Overexpression of GmPAP4 Enhances Symbiotic Nitrogen Fixation and Seed Yield in Soybean under Phosphorus-Deficient Condition.

Legume crops establish symbiosis with nitrogen-fixing rhizobia for biological nitrogen fixation (BNF), a process that provides a prominent natural nitrogen source in agroecosystems; and efficient nodulation and nitrogen fixation processes require a large amount of phosphorus (P). Here, a role of GmPAP4, a nodule-localized purple acid phosphatase, in BNF and seed yield was functionally characterized in whole transgenic soybean (Glycine max) plants under a P-limited condition. GmPAP4 was specifically expressed in the infection zones of soybean nodules and its expression was greatly induced in low P stress. Altered expression of GmPAP4 significantly affected soybean nodulation, BNF, and yield under the P-deficient condition. Nodule number, nodule fresh weight, nodule nitrogenase, APase activities, and nodule total P content were significantly increased in GmPAP4 overexpression (OE) lines. Structural characteristics revealed by toluidine blue staining showed that overexpression of GmPAP4 resulted in a larger infection area than wild-type (WT) control. Moreover, the plant biomass and N and P content of shoot and root in GmPAP4 OE lines were also greatly improved, resulting in increased soybean yield in the P-deficient condition. Taken together, our results demonstrated that GmPAP4, a purple acid phosphatase, increased P utilization efficiency in nodules under a P-deficient condition and, subsequently, enhanced symbiotic BNF and seed yield of soybean.

Allelic Variation in GmPAP14 Alters Gene Expression to Affect Acid Phosphatase Activity in Soybean.

Improvement in acid phosphatase (APase) activity is considered as an important approach to enhance phosphorus (P) utilization in crops. Here, GmPAP14 was significantly induced by low P (LP), and its transcription level in ZH15 (P efficient soybean) was higher than in NMH (P inefficient soybean) under LP conditions. Further analyses demonstrated that there were several variations in gDNA (G-GmPAP14Z and G-GmPAP14N) and the promoters (P-GmPAP14Z and P-GmPAP14N) of GmPAP14, which might bring about differential transcriptional levels of GmPAP14 in ZH15 and NMH. Histochemical staining measurements revealed that a stronger GUS signal was present in transgenic Arabidopsis with P-GmPAP14Z under LP and normal P (NP) conditions compared with the P-GmPAP14N plant. Functional research demonstrated that transgenic Arabidopsis with G-GmPAP14Z had a higher level of GmPAP14 expression than the G-GmPAP14N plant. Meanwhile, higher APase activity was also observed in the G-GmPAP14Z plant, which led to increases in shoot weight and P content. Additionally, validation of variation in 68 soybean accessions showed that varieties with Del36 displayed higher APase activities than the del36 plant. Thus, these results uncovered that allelic variation in GmPAP14 predominantly altered gene expression to influence APase activity, which provided a possible direction for research of this gene in plants.

GmSPX8, a nodule-localized regulator confers nodule development and nitrogen fixation under phosphorus starvation in soybean.

BACKGROUND: Biological nitrogen fixation (BNF) is an important nitrogen source for legume plants, and highly efficient nitrogen fixation requires sufficient phosphorus (P). However, the mechanism of maintaining nitrogen fixation of the legume nodules under low P concentration remains largely unknown. RESULTS: A nodule-localized SPX protein, GmSPX8, was discovered by transcriptome and functional analysis of its role in N2 fixation was characterized in soybean nodules. GmSPX8 was preferentially expressed in nodules and its expression was gradually increased during nodule development. And also the expression pattern was investigated using reporter gene ß-glucuronidase (GUS) driven by the promoter of GmSPX8. GmSPX8 was greatly induced and the GUS activity was increased by 12.2% under P deficiency. Overexpression of GmSPX8 in transgenic plants resulted in increased nodule number, nodule fresh weight and nitrogenase activity by 15.0%, 16.0%, 42.5%, subsequently leading to increased N and P content by 17.0% and 19.0%, while suppression of GmSPX8 showed significantly impaired nodule development and nitrogen fixation efficiency under low P stress. These data indicated that GmSPX8 conferred nodule development and nitrogen fixation under low P condition. By yeast two-hybrid screening, GmPTF1 was identified as a potential interacting protein of GmSPX8, which was further confirmed by BiFC, Y2H and pull down assay. Transcript accumulation of GmPTF1 and its downstream genes such as GmEXLB1 and EXPB2 were increased in GmSPX8 overexpressed transgenic nodules, and in the presence of GmSPX8, the transcriptional activity of GmPTF1 in yeast cells and tobacco leaves was greatly enhanced. CONCLUSIONS: In summary, these findings contribute novel insights towards the role of GmSPX8 in nodule development and nitrogen fixation partly through interacting with GmPTF1 in soybean under low P condition.

