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Plant Molecular Biology 26: 5-9, 1994.
© 1994 Kluwer Academic Publishers. Printed in Belgium.
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Plant hormones and nodulation: what's the connection?
Ann M. Hirsch and Yiwen Fang
Department of Biology, 405 Hilgard Avenue, University of California, Los Angeles, CA 90024-1606, USA
Received and accepted 13 June 1994
Ever since Thimann [20] proposed that auxin
plays a role in nodule development, considerable
effort has been expended to show whether phytohormones are involved. Because the hormones
are involved in other types of organogenesis, it
seems likely that they have a role in nodule development. However, important questions remain
unanswered: which hormones take part in nodule
formation, and how are they involved?
What's the evidence for hormone involvement?
Phytohormones are found in nodules.
There have been ample studies showing that four
of the hormones - auxin, cytokinin, gibberellin,
and ABA - can be isolated from nodules. All are
present in nodules at concentrations higher than
in uninfected roots (cited in [21]). However, it is
not clear which of the symbiotic partners is the
source of the hormones. It is particularly difficult
to elucidate the plant's involvement, especially for
auxin and cytokinin, because genes for the biosynthesis of these hormones have yet to be identified. Rhizobia can synthesize plant hormones,
but mutations in their auxin-producing genes do
not prevent nodulation, nor do N o d - mutants
fail to produce hormones (cited in [8]). Rhizobial
mutants defective in cytokinin production have
yet to be identified.
Inhibitory effects of phytohormones
Ethylene is a potent inhibitor of nodulation. Nonnodulating pea mutants, which are blocked at the
nodule primordium stage, can be restored to normal development if treated with AVG (aminoethoxyvinylglycine) or Ag +, which inhibit ethylene formation and action, respectively [7].
Another inhibitory role for plant hormones is
autoregulation, i.e., the control by the plant of the
number and size of its nodules (see [4] for review). Supernodulating legume mutants lack this
autoregulatory control, and copious nodulation
occurs. Grafting experiments between normal and
supernodulating plants show that the autoregulatory factor is shoot-derived and that it is elicited
by some sort of systemic messenger provided by
the root [4]. The autoregulatory factor is capable
of traversing graft junctions so it is likely that it
is a small molecule. Although at one time it was
speculated that the shoot-derived factor was
ABA, the autoregulatory molecule remains unidentified.
Libbenga et al. [ 12], studying pea root cortical
explants, found that cell divisions took place in
the pericycle if auxin only were added to the culture medium. If cytokinin and auxin were both
present, cell divisions occurred at positions where
nodules would emerge. Cytokinin could be replaced by a factor from the root stele. Smit et al.
[18] have found that Nod + rhizobia and mitogenic Nod factors inactivate this stele factor in a
normal pea plant, but not in a supernodulating
mutant, suggesting that the inactivation of the
stele factor may be part of the autoregulation response, and could possibly influence production
of the shoot-derived factor. The stele factor has
been identified (J. Kijne, pers. comm.).
Hormones or compounds that block hormone transport induce nodule-like structures
Hirsch etal. [9], extending the observations of
Allen etal. [1] and Torrey [21], found that inhibitors of polar auxin transport induced pseudonodules on alfalfa roots. These contained transcripts for two nodulin genes, thus demonstrating
that ENOD2 and Nms30 genes are developmentally rather than symbiotically regulated. This
observation has been repeated for other legumes
and for other early nodulin genes [14, 16].
Auxin and cytokinin frequently interact in organogenesis. The ratio of these two hormones
may be unbalanced by an exogenous auxin transport inhibitor. An increase in the cytokinin/auxin
ratio is suggested by reports where cytokinin applied to roots induced cortical cell divisions [2, 3,
14]. Similarly, Rhizobium meliloti transconjugants
carrying the tzs (trans-zeatin secretion) gene of
Agrobacterium tumefaciens, and presumably secreting cytokinin, induced bacteria-free nodules
containing M s E N O D 2 transcripts on alfalfa [5].
