Exploring the role of FBXL fbxl gene family in Soybean: Implications for plant height and seed size regulation.

F-box proteins constitute a significant family in eukaryotes and, as a component of the Skp1p-cullin-F-box complex, are considered critical for cellular protein degradation and other biological processes in plants. Despite their importance, the functions of F-box proteins, particularly those with C-terminal leucine-rich repeat (LRR) domains, remain largely unknown in plants. Therefore, the present study conducted genome-wide identification and in silico characterization of F-BOX proteins with C-terminal LRR domains in soybean (Glycine max L.) (GmFBXLs). A total of 45 GmFBXLs were identified. The phylogenetic analysis showed that GmFBXLs could be subdivided into ten subgroups and exhibited a close relationship with those from Arabidopsis thaliana, Cicer aretineum, and Medicago trunculata. It was observed that most cis-regulatory elements in the promoter regions of GmFBXLs are involved in hormone signalling, stress responses, and developmental stages. In silico transcriptome data illustrated diverse expression patterns of the identified GmFBXLs across various tissues, such as shoot apical meristem, flower, green pods, leaves, nodules, and roots. Overexpressing (OE) GmFBXL12 in Tianlong No.1 cultivar resulted in a significant difference in seed size, number of pods, and number of seeds per plant, indicated a potential increase in yield compared to wild type. This study offers valuable perspectives into the role of FBXLs in soybean, serving as a foundation for future research. Additionally, the identified OE lines represent valuable genetic resources for enhancing seed-related traits in soybean.

Identification of major quantitative trait loci and candidate genes for seed weight in soybean.

KEY MESSAGE: Four major quantitative trait loci for 100-seed weight were identified in a soybean RIL population under five environments, and the most likely candidate genes underlying these loci were identified. Seed weight is an important target of soybean breeding. However, the genes underlying the major quantitative trait loci (QTL) controlling seed weight remain largely unknown. In this study, a soybean population of 300 recombinant inbred lines (RILs) derived from a cross between PI595843 (PI) and WH was used to map the QTL and identify candidate genes for seed weight. The RIL population was genotyped through whole genome resequencing, and phenotyped for 100-seed weight under five environments. A total of 38 QTL were detected, and four major QTL, each explained at least 10% of the variation in 100-seed weight, were identified. Six candidate genes within these four major QTL regions were identified by analyses of their tissue expression patterns, gene annotations, and differential gene expression levels in soybean seeds during four developmental stages between two parental lines. Further sequence variation analyses revealed a C to T substitution in the first exon of the Glyma.19G143300, resulting in an amino acid change between PI and WH, and thus leading to a different predicted kinase domain, which might affect its protein function. Glyma.19G143300 is highly expressed in soybean seeds and encodes a leucine-rich repeat receptor-like protein kinase (LRR-RLK). Its predicted protein has typical domains of LRR-RLK family, and phylogenetic analyses reveled its similarity with the known LRR-RLK protein XIAO (LOC_Os04g48760), which is involved in controlling seed size. The major QTL and candidate genes identified in this study provide useful information for molecular breeding of new soybean cultivars with desirable seed weight.

Genome-wide characterization and functional analysis of class III peroxidase gene family in soybean reveal regulatory roles of GsPOD40 in drought tolerance.

Class III peroxidases (PODs) are plant-specific glycoproteins, that play essential roles in various plant physiological processes and defence responses. To date, scarce information is available about the POD gene family in soybean. Hence, the present study is the first comprehensive report about the genome-wide characterization of GmPOD gene family in soybean (Glycine max L.). Here, we identified a total of 124 GmPOD genes in soybean, that are unevenly distributed across the genome. Phylogenetic analysis classified them into six distinct sub-groups (A-F), with one soybean specific subgroup. Exon-intron and motif analysis suggested the existence of structural and functional diversity among the sub-groups. Duplication analysis identified 58 paralogous gene pairs; segmental duplication and positive/Darwinian selection were observed as the major factors involved in the evolution of GmPODs. Furthermore, RNA-seq analysis revealed that 23 out of a total 124 GmPODs showed differential expression between drought-tolerant and drought-sensitive genotypes under stress conditions; however, two of them (GmPOD40 and GmPOD42) revealed the maximum deregulation in all contrasting genotypes. Overexpression (OE) lines of GsPOD40 showed considerably higher drought tolerance compared to wild type (WT) plants under stress treatment. Moreover, the OE lines showed enhanced photosynthesis and enzymatic antioxidant activities under drought stress, resulting in alleviation of ROS induced oxidative damage. Hence, the GsPOD40 enhanced drought tolerance in soybean by regulating the key physiological and biochemical pathways involved in the defence response. Lastly, the results of our study will greatly assist in further functional characterization of GsPODs in plant growth and stress tolerance in soybean.

Identification of Novel Genomic Regions for Bacterial Leaf Pustule (BLP) Resistance in Soybean (Glycine max L.) via Integrating Linkage Mapping and Association Analysis.

