An pair of an atypical NLR encoding genes confer Asian soybean rust resistance in soybean.

Asian soybean rust (ASR), caused by Phakopsora pachyrhizi, is a devastating disease that is present in all major soybean-producing regions. The limited availability of resistant germplasm has resulted in a scarcity of commercial soybean cultivars that are resistant to the disease. To date, only the Chinese soybean landrace SX6907 has demonstrated an immune response to ASR. In this study, we present the isolation and characterization of Rpp6907-7 and Rpp6907-4, a gene pair that confer broad-spectrum resistance to ASR. Rpp6907-7 and Rpp6907-4 encode atypic nucleotide-binding leucine-rich repeat (NLR) proteins that are found to be required for NLR-mediated immunity. Genetic analysis shows that only Rpp6907-7 confers resistance, while Rpp6907-4 regulates Rpp6907-7 signaling activity by acting as a repressor in the absence of recognized effectors. Our work highlights the potential value of using Rpp6907 in developing resistant soybean cultivars.

RNA-Seq and Comparative Transcriptomic Analyses of Asian Soybean Rust Resistant and Susceptible Soybean Genotypes Provide Insights into Identifying Disease Resistance Genes.

Asian soybean rust (ASR), caused by Phakopsora pachyrhizi, is one of the most destructive foliar diseases that affect soybeans. Developing resistant cultivars is the most cost-effective, environmentally friendly, and easy strategy for controlling the disease. However, the current understanding of the mechanisms underlying soybean resistance to P. pachyrhizi remains limited, which poses a significant challenge in devising effective control strategies. In this study, comparative transcriptomic profiling using one resistant genotype and one susceptible genotype was performed under infected and control conditions to understand the regulatory network operating between soybean and P. pachyrhizi. RNA-Seq analysis identified a total of 6540 differentially expressed genes (DEGs), which were shared by all four genotypes. The DEGs are involved in defense responses, stress responses, stimulus responses, flavonoid metabolism, and biosynthesis after infection with P. pachyrhizi. A total of 25,377 genes were divided into 33 modules using weighted gene co-expression network analysis (WGCNA). Two modules were significantly associated with pathogen defense. The DEGs were mainly enriched in RNA processing, plant-type hypersensitive response, negative regulation of cell growth, and a programmed cell death process. In conclusion, these results will provide an important resource for mining resistant genes to P. pachyrhizi infection and valuable resources to potentially pyramid quantitative resistance loci for improving soybean germplasm.

Genome-Wide Identification of GmSPS Gene Family in Soybean and Expression Analysis in Response to Cold Stress.

Sucrose metabolism plays a critical role in development, stress response, and yield formation of plants. Sucrose phosphate synthase (SPS) is the key rate-limiting enzyme in the sucrose synthesis pathway. To date, genome-wide survey and comprehensive analysis of the SPS gene family in soybean (Glycine max) have yet to be performed. In this study, seven genes encoding SPS were identified in soybean genome. The structural characteristics, phylogenetics, tissue expression patterns, and cold stress response of these GmSPSs were investigated. A comparative phylogenetic analysis of SPS proteins in soybean, Medicago truncatula, Medicago sativa, Lotus japonicus, Arabidopsis, and rice revealed four families. GmSPSs were clustered into three families from A to C, and have undergone five segmental duplication events under purifying selection. All GmSPS genes had various expression patterns in different tissues, and family A members GmSPS13/17 were highly expressed in nodules. Remarkably, all GmSPS promoters contain multiple low-temperature-responsive elements such as potential binding sites of inducer of CBF expression 1 (ICE1), the central regulator in cold response. qRT-PCR proved that these GmSPS genes, especially GmSPS8/18, were induced by cold treatment in soybean leaves, and the expression pattern of GmICE1 under cold treatment was similar to that of GmSPS8/18. Further transient expression analysis in Nicotiana benthamiana and electrophoretic mobility shift assay (EMSA) indicated that GmSPS8 and GmSPS18 transcriptions were directly activated by GmICE1. Taken together, our findings may aid in future efforts to clarify the potential roles of GmSPS genes in response to cold stress in soybean.

