Exogenous melatonin enhances cell wall response to salt stress in common bean (Phaseolus vulgaris) and the development of the associated predictive molecular markers.

Common bean (Phaseolus vulgaris) is an important food crop; however, its production is affected by salt stress. Salt stress can inhibit seed germination, promote senescence, and modify cell wall biosynthesis, assembly, and architecture. Melatonin, an indole heterocycle, has been demonstrated to greatly impact cell wall structure, composition, and regulation in plants under stress. However, the molecular basis for such assumptions is still unclear. In this study, a common bean variety, "Naihua" was treated with water (W), 70 mmol/L NaCl solution (S), and 100 µmol/L melatonin supplemented with salt solution (M+S) to determine the response of common bean to exogenous melatonin and explore regulatory mechanism of melatonin against salt stress. The results showed that exogenous melatonin treatment alleviated salt stress-induced growth inhibition of the common bean by increasing the length, surface area, volume, and diameter of common bean sprouts. Moreover, RNA sequencing (RNA-seq) and real-time quantitative PCR (qRT-PCR) indicated that the cell wall regulation pathway was involved in the salt stress tolerance of the common bean enhanced by melatonin. Screening of 120 germplasm resources revealed that melatonin treatment improved the salt tolerance of more than 65% of the common bean germplasm materials. Melatonin also up-regulated cell wall pathway genes by at least 46%. Furthermore, we analyzed the response of the common bean germplasm materials to melatonin treatment under salt stress using the key genes associated with the synthesis of the common bean cell wall as the molecular markers. The results showed that two pairs of markers were significantly associated with melatonin, and these could be used as candidate markers to predict whether common bean respond to exogenous melatonin and then enhance salt tolerance at the sprouting stage. This study shows that cell wall can respond to exogenous melatonin and enhance the salt tolerance of common bean. The makers identified in this study can be used to select common bean varieties that can respond to melatonin under stress. Overall, the study found that cell wall could response melatonin and enhance the salt tolerance and developed the makers for predicting varieties fit for melatonin under stress in common bean, which may be applied in the selection or development of common bean varieties with abiotic stress tolerance.

Molecular mechanisms of flavonoid accumulation in germinating common bean (Phaseolus vulgaris) under salt stress.

Flavonoids are important secondary metabolites, active biomolecules in germinating beans, and have prominent applications in food and medicine due to their antioxidant effects. Rutin is a plant flavonoid with a wide biological activity range. In this study, flavonoid (rutin) accumulation and its related molecular mechanisms in germinating common bean (Phaseolus vulgaris) were observed at different time points (0-120 h) under salt stress (NaCl). The rutin content increased from germination onset until 96 h, after which a reducing trend was observed. Metabolome analysis showed that salt stress alters flavonoid content by regulating phenylpropanoid (ko00940) and flavonoid (ko00941) biosynthesis pathways, as well as their enzyme activities, including cinnamyl-alcohol dehydrogenase (CAD), peroxidase (POD), chalcone isomerase (CHI), and flavonol synthase (FLS). The RNA-seq and quantitative real-time PCR (qRT-PCR) analyses also showed that these two pathways were linked to changes in flavonoid content following salt treatment. These results reveal that salt stress effectively enhanced rutin content accumulation in germinating beans, hence it could be employed to enhance the functional quality of germinating common beans.

The bZIP Transcription Factor Family in Adzuki Bean (Vigna Angularis): Genome-Wide Identification, Evolution, and Expression Under Abiotic Stress During the Bud Stage.

Adzuki bean (Vigna angularis) is an important dietary legume crop that was first cultivated and domesticated in Asia. Currently, little is known concerning the evolution and expression patterns of the basic leucine zipper (bZIP) family transcription factors in the adzuki bean. Through the PFAM search, 72 bZIP members of adzuki bean (VabZIP) were identified from the reference genome. Most of them were located on 11 chromosomes and seven on an unknown chromosome. A comprehensive analysis, including evolutionary, motifs, gene structure, cis-elements, and collinearity was performed to identify VabZIP members. The subcellular localization results showed VabZIPs might locate on the nuclear. Quantitative real-time PCR (qRT-PCR) analysis of the relative expression of VabZIPs in different tissues at the bud stage revealed that VabZIPs had a tissue-specific expression pattern, and its expression was influenced by abiotic stress. These characteristics of VabZIPs provide insights for future research aimed at developing interventions to improve abiotic stress resistance.

Heat shock transcription factor (Hsf) gene family in common bean (Phaseolus vulgaris): genome-wide identification, phylogeny, evolutionary expansion and expression analyses at the sprout stage under abiotic stress.

