Characterisation of Medicago truncatula CLE34 and CLE35 in nitrate and rhizobia regulation of nodulation.

Legumes form a symbiosis with atmospheric nitrogen (N2 )-fixing soil rhizobia, resulting in new root organs called nodules that enable N2 -fixation. Nodulation is a costly process that is tightly regulated by the host through autoregulation of nodulation (AON) and nitrate-dependent regulation of nodulation. Both pathways require legume-specific CLAVATA/ESR-related (CLE) peptides. Nitrogen-induced nodulation-suppressing CLE peptides have not previously been investigated in Medicago truncatula, for which only rhizobia-induced MtCLE12 and MtCLE13 have been characterised. Here, we report on novel peptides MtCLE34 and MtCLE35 in nodulation control. The nodulation-suppressing CLE peptides of five legume species were classified into three clades based on sequence homology and phylogeny. This approached identified MtCLE34 and MtCLE35 and four new CLE peptide orthologues of Pisum sativum. Whereas MtCLE12 and MtCLE13 are induced by rhizobia, MtCLE34 and MtCLE35 respond to both rhizobia and nitrate. MtCLE34 was identified as a pseudogene lacking a functional CLE-domain. MtCLE35 was found to inhibit nodulation in a SUNN- and RDN1-dependent manner via overexpression analysis. Together, our findings indicate that MtCLE12 and MtCLE13 have a specific role in AON, while MtCLE35 regulates nodule numbers in response to both rhizobia and nitrate. MtCLE34 likely had a similar role to MtCLE35, but its function was lost due to a premature nonsense mutation.

GmYUC2a mediates auxin biosynthesis during root development and nodulation in soybean.

Auxin plays central roles in rhizobial infection and nodule development in legumes. However, the sources of auxin during nodulation are unknown. In this study, we analyzed the YUCCA (YUC) gene family of soybean and identified GmYUC2a as an important regulator of auxin biosynthesis that modulates nodulation. Following rhizobial infection, GmYUC2a exhibited increased expression in various nodule tissues. Overexpression of GmYUC2a (35S::GmYUC2a) increased auxin production in soybean, resulting in severe growth defects in root hairs and root development. Upon rhizobial infection, 35S::GmYUC2a hairy roots displayed altered patterns of root hair deformation and nodule formation. Root hair deformation occurred mainly on primary roots, and nodules formed exclusively on primary roots of 35S::GmYUC2a plants. Moreover, transgenic 35S::GmYUC2a composite plants showed delayed nodule development and a reduced number of nodules. Our results suggest that GmYUC2a plays an important role in regulating both root growth and nodulation by modulating auxin balance in soybean.

Triarabinosylation is required for nodulation-suppressive CLE peptides to systemically inhibit nodulation in Pisum sativum.

Legumes form root nodules to house beneficial nitrogen-fixing rhizobia bacteria. However, nodulation is resource demanding; hence, legumes evolved a systemic signalling mechanism called autoregulation of nodulation (AON) to control nodule numbers. AON begins with the production of CLE peptides in the root, which are predicted to be glycosylated, transported to the shoot, and perceived. We synthesized variants of nodulation-suppressing CLE peptides to test their activity using petiole feeding to introduce CLE peptides into the shoot. Hydroxylated, monoarabinosylated, and triarabinosylated variants of soybean GmRIC1a and GmRIC2a were chemically synthesized and fed into recipient Pisum sativum (pea) plants, which were used due to the availability of key AON pathway mutants unavailable in soybean. Triarabinosylated GmRIC1a and GmRIC2a suppressed nodulation of wild-type pea, whereas no other peptide variant tested had this ability. Suppression also occurred in the supernodulating hydroxyproline O-arabinosyltransferase mutant, Psnod3, but not in the supernodulating receptor mutants, Pssym29, and to some extent, Pssym28. During our study, bioinformatic resources for pea became available and our analyses identified 40 CLE peptide-encoding genes, including orthologues of nodulation-suppressive CLE peptides. Collectively, we demonstrated that soybean nodulation-suppressive CLE peptides can function interspecifically in the AON pathway of pea and require arabinosylation for their activity.

Legume nodulation: The host controls the party.

