N otizen
912
Calcium, Iron and Cobalt A ccum ulation in R oot
Hairs o f Soybean (Glycine max)
D ietrich W ern er, K lau s-P eter K uhlm ann
Botanisches Institut, Fachbereich Biologie der PhilippsUniversität Marburg, Lahnberge, D-3550 Marburg/L.
F rank G loystein, F riedrich-W . R ich ter
Fachbereich Physik der Philipps-Universität,
D-3550 Marburg/L.
Z. Naturforsch. 40c, 912—913 (1985);
received May 30/July 31, 1985
Calcium, Cobalt, Glycine max, Iron, Root-Hair
Root hairs of soybeans (Glycine max), target cells for
infection by Rhizobium japonicum accumulate iron more
than 10-fold, cobalt more than 8-fold and calcium more
than 7-fold compared to the other parts of the root system.
In root hairs of wheat (Triticum aestivum) a much smaller
accumulation of these elements was found. The symbiont
of Glycine max, Rhizobium japonicum, is known to have a
high requirement for iron, cobalt and calcium.
tion w hich is how ever not un d isp u ted [5, 6], M o re
th an 95% of the prim ary infections in the ro o t h air
also in effective host-sym biont com binations are
abortive. T he ro o t hair cells ap p aren tly recognize th e
R hizobium cells m ore as a p arasite than a sym biont.
T he sam e situation can be found in later stages of
nodule d ev elo p m en t, w here lysis of b acteroids in th e
vicinity of th e host cell nucleus was obvious w ith a
R hizobium japonicum m u tan t, ap p aren tly lacking a
defense m echanism against this attack [7], F rom
these observations it is assum ed th at th e invading
R hizobium can find in the ro o t hair a n u trie n t supply
which allows such a fast grow th of the b ac te ria , th a t
at least a sufficient n u m b er of infecting rh izo b ia su r­
vive th e attack by the invaded p lan t cell. A s n u trie n t
supply so far only the carbon/energy source has b een
co n sidered to be decisive. W e asked w h eth e r th e
ro o t h air of legum es m ight also be a source fo r m in ­
erals for w hich rhizobia have a special large re q u ire ­
m en t, as for calcium , iron and cobalt [8, 9].
Introduction
R o o t hairs of higher plants are form ing the largest
biological surface to the en v iro n m en t besides m icro­
organism s. A single m a tu re cereal p la n t can develop
b etw een 109 and IO10 ro o t hairs, w ith a surface in one
h ectar of cereals in the o rd e r of 20 to 200 km 2. D e ­
spite the ecological and physiological im portance of
this cell type, the special biochem istry of ro o t hairs is
alm ost com pletely unknow n, since they have not
b een isolated on a p rep a ra tiv e scale. W e have d e ­
veloped such a m eth o d , using the different fragility
of ro o t hairs and roots after freezing in liquid n itro ­
gen and brushing the ro o t hairs from th e roots. F rom
104 soybean seedlings, 109 ro o t hairs and abou t
100 mg ro o t hair pro tein could be isolated. T hree
ro o t hair specific p roteins have been se p arate d from
soybeans, presen t only in ra th e r sm all quantities or
absent in the o th e r ro o t tissue [1],
In m ost legum es, ro o t hairs are in addition to th eir
function for u ptake of w ater and n u trie n ts from the
soil and excretion of ro o t ex udates the ta rg e t cells for
infection by R hizobium species [2]. In the signal
chain of the R h izobium -legum e sym biosis especially
the prim ary events in the ro o t hair (signal u p tak e)
are unknow n on a biochem ical basis. A n exception is
the lectin-E PS/C PS/LPS hypothesis [3, 4] for recogniReprint requests to Prof. Dr. D. Werner.
Verlag der Zeitschrift für Naturforschung, D -7400 Tübingen
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Materials and Methods
Seeds of Glycine max cv. M aple A rrow w ere r e ­
ceived from A griculture C an ad a, O ttaw a R e sea rch
S tatio n , seeds of Triticum aestivum cv. K olibri from
v. L ochow -P etkus, N ortheim (G erm an y ). T h e seeds
w ere sterilized with 30% H 20 2 for 30 m in, th o r­
oughly w ashed and incubated fo r 10—20 h in sterile
tap w ater.
F o r g erm ination 60—80 seeds w ere in cu b ated on
large agar plates (30 x 40 cm ) covered w ith
alum inium foil w ith Vi co n cen trated H o ag lan d s m in ­
eral solution and 3% agar. T he te m p eratu re regim e
w as 25 °C for 14 h and 20 °C for 10 h in p h y to tro n s
w ith 50% hum idity.
A fter 4 to 8 days 5 —8 cm ro o ts w ere s e p a ra te d
from th e seeds and spread on m etal p lates, fro zen
w ith liquid nitrogen. T he flexible root hairs w ere im ­
m ediately frozen and could be se p arated from th e
m ain ro o ts w ith a thin brush.
R o o t hairs and o th e r parts of the roots w ere sto red
at —80 °C and later hom ogenized to a p o w d er by
using only plastic m aterial. T he m ineral com position
o f th e m aterial was d eterm in ed in th ree in d e p en d e n t
batches as described previously [11],
Results and Discussion
W ith ro o t hairs isolated on a p rep arativ e scale, we
analyzed the m ineral com position by th e P IX E
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913
Notizen
Element Soybean
root hair
K
S
Soybean root
Wheat root
hair
Table I. Mineral salt concentrations determined
by PIXE [10] in root hairs and roots of soybean
seedlings ( Glycine max cv. Maple Arrow) and
wheat seedlings ( Triticum aestivum ev. Kolibri),
8 days old, germinated on mineral agar with one
half concentrated Hoaglands solution. Concen­
trations in ng per g dry matter (ppm) ± standard
deviation.
