Naoko Ohkama-Ohtsu

OsNLP4 is a master regulator of nitrate signaling, but its ecological role in the root-associated bacterial communities is unknown. This study investigated how OsNLP4-mediated nitrate signaling influences root-associated microbial composition and root exudate metabolism. We found that the osnlp4 mutants exhibited distinct root-associated bacterial communities compared to the WT under non-nitrogen-fertilized conditions, with a significant enrichment of Bradyrhizobium and other bacterial taxa with potential roles in nitrogen cycling. Isolated Bradyrhizobium strains showed increased colonization in the osnlp4 mutant. Metabolomic analysis revealed increased secretion of sugars and phosphoric acid, along with a lower C:N ratio, in the osnlp4 mutant root exudates. Bacterial growth and chemotactic responses differed between WT- and osnlp4-derived root exudates, and sugars enriched in osnlp4 exudates elicited chemotactic responses in isolated Bradyrhizobium strains. These metabolic changes were associated with differential bacterial responses to WT and osnlp4 root exudates and may contribute to the altered root-associated bacterial community. Taken together, our results suggest that OsNLP4 links nitrate signaling and nitrogen metabolism with root-associated bacterial community assembly. Disruption of OsNLP4 reprograms root exudation and reshapes root-associated microbial communities, highlighting an association between nitrate signaling, nutrient allocation, and root-associated bacterial community assembly in rice.

Glutathione (GSH) is a central component of plant sulfur metabolism, best known for its roles in redox homeostasis, detoxification, and stress protection. Increasing evidence indicates that GSH also functions at the interface between primary and specialized sulfur metabolism. In plants, the best-characterized examples are concentrated in a limited number of lineages, particularly Brassicaceae and Allium species, where GSH is recruited into glucosinolate, camalexin, and S-alk(en)ylcysteine sulfoxide biosynthesis. Emerging observations further highlight the contribution of GSH degradation enzymes to sulfur transfer during specialized metabolite formation, linking GSH turnover to broader sulfur allocation networks. Beyond biosynthesis, GSH conjugation plays critical roles during specialized metabolite degradation by reducing the reactivity of intermediates, shaping immune-related metabolic outcomes, and enabling sulfur recycling through coordinated pathways of GSH degradation and resynthesis. Comparable GSH-dependent strategies in microbes and animals underscore the evolutionary conservation of GSH as a versatile metabolic scaffold. Collectively, these findings support a revised view in which specialized metabolism is embedded within dynamic GSH-dependent sulfur fluxes rather than functioning as an isolated sulfur sink. This review aims to integrate recent advances that highlight the role of GSH in connecting primary and specialized sulfur metabolism in response to environmental and developmental cues.

Glutathione (GSH) is a tripeptide that plays essential roles in redox regulation and stress responses across organisms. In Escherichia coli , the GSH-specific γ-glutamyl cyclotransferase (ChaC) has been characterized biochemically, yet its physiological role remains unclear. Moreover, ChaC has been annotated as a regulator of the Na⁺/H⁺ antiporter ChaA based on its genomic association, although experimental evidence supporting this function is limited. In this study, we investigated whether chaC and its co-transcribed gene, chaB , are involved in sodium transport or GSH metabolism. Gene expression analyses revealed that chaA , cha B, and chaC are upregulated under salt stress. Functional analyses using deletion mutants showed that loss of chaA reduced salt tolerance, whereas deletion of chaB enhanced tolerance and decreased intracellular sodium levels. In contrast, deletion of chaC had no significant effect on salt tolerance or sodium accumulation. Overexpression of cha genes further indicated that chaA , but not chaB or chaC , contributed to salt tolerance. Importantly, overexpression of chaC significantly reduced intracellular GSH levels, whereas chaB overexpression had no effect. These results indicate that ChaC primarily functions in GSH degradation rather than in cation transport, and that ChaB does not participate in GSH metabolism. Our findings clarify the distinct physiological roles of ChaC and ChaB and provide new insight into bacterial physiology regarding GSH metabolism and ion transport in E. coli .

Bacillus pumilus TUAT1, a gram-positive and spore-forming plant growth-promoting rhizobacterium, has been utilized as a biofertilizer due to its robust ability as spores to withstand environmental stresses and ensure long-term viability. This study investigated the mechanisms underlying the plant growth-promoting effects of spores and vegetative cells. Elemental analyses revealed that endospores are enriched in carbon, calcium, and manganese, which contribute to their protective properties, while vegetative cells are richer in nitrogen and phosphorus. Notably, both viable and dead spores and vegetative cells promoted the growth of Setaria viridis in natural soil. Microbial community analysis showed that bacterial alpha diversity was not changed across treatments, whereas beta diversity varied significantly, forming distinctly separated groups influenced by planting and inoculation. Fungal community analysis exhibited increased alpha diversity due to Setaria planting but no significant effects from bacterial treatments. Enrichment of Bdellovibrio spp., Bacteriovorax spp., and Pseudomonas spp. in soil inoculated with viable and dead vegetative cells and spores highlighted the capability of indirect mechanisms through microbial interactions rather than direct nutrient supply from bacterial residues. We believe that bacterial inoculants, including dead cells, modulate soil microbial communities to enhance plant growth, emphasizing their potential in sustainable agriculture.