Global demand to increase food production and simultaneously reduce synthetic nitrogen fertilizer inputs in agriculture are underpinning the need to intensify the use of legume crops. The symbiotic relationship that legume plants establish with nitrogen-fixing rhizobia bacteria is central to their advantage. This plant-microbe interaction results in newly developed root organs, called nodules, where the rhizobia convert atmospheric nitrogen gas into forms of nitrogen the plant can use. However, the process of developing and maintaining nodules is resource intensive; hence, the plant tightly controls the number of nodules forming. A variety of molecular mechanisms are used to regulate nodule numbers under both favourable and stressful growing conditions, enabling the plant to conserve resources and optimize development in response to a range of circumstances. Using genetic and genomic approaches, many components acting in the regulation of nodulation have now been identified. Discovering and functionally characterizing these components can provide genetic targets and polymorphic markers that aid in the selection of superior legume cultivars and rhizobia strains that benefit agricultural sustainability and food security. This review addresses recent findings in nodulation control, presents detailed models of the molecular mechanisms driving these processes, and identifies gaps in these processes that are not yet fully explained.

Author Correction: CLE peptide-encoding gene families in Medicago truncatula and Lotus japonicus, compared with those of soybean, common bean and Arabidopsis.

A correction to this article has been published and is linked from the HTML version of this paper. The error has not been fixed in the paper.

Arabinosylation Modulates the Growth-Regulating Activity of the Peptide Hormone CLE40a from Soybean.

Small post-translationally modified peptide hormones mediate crucial developmental and regulatory processes in plants. CLAVATA/ENDOSPERM-SURROUNDING REGION (CLE) genes are found throughout the plant kingdom and encode for 12-13 amino acid peptides that must often undergo post-translational proline hydroxylation and glycosylation with O-ß1,2-triarabinose moieties before they become functional. Apart from a few recent examples, a detailed understanding of the structure and function of most CLE hormones is yet to be uncovered. This is mainly owing to difficulties in isolating mature homogeneously modified CLE peptides from natural plant sources. In this study, we describe the efficient synthesis of a synthetic Araf3Hyp glycosylamino acid building block that was used to access a hitherto uninvestigated CLE hormone from soybean called GmCLE40a. Through the development and implementation of a novel in vivo root growth assay, we show that the synthetic triarabinosylated glycopeptide suppresses primary root growth in this important crop species.

CLE peptide-encoding gene families in Medicago truncatula and Lotus japonicus, compared with those of soybean, common bean and Arabidopsis.

CLE peptide hormones are critical regulators of many cell proliferation and differentiation mechanisms in plants. These 12-13 amino acid glycosylated peptides play vital roles in a diverse range of plant tissues, including the shoot, root and vasculature. CLE peptides are also involved in controlling legume nodulation. Here, the entire family of CLE peptide-encoding genes was identified in Medicago truncatula (52) and Lotus japonicus (53), including pseudogenes and non-functional sequences that were identified. An array of bioinformatic techniques were used to compare and contrast these complete CLE peptide-encoding gene families with those of fellow legumes, Glycine max and Phaseolus vulgaris, in addition to the model plant Arabidopsis thaliana. This approach provided insight into the evolution of CLE peptide families and enabled us to establish putative M. truncatula and L. japonicus orthologues. This includes orthologues of nodulation-suppressing CLE peptides and AtCLE40 that controls the stem cell population of the root apical meristem. A transcriptional meta-analysis was also conducted to help elucidate the function of the CLE peptide family members. Collectively, our analyses considerably increased the number of annotated CLE peptides in the model legume species, M. truncatula and L. japonicus, and substantially enhanced the knowledgebase of this critical class of peptide hormones.

Genome-wide annotation and characterization of CLAVATA/ESR (CLE) peptide hormones of soybean (Glycine max) and common bean (Phaseolus vulgaris), and their orthologues of Arabidopsis thaliana.

CLE peptides are key regulators of cell proliferation and differentiation in plant shoots, roots, vasculature, and legume nodules. They are C-terminally encoded peptides that are post-translationally cleaved and modified from their corresponding pre-propeptides to produce a final ligand that is 12-13 amino acids in length. In this study, an array of bionformatic and comparative genomic approaches was used to identify and characterize the complete family of CLE peptide-encoding genes in two of the world's most important crop species, soybean and common bean. In total, there are 84 CLE peptide-encoding genes in soybean (considerably more than the 32 present in Arabidopsis), including three pseudogenes and two multi-CLE domain genes having six putative CLE domains each. In addition, 44 CLE peptide-encoding genes were identified in common bean. In silico characterization was used to establish all soybean homeologous pairs, and to identify corresponding gene orthologues present in common bean and Arabidopsis. The soybean CLE pre-propeptide family was further analysed and separated into seven distinct groups based on structure, with groupings strongly associated with the CLE domain sequence and function. These groups provide evolutionary insight into the CLE peptide families of soybean, common bean, and Arabidopsis, and represent a novel tool that can aid in the functional characterization of the peptides. Transcriptional evidence was also used to provide further insight into the location and function of all CLE peptide-encoding members currently available in gene atlases for the three species. Taken together, this in-depth analysis helped to identify and categorize the complete CLE peptide families of soybean and common bean, established gene orthologues within the two legume species, and Arabidopsis, and provided a platform to help compare, contrast, and identify the function of critical CLE peptide hormones in plant development.

