The R2R3 MYB Transcription Factor MYB71 Regulates Abscisic Acid Response 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.


Introduction
MYB transcription factors are one of the largest transcription factor families in plants, and there are~200 genes in Arabidopsis encoding MYB transcription factors [1]. According to the number of N-terminal DNA binding domain repeats, MYB family transcription factors can be divided into four subfamilies (i.e., 1R or R3 MYBs, 2R or R2R3 MYBs, 3R or R1R2R3 MYBs and 4R or R1R2R3R4 MYBs), with R2R3 MYB proteins being the largest MYB transcription factor subfamily with 126 members in Arabidopsis [1,2].
As mentioned above, ABA regulates the expression of ABA responsive genes and thereby plant responses to abiotic stresses, suggest that ABA response genes are involved in the regulation of plant responses to abiotic stresses. Indeed, some ABA response genes have been shown to be involved in the regulation of plant abiotic stresses responses. For example, the expression level of the R2R3 MYB transcription gene MYB44 was increased in response to ABA treatment, and Arabidopsis transgenic plants overexpressing MYB44 showed an enhanced tolerance to drought and salt stresses [44], the heat shock factor gene HSFA6b is an ABA responsive gene, and it plays a role in regulating drought and salt tolerance [45], overexpression of AFBA1 (ABA-responsive FBA (F-box-associated) domaincontaining protein 1) enhanced plant drought [46], and the expression of bHLH transcription factor gene bHLH112 is induced by ABA, and it positively regulate drought and salt tolerance in Arabidopsis [47]. Yet the functions of most ABA responsive genes remain to be characterized. By analyzing uncharacterized ABA responsive genes, and examining their roles in regulating ABA responses first, we have successful identified several abiotic stress response regulator in Arabidopsis. For example, we found that the expression of two closely WD40 genes, ABA induced WD40-repeat 1 (AIW1) and AIW2, the non-DNA binding bHLH transcription factor gene Paclobutrazol Resistance 6 (PRE6), and a novel transcription factor family genes ABA-induced transcription repressors (AITRs) were all induced by ABA treatment [35,48,49], and then we showed that the aiw1 aiw2 mutants and the transgenic Arabidopsis plants overexpressing PRE6 showed increased sensitivity to ABA [48,49], whereas loss-of-function of AITR genes in Arabidopsis led to reduced sensitivity to ABA [35,36], suggest that these regulators regulate ABA response in Arabidopsis, indicating that they may be involved in the regulation of plant responses to abiotic stresses. Indeed, salt tolerance was reduced in Arabidopsis transgenic plants overexpressing AIW1 [48], Arabidopsis transgenic plants overexpressing PRE6 showed enhanced salt tolerance [49], and loss-of-function of AITR genes led to enhanced tolerance to abiotic stresses including drought as well as salt [35,36]. These results indicate that it is practicable to identify abiotic stress regulators by analyzing the roles of uncharacterized ABA responsive genes in regulating ABA responses in plants.
We report here the characterization of MYB71, a R2R3 MYB transcription factor in regulating ABA response in Arabidopsis. We found that the expression level of MYB71 is increased in response to ABA, MYB71 is able to activate reporter gene expression, and overexpression or mutation of MYB71 affected plant response to ABA.

Expression of MYB71 Is Induced by ABA Treatment
In the process of identifying abiotic stress regulators by examining the roles ABA response genes in regulating plant ABA responses as described previously [35], we found that the expression of the Arabidopsis R2R3 MYB transcription factor gene MYB71 was induced by ABA treatment ( Figure 1A). Since MYB71 is closely related to MYB79 and they formed a function uncharacterized small cluster together with MYB121 [2], we also examined the expression of MYB79 and MYB121 in response to ABA. As shown in Figure 1A, the expression levels of MYB79 and MYB121 increased sharply after the seedlings were treated with ABA.
cluding drought as well as salt [35,36]. These results indicate that it is practicable to identify abiotic stress regulators by analyzing the roles of uncharacterized ABA responsive genes in regulating ABA responses in plants.
We report here the characterization of MYB71, a R2R3 MYB transcription factor in regulating ABA response in Arabidopsis. We found that the expression level of MYB71 is increased in response to ABA, MYB71 is able to activate reporter gene expression, and overexpression or mutation of MYB71 affected plant response to ABA.

