Brett J. Ferguson

Molecular Analysis of Legume Nodule Development and Autoregulation

Legumes are highly important food, feed and biofuel crops. With few exceptions, they can enter into an intricate symbiotic relationship with specific soil bacteria called rhizobia. This interaction results in the formation of a new root organ called the nodule in which the rhizobia convert atmospheric nitrogen gas into forms of nitrogen that are useable by the plant. The plant tightly controls the number of nodules it forms, via a complex root-to-shoot-to-root signaling loop called autoregulation of nodulation (AON). This regulatory process involves peptide hormones, receptor kinases and small metabolites. Using modern genetic and genomic techniques, many of the components required for nodule formation and AON have now been isolated. This review addresses these recent findings, presents detailed models of the nodulation and AON processes, and identifies gaps in our understanding of these process that have yet to be fully explained.

Strigolactone acts downstream of auxin to regulate bud outgrowth in pea and Arabidopsis.

During the last century, two key hypotheses have been proposed to explain apical dominance in plants: auxin promotes the production of a second messenger that moves up into buds to repress their outgrowth, and auxin saturation in the stem inhibits auxin transport from buds, thereby inhibiting bud outgrowth. The recent discovery of strigolactone as the novel shoot-branching inhibitor allowed us to test its mode of action in relation to these hypotheses. We found that exogenously applied strigolactone inhibited bud outgrowth in pea (Pisum sativum) even when auxin was depleted after decapitation. We also found that strigolactone application reduced branching in Arabidopsis (Arabidopsis thaliana) auxin response mutants, suggesting that auxin may act through strigolactones to facilitate apical dominance. Moreover, strigolactone application to tiny buds of mutant or decapitated pea plants rapidly stopped outgrowth, in contrast to applying N-1-naphthylphthalamic acid (NPA), an auxin transport inhibitor, which significantly slowed growth only after several days. Whereas strigolactone or NPA applied to growing buds reduced bud length, only NPA blocked auxin transport in the bud. Wild-type and strigolactone biosynthesis mutant pea and Arabidopsis shoots were capable of instantly transporting additional amounts of auxin in excess of endogenous levels, contrary to predictions of auxin transport models. These data suggest that strigolactone does not act primarily by affecting auxin transport from buds. Rather, the primary repressor of bud outgrowth appears to be the auxin-dependent production of strigolactones.

Signaling Interactions During Nodule Development

Nitrogen fixing bacteria, collectively referred to as rhizobia, are able to trigger the organogenesis of a new organ on legumes, the nodule. The morphogenetic trigger is a Rhizobium-produced lipochitin-oligosaccharide called the Nod factor, which is necessary, and in some legumes sufficient, for triggering nodule development in the absence of the bacterium. Because plant development is substantially influenced by plant hormones, it has been hypothesized that plant hormones (mainly the classical hormones abscisic acid, auxin, cytokinins, ethylene and gibberellic acid) regulate nodule development. In recent years, evidence has shown that Nod factors might act in legumes by changing the internal plant hormone balance, thereby orchestrating the nodule developmental program. In addition, many nonclassical hormonal signals have been found to play a role in nodule development, some of them similar to signals involved in animal development. These compounds include peptide hormones, nitric oxide, reactive oxygen species, jasmonic acid, salicylic acid, uridine, flavonoids and Nod factors themselves. Environmental factors, in particular nitrate, also influence nodule development by affecting the plant hormone status. This review summarizes recent findings on the involvement of classical and nonclassical signals during nodule development with the aim of illustrating the multiple interactions existing between these compounds that have made this area so complicated to analyze.

