AtbZIP62 Acts as a Transcription Repressor to Positively Regulate ABA Responses 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.


Introduction
Due to their sessile nature, plants are constantly challenged by biotic stresses, such as various pathogens and abiotic stresses, including drought and high salinity [1]. To survive, plants had to respond properly to environmental stresses. The phytohormone abscisic acid (ABA) plays a key role in regulating plant responses to abiotic stresses [2,3]. The regulation of plant responses to abiotic stresses by ABA is usually linked to osmotic stress that can promote ABA accumulation [4,5]. Whereas ABA signal transduction leads to the activation/suppression of stress-related genes, affecting plant responses to abiotic stresses [6,7].

Expression of AtbZIP62 in Response to ABA and in Different Tissues and Organs
ABA regulates plant responses to abiotic stresses via activation/suppression of downstream genes [6,7]. Previous experiments have shown that the AtbZIP62 is involved in the regulation of plant responses to abiotic stresses such as drought and salinity [44,45]. To examine if ABA can regulate the expression of AtbZIP62, we compared the expression levels of AtbZIP62 in Arabidopsis seedlings treated with ABA and control samples. RNA was extracted from Arabidopsis seedlings treated with ABA or solvent for 4 h, and the expression of AtbZIP62 was determined using RT-PCR. As shown in Figure 1a, exogenous application of ABA significantly increased the expression level of AtbZIP62 when compared to the mock-treated control.
We also examine the expression patterns of AtbZIP62. RNA was isolated from different tissues and organs collected from Col wild-type Arabidopsis plants and used for RT-PCR analysis. As shown in Figure 1b, the transcripts of AtbZIP62 were detectable in all the tissues and organs examined with almost similar expression levels.
We also examine the expression patterns of AtbZIP62. RNA was isolated from different tissues and organs collected from Col wild-type Arabidopsis plants and used for RT-PCR analysis. As shown in Figure 1b, the transcripts of AtbZIP62 were detectable in all the tissues and organs examined with almost similar expression levels. (a) Expression of AtbZIP62 in response to ABA treatment. Col wild-type seedlings of10-day-old were treated for 4 h with 50 μM ABA, RNA was isolated and used for RT-PCR to examine the expression of AtbZIP62. ACT2 was used as a control for RT-PCR. (b) Expression pattern of AtbZIP62. Different tissues and organs were collected from the Col wild-type Arabidopsis plants. RNA was isolated and used for RT-PCR to examine the expression of AtbZIP62. ACT2 was used as a control gene for RT-PCR.

AtbZIP62 is a Transcription Repressor
As AtbZIP62 is a transcription factor whose transcriptional activities have not been tested, we examined the transcription activities using Arabidopsis protoplast transfection. We first examined the subcellular localization of AtbZIP62. Plasmids of GFP-AtbZIP62 and NLS-RFP, a nuclear marker gene construct, were co-transfected into Arabidopsis protoplasts, and the fluorescence of GFP and RFP were examined under a fluorescence microscope. As shown in Figure 2a, GFP fluorescence was predominantly observed in the nucleus.  (a) Expression of AtbZIP62 in response to ABA treatment. Col wild-type seedlings of10-day-old were treated for 4 h with 50 µM ABA, RNA was isolated and used for RT-PCR to examine the expression of AtbZIP62. ACT2 was used as a control for RT-PCR. (b) Expression pattern of AtbZIP62. Different tissues and organs were collected from the Col wild-type Arabidopsis plants. RNA was isolated and used for RT-PCR to examine the expression of AtbZIP62. ACT2 was used as a control gene for RT-PCR.