BACKGROUND: Common bean (Phaseolus vulgaris) is an essential crop with high economic value. The growth of this plant is sensitive to environmental stress. Heat shock factor (Hsf) is a family of antiretroviral transcription factors that regulate plant defense system against biotic and abiotic stress. To date, few studies have identified and bio-analyzed Hsfs in common bean. RESULTS: In this study, 30 Hsf transcription factors (PvHsf1-30) were identified from the PFAM database. The PvHsf1-30 belonged to 14 subfamilies with similar motifs, gene structure and cis-acting elements. The Hsf members in Arabidopsis, rice (Oryza sativa), maize (Zea mays) and common bean were classified into 14 subfamilies. Collinearity analysis showed that PvHsfs played a role in the regulation of responses to abiotic stress. The expression of PvHsfs varied across different tissues. Moreover, quantitative real-time PCR (qRT-PCR) revealed that most PvHsfs were differentially expressed under cold, heat, salt and heavy metal stress, indicating that PvHsfs might play different functions depending on the type of abiotic stress. CONCLUSIONS: In this study, we identified 30 Hsf transcription factors and determined their location, motifs, gene structure, cis-elements, collinearity and expression patterns. It was found that PvHsfs regulates responses to abiotic stress in common bean. Thus, this study provides a basis for further analysis of the function of PvHsfs in the regulation of abiotic stress in common bean.

GmNFYA13 Improves Salt and Drought Tolerance in Transgenic Soybean Plants.

NF-YA transcription factors function in modulating tolerance to abiotic stresses that are serious threats to crop yields. In this study, GmNFYA13, an NF-YA gene in soybean, was strongly induced by salt, drought, ABA, and H2O2, and suppressed by tungstate, an ABA synthesis inhibitor. The GmNFYA13 transcripts were detected in different tissues in seedling and flowering stages, and the expression levels in roots were highest. GmNFYA13 is a nuclear localization protein with self-activating activity. Transgenic Arabidopsis plants overexpressing GmNFYA13 with higher transcript levels of stress-related genes showed ABA hypersensitivity and enhanced tolerance to salt and drought stresses compared with WT plants. Moreover, overexpression of GmNFYA13 resulted in higher salt and drought tolerance in OE soybean plants, while suppressing it produced the opposite results. In addition, GmNFYA13 could bind to the promoters of GmSALT3, GmMYB84, GmNCED3, and GmRbohB to regulate their expression abundance in vivo. The data in this study suggested that GmNFYA13 enhanced salt and drought tolerance in soybean plants.

Genome- and Transcriptome-Wide Identification of C3Hs in Common Bean (Phaseolus vulgaris L.) and Structural and Expression-Based Analyses of Their Functions During the Sprout Stage Under Salt-Stress Conditions.

CCCH (C3H) zinc-finger proteins are involved in plant biotic and abiotic stress responses, growth and development, and disease resistance. However, studies on C3H genes in Phaseolus vulgaris L. (common bean) are limited. Here, 29 protein-encoding C3H genes, located on 11 different chromosomes, were identified in P. vulgaris. A phylogenetic analysis categorized the PvC3Hs into seven subfamilies on the basis of distinct features, such as exon-intron structure, cis-regulatory elements, and MEME motifs. A collinearity analysis revealed connections among the PvC3Hs in the same and different species. The PvC3H genes showed tissue-specific expression patterns during the sprout stage, as assessed by real-time quantitative PCR (RT-qPCR). Using RNA-sequencing and RT-qPCR data, PvC3Hs were identified as being enriched through Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analyses in binding, channel activity, and the spliceosome pathway. These results provide useful information and a rich resource that can be exploited to functionally characterize and understand PvC3Hs. These PvC3Hs, especially those enriched in binding, channel activity, and the spliceosome pathway will further facilitate the molecular breeding of common bean and provide insights into the correlations between PvC3Hs and salt-stress responses during the sprout stage.

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.

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.

QTL analysis of nodule traits and the identification of loci interacting with the type III secretion system in soybean.

Symbiotic nitrogen fixation is the main source of nitrogen for soybean growth. Since the genotypes of rhizobia and soybean germplasms vary, the nitrogen-fixing ability of soybean after inoculation also varies. A few studies have reported that quantitative trait loci (QTLs) control biological nitrogen fixation traits, even soybean which is an important crop. The present study reported that the Sinorhizobium fredii HH103 gene rhcJ belongs to the tts (type III secretion) cluster and that the mutant HH103ΩrhcJ can clearly decrease the number of nodules in American soybeans. However, few QTLs of nodule traits have been identified. This study used a soybean (Glycine max (L.) Merr.) 'Charleston' × 'Dongnong 594' (C × D, n = 150) recombinant inbred line (RIL). Nodule traits were analysed in the RIL population after inoculation with S. fredii HH103 and the mutant HH103ΩrhcJ. Plants were grown in a greenhouse with a 16-h light cycle at 26 °C and an 8-h dark cycle at 18 °C. Then, 4 weeks after inoculation, plants were harvested for evaluation of nodule traits. Through QTL mapping, 16 QTLs were detected on 8 chromosomes. Quantitative PCR (qRT-PCR) and RNA-seq analysis determined that the genes Glyma.04g060600, Glyma.18g159800 and Glyma.13g252600 might interact with rhcJ.

