Genome- and transcriptome-wide identification of trehalose-6-phosphate phosphatases (TPP) gene family and their expression patterns under abiotic stress and exogenous trehalose in soybean.

BACKGROUND: Trehalose-6-phosphate phosphatase (TPP) is an essential enzyme catalyzing trehalose synthesis, an important regulatory factor for plant development and stress response in higher plants. However, the TPP gene family in soybean has not been reported. RESULTS: A comprehensive analysis of the TPP gene family identified 18 GmTPPs classified into eight groups based on the phylogenetic relationships and the conservation of protein in six monocot and eudicot plants. The closely linked subfamilies had similar motifs and intron/exon numbers. Segmental duplication was the main driving force of soybean GmTPPs expansion. In addition, analysis of the cis-regulatory elements and promoter regions of GmTPPs revealed that GmTPPs regulated the response to several abiotic stresses. Moreover, RNA-seq and qRT-PCR analysis of the tissue-specific GmTPPs under different abiotic stresses revealed that most GmTPPs were associated with response to different stresses, including cold, drought, saline-alkali, and exogenous trehalose. Notably, exogenous trehalose treatment up-regulated the expression of most TPP genes under saline-alkali conditions while increasing the carbohydrate and trehalose levels and reducing reactive oxygen species (ROS) accumulation in soybean sprouts, especially in the saline-alkali tolerant genotype. Furthermore, the interaction network and miRNA target prediction revealed that GmTPPs interacted with abiotic stress response-related transcription factors. CONCLUSIONS: The findings in this study lay a foundation for further functional studies on TPP-based breeding to improve soybean development and stress tolerance.

Exogenous melatonin mitigates saline-alkali stress by decreasing DNA oxidative damage and enhancing photosynthetic carbon metabolism in soybean (Glycine max [L.] Merr.) leaves.

Saline-alkali stress (SS) is a common abiotic stress affecting crop cultivation worldwide, seriously inhibiting plant growth and biomass accumulation. Melatonin has been proven to relieve the inhibition of multiple abiotic stresses on plant growth. Therefore, soybean cultivars Heihe 49 (HH49, SS-tolerant) and Henong 95 (HN95, SS-sensitive) were pot-cultured in SS soil and then treated with 300 µM melatonin at the V1 stage, when the first trifoliate leaves were fully unfolded, to investigate if melatonin has an effect on SS. SS increased reactive oxygen species (ROS) accumulation in soybean leaves and thereby induced DNA oxidative damage. In addition, SS retarded cell growth and decreased the mesophyll cell size, chloroplast number, photosynthetic pigment content, which further reduced the light energy capture and electron transport rate in soybean leaves, and affected carbohydrate accumulation and metabolism. However, melatonin treatment reduced SS-induced ROS accumulation in the soybean leaves by increasing antioxidant content and oxidase activity. Effective removal of ROS reduced SS-induced DNA oxidative damage in the soybean leaf genome, which was represented by decreased random-amplified polymorphic DNA polymorphism, 8-hydroxy-20-deoxyguanine content, and relative density of apurinic/apyrimidinic-sites. Melatonin treatment also increased the volume of mesophyll cells, the numbers of chloroplast and starch grains, the contents of chlorophyll a and b and carotenoids in soybean seedling leaves treated with SS, thereby increasing the efficiency of effective light capture and electron transfer and improving photosynthesis. Subsequently, carbohydrate accumulation and metabolism in soybean leaves under SS were improved by melatonin treatment, which contributes to providing basic substances and energy for cell growth and metabolism, ultimately improving soybean SS tolerance.

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.

Exogenous melatonin enhances soybean (Glycine max (L.) Merr.) seedling tolerance to saline-alkali stress by regulating antioxidant response and DNA damage repair.

Saline-alkali (SA) stress induces excessive reactive oxygen species (ROS) accumulation in plant cells, resulting in oxidative damages of membranes, lipids, proteins, and nucleic acids. Melatonin has antioxidant protection effects in living organisms and thus has received a lot of attention. This study aimed to investigate the effect and regulating mechanism of melatonin treatment on soybean tolerance to SA stress. In this study, cultivars Heihe 49 (HH49, SA-tolerant) and Henong 95 (HN95, SA-sensitive) were pot-cultured in SA soil, then treated with MT (0-300 µM) at V1 stage. SA stress induced ROS accumulation and DNA damage in the seedling roots of both cultivars, causing G1/S arrest in HN95 and G2/M arrest in HH49. Melatonin treatment enhanced the activity of antioxidant enzymes in soybean seedling roots and reduced ROS accumulation. Additionally, melatonin treatment upregulated DNA damage repair genes, thus enhancing the reduction of DNA oxidative damage under SA stress. The effects of melatonin treatment were manifested as decreased RAPD polymorphism, 8-hydroxy-2'-deoxyguanine (8-OH-dG) level, and relative density of apurinic sites (AP-sites). Meanwhile, melatonin treatment partially alleviated the SA-induced G1/S arrest in HN95 and G2/M arrest in HH49, thus enhancing soybean seedling tolerance to SA stress.