Roles for Auxin, Cytokinin and Strigolactone in Regulating Shoot Branching

Many processes have been described in the control of shoot branching. Apical dominance is defined as the control exerted by the shoot tip on the outgrowth of axillary buds, whereas correlative inhibition includes the suppression of growth by other growing buds or shoots. The level, signaling, and/or flow of the plant hormone auxin in stems and buds is thought to be involved in these processes. In addition, RAMOSUS (RMS) branching genes in pea (Pisum sativum) control the synthesis and perception of a long-distance inhibitory branching signal produced in the stem and roots, a strigolactone or product. Auxin treatment affects the expression of RMS genes, but it is unclear whether the RMS network can regulate branching independently of auxin. Here, we explore whether apical dominance and correlative inhibition show independent or additive effects in rms mutant plants. Bud outgrowth and branch lengths are enhanced in decapitated and stem-girdled rms mutants compared with intact control plants. This may relate to an RMS-independent induction of axillary bud outgrowth by these treatments. Correlative inhibition was also apparent in rms mutant plants, again indicating an RMS-independent component. Treatments giving reductions in RMS1 and RMS5 gene expression, auxin transport, and auxin level in the main stem were not always sufficient to promote bud outgrowth. We suggest that this may relate to a failure to induce the expression of cytokinin biosynthesis genes, which always correlated with bud outgrowth in our treatments. We present a new model that accounts for apical dominance, correlative inhibition, RMS gene action, and auxin and cytokinin and their interactions in controlling the progression of buds through different control points from dormancy to sustained growth.

Inoculation- and Nitrate-Induced CLE Peptides of Soybean Control NARK-Dependent Nodule Formation

Systemic autoregulation of nodulation in legumes involves a root-derived signal (Q) that is perceived by a CLAVATA1-like leucine-rich repeat receptor kinase (e.g. GmNARK). Perception of Q triggers the production of a shoot-derived inhibitor that prevents further nodule development. We have identified three candidate CLE peptide-encoding genes (GmRIC1, GmRIC2, and GmNIC1) in soybean (Glycine max) that respond to Bradyrhizobium japonicum inoculation or nitrate treatment. Ectopic overexpression of all three CLE peptide genes in transgenic roots inhibited nodulation in a GmNARK-dependent manner. The peptides share a high degree of amino acid similarity in a 12-amino-acid C-terminal domain, deemed to represent the functional ligand of GmNARK. GmRIC1 was expressed early (12 h) in response to Bradyrhizobium-sp.-produced nodulation factor while GmRIC2 was induced later (48 to 72 h) but was more persistent during later nodule development. Neither GmRIC1 nor GmRIC2 were induced by nitrate. In contrast, GmNIC1 was strongly induced by nitrate (2 mM) treatment but not by Bradyrhizobium sp. inoculation and, unlike the other two GmCLE peptides, functioned locally to inhibit nodulation. Grafting demonstrated a requirement for root GmNARK activity for nitrate regulation of nodulation whereas Bradyrhizobium sp.-induced regulation was contingent on GmNARK function in the shoot.

Apical dominance and shoot branching. Divergent opinions or divergent mechanisms

Apical dominance is the term used to describe the control of the shoot tip over axillary bud outgrowth (e.g. [Cline, 1997][1]). It is best demonstrated via shoot tip removal (decapitation), which leads to apical dominance. Indeed, decapitation has been widely used to study bud outgrowth. In contrast

Nodulation Phenotypes of Gibberellin and Brassinosteroid Mutants of Pea

The initiation and development of legume nodules induced by compatible Rhizobium species requires a complex signal exchange involving both plant and bacterial compounds. Phytohormones have been implicated in this process, although in many cases direct evidence is lacking. Here, we characterize the root and nodulation phenotypes of various mutant lines of pea (Pisum sativum) that display alterations in their phytohormone levels and/or perception. Mutants possessing root systems deficient in gibberellins (GAs) or brassinosteroids (BRs) exhibited a reduction in nodule organogenesis. The question of whether these reductions represent direct or indirect effects of the hormone deficiency is addressed. For example, the application of GA to the roots of a GA-deficient mutant completely restored its number of nodules to that of the wild type. Grafting studies revealed that a wild-type shoot or root also restored the nodule number of a GA-deficient mutant. These findings suggest that GAs are required for nodulation. In contrast, the shoot controlled the number of nodules that formed in graft combinations of a BR-deficient mutant and its wild type. The root levels of auxin and GA were similar among these latter graft combinations. These results suggest that BRs influence a shoot mechanism that controls nodulation and that the root levels of auxin and GA are not part of this process. Interestingly, a strong correlation between nodule and lateral root numbers was observed in all lines assessed, consistent with a possible overlap in the early developmental pathways of the two organs.

