Autoregulation Of Nodulation Pathway Research Articles

The Defective in Autoregulation (DAR) gene of Medicago truncatula encodes a protein involved in regulating nodulation and arbuscular mycorrhiza

BackgroundLegumes utilize a long-distance signaling feedback pathway, termed Autoregulation of Nodulation (AON), to regulate the establishment and maintenance of their symbiosis with rhizobia. Several proteins key to this pathway have been discovered, but the AON pathway is not completely understood.ResultsWe report a new hypernodulating mutant, defective in autoregulation, with disruption of a gene, DAR (Medtr2g450550/MtrunA17_Chr2g0304631), previously unknown to play a role in AON. The dar-1 mutant produces ten-fold more nodules than wild type, similar to AON mutants with disrupted SUNN gene function. As in sunn mutants, suppression of nodulation by CLE peptides MtCLE12 and MtCLE13 is abolished in dar. Furthermore, dar-1 also shows increased root length colonization by an arbuscular mycorrhizal fungus, suggesting a role for DAR in autoregulation of mycorrhizal symbiosis (AOM). However, unlike SUNN which functions in the shoot to control nodulation, DAR functions in the root.ConclusionsDAR encodes a membrane protein that is a member of a small protein family in M. truncatula. Our results suggest that DAR could be involved in the subcellular transport of signals involved in symbiosis regulation, but it is not upregulated during symbiosis. DAR gene family members are also present in Arabidopsis, lycophytes, mosses, and microalgae, suggesting the AON and AOM may use pathway components common to other plants, even those that do not undergo either symbiosis.

The Phaseolus vulgaris Receptor-Like Kinase PvFER1 and the Small Peptides PvRALF1 and PvRALF6 Regulate Nodule Number as a Function of Nitrate Availability.

Legumes associate with Gram-negative soil bacteria called rhizobia, resulting in the formation of a nitrogen-fixing organ, the nodule. Nodules are an important sink for photosynthates for legumes, so these plants have developed a systemic regulation mechanism that controls their optimal number of nodules, the so-called autoregulation of nodulation (AON) pathway, to balance energy costs with the benefits of nitrogen fixation. In addition, soil nitrate inhibits nodulation in a dose-dependent manner, through systemic and local mechanisms. The CLE family of peptides and their receptors are key to tightly controlling these inhibitory responses. In the present study, a functional analysis revealed that PvFER1, PvRALF1, and PvRALF6 act as positive regulators of the nodule number in growth medium containing 0 mM of nitrate but as negative regulators in medium with 2 and 5 mM of nitrate. Furthermore, the effect on nodule number was found to be consistent with changes in the expression levels of genes associated with the AON pathway and with the nitrate-mediated regulation of nodulation (NRN). Collectively, these data suggest that PvFER1, PvRALF1, and PvRALF6 regulate the optimal number of nodules as a function of nitrate availability.

Inhibition of legume nodulation by Pi deficiency is dependent on the autoregulation of nodulation (AON) pathway.

Inhibition of nodule development is one of the main adverse effects of phosphate (Pi) deficiency in legumes. Despite all of the efforts made over the last decades to understand how root nodules cope with Pi deficiency, the molecular mechanisms leading to the reduction in nodule number under Pi deficiency remain elusive. In the present study, we provide experimental evidence indicating that Pi deficiency activates the autoregulation of nodulation (AON) pathway, leading to a reduction in nodule numbers in both common bean and soybean. A transcriptional profile analysis revealed that the expression of the AON-related genes PvNIN, PvRIC1, PvRIC2, and PvTML is upregulated under Pi deficiency conditions. The downregulation of the MYB transcription factor PvPHR1 in common bean roots significantly reduced the expression of these four AON-related genes. Physiological analyses indicated that Pi deficiency does not affect the establishment of the root nodule symbiosis in the supernodulation mutant lines Pvnark and Gmnark. Reciprocal grafting and split-roots analyses determined that the activation of the AON pathway was required for the inhibitory effect of Pi deficiency. Altogether, these data improve our understanding of the genetic mechanisms controlling the establishment of the root nodule symbiosis under Pi deficiency.

Autoregulation of nodulation pathway is dispensable for nitrate-induced control of rhizobial infection

ABSTRACTLegumes possess the autoregulation of nodulation (AON) pathway which is responsible for maintaining optimal root nodule number. In Lotus japonicus, AON comprises the CLE-HAR1-TML module, which plays an essential role in transmitting signals via root-to-shoot-to-root long-distance signaling. In addition to AON’s principal role of negatively regulating nodule number, a recent study revealed another in the systemic control of rhizobial infection. Nitrate also negatively regulates the pleiotropic phases of legume-Rhizobium symbioses, including rhizobial infection and nodule number. Nitrate signaling has recently been shown to use AON components such as CLE-RS2 and HAR1 to control nodule number. Here we consider the role of a loss-of-function mutation in CLE-RS1, -RS2 and TML in rhizobial infection in relation to nitrate. Our results agree with previous findings and support the hypothesis that AON is required for the control of rhizobial infection but not for its nitrate-induced control. Furthermore, we confirm that the tml mutants exhibit nitrate sensitivity that differs from that of cle-rs2 and har1. Hence, while the nitrate-induced control mechanism of nodule number uses AON components, an unknown pathway specific to nitrate may exist downstream of HAR1, acting in parallel with the HAR1> TML pathway.