Heterologous expression of the Glycine soja Kunitz-type protease inhibitor GsKTI improves resistance to drought stress and Helicoverpa armigera in transgenic Arabidopsis lines.

Kunitz-like protease inhibitors (KTIs) have been identified to play critical roles in insect defense, but evidence for their involvement in drought stress is sparse. The aim of this study was to identify and functionally characterize a Kunitz-like protease inhibitor, GsKTI, from the wild soybean (Glycine soja) variety ED059. Expression patterns suggest that drought stress and insect herbivory may induce GsKTI transcript levels. Transgenic Arabidopsis lines overexpressing GsKTI have been shown to exhibit enhanced drought tolerance by regulating the ABA signaling pathway and increasing xylem cell number. Transgenic Arabidopsis leaves overexpressing GsKTI interfered with insect digestion and thus had a negative effect on the growth of Helicoverpa armigera. It is concluded that GsKTI increases resistance to drought stress and insect attack in transgenic Arabidopsis lines.

The Bax inhibitor GmBI-1α interacts with a Nod factor receptor and plays a dual role in the legume-rhizobia symbiosis.

The gene networks surrounding Nod factor receptors that govern the symbiotic process between legumes and rhizobia remain largely unexplored. Here, we identify 13 novel GmNFR1α-associated proteins by yeast two-hybrid screening, and describe a potential interacting protein, GmBI-1α. GmBI-1α had the highest positive correlation with GmNFR1α in a co-expression network analysis, and its expression at the mRNA level in roots was enhanced by rhizobial infection. Moreover, GmBI-1α-GmNFR1α interaction was shown to occur in vitro and in vivo. The GmBI-1α protein was localized to multiple subcellular locations, including the endoplasmic reticulum and plasma membrane. Overexpression of GmBI-1α increased the nodule number in transgenic hairy roots or transgenic soybean, whereas down-regulation of GmBI-1α transcripts by RNA interference reduced the nodule number. In addition, the nodules in GmBI-1α-overexpressing plants became smaller in size and infected area with reduced nitrogenase activity. In GmBI-1α-overexpressing transgenic soybean, the elevated GmBI-1α also promoted plant growth and suppressed the expression of defense signaling-related genes. Infection thread analysis of GmBI-1α-overexpressing plants showed that GmBI-1α promoted rhizobial infection. Collectively, our findings support a GmNFR1α-associated protein in the Nod factor signaling pathway and shed new light on the regulatory mechanism of GmNFR1α in rhizobial symbiosis.

CRISPR/Cas9-Mediated Targeted Mutagenesis of GmUGT Enhanced Soybean Resistance Against Leaf-Chewing Insects Through Flavonoids Biosynthesis.

Leaf-chewing insects are important pests that cause yield loss and reduce seed quality in soybeans (Glycine max). Breeding soybean varieties that are resistant to leaf-chewing insects can minimize the need for insecticide use and reduce yield loss. The marker gene for QTL-M, Glyma.07g110300 (LOC100775351) that encodes a UDP-glycosyltransferase (UGT) is the major determinant of resistance against leaf-chewing insects in soybean; it exhibits a loss of function in insect-resistant soybean germplasms. In this study, Agrobacterium-mediated transformation introduced the CRISPR/Cas9 expression vector into the soybean cultivar Tianlong No. 1 to generate Glyma.07g110300-gene mutants. We obtained two novel types of mutations, a 33-bp deletion and a single-bp insertion in the GmUGT coding region, which resulted in an enhanced resistance to Helicoverpa armigera and Spodoptera litura. Additionally, overexpressing GmUGT produced soybean varieties that were more sensitive to H. armigera and S. litura. Both mutant and overexpressing lines exhibited no obvious phenotypic changes. The difference in metabolites and gene expression suggested that GmUGT is involved in imparting resistance to leaf-chewing insects by altering the flavonoid content and expression patterns of genes related to flavonoid biosynthesis and defense. Furthermore, ectopic expression of the GmUGT gene in the ugt72b1 mutant of Arabidopsis substantially rescued the phenotype of H. armigera resistance in the atugt72b1 mutant. Our study presents a strategy for increasing resistance against leaf-chewing insects in soybean through CRISPR/Cas9-mediated targeted mutagenesis of the UGT genes.

