AtERF71/HRE2, an Arabidopsis AP2/ERF Transcription Factor Gene, Contains Both Positive and Negative Cis-Regulatory Elements in Its Promoter Region Involved in Hypoxia and Salt Stress Responses

In the signal transduction network, from the perception of stress signals to stress-responsive gene expression, various transcription factors and cis-regulatory elements in stress-responsive promoters coordinate plant adaptation to abiotic stresses. Among the AP2/ERF transcription factor family, group VII ERF (ERF-VII) genes, such as RAP2.12, RAP2.2, RAP2.3, AtERF73/HRE1, and AtERF71/HRE2, are known to be involved in the response to hypoxia in Arabidopsis. Notably, HRE2 has been reported to be involved in responses to hypoxia and osmotic stress. In this study, we dissected HRE2 promoter to identify hypoxia- and salt stress-responsive region(s). The analysis of the promoter deletion series of HRE2 using firefly luciferase and GUS as reporter genes indicated that the −116 to −2 region is responsible for both hypoxia and salt stress responses. Using yeast one-hybrid screening, we isolated HAT22/ABIG1, a member of the HD-Zip II subfamily, which binds to the −116 to −2 region of HRE2 promoter. Interestingly, HAT22/ABIG1 repressed the transcription of HRE2 via the EAR motif located in the N-terminal region of HAT22/ABIG1. HAT22/ABIG1 bound to the 5′-AATGATA-3′ sequence, HD-Zip II-binding-like cis-regulatory element, in the −116 to −2 region of HRE2 promoter. Our findings demonstrate that the −116 to −2 region of HRE2 promoter contains both positive and negative cis-regulatory elements, which may regulate the expression of HRE2 in responses to hypoxia and salt stress and that HAT22/ABIG1 negatively regulates HRE2 transcription by binding to the HD-Zip II-binding-like element in the promoter region.


Introduction
Abiotic stresses have been shown to regulate the expression of genes with various functions in a variety of plants [1]. In the signal transduction network, from the perception of stress signals to stress-responsive gene expression, various transcription factors and cis-regulatory elements in the stress-responsive promoters are involved in the adaptation of plants to abiotic stresses. Transcription factors can control the expression of many target genes via the specific binding to the cis-regulatory element in the promoters of the respective target genes [2]. Several major transcription factor families that are activated in response to abiotic stresses have been identified in Arabidopsis (Arabidopsis thaliana), such as AP2/ERF, bZIP, zinc finger, WRKY, MYB, bHLH, and NAC families [3,4].
The AP2/ERF family is a large group of plant-specific transcription factors with 145 members in Arabidopsis, and these 145 genes are classified into the following four

Hypoxia-Responsive Positive Cis-Regulatory Element(s) of HRE2 Is Located in the −116 to −2 Region of Its Promoter
Previously, we have shown that the 180 bp promoter of HRE2 responds to hypoxia and salt treatment in Arabidopsis transgenic plants [35]. In this study, we performed a promoter-deletion analysis experiment to identify the hypoxia-and salt-responsive promoter region of HRE2. To this end, we generated constructs of firefly luciferase genes controlled by the −180 to +212, −116 to +212, −2 to +212, and +52 to +212 regions from the transcriptional start site of HRE2 promoter (Figure 1a). We then transformed each construct into Arabidopsis protoplasts, which were kept under hypoxic conditions during isolation and then transformation, and measured the firefly luciferase activity driven by the deletion series of HRE2 promoters. As a result, the −180 and −116 promoters showed high firefly luciferase activity, while −2 promoter showed approximately one-third the activity of that shown by the −180 and −116 promoters (Figure 1b). In addition, +52 promoter showed basal level of firefly luciferase activity (Figure 1b).