Expression of MYB71 Is Induced by ABA Treatment
In the process of identifying abiotic stress regulators by examining the roles ABA response genes in regulating plant ABA responses as described previously [35], we found that the expression of the Arabidopsis R2R3 MYB transcription factor gene MYB71 was induced by ABA treatment ( Figure 1A). Since MYB71 is closely related to MYB79 and they formed a function uncharacterized small cluster together with MYB121 [2], we also examined the expression of MYB79 and MYB121 in response to ABA. As shown in Figure 1A, the expression levels of MYB79 and MYB121 increased sharply after the seedlings were treated with ABA.
Amino acid sequence identify and similarity assays indicates that MYB71 shared 70.88% amino acid identify and 74.71% amino acid similarity, respectively with MYB79, by only 39.03% amino acid identify and amino acid similarity 45.72%, respectively, with MYB121 ( Figure 1B). Whereas amino acid sequence alignment shows that all of the three MYB proteins have a highly conserved R2R3 domain at theirs N-terminus, but a C-terminus with less amino acids conserved ( Figure 1C).  Amino acid sequence identify and similarity assays indicates that MYB71 shared 70.88% amino acid identify and 74.71% amino acid similarity, respectively with MYB79, by only 39.03% amino acid identify and amino acid similarity 45.72%, respectively, with MYB121 ( Figure 1B). Whereas amino acid sequence alignment shows that all of the three MYB proteins have a highly conserved R2R3 domain at theirs N-terminus, but a C-terminus with less amino acids conserved ( Figure 1C).

MYB71 Functions as a Transcription Activator
Considering that transcription factors may have transcriptional activation or repression functions, and should be functioned in nucleus, we therefore examined the subcellular localization and transcription activities of MYB71 by using Arabidopsis protoplast transient transfection assays.
To examine the subcellular localization of MYB71, plasmids of MYB71-GFP and the nucleus indicator gene NLS-RFP were co-transfected into Arabidopsis protoplasts, and the GFP and RFP fluorescence was observed under a cofocal microscope. As shown in Figure 2A, MYB71 was predominantly localized in the nucleus. Since MYB71, MYB79 and MYB121 formed a small cluster [2], we also examined the subcellular localization of MYB79 and MYB121, and found that both of them were also predominantly localized in the nucleus (Figure 2A).
Considering that transcription factors may have transcriptional activation or repression functions, and should be functioned in nucleus, we therefore examined the subcellular localization and transcription activities of MYB71 by using Arabidopsis protoplast transient transfection assays.
To examine the subcellular localization of MYB71, plasmids of MYB71-GFP and the nucleus indicator gene NLS-RFP were co-transfected into Arabidopsis protoplasts, and the GFP and RFP fluorescence was observed under a cofocal microscope. As shown in Figure 2A, MYB71 was predominantly localized in the nucleus. Since MYB71, MYB79 and MYB121 formed a small cluster [2], we also examined the subcellular localization of MYB79 and MYB121, and found that both of them were also predominantly localized in the nucleus (Figure 2A).
For MYB71 transcription activity examination, plasmids of the reporter gene Gal4:GUS and the effect gene GD-MYB71 or the control gene GD were co-transfected into Arabidopsis protoplasts, and GUS activities were measured by using a microplate reader. In this system, MYB71 is able to bind to the Gal4 promoter in the reporter gene via the fused GD domain. As shown in Figure 2B, expression of the reporter gene was activated by transfection of effector gene GD-MYB71, indicating that MYB71 function as a transcription activator. Similar, we also found that MYB79 and MYB121 function as transcription activators ( Figure 2B). were co-transfected, respectively with the reporter gene Gal4:GUS into protoplasts. The transfected protoplasts were incubated in dark for 20-22 h at room temperature, the GUS activities were measured by using a microplate reader. Data represent the mean ± SD of three replicates. * Significantly different from the control (p < 0.05).

