The basic region/leucine zipper (bZIP) transcription factor AtbZIP62 is involved in the regulation of plant responses to abiotic stresses, including drought and salinity stresses, NO₃ transport, and basal defense in Arabidopsis. It is unclear if it plays a role in regulating plant responses to abscisic acid (ABA), a phytohormone that can regulate plant abiotic stress responses via regulating downstream ABA-responsive genes. Using RT-PCR analysis, we found that the expression level of AtbZIP62 was increased in response to exogenously applied ABA. Protoplast transfection assays show that AtbZIP62 is predominantly localized in the nucleus and functions as a transcription repressor. To examine the roles of AtbZIP62 in regulating ABA responses, we generated transgenic Arabidopsis plants overexpressing AtbZIP62 and created gene-edited atbzip62 mutants using CRISPR/Cas9. We found that in both ABA-regulated seed germination and cotyledon greening assays, the 35S:AtbZIP62 transgenic plants were hypersensitive, whereas atbzip62 mutants were hyposensitive to ABA. To examine the functional mechanisms of AtbZIP62 in regulating ABA responses, we generated Arabidopsis transgenic plants overexpressing 35S:AtbZIP62-GR, and performed transcriptome analysis to identify differentially expressed genes (DEGs) in the presence and absence of DEX, and found that DEGs are highly enriched in processes including response to abiotic stresses and response to ABA. Quantitative RT-PCR results further show that AtbZIP62 may regulate the expression of several ABA-responsive genes, including USP, ABF2, and SnRK2.7. In summary, our results show that AtbZIP62 is an ABA-responsive gene, and AtbZIP62 acts as a transcription repressor to positively regulate ABA responses in Arabidopsis.

The plant hormone abscisic acid (ABA) is able to regulate the expression of ABA-responsive genes via signaling transduction, and thus plays an important role in regulating plant responses to abiotic stresses. Hence, characterization of unknown ABA response genes may enable us to identify novel regulators of ABA and abiotic stress responses. By using RT-PCR analysis, we found that the expression levels of ABA-induced Serine-rich Repressor 1 (ASR1)and ASR2, two closely related unknown function genes, were increased in response to ABA treatment. Amino acid sequence analyses show that ASR1 contains an L×L×L motif and both ASR1 and ASR2 are enriched in serine. Transfection assays in Arabidopsis leaf protoplasts show that ASR1 and ASR2 were predominantly localized in the nucleus and were able to repress the expression of the reporter gene. The roles of ASRs in regulating ABA responses were examined by generating transgenic Arabidopsis plants over-expressing ASR1 and ASR2, respectively, and CRISPR/Cas9 gene-edited single and double mutants for ASR1 and ASR2. In both the seed germination and cotyledon greening assays, ABA sensitivity remained largely unchanged in the over-expression transgenic plants and the single mutants of ASR1 and ASR2, but greatly increased ABA sensitivity was observed in the asr1 asr2 double mutants. In root elongation assays, however, decreased ABA sensitivity was observed in the 35S:ASR1 and 35S:ASR2 transgenic plants, whereas increased ABA sensitivity was observed in the asr1 and asr2 single mutants, and ABA sensitivity was further increased in the asr1 asr2 double mutants. Transcriptome analysis show that the differentially expressed genes (DEGs) down-regulated in the 35S:ASR1 transgenic plant seedlings, but up-regulated in the asr1 asr2 double mutant seedlings were highly enriched in processes including responses to plant hormones and stress stimuli. Taken together, our results show that ASR1 and ASR2 are closely related ABA response genes, ASR1 and ASR2 are serine-rich novel transcription repressors, and they negatively regulate ABA responses in Arabidopsis in a redundant manner.

