Plant Plhvsiol. ( 1966) 41, 760-76/3)
Effects of Calcium Deficiency
on
Symbiotic Nitrogen Fixation'
C. L. Banath2, E. A. N. Greenwood3, and J. F. Loneragan
Department of Soil Science and Plant Nutrition, Institute of Agriculture,
The University of Western Australia, Nedlands, Western Australia
Received November 3, 1965.
S'umuiarv . Thle mechanismi of the effect of mlild calciumii (leficiencv oni nlitrogell
metabolism of the symbiotic planttwas sttu(lied fromll the (listrib)ution of calciuimil alind of
nitrogeni anid carbohy-drate fractions in planlt organls.
.Nitrogenl conicelitrationis of all plant organis (lecreased w-ith calciumii deficiencv.
Adnitrogell or a calciumli salt inicreased niitrogeni concentrations. For
roots as well as whoc l)lants the effects of olne salt were largely rel)laced 1b the other.
'I'liese effects establish that calcitiuim deficiency (lecrease(I the stipply of fixe(d niitrogen
from
other organs.
nloduiles
weight of nodules wxas inldel)end(enlt of calcitimil it
follows that lno(diular efficiency was imnpaired.
Sinice niitrogeni concentrations of nlo(lules (lecreased ratiler thalln increased with calcium
deficienicv it is su--ested that calcium deficiency initerferes with the rate of redcItion
of nlitrogenl in the nlo(ltile rather thanl with the exl)ort of redtuce(d niitrogeni. Distribtition
of a-amilio, ali(le, and ammoinoiiilim niitrogenl are consistenit with thlis stiggestion.
Nitrogenl fixatiol \was nlo limiiite(d
tranlslocationi of carbollydrates to nou(iles as
calciul (leficielncv ha(l little effect oni the coincenitratioll of soltiule carbohy drates and
actually inlcreased1 the concentration of starch in niodules.
Calciulim (leficiency del)ressed the calciumii contenit of n1odules so that nitrogen fixation
niay have been inil)aire(l by iiia(le(Itiate calciumil for no(lular striutiure or metabolism.
(litionl of cither
a
to
Severe calciumi leficielicv restricts the amiioulit
of niitrogeni fixed 1y legumes throuighi restrictioni of
host pllant growth ni; itrogeni conlcenitrationls inicrease
an(l the planits do niot responl to fer.ilizer nlitrogen
Methods
Subterrtanean clover ( Trifoliizi sibt1erraulc11i L.
Alount
seeds
germiinated in aci(l
v-ar.
Barker)
ashied sai(l.
(1,7).
were
T'hey
watered
with the compl)ete
basal niutrienit described below pluls 2ni:r calcitlill
chloride alid 3 mtI amimioiumii niitrate, and(I N-ere in-
Oni the other hanld, whieni calciiium (leficicilxl is less
severe it miay have a specific effect oll the smilbliotic
sy-stenil] nitrogen concentrations (lecrease aiid( the
Plants resl)ond to iniorganiic niitrogen (5. 7).
Sinice calciumii deficiencv may interfere with the
tranisport of carbohydrates from shoot to root (6). it
has beeni suggested that mild calcium deficienlcy interferes with niitrogeni fixationi by decreasinig the rate
of tranisport of essenitial metabolites fromii shoot to
ere
with RhLobiL,o n trifolii strain TAI.
After 12 days, 6 plants wsere transferred to eacih
of 48 freely (Irained pots conltaininig 10 kg of aci(d
ocuilate(
washed
sand.
B3asal
anid treatmiielit
niltrielits
were
al)l)lied in solutions at the rate of 1 liter per pot per
app)lication. Applications wx-ere imiadle initially once
eachi xveek, then twice each week.
The comilpositionl of the basal ntitrienlt solutioni
(ppH 6.0) wvas as follows: 2 i1 m potassiuml chloride.
0.32 mm )otassiunm phosphate. 1 mim milaginesiuim sulfate, 25 Am boric acid, 15 1%m ferric citrate, 10
manganese sulfate. 1 ,u%m copper sulfate, 1 pcm zinlc
sulfate, 0.1 ,am cobalt chloride, 0.02 xm ammoniuimi
molvbdate. In addition, 0.5 im ammoniumil nitrate
was applied to all pots for /76 days after tranisplanitii1g.
