Asymmetric Redundancy of Soybean Nodule Inception (NIN) Genes in Root Nodule Symbiosis

Nodule Inception (NIN) is one of the most important root nodule symbiotic genes as it is required for both infection and nodule organogenesis in legumes. Unlike most legumes with a sole NIN gene, there are four putative orthologous NIN genes in soybean (Glycine max). Whether and how these NIN genes contribute to soybean-rhizobia symbiotic interaction remain unknown. In this study, we found that all four GmNIN genes are induced by rhizobia and that conserved CE and CYC binding motifs in their promoter regions are required for their expression in the nodule formation process. By generation of multiplex Gmnin mutants, we found that the Gmnin1a nin2a nin2b triple mutant and Gmnin1a nin1b nin2a nin2b quadruple mutant displayed similar defects in rhizobia infection and root nodule formation, Gmnin2a nin2b produced fewer nodules but displayed a hyper infection phenotype compared to wild type, while the Gmnin1a nin1b double mutant nodulated similar to wild type. Overexpression of GmNIN1a, GmNIN1b, GmNIN2a, and GmNIN2b reduced nodule numbers after rhizobia inoculation, with GmNIN1b overexpression having the weakest effect. In addition, overexpression of GmNIN1a, GmNIN2a, or GmNIN2b, but not GmNIN1b, produced malformed pseudo-nodule-like structures without rhizobia inoculation. In conclusion, GmNIN1a, GmNIN2a, and GmNIN2b play functionally redundant yet complicated roles in soybean nodulation. GmNIN1b, although expressed at a comparable level with the other homologs, plays a minor role in root nodule symbiosis. Our work provides insight into the understanding of the asymmetrically redundant function of GmNIN genes in soybean.

Asymmetric Redundancy of Soybean Nodule Inception (NIN) Genes in Root Nodule Symbiosis

NIN is one of the most important root nodule symbiotic genes as it is required for both infection and nodule organogenesis in legume. Unlike most legumes with a sole NIN gene, there are four putative NIN genes in soybean (Glycine max). Whether and how these orthologs NIN genes contribute to soybean-rhizobia symbiotic interaction remain unknown. In this study, we found that all four GmNIN genes are induced by rhizobia, and that conserved CE and CYC binding motifs in their promoter regions are required for their expression in the nodule formation process. By generation of multiplex Gmnin mutants, we found that Gmnin1a nin2a nin2b triple mutant and Gmnin1a nin1b nin2a nin2b quadruple mutant displayed similar defects in rhizobia infection and root nodule formation, Gmnin2a nin2b produced less nodules but displayed hyper infection phenotype than wild type, while a Gmnin1a nin1b double mutant nodulated as wild type. Overexpression of GmNIN1a, GmNIN1b, GmNIN2a, and GmNIN2b reduced nodule numbers after rhizobia inoculation, with GmNIN1b overexpression having the weakest effect. In addition, overexpression of GmNIN1a, GmNIN2a, or GmNIN2b, but not GmNIN1b, produced malformed pseudo-nodule like structures without rhizobia inoculation. In conclusion, GmNIN1a, GmNIN2a and GmNIN2b play functionally redundant yet complicated roles for soybean nodulation. GmNIN1b, although is expressed at comparable level with other homologs, plays a minor role in root nodule symbiosis. Our work provides insight into the understanding of asymmetrically redundant function of GmNIN genes in soybean.

Functional and comparative genomics reveals conserved noncoding sequences in the nitrogen-fixing clade

Nitrogen is one of the most inaccessible plant nutrients, but certain species have overcome this limitation by establishing symbiotic interactions with nitrogen-fixing bacteria in the root nodule. This root nodule symbiosis (RNS) is restricted to species within a single clade of angiosperms, suggesting a critical evolutionary event at the base of this clade, which has not yet been determined. While genes implicated in the RNS are present in most plant species (nodulating or not), gene sequence conservation alone does not imply functional conservation - developmental or phenotypic differences can arise from variation in the regulation of transcription. To identify putative regulatory sequences implicated in the evolution of RNS, we aligned the genomes of 25 species capable of nodulation. We detected 3,091 conserved noncoding sequences (CNS) in the nitrogen-fixing clade that are absent from outgroup species. Functional analysis revealed that chromatin accessibility of 452 CNS significantly correlates with the differential regulation of genes responding to lipo-chitooligosaccharides in Medicago truncatula. These included 38 CNS in proximity to 19 known genes involved in RNS. Five such regions are upstream of MtCRE1, Cytokinin Response Element 1, required to activate a suite of downstream transcription factors necessary for nodulation in M. truncatula. Genetic complementation of a Mtcre1 mutant showed a significant association between nodulation and the presence of these CNS, when they are driving the expression of a functional copy of MtCRE1. Conserved noncoding sequences, therefore, may be required for the regulation of genes controlling the root nodule symbiosis in M. truncatula.