Bacterial leaf pustule (BLP), caused by Xanthornonas axonopodis pv. glycines (Xag), is a worldwide disease of soybean, particularly in warm and humid regions. To date, little is known about the underlying molecular mechanisms of BLP resistance. The only single recessive resistance gene rxp has not been functionally identified yet, even though the genotypes carrying the gene have been widely used for BLP resistance breeding. Using a linkage mapping in a recombinant inbred line (RIL) population against the Xag strain Chinese C5, we identified that quantitative trait locus (QTL) qrxp-17-2 accounted for 74.33% of the total phenotypic variations. We also identified two minor QTLs, qrxp-05-1 and qrxp-17-1, that accounted for 7.26% and 22.26% of the total phenotypic variations, respectively, for the first time. Using a genome-wide association study (GWAS) in 476 cultivars of a soybean breeding germplasm population, we identified a total of 38 quantitative trait nucleotides (QTNs) on chromosomes (Chr) 5, 7, 8, 9,15, 17, 19, and 20 under artificial infection with C5, and 34 QTNs on Chr 4, 5, 6, 9, 13, 16, 17, 18, and 20 under natural morbidity condition. Taken together, three QTLs and 11 stable QTNs were detected in both linkage mapping and GWAS analysis, and located in three genomic regions with the major genomic region containing qrxp_17_2. Real-time RT-PCR analysis of the relative expression levels of five potential candidate genes in the resistant soybean cultivar W82 following Xag treatment showed that of Glyma.17G086300, which is located in qrxp-17-2, significantly increased in W82 at 24 and 72 h post-inoculation (hpi) when compared to that in the susceptible cultivar Jack. These results indicate that Glyma.17G086300 is a potential candidate gene for rxp and the QTLs and QTNs identified in this study will be useful for marker development for the breeding of Xag-resistant soybean cultivars.

Characterization and Rapid Gene-Mapping of Leaf Lesion Mimic Phenotype of spl-1 Mutant in Soybean (Glycine max (L.) Merr.).

In plants, lesion mimic mutants (LMMs) reveal spontaneous disease-like lesions in the absence of pathogen that constitutes powerful genetic material to unravel genes underlying programmed cell death (PCD), particularly the hypersensitive response (HR). However, only a few LMMs are reported in soybean, and no related gene has been cloned until now. In the present study, we isolated a new LMM named spotted leaf-1 (spl-1) from NN1138-2 cultivar through ethyl methanesulfonate (EMS) treatment. The present study revealed that lesion formation might result from PCD and excessive reactive oxygen species (ROS) accumulation. The chlorophyll content was significantly reduced but antioxidant activities, viz., superoxide dismutase (SOD), peroxidase (POD) and catalase (CAT), as well as the malondialdehyde (MDA) contents, were detected higher in spl-1 than in the wild-type. According to segregation analysis of mutant phenotype in two genetic populations, viz., W82×spl-1 and PI378692×spl-1, the spotted leaf phenotype of spl-1 is controlled by a single recessive gene named lm1. The lm1 locus governing mutant phenotype of spl-1 was first identified in 3.15 Mb genomic region on chromosome 04 through MutMap analysis, which was further verified and fine mapped by simple sequence repeat (SSR) marker-based genetic mapping. Genetic linkage analysis narrowed the genomic region (lm1 locus) for mutant phenotype to a physical distance of ~76.23 kb. By searching against the Phytozome database, eight annotated candidate genes were found within the lm1 region. qRT-PCR expression analysis revealed that, among these eight genes, only Glyma.04g242300 showed highly significant expression levels in wild-type relative to the spl-1 mutant. However, sequencing data of the CDS region showed no nucleotide difference between spl-1 and its wild type within the coding regions of these genes but might be in the non-coding regions such as 5' or 3' UTR. Hence, the data of the present study are in favor of Glyma.04g242300 being the possible candidate genes regulating the mutant phenotype of spl-1. However, further validation is needed to prove this function of the gene as well as its role in PCD, which in turn would be helpful to understand the mechanism and pathways involved in HR disease resistance of soybean.

Comparative Morpho-Physiological, Biochemical, and Gene Expressional Analyses Uncover Mechanisms of Waterlogging Tolerance in Two Soybean Introgression Lines.

Waterlogging is one of the key abiotic factors that severely impedes the growth and productivity of soybeans on a global scale. To develop soybean cultivars that are tolerant to waterlogging, it is a prerequisite to unravel the mechanisms governing soybean responses to waterlogging. Hence, we explored the morphological, physiological, biochemical, and transcriptional changes in two contrasting soybean introgression lines, A192 (waterlogging tolerant, WT) and A186 (waterlogging sensitive, WS), under waterlogging. In comparison to the WT line, waterlogging drastically decreased the root length (RL), shoot length (ShL), root fresh weight (RFW), shoot fresh weight (ShFW), root dry weight (RDW), and shoot dry weight (ShDW) of the WS line. Similarly, waterlogging inhibited soybean plant growth by suppressing the plant's photosynthetic capacity, enhancing oxidative damage from reactive oxygen species, and decreasing the chlorophyll content in the WS line but not in the WT line. To counteract the oxidative damage and lipid peroxidation, the WT line exhibited increased activity of antioxidant enzymes such as peroxidase (POD), superoxide dismutase (SOD), and catalase (CAT), as well as higher levels of proline content than the WS line. In addition, the expression of antioxidant enzyme genes (POD1, POD2, FeSOD, Cu/ZnSOD, CAT1, and CAT2) and ethylene-related genes (such as ACO1, ACO2, ACS1, and ACS2) were found to be up-regulated in WT line under waterlogging stress conditions. In contrast, these genes showed a down-regulation in their expression levels in the stressed WS line. The integration of morpho-physiological, biochemical, and gene expression analyses provide a comprehensive understanding of the responses of WT and WS lines to waterlogging conditions. These findings would be beneficial for the future development of soybean cultivars that can withstand waterlogging.