AtbZIP62 Is a Transcription Repressor
As AtbZIP62 is a transcription factor whose transcriptional activities have not been tested, we examined the transcription activities using Arabidopsis protoplast transfection. We first examined the subcellular localization of AtbZIP62. Plasmids of GFP-AtbZIP62 and NLS-RFP, a nuclear marker gene construct, were co-transfected into Arabidopsis protoplasts, and the fluorescence of GFP and RFP were examined under a fluorescence microscope. As shown in Figure 2a, GFP fluorescence was predominantly observed in the nucleus.
We also examine the expression patterns of AtbZIP62. RNA was isolated from different tissues and organs collected from Col wild-type Arabidopsis plants and used for RT-PCR analysis. As shown in Figure 1b, the transcripts of AtbZIP62 were detectable in all the tissues and organs examined with almost similar expression levels. (a) Expression of AtbZIP62 in response to ABA treatment. Col wild-type seedlings of10-day-old were treated for 4 h with 50 μM ABA, RNA was isolated and used for RT-PCR to examine the expression of AtbZIP62. ACT2 was used as a control for RT-PCR. (b) Expression pattern of AtbZIP62. Different tissues and organs were collected from the Col wild-type Arabidopsis plants. RNA was isolated and used for RT-PCR to examine the expression of AtbZIP62. ACT2 was used as a control gene for RT-PCR.

AtbZIP62 is a Transcription Repressor
As AtbZIP62 is a transcription factor whose transcriptional activities have not been tested, we examined the transcription activities using Arabidopsis protoplast transfection. We first examined the subcellular localization of AtbZIP62. Plasmids of GFP-AtbZIP62 and NLS-RFP, a nuclear marker gene construct, were co-transfected into Arabidopsis protoplasts, and the fluorescence of GFP and RFP were examined under a fluorescence microscope. As shown in Figure 2a, GFP fluorescence was predominantly observed in the nucleus.  Plasmids of the GD-AtbZIP62 effector, the LD-VP activator, and the LexA-Gal4:GUS reporter constructs were co-transfected into Arabidopsis protoplasts isolated from the Col wild-type plants, the transfected protoplasts were incubated in darkness for 20-22 h, and then GUS activity was assayed by using a microplate reader. Co-transfection of the GD construct plasmids served as a control. Data represent the mean ± SD of three replicates. * significantly different from the GD control (student's t-test, p < 0.001).
After showing that AtbZIP62 is a nucleus protein, we examined its transcriptional activities using a protoplast transfection system successfully used to study transcription repressors [48]. Plasmids of the reporter construct LexA-Gal4:GUS, the activator construct LD-VP, and the effector construct GD-AtbZIP62 or the control construct GD were cotransfected into Arabidopsis protoplasts. After overnight incubation of the transfected protoplasts in the dark, GUS activities were measured using a microplate reader. As shown in Figure 2b, co-transfection of LD-VP and GD activated the reporter gene, whereas cotransfection of LD-VP with GD-AtbZIP62 resulted in repressed expression of the reporter gene, suggesting that AtbZIP62 functions as a transcription repressor. CRISPR/Cas9 mediated genome editing was used to generate the atbzip62 mutants. Two target sites in the exon of AtbZIP62 were selected, and the corresponding sequence was used to generate the pHEE-FT-AtbZIP62 construct using the pHEE-FT vector [49]. The construct was transformed into the Col wild-type Arabidopsis plants. Editing status in early bolting T1 plants was examined, and Cas9-free homozygous mutants were isolated from T2 plants. Two independent homozygous mutants were chosen for further experiments. In the atbzip62-c1 mutant, a deletion of 825 nucleotides fragment occurred in AtbZIP62 (Figure 3a), resulting in a 275 amino acids deletion (Figure 3b). In the atbzip62-c2 mutant, a single nucleotide was inserted in AtbZIP62 (Figure 3a), leading to the substitution of a few amino acids and a premature stop in AtbZIP62 (Figure 3b). 20-22 h, and then GUS activity was assayed by using a microplate reader. Co-transfection of the GD construct plasmids served as a control. Data represent the mean ± SD of three replicates. * significantly different from the GD control (student's t-test, p < 0.001).
After showing that AtbZIP62 is a nucleus protein, we examined its transcriptional activities using a protoplast transfection system successfully used to study transcription repressors [48]. Plasmids of the reporter construct LexA-Gal4:GUS, the activator construct LD-VP, and the effector construct GD-AtbZIP62 or the control construct GD were co-transfected into Arabidopsis protoplasts. After overnight incubation of the transfected protoplasts in the dark, GUS activities were measured using a microplate reader. As shown in Figure 2b, co-transfection of LD-VP and GD activated the reporter gene, whereas co-transfection of LD-VP with GD-AtbZIP62 resulted in repressed expression of the reporter gene, suggesting that AtbZIP62 functions as a transcription repressor.

