)supplied
with NH 4 N0 3 (•).
(b)FractionofN03-derivedNinseedN,atmaturityofpeacv.Rondo,
inoculated with R. leguminosarum strainsP8(•),S310a (o),S313 (A),
and PRE ( 0 ) .
Seed yield
N dressings with KN^of pea plants ( c u l t i v a r Rondo), inoculated with d i f f e r ent Rhizobium s t r a i n s , raised seed dry weight ( F i g . 3a). With strains P8, S310a
and S313, seed yields were s i g n i f i c a n t l y enhanced (p < 0.001) by n i t r a t e supply
(1980). Strain PRE produced a high seed y i e l d in the 1980 experiment without
added n i t r a t e ; supply of the nitrogen compound gave only a low, weakly s i g n i f i cant (p < 0.05) additional seed y i e l d . However, i n the 1978 experiment, the seed
dry weight with PRE was much lower than in 1980, but i t was considerably enhanced by added N H ^ (p < 0.001). This points out that seasonal factors exert
an important influence on the y i e l d response to combined-nitrogen a p p l i c a t i o n .
102
<t
*
o—•
b
400
1200
1600 0
400
800
KN03,mg N/pot
Fig.4. Harvest indices (ratios of seed nitrogen to total plant nitrogen)of
pea-Rhisobium symbioses asaffected by split dressings of nitrate,
50% at sowing and 50%at podfill.
(a)Peacv. Rondo,inoculated with R. leguminosamm strains P8 (•),
S310a (o),S313 (A),and PRE( D ) .
(b)Peacvs Florix,early (x),Rondo,intermediate (O)» and Mercato,
late ( • ) , inoculated with R. leguminosarum PF„.
The contribution ofnitrate-derived nitrogen to the seedyield was higher in
pea plants inoculated with the ineffective strain P8than in nitrogen-fixing
plants (Fig.3b).
The harvest index of the various symbioses,which normally isameasureof
theefficiency of redistribution of nitrogen from vegetative parts and podwalls
to theseed was independent ofnitrate supply and of rhizobial strain (Figs
4a,b). However,with the ineffective strain P8the harvest index of theunfertilized plants deviated from those dressed with nitrate owing to nitrogen
deficiency sothat little nitrogen wasavailable for redistribution (Fig.4a,
Table 1).The harvest indexwasonly dependent on the pea variety as can be
seen bycomparing Figs 4aand b.
103
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104
Ei-t*
Nodule
formation
Additionofnitratetopeaplantscv. Rondoatsowinghadreduced nodulation
asitwas observedatpodfill. Numbersofnodulesonprimaryaswellason
lateral rootsofalmost allofthe symbioses testedhadconsiderably decreased
(Table 4).Thesamewas trueoftotal noduleweight perplant.Inthecaseof
weight pernodule theeffectofcombined nitrogen was much more variableas
concerns different symbioses.With R. leguminosarwn strain S313,which forms
large amountsofnodular tissue(upto40%oftotal root w t ) , weightper
noduleonprimary rootshaddroppedtoapproximately 1/3onthe nitrate-dressed
plantsascomparedtothe plants without added nitrate.Weight per noduleon
lateral rootswas also adversely affectedbynitrate supplyatsowingbutthe
reduction was much less pronounced thanitwas trueofthenodulesonprimary
roots.Inthe caseofstrain S310aandtoalesser extent PRE,weightper
noduleonprimary roots wasmuch higherwhen nitratehadbeen supplied. The
beneficial effectofnitrate supplyonweightofindividual nodulesonlateral
roots with thosetwostrains wasmuch less pronounced than thatonprimary
roots.
The second supplyofnitrateatthestartofpodfilling,has adversely
affected numbersandtotal weightofnodulesonboth primaryandlateral roots
as observedatmaturityofthe plants.Weight per noduleonlateral rootshad
only slightly decreasedascompared with thoseonprimary roots.Inthecase
of S310a thenoduleshadclearly increasedinweightbythedressing with
nitrate.
Nitrogenase activityoftheS313 nodules,measuredatpodfillinghad declined slightlyduetonitrate supplyatsowing,ascontrastedtothe S310aand
PRE nodules whose nitrogenase activity was considerably higher than thatof
nodules ofunfertilizedsymbioses.Nitrogen-fixing activityofthevarious
associations droppedtolowvalues during maturityofthe plants.Nodules from
plants dresseswith nitrate tendedtobemoreactive than thosewithout supply
of nitrate.Thiswasparticularly trueofthenodules formed withS313.
Carbohydrate
content
and amino acid composition.
A comparison of thecarbohydrate
contentsofthe rootsofineffectively nodulated (P8)andeffectively nodulated
(PRE)plants showed that thecontentsofethanol-insoluble carbohydrates
(mainly starch)inboth associations were equal,anddeclined slightly with
increased nitrate supply (Fig. 5).Ethanol-soluble carbohydrates (sugars)of
symbioseswith the effective strain PRE were lower than the ethanol-insoluble
105
200
400
600
800
KN0 3 .mg N/pot
Fig. 5.Ethanol-soluble (•,•) and ethanol-insoluble (o,0) carbohydrates in
rootsof pea plants (cv. Rondo),68days after sowing,inoculated with
R. legwrtinosavum strains P8 (•, o)and PRE (w ,0), asaffected by
nitrate supply at sowing.Values representmeansof triplicate
determinations.
carbohydrates. Insymbioseswith the ineffective P8strain,nitrate supply
caused adistinctmaximum in thecontent ofethanol-solublecarbohydrates,
which decreased to thevalue foundwith PREatthe highest level of nitrate
added. A similar phenomenon has been observed ineffectively nodulated soybean
plants (14).Itwasattributed toan increase inphotosynthetic activity of
the plant,without sufficient nitrogen being present for the utilization of
theadditonal sugars forgrowth,resulting inan increase in sugarconcentration.
No explanation can begivenwhy no increase inethanol-insoluble carbohydrates
('starch')was found along with the rise inethanol-soluble carbohydrates
since thecontents ofthese carbohydrates areusually related (seeChapter 3,
Fig. 2)
Theamino-acid content of thexylem exudatewas hardly affected by the
supply ofnitrate to theplants (Figs.6a,b),aswas found earlierwith peas
(6). The amount ofnitrogen present asamino acid inthe xylem sapof the
ineffective symbiosis Rondo x P8was lower than that of theeffective symbioses
Rondo xPRE,x S310a and x S313,but increased with nitrate supply (Fig.6a).
