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.

Genome resequencing reveals genetic loci and genes conferring resistance to SMV-SC8 in soybean.

KEY MESSAGE: A soybean natural population genotyped by resequencing and another RIL population genotyped by SoySNP6K were used to explore consistent genetic loci and genes under greenhouse- and field-conditions for SMV-SC8 resistance. Soybean mosaic virus (SMV) is a member of the genus Potyvirus that occurs in all soybean-growing areas of the world and causes serious losses of yield and seed quality. In this study, a natural population composed of 209 accessions resequenced at an average depth of 18.44 × and another RIL population containing 193 lines were used to explore genetic loci and genes conferring resistance to SMV-SC8. There were 3030 SNPs significantly associated with resistance to SC8 on chromosome 13 in the natural population, among which 327 SNPs were located within an ~ 0.14 Mb region (from 28.46 to 28.60 Mb) of the major QTL qRsc8F in the RIL population. Two genes among 21 candidate genes, GmMACPF1 and GmRad60, were identified in the region of consistent linkage and association. Compared to the mock control, the changes in the expression of these two genes after inoculation with SC8 differed between resistant and susceptible accessions. More importantly, GmMACPF1 was shown to confer resistance to SC8 by significantly decreasing virus content in soybean hairy roots overexpressing this gene. A functional marker, FMSC8, was developed based on the allelic variation of GmMACPF1, and a high coincidence rate of 80.19% between the disease index and marker genotype was identified in the 419 soybean accessions. The results provide valuable resources for studies on the molecular mechanism of SMV resistance and genetic improvement in 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.

Identification of closely associated SNPs and candidate genes with seed size and shape via deep re-sequencing GWAS in soybean.

KEY MESSAGE: A soybean natural population was genotyped by deep re-sequencing and phenotyped for six seed size- and shape-related traits under six environments to identify closely associated SNPs and candidate genes. Seed size and shape are important determining factors for soybean yield formation, while their genetic basis and molecular mechanism are still largely unknown, which seriously constrains the increasing of soybean yield at present. In view of this, a natural population was genotyped via the deep re-sequencing technique (~ 20 ×) and phenotyped for six related traits under six environments. In total, 154 SNPs were closely associated with seed length across diverse environments, and 323, 483, 565, 394 and 2038 SNPs were closely associated with seed width, seed diameter, seed circumference, seed area and ratio of length to width under multiple environments. Moreover, 98.70%, 96.28%, 48.24%, 85.13%, 97.21% and 98.58% of them were further demonstrated by the BLUP and mean values of the related traits. Furthermore, 218 genes flanking the associated SNPs on chromosomes 6 and 10 were analyzed for DNA mutations and RNA expressions through SNP alleles and transcriptome data, simultaneously. The candidate genes, Glyma.10G035200 (Sn1-specific diacylglycerol lipase), Glyma.10G035400 (transcription factor) and Glyma.10G058200 (phenylalanine ammonia-lyase), were discovered to relate with the seed size and shape for their different DNA sequences or differential RNA expressions among soybean varieties at five seed developmental stages. Thus, these closely associated SNPs and related genes provide novel insights and useful information for the seed size and shape genetic basis dissection and breeding improvement in soybean.

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.

A Nodule-Localized Small Heat Shock Protein GmHSP17.1 Confers Nodule Development and Nitrogen Fixation in Soybean.

Small heat shock proteins (sHSPs) are ubiquitous proteins present in all organisms. The sHSPs are not only upregulated under heat shock as well as other stresses but also are expressed in unstressed cells, indicating quite diverse functions of sHSPs. However, there is little known about the role of sHSPs in nodulation and nitrogen fixation in soybean. In this study, we cloned a candidate protein of sHSP, GmHSP17.1, from proteome of nodule and analyzed its function in soybean nodulation. We found that GmHSP17.1 was a cytosolic protein and preferentially expressed during nodule development. An overexpression of GmHSP17.1 in composite transgenic plants showed increases in nodule number, fresh weight, nodule size, area of infection cells, and nitrogenase activity, and subsequently promoted the content of nitrogen and growth of soybean plants. While GmHSP17.1 RNA interference (RNAi) lines showed significantly impaired nodule development and nitrogen fixation efficiency. Through liquid chromatography-tandem mass spectrometry (LC-MS/MS), GmRIP1 was identified as the first potential target of GmHSP17.1, and was shown to be specifically expressed in soybean nodules. The interaction between GmHSP17.1 and GmRIP1 was further confirmed by yeast-two hybrid (Y2H), bimolecular fluorescence complementation (BiFC) in vivo and pull-down assay in vitro. Furthermore, peroxidase activity was markedly increased in GmHSP17.1 overexpressed nodules and decreased in RNAi lines. As a result, the reactive oxygen species (ROS) content greatly decreased in GmHSP17.1 overexpression lines and increased in suppression lines. Taken together, we conclude that GmHSP17.1 plays an important role in soybean nodulation through interacting with GmRIP1. Our results provide foundation for studying the mechanism of nitrogen fixation and for the genetics improvement of legume plants.