Identification of Soybean Genes Related to Soybean Seed Protein Content Based on Quantitative Trait Loci Collinearity Analysis.

Increasing the protein content of soybean seeds through a higher ratio of glycinin is important for soybean breeding and food processing; therefore, the integration of different quantitative trait loci (QTLs) is of great significance. In this study, we investigated the collinearity of seed protein QTLs. We identified 192 collinear protein QTLs that formed six hotspot regions. The two most important regions had seed protein 36-10 and seed protein 36-20 as hub nodes. We used a chromosome segment substitution line (CSSL) population for QTL validation and identified six CSSL materials with collinear QTLs. Five materials with higher protein and glycinin contents in comparison to the recurrent parent were analyzed. A total of 13 candidate genes related to seed protein from the QTL hotspot intervals were detected, 8 of which had high expression in mature soybean seeds. These results offer a new analysis method for molecular-assisted selection (MAS) and improvement of soybean product quality.

Identification of Soybean Genes Whose Expression is Affected by the Ensifer fredii HH103 Effector Protein NopP.

In some legume⁻rhizobium symbioses, host specificity is influenced by rhizobial nodulation outer proteins (Nops). However, the genes encoding host proteins that interact with Nops remain unknown. We generated an Ensifer fredii HH103 NopP mutant (HH103ΩNopP), and analyzed the nodule number (NN) and nodule dry weight (NDW) of 10 soybean germplasms inoculated with the wild-type E. fredii HH103 or the mutant strain. An analysis of recombinant inbred lines (RILs) revealed the quantitative trait loci (QTLs) associated with NopP interactions. A soybean genomic region containing two overlapping QTLs was analyzed in greater detail. A transcriptome analysis and qRT-PCR assay were used to identify candidate genes encoding proteins that interact with NopP. In some germplasms, NopP positively and negatively affected the NN and NDW, while NopP had different effects on NN and NDW in other germplasms. The QTL region in chromosome 12 was further analyzed. The expression patterns of candidate genes Glyma.12g031200 and Glyma.12g073000 were determined by qRT-PCR, and were confirmed to be influenced by NopP.

Meta-analysis and transcriptome profiling reveal hub genes for soybean seed storage composition during seed development.

Soybean is an important crop providing edible oil and protein source. Soybean oil and protein contents are quantitatively inherited and significantly affected by environmental factors. In this study, meta-analysis was conducted based on soybean physical maps to integrate quantitative trait loci (QTLs) from multiple experiments in different environments. Meta-QTLs for seed oil, fatty acid composition, and protein were identified. Of them, 11 meta-QTLs were located on hot regions for both seed oil and protein. Next, we selected 4 chromosome segment substitution lines with different seed oil and protein contents to characterize their 3 years of phenotype selection in the field. Using strand-specific RNA-sequencing analysis, we profile the time-course transcriptome patterns of soybean seeds at early maturity, middle maturity, and dry seed stages. Pairwise comparison and K-means clustering analysis revealed 7,482 differentially expressed genes and 45 expression patterns clusters. Weighted gene coexpression network analysis uncovered 46 modules of gene expression patterns. The 2 most significant coexpression networks were visualized, and 7 hub genes were identified that were involved in soybean oil and seed storage protein accumulation processes. Our results provided a transcriptome dataset for soybean seed development, and the candidate hub genes represent a foundation for further research.

SNP-SNP Interaction Analysis on Soybean Oil Content under Multi-Environments.

Soybean oil content is one of main quality traits. In this study, we used the multifactor dimensionality reduction (MDR) method and a soybean high-density genetic map including 5,308 markers to identify stable single nucleotide polymorphism (SNP)-SNP interactions controlling oil content in soybean across 23 environments. In total, 36,442,756 SNP-SNP interaction pairs were detected, 1865 of all interaction pairs associated with soybean oil content were identified under multiple environments by the Bonferroni correction with p <3.55×10-11. Two and 1863 SNP-SNP interaction pairs detected stable across 12 and 11 environments, respectively, which account around 50% of total environments. Epistasis values and contribution rates of stable interaction (the SNP interaction pairs were detected in more than 2 environments) pairs were detected by the two way ANOVA test, the available interaction pairs were ranged 0.01 to 0.89 and from 0.01 to 0.85, respectively. Some of one side of the interaction pairs were identified with previously research as a major QTL without epistasis effects. The results of this study provide insights into the genetic architecture of soybean oil content and can serve as a basis for marker-assisted selection breeding.