High-resolution DNA methylome reveals that demethylation enhances adaptability to continuous cropping comprehensive stress in soybean.

BACKGROUND: Continuous cropping stress involves such factors as biological barriers, allelopathic autotoxicity, deterioration of soil physicochemical properties, and soil fertility imbalance and is regarded as a kind of comprehensive stress limiting soybean yield and quality. Genomic DNA methylation is an important regulatory mechanism for plants to resist various environmental stresses. Therefore, it is especially worthwhile to reveal genomic methylation characteristics under stress and clarify the relationship between DNA methylation status and continuous cropping stress adaptability in soybean. RESULTS: We generated a genome-wide map of cytosine methylation induced by this kind of comprehensive stress in a tolerant soybean variety (Kang Xian 2, KX2) and a sensitive variety (He Feng, HF55) using whole-genome bisulfite sequencing (WGBS) technology. The expression of DNA demethylase genes was detected using real-time quantitative PCR (qRT-PCR). The functions of differentially methylated genes (DMGs) involved in stress response in biochemical metabolism and genetic information transmission were further assessed based on Gene Ontology (GO) annotation and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis. The results showed that genomic DNA demethylation was closely related to continuous cropping comprehensive stress adaptability in soybean, which was further verified by the increasing expression of DNA demethylases ROS1 and DML. The demethylation of mCpG and mCpHpG (mCpApG preferred) contexts was more critical, which mainly occurred in gene-regulatory regions at the whole-chromosome scale. Moreover, this kind of stress adaptability may be related to various stress responders generated through strengthened glucose catabolism and amino acid and fatty acid anabolism, as well as fidelity transmission of genetic information. CONCLUSIONS: Genomic DNA demethylation was closely associated with continuous cropping comprehensive stress adaptability, highlighting the promising potential of screening continuous cropping-tolerant cultivars by DNA methylation index and further exploring the application of DNA demethylases in soybean breeding.

Genome-wide analysis of the CML gene family and its response to melatonin in common bean (Phaseolus vulgaris L.).

Calmodulin-like proteins (CML) are important calcium signal transduction proteins in plants. CML genes have been analyzed in several plants. However, little information on CML in Phaseolus vulgare is available. In this study, we identified 111 PvCMLs distributed on eleven chromosomes. Phylogenetic analysis classified them into seven subfamilies. Cis-acting element prediction showed that PvCML contained elements related to growth and development, response to abiotic stress and hormones. Moreover, the majority of PvCMLs showed different expression patterns in most of the nine tissues and developmental stages which indicated the role of PvCML in the growth and development of common bean. Additionally, the common bean was treated with melatonin by seed soaking, and root transcriptome at the 5th day and qRT-PCR of different tissue at several stages were performed to reveal the response of PvCML to the hormone. Interestingly, 9 PvCML genes of subfamily VI were detected responsive to exogenous melatonin, and the expression dynamics of nine melatonin response PvCML genes after seed soaking with melatonin were revealed. Finally, the protein interaction network analysis of nine melatonin responsive PvCMLs was constructed. The systematic analysis of the PvCML gene family provides theoretical support for the further elucidation of their functions, and melatonin response molecular mechanism of the CML family in P. vulgaris.

Genome-wide identification of the soybean cytokinin oxidase/dehydrogenase gene family and its diverse roles in response to multiple abiotic stress.

Cytokinin oxidase/dehydrogenase (CKX) irreversibly degrades cytokinin, regulates growth and development, and helps plants to respond to environmental stress. Although the CKX gene has been well characterized in various plants, its role in soybean remains elusive. Therefore, in this study, the evolutionary relationship, chromosomal location, gene structure, motifs, cis-regulatory elements, collinearity, and gene expression patterns of GmCKXs were analyzed using RNA-seq, quantitative real-time PCR (qRT-PCR), and bioinformatics. We identified 18 GmCKX genes from the soybean genome and grouped them into five clades, each comprising members with similar gene structures and motifs. Cis-acting elements involved in hormones, resistance, and physiological metabolism were detected in the promoter regions of GmCKXs. Synteny analysis indicated that segmental duplication events contributed to the expansion of the soybean CKX family. The expression profiling of the GmCKXs genes using qRT-PCR showed tissue-specific expression patterns. The RNA-seq analysis also indicated that GmCKXs play an important role in response to salt and drought stresses at the seedling stage. The responses of the genes to salt, drought, synthetic cytokinin 6-benzyl aminopurine (6-BA), and the auxin indole-3-acetic acid (IAA) at the germination stage were further evaluated by qRT-PCR. Specifically, the GmCKX14 gene was downregulated in the roots and the radicles at the germination stage. The hormones 6-BA and IAA repressed the expression levels of GmCKX1, GmCKX6, and GmCKX9 genes but upregulated the expression levels of GmCKX10 and GmCKX18 genes. The three abiotic stresses also decreased the zeatin content in soybean radicle but enhanced the activity of the CKX enzymes. Conversely, the 6-BA and IAA treatments enhanced the CKX enzymes' activity but reduced the zeatin content in the radicles. This study, therefore, provides a reference for the functional analysis of GmCKXs in soybean in response to abiotic stresses.