Generation of the 35S:AtbZIP62 and the 35S:AtbZIP62-GR Transgenic Plants, and the Gene-Edited atbzip62 Mutants
To examine the functions of AtbZIP62, we generated plants overexpressing AtbZIP62 under the control of 35S promoter (35S:AtbZIP62) and CRISPR/Cas9 gene-edited atbzip62 mutants.
CRISPR/Cas9 mediated genome editing was used to generate the atbzip62 mutants. Two target sites in the exon of AtbZIP62 were selected, and the corresponding sequence was used to generate the pHEE-FT-AtbZIP62 construct using the pHEE-FT vector [49]. The construct was transformed into the Col wild-type Arabidopsis plants. Editing status in early bolting T1 plants was examined, and Cas9-free homozygous mutants were isolated from T2 plants. Two independent homozygous mutants were chosen for further experiments. In the atbzip62-c1 mutant, a deletion of 825 nucleotides fragment occurred in At-bZIP62 (Figure 3a), resulting in a 275 amino acids deletion ( Figure 3b). In the atbzip62-c2 mutant, a single nucleotide was inserted in AtbZIP62 (Figure 3a), leading to the substitution of a few amino acids and a premature stop in AtbZIP62 (Figure 3b). Total RNA was isolated from seedlings of 10-day-old homozygous transgenic plants and used for RT-PCR analysis to examine the expression of AtbZIP62. ACT2 was used as a control.
The 35S:AtbZIP62 overexpression transgenic plants were generated by transforming the Col wild-type Arabidopsis plants with pPZP211-35S:AtbZIP62. In the T3 generation, homozygous plants were selected, and the expression level of AtbZIP62 in homozygous transgenic plants was examined. The #22 and #33, two independent lines with similar expression levels of the AtbZIP62 gene (Figure 3c), were selected and used for the following experiments. To examine genes that may be regulated by AtbZIP62, we generated 35S:AtbZIP62-GR transgenic plants, in which the AtbZIP62 protein was fused with the glucocorticoid receptor (GR) (Figure 3d).

AtbZIP62 Positively Regulates ABA Response in Arabidopsis
Having shown that AtbZIP62 is an ABA-responsive gene (Figure 1a), we further explored the functions of AtbZIP62 in regulating ABA responses in Arabidopsis by using the overexpression transgenic plants and mutants generated. ABA-inhibited seed germination and cotyledon greening assays were used to examine ABA responses of the Col wild-type, the 35S:AtbZIP62 transgenic plants, and the atbzip62 mutants. In the seed germination assays, we found that seed germination of different genotypes was similar in the absence of ABA, i.e., almost all the seeds germinated 36 h after the plates were transferred to the growth room ( Figure 4). In the presence of ABA, however, lower germination rates were observed for seeds of the 35S:AtbZIP62 transgenic plants, whereas higher germination rates were observed for seeds of the atbzip62 mutants at all the time points examined (Figure 4), indicating that ABA sensitive was reduced in the atbzip62 mutants but increased in the 35S:AtbZIP62 transgenic plants.   kept at 4 • C and in darkness for 2 days, and then transferred to a growth room. The number of germinated seeds was counted every 12 h after the transfer until all the seeds were germinated. The percentage of seed germination was then calculated. Data represent the mean ± SD of three replicates.
Similar results were obtained in the cotyledon greening assays. As shown in Figure 5a, in the presence of ABA, the numbers of green seedlings of the 35S:AtbZIP62 transgenic overexpression plants were lower than those of Col wild type plants, while that of the atbzip62 mutant plants were clearly higher than those of Col wild type plants. Quantitative results show that at the present of ABA, the cotyledon greening rate for the Col wild type was~50%, that for the 35S:AtbZIP62 transgenic plants was~20%, whereas that for the atbzip62 mutants was~100% (Figure 5b). These results suggest that AtbZIP62 positively regulates ABA responses in Arabidopsis. . Effect of ABA on seed germination of the Col wild type, the 35S:AtbZIP62 transgenic plants, and the atbzip62 mutants. Sterilized seeds were sown on 1/2 MS plates with or without 1 μM ABA, kept at 4 °C and in darkness for 2 days, and then transferred to a growth room. The number of germinated seeds was counted every 12 h after the transfer until all the seeds were germinated. The percentage of seed germination was then calculated. Data represent the mean ± SD of three replicates.
Similar results were obtained in the cotyledon greening assays. As shown in Figure  5a, in the presence of ABA, the numbers of green seedlings of the 35S:AtbZIP62 transgenic overexpression plants were lower than those of Col wild type plants, while that of the atbzip62 mutant plants were clearly higher than those of Col wild type plants. Quantitative results show that at the present of ABA, the cotyledon greening rate for the Col wild type was ~50%, that for the 35S:AtbZIP62 transgenic plants was ~20%, whereas that for the at-bzip62 mutants was ~100% (Figure 5b). These results suggest that AtbZIP62 positively regulates ABA responses in Arabidopsis. (b) Quantitative assays of cotyledon greening of the Col wild type, the 35S:AtbZIP62 transgenic plants, and the atbzip62 mutants in the presence of 1 µM ABA. Seedlings with green cotyledons were counted 8 days after the transfer, and the percentage of green seedlings was calculated. Data represent means ± SD of three replicates. * significantly different from that of the Col wild type (student's t-test, p < 0.005).

