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.

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.

The abscisic acid (ABA) signaling pathway is crucial for regulating downstream ABA-responsive genes, which influence plant responses to ABA and abiotic stresses. However, many ABA-responsive genes remain poorly characterized. This study reports on the identification and characterization of ABA up-regulated EAR motif-containing proteins (AtAUEs), a novel family of EAR motif-containing proteins in Arabidopsis thaliana. From a previous transcriptome dataset, AtAUEs were identified as a family of unknown-function ABA-response genes with only five members, and the up-regulation of AtAUEs by ABA was further confirmed by quantitative RT-PCR (qRT PCR). All AtAUEs contain at least one LxLxL EAR motif and can repress reporter gene expression in Arabidopsis protoplasts. We generated CRISPR/Cas9 gene-edited ataue1, ataue2 and ataue3 single, ataue1 ataue2 (ataue12) double, and ataue1 ataue2 ataue3 (ataue123) triple mutants, as well as transgenic plants overexpressing AtAUE1, and examined their ABA sensitivity. We found that the single and double mutants displayed wild-type responses to ABA treatment, while the ataue123 triple mutants showed increased sensitivity in seed germination and cotyledon greening assays but decreased sensitivity to ABA treatment in root elongation assays. Conversely, the 35S:AtAUE1 showed decreased sensitivity in seed germination and cotyledon greening assays but increased sensitivity to ABA treatment in root elongation assays. The qRT PCR results show that the expression level of ABI5 was increased in the ataue123 mutants and decreased in the 35S:AtAUE1 plants. These findings suggest that AtAUEs function redundantly to regulate ABA responses in Arabidopsis, likely by modulating the expression of key regulatory genes in ABA-signaling pathway.

The modulation of plant responses to abscisic acid (ABA) and/or abiotic stresses can be manipulated by the expression of ABA-responsive genes, which is affected by phytohormone ABA. While some ABA-responsive genes have been shown to regulate plant responses to ABA and/or abiotic stresses, the functions of numerous ABA-responsive genes remain unknown. Therefore, characterizing these unstudied genes would provide a practical way to identify novel regulators of plant adaptations to ABA and/or abiotic stresses. Here, we characterized four closely related unstudied ABA-responsive genes in Arabidopsis thaliana, named Arabidopsis thaliana ABA-up regulated genes (AtAUGs). We found that ABA treatment induces AtAUGs expression level, and our results in transfected protoplasts show that AtAUGs exhibit nucleus localization and downregulate the co-transfected reporter expression level. The results of ABA sensitivity assays, including seed germination, cotyledon greening, and root extension assay show that transgenic plants overexpressing AtAUGs had increased sensitivity, but ataugs mutants generated by isolating T-DNA insertion lines or through CRISPR/Cas9 gene-editing of AtAUGs had decreased sensitivity; in addition, the greatest decrease in ABA sensitivity was observed in the ataug1 ataug2 ataug3 ataug4 (ataug1234) quadruple mutants. The qRT-PCR results show that the expression levels of several Type 2C Protein Phosphatase (PP2C) genes, the key negative regulator genes of ABA signaling including PP2CA, Hypersensitive to ABA 1 (HAB1), HAB2, Highly ABA-Induced PP2C protein 3 (HAI3), ABA-Hypersensitive Germination 1 (AHG1), and ABA Insensitive 2 (ABI2) decreased in 35S:AtAUGs transgenic plants, but increased in the ataug1234 quadruple mutants. Taken together, these results suggest that AtAUGs are ABA-responsive genes, and AtAUGs positively regulate ABA responses in a redundant manner, by downregulating the expression of crucial negative regulator genes in ABA signaling.