Generation of the 35S:MYB71 Transgenic Plants and the myb71 Mutants
In order to study the roles of MYB71 in regulating ABA responses in Arabidopsis, we generated transgenic overexpression plants and gene edited mutants for MYB71. Transgenic plants overexpressing MYB71 were generated by transforming the Col wild type plants with the 35S:MYB71 construct, and selecting homozygous transgenic plants in the T3 generation. Four different lines of homozygous transgenic plants were obtained, and RT-PCR results show that the expression level of MYB71 was increased in all of the four lines when compared with that in the Col wild type plants, with line #7 has the highest expression level ( Figure 3A). Transgenic plants overexpressing MYB71-GR fusion gene For MYB71 transcription activity examination, plasmids of the reporter gene Gal4:GUS and the effect gene GD-MYB71 or the control gene GD were co-transfected into Arabidopsis protoplasts, and GUS activities were measured by using a microplate reader. In this system, MYB71 is able to bind to the Gal4 promoter in the reporter gene via the fused GD domain. As shown in Figure 2B, expression of the reporter gene was activated by transfection of effector gene GD-MYB71, indicating that MYB71 function as a transcription activator. Similar, we also found that MYB79 and MYB121 function as transcription activators ( Figure 2B).

Generation of the 35S:MYB71 Transgenic Plants and the MYB71 Mutants
In order to study the roles of MYB71 in regulating ABA responses in Arabidopsis, we generated transgenic overexpression plants and gene edited mutants for MYB71. Transgenic plants overexpressing MYB71 were generated by transforming the Col wild type plants with the 35S:MYB71 construct, and selecting homozygous transgenic plants in the T3 generation. Four different lines of homozygous transgenic plants were obtained, and RT-PCR results show that the expression level of MYB71 was increased in all of the four lines when compared with that in the Col wild type plants, with line #7 has the highest expression level ( Figure 3A). Transgenic plants overexpressing MYB71-GR fusion gene was also generated in order to identify genes that may be regulated by MYB71 ( Figure 3B). was also generated in order to identify genes that may be regulated by MYB71 ( Figure  3B). There is one T-DNA insertion line for MYB71 available from TAIR, we therefore isolated the single mutant MYB71-1 from the T-DNA insertion line ( Figure 4A).  There is one T-DNA insertion line for MYB71 available from TAIR, we therefore isolated the single mutant myb71-1 from the T-DNA insertion line ( Figure 4A). was also generated in order to identify genes that may be regulated by MYB71 ( Figure  3B). There is one T-DNA insertion line for MYB71 available from TAIR, we therefore isolated the single mutant MYB71-1 from the T-DNA insertion line ( Figure 4A).  Since there is only one T-DNA insertion mutant, we decided to generate gene edited mutants for MYB71 by using CRISPR/Cas9 gene editing, a method that can be used to generate transgene-free mutants [50][51][52][53]. Two overlapped target sequences near the start the condon of MYB71 was selected ( Figure 4B), and gene editing construct was generated and used for plant transformation. By sequencing the MYB71 gene in the transgenic plants, and isolated transgene-free mutants based on the PCR amplification of Cas9 vector fragment, we successfully obtained two transgene-free MYB71 mutants (i.e., myb71-c1 and myb71-c2).
In the myb71-c1 mutant, a single nucleotide insertion was occurred in the first target sequence, whereas in the myb71-c2 mutant, an 11 bp deletion was occurred in the second target sequence ( Figure 4B). Both the single nucleotide insertion and the 11 bp deletion in MYB71 gene resulted in a few amino acid substitutions and premature stops for MYB71 ( Figure 4C). The predicated proteins encoded by the mutated MYB71 gene in both the my71-c1 and myb71-c2 mutants contain only~20 amino acids before the R2R3 domain in MYB71, whereas the predicated truncated MYB71 protein in the T-DNA insertion mutant myb71-1 contains the entire R2R3 domain and part of the C-terminal domain ( Figure 4C). Sterilized seeds of the Col wild type, the transgenic overexpression plants and gene edited mutants of MYB71 generated were plated on 1/2 MS plates with or without 1 µM ABA, and seeds germination were counted at indicated and percentage of germination was calculated. We also included the MYB71 T-DNA insertion mutant in our assays. As shown in Figure 5A, in the control plates, almost all of the seeds including seeds of the Col wild type, the 35S:MYB71 transgenic plants, the gene edited mutants myb71-c1 and myb71-c2, and the T-DNA insertion mutant myb71-1 germinated 36 h after the plates were transferred to a growth room. On the other hand, germination of the seeds of all of the plants was inhibited in the ABA-containing plates. However, lower germination rate was observed for the 35S:MYB71 transgenic plant seeds, whereas higher germinated rate was observed for the MYB71 mutant seeds when compared with that of the Col wild type seeds ( Figure 5A). There results suggest that the MYB71 mutants were less sensitive but the 35S:MYB71 transgenic plants were more sensitive to ABA. Similar, the cotyledon greening assay results also indicate that the MYB71 mutants were less sensitive, whereas the 35S:MYB71 transgenic plants were more sensitive to ABA ( Figure 5B). These results suggest that MYB71 is a positive regulator of ABA response in Arabidopsis.