A 2 X 6 factorial conibination of 2 treatmienits
Nwas arraniged in 4 ranidomlized blocks as followvs:
Calciunm chloride, 0, 0.3, 0.6. 0.9. 1.2. or 1.5 ilm fromii
transplanting to (laxv 63 xwhen all levels were halved.
A11inmonl1tinm niltrate, zero or ; 1inim applied fromii 7/6
days after transplantinig.
nlodule (7).
The experimiielit reporte(l in this paper was unidertakeni to examinie the effects of calcitiuim deficiencv
oii carbohydrate anicl nitrogeni fractions of (liffereint
parts of the symibiotic plant to clarifv the nature of
the effect of calcium deficiency oil symbiotic nitrogen
fixationi.
W
\\ork
sul)p)ortedl by Whlieat Researchl Commlittee of
\V'estern
Australia.
2 Present address:
tonl,2 Tasmania.
Departmenit of Agriculture, ILauncesDiv-isioni of Planit IndustrV, Westerni
C.S.I.R.O.,
Australiacn Regional Laboratory, Nedlands, WVestern Aus-
tralia.
760
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761
calcium chloride alleviated these nitrogen-deficiency
BANATII ET AL-,.CALCIlUMt AND NITROGEN FIXATION7
All plailts xver e harvested 95 days after transplantiing when nlitro geen deficiency symiiptoms appeared
in the appropriate treatmenits. Plants treated with
0.3 anid 1.5 mim cal cium chloride both in the presence
and absence of am moniumii niitrate were divided into
leaf, petiole, stem. root, and niodules. Small subsamples were take ni for determiniation of moisture
conitent. About 1C g of each of the organis except
nodules were extratcte(l with 75 mil of boiling 80 %
(v/v) ethaniol in a blenidor. About 200 mg of
niodule tissue was gground with sand in a mortar and
pestle l)rior to extiraction xvith 80 % ethanol. Alcoholic extracts alnd fresh material were stored at -300.
Calciumii was de termined in acid digests by atomic
albsorption spectrolplhotoni)etry (3).
Total nlitrog,enl was determined 1b iimicro-Kjeldahl
digestioni anid (listiillationi in a Parinas-\Vagnier still
(9) .
Fr-ee ninhvd(rin nitrogell xas measuire(l onl the
alcolholic extracts by the miietlho(d of Yemmii anid
Cocking (13). Alnmliomumtim anid amiiide niitrogell were
vacuum (listille(l 11) and (leteriflinled b1 nlesslerizatiOll.
Total soluble c arbohvdrates xvere (letermiile(l onl
the alcolholic extralCtS tisilig the alnthronie imietlhod of
Murphy (10) wit h the imiodificationls that samples
xvere layered abov{e the anitlhronie reagenlt prior to
Imlixilng an1(l01)xD , as rea(l at 620 niu.
Starch was de ternliie(l b1y perchloric acid hb(lrolvsis of the resii(lec frolmi alcoholic extractioni. folloxed b)v the ailtlhi ronie mietho(d (8).
Resu
D)
I
1'eiglhts.
its and Discussion
Calciuml chlloridle
inlcreasedl the dry
weight of roots aii dI tops; amlmonliumll nitrate appl)liedl
had
effect (table aI
late in the exl)eril
Planits groxvi c nllcalciumlll chloride conlcenltrationls
of 0.9 m.xm or iisorce in the nutitrienit miieditumi produce(d
-\
maximumiii dry xx-eilght anld shlowed nlO symlptomlls.
decreased
conicenitrationi of
Id xveit
about 20 % andI gnave synmltoms of miild
calcium deficienicy (reddenii ig of okler leaves). WShen llO
aniimoilitumii niitrate was added, the leaves of these
)lants xxere vellox ish-greenl, indicative of nitrogenl
deficiency. Additi iOnl of amllmoniumll nlitrate or of
dlenpt nmo
mav
be
Table I. Effects of Calciuml Chloride anid q,ninionion,
Nitraic (N) on the Yield of Subterranean Clover
Yield is calculateAd at dry weight in g per 100 plants.