Identification of the Important Genes of Bradyrhizobium diazoefficiens 113-2 Involved in Soybean Nodule Development and Senescence.

Legume nodule development and senescence directly affect nitrogen fixation efficiency and involve a programmed series of molecular events. These molecular events are carried out synchronously by legumes and rhizobia. The characteristics and molecular mechanisms of nitrogen fixation at soybean important developmental stages play critical roles in soybean cultivation and fertilizer application. Although the gene expression of soybean were analyzed in nodules at five important soybean developmental stages, information on the expression of rhizobial genes in these nodule samples is limited. In the present study, we investigated the expression of Bradyrhizobium diazoefficiens 113-2 genes in the nodule samples from five developmental stages of soybean (Branching stage, flowering stage, fruiting stage, pod stage and harvest stage). Similar gene expression patterns of B. diazoefficiens 113-2 were existed during optimal symbiotic functioning, while different expression patterns were found among early nodule development, nitrogen fixation progress and nodule senescence. Besides, we identified 164 important different expression genes (DEGs) associated with nodule development and senescence. These DEGs included those encoding nod, nif, fix proteins and T3SS secretion system-related proteins, as well as proteins involved in nitrogen metabolism, ABC transporters and two-component system pathways. Gene Ontology, KEGG pathway and homology analysis of the identified DEGs revealed that most of these DEGs are uncharacterized genes associated with nodule development and senescence, and they are not core genes among the rhizobia genomes. Our results provide new clues for the understanding of the genetic determinants of soil rhizobia in nodule development and senescence, and supply theoretical basis for the creation of high efficiency soybean cultivation technology.

Suppression of LjBAK1-mediated immunity by SymRK promotes rhizobial infection in Lotus japonicus.

An important question in biology is how organisms can associate with different microbes that pose no threat (commensals), pose a severe threat (pathogens), and those that are beneficial (symbionts). The root nodule symbiosis serves as an important model system for addressing such questions in the context of plant-microbe interactions. It is now generally accepted that rhizobia can actively suppress host immune responses during the infection process, analogous to the way in which plant pathogens can evade immune recognition. However, much remains to be learned about the mechanisms by which the host recognizes the rhizobia as pathogens and how, subsequently, these pathways are suppressed to allow establishment of the nitrogen-fixing symbiosis. In this study, we found that SymRK (Symbiosis Receptor-like Kinase) is required for rhizobial suppression of plant innate immunity in Lotus japonicus. SymRK associates with LjBAK1 (BRASSINOSTEROID INSENSITIVE 1-Associated receptor Kinase 1), a well-characterized positive regulator of plant innate immunity, and directly inhibits LjBAK1 kinase activity. Rhizobial inoculation enhances the association between SymRK and LjBAK1 in planta. LjBAK1 is required for the regulation of plant innate immunity and plays a negative role in rhizobial infection in L. japonicus. The data indicate that the SymRK-LjBAK1 protein complex serves as an intersection point between rhizobial symbiotic signaling pathways and innate immunity pathways, and support that rhizobia may actively suppress the host's ability to mount a defense response during the legume-rhizobium symbiosis.

Characteristics and Research Progress of Legume Nodule Senescence.

Delaying the nodule senescence of legume crops can prolong the time of nitrogen fixation and attenuate the lack of fertilizer in the later stage of legume crop cultivation, resulting in improved crop yield and reduced usage of nitrogen fertilizer. However, effective measures to delay the nodule senescence of legume crops in agriculture are relatively lacking. In the present review, we summarized the structural and physiological characteristics of nodule senescence, as well as the corresponding detection methods, providing technical support for the identification of nodule senescence phenotype. We then outlined the key genes currently known to be involved in the regulation of nodule senescence, offering the molecular genetic information for breeding varieties with delayed nodule senescence. In addition, we reviewed various abiotic factors affecting nodule senescence, providing a theoretical basis for the interaction between molecular genetics and abiotic factors in the regulation of nodule senescence. Finally, we briefly prospected research foci of nodule senescence in the future.

Critical role of cysteine-266 of SIE3 in regulating the ubiquitination and degradation of SIP1 transcription factor in Lotus japonicus.

