Oceanobacillus picturae alleviates cadmium stress and promotes growth in soybean seedlings.

Cadmium (Cd) is a heavy metal that significantly impacts human health and the environment. Microorganisms play a crucial role in reducing heavy metal stress in plants; however, the mechanisms by which microorganisms enhance plant tolerance to Cd stress and the interplay between plants and microorganisms under such stress remain unclear. In this study, Oceanobacillus picturae (O. picturae) was isolated for interaction with soybean seedlings under Cd stress. Results indicated that Cd treatment alone markedly inhibited soybean seedling growth. Conversely, inoculation with O. picturae significantly improved growth indices such as plant height, root length, and fresh weight, while also promoting recovery in soil physiological indicators and pH. Metabolomic and transcriptomic analyses identified 157 genes related to aspartic acid, cysteine, and flavonoid biosynthesis pathways. Sixty-three microbial species were significantly associated with metabolites in these pathways, including pathogenic, adversity-resistant, and bioconductive bacteria. This research experimentally demonstrates, for the first time, the growth-promoting effect of the O. picturae strain on soybean seedlings under non-stress conditions. It also highlights its role in enhancing root growth and reducing Cd accumulation in the roots under Cd stress. Additionally, through the utilization of untargeted metabolomics, metagenomics, and transcriptomics for a multi-omics analysis, we investigated the impact of O. picturae on the soil microbiome and its correlation with differential gene expression in plants. This innovative approach unveils the molecular mechanisms underlying O. picturae's promotion of root growth and adaptation to Cd stress.

Integrated Transcriptomic and Proteomic Characterization of a Chromosome Segment Substitution Line Reveals the Regulatory Mechanism Controlling the Seed Weight in Soybean.

Soybean is the major global source of edible oils and vegetable proteins. Seed size and weight are crucial traits determining the soybean yield. Understanding the molecular regulatory mechanism underlying the seed weight and size is helpful for improving soybean genetic breeding. The molecular regulatory pathways controlling the seed weight and size were investigated in this study. The 100-seed weight, seed length, seed width, and seed weight per plant of a chromosome segment substitution line (CSSL) R217 increased compared with those of its recurrent parent 'Suinong14' (SN14). Transcriptomic and proteomic analyses of R217 and SN14 were performed at the seed developmental stages S15 and S20. In total, 2643 differentially expressed genes (DEGs) and 208 differentially accumulated proteins (DAPs) were detected at S15, and 1943 DEGs and 1248 DAPs were detected at S20. Furthermore, integrated transcriptomic and proteomic analyses revealed that mitogen-activated protein kinase signaling and cell wall biosynthesis and modification were potential pathways associated with seed weight and size control. Finally, 59 candidate genes that might control seed weight and size were identified. Among them, 25 genes were located on the substituted segments of R217. Two critical pathways controlling seed weight were uncovered in our work. These findings provided new insights into the seed weight-related regulatory network in soybean.

Natural variation in Fatty Acid 9 is a determinant of fatty acid and protein content.

Soybean is one of the most economically important crops worldwide and an important source of unsaturated fatty acids and protein for the human diet. Consumer demand for healthy fats and oils is increasing, and the global demand for vegetable oil is expected to double by 2050. Identification of key genes that regulate seed fatty acid content can facilitate molecular breeding of high-quality soybean varieties with enhanced fatty acid profiles. Here, we analysed the genetic architecture underlying variations in soybean seed fatty acid content using 547 accessions, including mainly landraces and cultivars from northeastern China. Through fatty acid profiling, genome re-sequencing, population genomics analyses, and GWAS, we identified a SEIPIN homologue at the FA9 locus as an important contributor to seed fatty acid content. Transgenic and multiomics analyses confirmed that FA9 was a key regulator of seed fatty acid content with pleiotropic effects on seed protein and seed size. We identified two major FA9 haplotypes in 1295 resequenced soybean accessions and assessed their phenotypic effects in a field planting of 424 accessions. Soybean accessions carrying FA9H2 had significantly higher total fatty acid contents and lower protein contents than those carrying FA9H1 . FA9H2 was absent in wild soybeans but present in 13% of landraces and 26% of cultivars, suggesting that it may have been selected during soybean post-domestication improvement. FA9 therefore represents a useful genetic resource for molecular breeding of high-quality soybean varieties with specific seed storage profiles.