106
200
400
600
800
KN03.mg N/pot
Fig.6.Amino-acid nitrogen inthe xyleniexudate of pea (cv.Rondo) inoculated
with R. legwninosarum strains P8 (•),S310a (o),S313 ( A ) , and
PRE ( 0 ) , asaffected bynitrate supply at sowing.Analyses made
68daysafter sowing.
(a)Total amino-acid Nconcentration.
(b)RatioofAsp.-N toAsn.-N.
A similar slight response tonitrate occurred in theexudate of pea plants
associated with the poorly N2-assimilating R. leguminosarum strain S310a.The
only difference intheamino acid pattern between the strains (notshown)was
formed by theaspartic acid and asparagine concentrations aswas found earlier
inthis laboratory (22).With the ineffective strain P8,nitrogen wastransported intheplantasaspartic acid ratherthanasasparagine,whereas the
reversewas truewith the effective strains (Fig.6b).These twoamino acids
togetheraccounted for 58-89%ofthe total amino-acid nitrogen ofthexylem
exudate ofall symbioses.
107
DISCUSSION
Differential response to combined nitrogen of pea plants inoculated with
different rhizobial
strains
Seed yieldofpeaplantswas increasedbythe supplyofcombined nitrogen,
evenwith strain PREwhich possessesahigh nitrogen-fixing capacity without
added nitrate (Table 2,Fig.7b).Nitrate uptakeandutilizationbyplants
inoculated with strainswithalownitrogen-fixing capacity (S313,S310a)were
higher thanitwas trueinplants inoculated with strainPRE(Table 2).Athigh
800
1200
1600
KN03,mg N/pot
Fig. 7(a).Dryweightofshoots (=leaves+stem)atmaturityofpeaplants,
inoculated with R. leguminosarum strainsP8(•),S310a (o),S313(A),
andPRE( D ) ,asaffected bynitrate supply. Valuesare meansof
4 potsof8plants each,
(b). Nitrogen fixation during growthofpeaplants,inoculated with
R. leguminosarum strains S310a (o),S313 (A),andPRE( D ) .Values
derived from Table2.
108
nitrate supply,seedyields with S313andS310a were higher than those with
PRE (Fig.3a).Thepeacv.Rondo association with strainPREfixed the highest
amountsofnitrogenatlow nitrate supply (Table 2,Fig.7b).Athigh levels
ofadded nitrate,theyieldoffixed nitrogen decreased sharplyto31%ofthe
valuewithout added nitrate.Theshootdryweightofthis symbiosis increased
slightly,duetonitrate supply (Fig.7a).Incontrast,theshoot dry weight
of plants inoculated with strain S310a rose steeply when nitrate was added.
The poornitrogen-fixing capacityofthepeasymbiosis with S310a was enhanced
from 100to138%atanitrate supplyof800mgN/pot,butfell to57%ofthe
initial value when higheramountsofnitrate were supplied. Nitrogen fixation
with themoderately effective strain S313was only loweredto54%athigh
nitrate supply.Theshoot dryweightofthis symbiosis responded toadded
nitratetoalower degree than with S310a butmore than with PRE.
The dissimilar responsetothe supplyofcombined nitrogen with highly
effectiveandmoderately effective rhizobial strains isexplained byassuming
theoccurrenceofdifferent levelsofC-andN-limited growth simultaneously.
A symbiosis growing underNlimitation might reacttocombined nitrogen supply
by increased photosynthesis,withanenhanced nitrogen fixationasanindirect
result.However,alsoasymbiosis growing underClimitation might reactto
combinedNsupply,assimultaneouslywithaClimitation nitrogen fixation
islimited owingtoashortageofcarbohydrates.Asimilar concept has recently
<
nitrogen
n i t r o g e n fif i x a t i o n
I
(a)
I* .
photosynt hesis
^photosynthesis^^
combined^.
1
- ^ s e e d yield
nitrogen-»,* „ „
^^*
'kl
fixation
Fig. 8. Model of relations between combined nitrogen supply, photosynthesis
and nitrogen fixation with regard to seed yield of plants.
(a)Vegetative phase.
(b)Generative phase.
109
been proposed by Phillips and co-workers (16,23).These relations are visualized in the summary models presented in Fig.8for thevegetative and the
generative growth phases.
Themodel of Fig.8a for vegetative growth isbased upon the assumption that
growth of legumes in soil in this phase is principiallyN-limited. There is
support from literature tojustify this assumption from fertilizer trials
{e.g. 12,15,24),but ithas also been shown that under such conditions the
available amount of nitrogenase is limiting nitrogen fixation (Chapter 1,pp.
21 -34)Themodel of Fig.8b forgenerative growth depends on the assumption
that principally aC limitation exists during this growth phase.This assumption
isalso supported by experimental evidence, e.g. the response tocarbon dioxide
enrichment of theatmosphere (9)and the response toa higher light intensity
(3). Furthermore by the observation that during the generative growth phase,an
excess ofnitrogenase is present in the nodules,not used in nitrogen fixation
(Chapter 1)
Inorder to provide a quantitative description of the effect of carbon and
nitrogen limitation on growth ofthe various symbioses studied,a pathway
analysis wasmade according to themodels of Fig.8.The results of this
analysis using the data ofTable 2for nitrogen fixation and combined nitrogen
uptake (columns8 and 7,respectively )and Figs 3and 7for seed yield and
shootweight,respectively, are listed inTable 5. Photosynthesis has been
roughly estimated by using dryweights of plantmass with photosynthetic
activity, i.e. shoot,leaves and pod walls. This is inaccurate because photosynthetic activity per unit of leafmass differswith rhizobial strain (5;
Chapter 3,Table 4)and changes with age,but it is suitable for anapproximate evaluation of the relationship between thevariables.
The path regressions of Table 5are derived from a series of linear multiple
regression equations,assuming a causal order as shown inFig.8. Inpath
regressions,the influence ofvariables which intervene according to thecausal
order assumed isaccounted for.Forexample,the path regression ofphotosynthesis (dependent variable)oncombined nitrogen (independent variable)
during the vegetative period (Table 5, item 3) includes the direct effect of
combined nitrogen on photosynthesis aswell as the indirect effect via nitrogen
fixation.