A GmNINa-miR172c-NNC1 Regulatory Network Coordinates the Nodulation and Autoregulation of Nodulation Pathways in Soybean

Symbiotic root nodules are root lateral organs of plants in which nitrogen-fixing bacteria (rhizobia) convert atmospheric nitrogen to ammonia. The formation and number of nodules in legumes are precisely controlled by a rhizobia-induced signal cascade and host-controlled autoregulation of nodulation (AON). However, how these pathways are integrated and their underlying mechanisms are unclear. Here, we report that microRNA172c (miR172c) activates soybean (Glycine max) Rhizobia-Induced CLE1 (GmRIC1) and GmRIC2 by removing the transcriptional repression of these genes by Nodule Number Control 1 (NNC1), leading to the activation of the AON pathway. NNC1 interacts with GmNINa, the soybean ortholog of Lotus NODULE INCEPTION (NIN), and hampers its transcriptional activation of GmRIC1 and GmRIC2. Importantly, GmNINa acts as a transcriptional activator of miR172c. Intriguingly, NNC1 can transcriptionally repress miR172c expression, adding a negative feedback loop into the NNC1 regulatory network. Moreover, GmNINa interacts with NNC1 and can relieve the NNC1-mediated repression of miR172c transcription. Thus, the GmNINa-miR172c-NNC1 network is a master switch that coordinately regulates and optimizes NF and AON signaling, supporting the balance between nodulation and AON in soybean.

Unraveling new molecular players involved in the autoregulation of nodulation in Medicago truncatula.

The number of legume root nodules resulting from a symbiosis with rhizobia is tightly controlled by the plant. Certain members of the CLAVATA3/Embryo Surrounding Region (CLE) peptide family, specifically MtCLE12 and MtCLE13 in Medicago truncatula, act in the systemic autoregulation of nodulation (AON) pathway that negatively regulates the number of nodules. Little is known about the molecular pathways that operate downstream of the AON-related CLE peptides. Here, by means of a transcriptome analysis, we show that roots ectopically expressing MtCLE13 deregulate only a limited number of genes, including three down-regulated genes encoding lysin motif receptor-like kinases (LysM-RLKs), among which are the nodulation factor (NF) receptor NF Perception gene (NFP) and two up-regulated genes, MtTML1 and MtTML2, encoding Too Much Love (TML)-related Kelch-repeat containing F-box proteins. The observed deregulation was specific for the ectopic expression of nodulation-related MtCLE genes and depended on the Super Numeric Nodules (SUNN) AON RLK. Moreover, overexpression and silencing of these two MtTML genes demonstrated that they play a role in the negative regulation of nodule numbers. Hence, the identified MtTML genes are the functional counterpart of the Lotus japonicus TML gene shown to be central in the AON pathway. Additionally, we propose that the down-regulation of a subset of LysM-RLK-encoding genes, among which is NFP, might contribute to the restriction of further nodulation once the first nodules have been formed.

A member of the ALOG gene family has a novel role in regulating nodulation in Lotus japonicus.

Legumes can control the number of symbiotic nodules that form on their roots, thus balancing nitrogen assimilation and energy consumption. Two major pathways participate in nodulation: the Nod factor (NF) signaling pathway which involves recognition of rhizobial bacteria by root cells and promotion of nodulation, and the autoregulation of nodulation (AON) pathway which involves long-distance negative feedback between roots and shoots. Although a handful of genes have a clear role in the maintenance of nodule number, additional unknown factors may also be involved in this process. Here, we identify a novel function for a Lotus japonicus ALOG (Arabidopsis LSH1 and Oryza G1) family member, LjALOG1, involved in positively regulating nodulation. LjALOG1 expression increased substantially after inoculation with rhizobia, with high levels of expression in whole nodule primordia and in the base of developing nodules. The ljalog1 mutants, which have an insertion of the LORE1 retroelement in LjALOG1, had significantly fewer nodules compared with wild type, along with increased expression of LjCLE-RS1 (L. japonicus CLE Root Signal 1), which encodes a nodulation suppressor in the AON pathway. In summary, our findings identified a novel factor that participates in controlling nodulation, possibly by suppressing the AON pathway.

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.

The Art of Self-Control - Autoregulation of Plant-Microbe Symbioses.

Plants interact with diverse microbes including those that result in nutrient-acquiring symbioses. In order to balance the energy cost with the benefit gained, plants employ a systemic negative feedback loop to control the formation of these symbioses. This is particularly well-understood in nodulation, the symbiosis between legumes and nitrogen-fixing rhizobia, and is known as autoregulation of nodulation (AON). However, much less is understood about the autoregulation of the ancient arbuscular mycorrhizal symbioses that form between Glomeromycota fungi and the majority of land plants. Elegant physiological studies in legumes have indicated there is at least some overlap in the genes and signals that regulate these two symbioses but there are major gaps in our understanding. In this paper we examine the hypothesis that the autoregulation of mycorrhizae (AOM) pathway shares some elements with AON but that there are also some important differences. By reviewing the current knowledge of the AON pathway, we have identified important directions for future AOM studies. We also provide the first genetic evidence that CLV2 (an important element of the AON pathway) influences mycorrhizal development in a non-legume, tomato and review the interaction of the autoregulation pathway with plant hormones and nutrient status. Finally, we discuss whether autoregulation may play a role in the relationships plants form with other microbes.