The value of biodiversity in legume symbiotic nitrogen fixation and nodulation for biofuel and food production.

Much of modern agriculture is based on immense populations of genetically identical or near-identical varieties, called cultivars. However, advancement of knowledge, and thus experimental utility, is found through biodiversity, whether naturally-found or induced by the experimenter. Globally we are confronted by ever-growing food and energy challenges. Here we demonstrate how such biodiversity from the food legume crop soybean (Glycine max L. Merr) and the bioenergy legume tree Pongamia (Millettia) pinnata is a great value. Legume plants are diverse and are represented by over 18,000 species on this planet. Some, such as soybean, pea and medics are used as food and animal feed crops. Others serve as ornamental (e.g., wisteria), timber (e.g., acacia/wattle) or biofuel (e.g., Pongamia pinnata) resources. Most legumes develop root organs (nodules) after microsymbiont induction that serve as their habitat for biological nitrogen fixation. Through this, nitrogen fertiliser demand is reduced by the efficient symbiosis between soil Rhizobium-type bacteria and the appropriate legume partner. Mechanistic research into the genetics, biochemistry and physiology of legumes is thus strategically essential for future global agriculture. Here we demonstrate how molecular plant science analysis of the genetics of an established food crop (soybean) and an emerging biofuel P. pinnata feedstock contributes to their utility by sustainable production aided by symbiotic nitrogen fixation.

The structure and activity of nodulation-suppressing CLE peptide hormones of legumes.

Legumes form a highly-regulated symbiotic relationship with specific soil bacteria known as rhizobia. This interaction results in the de novo formation of root organs called nodules, in which the rhizobia fix atmospheric di-nitrogen (N2) for the plant. Molecular mechanisms that regulate the nodulation process include the systemic 'autoregulation of nodulation' and the local nitrogen-regulation of nodulation pathways. Both pathways are mediated by novel peptide hormones called CLAVATA/ESR-related (CLE) peptides that act to suppress nodulation via negative feedback loops. The mature peptides are 12-13 amino acids in length and are post-translationally modified from the C-terminus of tripartite-domain prepropeptides. Structural redundancy between the prepropeptides exists; however, variations in external stimuli, timing of expression, tissue specificity and presence or absence of key functional domains enables them to act in a specific manner. To date, nodulation-regulating CLE peptides have been identified in Glycine max (L.) Merr., Medicago truncatula Gaertn., Lotus japonicus (Regel) K.Larsen and Phaseolus vulgaris L. One of the L. japonicus peptides, called LjCLE-RS2, has been structurally characterised and found to be an arabinosylated glycopeptide. All of the known nodulation CLE peptides act via an orthologous leucine rich repeat (LRR) receptor kinase. Perception of the peptide results in the production of a novel, unidentified inhibitor signal that acts to suppress further nodulation events. Here, we contrast and compare the various nodulation-suppressing CLE peptides of legumes.

The soybean (Glycine max) nodulation-suppressive CLE peptide, GmRIC1, functions interspecifically in common white bean (Phaseolus vulgaris), but not in a supernodulating line mutated in the receptor PvNARK.

Legume plants regulate the number of nitrogen-fixing root nodules they form via a process called the Autoregulation of Nodulation (AON). Despite being one of the most economically important and abundantly consumed legumes, little is known about the AON pathway of common bean (Phaseolus vulgaris). We used comparative- and functional-genomic approaches to identify central components in the AON pathway of common bean. This includes identifying PvNARK, which encodes a LRR receptor kinase that acts to regulate root nodule numbers. A novel, truncated version of the gene was identified directly upstream of PvNARK, similar to Medicago truncatula, but not seen in Lotus japonicus or soybean. Two mutant alleles of PvNARK were identified that cause a classic shoot-controlled and nitrate-tolerant supernodulation phenotype. Homeologous over-expression of the nodulation-suppressive CLE peptide-encoding soybean gene, GmRIC1, abolished nodulation in wild-type bean, but had no discernible effect on PvNARK-mutant plants. This demonstrates that soybean GmRIC1 can function interspecifically in bean, acting in a PvNARK-dependent manner. Identification of bean PvRIC1, PvRIC2 and PvNIC1, orthologues of the soybean nodulation-suppressive CLE peptides, revealed a high degree of conservation, particularly in the CLE domain. Overall, our work identified four new components of bean nodulation control and a truncated copy of PvNARK, discovered the mutation responsible for two supernodulating bean mutants and demonstrated that soybean GmRIC1 can function in the AON pathway of bean.