The decrease of nitrogen fixation due tocombined nitrogen supply was
significantly different between the Rhizobiwn strains (Table 5,items 1and 6)
in the sequence PRE >S313 > S310a.During the vegetative growth phase,photosynthesis with strain PRE appeared to bemainly dependent on nitrogen fixation,
110
Table 5. Path regressions of nitrogen fixation,photosynthesis and seed yield
on combined nitrogen 1)
Strain of microsymbiont
Path regression
Vegetative growth (Fig.8a)
1N„ fixation on combinedN
2 Photosynthesis on N ? fixation
3 Photosynthesis oncombined N
4 Fit ofmultiple regressions (R )
Generative growth (Fig. 8b)
5 Photosynthesis on combinedN
6 N„ fixation on combined N
7 Seedyield on combinedN
8 N~ fixation on photosynthesis
9 Seed yield on photosynthesis
10Seedyield on N ? fixation
11Fitof multiple regressions {Rù)
P8
S313
0
0
30 28 a
-0 48
16 67
14 00
0.69
0.85
I
[
<)
21 o o r
0
19 oi5*
0
-0 28 b
0
0 91
S310a
**
b
**
b
**
b
**
6.58 a
**
-0.51 b
**
4.71 a
**
0.06 a
PRE
**
-0 23 c
**
15 93 b
**
18 09 b
0.77
b*
**
-0.27 a
**
11.75 b
**
0.05 a
0.93 a
22.18 a
0.86
12.33 b
0.95
**
**
**
a
**
a
o
**
4.12 d
10.95
0.78 a
**
-0 73
25 51
6 28
0.75
-0.78 a
**
3.08 a
-0.00 b
-0-33 b
**
22.40 a
0.77
Path regressions from multiple linear regression equations (units mg)based
upon 25cases per strain in each part of the growth period. Values inone row
followed by the same letter are not significantly different at the 0.05 level
inatwo-sided t-test.Asterisks indicate that the path regression isnon-zero
(*, 0.05 level;**, 0.01 level)according toanFtest.
whereas with strains S313 and S310a combined nitrogen and nitrogen fixation
influenced photosynthesis toan equal extent (Table 5,items 2and3 ) .
Photosynthesis during thegenerative growth period was increased by combined
nitrogen in the sequence P8>S310a >S313 > PRE (item 5). With strains S310a
and S313,an enhanced photosynthesis due to theaddition of nitrate significantly increased nitrogen fixation (item 8)and seed yield (item 9). Seed
yield was not significantly correlated with photosynthesis with strains PRE
and P8owing tothedominating effect of nitrogen fixation in PREand combined
nitrogen inP8.
111
The fit of themultiple regressions was reasinably good (items 4and11),
notwithstanding the fact that the assumption of linear relationship was not
always justified. Forexample,the relation between nitrogen fixation and
combined nitrogen contained a significant square power termwith strain S310a
(cf.Fig. 7b),,which was left outof consideration.
The results presented in this paper show that pea plants inoculated with
strains ofif. leguminosarum with adifferent nitrogen-fixing capacity respond
to combined nitrogen inadifferent way aswas found before ina field trial
with these strains (10).At high dressings more nitrate is taken up by plants
living in symbiosis with a strain ofmoderate nitrogen-fixing capacity than by
plants with a highly effective strain (Table 2 ) .Photosynthesis isenhanced by
added nitrate inplants inoculated with a less effective strain.Therefore,at
high nitrate dressings of these strains a higher level of nitrogen fixation is
found than with a highly effective strain at high nitrate dressings.An
increased nitrate uptake,photosynthesis and nitrogen fixation account for
the high seed production of less effective strains as compared with highly
effective strains at high nitrate dressings. Consequently, the less effective
strains possess a nitrogen-fixing capacity that is less sensitive to combined
nitrogen. Selection ofsuch strains for useas inoculant,isnot likely to
produceyield increases with economically feasible fertilizer dressings.
REFERENCES
1.Akkermans,A.D.L. 1971.Nitrogen fixation and nodulationof Alnus and Hippopha
under natural conditions.Thesis,Univ. Leiden,The Netherlands.
2.Alios,H.F.,and Bartholomew,W.V. 1959.Replacement of symbiotic nitrogen
fixation by available nitrogen. Soil Sei. 87:61-66.
3.Antoniw,L.D.,and Sprent,J.L. 1978.Growth and nitrogen fixation of Phaseoli
vulgaris L.at two irradiances. IINitrogen fixation.Ann.Bot.42:399-410.
4.Bethlenfalvay,G.J.,Abu-Shakra,S.S.,and Phillips,D.A. 1978. Interdependent
ofnitrogen nutrition and photosynthesis in Pisum sativum L.1.Effect of combined nitrogen on symbiotic nitrogen fixation and photosynthesis. PlantPhysii
62: 127-130.
5.Bethlenfalvay,G.J.,Abu-Shakra,S.S.,and Phillips,D.A. 1978.Interdependem
of nitrogen nutrition and photosynthesis in Pisum sativum L.2.Host plant
response to nitrogen fixation by Rhizobium strains.Plant Physiol.62:131-13:
6. Cowan,M.C. 1979.The effect of nitrogen source on levels ofamino acids in
peas. Plant and Soil 51:279-282.
7.Ferraris,M.M.,and Proksch,G. 1972.Calibration methods and instrumentation
foroptical 15 N determinations with electrodelessdischarge tubes.Anal.Chim
Acta 59:177-185.
112
8. Fried,M.,and Middelboe,V. 1977.Measurement of amount of nitrogen fixed
by a legume crop. Plant and Soil 47:713-715.
9. Hardy. R.W.F,and Havelka,U.D. 1976.Photosynthesis as amajor factor limiting
nitrogen fixation by field-grown legumes with emphasis on soybeans,pp.421-439
in Symbiotic nitrogen fixation inplants (ed. P.S.Nutman), IBP 7,Cambridge
Un. Press,Cambridge.
10.Hiele,F.J.H. van. 1960.Enting van lupinen,erwten en stambonen metverschillende Rhizobiumstammen;veldproeven. Verslagen van Landbouwk.Onderzoekingen
66.19 Pudoc Wageningen,TheNetherlands.
11.Houwaard,F. 1978. Influence of ammonium chloride on the nitrogenase activity
of nodulated pea plants {visum sativum). Appl. Envir.Microbiol.35:1061-1065.
12.Mulder,E.G. 1949. Investigations of thenitrogen nutrition of peaplants.
Plant and Soil 1:179-212.
13. Orcutt,F.S.,and Fred,E.B. 1935.Light intensity as an inhibiting factor
in the fixation of atmospheric nitrogen by Manchu soybeans.J.Am. Soc.
Agron. 27:550-558.
14. Orcutt,F.S.,and Wilson,P.W. 1935.Theeffect of nitrate-nitrogen on the
carbohydrate metabolism of inoculated soybeans.Soil Sei.: 39:289-296.
15. Pate,J.S.,and Dart,P.J. 1961.Modulation studies in legumes.IVThe
influence of inoculum strain and timeof application ofammonium nitrate
to symbiotic response. Plant and Soil 15:329-346.
