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Vegetable soybean is one of the most important vegetables in China, and the demand for this vegetable has markedly increased worldwide over the past two decades. Here, we present a high-quality de novo genome assembly of the vegetable soybean cultivar Zhenong 6 (ZN6), which is one of the most popular cultivars in China. The 20 pseudochromosomes cover 94.57% of the total 1.01 Gb assembly size, with contig N50 of 3.84 Mb and scaffold N50 of 48.41 Mb. A total of 55 517 protein-coding genes were annotated. Approximately 54.85% of the assembled genome was annotated as repetitive sequences, with the most abundant long terminal repeat transposable elements. Comparative genomic and phylogenetic analyses with grain soybean Williams 82, six other Fabaceae species and Arabidopsis thaliana genomes highlight the difference of ZN6 with other species. Furthermore, we resequenced 60 vegetable soybean accessions. Alongside 103 previously resequenced wild soybean and 155 previously resequenced grain soybean accessions, we performed analyses of population structure and selective sweep of vegetable, grain, and wild soybean. They were clearly divided into three clades. We found 1112 and 1047 genes under selection in the vegetable soybean and grain soybean populations compared with the wild soybean population, respectively. Among them, we identified 134 selected genes shared between vegetable soybean and grain soybean populations. Additionally, we report four sucrose synthase genes, one sucrose-phosphate synthase gene, and four sugar transport genes as candidate genes related to important traits such as seed sweetness and seed size in vegetable soybean. This study provides essential genomic resources to promote evolutionary and functional genomics studies and genomically informed breeding for vegetable soybean.

Plant MAPK cascades have been implicated in various developmental processes and in response to stresses. MAPKKs, a convergent linking point between MAPKKKs and MAPKs, play pivotal roles in MAPK cascades to regulate various stress responses in plants. Few MAPKKs have been functionally characterized in Brachypodium distachyon, a new monocot model plant. In this study, we cloned and characterized a group A MAPKK gene, designated as BdMKK6.2, from B. distachyon. Quantitative RT-PCR analysis revealed that BdMKK6.2 expression was upregulated by cold, heat, and H2O2 treatments but downregulated by PEG and ABA treatments. Transient expression of BdMKK6.2 in onion epidermal cells suggested that it was exclusively localized in the cytoplasm. Overexpression of BdMKK6.2 in transgenic tobacco plants increased their sensitivity to osmotic stress during seed germination. Moreover, BdMKK6.2 overexpression resulted in reduced tolerance to drought stress. Physiological-biochemical analyses showed that BdMKK6.2-overexpressing plants had decreased survival rates but increased water loss rates, ion leakage, malondialdehyde content, and reactive oxygen species accumulation under drought condition. In addition, the transcriptional levels of two ROS-producing genes NtRbohD and NtRbohF were upregulated, but a stress-responsive gene NtNCED1 was downregulated in BdMKK6.2-overexpressing plants compared to wild type plants. These results indicate that BdMKK6.2 is a negative regulator of drought stress response through influencing ROS homeostasis and transcription of stress-responsive gene.

Six foxtail millet ASR genes were regulated by various stress-related signals. Overexpression of ASR1 increased drought and oxidative tolerance by controlling ROS homeostasis and regulating oxidation-related genes in tobacco plants. Abscisic acid stress ripening (ASR) proteins with ABA/WDS domains constituted a class of plant-specific transcription factors, playing important roles in plant development, growth and abiotic stress responses. However, only a few ASRs genes have been characterized in crop plants and none was reported so far in foxtail millet (Setaria italic), an important drought-tolerant crop and model bioenergy grain crop. In the present study, we identified six foxtail millet ASR genes. Gene structure, protein alignments and phylogenetic relationships were analyzed. Transcript expression patterns of ASR genes revealed that ASRs might play important roles in stress-related signaling and abiotic stress responses in diverse tissues in foxtail millet. Subcellular localization assays showed that SiASR1 localized in the nucleus. Overexpression of SiASR1 in tobacco remarkably increased tolerance to drought and oxidative stresses, as determined through developmental and physiological analyses of germination rate, root growth, survival rate, relative water content, ion leakage, chlorophyll content and antioxidant enzyme activities. Furthermore, expression of SiASR1 modulated the transcript levels of oxidation-related genes, including NtSOD, NtAPX, NtCAT, NtRbohA and NtRbohB, under drought and oxidative stress conditions. These results provide a foundation for evolutionary and functional characterization of the ASR gene family in foxtail millet.

Drought-induced (Di19) proteins played important roles in plant growth, development, and abiotic stress responses. In the present study, a total of seven Di19 genes were identified in soybean. Each soybean Di19 gene showed specific responses to salt, drought, oxidative, and ABA stresses based on expression profiles. With a relatively higher transcript level among Di19 members under four stress treatments, GmDi19-5 was selected for detailed analysis. Inhibitor assays revealed that ABA inhibitor (Fluridone) or H2O2 inhibitor (DMTU) was involved in the drought- or salt-induced transcription of GmDi19-5. The GUS activity driven by the GmDi19-5 promoter was induced by salt, PEG, ABA, and MV treatments and tended to be accumulated in the vascular bundles and young leaves. A subcellular localization assay showed that GmDi19-5 protein localized in the nucleus. Further investigation showed that GmDi19-5 protein was involved in the interaction with GmLEA3.1. Overexpression of GmDi19-5 increased sensitivity of transgenic Arabidopsis plants to salt, drought, oxidative, and ABA stresses and regulated expression of several ABA/stress-associated genes. This present investigation showed that GmDi19-5 functioned as a negative factor under abiotic stresses and was involved in ABA and SOS signaling pathway by altering transcription of stress-associated genes.

The expression of BdWRKY36 was upregulated by drought treatment. BdWRKY36 -overexpressing transgenic tobacco increased drought tolerance by controlling ROS homeostasis and regulating transcription of stress related genes. WRKY transcription factor plays important roles in plant growth, development and stress response. However, the function of group IIe WRKYs is less known. In this study, we cloned and characterized a gene of group IIe WRKY, designated as BdWRKY36, from Brachypodium distachyon. Transient expression of BdWRKY36 in onion epidermal cell suggested its localization in the nucleus. Transactivation assay revealed that the C-terminal region, instead of full length BdWRKY36, had transcriptional activity. BdWRKY36 expression was upregulated by drought. Overexpression of BdWRKY36 in transgenic tobacco plants resulted in enhanced tolerance to drought stress. Physiological–biochemical indices analyses showed that BdWRKY36-overexpressing tobacco lines had lesser ion leakage (IL) and reactive oxygen species (ROS) accumulation, but higher contents of chlorophyll, relative water content (RWC) and activities of antioxidant enzyme than that in control plants under drought condition. Meanwhile expression levels of some ROS-scavenging and stress-responsive genes were upregulated in BdWRKY36-overexpressing tobacco lines under drought stress. These results demonstrate that BdWRKY36 functions as a positive regulator of drought stress response by controlling ROS homeostasis and regulating transcription of stress related genes.