ROOT DETERMINED NODULATION1 Is Required for M. truncatula CLE12, But Not CLE13, Peptide Signaling through the SUNN Receptor Kinase.

The combinatorial interaction of a receptor kinase and a modified CLE peptide is involved in several developmental processes in plants, including autoregulation of nodulation (AON), which allows legumes to limit the number of root nodules formed based on available nitrogen and previous rhizobial colonization. Evidence supports the modification of CLE peptides by enzymes of the hydroxyproline O-arabinosyltransferase (HPAT/RDN) family. Here, we show by grafting and genetic analysis in Medicago truncatula that, in the AON pathway, RDN1, functioning in the root, acts upstream of the receptor kinase SUNN, functioning in the shoot. As expected for a glycosyltransferase, we found that RDN1 and RDN2 proteins are localized to the Golgi, as was shown previously for AtHPAT1. Using composite plants with transgenic hairy roots, we show that RDN1 and RDN2 orthologs from dicots as well as a related RDN gene from rice (Oryza sativa) can rescue the phenotype of rdn1-2 when expressed constitutively, but the less related MtRDN3 cannot. The timing of the induction of MtCLE12 and MtCLE13 peptide genes (negative regulators of AON) in nodulating roots is not altered by the mutation of RDN1 or SUNN, although expression levels are higher. Plants with transgenic roots constitutively expressing MtCLE12 require both RDN1 and SUNN to prevent nodule formation, while plants constitutively expressing MtCLE13 require only SUNN, suggesting that the two CLEs have different requirements for function. Combined with previous work, these data support a model in which RDN1 arabinosylates MtCLE12, and this modification is necessary for the transport and/or reception of the AON signal by the SUNN kinase.

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.

Common and divergent roles of plant hormones in nodulation and arbuscular mycorrhizal symbioses

All of the classical plant hormones have been suggested to influence nodulation, including some that interact with the Autoregulation of Nodulation (AON) pathway. Leguminous plants strictly regulate the number of nodules formed through this AON pathway via a root-shoot-root loop that acts to suppress excessive nodulation. A related pathway, the Autoregulation of Mycorrhization (AOM) pathway controls the more ancient, arbuscular mycorrhizal (AM) symbiosis. A comparison of the published responses to the classical hormones in these 2 symbioses shows that most influence the symbioses in the same direction. This may be expected if they affect the symbioses via common components of these symbiotic regulatory pathways. However, some hormones influence these symbioses in opposite directions, suggesting a more complex relationship, and probably one that is not via the common components of these pathways. In a recent paper we showed, using a genetic approach, that strigolactones and brassinosteroids do not act downstream of the AON genes examined and argued that they probably act independently to promote nodule formation. Recently it has been shown that the control of nodulation via the AON pathway involves mobile CLE peptide signals. It is therefore suggested that a more direct avenue to determine if the classical hormones play a direct role in the autoregulatory pathways is to further examine whether CLE peptides and other components of these processes can influence, or be influenced by, the classical hormones. Such studies and other comparisons between the nodulation and mycorrhizal symbioses should allow the role of the classical hormones in these critical symbioses to be rapidly advanced.

The potential roles of strigolactones and brassinosteroids in the autoregulation of nodulation pathway

The number of nodules formed on a legume root system is under the strict genetic control of the autoregulation of nodulation (AON) pathway. Plant hormones are thought to play a role in AON; however, the involvement of two hormones recently described as having a largely positive role in nodulation, strigolactones and brassinosteroids, has not been examined in the AON process. A genetic approach was used to examine if strigolactones or brassinosteroids interact with the AON system in pea (Pisum sativum). Double mutants between shoot-acting (Psclv2, Psnark) and root-acting (Psrdn1) mutants of the AON pathway and strigolactone-deficient (Psccd8) or brassinosteroid-deficient (lk) mutants were generated and assessed for various aspects of nodulation. Strigolactone production by AON mutant roots was also investigated. Supernodulation of the roots was observed in both brassinosteroid- and strigolactone-deficient AON double-mutant plants. This is despite the fact that the shoots of these plants displayed classic strigolactone-deficient (increased shoot branching) or brassinosteroid-deficient (extreme dwarf) phenotypes. No consistent effect of disruption of the AON pathway on strigolactone production was found, but root-acting Psrdn1 mutants did produce significantly more strigolactones. No evidence was found that strigolactones or brassinosteroids act downstream of the AON genes examined. While in pea the AON mutants are epistatic to brassinosteroid and strigolactone synthesis genes, we argue that these hormones are likely to act independently of the AON system, having a role in the promotion of nodule formation.