Hypoxia-Responsive Positive Cis-Regulatory Element(s) of HRE2 Is Located in the −116 to −2 Region of Its Promoter
Previously, we have shown that the 180 bp promoter of HRE2 responds to hypoxia and salt treatment in Arabidopsis transgenic plants [35]. In this study, we performed a promoter-deletion analysis experiment to identify the hypoxia-and salt-responsive promoter region of HRE2. To this end, we generated constructs of firefly luciferase genes controlled by the −180 to +212, −116 to +212, −2 to +212, and +52 to +212 regions from the transcriptional start site of HRE2 promoter (Figure 1a). We then transformed each construct into Arabidopsis protoplasts, which were kept under hypoxic conditions during isolation and then transformation, and measured the firefly luciferase activity driven by the deletion series of HRE2 promoters. As a result, the −180 and −116 promoters showed high firefly luciferase activity, while −2 promoter showed approximately one-third the activity of that shown by the −180 and −116 promoters (Figure 1b). In addition, +52 promoter showed basal level of firefly luciferase activity (Figure 1b).
We further confirmed this result by measuring GUS activity controlled by the same promoter deletion series of HRE2 as that used in the firefly luciferase reporter assay (Figure 1c). For this, 12-day-old transgenic plants were subjected to hypoxia, and histochemical GUS assay was performed. As a result, the −180 and −116 promoters showed high GUS activity in the cotyledons, whereas −2 and +52 promoter regions showed no GUS activity under hypoxic conditions (Figure 1d). These results demonstrated that the 115 bp of HRE2 promoter, namely the −116 to −2 region, includes positive cis-regulatory element(s) involved in the response to hypoxia. Relative firefly luciferase activity in Arabidopsis protoplasts. Transformation efficiency was normalized using Nano luciferase activity. Normalized firefly luciferase activity of negative control was set as 1. Empty reporter plasmid was used for the negative control. Data are shown as means ± S.D. (n = 3). Different letters display significant differences (p < 0.05). NC, negative control. (c) A schematic map of vector for HRE2 promoter deletion series analysis. (d) Histochemical GUS assay of Arabidopsis T2 transgenic plants carrying the deletion series of HRE2 promoter at 12 days after germination (DAG) under short-day (SD) conditions. GUS activity was observed in at least 15 transgenic plants for each construct; representative staining results are shown here. In (a,c), PHRE2 indicates promoter of HRE2. Relative firefly luciferase activity in Arabidopsis protoplasts. Transformation efficiency was normalized using Nano luciferase activity. Normalized firefly luciferase activity of negative control was set as 1. Empty reporter plasmid was used for the negative control. Data are shown as means ± S.D. (n = 3). Different letters display significant differences (p < 0.05). NC, negative control. (c) A schematic map of vector for HRE2 promoter deletion series analysis. (d) Histochemical GUS assay of Arabidopsis T 2 transgenic plants carrying the deletion series of HRE2 promoter at 12 days after germination (DAG) under shortday (SD) conditions. GUS activity was observed in at least 15 transgenic plants for each construct; representative staining results are shown here. In (a,c), P HRE2 indicates promoter of HRE2.
We further confirmed this result by measuring GUS activity controlled by the same promoter deletion series of HRE2 as that used in the firefly luciferase reporter assay ( Figure 1c). For this, 12-day-old transgenic plants were subjected to hypoxia, and histochemical GUS assay was performed. As a result, the −180 and −116 promoters showed high GUS activity in the cotyledons, whereas −2 and +52 promoter regions showed no GUS activity under hypoxic conditions (Figure 1d). These results demonstrated that the 115 bp of HRE2 promoter, namely the −116 to −2 region, includes positive cis-regulatory element(s) involved in the response to hypoxia.
Next, we validated the hypoxic response of HRE2 promoter in Arabidopsis plants. To this end, we generated Arabidopsis transgenic plants harboring firefly luciferase gene controlled by the −180 to +212 region from the transcriptional start site of HRE2 promoter (Figure 2a). We then analyzed firefly luciferase activity in 15-day-old seedlings after hypoxia treatment. We observed that the promoter activity of the −180 promoter was highly increased after hypoxia treatment (Figure 2b), indicating that the −180 promoter of HRE2 with 5 -UTR is responsive to hypoxia in both protoplasts and plants. Next, we validated the hypoxic response of HRE2 promoter in Arabidopsis plants. To this end, we generated Arabidopsis transgenic plants harboring firefly luciferase gene controlled by the −180 to +212 region from the transcriptional start site of HRE2 promoter (Figure 2a). We then analyzed firefly luciferase activity in 15-day-old seedlings after hypoxia treatment. We observed that the promoter activity of the −180 promoter was highly increased after hypoxia treatment (Figure 2b), indicating that the −180 promoter of HRE2 with 5′-UTR is responsive to hypoxia in both protoplasts and plants.