However, work by Kondorosi et al. [ 11 ] indicates that auxin-sensitive variants (such plants
frequently contain a higher concentration of
auxin) ofMedicago varia formed significantly more
nodules than a less auxin-sensitive line. The nodules also developed earlier than the nodules on
the normal line. In addition, plants transformed
with the rol genes from Agrobacterium rhizogenes
were affected in their nodulation ability. Plants
containing rolABC, and particularly rolB, nodulated faster and had more nodules than untransformed plants.
Nevertheless, nodule development is not required for early nodulin gene expression. Dehio
and deBruijn [6] have demonstrated that exogenous benzyladenine induced the expression of
SrENOD2 in Sesbania roots, and we (Y. Fang,
S. Asad, and A.M. Hirsch, unpublished results)
have similarly shown that benzylaminopurine
elicits the expression in alfalfa roots of an
ENOD2-1ike gene, and also ENOD12 and
ENOD40 genes. The E N O D genes are thus the
first reported cytokinin-responsive genes that are
not activated by other hormones, such as auxin,
gibberellin, and ethylene, or various environmental factors, such as light or nitrate. Moreover,
gene expression is induced very quickly after cytokinin application. The ENOD40 gene is expressed within 6 h, the same time needed to detect
ENOD40 transcripts on northern blots after
treating alfalfa roots with purified Nod factor (Y.
Fang, S. Asad, and A.M. Hirsch, unpublished
results).
What's the relationship between hormones and
nodule development?
There are at least two explanations for the connection between Nod factor, the external signal,
and nodule organogenesis, the internal response.
One hypothesis is that the Nod factor acts as a
hormone, activating cell divisions and nodule primordia formation. Endogenous Nod factor-like
molecules may exist in uninfected plants [19].
Transgenic tobacco plants containing either R.
meliloti nodA or nodB or both genes under the
control of diverse promoters displayed distinct
phenotypes which are compatible with modified
levels of endogenous hormones [17]. NodA is
involved in attaching a fatty acid acyl chain to a
tetrasaccharide precursor [15] and NodB is a
deacetylase [ 10]. NodA and NodB probably perform the same functions in the transgenic plants,
indicating that substrates for these proteins must
be present within the plant cells.
The difficulty with this hypothesis is that lipochitosans have not been identified in plants except in secondary cell walls (cited in [19]). In
addition, such molecules are not restricted to
legumes; yet, only legumes are nodulated. If
Rhizobium inoculation triggers the release or increased synthesis of an endogenous Nod factorlike molecule, why don't all plants curl their root
hairs and undergo mitoses upon inoculation with
Rhizobium? One argument is that only the legumes
synthesize Nod factor-like molecules in their roots
whereas in non-legumes, the endogenous Nod
factors are produced only in the aerial parts of the
plant.
A second and more conservative hypothesis,
apex
~--~
basipetal transport of auxin
during pod fill
nodulation stops
autoregulatory factor
•
autoregulati~,/~
nodule primordium
\
~
Cauxin
"~~ ~
• . cytokinin
r h'z°b'a ~ gi bberellins] ~
L Nod factor ~'-':''J- ~
~-2 .,~,ethylene
~ ~L~-v~-.- ~*" cytokinin
~~'~
"W~" auxin
nodu e
~(~.~tk~_.~"
mt "~\\ ~\'~'- shepherd'scrook
a t
cytokinin production
Fig. I. Diagram showing the various plant growth regulators thought to be involved with nodulation. Cytokinin is produced in all
the root tips, and auxin, produced in the shoot tip, is basipetally transported down the stem. Rhizobia synthesize Nod factor which
affects both root hair deformation and cell divisions that foreshadow the nodule primordia. Although rhizobia also produce auxin,
cytokinins, and gibberellins, it is not clear whether any of these are required for nodule primoria formation. Exogenous cytokinin
can elicit nodule formation, and nodules contain high levels of cytokinin and auxin. Although auxin is involved in some way with
nodulation, its role is as yet unclear. Ethylene and a shoot-derived autoregulatory factor inhibit nodule development. The stimulus to produce the shoot-derived factor is synthesized in the root and transferred to the shoot. During pod fill, nodulation stops.
relying only on the k n o w n plant hormones, states
that there is a change in the endogenous h o r m o n e
balance brought about by N o d factor treatment.