Meta-Analysis and Multiomics of a Chromosome Segment Substitution Line Reveal Candidate Genes Associated with Seed Hardness in Soybean.

Soybean seed hardness is a key trait that influences planting, nutritional quality, and postharvest processing, but its genetic and molecular mechanisms remain to be clarified. We used meta-analysis to detect 17 meta-quantitative trait locus (QTLs) for soybean seed hardness. We then identified a hard-seeded chromosome segment substitution line, R75, with fragments introduced from hard-seeded wild germplasm in four of the meta-QTL intervals. Observations of the seed coat ultrastructure revealed thicker palisade tissue in R75 than in its soft-seeded recurrent parent. Transcriptomics and proteomics of R75 and its recurrent parent revealed multiple candidate genes associated with seed hardness. Fifty-seven were located on homozygous introduced fragments, 26 in meta-QTL intervals, and one in both (Glyma.02G268600). Five initial candidates were selected for KASP marker development on the basis of their predicted functions and nonsynonymous SNPs. The selection efficiency of the markers was as high as 90% for nonhard lines and 43% for hard lines in the chromosome segment substitution line (CSSL) population.

The impact of GmTSA and GmALS on soybean salt tolerance: uncovering the molecular landscape of amino acid and secondary metabolism pathways.

KEY MESSAGE: GmTSA and GmALS were screened out for salt stress in soybean and explore the poteintial amino acid secondary metabolism pathways. Soybean (Glycine max L.) is an oil and protein crop of global importance, and salinity has significant effects on soybean growth. Here, a population of soybean chromosome segment substitution lines was screened to identify highly salt-tolerant lines. In total, 24 quantitative trait loci (QTLs) on seven chromosomes were associated with salt tolerance, and CSSL_R71 was selected for further analysis. Although numerous genes were differentially expressed in CSSL_R71 in response to salt statically no differently, transcript levels of classical salt-response genes, including those of the salt overly sensitive pathway. Rather, salt tolerance in CSSL_R71 was associated with changes in amino acid and lipid metabolism. In particular, changes in p-coumaric acid, shikimic acid, and pyrrole-2-carboxylic acid levels accompanied salt tolerance in CSSL_R71. Eleven differentially expressed genes (DEGs) related to amino acid and secondary metabolism were identified as candidate genes on the substituted chromosome fragment. Six of these showed differences in coding sequence between the parental genotypes. Crucially, overexpression of GmTSA (Glyma.03G158400, tryptophan synthase) significantly enhanced salt tolerance in soybean hairy roots, whereas overexpression of GmALS (Glyma.13G241000, acetolactate synthase) decreased salt tolerance. Two KASP markers were developed for GmALS and used to genotype salt-tolerant and salt-sensitive lines in the CSSL population. Non-synonymous mutations were directly associated with salt tolerance. Taken together, these data provide evidence that changes in amino acid and secondary metabolism have the potential to confer salt tolerance in soybean.

An analysis of differentially expressed and differentially m6A-modified transcripts in soybean roots treated with lead.

Lead is one of the most common toxic heavy metal pollutants in nature, and exposure to lead can cause serious toxicity to many organisms. In this study, we collected root growth data from soybean plants exposed to lead for seven days and confirmed that lead significantly inhibited root growth. We performed a transcriptome-wide m6A methylation analysis to study the response of soybean RNA methylation groups to lead. The m6A modified regions were enriched near the 3'UTR region and stop codon, and m6A methylation was positively correlated with transcript abundance. In the presence of lead, the transcriptome range of m6A RNA methylation peaks increased, and we identified 1144 m6A modification peaks and 1094 differentially expressed genes. The integration of m6A methylation and transcriptomic results enabled us to identify 16 candidate genes whose transcripts were differentially methylated and differentially expressed under lead stress. Annotation results suggest that these genes may promote abiotic stress tolerance by impacting lead uptake, transport, and accumulation through ROS pathways, enzymes, transporters, and hormones. These results provide candidate genes for future studies of lead stress tolerance mechanisms in soybean roots and provide genetic resources for studying plant heavy metal stress in soybean breeding.