MYB71 Affected Genes Are Enriched in Plant Response to Environmental Stimuli
Since MYB71 is a transcription factor, to explore the functional mechanism of MYB71, we decide to identify the genes that may be regulated by MYB71. To do that, we generated transgenic plants overexpressing MYB71-GR, in which the MYB71 protein was fused with the glucocorticoid receptor (GR). Under normal condition, the fusion proteins cannot get into the nucleus, therefore are not functional. However, when the transgenic plants were treated with dexamethasone (DEX), the fusion proteins can move into nucleus, and MYB71 is able to regulate the expression of its downstream genes.

MYB71 Affected Genes Are Enriched in Plant Response to Environmental Stimuli
Since MYB71 is a transcription factor, to explore the functional mechanism of MYB71, we decide to identify the genes that may be regulated by MYB71. To do that, we generated transgenic plants overexpressing MYB71-GR, in which the MYB71 protein was fused with the glucocorticoid receptor (GR). Under normal condition, the fusion proteins cannot get into the nucleus, therefore are not functional. However, when the transgenic plants were treated with dexamethasone (DEX), the fusion proteins can move into nucleus, and MYB71 is able to regulate the expression of its downstream genes.
We therefore treated the 35S:MYB71-GR transgenic seedlings with DEX, isolated RNA from the treated and control seedlings, and performed transcriptome analysis. Since MYB71 functions as a transcription activator ( Figure 2B), we thus focused on the genes that were upregulated in the DEX treated seedlings. We found that only 26 genes were upregulated at least two folds in the DEX treated seedlings (Table S1).
GO (Gene Ontology) analysis show that the 26 upregulated genes are involved in multiple biological processes including plant growth and development, signal transduc- We therefore treated the 35S:MYB71-GR transgenic seedlings with DEX, isolated RNA from the treated and control seedlings, and performed transcriptome analysis. Since MYB71 functions as a transcription activator ( Figure 2B), we thus focused on the genes that were upregulated in the DEX treated seedlings. We found that only 26 genes were upregulated at least two folds in the DEX treated seedlings (Table S1).
GO (Gene Ontology) analysis show that the 26 upregulated genes are involved in multiple biological processes including plant growth and development, signal transduction, and secondary metabolic ( Figure 6). However, a majority of the genes are involved in biological processes related to environmental stimuli. For example, 18 genes are involved in response to stress, 13 to chemical, 12 to biotic stimulus and external stimulus, and 9 to abiotic stimulus ( Figure 6). tion, and secondary metabolic ( Figure 6). However, a majority of the genes are involved in biological processes related to environmental stimuli. For example, 18 genes are involved in response to stress, 13 to chemical, 12 to biotic stimulus and external stimulus, and 9 to abiotic stimulus ( Figure 6).