Genetic loci and causal genes for seed fatty acids accumulation across multiple environments and genetic backgrounds in soybean.

Soybean is a major oil crop in the world, and fatty acids are the predominant components for oil bio-synthesis and catabolism metabolisms and also are the most important energy resources for organisms. In view of this, two recombinant inbred line (RIL) populations (ZL-RIL and ZQ-RIL) and one natural population were evaluated for five individual seed fatty acid contents (palmitic acid, stearic acid, oleic acid, linoleic acid, and linolenic acid) under four different environments, simultaneously. In total, sixteen additive QTL clusters were identified in ZL-RIL population, and fifteen were stably expressed across multiple environments or had pleiotropic effects on various fatty acid contents. Furthermore, five and five of these 16 QTL clusters were verified in ZQ-RIL population and natural population, respectively. Among these consistent and stable QTL clusters, one QTL cluster controlling fatty acid on chromosome 5 with pleiotropic effect was identified under all of the environments in ZL-RIL and ZQ-RIL populations and also was validated in the natural population. Meanwhile, another stable QTL cluster was detected on chromosome 9 with pleiotropic effect under multiple environments in ZL-RIL population and was further verified by the natural population. More importantly, some causal genes, such as the genes on chromosome 9, involving in the fatty acid catabolism process were found in these stable QTL clusters, and some of them, such as Gm09G042000, Gm09G041500, and Gm09G047200 on chromosome 9, showed different expressions in ZL-RIL parents (Zheng92116 and Liaodou14) based on the transcriptome sequencing analysis at different seed developmental stages. Thus, the study results provided insights into the genetic basis and molecular markers for regulating seed fatty acid contents in soybean breeding program. Supplementary Information: The online version contains supplementary material available at 10.1007/s11032-021-01227-y.

GmPAP4, a novel purple acid phosphatase gene isolated from soybean (Glycine max), enhanced extracellular phytate utilization in Arabidopsis thaliana.

KEY MESSAGE: GmPAP4 , a novel plant PAP gene in soybean, has phytase activity. Over-expressing GmPAP4 can enhance Arabidopsis growth when phytate is the sole P source in culture. Phosphorus (P) is an important macronutrient for plant growth and development. However, most of the total P in soils is fixed into organic phosphate (Po). Purple acid phosphatase (PAP) can hydrolyze Po in the soil to liberate inorganic phosphate and enhance plant P utilization. We isolated a novel PAP gene, GmPAP4, from soybean (Glycine max). It had an open reading frame of 1,329 bp, encoding 442 amino acid residues. Sequence alignment and phylogenetics analysis indicated that GmPAP4 was similar to other plant PAPs with large molecular masses. Quantitative real-time PCR analysis showed that the induced expression of GmPAP4 was greater in P-efficient genotype Zhonghuang15 (ZH15) than in P-inefficient genotype Niumaohuang (NMH) during the periods of flowering (28-35 days post phytate stress; DPP) and pod formation (49-63 DPP). Moreover, peak expression, at 63 DPP, was about 3-fold higher in 'ZH15' than in 'NMH'. Sub-cellular localization showed that GmPAP4 might be on plasma membrane or in cytoplasm. Over-expressing GmPAP4 in Arabidopsis resulted in significant rises in P acquisition and utilization compared with the wild-type (WT). Under phytate condition, transgenic Arabidopsis plants showed increases of approximately 132.7 % in dry weight and 162.6 % in shoot P content compared with the WT. Furthermore, when phytate was added as the sole P source in cultures, the activity of acid phosphatase was significantly higher in transgenic plants. Therefore, GmPAP4 is a novel PAP gene that functions in plant's utilization of organic phosphate especially under phytate condition.