Changes in the m6A RNA methylome accompany the promotion of soybean root growth by rhizobia under cadmium stress.

Cadmium (Cd) is the most widely distributed heavy metal pollutant in soil and has significant negative effects on crop yields and human health. Rhizobia can enhance soybean growth in the presence of heavy metals, and the legume-rhizobia symbiosis has been used to promote heavy-metal phytoremediation, but much remains to be learned about the molecular networks that underlie these effects. Here, we demonstrated that soybean root growth was strongly suppressed after seven days of Cd exposure but that the presence of rhizobia largely eliminated this effect, even prior to nodule development. Moreover, rhizobia did not appear to promote root growth by limiting plant Cd uptake: seedlings with and without rhizobia had similar root Cd concentrations. Previous studies have demonstrated a role for m6A RNA methylation in the response of rice and barley to Cd stress. We therefore performed transcriptome-wide m6A methylation profiling to investigate changes in the soybean RNA methylome in response to Cd with and without rhizobia. Here, we provide some of the first data on transcriptome-wide m6a RNA methylation patterns in soybean; m6A modifications were concentrated at the 3' UTR of transcripts and showed a positive relationship with transcript abundance. Transcriptome-wide m6A RNA methylation peaks increased in the presence of Cd, and the integration of m6A methylome and transcriptome results enabled us to identify 154 genes whose transcripts were both differentially methylated and differentially expressed in response to Cd stress. Annotation results suggested that these genes were associated with Ca2+ homeostasis, ROS pathways, polyamine metabolism, MAPK signaling, hormones, and biotic stress responses. There were 176 differentially methylated and expressed transcripts under Cd stress in the presence of rhizobia. In contrast to the Cd-only gene set, they were also enriched in genes related to auxin, jasmonic acid, and brassinosteroids, as well as abiotic stress tolerance. They contained fewer genes related to Ca2+ homeostasis and also included candidates with known functions in the legume-rhizobia symbiosis. These findings offer new insights into how rhizobia promote soybean root growth under Cd stress; they provide candidate genes for research on plant heavy metal responses and for the use of legumes in phytoremediation.

Transcriptome-wide N6-methyladenosine (m6A) methylation in soybean under Meloidogyne incognita infection.

N6-methyladenosine (m6A) is a reversible epigenetic modification of mRNA and other RNAs that plays a significant role in regulating gene expression and biological processes. However, m6A abundance, dynamics, and transcriptional regulatory mechanisms remain unexplored in the context of soybean resistance to Meloidogyne incognita. In this study, we performed a comparative analysis of transcriptome-wide m6A and metabolome profiles of soybean root tissues with and without M. incognita infection. Global m6A hypermethylation was widely induced in response to M. incognita infection and was enriched around the 3' end of coding sequences and in 3' UTR regions. There were 2069 significantly modified m6A sites, 594 differentially expressed genes, and 103 differentially accumulated metabolites between infected and uninfected roots, including coumestrol, psoralidin, and 2-hydroxyethylphosphonate. Among 101 m6A-modified DEGs, 34 genes were hypomethylated and upregulated, and 39 genes were hypermethylated and downregulated, indicating a highly negative correlation between m6A methylation and gene transcript abundance. A number of these m6A-modified DEGs, including WRKY70, ERF60, POD47 and LRR receptor-like serine/threonine-protein kinases, were involved in plant defense responses. Our study provides new insights into the critical role of m6A modification in early soybean responses to M. incognita. Supplementary Information: The online version contains supplementary material available at 10.1007/s42994-022-00077-2.

Construction of Chromosome Segment Substitution Lines and Inheritance of Seed-Pod Characteristics in Wild Soybean.