MAIN CONCLUSION: A conserved cysteine residue (C266)-mediated homo-dimerization of SIE3 is required for the ubiquitination and degradation of SIP1 transcription factor in Lotus japonicas CTLH/CRA/RING-containing proteins have been shown to possess E3-ligase activities and are crucial for the regulation of numerous cellular signaling pathways. In our previous studies, SIE3 (SymRK-Interacting E3 ubiquitin ligase), a CTLH/CRA/RING-containing protein from Lotus japonicus, has been shown to associate with both Symbiosis Receptor Kinase (SymRK) and SIP1 (SymRK interacting protein 1) transcription factor, and ubiquitinate SymRK (Yuan et al. Plant Physiol 160 (1):106-117, 2012; Feng et al. Front Plant Sci 11: 795, 2020). Besides, we previously also demonstrated that the residue, cysteine-266 in the CRA (CT11-RanBPM) domain is required for homodimerization of SIE3 and cysteine-266 residue-mediated homodimerization is important for the symbiosic function of SIE3 (Feng et al. 2020). In this report, SIE3 was shown to induce the ubiquitination and degradation of SIP1. The cysteine-266 residue is essential for the E3-ligase activity and is highly conserved in the SIE3-like proteins. Our works refined the working model that homodimerization of SIE3 is required for ubiquitin-related degradation of SIP1 and found a conserved cysteine residue plays a key role in the activity of a plant dimeric E3 ligase.

Genome-Wide Investigation and Expression Profiling Under Abiotic Stresses of a Soybean Unknown Function (DUF21) and Cystathionine-ß-Synthase (CBS) Domain-Containing Protein Family.

Cystathionine-ß-synthase (CBS) domain-containing proteins (CDCPs) constitute a large family in plants, and members of this family have been implicated in a variety of biological processes. However, the precise functions and the underlying mechanisms of most members of this family in plants remain to be elucidated. CBSDUF proteins belong to the CDCP superfamily, which contains one domain of unknown function (DUF21) and an N terminus that is adjacent to two intracellular CBS domains. In this study, a comprehensive genome database analysis of soybean was performed to investigate the role(s) of these CBSDUFs and to explore their nomenclature, classification, chromosomal distribution, exon-intron organization, protein structure, and phylogenetic relationships; the analysis identified a total of 18 putative CBSDUF genes. Using specific protein domains and phylogenetic analysis, the CBSDUF gene family was subdivided into eight groups. The soybean CBSDUF genes showed an uneven distribution on 12 chromosomes of Glycine max. RNA-seq transcriptome data from different tissues in public databases revealed tissue-specific and differential expression profiles of the GmCBSDUFs, and qPCR analysis revealed that certain groups of soybean CBSDUFs are likely involved in specific stress responses. In addition, GmCBSDUF3 transgenic Arabidopsis was subjected to phenotypic analysis under NaCl, PEG, and ABA stress treatments. The overexpression of GmCBSDUF3 could enhance tolerance to drought and salt stress in Arabidopsis. This study presents a first comprehensive look at soybean CBSDUF proteins and provides valuable resources for functionally elucidating this protein subgroup within the CBS domain-containing protein family.

Overexpression of GmMYB14 improves high-density yield and drought tolerance of soybean through regulating plant architecture mediated by the brassinosteroid pathway.