- N
+ N
P 0.05
N
+ N
P 0.05
0.3
0.6
0.9
1.2
1.5
Tops
352
442
481
476
456
12
360
474
477
435
21
465
= 32 excludhiuig values for zero calciunm chlor.de
Roots and nodules
191
214
4
201
211
223
194
214
214
20)1
241
6
= 23 exclucalinlg values for zero calciumchloride
mtm CaC12
0
symptoms.
Planits given 0.3 nm\t calcium chloride are referred
to as mildly calcium deficient. Planits giveni 1.5 mli
calciulmi chloride were taken as conitrols. These 2
calcium treatmlent"s both with and without ammonium
nitrate were choseni for more detailed study.
T'he effects of anmnonium nitrate treatnmelnt and
calciumil deficiency onl the dry weights of leaves,
petioles, stems, anid roots were similar to their effects
on the whole pllalt (table II). Their effects oln
nodule dry weight were in marked colntrast alnd are
discussed separately.
Total NitrogeN. Mild calciumii deficienlcy depresse(d niitrogeni colncentrations in all plant organiis
x-heni ammiloniiumii iiitrate was not applied (table II).
Additioni of ammiioniium niitrate or of calciumll chloride
increase(d the niitrogeni concentrationis. For the wlvole
l)lalnt the effect of the additionl of ammiiiioniitumii nlitrate
was miuiichl greater in the calciumii deficienlt )lants than
in the conitrols so that each salt largely replaced the
other in its effect oni niitrogeni concentrations (interactioni niegative and signiificant at P < 0.01). Calciumiii allnd niitrogenl treatmiients lproducecl siimilar effects
(table II) anid inlteractioni (P < 0.01() oni niitrogeni
conicenitrationi of roots. These results clearly establish that the nlitrogeni deficiency iniduced 1by calcituilm
leficieiicv in the tops of subterraneani clover plants
in this anld other (5, 7) studies arose from the failure
of the niodtules to suplylv adequate niitrogeln to other
m.lild
organis.
Tlhe effect of calcium (leficiencv il (lepressili,g
the sul)l)l of fixe(d niitrogeni fromii niodules cotldl arise
fromi a decrease in the overall rate of reductioni of
nitrogeni in the nodlule, from failture to metabolize
the redlucedl nitrogen, or from inhibitioni of tranAslo-
cationl of the reduced nitrogen from the nodtule. The
first alterniative should result in depletioni of nitrogen
in the nio(lule; the last 2 in accumulationi. As mild
calciumii deficiencyv did nlot increase the concenitration
of nitrogen in the nodule but may eveni have de-
(table
creased it
II), it is suggested that the (leficiencv in somiie wax limits the rate of nitrogen reduction
ilnthe nodule. Unfortuniately. although supplemen-
tary data are consistenit with this hypothesis, thex
do not establish it.
The effect of mild calcium deficienicy in decreasing the nitrogein concentrationi of planit organs wvas
least in the nodules and increased progressively xvith
increasing distance from the niodules. This phenomenon is consistent with the l)roposal that the deficiency decreases nitrogen redulctioni in the nodule
if it is accepted that the organ wvhich is closest to
the source of a limited nitrogeln supp)ly has a comilpetitive advanitage over more distal organs for its
i
needs. The same distribution pattern would
nitrogeni
not be expected to hold for readily translocatable
formiis of niitrogen such as the amino acidIs.