This change may c o m e about either by altering
the effective concentrations of the endogenous
p h y t o h o r m o n e s or by altering h o r m o n e sensitivity through the expression o f different classes of
h o r m o n e receptors or binding proteins. In addition to the experiments described earlier, evidence
suggesting that a perturbed h o r m o n e balance m a y
be d o w n s t r e a m of N o d factor perception is supported by the existence o f spontaneously nodulating alfalfa genotypes, which form nodules in
the absence o f rhizobia and without an apparent
external trigger [22]. Also, when the nonnodulating alfalfa line M N 1 0 0 8 [13] is treated
with an auxin transport inhibitor, pseudonodules
form, indicating that cell divisions can be
8
uncoupled from Nod factor perception (Y. Fang,
S. Asad, and A.M. Hirsch, unpublished results).
The roots of MN1008 contain ENOD40 transcripts after treatment with cytokinin (Y. Fang, S.
Asad and A.M. Hirsch unpublished results).
The two alternatives presented above are not
mutually exclusive. The genotype which spontaneously forms nodules may overexpress an endogenous Nod factor-like molecule. Similarly, a
nodB gene product may deacylate or a nodA
gene product may attach a lipid to some other
target molecule, for example, producing a lipidconjugated plant hormone. This molecule, when
ectopically expressed, results in a phenocopy of a
plant treated with an excess of plant hormones.
Summary and future prospects
Figure 1 summarizes the various ways the phytohormones and other factors may control nodulation. Experimental data implicate all of the
known hormones in nodulation. Studies of nodule development and hormone action has progressed rapidly in the past five years. The challenge now is to determine whether nodule
organogenesis involves the traditional plant hormones or novel ones. In addition, the study of the
hormone-responsive early nodulin genes may help
elucidate the link between the external (Nod factor; Rhizobium) signals and the internal ones (hormones, novel factors?).
Acknowledgements
We are very grateful to Tom LaRue for helpful
discussions and comments on the manuscript.
Thanks also to Bettina Niner and Stefan J.
Kirchanski for their help. Margaret Kowalczyk is
thanked for preparing the figure. The research in
AMH's laboratory is funded by NSF and USDA
grants.
References
1. Allen EK, Allen ON, Newman AS: Pseudonodulation of
leguminous plants induced by 2-bromo-3,5-dichlorobenzoic acid. Am J Bot 40:429-435 (1953).
2. Arora N, Skoog F, Allen ON: Kinetin-induced pseudonodules on tobacco roots. Am J Bot 46:610-613 (1959).
3. Bauer WB, Bhuvaneswari TV, Calvert HE, Law IJ, Malik
NSA, Vesper SJ: Recognition and infection by slowgrowing rhizobia. In: Evans H J, Bottomley PJ, Newton
WE (eds) Nitrogen Fixation Research Progress, pp. 247253. Martinus Nijhoff Publishers, Dordrecht (1985).
4. Caetano-Anoll~s G, GresshoffPM: Plant genetic control
of nodulation. Annu Rev Microbiol 45:345-382 (1991).
5. Cooper JB, Long SR: Morphogenetic rescue of Rhizobium meliloti nodulation mutants by trans-zeatin secretion. Plant Cell 6:215-225 (1994).
6. Dehio C, deBruijn FJ: The early nodulin gene SrEnod2
from Sesbania rostrata is inducible by cytokinin. Plant J
2:117-128 (1992).
7. Guinel FC, LaRue TA: Light microscopy study of nodule initiation in Pisum sativum L. cv. Sparkle and its lownodulation mutant E2 (sym 5). Plant Physiol 97: 12061211 (1991).
8. Hirsch AM: Tansley Review No. 40. Developmental
biology of legume nodulation. New Phytol 122:211-237
(1992).