Suppression of hypernodulation in soybean by a leaf-extracted, NARK- and Nod factor-dependent, low molecular mass fraction

*Legumes regulate the number of nodules they form via a process called autoregulation of nodulation (AON). This involves a shoot-derived inhibitor (SDI) molecule that is synthesized in the shoots and is transported down to the roots where it inhibits further nodule development. *To characterize SDI, we developed a novel feeding bioassay. This involved feeding aqueous leaf extracts directly into the petiole of hypernodulating and supernodulating nark mutant plants of Glycine max (soybean). These mutants normally exhibit an increased nodulation phenotype because SDI is not produced and thus AON is nonfunctional. *Feeding wild-type leaf extracts presumed to contain SDI was successful in suppressing the increased nodulation phenotype, whereas feeding with Gmnark leaf extracts did not. Suppression activity was inoculation-dependent, Nod factor-dependent, required GmNARK activity, and was heat-, Proteinase K- and ribonuclease A-resistant. Wild-type extracts maintained suppressive activity even at a ninefold dilution. Sinorhizobium meliloti-inoculated Medicago truncatula leaf extracts from wild-type, but not from supernodulating mutant Mtsunn, suppressed hypernodulation in soybean. *Our results demonstrate that the petiole feeding bioassay is an efficient and effective technique to introduce aqueous extracts into plants. They also demonstrate that SDI is a small compound with an apparent molecular mass of < 1000 Da and is unlikely to be a protein or an RNA molecule.

Phytohormone Regulation of Legume-Rhizobia Interactions

The symbiosis between legumes and nitrogen fixing bacteria called rhizobia leads to the formation of root nodules. Nodules are highly organized root organs that form in response to Nod factors produced by rhizobia, and they provide rhizobia with a specialized niche to optimize nutrient exchange and nitrogen fixation. Nodule development and invasion by rhizobia is locally controlled by feedback between rhizobia and the plant host. In addition, the total number of nodules on a root system is controlled by a systemic mechanism termed ’autoregulation of nodulation’. Both the local and the systemic control of nodulation are regulated by phytohormones. There are two mechanisms by which phytohormone signalling is altered during nodulation: through direct synthesis by rhizobia and through indirect manipulation of the phytohormone balance in the plant, triggered by bacterial Nod factors. Recent genetic and physiological evidence points to a crucial role of Nod factor-induced changes in the host phytohormone balance as a prerequisite for successful nodule formation. Phytohormones synthesized by rhizobia enhance symbiosis effectiveness but do not appear to be necessary for nodule formation. This review provides an overview of recent advances in our understanding of the roles and interactions of phytohormones and signalling peptides in the regulation of nodule infection, initiation, positioning, development, and autoregulation. Future challenges remain to unify hormone–related findings across different legumes and to test whether hormone perception, response, or transport differences among different legumes could explain the variety of nodules types and the predisposition for nodule formation in this plant family. In addition, the molecular studies carried out under controlled conditions will need to be extended into the field to test whether and how phytohormone contributions by host and rhizobial partners affect the long term fitness of the host and the survival and competition of rhizobia in the soil. It also will be interesting to explore the interaction of hormonal signalling pathways between rhizobia and plant pathogens.

Soybean miR172c Targets the Repressive AP2 Transcription Factor NNC1 to Activate ENOD40 Expression and Regulate Nodule Initiation

Induction of miR172c in response to rhizobial inoculation activates ENOD40 expression and nodule formation by repressing an AP2/ERF transcription factor, called NNC1, that acts to inhibit ENOD40 expression. MicroRNAs are noncoding RNAs that act as master regulators to modulate various biological processes by posttranscriptionally repressing their target genes. Repression of their target mRNA(s) can modulate signaling cascades and subsequent cellular events. Recently, a role for miR172 in soybean (Glycine max) nodulation has been described; however, the molecular mechanism through which miR172 acts to regulate nodulation has yet to be explored. Here, we demonstrate that soybean miR172c modulates both rhizobium infection and nodule organogenesis. miR172c was induced in soybean roots inoculated with either compatible Bradyrhizobium japonicum or lipooligosaccharide Nod factor and was highly upregulated during nodule development. Reduced activity and overexpression of miR172c caused dramatic changes in nodule initiation and nodule number. We show that soybean miR172c regulates nodule formation by repressing its target gene, Nodule Number Control1, which encodes a protein that directly targets the promoter of the early nodulin gene, ENOD40. Interestingly, transcriptional levels of miR172c were regulated by both Nod Factor Receptor1α/5α-mediated activation and by autoregulation of nodulation-mediated inhibition. Thus, we established a direct link between miR172c and the Nod factor signaling pathway in addition to adding a new layer to the precise nodulation regulation mechanism of soybean.