Free Ninhvdri,, Nitrogen Concentrations. For
the xv-hole l)lan;t the main effects and interactionls of
calcium chloride and anionium nitrate on the con-
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762
PLANST PHY\SIOLOGY
Table 11. Effcts of Calcium Chliridc antd .-,nimoniuni Aitratc (A) on
of Snbtcrrancan C'lovcr
CaCl.,
nim
Whole plant
0.3
1.5
Leaves
0.3
1.5
552
554
149
160
Petioles
0.3
1.5
Dry
N
+ N
P = 0.05
2.98
3.56
N
3.28
3.56
0.11
P
0.05
=
wt in
188
0.133
0.151
0.185
0.202
0.013
g
77
81
182
16
Nitrogeni
65
- N
1
679
677
D)ryIMat.ter Yield (and Com position
Stems
0.3
1.
per 100
101
R(x)ts
0.3
1.5
Nodules
0.3
1 .5
191
188
9.7
5.8
plaiits
125
97
167
156
120
213
233
19
9
9.5
7.3
27
1.3
percelntage of dry wt
as
3.55
4.01
1.61
1.84
3.94
4.05
1.91
1.97
2.69
3.03
3.12
3.32
3.33
3.72
3.66
4.()(
0.11
0.17
0.30
0.18
Free ninhyd(rini niitrogeni as percentage dry wt
0.089
0.097
0.049 0.!059
0.181
0.189
0.163
0.209
0.113
0.120
0.070 0.079
0.226
0.263
0.267
0.267
0.013
0.044
0.011
0.015
A\mmonium + ami(le niitrogeni
percentage dlry wt
6.77
6.81
7.06
6.78
0.32
0.239
0.236
0.270
0.247
0.027
as
N
-
...
...
+ N
P = 0.05
0.046
0.060
0.217
.0.069
0.078
0.208
0.016
...
...
...
niitrogeni
...
... ...
...
as
p)ercentage
dry
wt
...
...
...
...
...
* -*
...
...
...
...
...
0.0622
0.0641
0.0518
0.0472
P = 0.05
0.0218
Calciumii
N
+ N'
P
-
0.257
.086
AmllillOllium
N
0.255
0.1I5
0.37
0.15
0.39
0.05
N
+ N
P = 0.05
12.0
12.3
N
0
+
I' = 0.()5
...
13.2
13.6
...
...
0.17
0.17
0.68
0.76
as
percentage
0.19
0.21
(Iry wt
0.45
0.46
0.13
0.13
0.28
0.11
(.1(
(1.29
0.05
0.04
0.02
Soluble carbohydrates as lpercentage dry wt
10.7
9.9
19.7
17.2
18.2
16.6
11.0
10.7
18.4
20.7
17.1
20.2
0.6
1.2
3.0
Starch as p)ercentage dIry xvt
...
...
...
*-...
...
...
...
*--.*--.--.
0.13
0.2(0
0.22
0.12
0.02
0.24
0.05
7.4
10.9
8.0
9.0
4.2
4.1
0.7
...
0.25
4.()
4.7
1.4
2.16
2.24
2.05
2.23
3.53
2.66
0.32
2.97
2.09
0.44
cenitrationi of free ninlhvdrini nitrogeni wvere simiiilar
In fact addition of aImoniliumii nitrate wN-ithotit calciuIlm
to their effects on total niitrogen (table II). But
by conltrast to its effect on total nitrogen concenltrationi, mild calcitulli deficielncy lowered the free ninhvdrin iitrogeni concenitrationi of the roots much
n:ore miiarkedly than that of other plant organs.
clhloride inicreasedl the free ninihvdlriin nitrogeni to
high levels. Additioni of calciumiii chloride with amii-
However,
uinilike
total
nitrogen
which
is
mainly
structural i l)rotein. free ninhvdrinl nitrogeln is eCs< entiallv labile in metabolic 1)ools and in transit.
Nitrogeln in traansit can be expected to b)e very reslponsive to the rate of nitrogen sutpply from the
niodulle. Therefore an+ (lecrease in the rate of re(luctioin of niitrogeln in the nodules should produce
marked (lecreases in the conicenitrations of free ninhydrin 1iitrogeni in the roots and stems. The large
effect of mild calcium deficiency on free ninhydrin
nitrogeni of the plant roots is thus consistent with
ani effect of this treatmiient in decreasinig the rate
of reduictioln of niitrogeni in the nodules.
Support for this hvpothesis is given bv the changes
plro(luced in free ninihvdrin nitrogen by additioln of
coml)illed nitrogen. If mild calcium deficiency interfered wvith the transfer of reduced nitrogeni to
carbohydrate acceptors, addition of ammllonium nitrate
to (leficienit planits -ould not be expected to inicrease
the free ninhvdrin niitrogeni of the roots anid inodlules.
monitimii nitrate
hadl
signlificant at P <
nio
a(lditionial effect (initeraction
0.01).