Some of the Genes Regulated by MYB71 Are Related to Plant Response to Environmental Stimuli and Seed Germination
Among the 26 upregulated genes in DEX treated 35S:MYB71-GR transgenic seedlings, we found several genes have been functional characterized, for example, LURP1 and CYP82C2 are involved in pathogen defense [54][55][56], PER4 has been shown to regulated seed germination [57,58], and At1g14550 is a tandem repeat gene of PER4, and encodes a protein closely related to PER4 [57]. We therefore take these genes as examples to compare their expression in DEX treated and control seedlings. As shown in Figure 7, the expression levels of all of the four genes were clearly increased in DEX treated seedlings.

Some of the Genes Regulated by MYB71 Are Related to Plant Response to Environmental Stimuli and Seed Germination
Among the 26 upregulated genes in DEX treated 35S:MYB71-GR transgenic seedlings, we found several genes have been functional characterized, for example, LURP1 and CYP82C2 are involved in pathogen defense [54][55][56], PER4 has been shown to regulated seed germination [57,58], and At1g14550 is a tandem repeat gene of PER4, and encodes a protein closely related to PER4 [57]. We therefore take these genes as examples to compare their expression in DEX treated and control seedlings. As shown in Figure 7, the expression levels of all of the four genes were clearly increased in DEX treated seedlings. tion, and secondary metabolic ( Figure 6). However, a majority of the genes are involved in biological processes related to environmental stimuli. For example, 18 genes are involved in response to stress, 13 to chemical, 12 to biotic stimulus and external stimulus, and 9 to abiotic stimulus ( Figure 6).

Some of the Genes Regulated by MYB71 Are Related to Plant Response to Environmental Stimuli and Seed Germination
Among the 26 upregulated genes in DEX treated 35S:MYB71-GR transgenic seedlings, we found several genes have been functional characterized, for example, LURP1 and CYP82C2 are involved in pathogen defense [54][55][56], PER4 has been shown to regulated seed germination [57,58], and At1g14550 is a tandem repeat gene of PER4, and encodes a protein closely related to PER4 [57]. We therefore take these genes as examples to compare their expression in DEX treated and control seedlings. As shown in Figure 7, the expression levels of all of the four genes were clearly increased in DEX treated seedlings.