AtbZIP62 Regulated Genes Are Enriched in Response to ABA
To explore the functional mechanism of AtbZIP62 in regulating ABA response in Arabidopsis, we decided to investigate the downstream gene whose response to ABA may be regulated by AtbZIP62. We, therefore, generated the 35S:AtbZIP62-GR transgenic plants ( Figure 3d) and used them for transcriptome analysis.
In transgenic plants, the fusion proteins cannot enter the nucleus in normal conditions and are not functional, but they can enter the nucleus when treated with dexamethasone (DEX), and AtbZIP62 can regulate the expression of its downstream genes. We, therefore, performed comparative transcriptome analysis with 10-day-old seedlings of the 35S:AtbZIP62-GR plants treated with 50 µM ABA and 50 µM ABA + 10 µM DEX for 4 h. Since AtbZIP62 functions as a transcriptional repressor (Figure 2b), we focus on the genes that were down-regulated in ABA + DEX-treated seedlings.
A total of 231 different expressed genes (DEGs) were found down-regulated by ABA + DEX treatment in 35S:AtbZIP62-GR transgenic plant seedlings (Table S1). Gene ontology (GO) term enrichment analysis reveals that these genes were enriched in several different processes such as transport, including nitrate transport, chloride transmembrane transport, calcium ion transmembrane transport, biosynthetic process (including ethy-lene biosynthetic, thiamine biosynthetic, and cinnamic acid biosynthetic), and fatty acid biosynthetic, regulation of DNA-template transcription, regulation of seed germination, glucose-mediated signaling pathway, and responses to environmental stimuli including wounding cold and salt stress. However, the genes were mostly enriched in response to ABA ( Figure 6, Table S1).
Since AtbZIP62 functions as a transcriptional repressor (Figure 2b), we focus on the genes that were down-regulated in ABA + DEX-treated seedlings.
A total of 231 different expressed genes (DEGs) were found down-regulated by ABA + DEX treatment in 35S:AtbZIP62-GR transgenic plant seedlings (Table S1). Gene ontology (GO) term enrichment analysis reveals that these genes were enriched in several different processes such as transport, including nitrate transport, chloride transmembrane transport, calcium ion transmembrane transport, biosynthetic process (including ethylene biosynthetic, thiamine biosynthetic, and cinnamic acid biosynthetic), and fatty acid biosynthetic, regulation of DNA-template transcription, regulation of seed germination, glucose-mediated signaling pathway, and responses to environmental stimuli including wounding cold and salt stress. However, the genes were mostly enriched in response to ABA ( Figure 6, Table S1).