16. Phillips,D.A., De Jong,T.M., and Williams,L.E. 1980.Carbon and nitrogen
limitations on symbiotically grown soybean seedlings,pp. 117-120 in Current
perspectives in nitrogen fixation (eds.A.H.Gibson and W.E.Newton) Elsevier
Amsterdam,The Netherlands
17. Rautenberg,F.,and Kuhn,G. 1864.Vegetationsversuche imSommer 1863.
J. Landwirt. 12:107-140.
.
18. Small,J.G.C.,and Leonard,O.A. 1969.Translocation of 4C-labeledphotosynthate innodulated legumed as influenced by nitrate nitrogen.Amer. J.
Bot. 56:187-194.
19.Thornton,G.D. 1956.Theeffect offertilizernitrogen on nodulation,growth
and nitrogen content of several legumes grown in sandy soils.Proc. Soil and
Crop Sei. Soc.Florida 16:146-151.
20. Trevelyan,W.E.,and Harrison,J.S. 1952.Studies onyeast metabolism.
IFractionation and microdetermination of cell carbohydrates.Biochem. J.
50:298-303.
21. Welch,L.F.,Boone,L.V., Chambliss,C G . , Christansen,A.T.,Mulvaney, D.L.,
Oldham,M.G.,and Pendleton,J.W. 1973.Soybean yieldswith direct and
residual nitrogenfertilization.Agron.J. 65:547-550.
22. Wieringa,K.T.,and Bakhuis,J.A. 1957.Chromatography as ameans of selecting effective strains of Rhizobia. Plant and Soil 8:254-262.
23. Williams,L.E.,and Phillips,D.A. 1980.Effect of irriadiance on development
of apparent nitrogen fixation and photosynthesis in soybean.Plant Physiol.
66:968-972.
24. Wilson,P.W. 1940.The biochemistry of symbiotic nitrogen fixation.Un.of
Wisconsin Press,Madison,U.S.A.
113
GENERAL DISCUSSION
Potential nitrogen fixation
The in vitro nitrogenase assay (Ch.1)ofisolated bacteroids,treated with
EDTAandtoluene,andsupplied withATPandsodium dithionite,appeared tobe
a valuable methodforobtaining informationonnitrogenase utilizationin
legume-rhizobia symbioses.Themethod was used inexperiments with various
host plants inoculated with R. leguminosarum strainsofdifferent origin viz.
PRE,The Netherlands (Chs1,3 ) ;S310a,Sweden (Ch.3);RB1,Turkey (Ch.1 ) ;
TOM,TurkeyandHIM,Pakistan (van Mil,unpublished results). However,with
some strains, e.g. PF ?andS313,this assaydidnotprovide satisfactory
results,rendering these strains unsuitableforthe purposeofdetermining
nitrogenase utilization.
TheEDTA-toluenemethod usually yields valuesofin vitro nitrogenase
activity that are higher than thoseofin vivo activity,which enabledusto
develop theconceptofpotential nitrogen fixation.Theelectron donor used,
sodium dithionite,wasrecently showntobenotasefficient ingenerating
nitrogenase activityasamixtureofflavodoxin+ferredoxin I(25).Therefore,
the EDTA-toluene method might underestimate potential nitrogen fixation.
Accordingtotheresults obtained with ourassay,peaplants inoculated with
R. leguminosarum strains PREandS310a,andfield bean plants inoculated with
RB1 utilized their full potential nitrogen fixation only duringthevegetative
phase,when grown insoil intheopenair(Chs 1 , 3 ) .Actual nitrogen fixation
was foundtobeconsistently lower than potential nitrogen fixation inthe
generative phase,irrespectiveofshoot massorofshoottoroot ratio (Ch.1).
This discrepancy between actualandpotential nitrogen fixation was widened
by theadditionofnitrate,which lowered actual nitrogen fixation,whereas
potential nitrogen fixation remained unaffected for10days (Ch. 1).Boththe
developmentofgenerative organs (13)andthe additionofcombined nitrogen
(10, 19)causeachangeinphotosynthate translocation pattern from the shoot
115
totheroot,resultinginalower carbohydrate supplyofthe nodules. The
applicationofbenzyladeninetothe nodules eliminatesthe adverse effectof
added nitrateonactual nitrogen fixation (Ch.2 ) ,probablybyincreasing
carbohydrate supplytothenodules.From the data presented inChs1and2
the conclusioncanbedrawn thatinthevegetative phase,nitrogen fixation
is limitedbythenitrogenase contentofthe bacteroids,whereasinthe
generative phase,aninadequate supplyofcarbohydrates from the host plant
to themicro-symbiontcausesasuboptimal rateofnitrogen fixation.Asthese
results were also obtained infield-grown bean plants (Ch.1)itislikely that
this conclusion also holds under circumstances relevanttoagriculture.
Regulation
of nodule
formation
Despite thedifferences innodule-formation characteristics betweenthe
pea-Rhisobium symbioses investigated,themaximum amountofnodules per plant
were obtained atthebeginningofthe pod-filling period (Ch.5;cf.field
beans,Ch. 1).The processofnodule formationcanberegardedasacontinuous
adaptationofnodularmasstotheincreasing shootmass (Ch. 1),butit is
surprising that increaseofnodular weight occurs preferentially tothe complete
utilizationofthe nitrogenase presentinthe bacteroids.Nodule formation
probably occursatlower levelsofcarbohydrate supplythan thefunctioningof
nitrogenaseatoptimum rate (28).Thesamewas found inexperiments with
enhanced photosynthesis, e.g. bycarbon dioxide enrichmentofthe atmosphere
(8, 16),byincreased light intensity (1)orbygraftingoftwoshootsonone
root system (22).Nodule activity was raised immediately after these treatments,
butlateronnodular mass grew higherand accountedfor themajor partofthe
increased nitrogen fixationper plant.
An explanationofthese phenomena mightbeofferedbytheobservation that
uninfected cellsofthenodules rather than infected cellsare supplied with
photosynthates (12).This might pointtoalower synthesisofphotoassimilateattracting phytohormonesincells infected with rhizobia thaninuninfected
cells, e.g. thenodularmeristematic tissue.
Hydrogen production
and hydrogenase
activity
Gradually less hydrogen was foundtobeproducedbynodulesinairrelative
to total nitrogenase activity during ontogenesisofboth beanandpea plants,
giving risetoanincreased relative efficiency uponstarting ofpod filling
(Chs 4,5;3 ) .Anenhanced light intensity was showntostimulate nitrogenase
activity,buthydrogen production was raisedtoahigher degree,resultingin
116
a decrease of the relative efficiency (4).The addition ofcombined nitrogen
to pea plants caused a lower actual nitrogenase activity coupled with a higher
relative efficiency than thatofcontrol plants without added nitrate,whereas
treatment ofnodules with benzyladenine produced opposite results (Ch.2 ) .