Discussion
ABA is an important hormone that can trigger plants responses to various abiotic stresses, including salinity, drought, high temperature, chilling, and oxidative stress [59,60].
Since there is a close relationship between ABA and abiotic stress tolerance in plants, examining the roles of ABA response genes in regulating plant responses to ABA will help to elucidate the mechanisms of plant abiotic stress tolerance [47]. R2R3 MYB proteins are the largest subfamily of the MYB transcription factor family in Arabidopsis with multiple functions [1,2]. Several R2R3 MYBs such as MYB32, MYB37, MYB44, have been shown to be involved in the regulation of ABA responses [22,44,61]. In this study, we provide evidence here that MYB71 is an ABA responsive gene, and MYB71 positively regulates ABA response in Arabidopsis.
Our data show that the expression level of MYB71 increased dramatically in response to ABA treatment, in addition, its closely related R2R3 MYB genes MYB79 and MYB121 are also ABA responsive genes (Figure 1). By generating 35S:MYB71 transgenic plants and gene edited MYB71 mutants (Figures 3 and 4), and examining their responses to ABA by using seed germination and seedling greening experiments, we found that ABA sensitively was increased in the 35S:MYB71 transgenic plant, but decreased in the MYB71 mutants ( Figure 5). We also noted that, among the three different lines of the 35S:MYB71 transgenic plants, highest ABA sensitivity was observed in line #7 ( Figure 5), and the highest expression level of MYB71 was also observed in line #7 (Figure 3). On the other hand, the gene edited MYB71 mutants showed higher ABA tolerance than the T-DNA insertion mutant myb71-1 ( Figure 5). Considering that both of the gene edited MYB71 mutants are likely loss-of-function mutants, as the predicated ORF (open reading frame) of MYB71 in the mutants only encodes about 20 amino acids before the R2R3 MYB domain in the MYB71 protein, whereas the T-DNA insertion mutant myb71-1 may not be a loss-offunction as the CDS (coding sequence) before the T-DNA insertion encodes the entire R2R3 MYB domain and part of the C-terminal domain (Figure 4), there results further confirmed that MYB71 is a positive regulator of ABA response.
It has been shown that some of the R2R3 MYB transcription factor whose gene expression can be regulated by ABA may play a feedback regulating role in ABA signaling. For example, MYB44 is able to suppress the expression of PP2C genes [44], MYB32 positively regulated the expression of ABSCISIC ACID-INSENSITIVE 3 (ABI3), ABI4 and ABI5 [22], and MYB37 is also able to regulate the expression of some ABA signaling key regulator genes as well as some other ABA responsive genes [61]. By using a DEX inducible system, we try to identify genes that may be regulated by MYB71, a total of 26 genes were found to be unregulated no less than 2 folds in the DEX treated 35S:MYB71-GR transgenic plants (Table S1). Considering that MYB71 functions as a transcription activator (Figure 2), these genes are likely regulated by MYB71, and some of them may be directly targets of MYB71. However, none of them are ABA signaling key regulator genes, indicating that MYB71 may not able to regulate the expression of ABA signaling key regulator genes. Therefore, it will be of interest to investigate how MYB71 may regulate ABA response in Arabidopsis.
By charactering unknown function ABA responsive genes, we have successfully identified several different types of transfection factors as abiotic stress response regulators in Arabidopsis, including the WD40 transcription factors AIW1 and AIW2 [48], the non-DNA binding bHLH transcription factor PRE6 [49], and the novel transcription factor family AITRs [35]. Similar to these transcription factors, the expression of MYB71 is regulated by ABA (Figure 1), and MYB71 is involved in the regulation of ABA responses in Arabidopsis ( Figure 5). GO analysis of the genes that may regulated by MYB71 also show that most of the genes are involved in the process of response to environmental stimuli, including abiotic stresses, suggest that MYB71 may be involved in the regulation of plant responses to abiotic stresses. However, further evidence is required to support a role of MYB71 in regulating plant abiotic stress responses.
Furthermore, as the expression of both MYB79 and MYB121 are also induced by ABA, MYB71 is closely related to MYB79, and they shared high amino acid identity and similarity at full length amino acid level, and MYB121 also shared high amino acid identity and similarity with MYB71 and MYB79 at the R2R3 repeat domain, but not the C-terminus ( Figure 1). It will be of interest to examine if MYB79 and MYB121 are involved in regulating plant responses to ABA and abiotic stresses, and if they have similar functions as MYB71.

Plant Materials and Growth Conditions
The wild-type Columbia-0 (Col) ecotype Arabidopsis (Arabidopsis thaliana) was used for protoplasts isolation, and for plant transformed to generate transgenic plants and gene edited mutants. Seeds of the SALK_013451 T-DNA insertion line in Col background were obtained from ABRC (Arabidopsis Biological Resource Center), and used to isolate the myb71-1 mutant.
To growth plants for transformation and seeds production, seeds were soaked with sterile water and kept at 4 • C in darkness for 2-3 days, then sown into the soil pots directly and kept in a growth room. The pots were covered with plastic covers for 3-4 days to facilitate generation. The growth room was set with a cycle of 16 h for light with light density at approximately 125 µmol m −2 s −1 and 8h for darkness, the temperature in the growth room was set at 22 • C.