Dysfunction of GmVPS8a causes compact plant architecture in soybean.

Vacuolar Protein Sorting 8 (Vps8) protein is a specific subunit of the class C core vacuole/endosome tethering (CORVET) complex that plays a key role in endosomal trafficking in yeast (Saccharomyces cerevisiae). However, its functions remain largely unclear in plant vegetative growth. Here, we identified a soybean (Glycine max) T4219 mutant characterized with compact plant architecture. Map-based cloning targeted to a candidate gene GmVPS8a (Glyma.07g049700) and further found that two nucleotides deletion in the first exon of GmVPS8a causes a premature termination of the encoded protein in the T4219 mutant. Its functions were validated by CRISPR/Cas9-engineered mutation in the GmVPS8a gene that recapitulated the T4219 mutant phenotypes. Furthermore, NbVPS8a-silenced tobacco (Nicotiana benthamiana) plants exhibited similar phenotypes to the T4219 mutant, suggesting its conserved roles in plant growth. The GmVPS8a is widely expressed in multiple organs and its protein interacts with GmAra6a and GmRab5a. Combined analysis of transcriptomic and proteomic data revealed that dysfunction of GmVPS8a mainly affects pathways on auxin signal transduction, sugar transport and metabolism, and lipid metabolism. Collectively, our work reveals the function of GmVPS8a in plant architecture, which may extend a new way for genetic improvement of ideal plant-architecture breeding in soybean and other crops.

Combining Fine Mapping, Whole-Genome Re-Sequencing, and RNA-Seq Unravels Candidate Genes for a Soybean Mutant with Short Petioles and Weakened Pulvini.

A short petiole is an important agronomic trait for the development of plant ideotypes with high yields. However, the genetic basis underlying this trait remains unclear. Here, we identified and characterized a novel soybean mutant with short petioles and weakened pulvini, designated as short petioles and weakened pulvini (spwp). Compared with the wild type (WT), the spwp mutant displayed shortened petioles, owing to the longitudinally decreased cell length, and exhibited a smaller pulvinus structure due to a reduction in motor cell proliferation and expansion. Genetic analysis showed that the phenotype of the spwp mutant was controlled by two recessive nuclear genes, named as spwp1 and spwp2. Using a map-based cloning strategy, the spwp1 locus was mapped in a 183 kb genomic region on chromosome 14 between markers S1413 and S1418, containing 15 annotated genes, whereas the spwp2 locus was mapped in a 195 kb genomic region on chromosome 11 between markers S1373 and S1385, containing 18 annotated genes. Based on the whole-genome re-sequencing and RNA-seq data, we identified two homologous genes, Glyma.11g230300 and Glyma.11g230600, as the most promising candidate genes for the spwp2 locus. In addition, the RNA-seq analysis revealed that the expression levels of genes involved in the cytokinin and auxin signaling transduction networks were altered in the spwp mutant compared with the WT. Our findings provide new gene resources for insights into the genetic mechanisms of petiole development and pulvinus establishment, as well as soybean ideotype breeding.

An APETALA2/ethylene responsive factor transcription factor GmCRF4a regulates plant height and auxin biosynthesis in soybean.

Plant height is one of the key agronomic traits affecting soybean yield. The cytokinin response factors (CRFs), as a branch of the APETALA2/ethylene responsive factor (AP2/ERF) super gene family, have been reported to play important roles in regulating plant growth and development. However, their functions in soybean remain unknown. This study characterized a soybean CRF gene named GmCRF4a by comparing the performance of the homozygous Gmcrf4a-1 mutant, GmCRF4a overexpression (OX) and co-silencing (CS) lines. Phenotypic analysis showed that overexpression of GmCRF4a resulted in taller hypocotyls and epicotyls, more main stem nodes, and higher plant height. While down-regulation of GmCRF4a conferred shorter hypocotyls and epicotyls, as well as a reduction in plant height. The histological analysis results demonstrated that GmCRF4a promotes epicotyl elongation primarily by increasing cell length. Furthermore, GmCRF4a is required for the expression of GmYUCs genes to elevate endogenous auxin levels, which may subsequently enhance stem elongation. Taken together, these observations describe a novel regulatory mechanism in soybean, and provide the basis for elucidating the function of GmCRF4a in auxin biosynthesis pathway and plant heigh regulation in plants.