AtbZIP62 Regulates ABA-Induced Expression of Some ABA and Abiotic Stress Response Regulator Genes
Among the DEGs down-regulated in ABA + DEX treated 35S:AtbZIP62-GR transgenic plant seedlings, several genes, including USP, SnRK2.7, and ABF2, have been well studied and are known to be related to ABA and abiotic stress responses. The expression of USP, a universal stress protein gene, is induced by ABA, and ABA sensitivity in the atusp mutant was increased, as indicated by seed germination assays [50]. SnRK2.7 responds strongly to osmotic stress and ABA [51], whereas ABF2 is a master regulator of ABA signaling, and the expression of ABF2 is induced by ABA [33]. We, therefore, examined their expression in ABA and ABA + DEX treated 35S:AtbZIP62-GR transgenic plant seedlings using qRT-PCR. As shown in Figure 7a, ABA response of all three genes was reduced, consistent with the transcriptome data (Table S1). We also examined the ABA-induced expression of USP, SnRK2.7, and ABF2 in the 35S:AtbZIP62 transgenic plant and the atbzip62 mutant seedlings, and found that ABA-induced expression of all three genes was reduced in the 35S:AtbZIP62 transgenic plant seedlings (Figure 7b). ABA signaling, and the expression of ABF2 is induced by ABA [33]. We, therefore, examined their expression in ABA and ABA + DEX treated 35S:AtbZIP62-GR transgenic plant seedlings using qRT-PCR. As shown in Figure 7a, ABA response of all three genes was reduced, consistent with the transcriptome data (Table S1). We also examined the ABAinduced expression of USP, SnRK2.7, and ABF2 in the 35S:AtbZIP62 transgenic plant and the atbzip62 mutant seedlings, and found that ABA-induced expression of all three genes was reduced in the 35S:AtbZIP62 transgenic plant seedlings (Figure 7b). μM ABA in the presence and absence of 10 μM DEX for 4 h. RNA was then isolated and used for qRT-PCR analysis. ACT2 was used as an inner control, and fold changes were calculated by comparing the expression levels of the corresponding genes in ABA-treated with control seedlings in the presence and absence of DEX, respectively. Data represent the mean ± SD of three replicates. * significantly different from ABA-treated seedlings (student's t-test, p < 0.005). (b) ABA-induced expression of USP, SnRK2.7, and ABF2 in the 35S:AtbZIP62 transgenic plant and atbzip62 mutant seedlings. Seedlings of 10-days-old Col wild type, the 35S:AtbZIP62 transgenic plants, and the ztbzip62 mutants were treated with 50 μM ABA for 4 h. RNA was then isolated and subjected to qRT-PCR analysis. ACT2 was used as an inner control, and fold changes were calculated by comparing the expression levels of the corresponding genes in ABA-treated with control seedlings. Data represent the mean ± SD of three replicates. * significantly different from that in the Col wild-type seedlings (student's t-test, p < 0.05).

Discussion
The bZIP transcription factor family is one of the largest transcription factor plant families and is involved in the regulation of a wide range of biological activities, including plant growth, development, and responses to abiotic and biotic stresses [22]. So far, several bZIP transcription factors, especially those from group A, including bZIP36/ABF2, bZIP37/ABF3, and bZIP38/ABF4 have been shown to be involved in the regulation of plant abiotic stress responses via regulating ABA responses [15,52,53]. It has been previously shown that AtbZIP62, a group V bZIP transcription factor [54], regulates plant responses ACT2 was used as an inner control, and fold changes were calculated by comparing the expression levels of the corresponding genes in ABA-treated with control seedlings in the presence and absence of DEX, respectively. Data represent the mean ± SD of three replicates. * significantly different from ABA-treated seedlings (student's t-test, p < 0.005). (b) ABA-induced expression of USP, SnRK2.7, and ABF2 in the 35S:AtbZIP62 transgenic plant and atbzip62 mutant seedlings. Seedlings of 10-days-old Col wild type, the 35S:AtbZIP62 transgenic plants, and the ztbzip62 mutants were treated with 50 µM ABA for 4 h. RNA was then isolated and subjected to qRT-PCR analysis. ACT2 was used as an inner control, and fold changes were calculated by comparing the expression levels of the corresponding genes in ABA-treated with control seedlings. Data represent the mean ± SD of three replicates. * significantly different from that in the Col wild-type seedlings (student's t-test, p < 0.05).