Depodding of field beans reduced nitrogenase activity butenhanced relative
efficiency.Application ofbenzyladenine to the lower leaves decreased relative
efficiency,but increased nitrogenase activity (van Mil,unpublished results).
From these results itmight beconcluded that a high production of hydrogen
by the nitrogenase system isa sign ofan excess ofenergy,whereas under
circumstances ofa low energy supply of the nodules,hydrogen production would
decline more sharply than total nitrogenase activity. However,thereare also
reports according towhich short-term changes in energy supply would have no
impact on relative efficiency when influencing total nitrogenase activity (1.8)
orwhere a rise innoduleactivity was associated with a rise in relative
efficiency (2,9 ) .Furthermore,experiments with isolated nitrogenase have
convincingly shown that electrons are allocated to hydrogen production rather
than tonitrogen fixation under low energy supply (21).No explanation can be
given of thewidely differing results ofthe experiments with isolated nitrogenase and those obtained in the present investigation with nodulated pea plants
growing in soils,as to the influence ofenergy supply ofthe nitrogenase system
on hydrogen production and nitrogen fixation.Thereforemore research needs to
be done toelucidate this effect.
Hydrogenase activity of R. leguminosarwn strain S310a (Hup )was notassociated with a high level of nitrogenase utilization,nor did itprevent ordelay
nodule senescence as compared with strain PRE (Hup"). As hydrogen oxidation
in plants with S310a would only provide amaximum of 0.6-4.7%ofATP supply to
nitrogenase (Ch.4) {i.e. less than 0.6% of the energy gained innetphotosynthesis),noamazing resultscan beexpected. Evans and co-workers (7)reported an energy gain by hydrogen oxidation in soybeans of 9.1% ofATP supply
tonitrogenase,which would account fora rise in dry matter production of21%
ascompared with soybeans inoculated with a Hup strain after an exponential
growth period for 25 dayswith a 9.1% enhanced relative growth rate.As ahigh
dry matter production ingeneraliscoupled with a high nodularmass (Chs 1and
5 ) , plants inoculated with Hup bacteria would releasemore Hup bacteria
upon senescence than plants inoculated with Hup"bacteria.These Hup bacteria
might also be benefitted by hydrogenase activitywhen occurring inthe soil in
a free-living state (6),which would offer them anotherconsiderable advantage
117
overHup bacteria.However,the fact that the vastmajorityofnaturally
occurring strainsisHup" (Ch.5;14)indicatesthatatleast oneofthese
statementsissubstantially incorrect.
Interactions
between C and N limitations
of growth
A 'starter dose'ofcombined nitrogenmayincrease nitrogen fixation via
increased photosynthesis (seeGeneral Introduction).InCh.6thelownitrogenfixing capacityofpea plants inoculated with R. leguminosarum strain S310a
was showntobeenhancedbythe supplyofnitrateupto800mgNper pot,
correlated withariseinleafdryweight.Incontrast,thehighnitrogenfixing capacityofpea plants inoculated with strain PREwas reducedbyadded
nitrate,theleafdry weight hardly being altered.Netphotosynthesis,as
calculated from respiration measurementsofroot systemsandofplant-growth
data (Ch. 3 ) , was increasedtoagreater extentbynitrate supplyinpeaplants
inoculated with themoderately effective strain S313 thaninplantswiththe
highly effective strain PRE.The generationofalowrateofalternative
respiratory activity(Ch.3)duetoadded nitrateeveninplants inoculated
with highly effective Rhizobium strainsmight pointtoanNlimitationof
growth. The potential nitrogen fixation,however,inpeaplantsofthe same
age, kept under identical conditions wasnot fully utilized (Ch. 2 ) , which
indicatesaC-limited growth.Toexplain thisapparent contradiction,wemight
assume thatCandNlimitations occur simultaneously innodulesandinwhole
plants. For example,thenodulesmightbeC-limited,with partofthe potential
nitrogenfixationnot utilized,which would resultinanN-limited growthof
thewhole plant.Asimilarapproach has been advocatedbyPhillipsand co-workers
(15,26).
During generative growth leguminous plantsare C-limited,ascanbeconcluded
from experimentswith C0„ enrichment.which leadstoahighernitrogen fixation
andahigheryield (8).However,also combined-nitrogen dressings duringthe
pod-filling periodmayresultinanenhanced yield.Combined-nitrogen supply
increased seed yieldofadeterminate soybean cultivar,whereas seedyieldof
an indeterminate cultivarwas not affected (11).This pointstoanimportant
influenceofthe rateofseed productiononphotosynthesis,nitrogen fixation
andyield.
Inthe present investigation seedyieldofpeaplants cv. Rondo (pod-filling
period25days)respondedtonitrateadded duringthe pod-filling period (Ch. 6),
whereas seedyieldoffield beans (pod-filling period35days)wasnotraised
(van Mil,unpublished results;17).Duetotherapid seed formationinpea,
118
nitrogen fixation declined more sharply thaninfield beans during ontogenesis
ofplants grown withoutadded nitrate (Chs1 , 4 ) .
Thus,ifthe rateofseed productionishigh,the nitrogen demandofthe
seedwill alsobehigh.Atthe same time nitrogen fixationislimitedbythe
reduced carbohydrate supply,andconsequently,nitrogenous compoundsare
mobilized from vegetative tissueand transportedtothegrowing seeds.Photosynthesisisreduced,whichinturn leadstoalower nitrogen fixation,
eventuallyresulting inthe'self-destruction'ofthe plant (19).Insucha
case,combined-nitrogen dressings during thegenerative growth phase,preferably
as foliar sprayofurea because the nitrate-absorption capacityofthe rootsis
reduced (5,23,24,27),mayprevent self-destruction bylengtheningtheperiod
of high photosynthetic activityandthusmayincreaseyield.
Iftherateofseed growth islow(asinfield bean),theself-destructive
traitsareless pronouncedandcombined nitrogen supplyatmid seasonhasno
effectonyield(17).
Prospects for agriculture and research
Inagricultural practice there seemstobenoclear-cut approachasto
increaseinyieldsofleguminous cropsbycombined-nitrogen dressings,except
with suboptimum symbiosesorwhenanextremely lowquantityofsoil nitrogen
isavailable (seeGeneral Introduction).Theyieldofpeaswitharelatively
short pod-filling period mightbeincreased bycombined-nitrogen dressings
during that period,although thereistheriskoflodging.With peas grownfor
thecanning industryyield increases,ifany,will below becausealarge
amountofnitrogenisretained intheleavesduetotheearly harvest.
