Abscisic acid (ABA) regulates plant responses to abiotic stresses via regulating the expression of downstream genes, yet the functions of many ABA responsive genes remain unknown. We report here the characterization of MYB71, a R2R3 MYB transcription factor in regulating ABA responses in Arabidopsis. RT-PCR results show that the expression level of MYB71 was increased in response to ABA treatment. Arabidopsis protoplasts transfection results show that MYB71 was specifically localized in nucleus and it activated the Gal4:GUS reporter gene when recruited to the Gal4 promoter by a fused DNA binding domain GD. Roles of MYB71 in regulating plant response to ABA were analyzed by generating Arabidopsis transgenic plants overexpression MYB71 and gene edited mutants of MYB71. The results show that ABA sensitivity was increased in the transgenic plants overexpression MYB71, but decreased in the MYB71 mutants. By using a DEX inducible system, we further identified genes are likely regulated by MYB71, and found that they are enriched in biological process to environmental stimuli including abiotic stresses, suggesting that MYB71 may regulate plant response to abiotic stresses. Taken together, our results suggest that MYB71 is an ABA responsive gene, and MYB71 functions as a transcription activator and it positively regulates ABA response in Arabidopsis.

Tobacco is a cash crop throughout the world, and its growth and development are affected by abiotic stresses including drought stress; therefore, drought-tolerant breeding may help to improve tobacco yield and quality under drought stress conditions. Considering that the plant hormone ABA (abscisic acid) is able to regulate plant responses to abiotic stresses via activating ABA response genes, the characterization of ABA response genes may enable the identification of genes that can be used for molecular breeding to improve drought tolerance in tobacco. We report here the identification of NtAITRs (Nicotiana tabacum ABA-induced transcription repressors) as a family of novel regulators of drought tolerance in tobacco. Bioinformatics analysis shows that there are a total of eight NtAITR genes in tobacco, and all the NtAITRs have a partially conserved LxLxL motif at their C-terminus. RT-PCR results show that the expression levels of at least some NtAITRs were increased in response to ABA and drought treatments, and NtAITRs, when recruited to the Gal4 promoter via a fused GD (Gal4 DNA-binding domain), were able to repress transcription activator LD-VP activated expression of the LexA-Gal4-GUS reporter gene. Roles of NtAITRs in regulating drought tolerance in tobacco were analyzed by generating CRISPR/Cas9 gene-edited mutants. A total of three Cas9-free ntaitr12356 quintuple mutants were obtained, and drought treatment assays show that drought tolerance was increased in the ntaitr12356 quintuple mutants. On the other hand, results of seed germination and seedling greening assays show that ABA sensitivity was increased in the ntaitr12356 quintuple mutants, and the expression levels of some ABA signaling key regulator genes were altered in the ntaitr12356-c3 mutant. Taken together, our results suggest that NtAITRs are ABA-responsive genes, and that NtAITRs function as transcription repressors and negatively regulate drought tolerance in tobacco, possibly by affecting plant ABA response via affecting the expression of ABA signaling key regulator genes.

Anthocyanins are an important group of water-soluble and non-toxic natural pigments with antioxidant and anti-inflammatory properties that can be found in flowers, vegetables, and fruits. Anthocyanin biosynthesis is regulated by several different types of transcription factors, including the WD40-repeat protein Transparent Testa Glabra 1 (TTG1), the bHLH transcription factor Transparent Testa 8 (TT8), Glabra3 (GL3), Enhancer of GL3 (EGL3), and the R2R3 MYB transcription factor Production of Anthocyanin Pigment 1 (PAP1), PAP2, MYB113, and MYB114, which are able to form MYB-bHLH-WD40 (MBW) complexes to regulate the expression of late biosynthesis genes (LBGs) in the anthocyanin biosynthesis pathway. Nasturtium (Tropaeolum majus) is an edible flower plant that offers many health benefits, as it contains numerous medicinally important ingredients, including anthocyanins. By a comparative examination of the possible anthocyanin biosynthesis regulator genes in nasturtium varieties with different anthocyanin contents, we found that TmPAP2, an R2R3 MYB transcription factor gene, is highly expressed in “Empress of India”, a nasturtium variety with high anthocyanin content, while the expression of TmPAP2 in Arabidopsis led to the overproduction of anthocyanins. Protoplast transfection shows that TmPAP2 functions as a transcription activator; consistent with this finding, some of the biosynthesis genes in the general phenylpropanoid pathway and anthocyanin biosynthesis pathway were highly expressed in “Empress of India” and the 35S:TmPAP2 transgenic Arabidopsis plants. However, protoplast transfection indicates that TmPAP2 may not be able to form an MBW complex with TmGL3 and TmTTG1. These results suggest that TmPAP2 may function alone as a key regulator of anthocyanin biosynthesis in nasturtiums.

The miRNA172–APETALA2 (AP2) regulatory module is a conserved mechanism governing floral development in plants. Disruption of the miR172 target sites in AP2 genes has been shown to be key to the domestication of double flowers in ornamental species. Camellia japonica, a woody ornamental plant with diverse floral forms, serves as an important model for studying double-flower formation. In this study, we characterized two AP2-like transcription factors, CjAP2-1 and CjAP2-2, which possess evolutionarily conserved miR172-binding sites and exhibit broad expression across floral tissues. To investigate the role of the miR172–AP2 module in C. japonica, we identified four members of the miR172 family and demonstrated that miR172 is directly involved in the cleavage of CjAP2-1 and CjAP2-2 transcripts. Through bulked amplicon sequencing of cultivars with diverse floral forms, we uncovered natural variations at the miR172-binding sites of CjAP2-1 and CjAP2-2, which can potentially disrupt miR172-mediated mRNA cleavage. We showed that two dinucleotide mutations (CjAP2-1-mut5 and CjAP2-1-mut9) significantly reduced the miR172-mediated repression of CjAP2-1 transcripts. Functional analysis in Arabidopsis revealed that overexpression of the CjAP2-1-mut5 variant caused significant floral abnormalities, including ectopic formation of reproductive organs, loss of floral determinacy, and fusion of floral organs. Further analysis of downstream genes indicated that key regulators of floral homeotic and meristem activity were markedly altered in the transgenic plants. Our findings demonstrate that perturbations in the miR172–AP2 regulatory relationship underlie the formation of double flowers in C. japonica by altering floral meristem determinacy and organ identity.