Genetic populations provide the basis for genetic and genomic research, and chromosome segment substitution lines (CSSLs) are a powerful tool for the fine mapping of quantitative traits, new gene mining, and marker-assisted breeding. In this study, 213 CSSLs were obtained by self-crossing, backcrossing, and marker-assisted selection between cultivated soybean (Glycine max [L.] Merr.) variety Suinong14 (SN14) and wild soybean (Glycine soja Sieb. et Zucc.) ZYD00006. The genomes of these 213 CSSLs were resequenced and 580,524 single-nucleotide polymorphism markers were obtained, which were divided into 3,780 bin markers. The seed-pod-related traits were analyzed by quantitative trait locus (QTL) mapping using CSSLs. A total of 170 QTLs were detected, and 32 QTLs were detected stably for more than 2 years. Through epistasis analysis, 955 pairs of epistasis QTLs related to seed-pod traits were obtained. Furthermore, the hundred-seed weight QTL was finely mapped to the region of 64.4 Kb on chromosome 12, and Glyma.12G088900 was identified as a candidate gene. Taken together, a set of wild soybean CSSLs was constructed and upgraded by a resequencing technique. The seed-pod-related traits were studied by bin markers, and a candidate gene for the hundred-seed weight was finely mapped. Our results have revealed the CSSLs can be an effective tool for QTL mapping, epistatic effect analysis, and gene cloning.

Multi-Omics Techniques for Soybean Molecular Breeding.

Soybean is a major crop that provides essential protein and oil for food and feed. Since its origin in China over 5000 years ago, soybean has spread throughout the world, becoming the second most important vegetable oil crop and the primary source of plant protein for global consumption. From early domestication and artificial selection through hybridization and ultimately molecular breeding, the history of soybean breeding parallels major advances in plant science throughout the centuries. Now, rapid progress in plant omics is ushering in a new era of precision design breeding, exemplified by the engineering of elite soybean varieties with specific oil compositions to meet various end-use targets. The assembly of soybean reference genomes, made possible by the development of genome sequencing technology and bioinformatics over the past 20 years, was a great step forward in soybean research. It facilitated advances in soybean transcriptomics, proteomics, metabolomics, and phenomics, all of which paved the way for an integrated approach to molecular breeding in soybean. In this review, we summarize the latest progress in omics research, highlight novel findings made possible by omics techniques, note current drawbacks and areas for further research, and suggest that an efficient multi-omics approach may accelerate soybean breeding in the future. This review will be of interest not only to soybean breeders but also to researchers interested in the use of cutting-edge omics technologies for crop research and improvement.

SoySNP618K array: A high-resolution single nucleotide polymorphism platform as a valuable genomic resource for soybean genetics and breeding.

Innovations in genomics have enabled the development of low-cost, high-resolution, single nucleotide polymorphism (SNP) genotyping arrays that accelerate breeding progress and support basic research in crop science. Here, we developed and validated the SoySNP618K array (618,888 SNPs) for the important crop soybean. The SNPs were selected from whole-genome resequencing data containing 2,214 diverse soybean accessions; 29.34% of the SNPs mapped to genic regions representing 86.85% of the 56,044 annotated high-confidence genes. Identity-by-state analyses of 318 soybeans revealed 17 redundant accessions, highlighting the potential of the SoySNP618K array in supporting gene bank management. The patterns of population stratification and genomic regions enriched through domestication were highly consistent with previous findings based on resequencing data, suggesting that the ascertainment bias in the SoySNP618K array was largely compensated for. Genome-wide association mapping in combination with reported quantitative trait loci enabled fine-mapping of genes known to influence flowering time, E2 and GmPRR3b, and of a new candidate gene, GmVIP5. Moreover, genomic prediction of flowering and maturity time in 502 recombinant inbred lines was highly accurate (>0.65). Thus, the SoySNP618K array is a valuable genomic tool that can be used to address many questions in applied breeding, germplasm management, and basic crop research.

Identification of Finely Mapped Quantitative Trait Locus and Candidate Gene Mining for the Three-Seeded Pod Trait in Soybean.

The three-seeded pod number is an important trait that positively influences soybean yield. Soybean variety with increased three-seeded pod number contributes to the seed number/plant and higher yield. The candidate genes of the three-seeded pod may be the key for improving soybean yield. In this study, identification and validation of candidate genes for three-seeded pod has been carried out. First, a total of 36 quantitative trait locus (QTL) were detected from the investigation of recombinant inbred lines including 147 individuals derived from a cross between Charleston and Dongning 594 cultivars. Five consensus QTLs were integrated. Second, an introgressed line CSSL-182 carrying the target segment for the trait from the donor parent was selected to verify the consensus QTL based on its phenotype. Third, a secondary group was constructed by backcrossing with CSSL-182, and two QTLs were confirmed. There were a total of 162 genes in the two QTLs. The mining of candidate genes resulted in the annotation of eight genes with functions related to pod and seed sets. Finally, haplotype analysis and quantitative reverse transcriptase real-time PCR were carried to verify the candidate genes. Four of these genes had different haplotypes in the resource group, and the differences in the phenotype were highly significant. Moreover, the differences in the expression of the four genes during pod and seed development were also significant. These four genes were probably related to the development process underlying the three-seeded pod in soybean. Herein, we discuss the past and present studies related to the three-seeded pod trait in soybean.