The recommendationof'super-strains'with hydrogenase,does notseemto
offer unequivocally good prospects,ashydrogenase-containing strainsmay
have disadvantages (Chs 4, 5 ) .
Thus,perspectives forenhancing theyieldoflegumecropswill beona
long term,andinthefieldofresearch.Theinteractions betweenC andN
limitation need elucidation.Toremovethe'yield barrier',attention should
be focusedonincreasingthephotosynthetic capacityofthe plant,asthe
nodular tissue seemstobeformedinresponsetotheneedsoftheplant.
Another fruitful approach might involvearenewed interestinthe roleofplant
growth regulators,asthesupplyofthe noduleswith photosynthatesisof
crucial importancetonitrogen fixation.StudiesonC-and N-limited growth
in legumes with different ratesofseed production {e.g. peaandfield bean)
119
and with different nitrogen requirements for seed production (e.g. pea and
clover)would provide still another valuable extension of the investigations
reported in this thesis.
REFERENCES
1.Antoniw,L.D., and Sprent,J.I. 1978.Growth and nitrogen fixation of
Phaseolus vulgaris L. at two irradiances. IINitrogen fixation.Ann.Bot.
42: 399-410
2. Bethlenfalvay,G.J., Abu-Shakra,S.S.,and Phillips,D.A. 1978.Interdependence ofnitrogen nutrition and photosynthesis in visum sativum L. IEffect
of combined nitrogen on symbiotic nitrogen fixation and photosynthesis.
Plant Physiol.62:127-130.
3.Bethlenfalvay,G.J., and Phillips,D.A. 1977.Ontogenetic interactions betweei
photosynthesis and symbiotic nitrogen fixation in peas. Plant Physiol.60:
419-421.
4.Bethlenfalvay,G.J., and Phillips,D.A. 1977.Effect of light intensity on
efficiency of carbon dioxide and nitrogen reduction in visum sativum L.
Plant Physiol.60:868-871.
5. Eaglesham,A.J.R.,Minchin,F.R., Summerfield,R.J.,Dart,P.J., Huxley, P.A.
and Day,J.M. 1977.Nitrogen nutrition of cowpea [vigna unguioulata) .
Ill Distribution ofnitrogen within effectively nodulated plants.Exptl.
Agric. 13:369-380.
6. Evans,H.J., Emerich,D.W., Lepo,J.E.,Maier,R.J., Carter,K.R., Hanus,F.J.
and Russell,S.A. 1980.The role of hydrogenase in nodule bacteroids and
free-living rhizobia,pp.55-81 in Nitrogen fixation (eds.W.D.P.Stewart
and J.R. Gallon),Acad.Press,London
7. Evans,H.J., Purohit,K.,Cantrell,M.A.,Eisbrenner,G., Russell, S.A.,
Hanus,F.J., and Lepo,J.E. 1980.Hydrogen losses and hydrogenases in
nitrogen-fixing systems,pp.84-96 in Current perspectives in nitrogen
fixation (eds.A.H.Gibson and W.E.Newton) Elsevier/North-Holland,Amsterdam
8.Hardy,R.W.F.,and Havelka,U.D. 1976.Photosynthate asamajor factor
limiting nitrogen fixation by field-grown legumeswith emphasis on soybeans,
pp. 421-439 in Symbiotic nitrogen fixation in plants (ed. P.S.Nutman)
IBP 7,Cambridge Un. Press,Cambridge.
9. Lambers,H.,Layzell,D.B.,and Pate,J.S. 1980.Efficiency and regulation
of root respiration in a legume:effects of the N source. Phys. Plant.50:
319-325,
10. Latimore,M.,Giddens,J., and Ashley,D.A. 1977.Effect of ammonium and
nitrate nitrogen upon photosynthate supply and nitrogen fixation by soybeans.
Crop Sei. 17:399-404.
11. Lawn,R.J.,and Brun,W.A. 1974.Symbiotic nitrogen fixation insoybeans.
Ill Effect of supplemental nitrogen and intervarietal grafting. Crop Sei.14:
22-25.
12. Lawrie,A.C.,and Wheeler,C.T. 1973.The supply of photosynthetic assimilate
to nodules of visum sativum L. in relation to the fixation of nitrogen.
New Phytol. 72:1341-1348.
13.Lawrie,A.C.,and Wheeler,C.T. 1974.The effects of flowering and fruit
formation on the supply of photosynthetic assimilates to the nodules of
Visum sativum L. in relation to the fixation of nitrogen.New Phytol.73:
1119-1127.
120
14. Lim,S.T.,Andersen,K.,Tait,R.,andValentine,R.C. 1980.Genetic
engineering inagriculture: hydrogen uptake (hup)genes.TIBS,June,167-170.
15. Phillips,D.A., DeJong,T.M.,andWilliams,L.E. 1981.Carbonandnitrogen
limitationsonsymbiotically-grown soybean seedlings,pp. 117-120 in Current
perspectives innitrogen fixation (eds.A.H.GibsonandW.E.Newton)
Elsevier/North-Hol1and,Amsterdam.
16. Phillips,D.A., Newell,K.D.,Hassell,S.A.,andFelling,C E . 1976. The
effectofCCL enrichmentonroot nodule developmentandsymbiotic N~
reduction inVisum sativum L.Am.J.Bot.63:356-362.
17. Richards,J.E.,andSoper,R.J. 1979. EffectofNfertilizeron yield, protein
content,andsymbioticNfixation infababeans.Agron.J.71:807-811.
18. Sheikholeslam,S.N., Fishbeck,K.A.,andPhillips,D.A. 1980.Effectof
irradianceonpartitioning ofphotosynthatetopearootnodules.Bot.Gaz.
141: 48-52.
19. Sinclair,T.R.,andWit,C T . de. 1976.Analysisofthecarbonandnitrogen
limitationstosoybean yield.Agron.J.68:319-324.
..
20. Small,J.G.C.,andLeonard,O.A. 1969.TranslocationofC -labeled photosynthateinnodulated legumesasinfluenced bynitrate nitrogen.Am.J. Bot.
56: 187-194.
21. Stiefel,E.J.,Burgess,B.K.,Wherland,D.,Newton,W.E.,Corbon,J.L.,and
Watt,G.D. 1980. Azotobaatev vinelandii
biochemistry: ^(DgîNo relationships
of nitrogenaseandsome aspectsofiron metabolism,pp.21Ï-222 in Nitrogen
fixation,vol.1(eds.W.E.NewtonandW.H.Orme-Johnson),Un.ParkPress
Baltimore.