ABA Treatment, RNA Isolation and RT-PCR
To examine the response of MYB71 (At3g24310), MYB79 (At4g13480) and MYB121 (At3g30210) to ABA, 10-day-old Col wild type seedlings grown in 1/2 MS plates were collected and treated with 50 µM ABA solution by shaking in darkness for 4 h, and the seedlings treated with solvent methanol were used as a control. The seedlings were then frozen in liquid nitrogen, and use for RNA isolation as described previously [18,35,62,63].
To examine the expression levels of MYB71 in the transgenic plants, 10-day-old seedlings of the T3 homozygous transgenic plants grown in 1/2 MS plates were collected, frozen in liquid nitrogen and used for RNA isolation.
Total RNA was isolated by using an EasyPure PlantRNA Kit (TransGen Biotech, Beijing, China) according to the manufacturer's instruction, DNA removal was conducted by treat the RNA isolated with DNase I for 15 min, and cDNA was synthesized by using an EasyScript First-strand DNA Synthesis Super Mix kit (TransGen Biotech, Beijing, China). If brief, 2 µg RNA was mixed with Reaction Mix, oligo(dT) 18 , Enzyme Mix and Rnase-free water, incubated at 42 • C for 30 min and then 85 • C for 5 s.

Constructs
To generate constructs for transcription activity assays, the full length ORF sequences of MYB71, MYB79 and MYB121 were amplified, respectively by RT-PCR, and used to the generation of GD-MYB71, GD-MYB79 and GD-MYB121 by cloning into the N-terminal GD tag-containing pUC19 vector under the control of the CaMV double 35S promoter [64,65]. The Gal4 DNA binding domain control construct GD and the Gal4:GUS reporter construct used for protoplast transfection were as described previously [66,67].
To generated constructs for the subcellular localization assay, the GD tag in the GD-MYB71, GD-MYB79 and GD-MYB121 constructs were replaced with a GFP tag to generated GFP-MYB71, GFP-MYB79 and GFP-MYB121 constructs respectively. The NLS-RFP nuclear indicator was as described previously [68].
To generate 35S:MYB71 construct for plant transformation, the GD tag in the GD-MYB71 construct was replaced with a HA tag. To generate the 35S:MYB71-GR construct for plant transformation, the full-length ORF of MYB71 without stop condon was PCR amplified and cloned into the N-terminal HA tag-and C-terminal GR tag-containing pUC19 vector. The 35S:MYB71 and 35S:MYB71-GR constructs generated were then digested with corresponding restriction enzymes and cloned into the binary vector pPZP211 [69].

Plant Transformation, Transgenic Plant Selection and Cas9-Free Mutant Isolation
The Col wild type plants about 5-week-old with a few flowers in the main inflorescence were transformed by the floral dip method [71]. The plants were transformed via GV3101 agrobacterium cells with the 35S:MYB71, 35S:MYB71-GR and the CRISPR/Cas9 constructs to obtain overexpression transgenic plants and gene edited mutants for MYB71, respectively. T1 seeds were collected from transgenic plants selected.
To select transgenic plants, the T1 seeds collected were sown on the plates with proper antibiotic. For overexpression transgenic plants selection, T1 seeds were sown on 1/2 MS plates containing 100 µg/mL Carbenicillin and 50 µg/mL Kanamycin. For gene editing transgenic plants selection, T1 seeds were sown on 1/2 MS plates containing 30 µg/mL Hygromycin and 100 µg/mL Carbenicillin.
To identify 35S:MYB71 and 35S:MYB71-GR homozugous transgenic plants, T2 seeds from T1 transgenic plants were sown on 1/2 MS plates with 25 µg/mL Kanamycin to selected transgenic plants with a single T-DNA insertion (3:1 segregation of transgenic and wild type seedlings), and T3 seeds from T2 plants with a single T-DNA insertion were selected on 1/2 MS plates with 25 µg/mL Kanamycin to identify homozygous transgenic plants. The expression level of MYB71 in the homozygous transgenic plants was examined by RT-PCR to identify overexpression transgenic plants.
To identify transgene-free gene edited mutants for MYB71, gene editing status of MYB71 in T1 plants was examined by amplifying and sequencing the genomic sequence of MYB71. T2 seeds from gene edited T1 plants were sown in soil pots, and transgene-free T2 plants were identified by amplifying Cas9 fragment as described previously [36,72]. Gene editing status of MYB71 in the transgene-free T2 plants was then examined to identify homozygous mutants.

