Both seed size and abiotic stress tolerance are important agronomic traits in crops. In Arabidopsis, two closely related transcription repressors DPA4 (Development-Related PcG Target in the APEX4)/NGAL3 and SOD7 (Suppressor of da1-1)/NGAL2 (NGATHA-like protein) function redundantly to regulate seed size, which was increased in the dpa4 sod7 double mutants. Whereas ABA-induced transcription repressors (AITRs) are involved in the regulation of ABA signaling and abiotic stress tolerance, Arabidopsis aitr2 aitr5 aitr6 (aitr256) triple mutant showed enhanced tolerance to drought and salt. Here we performed CRISPR/Cas9 genome editing to disrupt DPA4 and SOD7 in aitr256 mutant, trying to integrate seed size and abiotic stress tolerance traits in Arabidopsis, and also to examine whether DPA4 and SOD7 may regulate other aspects of plant growth and development. Indeed, seed size was increased in the dpa4 sod7 aitr256 quintuple mutants, and enhanced tolerance to drought was observed in the mutants. In addition, we found that shoot branching was affected in the dpa4 sod7 aitr256 mutants. The mutant plants failed to produce secondary branches, and flowers/siliques were distributed irregularly on the main stems of the plants. Floral organ number and fertility were also affected in the dpa4 sod7 aitr256 mutant plants. To examine if these phenotypes were dependent on loss-of-function of AITRs, dpa4 sod7 double mutants were generated in Col wild type background, and we found that the dpa4 sod7 mutant plants showed a phenotype similar to the dpa4 sod7 aitr256 quintuple mutants. Taken together, our results indicate that the integration of seed size and abiotic stress tolerance traits by CRISPR/Cas9 editing was successful, and our results also revealed a role of DPA4 and SOD7 in the regulation of inflorescence architecture in Arabidopsis.

Trichome formation in Arabidopsis is regulated by several key regulators, and plants hormones such as gibberellin, salicylic acid, jasmonic acid and cytokinins have been shown to regulate trichome formation by affecting the transcription or activities of the key regulators. We report here the identification of two abscisic acid (ABA) responsive genes, SMALLER TRICHOMES WITH VARIABLE BRANCHES (SVB) and SVB2 as trichome formation regulator genes in Arabidopsis. The expression levels of SVB and SVB2 were increased in response to ABA treatment, their expression levels were reduced in the ABA biosynthesis mutant aba1-5, and they have similar expression pattern. In addition to the trichome defects reported previously for the svb single mutant, we found that even though the trichome numbers were largely unaffected in both the svb and svb2 single mutants generate by using CRISPR/Cas9 gene editing, the trichome numbers were greatly reduced in the svb svb2 double mutants. On the other hand, trichome numbers were increased in SVB or SVB2 overexpression plants. RT-PCR results show that the expression of the trichome formation key regulator gene ENHANCER OF GLABRA3 (EGL3) was affected in the svb svb2 double mutants. Our results suggest that SVB and SVB2 are ABA responsive genes, and SVB and SVB2 function redundantly to regulate trichome formation in Arabidopsis.

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.

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 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.