22. Streeter,J.G. 1974.Growthoftwosoybean shootsonasingle root.J. Exp.
Bot. 25:189-198.
23. Thibodeau,P.S.,andJaworski,E.G. 1975.Patternsofnitrogen utilization
inthesoybean. Planta 127:133-145.
24. Vasilas,B.L.,Legg,J.O.,andWolf,D.C. 1980.Foliarfertilizationof soybeans: absorptionandtranslocationof15|\|_]abe-|ec)u r e a . Agron.J. 72:
271-275.
25. Veeger,C ,Laane,C ,Scherings,G., Matz,L.,Haaker,H.,andZeelandWolbers,L.van. 1980.Membrane energizationandnitrogen fixationin
Azotobaoter
vinelandii
and Rhizobium
leguminosarum,
pp. 111-137 in Nitrogen
fixation,vol.1(eds.W.E.NewtonandW.H. Orme-Johnson),Un. Park Press
Baltimore.
26. Williams,L.E.,andPhillips,D.A. 1980.Effectofirradianceondevelopment
ofapparent nitrogen fixationandphotosynthesisinsoybean. Plant Physiol.
66:968-972.
27. Wych,R.D.,andRains,D.W. 1979.Nitrate absorptionandacetylene reduction
by soybeans during reproductive development. Physiol. Plant.47:200-204.
28. Yoshida,S.,andYatazawa,M.1977.Direct evidenceofsucrose effecton
nodule formationandsymbiotic nitrogen fixation inleguminous plantsas
revealedbythe useofexcised root culture methodandacetylene reduction
method.J.Japan Grass!. Sei. 23: 14-17.
121
SUMMARY
Actual nitrogen fixation of pea and field-bean plants,grown in soil in the
open air,was determined as the acetylene reduction ofnodulated roots.During the
major part of the vegetative growth of these plants,actual nitrogen fixation was
equal tothe potential maximum nitrogenase activity of the bacteroids present in
the nodules.This means that increase of the actual nitrogen fixation could be
achieved only ifthe potential nitrogenase activity of the bacteroids would be
enhanced or ifmore noduleswould be present.During thegenerative growth phase,
the potential nitrogenase activity of the bacteroids was notentirely utilized
irrespective ofthe shootmass of the host plant orthe nitrogen-fixing capacity
ofthe Rhizobium microsymbiont.
The addition of nitrate tonodulated plants sharply reduced actual nitrogen
fixation but did not affect potential nitrogen fixation within 10days.The
nitrate effectwas temporarily eliminated by treatment of the root noduleswith
benzyladenine,a synthetic plant-growth regulatorwith a photosynthate-attracting
action. It isconcluded that incomplete utilization of thenitrogenase present in
the bacteroids iscaused byan inadequatecarbohydrate supply of the nodules upon
supply of the leguminous plantwith nitrate.
In root systems of pea plantsgrowing in symbiosis with R. legvminosarum
strains of poor nitrogen-fixing capacity,theN-limited growth led toanaccumulation of carbohydrates.Upon the supply of such plantswith nitrate,the carbohydrate level was decreased concomitantly toan increase inalternative {i.e.
cyanide-resistant) respiration.Low rates of alternative respiration were found
when nitratewas added to pea plants inoculated with highly effectivestrains,
as theN-limitation of the plantswas less severe.The carbohydrates respired
in the nodules amounted to 6.3 mg C/mg N fixed with amoderately effective strain
but only 3.1 mg C/mg N fixed with ahighly effective strain.
Some rhizobial strains possess a hydrogenase that iscapable of recirculating
part of the hydrogen evolved inair by nitrogenase simultaneously with nitrogen
fixation. However,the energy gain by hydrogen oxidation was very low,
viz.
123
0.6-4.3%of thecosts ofnitrogen fixation.As hydrogen oxidation did not
delay nodule senescence or increase nitrogenase utilization,the presence of
hydrogenase in rhizobial strains seems to bean unimportant factor indetermining the nitrogen-fixing capacity ofthesymbiosis.
Strains of R. legvminosawm with a low nitrogen-fixing capacity showeda
distinctly different nodule formation pattern on primary and lateral roots of
pea plants ascompared with highly effective strains.Theestimates of theyieldo1
fixed nitrogen,derived from the acetylene-reduction method,of plants inoculated
with rhizobial strains of different nitrogen-fixing capacity,equally deteriorated during thegrowth period. When rates of hydrogen production inairwere
subtracted from the acetylene reduction rates,the estimates ofnitrogen fixation
were severely biased infavour ofahydrogenase-containing strain.
Nitrogen fixation of pea plants infected with ahighly effective strain of
R. leguminosarum was decreased by the supply ofcombined nitrogen toagreater
extent than that of plantswith amoderately effective strain. In plants inoculated with a strain of poornitrogen-fixing capacity,nitrogen fixation per plant
was even stimulated bya low dose ofcombined nitrogen.This increase wasprobably due toan enhanced photosynthetic capacity which counteracted the adverse
effect ofcombined nitrogen. Seedyields were increased bynitrate dressings,
regardless of the rhizobial strain. Seed yields of plants inoculated withmoderately effective strains were slightly higher than those ofhighly effective
strains at high levels ofcombined nitrogen,owing to higher nitrate uptake,
higher nitrogen fixation and increased photosynthesis.
124
SAMENVATTING
Vlinderbloemige gewassen zoals erwtentuinboon (veldboon)zijninstaatom
stikstof uitdeluchttebinden dooreen samenwerking (symbiose)aantegaan
met bacteriënvanhet geslacht Bh-Lzobium. Deze bacteriën dringendewortel
binnenviadewortelharenenveroorzaken daardevorming vanwortelknollen.De
stikstofbindende bacteriënindeze knollen hebbeneengedaanteverandering
ondergaanvanstaafvorm naar vertakteofknotsvorm (bacteroïden).
Tijdensdesymbiose voorzietdewaardplantdebacteroïdenvansuikers,
verkregen doordefotosynthese.Bijerwtentuinboondieinpotten met grond
indeopen luchtwerden geteeld wastotdebloeidemaximale stikstofbinding
vandebacteroïden (de potentiële stikstofbinding)debeperkende factorvande
stikstofbindingvandeplanten (actuele stikstofbinding).Indeze periodekan
alleen door betere knollenofdoormeer knollendestikstofbindingvande
planten worden verhoogd.Ditisintegenstelling totdeperiodevanbloeien
peulvorming waarinhetstikstofbindend vermogenvandebacteroïden meestalaanzienlijk hogerisdandewerkelijke stikstofbinding(potentiële groter danactuele
stikstofbinding).Indeze periode krijgendeknollenteweinig suikersvande
plantomoptimaal tefunctioneren omdat een aanzienlijk deelvandeprodukten
vandefotosynthese voorzaadvorming wordtgebruikt.
