SHORT-ROOT paralogs mediate feedforward regulation of D-type cyclin to promote nodule formation in soybean

Nitrogen fixation in soybean takes place in root nodules that arise from de novo cell divisions in the root cortex. Although several early nodulin genes have been identified, the mechanism behind the stimulation of cortical cell division during nodulation has not been fully resolved. Here we provide evidence that two paralogs of soybean SHORT-ROOT (GmSHR) play vital roles in soybean nodulation. Expression of GmSHR4 and GmSHR5 (GmSHR4/5) is induced in cortical cells at the beginning of nodulation, when the first cell divisions occur. The expression level of GmSHR4/5 is positively associated with cortical cell division and nodulation. Knockdown of GmSHR5 inhibits cell division in outer cortical layers during nodulation. Knockdown of both paralogs disrupts the cell division throughout the cortex, resulting in poorly organized nodule primordia with delayed vascular tissue formation. GmSHR4/5 function by enhancing cytokinin signaling and activating early nodulin genes. Interestingly, D-type cyclins act downstream of GmSHR4/5, and GmSHR4/5 form a feedforward loop regulating D-type cyclins. Overexpression of D-type cyclins in soybean roots also enhanced nodulation. Collectively, we conclude that the GmSHR4/5-mediated pathway represents a vital module that triggers cytokinin signaling and activates D-type cyclins during nodulation in soybean.

Interactions between hydrogen sulfide and rhizobia modulate the physiology and metabolism during water deficiency-induced oxidative defense in soybean

Hydrogen sulphide (H2S), as a new gas signal molecule, participates in the regulation of a variety of abiotic stresses in plants. However, it was unclear how H2S and rhizobia can together to affect the adaptation of soybean to water deficiency. Here, the adaptation mechanism of H2S and rhizobia in soybean to water deficiency was studied. Our results showed that H2S and rhizobia jointly enhanced leaf chlorophyll content, the relative water content (RWC) and caused an increase biomass in soybean under water deficiency. Besides, under water deficiency, H2S enhanced biomass by affecting nodule numbers and nitrogenase activity during the growth of soybean. The expression of soybean nodulation marker genes including early nodulin 40 (GmENOD40), ERF required for nodulation (GmERN), and nodulation inception genes were up-regulated by H2S and rhizobia in nodules. Moreover, the combined effect of H2S and rhizobia were proved to affect the enzyme activities and gene expression level of antioxidant, as well as osmotic protective substance under water deficiency. In addition, the metabolomics results provided that the changes of lipids and lipid-like molecules were remarkably promoted by the combined effect of H2S and rhizobia. Thus, H2S and rhizobia synergistically subsided the oxidative damage by increasing the accumulation of metabolites and strengthening the antioxidant capacity under water deficiency.

Salt stress enhances early symbiotic gene expression in Medicago truncatula and induces a stress-specific set of rhizobium-responsive genes.

Salt stress is a major agricultural concern inhibiting not only plant growth but also the symbiotic association between legume roots and the soil bacteria rhizobia. This symbiotic association is initiated by a molecular dialogue between the two partners, leading to the activation of a signaling cascade in the legume host and ultimately the formation of nitrogen-fixing root nodules. Here we show that a moderate salt stress increases the responsiveness of early symbiotic genes in Medicago truncatula to its symbiotic partner, Sinorhizobium meliloti, while conversely, inoculation with S. meliloti counteracts salt-regulated gene expression, restoring one-third to control levels. Our analysis of Early Nodulin 11 shows that salt-induced expression is dynamic, Nod-factor dependent, and requires the ionic, but not the osmotic, component of salt. We demonstrate that salt stimulation of rhizobium-induced gene expression requires NSP2, which functions as a node to integrate the abiotic and biotic signals. In addition, our work reveals that inoculation with Sinorhizobium meliloti succinoglycan mutants also hyperinduces ENOD11 expression in the presence or absence of salt, suggesting a possible link between rhizobial exopolysaccharide and the plant response to salt stress. Finally, we identify an accessory set of genes that are induced by rhizobium only under conditions of salt stress and have not been previously identified as being nodulation-related genes. Our data suggests that interplay of core nodulation genes with different accessory sets, specific for different abiotic conditions, function to establish the symbiosis. Together, our findings reveal a complex and dynamic interaction between plant, microbe, and environment.