Wheat root
11740 ± 2450 12840 ± 2640 4670 ± 1010 4780 ± 990
530 ± 165
560 ± 170 180 ±
55 190 ± 60
P
1326 ±
410
2590 ±
790
526 ±
165 1570 ± 475
Fe
Co
Ca
414 ±
7.9 ±
2200 ±
138
3.8
460
31 ±
0.88 ±
287 ±
5
0.4
70
120 ±
2.6 ±
246 ±
35
0.8
60
44 ±
1.3 ±
288 ±
26
1.1
70
Mo
3.1 ±
0.5
5.4
0.7
0.6 ± 0.12
0.5 ±
0.3
±
m eth o d [10, 11]. T he potassium and the sulfur con­
te n t in ro o t hairs of soybean is very sim ilar to the
o th e r p arts of the ro o t system (Table I). T his is also
ob serv ed in th e com p ariso n of root hairs and roots
from w h eat, although th e absolute concentrations
are low er by a facto r o f th ree . T he stan d ard devia­
tions for the m a cro -n u trien ts d eterm ined by the
P IX E m e th o d are com paratively large. H ow ever,
especially the q u an tita tiv e ratio betw een the various
elem en ts is very ac cu rate, due to a continuous co u n t­
ing of the sam ples for 20 m in w ith m onitoring all
elem en ts at th e sam e tim e [10]. T he phosphorus con­
cen tratio n in the ro o t hairs of soybean is only about
half th e figure from th e to ta l ro o t system . A p p a re n t­
ly ro o t hairs do not serve as storage cells for phos­
p h ate. T he m ost re m a rk a b le difference in m ineral
accum ulation in ro o t hairs o f soybean was found with
iron, cobalt and calcium (T able I). T he iron concen­
tratio n of ab o u t 400 ppm is about ten tim es as large
as in the o th e r parts of th e ro o t system in 8 days old
[1] D. Werner and M. Rohm, in: Genetic Engineering of
Plants and Microorganisms Important for Agriculture
(N. de Nettancourt and E. Magnien, eds.), M. Nijhoff/
Dr. W. Junk Publ., The Hague 1985.
[2] D. Werner and A. Krotzky, Funkt. Biol. Med. 2, 31
(1983).
[3] F. B. Dazzo, G. L. Truchet, and E. M. Hrabak, in:
Advances in Nitrogen Fixation Research, p. 413 (D.
Veeger and W. E. Newton, eds.), M. Nijhoff/Dr. W.
Junk Publ., The Hague 1984.
[4] W. Kamberger, FEMS Microbiol. Lett. 6, 361 (1979).
[5] A. Quispel, J. W. Kijne, A. A. N. van Brussel, E.
Pees, C. A. Wijffelman, and A. J. P. Burggraaf, in:
Advances in Nitrogen Fixation Research, p. 381 (D.
Veeger and W. E. Newton, eds.), M. Nijhoff/Dr. W.
Junk Publ., The Hague 1984.
[6] J. G. Robertson, P. Lyttleton. and C. E. Pankhurst,
seedlings. The accum ulation facto r for cobalt and
calcium is betw een 6 and 8 in ro o t hairs co m p ared to
roots. It is in terestin g to note th a t m olybdenum a
co m p o n en t of n itra te red u ctase an d of nitro g en ase
co m p o n en t II (M o -F e-p ro tein ) is p rese n t in sim ilar
q u an tities in ro o t hairs and ro o ts. T he en rich m en t
factors for calcium , iron and co b alt and also th e ab ­
solute co n cen tratio n s in ro o t hairs o f w h eat are sig­
nificantly sm aller co m p ared to th e soybean. From
th ese results we conclude th at R hizobium japonicum
can find after infection of a soybean ro o t h air not
only a supply of carb o n and energy so u rce, b u t also a
supply of calcium , iron and co b alt, for w hich a high
req u irem en t by this b acterium is estab lish ed [8, 9],
Acknow ledgem ents
T his w ork was su p p o rted by th e C E C p ro g ram m e
“G en etic engineering o f plants and m icroorganism s
im p o rtan t for ag ric u ltu re” . W e also th an k M rs. L.
K arn er for p rep arin g the m anuscript.
[7]
[8]
[9]
[10]
[11]
in: Current Perspectives in Nitrogen Fixation, p. 280
(A . H. Gibson and W. E. Newton, eds.), Australian
Academy of Science, Canberra 1981.
D. Werner, E. Mörschel, R. Kort, R. B. Mellor, and
S. Bassarab, Planta 162, 8 (1984).
H. J. Evans and S. Russel, in: The Chemistry and
Biochemistry of Nitrogen Fixation, p. 191 (J. Post­
gate, ed.), Plenum Press, London 1971.
M. J. Trinick, in: Nitrogen Fixation, Vol. 2,
Rhizobium, p. 76 (W. J. Broughton, ed.), Clarendon
Press, Oxford 1982.
J. Hasselmann, W. Koenig, F. W. Richter, U. Steiner,
U. Wätjen, J. C. Bode, and W. Ohta, Nuclear Instru­
ments and Methods 142, 163 (1977).
K.-P. Kuhlmann, R. Stripf. W. Wätjen, F. W. Rich­
ter, F. Gloystein, and D. Werner, Angew. Bot. 56,
315 (1 9 8 2 )/
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