De toevoegingvannitraat aan planten metwortelknollen leidt toteendaling
vandestikstofbinding doordatdetoevoervan suikers naardewortelknollen
vermindert. Diteffectkantijdelijk worden tegengegaan wanneerdewortelknollen
tegelijk metdenitraatgift behandeld wordenmetbenzyladenine,eensynthetisch
plantehormoondat suikerskan aantrekken.
Tuinbonen ontwikkelen zich veel forserdan erwtenenbindenook veel meer
stikstof.Destikstofbindingvanerwtentuinboon bereikt haarmaximale hoogte
aan hetbeginvandepeulvullingsfase.Ditkomt doordat steeds nieuwe actieve
wortelknollen worden gevormd.Hetisechter vreemddatindesymbiose van plant
enbacteriedestikstofbinding eerderwordt verhoogd doormeer knolweefselte
125
maken dan doorhet stikstofbindend vermogen van debactero'fden volledig te
benutten.
Tijdens de peulvullingsfase daalt de stikstofbinding.Het sterkst gebeurt
dit bij deerwten dieeen korte peulvullingsduur hebben,hetminst bij tuinbonen
enerwten meteen lange peulvullingsduur.
Nietalle bacteriestammen hebben een evengroot vermogen om stikstof tebinden
in symbiosemet een bepaaldewaardplant.Erwteplanten diemet eenmatig of
slecht stikstofbindende bacteriestam worden geënt groeien bij stikstofgebrek.
Er isdan teweinig stikstof in deplant aanwezig waardoor dedoor defotosynthese gevormde suikers niet volledig worden benut voor degroei,maarworden
opgehoopt indeerwteplanten.Wanneer deze planten dan van nitraatworden voorzien,worden de suikers zodanig verademd datweinig energiewordt verkregen
(verspillende ademhaling). Inplanten diemeteen goed stikstofbindende
R. leguminosarum stam zijn geënt worden minder suikers opgehoopt. Daardoor
treedtminder verspillende ademhaling opals nitraatwordt toegevoegd.
Tijdens de stikstofbinding vormen de bacteroïden naastammoniak ook waterstof
die in de lucht verdwijnt,waardoor energie verloren gaat.Sommige Rhizdbivmstammen vormen nietalleen waterstof,maar kunnen deze verbinding zelf weer
opnemen.Op dezemanier zou een deel van deverloren gegane energie weer kunnen
worden teruggewonnen. Inons onderzoek bleek echter,dat dit waterstofopnemend
vermogen van weinig belang isom de stikstofbinding en de groei van de planten
tebevorderen.
Erwteplanten die insymbiose meteen goede R. leguminosarum stamgroeien,
binden meer stikstof en produceren meer bladmateriaal en zaad dan planten die
met een slechte ofmatige stam knollen vormen.Wanneer nitraatwordt gegeven
aan planten geëntmet een goede stam,wordt de stikstofbinding verlaagd maar de
zaadopbrengst vaak iets verhoogd doorde ruime stikstofvoorziening alsgevolg
van de nitraatbemesting.Bij planten diemet een matige stam zijn geënt daalt
de stikstofbinding minder dan het geval was bij een goede stammaar de reactie
op toegediende kunstmeststikstof isgroter.Er kan danmeer bladworden gevormd
waardoor de fotosynthesewordt verhoogd en hetnadelig effect van nitraat op de
stikstofbinding kanworden tegengegaan. Bij planten die dooreen slechte
Rhizobium-stam zijn geïnfecteerd kaneen lage nitraatgift op dezewijze zelfs
de stikstofbinding verhogen.Bij een hogenitraatgift kan de zaadproductie
van dergelijke planten iets hoger zijn dan die van plantenmet een goede stam
omdat de laatste in dat geval minder nitraat opnemen,minder stikstof binden
126
en daardoor een lagere fotosynthese hebben.
Voorde 1andbouwpraktijk kunnen de in ditonderzoek verkregen resultaten niet rechtstreeks gebruiktworden.Bijerwten zal ineen beperktaantal
gevallen een kleine stikstofbemesting bijhetzaaien een gunstige uitwerking
kunnen hebben opde bladvorming en uiteindelijk ook opdezaadopbrengst.In
principe zou een bemesting tijdens de peulvullingsfase,bijvoorbeeld dooreen
bespuiting met ureum,een opbrengstverhoging kunnen geven,maar vanuit de
praktijk kunnen bezwaren ingebrachtworden tegen een bemesting van een te
velde staand gewas.
Hetvoornaamste resultaat van dit onderzoek iswel dat de opbrengst van
vlinderbloemigegewassen verhoogd kanworden als de fotosynthese verhoogdwordt.
Erworden dan extra wortelknol!en gevormd waardoor indirect een hogere stikstofbinding kan worden verkregen. Er isop ditmoment teweinig bekend omtrent het
verband tussen fotosynthese en stikstofbinding tijdens de groeicyclus van de
peulvruchten.Naderonderzoek hierover isdan ook noodzakelijk om de opbrengsten
vandezegewassen te kunnen verhogen.VoorNederland kan verwachtworden dat
debelangstellingvoor peulvruchten zal toenemen als deenergieprijzen blijven
stijgen. De prijs van stikstofmeststoffen zal er dan toe leiden dat devlinderbloemige gewassen,dieweinig of geen stikstofmeststof nodig hebben,zich uitbreiden ten koste van gewassen dieeen hoge stikstofgift nodig hebben,zekerals
de opbrengsten verhoogd kunnen worden.Tedenken valt bijvoorbeeld aan het
vervangen van gras als eiwitrijk ruwvoeder door veldbonen ofaan het vervangen
van vlees voormenselijke consumptie door peulvruchten.
Voorontwikkelingslanden zijn hogere opbrengsten van peulvruchten,dieals
voedselgewas verbouwd worden,noodzakelijk,maar niet toereikend,om hetbestaande eiwittekort in de voeding op teheffen.
127
CURRICULUM VITAE
5 september 1951 geborenteZwolle
1963-1969Gymnasium $teGroningen
1969-1977Landbouwhogeschool teWageningen
Studierichting Milieuhygiëne
Doctoraalvakken Waterzuivering, Microbiologie,
KolloïdchemieenInternationale betrekkingen
en ontwikkelingen
1977-1980Wetenschappelijk assistent Laboratorium voor Microbiologie
vandeLandbouwhogeschool teWageningen
1981 Docent MilieukundeenChemie aande
Rijks Hogere Landbouwschool teGroningen
128

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