Discussion
The bZIP transcription factor family is one of the largest transcription factor plant families and is involved in the regulation of a wide range of biological activities, including plant growth, development, and responses to abiotic and biotic stresses [22]. So far, several bZIP transcription factors, especially those from group A, including bZIP36/ABF2, bZIP37/ABF3, and bZIP38/ABF4 have been shown to be involved in the regulation of plant abiotic stress responses via regulating ABA responses [15,52,53]. It has been previously shown that AtbZIP62, a group V bZIP transcription factor [54], regulates plant responses to abiotic stresses such as drought and salinity [44,45]. We provide evidence here that AtbZIP62 is an ABA-responsive gene, and AtbZIP62 regulates ABA responses in Arabidopsis.
It has been shown that the expression of AtbZIP36 and AtbZIP38 genes were induced by high-salt and ABA treatments [55]. Our data indicate that the expression level of AtbZIP62 is significantly increased in response to exogenous ABA treatment (Figure 1a). To understand the roles of the AtbZIP62 gene in Arabidopsis, we analyzed the expression pattern of the AtbZIP62 in different tissues and organs, and found that AtbZIP62 expressed at almost similar levels in all the tissues and organs examined (Figure 1b). The results suggest that AtbZIP62 may not play a particular role in plant growth and development. Protoplast transfection assays indicated that AtbZIP62 is a nuclear protein (Figure 2a) and can reduce the expression of the reporter gene activated by a transcription activator, indicating that AtbZIP62 is a transcription repressor (Figure 2b). This observation is consistent with the observation that most bZIP proteins are exclusively localized in the nucleus to exert their function as transcription factors [56].
It has been shown that ABA is involved in regulating seed dormancy, seed germination, and seedling growth [57] and that abiotic stresses can induce ABA biosynthesis and trigger ABA-dependent signaling transduction [58]. So far, most bZIPs that bind to ABREs, such as ABI5, ABRE1/ABF2, ABRE2/ABF4, and ABF1, are reported to belong to group A in bZIP transcription factors in Arabidopsis, and their gene expression is strongly induced by ABA and abiotic stresses [59,60]. A previous study investigated that AREB1 and AREB2 are the most well-known bZIP transcription factors that act as fundamental components in regulating ABRE-dependent gene expression. Overexpression analysis reveals that ABF2, ABF3, ABF4, and ABI5 transgenic plants are more sensitive to ABA during seed germination and later growth stage [61,62]. Our results suggest that group V bZIP transcript factor AtbZIP62 also positively regulates ABA response in Arabidopsis, as the transgenic plants overexpressing AtbZIP62 showed a hypersensitivity, while the atbzip62 mutant showed a hyposensitivity response to ABA in both seed germination and green cotyledon assays (Figures 4 and 5a,b).
Previous research has revealed that Arabidopsis bZIPs play various roles in plant growth, environmental signaling, and stress response [26]. Transcriptomic analysis revealed that DEGs downregulated in ABA + DEX treated 35S:AtbZIP62-GR transgenic plant seedlings enriched in biological processes such as response to abscisic acid, wounding, and abiotic stimulus ( Figure 6, Table S1). ABA-responsive genes within these clusters, including USP, SnR2.7, and ABF2, whose expression levels in response to ABA are significantly reduced in the 35S:AtbZIP62-GR transgenic plants treated with DEX ( Figure 7a). Therefore, it can be speculated that AtbZIP62 overexpression affected ABA-regulated gene expression, which, in turn, affected plant response to abiotic stresses, as reported previously [44,45]. In addition, the results shown in Figure 7b indicate that the expression of USP, SnRK2.7, and ABF2 in response to ABA was affected in the AtbZIP62 overexpress lines and the atbzip62 mutants. Considering that USP and ABF2 are regulated by phytohormones and involved in regulating seed germination in Arabidopsis [42,50], it is likely that AtbZIP62 regulates seed germination via regulating the expression of USP and ABF2.