Enhanced production of seed oil with improved fatty acid composition by overexpressing NAD+ -dependent glycerol-3-phosphate dehydrogenase in soybean.

There is growing interest in expanding the production of soybean oils (mainly triacylglycerol, or TAG) to meet rising feed demand and address global energy concerns. We report that a plastid-localized glycerol-3-phosphate dehydrogenase (GPDH), encoded by GmGPDHp1 gene, catalyzes the formation of glycerol-3-phosphate (G3P), an obligate substrate required for TAG biosynthesis. Overexpression of GmGPDHp1 increases soybean seed oil content with high levels of unsaturated fatty acids (FAs), especially oleic acid (C18:1), without detectably affecting growth or seed protein content or seed weight. Based on the lipidomic analyses, we found that the increase in G3P content led to an elevated diacylglycerol (DAG) pool, in which the Kennedy pathway-derived DAG was mostly increased, followed by PC-derived DAG, thereby promoting the synthesis of TAG containing relatively high proportion of C18:1. The increased G3P levels induced several transcriptional alterations of genes involved in the glycerolipid pathways. In particular, genes encoding the enzymes responsible for de novo glycerolipid synthesis were largely upregulated in the transgenic lines, in-line with the identified biochemical phenotype. These results reveal a key role for GmGPDHp1-mediated G3P metabolism in enhancing TAG synthesis and demonstrate a strategy to modify the FA compositions of soybean oils for improved nutrition and biofuel.

Fine mapping of a QTL locus (QNFSP07-1) and analysis of candidate genes for four-seeded pods in soybean.

Soybean [Glycine max (L.) Merr.] is an important grain and oil crop in the world, and it is the main source of high-quality protein. The number of four-seeded pods is a quantitative trait in soybean and is closely related to yield in terms of breeding. Therefore, it is of great significance to study the inheritance of four-seed pods and to excavate related genes for improving soybean yield. In this study, individuals with high ratio of four-seed pods which from chromosome segment substitution lines (CSSLs) that can be stably inherited were selected as the parent, and Suinong 14 (SN14) was used as recurrent parent to construct secondary mapping population via marker-assisted selection. From 2006 to 2017, QTL analysis was performed using secondary mapping populations, and the initial QTL mapping interval was 0.67 Mb and was located on Gm07. Based on the initial QTL mapping results, individuals that were heterozygous at the interval (36,116,118-37,399,738 bp) were screened in 2018, and the heterozygous individuals were subjected to inbreeding to obtain 13 F3 populations, with a target interval of 321 kb. Gene annotation was performed on the fine mapping interval, and 27 genes were obtained. Among 27 genes, Glyma.07G200900 and Glyma.07G201200 were identified as candidate genes. qRT-PCR was used to measure the expression of the 2 candidate genes at different developmental stages of soybean, and the expression levels of the 2 candidate genes in terms of cell division (axillary buds, COTs, EMs) were higher than those in terms of cell expansion (MM, LM), and these genes play a positive regulatory role in the formation of four-seeded pods. Haplotype analysis of 2 candidate genes which shows that Glyma.07G201200 has two excellent haplotypes, and the significance level between the two excellent haplotypes at p < 0.05. Those results provide the information for gene map-based cloning and molecular marker-assisted breeding of the number of four-seeded pod in soybean. Supplementary Information: The online version contains supplementary material available at 10.1007/s11032-021-01265-6.

Responses of Soybean Genes in the Substituted Segments of Segment Substitution Lines Following a Xanthomonas Infection.

