Summary Though root architecture modifications may be critically important for improving phosphorus (P) efficiency in crops, the regulatory mechanisms triggering these changes remain unclear. In this study, we demonstrate that genotypic variation in GmEXPB2 expression is strongly correlated with root elongation and P acquisition efficiency, and enhancing its transcription significantly improves soybean yield in the field. Promoter deletion analysis was performed using 5′ truncation fragments (P1–P6) of GmEXPB2 fused with the GUS gene in soybean transgenic hairy roots, which revealed that the P1 segment containing three E‐box elements significantly enhances induction of gene expression in response to phosphate (Pi) starvation. Further experimentation demonstrated that GmPTF1, a basic‐helix‐loop‐helix transcription factor, is the regulatory factor responsible for the induction of GmEXPB2 expression in response to Pi starvation. In short, Pi starvation induced expression of GmPTF1, with the GmPTF1 product directly binding to the E‐box motif in the P1 region of the GmEXPB2 promoter. Plus, both GmPTF1 and GmEXPB2 highly expressed in lateral roots, and were significantly enhanced by P deficiency. Further work with soybean stable transgenic plants through RNA sequencing analysis showed that altering GmPTF1 expression significantly impacted the transcription of a series of cell wall genes, including GmEXPB2, and thereby affected root growth, biomass and P uptake. Taken together, this work identifies a novel regulatory factor, GmPTF1, involved in changing soybean root architecture partially through regulation of the expression of GmEXPB2 by binding the E‐box motif in its promoter region.

Background and Aims Efficient biological nitrogen fixation (BNF) requires leghaemoglobin (Lb) to modulate oxygen pressure in nodules. Excess N supply severely inhibits BNF through effects on Lb during nodulation. As yet, a systematic identification and characterization of Lb-encoding genes in soybean has not been reported. Methods The effects of N on BNF were studied in soybean plants inoculated with rhizobia and exposed to excess or low N availability in hydroponic cultures. To identify soybean Lb proteins, BLAST searches were performed on the Phytozome website. Bioinformatic analysis of identified GmLbs was then carried out to investigate gene structure, protein homology and phylogenetic relationships. Finally, quantitative real-time PCR was employed to analyse the expression patterns of soybean Lb genes in various tissues and in response to high N availability. Key Results Excess N significantly accelerated nodule senescence and the production of green Lb in nodules. In total, seven haemoglobin (Hb) genes were identified from the soybean genome, with these Hb genes readily split into two distinct clades containing predominantly symbiosis-associated or non-symbiotic Hb members. Expression analysis revealed that all of the symbiosis-associated Lbs except GmLb5 were specifically expressed in nodules, while the non-symbiotic GmHbs, GmHb1 and GmHb2, were predominantly expressed in leaves and roots, respectively. Among identified GmLbs, GmLb1–4 are the major Lb genes acting in soybean nodulation, and each one is also significantly suppressed by exposure to excess N. Conclusions Taken together, the results show that excess N inhibits BNF by reducing nodule formation, Lb concentration and nitrogenase activity. The characteristics of the entire Hb family were analysed, and we found that GmLb1–4 are closely associated with nodule development and N2 fixation. This works forms the basis for further investigations of the role of Lbs in soybean nodulation.

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Legume biological nitrogen fixation (BNF) is the most important N source in agricultural ecosystems. Nodule organogenesis from the primordia to the development of mature nodules with the ability to fix N 2 largely determines BNF capacity. However, nodule growth is often limited by low phosphorus (P) availability, while the mechanisms underlying nodule development responses to P deficiency remain largely unknown. In this study, we found that nodule enlargement is severely inhibited by P deficiency, as reflected by the smaller individual nodule size from a soybean core collection in the field. Wide-ranging natural diversity in nodule size was further identified in soybeans reared in low P soils, with the FC-1 genotype outperforming FC-2 in assessments of nodulation under low P conditions. Among β-expansin members, GmINS1 expression is most abundantly enhanced by P deficiency in FC-1 nodules, and its transcript level is further displayed to be tightly associated with nodule enlargement. Four single nucleotide polymorphisms discovered in the GmINS1 promoter distinguished the FC-1 and FC-2 genotypes and accounted for the differential expression levels of GmINS1 responses to P deficiency. GmINS1 overexpression led to increases in nodule size, infected cell abundance, and N 2 fixation capacity and subsequently promoted increases in N and P content, soybean biomass, and yield. Our findings provide a candidate gene for optimizing BNF capacity responses to low P stress in soybean molecular breeding programs.

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