Hydrogen Sulfide Promotes Nodulation and Nitrogen Fixation in Soybean–Rhizobia Symbiotic System

The rhizobium–legume symbiotic system is crucial for nitrogen cycle balance in agriculture. Hydrogen sulfide (H2S), a gaseous signaling molecule, may regulate various physiological processes in plants. However, whether H2S has regulatory effect in this symbiotic system remains unknown. Herein, we investigated the possible role of H2S in the symbiosis between soybean (Glycine max) and rhizobium (Sinorhizobium fredii). Our results demonstrated that an exogenous H2S donor (sodium hydrosulfide [NaHS]) treatment promoted soybean growth, nodulation, and nitrogenase (Nase) activity. Western blotting analysis revealed that the abundance of Nase component nifH was increased by NaHS treatment in nodules. Quantitative real-time polymerase chain reaction data showed that NaHS treatment upregulated the expressions of symbiosis-related genes nodA, nodC, and nodD of S. fredii. In addition, expression of soybean nodulation marker genes, including early nodulin 40 (GmENOD40), ERF required for nodulation (GmERN), nodulation signaling pathway 2b (GmNSP2b), and nodulation inception genes (GmNIN1a, GmNIN2a, and GmNIN2b), were upregulated. Moreover, the expressions of glutamate synthase (GmGOGAT), asparagine synthase (GmAS), nitrite reductase (GmNiR), ammonia transporter (GmSAT1), leghemoglobin (GmLb), and nifH involved in nitrogen metabolism were upregulated in NaHS-treated soybean roots and nodules. Together, our results suggested that H2S may act as a positive signaling molecule in the soybean–rhizobia symbiotic system and enhance the system’s nitrogen fixation ability.

Hydrogen sulfide promotes nodulation and nitrogen fixation in soybean-rhizobia symbiotic system

The rhizobium-legume symbiotic system is crucial for nitrogen cycle balance in agriculture. Hydrogen sulfide (H2S), a gaseous signaling molecule, may regulate various physiological processes in plants. However, whether H2S has regulatory effect in this symbiotic system remains unknown. Herein, we investigated the possible role of H2S in the symbiosis between soybean (Glycine max) and rhizobium (Sinorhizobium fredii). Our results demonstrated that exogenous H2S donor (sodium hydrosulfide, NaHS) treatment promoted soybean growth, nodulation and nitrogenase (Nase) activity. Western blotting analysis revealed that the abundance of nitrogenase component nifH was increased by NaHS treatment in nodules. Quantitative real-time PCR data showed that NaHS treatment up-regulated the expressions of symbiosis-related genesnodCandnodDofS. fredii. Besides, expression of soybean nodulation marker genes including early nodulin 40 (GmENOD40), ERF required for nodulation (GmERN), nodulation signaling pathway2b (GmNSP2b) and nodulation inception genes (GmNIN1a, GmNIN2aandGmNIN2b) were up-regulated. Moreover, the expressions of glutamate synthase (GmGS), nitrite reductase (GmNiR), ammonia transporter (GmSAT1), andnifHinvolved in nitrogen metabolism were up-regulated in NaHS-treated soybean roots and nodules. Together, our results suggested that H2S may act as a positive signaling molecule in soybean-rhizobia symbiotic system and enhance their nitrogen fixation ability.HighlightWe demonstrated for the first time that H2S as a signaling molecule may promote the establishment of symbiotic relationship and nitrogen fixation ability in the soybean-rhizobia symbiotic system.

Heterologous Expression of Rhizobial CelC2 Cellulase Impairs Symbiotic Signaling and Nodulation in Medicago truncatula

The infection of legume plants by rhizobia is tightly regulated to ensure accurate bacterial penetration, infection, and development of functionally efficient nitrogen-fixing root nodules. Rhizobial Nod factors (NF) have key roles in the elicitation of nodulation signaling. Infection of white clover roots also involves the tightly regulated specific breakdown of the noncrystalline apex of cell walls in growing root hairs, which is mediated by Rhizobium leguminosarum bv. trifolii cellulase CelC2. Here, we have analyzed the impact of this endoglucanase on symbiotic signaling in the model legume Medicago truncatula. Ensifer meliloti constitutively expressing celC gene exhibited delayed nodulation and elicited aberrant ineffective nodules, hampering plant growth in the absence of nitrogen. Cotreatment of roots with NF and CelC2 altered Ca2+ spiking in root hairs and induction of the early nodulin gene ENOD11. Our data suggest that CelC2 alters early signaling between partners in the rhizobia-legume interaction.