Bacterial blight, which is one of the most common soybean diseases, is responsible for considerable yield losses. In this study, a novel Xanthomonas vasicola strain was isolated from the leaves of soybean plants infected with bacterial blight under field conditions. Sequencing the X. vasicola genome revealed type-III effector-coding genes. Moreover, the hrpG deletion mutant was constructed. To identify the soybean genes responsive to HrpG, two chromosome segment substitution lines (CSSLs) carrying the wild soybean genome, but with opposite phenotypes following Xanthomonas inoculations, were used to analyze gene expression networks based on RNA sequencing at three time points after inoculations with wild-type Xanthomonas or the hrpG deletion mutant. To further identify the hub genes underlying soybean responses to HrpG, the genes located on the substituted chromosome segments were examined. Finally, a combined analysis with the QTLs for resistance to Xanthomonas identified 35 hub genes in the substituted chromosomal segments that may help regulate soybean responses to Xanthomonas and HrpG. Furthermore, two candidate genes in the CSSLs might play pivotal roles in response to Xanthomonas.

QTL Mapping and Data Mining to Identify Genes Associated With the Sinorhizobium fredii HH103 T3SS Effector NopD in Soybean.

In some legume-rhizobium symbioses, host specificity is influenced by rhizobial type III effectors-nodulation outer proteins (Nops). However, the genes encoding host proteins that interact with Nops remain unknown. In this study, we aimed to identify candidate soybean genes associated with NopD, one of the type III effectors of Sinorhizobium fredii HH103. The results showed that the expression pattern of NopD was analyzed in rhizobia induced by genistein. We also found NopD can be induced by TtsI, and NopD as a toxic effector can induce tobacco leaf death. In 10 soybean germplasms, NopD played a positively effect on nodule number (NN) and nodule dry weight (NDW) in nine germplasms, but not in Kenjian28. Significant phenotype of NN and NDW were identified between Dongnong594 and Charleston, Suinong14 and ZYD00006, respectively. To map the quantitative trait locus (QTL) associated with NopD, a recombinant inbred line (RIL) population derived from the cross between Dongnong594 and Charleston, and chromosome segment substitution lines (CSSLs) derived from Suinong14 and ZYD00006 were used. Two overlapping conditional QTL associated with NopD on chromosome 19 were identified. Two candidate genes were identified in the confident region of QTL, we found that NopD could influence the expression of Glyma.19g068600 (FBD/LRR) and expression of Glyma.19g069200 (PP2C) after HH103 infection. Haplotype analysis showed that different types of Glyma.19g069200 haplotypes could cause significant nodule phenotypic differences, but Glyma.19g068600 (FBD/LRR) was not. These results suggest that NopD promotes S. fredii HH103 infection via directly or indirectly regulating Glyma.19g068600 and Glyma.19g069200 expression during the establishment of symbiosis between rhizobia and soybean plants.

Analysing the phenotype development of soybean plants using low-cost 3D reconstruction.

With the development of digital agriculture, 3D reconstruction technology has been widely used to analyse crop phenotypes. To date, most research on 3D reconstruction of field crops has been limited to analysis of population characteristics. Therefore, in this study, we propose a method based on low-cost 3D reconstruction technology to analyse the phenotype development during the whole growth period. Based on the phenotypic parameters extracted from the 3D reconstruction model, we identified the "phenotypic fingerprint" of the relevant phenotypes throughout the whole growth period of soybean plants and completed analysis of the plant growth patterns using a logistic growth model. The phenotypic fingerprint showed that, before the R3 period, the growth of the five varieties was similar. After the R5 period, the differences among the five cultivars gradually increased. This result indicates that the phenotypic fingerprint can accurately reveal the patterns of phenotypic changes. The logistic growth model of soybean plants revealed the time points of maximum growth rate of the five soybean varieties, and this information can provide a basis for developing guidelines for water and fertiliser application to crops. These findings will provide effective guidance for breeding and field management of soybean and other crops.

RNA Sequencing-Associated Study Identifies GmDRR1 as Positively Regulating the Establishment of Symbiosis in Soybean.