Plant Materials and Growth Conditions
The Columbia-0 (Col) wild-type Arabidopsis (Arabidopsis thaliana) stored in our lab was used as a control for gene expression pattern and ABA sensitivity assays, for protoplasts isolation, and for plant transformation to generate transgenic overexpression plants and gene-edited mutants.
For ABA treatment, gene expression analysis, transcriptome analysis, seed germination, and cotyledon greening assays, seeds of the Col wild-type, the AtbZIP62 and AtbZIP62-GR transgenic plants, and the atbzip62 mutants were surface sterilized, rinsed with sterilized water, and then sowed on 1 2 MS (Murashige and Skoog) plates containing 1% sucrose and 0.6% agar. The plates were incubated in darkness at 4 • C for two days before being transferred to a growth room. For gene expression pattern assay, protoplast isolation, and plant transformation, the Col wild-type seeds were germinated and grown in soil pots in a growth room. The growth room conditions were set at 22 • C with a light density of~120 µmol m −2 s −1 under 16/8 h light/dark conditions.

DNA and RNA Isolation, RT-PCR, and Quantitative RT-PCR (qRT-PCR)
DNA and total RNA were extracted, and cDNA was synthesized as previously described [63,64]. In brief, the total RNA in the samples collected was isolated by using the EasyPure Plant RNA Kit (TransGene Biotech, Beijing, China), and cDNA was synthesized by using the EasyScript FirstStrand DNA Synthesis Super Mix (TransGen Biotech, Beijing, China) according to the manufacturer's protocols. DNA was isolated using a DNA isolation buffer, as described previously [65].
For expression pattern assay of AtbZIP62, tissues and organs were collected from Col wild-type Arabidopsis plants grown in soil pots. To examine the expression of AtbZIP62 in response to ABA treatment, ABA-treated and control seedlings were collected. To examine the expression of AtbZIP62 in the overexpression of transgenic plants, seedlings of homozygous transgenic plants were collected. The collected samples were frozen in liquid N 2 immediately and stored at −80 • C for RNA isolation.
To examine gene editing status, DNA was extracted from the leaves of T1 or T2 transgenic plants. DNA isolated from T1 transgenic plants was used for PCR amplification of AtbZIP62 genome sequences. The PCR products were isolated and sequenced, and the sequencing results were aligned with the wild-type AtbZIP62 sequence to select the gene-edited T1 transgenic plants. DNA isolated from T2 progeny of the altered T1 plants was used for PCR amplification to isolate Cas9-free homozygous mutants. The primers used to amplify the AtbZIP62 genomic sequence are AtbZIP62-F 5 -ATGGAGTTGGAGCCTATATCA-3 and AtbZIP62-R 5 -TTATCCGACCTGCATCC-3 . The primers used for PCR amplification of Cas9 have been described previously [66].
To generate GD-AtbZIP62 and GFP-AtbZIP62 constructs, the full-length open reading frame (ORF) of AtbZIP62 was RT-PCR amplified using RNA isolated from 10-day-old Col seedlings as described previously [63,64] and cloned into the pUC19 vector under the control of the CaMV 35S promoter, and in frame with an N-terminal GD tag for transcriptional activity assay or a GFP tag for subcellular localization assay.
To generate 35S:AtbZIP62 constructs, the full-length ORF sequence of AtbZIP62 was amplified and cloned into the pUC19 vector with an in-frame N-terminal HA tag and under the control of the 35S promoter [68,71]. To generate the 35S:AtbZIP62-GR construct for plant transformation, the full-length ORF of AtbZIP62 without stop codon was PCR amplified and cloned in frame with an N-terminal HA tag a C-terminal GR tag into pUC19 vector. The pUC19-35S:AtbZIP62 and pUC19-35S:AtbZIP62-GR constructs were digested with corresponding restriction enzymes and then cloned, respectively, into the binary vector pPZP211 [72]. The primers (underlines indicate enzyme sites) used to amplify AtbZIP62 were 5 -CAACATATGGAGTTGGAGCCTATATCA-3 and 5 -CAAGAGCTCTTATCCGACC TGCATCC-3, the reverse primer used to amplify AtbZIP62 without stop codon was 5 -CAAGAGCTCTCCGACCTGCATCCGAC-3 .
For the subcellular localization assay, plasmids of the effector construct GFP-AtbZIP62 and nuclear indicator construct NLS-RFP were co-transfected into the Arabidopsis protoplasts. For the transcriptional activity assays, the reporter plasmid construct LexA-Gal4:GUS, the activator construct LD-VP, and the effector construct GD-AtbZIP62, or the control construct GD, were co-transfected into Arabidopsis protoplasts. The transfected Arabidopsis protoplasts were incubated at room temperature for 18-22 h in darkness. Fluorescence of GFP and RFP was observed under a fluorescent microscope (Olympus, Tokyo, Japan), and GUS activities were measured using a Synergy HT fluorescence microplate reader (BioTEK, Vermont, USA).