MYB transcription factors (TFs) have been reported to regulate the biosynthesis of secondary metabolites, as well as to mediate plant adaption to abiotic stresses, including drought. However, the roles of MYB TFs in regulating plant architecture and yield potential remain poorly understood. Here, we studied the roles of the dehydration-inducible GmMYB14 gene in regulating plant architecture, high-density yield and drought tolerance through the brassinosteroid (BR) pathway in soybean. GmMYB14 was shown to localize to nucleus and has a transactivation activity. Stable GmMYB14-overexpressing (GmMYB14-OX) transgenic soybean plants displayed a semi-dwarfism and compact plant architecture associated with decreased cell size, resulting in a decrease in plant height, internode length, leaf area, leaf petiole length and leaf petiole angle, and improved yield in high density under field conditions. Results of the transcriptome sequencing suggested the involvement of BRs in regulating GmMYB14-OX plant architecture. Indeed, GmMYB14-OX plants showed reduced endogenous BR contents, while exogenous application of brassinolide could partly rescue the phenotype of GmMYB14-OX plants. Furthermore, GmMYB14 was shown to directly bind to the promoter of GmBEN1 and up-regulate its expression, leading to reduced BR content in GmMYB14-OX plants. GmMYB14-OX plants also displayed improved drought tolerance under field conditions. GmBEN1 expression was also up-regulated in the leaves of GmMYB14-OX plants under polyethylene glycol treatment, indicating that the GmBEN1-mediated reduction in BR level under stress also contributed to drought/osmotic stress tolerance of the transgenic plants. Our findings provided a strategy for stably increasing high-density yield and drought tolerance in soybean using a single TF-encoding gene.

Whole-Genome Sequencing of Bradyrhizobium diazoefficiens 113-2 and Comparative Genomic Analysis Provide Molecular Insights Into Species Specificity and Host Specificity.

In the present study, we sequenced the complete genome of Bradyrhizobium diazoefficiens 113-2. The genomic characteristics of six selected rhizobial strains (two fast-growing rhizobia, two medium-slow-growing rhizobia and two slow-growing rhizobia) with four different legume hosts were analyzed by comparative genomic analysis. Genomes of B. diazoefficiens 113-2 and B. diazoefficiens USDA110 were found to share a large synteny blocks and a high ANI value, supporting 113-2 as a strain of B. diazoefficiens. 5,455 singletons and 11,656 clusters were identified among the six rhizobia genomes, and most of the pair-wise comparisons clusters were shared by the two genomes of strains in the same genus. Similar genus-specific gene numbers in the assigned COG functional terms were present in the two strains of the same genus, while the numbers were decreased with the increase of growth rate in most of the COG terms. KEGG pathway analysis of B. diazoefficiens 113-2 suggested that the rhizobial genes in ABC transporters and Two-Component system were mainly species-specific. Besides, the candidate genes related to secretion system and surface polysaccharides biosynthesis in the genomes of the six strains were explored and compared. 39 nodulation gene families, 12 nif gene families and 10 fix gene families in the genomes of these six strains were identified, and gene classes in most of gene families and the types and total gene numbers of gene families were substantially different among these six genomes. We also performed synteny analyses for above-mentioned nod, nif, and fix gene groupings, and selected NodW, NolK, NoeJ, NifB, FixK, and FixJ gene families to perform phylogeny analyses. Our results provided valuable molecular insights into species specificity and host specificity. The genetic information responsible for host specificity will play important roles in expanding the host range of rhizobia among legumes, which might provide new clues for the understanding of the genetic determinants of non-legume-rhizobium symbiosis.

Genome-wide survey of soybean papain-like cysteine proteases and their expression analysis in root nodule symbiosis.

BACKGROUND: Plant papain-like cysteine proteases (PLCPs) are a large class of proteolytic enzymes and play important roles in root nodule symbiosis (RNS), while the whole-genome studies of PLCP family genes in legume are quite limited, and the roles of Glycine max PLCPs (GmPLCPs) in nodulation, nodule development and senescence are not fully understood. RESULTS: In the present study, we identified 97 GmPLCPs and performed a genome-wide survey to explore the expansion of soybean PLCP family genes and their relationships to RNS. Nineteen paralogous pairs of genomic segments, consisting of 77 GmPLCPs, formed by whole-genome duplication (WGD) events were identified, showing a high degree of complexity in duplication. Phylogenetic analysis among different species showed that the lineage differentiation of GmPLCPs occurred after family expansion, and large tandem repeat segment were specifically in soybean. The expression patterns of GmPLCPs in symbiosis-related tissues and nodules identified RNS-related GmPLCPs and provided insights into their putative symbiotic functions in soybean. The symbiotic function analyses showed that a RNS-related GmPLCP gene (Glyma.04G190700) really participate in nodulation and nodule development. CONCLUSIONS: Our findings improved our understanding of the functional diversity of legume PLCP family genes, and provided insights into the putative roles of the legume PLCPs in nodulation, nodule development and senescence.