The molecular events underpinning cultivar differences in melatonin counteracting salt damage in Phaseolus vulgaris.

Melatonin (N -acetyl-5-methoxytryptamine) plays important roles in multiple stress responses, especially under salt stress. However, cultivar differences in melatonin mediated salt stress tolerance are unclear. Phaseolus vulgaris L. (common bean) cultivars Jiyin 1 (JY, salt-tolerant) and Xuliyabai (XL, salt-sensitive) were used in this study. Exogenous melatonin significantly improved root growth under salt stress in JY, but had little effect on XL. Physiology analysis showed significant differences in activities of antioxidant enzymes (superoxide, SOD; and catalase, CAT) and malondialdehyde content between JY and XL. Meanwhile, the change of ABA content in JY and XL root was opposite in salt plus melatonin treatment. Comparative root transcriptomes of JY and XL revealed 3505 and 668 differentially expressed genes (DEGs) regulated by salt stress and melatonin. The most enriched melatonin-responsive genes under salt stress are mainly involved in regulation of transcription, oxidation-reduction process, transcription factor activity, oxidoreductase activity. In addition, melatonin induced more obvious changes of DEGs in JY than that in XL under salt condition. Melatonin also significantly induced 41 DEGs only in JY, including signal transduction genes, transcription factors, ubiquitin protein ligases, ion homeostasis and osmotic adjustment genes etc. This study uncovered the molecular mechanism of cultivar difference of melatonin response under salt stress in common bean.

Identification and Expression Analysis of bZIP Members under Abiotic Stress in Mung Bean (Vigna radiata).

The main aim of this study was to identify the bZIP family members in mung bean and explore their expression patterns under several abiotic stresses, with the overarching goal of elucidating their biological functions. Results identified 75 bZIP members in mung bean, which were unevenly distributed in the chromosomes (1-11), and all had a highly conserved bZIP domain. Phylogenetic analysis divided the members into 10 subgroups, with members in the same subgroup having similar structure and motif. The cis-acting elements in the promoter region revealed that most of the bZIP members might have the connection with abscisic acid, ethylene, and stress responsive elements. The transcriptome data demonstrated that bZIP members could respond to salt stress at different degrees in leaves, but the expression patterns could vary at different time points under stress. Differentially expressed genes (DEGs), such as VrbZIP12, VrbZIP37, and VrZIP45, were annotated into the plant hormone signal transduction pathway, which might be regulated the expression of abiotic stress-related gene (ABF). Quantitative real-time polymerase chain reaction (qRT-PCR) was applied to determine the expression of bZIP members in roots and leaves under drought, alkali, and low-temperature stress. Results showed that bZIP members respond differently to diverse stresses, and their expression was tissue-specific, which suggests that they may have different regulatory mechanism in different tissues. Overall, this study will provide a reference for further research on the functions of bZIP members in mung bean.

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.

RNA-Seq Analysis Demonstrates Different Strategies Employed by Tiger Nuts (Cyperus esculentus L.) in Response to Drought Stress.

Drought stress, an important abiotic stress, has affected global agricultural production by limiting the yield and the quality of crops. Tiger nuts (Cyperus esculentus L.) are C4 crops in the Cyperaceae family, which have high-quality wholesome ingredients. However, data on mechanisms underlying the response of tiger nuts to drought stress are few. Here, the variety of Jisha 1 and 15% polyethylene glycol (PEG; a drought stress simulator) were used to study the mechanisms of stress response in tiger nuts. Our evaluation of the changes in physiological indicators such as electrolyte leakage (El), malondialdehyde (MDA), hydrogen peroxide (H2O2), superoxide anion (O2-) and activities of reactive oxygen species (ROS) showed that 12 h was the most suitable time point to harvest and analyze the response to drought stress. Thereafter, we performed transcriptome (RNA-Seq) analysis in the control (CK) and stress treatment groups and showed that there was a total of 1760 differentially expressed genes (DEGs). Gene Ontology (GO) analysis showed that the DEGs were enriched in abscisic acid (ABA) terms, and pathways such as starch and sucrose metabolism (ko00500), phenylpropanoid biosynthesis (ko00940) and plant hormone signal transduction (ko04075) were significantly enriched in the Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis. In addition, quantitative real-time PCR (qRT-PCR) analysis of the DEGs demonstrated an upregulation of ABA and lignin content, as well as enzyme activities in enriched pathways, which validated the RNA-Seq data. These results revealed the pathways and mechanisms adopted by the tiger nuts in response to drought stress.

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.