Plant Transformation and Transgenic Plants Selection
Col wild-type plants about 5-week-old and with several mature flowers on the main inflorescence stems were used for transformation by using the floral dip method [77]. The plants were transformed with the 35S:AtbZIP62, the 35S:AtbZIP62-GR, and the CRISPR/Cas9 constructs via GV3101 agrobacterium cells to obtain transgenic overexpression plants, as well as gene-edited mutants for gene AtbZIP62.
To select transgenic plants, T1 seeds were collected from the transformed plants and plated on 1 2 MS plates containing 100 µg/mL carbenicillin and 50 µg/mL kanamycin, and the transgenic plants selected were transplanted to soil pots and grown in a growth room. For 35S:AtbZIP62 and 35S:AtbZIP62-GR overexpression plants, multiple homozygous lines were obtained, and the ones with high expression levels of AtbZIP62 were used for the experiments.
The atbzip62 Cas9-free gene-edited mutants were obtained by following the procedure described previously [49]. Briefly, the editing status of the AtbZIP62 gene in T1 plants with early flowering phenotypes was examined. T2 seeds were collected from gene-edited T1 plants and sowed directly into soil pots, and the editing status of the AtbZIP62 gene in the normal flowering (Cas9-free) was examined to identify homozygous mutants. Cas9-free status was further confirmed by amplifying the Cas9 fragment in the homozygous mutants.

ABA Treatment
To examine the expression of AtbZIP62 in response to ABA treatment, seedlings of 10-day-old Col wild-type plants were transferred to a 50 mL falcon tube containing 50 µM ABA and shaken in the dark for 4 h. To examine the expression of USP, SnRK2.7, and ABF2 in Col wild-type, the 35S:AtbZIP62 transgenic plants and the atbzip62 mutant, seedlings of 10-day-old Col wild type, the 35S:AtbZIP62 transgenic plants and the atbzip62 mutants were treated with 50 µM ABA for 4 h.

Seed Germination and Cotyledon Greening Assays
For phenotypic analysis, seeds were germinated and grown on 1 2 MS supplemented with or without 1 µm ABA. Seed germination was judged based on the emergence of an embryonic axis protrusion. Cotyledon greening was judged based on observing cotyledon expansion and turning green. Three independent experiments were carried out, and similar results were obtained.

Transcriptome Analysis
For transcriptome analysis, 10-day-old seedlings of the 35S:AtbZIP62-GR transgenic plants were transferred to a falcon tube and treated for 4 h with 50 µM ABA or 50 µM ABA + 10 µM DEX, frozen in liquid N 2 and sent to the company, Beijing Genomics Institute for transcriptome sequencing and analysis.
Down-regulated DEGs in ABA + DEX treated vs. ABA treated seedlings were subject to functional Gene Ontology (GO) under the term of biological process. The pHyper function in R software was used and estimated by hypergeometric test for the gene ontology (GO) annotation enrichment of DEGs analysis. GO terms with a Q value (corrected p-value) equal to 0.05 were considered significantly enriched.

Conclusions
Our results in this study show that AtbZIP62 is an ABA-responsive gene. AtbZIP62 functions as a transcription repressor and positively regulates ABA responses in Arabidopsis, possibly via regulating the expression of some ABA signaling regulator genes.