A GATA Transcription Factor from Soybean (Glycine max) Regulates Chlorophyll Biosynthesis and Suppresses Growth in the Transgenic Arabidopsis thaliana.

Chlorophyll plays an essential role in photosynthetic light harvesting and energy transduction in green tissues of higher plants and is closely related to photosynthesis and crop yield. Identification of transcription factors (TFs) involved in regulating chlorophyll biosynthesis is still limited in soybean (Glycine max), and the previously identified GmGATA58 is suggested to potentially modulate chlorophyll and nitrogen metabolisms, but its complete function is still unknown. In this study, subcellular localization assay showed that GmGATA58 was localized in the nucleus. Histochemical GUS assay and qPCR assay indicated that GmGATA58 was mainly expressed in leaves and responded to nitrogen, light and phytohormone treatments. Overexpression of GmGATA58 in the Arabidopsis thaliana ortholog AtGATA21 (gnc) mutant complemented the greening defect, while overexpression in Arabidopsis wild-type led to increasing chlorophyll content in leaves through up-regulating the expression levels of the large of chlorophyll biosynthetic pathway genes, but suppressing plant growth and yield, although the net photosynthetic rate was slightly improved. Dual-luciferase reporter assay also supported that GmGATA58 activated the transcription activities of three promoters of key chlorophyll biosynthetic genes of soybean in transformed protoplast of Arabidopsis. It is concluded that GmGATA58 played an important role in regulating chlorophyll biosynthesis, but suppressed plant growth and yield in transgenic Arabidopsis.

The Lotus japonicus Ubiquitin Ligase SIE3 Interacts With the Transcription Factor SIP1 and Forms a Homodimer.

The symbiosis receptor kinase SymRK plays an essential role in symbiotic signal transduction and nodule organogenesis. Several proteins bind to SymRK, but how the symbiosis signals are transduced from SymRK to downstream components remains elusive. We previously demonstrated that both SymRK interacting protein 1 (SIP1, an ARID-type DNA-binding protein) and SymRK interacting E3 ligase [SIE3, a RING (Really Interesting New Gene)-containing E3 ligase] interact with SymRK to regulate downstream cellular responses in Lotus japonicus during the legume-rhizobia symbiosis. Here, we show that SIE3 interacts with SIP1 in both yeast cells and Nicotiana benthamiana. SIE3 associated with itself and formed a homodimer. The cysteine 266 residue was found to be essential for SIE3 dimerization and for promoting nodulation in transgenic hairy roots of L. japonicus. Our findings provide a foundation for further investigating the regulatory mechanisms of the SymRK-mediated signaling pathway, as well as the biological function of E3 ligase dimerization in nodule organogenesis.

Identification of soybean drought-tolerant genotypes and loci correlated with agronomic traits contributes new candidate genes for breeding.

KEY MESSAGE: Drought tolerance level of 136 soybean genotypes, the correlations among traits were evaluated, and several important drought-tolerant genotypes, traits, SNPs and genes were possibly useful for soybean genetic breeding. Drought is an adverse environmental factor affecting crops growth, development, and yield. Promising genotypes and genes with improved tolerance to drought are probably effective ways to alleviate the situation. In this study, our main task was to determine drought tolerance level of 136 soybean genotypes, the correlations among physiological and agronomic traits under drought, and drought-tolerant single nucleotide polymorphism (SNPs) and genes. In this study, twenty-six varieties were identified as excellent tolerant genotypes to stress among which S14, S93 and S135 with high drought-tolerant index (DTI > 1.3) and yield (Y > 300 kg). Fourteen varieties were identified as drought-sensitive genotypes, such as S25, S45 and S58, with low drought-tolerant index (DTI < 0.5). 422 SNPs and 302 genes correlated with seed number per plant (SNPP), maturity (M), number of seeds per pod (NSPP), node number of main stem (NNMS), Stem diameter (SD) and pull stem (PS) were detected under well-watered and drought conditions by genome-wide association study (GWAS). Among them, we found SNPs (Chr 3:1758920-1958934) between drought-tolerant and sensitive genotypes. 13 genes (Glyma.03G017800, Glyma.03G018000, Glyma.03G018200, Glyma.03G018400, Glyma.03G018500, Glyma.03G018600, Glyma.03G018700, Glyma.03G018800, Glyma.03G018900, Glyma.03G019000, Glyma.03G019100, Glyma.03G019200, Glyma.03G019300) correlated with NNMS were detected. By qRT-PCR, the expression level of Glyma.03G018000 and Glyma.03G018900 in drought-tolerant varieties was significantly increased, but low or no expression in sensitive varieties under drought stress. This study provides important drought-tolerant genotypes, traits, SNPs and potential genes, possibly useful for soybean genetic breeding.