Discovery of genetic loci and causal genes for seed germination via deep re-sequencing in soybean.

High seed germination is crucial for mechanical sowing, seedling establishment, growth potential, multiple resistances, and the formation of yield and quality. However, few genetic loci and candidate genes conferring seed germination were explored in soybean at present. In view of this, a natural population containing 199 accessions was assessed for the germination potential (GP) and germination rate (GR) and also was re-sequenced at the average sequencing depth of 18.4 × for each accession. In total, 5,665,469 SNPs were obtained for association analysis, and 470 SNPs in 55 loci on 18 chromosomes were identified to associate with seed germination. Of them, 85 SNPs on chromosomes 1, 10, and 14 were associated with mean value and BLUP value for GP and GR, simultaneously. Moreover, 324 SNPs (68.9% of the total) in four loci were located on chromosome 14 for seed germination, of which 11 SNPs were located in the exons, 30 in introns, 17 in 5'UTR or 3'UTR, and 46 in upstream or downstream. Based on these, 131 candidate genes flanking the associated SNPs were analyzed for gene annotation, SNP mutation, and RNA expression, and three causal genes, Glyma.14G069800 (RNA-binding protein), Glyma.14G071400 (bZIP transcription factor), and Glyma.17G033200 (nucleic acid-binding protein), were screened out and might be responsible for the seed germination. The closely associated SNPs and causal genes provided an important resource and dissecting of genetic basis for seed germination improvement in soybean. Supplementary Information: The online version contains supplementary material available at 10.1007/s11032-022-01316-6.

A small heat shock protein, GmHSP17.9, from nodule confers symbiotic nitrogen fixation and seed yield in soybean.

Legume-rhizobia symbiosis enables biological nitrogen fixation to improve crop production for sustainable agriculture. Small heat shock proteins (sHSPs) are involved in multiple environmental stresses and plant development processes. However, the role of sHSPs in nodule development in soybean remains largely unknown. In the present study, we identified a nodule-localized sHSP, called GmHSP17.9, in soybean, which was markedly up-regulated during nodule development. GmHSP17.9 was specifically expressed in the infected regions of the nodules. GmHSP17.9 overexpression and RNAi in transgenic composite plants and loss of function in CRISPR-Cas9 gene-editing mutant plants in soybean resulted in remarkable alterations in nodule number, nodule fresh weight, nitrogenase activity, contents of poly ß-hydroxybutyrate bodies (PHBs), ureide and total nitrogen content, which caused significant changes in plant growth and seed yield. GmHSP17.9 was also found to act as a chaperone for its interacting partner, GmNOD100, a sucrose synthase in soybean nodules which was also preferentially expressed in the infected zone of nodules, similar to GmHSP17.9. Functional analysis of GmNOD100 in composite transgenic plants revealed that GmNOD100 played an essential role in soybean nodulation. The hsp17.9 lines showed markedly more reduced sucrose synthase activity, lower contents of UDP-glucose and acetyl coenzyme A (acetyl-CoA), and decreased activity of succinic dehydrogenase (SDH) in the tricarboxylic acid (TCA) cycle in nodules due to the missing interaction with GmNOD100. Our findings reveal an important role and an unprecedented molecular mechanism of sHSPs in nodule development and nitrogen fixation in soybean.

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.

GmPAP12 Is Required for Nodule Development and Nitrogen Fixation Under Phosphorus Starvation in Soybean.