Anmnionizunt anid Aninde Aitrogen. MAild calcium
dleficiencV deplressed aimimoiniumil anl(l amide conicenltrationis of the no(dtiles as would he expected if it influelnces symiibiotic nitrogen fixation by decreasinig the
rate of niitrogeni reductioln. 'I'he (lecrease was sturprisinglv smiiall anid miay exveni have ariseni bv clhanice. Such
a small effect is onlyN conisistenit with anl effect of calciumil deficieIncy' On1 nlitrogeni reductionl if. as has
been postulated (2). the pool of ammliioniumii in the
niodule in rapid equilibrium witlh molecular niitrogen
is onlv a smiiall proportion of the total niodule ammoiniumii.
\Todiles.
Any effect of mild calciumii deficienicv
in limitinig nitrogen reductioln
caniniot
be ascrilsed to
less total inodular tissue since this
independent
of calciumni treatmlienit whenl no amimoliumiiii nlitrate wvas
applied (table II).
Calciut Z1 Conccntrations. 'The ioodules of miiildlyx
calciumil deficienit plants. hladl a lowx er colncenitration
of calciumii thani those of conitrol plants (table 11)
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was
BANATH ET AL.-CALCIUNM
so that calciumii miay have been iniadequate for lnodular structure or metabolism.
On the other hand calcium deficiency depressed
the calcium concentrations of aerial organs to an
even greater extent (roots 86 %, nodules 80 %, stems
46 %, petioles 42 %, leaves 25 % of control plants)
so that it may have seriously interfered wvith the
supply of metabolites from plant tops to nodules
as has been previously suggested (7). Alternatively,
the large effects of calcium treatments on the calciumi1 concentrations of the aerial parts of the plant
may reflect the inability of excess calcium to re-cycle
in the plant.
Carbohydrates. Carbohydrates appear to be the
major miietabolite tranislocated to the nodule where
they are probably the ultimate source of carbon skeletonls in ammonia metabolism and of reductant in nitrogen reduction. Certainly, wheni sucrose is fed to
excised roots carryinig niodules, 15N., is fixed (12).
Translocation of carbohydrates to nodules of
calcium deficient plants was not limiting for nodule
mletabolism in the present experiment since the nodules
lhad conlcentrations of soluble carbohydrates almnost
as high as the controls and they had appreciably
hiigher concentrations of starch (table II).
The data therefore indicate that, if calcium deficiency interferes with nitrogen reduction by effects
on carbohydrate metabolism, it does so within the
niodule itself rather than through tranisport of carbohydrate to the nodule. However, supply of other
metabolites may have been affected.
The accumulation of starch miay reflect inability
of the mildly calciumii deficient lnodule to mobilize it
for niitrogen reductioni. In fact, miianyv plant and
bacterial a-amylases are activated by calciumii (4).
But detail of the metabolism of starch inl nodules is
nlot known and its role in nitrogen reduction may not
be important. If this is so, calcium must function
in some other phase of the symbiosis.
AND NITROGEN FIXATION7
763
Literature Cited
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bacteria in relation to fertility levels of the soil.
Proc. Soil Sci. Soc. Am. 2: 315-27.
2. APRISON, M. H., W. E. MAGEE AND R. H. BUIERIS.
1954. Nitrogen fixation by excised soy bean root
nodules. J. Biol. Chem. 208: 29-39.
3. DAVID, D. J. 1959. Determination of calcium. in
4.
D.
6.
7.
8.
9.
10.
1 1.
Acknowledgments
12.
The authlors thia.nk Dr. D. W. Goodall for advice onl
statistical methods, Mrs. C. L. Banath for help with
statistical calculations, and Mr. Z. V. Titmanis for
ammonia, amide, and starch analyses.
13.
plant material by atomic absorption spectrophotometry. Analyst 84: 536-45.
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GREENWOOD, E. A. N. AND E. G. HALLSWORTH.
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