Aboveground plant-to-plant communication reduces root nodule symbiosis and soil nutrient concentrations

Aboveground communication between plants is well known to change defense traits in leaves, but its effects on belowground plant traits and soil characteristics have not been elucidated. We hypothesized that aboveground plant-to-plant communication reduces root nodule symbiosis via induction of bactericidal chemical defense substances and changes the soil nutrient environment. Soybean plants were exposed to the volatile organic compounds (VOCs) from damaged shoots of Solidago canadensis var. scabra, and leaf defense traits (total phenolics, saponins), root saponins, and root nodule symbiosis traits (number and biomass of root nodules) were measured. Soil C/N ratios and mineral concentrations were also measured to estimate the effects of resource uptake by the plants. We found that total phenolics were not affected. However, plants that received VOCs had higher saponin concentrations in both leaves and roots, and fewer root nodules than untreated plants. Although the concentrations of soil minerals did not differ between treatments, soil C/N ratio was significantly higher in the soil of communicated plants. Thus, the aboveground plant-to-plant communication led to reductions in root nodule symbiosis and soil nutrient concentrations. Our results suggest that there are broader effects of induced chemical defenses in aboveground plant organs upon belowground microbial interactions and soil nutrients, and emphasize that plant response based on plant-to-plant communications are a bridge between above- and below-ground ecosystems.

An NADPH oxidase regulates carbon metabolism and the cell cycle during root nodule symbiosis in common bean (Phaseolus vulgaris)

Background Rhizobium–legume symbiosis is a specific, coordinated interaction that results in the formation of a root nodule, where biological nitrogen fixation occurs. NADPH oxidases, or Respiratory Burst Oxidase Homologs (RBOHs) in plants, are enzymes that generate superoxide (O2•−). Superoxide produces other reactive oxygen species (ROS); these ROS regulate different stages of mutualistic interactions. For example, changes in ROS levels are thought to induce ROS scavenging, cell wall remodeling, and changes in phytohormone homeostasis during symbiotic interactions. In common bean (Phaseolus vulgaris), PvRbohB plays a key role in the early stages of nodulation. Results In this study, to explore the role of PvRbohB in root nodule symbiosis, we analyzed transcriptomic data from the roots of common bean under control conditions (transgenic roots without construction) and roots with downregulated expression of PvRbohB (by RNA interference) non-inoculated and inoculated with R. tropici. Our results suggest that ROS produced by PvRBOHB play a central role in infection thread formation and nodule organogenesis through crosstalk with flavonoids, carbon metabolism, cell cycle regulation, and the plant hormones auxin and cytokinin during the early stages of this process. Conclusions Our findings provide important insight into the multiple roles of ROS in regulating rhizobia–legume symbiosis.

Non-specific Lipid Transfer Proteins in Legumes and Their Participation During Root-Nodule Symbiosis

Non-specific lipid transfer proteins (LTPs) constitute a large protein family in plants characterized by having a tunnel-like hydrophobic cavity, which allows them to transfer different lipid molecules. LTPs have been studied in various model plants including those of agronomic interest. Recent studies have demonstrated that LTPs play key functions in both biotic and abiotic stress. In plants-pathogen interaction, they act as either positive or negative regulators of defense responses. However, little is known about the roles of LTPs in symbiotic interactions, especially in root nodule symbiosis. Here, we performed a broad genome analysis of LTP family members in legumes and other important model plants, focusing on their possible roles in legume-rhizobium symbiosis. In silico analysis showed that legumes contain large LTP families, with at least 70 LTP members clustered into four clades. Although the structures of LTP genes and proteins are conserved among species, differences were observed between clades from different species. LTPs are widely expressed in different plant tissues. In general, genes of the LTP1 and LTP2 classes are highly expressed in shoot and reproductive tissues in all analyzed species. Furthermore, genes of the different classes are also expressed in roots inoculated with rhizobia and nodules of legumes. RT-qPCR expression profile analysis of seven PvLTP genes in common bean (Phaseolus vulgaris) revealed that these genes are differentially expressed during the early and late stages of nodulation and they are genetically regulated by PvRbohA. These findings provide insight into the putative roles of LTP family members in legume-rhizobium symbiosis and their possible interactions with RBOH-dependent ROS production.

Genome-wide survey of soybean papain-like cysteine proteases and their expression analysis in root nodule symbiosis

Background Plant papain-like cysteine proteases (PLCPs) are a large class of proteolytic enzymes and play important roles in root nodule symbiosis (RNS), while the whole-genome studies of PLCP family genes in legume are quite limited, and the roles of Glycine max PLCPs (GmPLCPs) in nodulation, nodule development and senescence are not fully understood. Results In the present study, we identified 97 GmPLCPs and performed a genome-wide survey to explore the expansion of soybean PLCP family genes and their relationships to RNS. Nineteen paralogous pairs of genomic segments, consisting of 77 GmPLCPs, formed by whole-genome duplication (WGD) events were identified, showing a high degree of complexity in duplication. Phylogenetic analysis among different species showed that the lineage differentiation of GmPLCPs occurred after family expansion, and large tandem repeat segment were specifically in soybean. The expression patterns of GmPLCPs in symbiosis-related tissues and nodules identified RNS-related GmPLCPs and provided insights into their putative symbiotic functions in soybean. The symbiotic function analyses showed that a RNS-related GmPLCP gene (Glyma.04G190700) really participate in nodulation and nodule development. Conclusions Our findings improved our understanding of the functional diversity of legume PLCP family genes, and provided insights into the putative roles of the legume PLCPs in nodulation, nodule development and senescence.