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 genes nodC and nodD of S. 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, GmNIN2a and GmNIN2b) were up-regulated. Moreover, the expressions of glutamate synthase (GmGS), nitrite reductase (GmNiR), ammonia transporter (GmSAT1), and nifH involved 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.

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Hydrogen sulfide (H 2 S) is emerging as an important signalling molecule that regulates plant growth and abiotic stress responses. However, the roles of H 2 S in symbiotic nitrogen (N) assimilation and remobilization have not been characterized. Therefore, we examined how H 2 S influences the soybean (Glycine max)/rhizobia interaction in terms of symbiotic N fixation and mobilization during N deficiency-induced senescence. H 2 S enhanced biomass accumulation and delayed leaf senescence through effects on nodule numbers, leaf chlorophyll contents, leaf N resorption efficiency, and the N contents in different tissues. Moreover, grain numbers and yield were regulated by H 2 S and rhizobia, together with N accumulation in the organs, and N use efficiency. The synergistic effects of H 2 S and rhizobia were also demonstrated by effects on the enzyme activities, protein abundances, and gene expressions associated with N metabolism, and senescence-associated genes (SAGs) expression in soybeans grown under conditions of N deficiency. Taken together, these results show that H 2 S and rhizobia accelerate N assimilation and remobilization by regulation of the expression of SAGs during N deficiency-induced senescence. Thus, H 2 S enhances the vegetative and reproductive growth of soybean, presumably through interactions with rhizobia under conditions of N deficiency. K E Y W O R D S assimilation, hydrogen sulfide (H 2 S), nitrogen, remobilization, rhizobia, soybean (Glycine max)

Background: Hydrogen sulphide (H 2 S) is involved in regulating physiological processes in plants. We investigated how H 2 S ameliorates iron (Fe) deficiency in soybean (Glycine max L.) seedlings. Multidisciplinary approaches including physiological, biochemical and molecular, and transcriptome methods were used to investigate the H 2 S role in regulating Fe availability in soybean seedlings. Results: Our results showed that H 2 S completely prevented leaf interveinal chlorosis and caused an increase in soybean seedling biomass under Fe deficiency conditions. Moreover, H 2 S decreased the amount of root-bound apoplastic Fe and increased the Fe content in leaves and roots by regulating the ferric-chelate reductase (FCR) activities and Fe homeostasis-and sulphur metabolism-related gene expression levels, thereby promoting photosynthesis in soybean seedlings. In addition, H 2 S changed the plant hormone concentrations by modulating plant hormone-related gene expression abundances in soybean seedlings grown in Fe-deficient solution. Furthermore, organic acid biosynthesis and related genes expression also played a vital role in modulating the H 2 Smediated alleviation of Fe deficiency in soybean seedlings. Conclusion: Our results indicated that Fe deficiency was alleviated by H 2 S through enhancement of Fe acquisition and assimilation, thereby regulating plant hormones and organic acid synthesis in plants.

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