In soybean (Glycine max)-rhizobium interactions, the type III secretion system (T3SS) of rhizobium plays a key role in regulating host specificity. However, the lack of information on the role of T3SS in signaling networks limits our understanding of symbiosis. Here, we conducted an RNA sequencing analysis of three soybean chromosome segment substituted lines, one female parent and two derived lines with different chromosome-substituted segments of wild soybean and opposite nodulation patterns. By analyzing chromosome-linked differentially expressed genes in the substituted segments and quantitative trait loci (QTL)-assisted selection in the substituted-segment region, genes that may respond to type III effectors to mediate plant immunity-related signaling were identified. To narrow down the number of candidate genes, QTL assistant was used to identify the candidate region consistent with the substituted segments. Furthermore, one candidate gene, GmDRR1, was identified in the substituted segment. To investigate the role of GmDRR1 in symbiosis establishment, GmDRR1-overexpression and RNA interference soybean lines were constructed. The nodule number increased in the former compared with wild-type soybean. Additionally, the T3SS-regulated effectors appeared to interact with the GmDDR1 signaling pathway. This finding will allow the detection of T3SS-regulated effectors involved in legume-rhizobium interactions.

Genome-Wide Analysis of the Glucose-6-Phosphate Dehydrogenase Family in Soybean and Functional Identification of GmG6PDH2 Involvement in Salt Stress.

Glucose-6-phosphate dehydrogenase (G6PDH) is known as a critical enzyme responsible for nicotinamide adenine dinucleotide phosphate (NADPH) generation in the pentose phosphate pathway (PPP), and has an essential function in modulating redox homeostasis and stress responsiveness. In the present work, we characterized the nine members of the G6PDH gene family in soybean. Phylogenic analysis and transit peptide prediction showed that these soybean G6PDHs are divided into plastidic (P) and cytosolic (Cy) isoforms. The subcellular locations of five GmG6PDHs were further verified by confocal microscopy in Arabidopsis mesophyll protoplasts. The respective GmG6PDH genes had distinct expression patterns in various soybean tissues and at different times during seed development. Among them, the Cy-G6PDHs were strongly expressed in roots, developing seeds and nodules, while the transcripts of P-G6PDHs were mainly detected in green tissues. In addition, the activities and transcripts of GmG6PDHs were dramatically stimulated by different stress treatments, including salt, osmotic and alkali. Notably, the expression levels of a cytosolic isoform (GmG6PDH2) were extraordinarily high under salt stress and correlated well with the G6PDH enzyme activities, possibly implying a crucial factor for soybean responses to salinity. Enzymatic assay of recombinant GmG6PDH2 proteins expressed in Escherichia coli showed that the enzyme encoded by GmG6PDH2 had functional NADP+-dependent G6PDH activity. Further analysis indicated overexpression of GmG6PDH2 gene could significantly enhance the resistance of transgenic soybean to salt stress by coordinating with the redox states of ascorbic acid and glutathione pool to suppress reactive oxygen species generation. Together, these results indicate that GmG6PDH2 might be the major isoform for NADPH production in PPP, which is involved in the modulation of cellular AsA-GSH cycle to prevent the oxidative damage induced by high salinity.

Analysis of miRNAs Targeted Storage Regulatory Genes during Soybean Seed Development Based on Transcriptome Sequencing.

Soybeans are an important cash crop and are widely used as a source of vegetable protein and edible oil. MicroRNAs (miRNA) are endogenous small RNA that play an important regulatory role in the evolutionarily conserved system of gene expression. In this study, we selected four lines with extreme phenotypes, as well as high or low protein and oil content, from the chromosome segment substitution line (CSSL) constructed from suinong (SN14) and ZYD00006, and planted and sampled at three stages of grain development for small RNA sequencing and expression analysis. The sequencing results revealed the expression pattern of miRNA in the materials, and predicted miRNA-targeted regulatory genes, including 1967 pairs of corresponding relationships between known-miRNA and their target genes, as well as 597 pairs of corresponding relationships between novel-miRNA and their target genes. After screening and annotating genes that were targeted for regulation, five specific genes were identified to be differentially expressed during seed development and subsequently analyzed for their regulatory relationship with miRNAs. The expression pattern of the targeted gene was verified by Real-time Quantitative PCR (RT-qPCR). Our research provides more information about the miRNA regulatory network in soybeans and further identifies useful genes that regulate storage during soy grain development, providing a theoretical basis for the regulation of soybean quality traits.