CRISPR/Cas9-mediated targeted mutagenesis of GmSPL9 genes alters plant architecture in soybean.

BACKGROUND: The plant architecture has significant effects on grain yield of various crops, including soybean (Glycine max), but the knowledge on optimization of plant architecture in order to increase yield potential is still limited. Recently, CRISPR/Cas9 system has revolutionized genome editing, and has been widely utilized to edit the genomes of a diverse range of crop plants. RESULTS: In the present study, we employed the CRISPR/Cas9 system to mutate four genes encoding SQUAMOSA PROMOTER BINDING PROTEIN-LIKE (SPL) transcription factors of the SPL9 family in soybean. These four GmSPL9 genes are negatively regulated by GmmiR156b, a target for the improvement of soybean plant architecture and yields. The soybean Williams 82 was transformed with the binary CRISPR/Cas9 plasmid, assembled with four sgRNA expression cassettes driven by the Arabidopsis thaliana U3 or U6 promoter, targeting different sites of these four SPL9 genes via Agrobacterium tumefaciens-mediated transformation. A 1-bp deletion was detected in one target site of the GmSPL9a and one target site of the GmSPL9b, respectively, by DNA sequencing analysis of two T0-generation plants. T2-generation spl9a and spl9b homozygous single mutants exhibited no obvious phenotype changes; but the T2 double homozygous mutant spl9a/spl9b possessed shorter plastochron length. In T4 generation, higher-order mutant plants carrying various combinations of mutations showed increased node number on the main stem and branch number, consequently increased total node number per plants at different levels. In addition, the expression levels of the examined GmSPL9 genes were higher in the spl9b-1 single mutant than wild-type plants, which might suggest a feedback regulation on the expression of the investigated GmSPL9 genes in soybean. CONCLUSIONS: Our results showed that CRISPR/Cas9-mediated targeted mutagenesis of four GmSPL9 genes in different combinations altered plant architecture in soybean. The findings demonstrated that GmSPL9a, GmSPL9b, GmSPL9c and GmSPL9 function as redundant transcription factors in regulating plant architecture in soybean.

GmSYP24, a putative syntaxin gene, confers osmotic/drought, salt stress tolerances and ABA signal pathway.

As major environment factors, drought or high salinity affect crop growth, development and yield. Transgenic approach is an effective way to improve abiotic stress tolerance of crops. In this study, we comparatively analyzed gene structures, genome location, and the evolution of syntaxin proteins containing late embryogenesis abundant (LEA2) domain. GmSYP24 was identified as a dehydration-responsive gene. Our study showed that the GmSYP24 protein was located on the cell membrane. The overexpression of GmSYP24 (GmSYP24ox) in soybean and heteroexpression of GmSYP24 (GmSYP24hx) in Arabidopsis exhibited insensitivity to osmotic/drought and high salinity. However, wild type soybean, Arabidopsis, and the mutant of GmSYP24 homologous gene of Arabidopsis were sensitive to the stresses. Under the abiotic stresses, transgenic soybean plants had greater water content and higher activities of POD, SOD compared with non-transgenic controls. And the leaf stomatal density and opening were reduced in transgenic Arabidopsis. The sensitivity to ABA was decreased during seed germination of GmSYP24ox and GmSYP24hx. GmSYP24hx induced up-regulation of ABA-responsive genes. GmSYP24ox alters the expression of some aquaporins under osmotic/drought, salt, or ABA treatment. These results demonstrated that GmSYP24 played an important role in osmotic/drought or salt tolerance in ABA signal pathway.