9. Hirsch AM, Bhuvaneswari TV, Torrey JG, Bisseling T:
Early nodulin genes are induced in alfalfa outgrowths
elicited by auxin transport inhibitors. Proc Natl Acad Sci
USA 86:1244-1248 (1989).
10. John M, R0hrig H, Schmidt J, Wieneke U, Schell J:
Rhizobium NodB protein involved in nodulation signal
synthesis is a chitooligosaccharide deacetylase. Proc Natl
Acad Sci USA 90:625-629 (1993).
11. Kondorosi E, Schultze M, Savoure A, Hoffmann B,
Dudits D, Pierre M, Allison L, Bauer P, Kiss GB,
Kondorosi A: Control of nodule induction and plant cell
growth by Nod factors. In: Nester EW, Verma DPS
(eds) Advances in Molecular-Genetics of Plant-Microbe
Interactions, pp. 143-150. Kluwer Academic Publishers,
Dordrecht (1993).
12. Libbenga KR, van Iren F, Bogers RJ, Schraag-Lamers
MF: The role of hormones and gradients in the initiation
of cortex proliferation and nodule formation in Pisum
sativum L. Planta 114:29-39 (1973).
13. Peterson MA, Barnes DK: Inheritance of ineffective
nodulation and non-nodulationtraits in alfalfa. Crop Sci
21:611-616 (1981).
14. Relid B, Talmont F, Kopcinska J, Golinowski W, Prom6,
Broughton WJ: Biological activity of Rhizobium sp.
NGR234 Nod-factors on Macroptilium atropurpureum.
Mol Plant-Microbe Interact 6:764-774 (1993).
15. ROhrig H, Schmidt J, Wieneke U, Kondorosi, Earlier I,
Schell J, John M: Biosynthesis of lipooligosaccharide
nodulation factors: Rhizobium NodA protein is involved
in N-acylation of the chitooligosaccharide backbone. Proc
Natl Acad Sci USA. 91, 3122-3126 (1994).
16. Scheres B, McKhann HI, Zalensky A, LObler M,
Bisseling T, Hirsch AM: The PsENOD12 gene is expressed at two different sites in Afghanistan pea pseudo-
nodules induced by auxin transport inhibitors. Plant
Physiol 100:1649-1655 (1992).
17. Schmidt J, R~3hrig H, John M, Wieneke U, Stacey G,
Koncz C, Schell J: Alteration of plant growth and development by Rhizobium nodA and nodB genes involved in
the synthesis of oligosaccharide signal molecules. Plant J
4:651-658 (1993).
18. Smit G, van Brussel TAN, Kijne JW: Inactivation of a
root factor by ineffective Rhizobium: a molecular key
to autoregulation of nodulation in Pisum sativum. In:
Palacios R, Mora J, Newton WE (eds) New Horizons in
Nitrogen Fixation, p. 371. Kluwer Academic Publishers,
Dordrecht (1993).
19. Spaink HP, Aarts A, Bloemberg GV, Folch J, Geiger
O, Schlaman HRM, Thomas-Oates JE, van Brussel
AAN, van de Sande K, van Spronsen P, Wijtjes AHM,
Lugtenberg BJ J: Rhizobial lipo-oligosaccharide signals:
their biosynthesis and their role in the plant. In: Nester
EW, Verma DPS (eds) Advances in Molecular-Genetics
of Plant-Microbe Interactions, pp. 151-162. Kluwer Academic Publishers, Dordrecht (1993).
20. Thimann KV: On the physiology of the formation of nodules on legume roots. Proc Natl Acad Sci USA 22:511514 (1936).
21. Torrey JG: Endogenous and exogenous influences on the
regulation of lateral root formation. In: Jackson MB (ed)
New Root Formation in Plants and Cuttings, pp. 31-66.
Martinus Nijhoff Publishers, Dordrecht (1986).
22. Truchet G, Barker DG, Camut S, deBilly F, Vasse J,
Huguet T: Alfalfa nodulation in the absence of Rhizobium.
Mol Gen Genet 219:65-68 (1989).