Soil salt-alkalization seriously impacts crop growth and productivity worldwide. Breeding and applying tolerant varieties is the most economical and effective way to address soil alkalization. However, genetic resources for breeders to improve alkali tolerance are limited in mung bean. Here, a genome-wide association study (GWAS) was performed to detect alkali-tolerant genetic loci and candidate genes in 277 mung bean accessions during germination. Using the relative values of two germination traits, 19 QTLs containing 32 SNPs significantly associated with alkali tolerance on nine chromosomes were identified, and they explained 3.6 to 14.6% of the phenotypic variance. Moreover, 691 candidate genes were mined within the LD intervals containing significant trait-associated SNPs. Transcriptome sequencing of alkali-tolerant accession 132–346 under alkali and control conditions after 24 h of treatment was conducted, and 2565 DEGs were identified. An integrated analysis of the GWAS and DEGs revealed six hub genes involved in alkali tolerance responses. Moreover, the expression of hub genes was further validated by qRT-PCR. These findings improve our understanding of the molecular mechanism of alkali stress tolerance and provide potential resources (SNPs and genes) for the genetic improvement of alkali tolerance in mung bean.

Trichome formation in Arabidopsis is regulated by a MBW complex formed by MYB, bHLH and WD40 transcriptional factors, which can activate GLABRA2 (GL2) and the R3 MYB transcription factor genes. GL2 promotes trichome formation, whereas R3 MYBs are able to block the formation of the MBW complex. It has been reported that the C2H2 transcription factor GIS (GLABROUS INFLORESCENCE STEMS) functions upstream of the MBW activator complex to regulate trichome formation, and that the expression of TCL1 is not regulated by the MBW complex. However, gis and the R3 MYB gene mutant tcl1 (trichomeless 1) have opposite inflorescence trichome phenotypes, but their relationship in regulating trichome formation remained unknown. By generating and characterization of the gis tcl1 double mutant, we found that trichome formation in the gis tcl1double and the tcl1 single mutants were largely indistinguishable, but the trichome formation in the 35S:TCL1/gis transgenic plant was similar to that in the gis mutant. By using quantitative RT-PCR analysis, we showed that expression level of GIS was increased in the triple mutant tcl1 try cpc, but the expression level of TCL1 was not affected in the gis mutant. On the other hand, trichome morphology in both gis tcl1 and 35S:TCL1/gis plants was similar to that in the gis mutant. In summary, our results indicate that GIS may work downstream of TCL1 to regulate trichome formation, and GIS has a dominant role in controlling trichome morphology.

The plant hormone ABA (abscisic acid) regulates plant responses to abiotic stresses by regulating the expression of ABA response genes. However, the functions of a large portion of ABA response genes have remained unclear. We report in this study the identification of ASDs (ABA-inducible signal peptide-containing DUF538 proteins), a subgroup of DUF538 proteins with a signal peptide, as the regulators of plant responses to ABA in Arabidopsis. ASDs are encoded by four closely related DUF538 genes, with ASD1/ASD2 and ASD3/ASD4 being two pairs of duplicated tandemly repeated genes. The quantitative RT-PCR (qRT-PCR) results showed that the expression levels of ASDs increased significantly in response to ABA as well as NaCl and mannitol treatments, with the exception that the expression level of ASD2 remained largely unchanged in response to NaCl treatment. The results of Arabidopsis protoplast transient transfection assays showed that ASDs were localized on the plasma membrane and in the cytosol and nucleus. When recruited to the promoter of the reporter gene via a fused GD domain, ASDs were able to slightly repress the expression of the co-transfected reporter gene. Seed germination and cotyledon greening assays showed that ABA sensitivity was increased in the transgenic plants that were over-expressing ASD1 or ASD3 but decreased in the transgenic plants that were over-expressing ASD2 or ASD4. On the other hand, ABA sensitivity was increased in the CRISPR/Cas9 gene-edited asd2 single mutants but decreased in the asd3 single mutants. A transcriptome analysis showed that differentially expressed genes in the 35S:ASD2 transgenic plant seedlings were enriched in several different processes, including in plant growth and development, the secondary metabolism, and plant hormone signaling. In summary, our results show that ASDs are ABA response genes and that ASDs are involved in the regulation of plant responses to ABA in Arabidopsis; however, ASD1/ASD3 and ASD2/ASD4 have opposite functions.

EAR (Ethylene-responsive element binding factor-associated Amphiphilic Repression) motif-containing transcription repressors have been shown to regulate plant growth and development, and plant responses to plant hormones and environmental stresses including biotic and abiotic stresses. However, the functions of most EAR-motif-containing proteins remain largely uncharacterized. The plant hormone abscisic acid (ABA) also plays important roles in regulating plant responses to abiotic stresses via activation/repression of ABA-responsive genes. We report here the identification and functional characterization of two ABA-responsive EAR motif-containing protein genes, AtEAU1 (Arabidopsis thaliana EAR motif-containing ABAUp-regulated 1) and AtEAU2. Quantitative RT-PCR results show that the expressions of AtEAU1 and AtEAU2 were increased by ABA treatment, and were decreased in the ABA biosynthesis mutant aba1-5. Assays in transfected Arabidopsis protoplasts show that both AtEAU1 and AtEAU2 were specifically localized in the nucleus, and when recruited to the promoter region of the reporter gene by a fused DNA binding domain, repressed reporter gene expression. By using T-DNA insertion mutants and a gene-edited transgene-free mutant generated by CRISPR/Cas9 gene editing, we performed ABA sensitivity assays, and found that ABA sensitivity in the both ateau1 and ateau2 single mutants was increased in seedling greening assays. ABA sensitivity in the ateau1 ateau2 double mutants was also increased, but was largely similar to the ateau1 single mutants. On the other hand, all the mutants showed a wild type response to ABA in root elongation assays. Quantitative RT-PCR results show that the expression level of PYL4, an ABA receptor gene was increased, whereas that of ABI2, a PP2C gene was decreased in the ateau1 and ateau1 single, and the ateau1 ateau2 double mutants. In summary, our results suggest that AtEAU1 and AtEAU2 are ABA-response genes, and AtEAU1 and AtEAU2 are novel EAR motif-containing transcription repressors that negatively regulate ABA responses in Arabidopsis, likely by regulating the expression of some ABA signaling key regulator genes.

Mung bean (Vigna radiata) plays a significant role in agricultural trade, food processing and utilization, and cropping structure adjustment due to its abundant nutritional components, medicine-food homology, capacity for nitrogen fixation, and soil improvement. The low yield level is a crucial limitation factor in the mung bean industry, while heterosis is an efficient path for increasing crop yields. The flexible utilization of male sterile mung bean materials may solve this pressing demand in the industry. This study identified a completely male-sterile mutant, vrnp 1, in the EMS-mutagenized mung bean cultivar Jilv 10 population, which is controlled by a single recessive nuclear gene. Furthermore, we employed a series of microscopical and histological techniques and observed that the tapetal cells in the vrnp 1 mutant did not perform as expected when reaching stage 8 of anther development, notably exhibiting a delay in entering PCD. This was accompanied by a failure to deposit cell wall components onto the pollen wall, culminating in a complete absence of mature pollen and the manifestation of male sterility. In conclusion, the vrnp 1 mutant could potentially serve as a promising candidate for male sterility in exploiting hybrid vigor in mung bean. Our research may elucidate how the delayed initiation of programmed cell death in tapetal cells contributes to a factor implicated in mung bean male sterility. Furthermore, the phenotypic data collected during pivotal developmental phases may have contributed to a better grasp of mung bean microspores and anther development.