DNA Isolation and PCR
To confirm the T1 35S:MYB71 and 35S:MYB71-GR transgenic plants obtained, DNA was isolated from leaves of T1 plants, a few days after the seedling were transferred into soil pots, by using DNA isolation buffer as described previously [66], and used for PCR analysis. The HA primer 5 -CCATGGGATACCCTTACGATG-3 and the reverse primer used to amplify MYB71 CDS were used to confirm the 35S:MYB71 and 35S:MYB71-GR T1 transgenic plants.
To examine gene editing status of the T1 transgenic plants of the gene editing transgenic plants, and the T2 transgene-free plants, DNA was isolated from leaves of T1 plants and T2 plants a few days after the seedling were transferred into soil pots, and genome sequence of MYB71 was PCR amplified, for 32 cycles with an annealing tem-perature of 56 • C by using primers 5 -AGGTTTCACAAGACAGAGAGAC-3 and 5 -CAAGAGCTCTTAACAGAAGGGAATGACCATGTTC-3 and sequenced.

Plasmid DNA Isolation, Protoplasts Isolation, Transfection, GFP Observation and GUS Activity Assays
The plasmids DNA including the reporter and effector constructs, the GFP and NLS-RFP subcellular localization constructs were isolated by using a GoldHi Endo Free Plasmid Maxi Kit (CWBIO, Beijing, China) from transformed E. coli cells, and DNA concentration was measured by using a NanoDrop (Thermo, Waltham, MA, USA) machine.
Rosette leaves from~3 to 4 weeks old Col wild type plants were used to isolation protoplasts, and protoplasts were transfected by using a procedure described previously [64,73,74]. For transcription activity assays, the GD, GD-MYB71, GD-MYB79 and GD-MYB121 effects were co-trasfected, respectively, with the Gal4:GUS reporter construct plasmids DNA into the protoplasts isolated, and the transfected protoplasts were incubated at room temperature for 20-22 h under darkness. GUS activities were then assayed by using a Synergy TM HT fluorescence microplate reader (BioTEK, VT, USA).
For the subcellular localization assay, plasmids of MYB71-GFP, MYB79-GFP and MYB121-GFP were co-transfected respectively with NLS-RFP, and the transfected protoplasts were incubated at room temperature for 18-20 h under darkness. GFP and RFP fluorescence were then observed under a confocal microscope (Olympus, Tokyo, Japan).

ABA Response Assays
ABA inhibited seed germination and cotyledon greening were used to examine ABA response of the overexpression transgenic plants and gene edited mutants of MYB71, by using a procedure described previously [35,[75][76][77]. Briefly, seeds of the Col wild type, the 35:MYB71 overexpression transgenic plants and MYB71 mutants were sterilized and sowed on the 1/2 MS plates containing 1 µM ABA, or the solvent methanol as control. The plates were kept at 4 • C for 2 days in darkness and then transferred into the growth room. The numbers of seeds germinated were counted every 12 h, and photographs were taken 12 days after the transfer.

Transcriptome Analysis
35S:MYB71-GR seeds were sown on 1/2 MS plates and kept for 2 days at 4 • C in darkness. Then the plates were transferred into the growth room and grown for 10 days. The 35S:MYB71-GR seedlings were then treated with 10 µM DEX or solvent only as control by shaking for 4 h. The seedlings were then frozen in liquid nitrogen, and send to the company (Beijing Genomics Institute, Shenzhen, China) for transcriptome sequencing and analysis. A potion of RNA isolated was returned by the company for result verification purpose. Genes upregulated at least 2 folds by DEX treatment were subject to functional categorization on TAIR Gene Ontology (GO) Annotation (https://www.arabidopsis.org/ tools/bulk/go/index.jsp, accessed on 6 April 2021) under the term of biological process.

Conclusions
Our results in this research show that the MYB71 is an ABA responsive gene, MYB71 functions as a transcription activator, and it regulates plant ABA response in Arabidopsis.