Nodulation process in legume plants is essential for biological nitrogen fixation during which process a large amount of phosphorus (P) is required. Under P deficiency, nodule formation is greatly affected, and induction of purple acid phosphatases (PAPs) is an adaptive strategy for nodules to acquire more P. However, regulation roles of PAPs in nodules remain largely understood. In this study, by transcriptome sequencing technology, five PAP genes were found to be differentially expressed, which led to the greatly increased acid phosphatase (APase) and phytase activities in soybean mature nodules under P starvation conditions; and among the five PAP genes, GmPAP12 had the highest transcript level, and RT-PCR indicated expression of GmPAP12 was gradually increasing during nodule development. GUS activity driven by GmPAP12 promoter was also significantly induced in low phosphorus conditions. Further functional analysis showed that under low phosphorus stress, overexpression of GmPAP12 resulted in higher nodule number, fresh weight, and nitrogenase activity as well as the APase activity than those of control plant nodules, whereas the growth performance and APase activity of nodules on hairy roots were greatly lower when GmPAP12 was suppressed, indicating that GmPAP12 may promote P utilization in soybean nodules under low P stress, which thus played an important role in nodulation and biological nitrogen fixation. Moreover, P1BS elements were found in the promoter of GmPAP12, and yeast one-hybrid experiment further proved the binding of P1BS by transcription factor GmPHR1 in the promoter of GmPAP12. At last, overexpression and suppression of GmPHR1 in nodules indeed caused highly increased and decreased expression of GmPAP12, respectively, indicating that GmPAP12 is regulated by GmPHR1 in soybean nodules. Taken together, these data suggested that GmPAP12 was a novel soybean PAP involved in the P utilization and metabolism in soybean root nodules and played an important role in the growth and development of root nodules and biological nitrogen fixation.

GmEXLB1, a Soybean Expansin-Like B Gene, Alters Root Architecture to Improve Phosphorus Acquisition in Arabidopsis.

Expansins comprise four subfamilies, α-expansin (EXPA), ß-expansin (EXPB), expansin-like A (EXLA), and expansin-like B (EXLB), which are involved in the regulation of root development and growth under abiotic stress. To date, few EXLB genes have been shown to respond to low phosphorus (P) in plants. In this study, we identified an EXLB gene, GmEXLB1, by analyzing the transcription profiles of GmEXLBs in soybean. Quantitative analysis showed that GmEXLB1 was expressed and induced in the lateral roots of soybean under low P conditions. The observation of ß-glucuronidase staining in transgenic Arabidopsis suggested that GmEXLB1 might be associated with lateral root emergence. GmEXLB1 overexpression altered the root architecture of transgenic Arabidopsis by increasing the number and length of lateral roots and the length of primary roots under low P conditions. Additionally, the length of the elongation zone and the average cell length in the elongation zone were increased in transgenic Arabidopsis. Increases in biomass and P content suggested that GmEXLB1 overexpression enhanced P acquisition in Arabidopsis. Overall, we conclude that GmEXLB1 expression is induced in soybean under low P conditions, and the overexpression of GmEXLB1 improves P acquisition by regulating root elongation and architecture in Arabidopsis, which provides a possible direction for research of the function of this gene in soybean.

The Soybean Purple Acid Phosphatase GmPAP14 Predominantly Enhances External Phytate Utilization in Plants.

Induction and secretion of acid phosphatases (APases) is considered to be an important strategy for improving plant growth under conditions of low inorganic phosphate (Pi). Purple acid phosphatases (PAPs), are an important class of plant APases that could be secreted into the rhizosphere to utilize organic phosphorus (Po) for plant growth and development. To date, only a few members of the PAP family have been identified in soybean. In this paper, we identified a secreted PAP in soybean, GmPAP14, and investigated its role in utilizing external phytate, the main form of organic phosphorus in the soil. An analysis of its expression and promoter showed that GmPAP14 was mainly expressed in the root and was strongly induced following Po treatment, during which its expression expanded from meristematic to maturation zones and root hairs. In vitro enzyme assays indicated that GmPAP14 had a relatively high phytase activity. Furthermore, GmPAP14 overexpression increased secreted APase activities and phytase activities, leading to the improved use of external plant phytate, higher phosphorus content, and increased shoot weight. Thus, these results confirmed that GmPAP14 is an important gene induced in response to Po, and that it predominantly participates in utilizing external Po to enhance plant growth and development.

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.