The plant hormone ABA (abscisic acid) is able to regulate plant responses to abiotic stresses via regulating the expression of ABA response genes. BIC1 (Blue-light Inhibitor of Cryptochromes 1) and BIC2 have been identified as the inhibitors of plant cryptochrome functions, and are involved in the regulation of plant development and metabolism in Arabidopsis . In this study, we report the identification of BIC2 as a regulator of ABA responses in Arabidopsis . RT-PCR (Reverse Transcription-Polymerase Chain Reaction) results show that the expression level of BIC1 remained largely unchanged, but that of BIC2 increased significantly in response to ABA treatment. Transfection assays in Arabidopsis protoplasts show that both BIC1 and BIC2 were mainly localized in the nucleus, and were able to activate the expression of the co-transfected reporter gene. Results in seed germination and seedling greening assays show that ABA sensitivity was increased in the transgenic plants overexpressing BIC2, but increased slightly, if any, in the transgenic plants overexpressing BIC1. ABA sensitivity was also increased in the bic2 single mutants in seedling greening assays, but no further increase was observed in the bic1 bic2 double mutants. On the other hand, in root elongation assays, ABA sensitivity was decreased in the transgenic plants overexpressing BIC2, as well as the bic2 single mutants, but no further decrease was observed in the bic1 bic2 double mutants. By using qRT-PCR (quantitative RT-PCR), we further examined how BIC2 may regulate ABA responses in Arabidopsis , and found that inhibition of ABA on the expression of the ABA receptor genes PYL4 (PYR1-Like 4) and PYL5 were decreased, but promotion of ABA on the expression of the protein kinase gene SnRK2.6 (SNF1-Related Protein Kinases 2.6) was enhanced in both the bic1 bic2 double mutants and 35S:BIC2 overexpression transgenic plants. Taken together, our results suggest that BIC2 regulates ABA responses in Arabidopsis possibly by affecting the expression of ABA signaling key regulator genes.

NF-Y is a class of heterotrimeric transcription factor composed of three subunits; NF-YA, NF-YB, and NF-YC. This complex binds to the CCAAT box found in eukaryotic promoters and is involved in the plant development and proliferation at various stages. Although many studies were conducted on NF-Y gene family in various species, but no study has been reported yet in switchgrass (Panicum virgatum L.). In this study, 47 PvNF-Y genes (17 PvNF-YA, 18 PvNF-YB, and 12 PvNF-YC) have been identified and named according to their subfamily. Chromosome location analysis revealed that all 47 PvNF-Y genes are randomly distributed across nine chromosomes. Moreover, multiple sequence alignment showed the DNA-binding domain and NF-YA/NFYB interacting domains flanking with non-conserved domains. In addition, prediction of functional similarities among PvNF-Ys genes phylogenetic tree was constructed corresponding to Arabidopsis. The gene structure, conserved domains and motifs analysis of PvNF-Ys genes demonstrated their specificity and functional conservation. Cis-regulatory elements analysis identified numerous key CREs that are significantly associated with light, hormone, stress and plant development responses. Expression profiling indicated higher expression levels of many PvNF-YA genes during drought and heat stress. Additionally, qRT-PCR analysis showed that some PvNF-Ys genes have high expression level in root. In conclusion, the findings of this study could provide a foundation for further cloning and functional analysis of NF-Y genes in switchgrass.

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.

Background: The WRKY gene family is identified as one of the most prominent transcription factor families in plants and is involved in various biological processes such as metabolism, growth and development, and response to biotic and abiotic stresses. In many plant species, the WRKY gene family was widely studied and analyzed but little to no information for Fortunella hindsii . However, the completion of the whole genome sequencing of Fortunella hindsii allowed us to investigate the genome-wide analysis of WRKY proteins. Objective: The main objective of this study was to analyze and identify the WRKY gene family in Fortunella hindsii genome. Methodology: Various bioinformatics approaches have been used to conduct this study. Results: We constituted 46 members of the Fortunella hindsii WRKY gene family, which were unevenly distributed on all nine chromosomes. The phylogenetic relationship of predicted WRKY proteins of Fortunella hindsii with the WRKY proteins of Arabidopsis showed that 46 FhWRKY genes were divided into three main groups (G1, G2, G3) with five subgroups (2A, 2B, 2C, 2D, and 2E) of G2 group. Domain, conserved motif identification, and gene structure were conducted and the results found that these FhWRKY proteins have conserved identical characteristics within groups and maintain differences between groups. In silico subcellular localization, results showed that FhWRKY genes are located in the nucleus. The cis -regulatory element analysis identified several key CREs that are significantly associated with light, hormone responses, and stress. The gene ontology analysis of these predicted FhWRKY genes showed that these genes are significantly enriched in sequence-specific DNA binding, transcriptional activity, cellular biosynthesis, and metabolic processes. Conclusion: Therefore, overall, our results provided an excellent foundation for further functional characterization of WRKY genes with an aim of Fortunella hindsii citrus crop improvement.