Soybeans have been cultivated in Poland for more than 140 years. However, Poland’s cold and water-deficient climatic conditions hinder soybean cultivation. Although the availability of suitable soybean varieties in Poland contributes to meeting the demand for soybean production, it is important to identify rhizobial inoculants in Polish soils suitable for soybean cultivation. In this study, we cultivated soybean varieties (Abelina, Merlin, and Sultana) grown in soils taken from four regions in Poland and isolated 330 strains from soybean root nodules. 16S rRNA gene sequencing identified 49 strains of highly stress-tolerant nodule-associated bacteria, including Bradyrhizobium, Rhizobium, Ensifer, Tardiphaga, and Ralstonia spp. Several isolates exhibited positive effects on soybean growth under cold and water-deficient conditions. In particular, the isolate Bradyrhizobium japonicum PSN49, which is phylogenetically similar to B. japonicum USDA 123, increased plant biomass and nodule formation in the soybean cultivar Abelina under abiotic stress conditions due to its high nitrogen-fixing activity. Whole-genome comparisons between PSN49 and other Bradyrhizobium strains revealed that trehalose biosynthesis genes and cold shock proteins contributed to cold stress tolerance. These findings and the strains identified in this study will enhance soybean production and deepen the understanding of the soybean–rhizobium relationship in Poland.

γ-Glutamyl peptidase 1 (GGP1) plays a dual role in primary and secondary sulfur metabolism in Arabidopsis thaliana . During glutathione (GSH) turnover, GGP1 hydrolyzes the isopeptide bond of GSH to degrade the tripeptide into Glu and Cys-Gly. During glucosinolate and camalexin biosynthesis, GGP1 processes GSH conjugates, which have a large substituent at the thiol side chain, by hydrolyzing the same isopeptide bond of γ-Glu. In the present study, we determined the crystal structures of the following GGP1 forms: ligand-free, Glu complex, covalent γ-Glu intermediate, and disulfide-linked S-S inactive forms. The intermediate structure, in which γ-Glu is covalently linked to the nucleophile C100, was trapped by mutating the catalytic His to Asn (H192N). In the Glu complex and γ-Glu intermediate structures, Glu bound to the S1 subsite is extensively recognized by several hydrogen bonds. The substrate recognition of the Cys-Gly moiety at the S1’ and S2’ subsites was revealed by modeling GSH in the active site. Mutational analysis indicated that R206 plays an important role in substrate binding by forming a salt bridge with Gly at the S2’ subsite. An open pocket is present beyond the thiol side chain of Cys in the S1’ subsite, which contributed to the dual activity of GGP1 toward GSH and GSH conjugates. The S-S inactive structure was obtained by soaking GGP1 crystals in Cys-Gly, and the catalytic cysteine (C100) partially formed a disulfide bond with a neighboring C154 residue. The partial inactivation of GGP1 in the presence of a pro-oxidant (Cys-Gly) has revealed its possible role in oxidative stress regulation in Arabidopsis .

Biofertilizers are promising technologies for achieving sustainable agriculture. However, high-temperature tolerance is a constraint that limits the function of microbial inoculants. To characterize the genetic changes responsible for the high-temperature tolerance of rhizobia, mutant screening was performed using Bradyrhizobium diazoefficiens USDA110. The wild-type cells were mutagenized with carbon-ion irradiation, and two mutant strains, designated M10 and M14, were obtained after a three-day heat-shock treatment at 43 °C. In particular, M14 showed superior growth at 36 °C, at which temperature growth of the wild type was extremely slow, whereas M14 grew more slowly than the wild type at 32 °C. Whole-genome sequencing revealed that M10 had seven point mutations, whereas M14 had eight point mutations together with a 1.27 Mb inversion. RNA sequencing showed that the number of differentially expressed genes greatly exceeded the actual number of induced mutations. In M14, a gene cluster associated with pyruvate metabolism was markedly downregulated, probably because of disjunction with the promoter region after inversion, and was considered to be the cause of the slow growth rate of M14 at 32 °C. Notably, transmembrane proteins, including porins, were enriched among the genes upregulated in both M10 and M14. M14 was confirmed to retain symbiotic functions with soybeans. These results indicate that high-temperature tolerance was conferred by random mutagenesis while the symbiotic functions of rhizobia was maintained.