The −116 to −2 Region of HRE2 Promoter Includes Positive Cis-Regulatory Element(s) Responsible for Responses to Salt Stress as Well as Hypoxia
HRE2 is known to respond to salt stress and hypoxia [35]. To identify the salt stressresponsive promoter region of HRE2, we transformed the same HRE2 promoter deletion constructs as those used in the hypoxia-response experiments into Arabidopsis protoplasts under normal or salt stress conditions, and then analyzed the firefly luciferase activity ( Figure 3a). The firefly luciferase activities of the −180 and −116 promoters were observed to have increased almost 1.6-fold under salt treatment condition compared to that under normal conditions, while the firefly luciferase activities of the −2 and +52 promoters did not show any response to the salt treatment ( Figure 3b).
We also analyzed Arabidopsis transgenic plants harboring GUS controlled by the deletion series of HRE2 promoter (Figure 3c). The results of the histochemical GUS assay showed that the −180 and −116 promoters showed high GUS activity in cotyledons and roots under salt stress conditions, whereas −2 and +52 promoters showed no GUS activity under these conditions (Figure 3d). These results indicated that the −116 to −2 region of HRE2 promoter is positively involved in the response to salt stress as well as hypoxia.  HRE2 is known to respond to salt stress and hypoxia [35]. To identify the salt stressresponsive promoter region of HRE2, we transformed the same HRE2 promoter deletion constructs as those used in the hypoxia-response experiments into Arabidopsis protoplasts under normal or salt stress conditions, and then analyzed the firefly luciferase activity ( Figure 3a). The firefly luciferase activities of the −180 and −116 promoters were observed to have increased almost 1.6-fold under salt treatment condition compared to that under normal conditions, while the firefly luciferase activities of the −2 and +52 promoters did not show any response to the salt treatment ( Figure 3b).
We also analyzed Arabidopsis transgenic plants harboring GUS controlled by the deletion series of HRE2 promoter (Figure 3c). The results of the histochemical GUS assay showed that the −180 and −116 promoters showed high GUS activity in cotyledons and roots under salt stress conditions, whereas −2 and +52 promoters showed no GUS activity under these conditions (Figure 3d). These results indicated that the −116 to −2 region of HRE2 promoter is positively involved in the response to salt stress as well as hypoxia.

Reconfirmation of the Positive Response of the −116 to −2 Region of HRE2 Promoter to Hypoxia
To reconfirm the positive response of the −116 to −2 region of HRE2 promoter to hypoxia, we generated a construct containing the firefly luciferase gene controlled by tandem repeats of the −116 to −2 region of HRE2 promoter and transformed it into Arabidopsis protoplasts ( Figure 4a). The longest HRE2 promoter, namely the −180 promoter, was used as the positive control ( Figure 4a). Tandem repeats of the −116 to −2 region of HRE2 promoter showed firefly luciferase activity similar to that of the −180 promoter (Figure 4b), demonstrating that the −116 to −2 region of HRE2 promoter includes positive cis-regulatory element(s) responsible for hypoxia response.   To reconfirm the positive response of the −116 to −2 region of HRE2 promoter to hypoxia, we generated a construct containing the firefly luciferase gene controlled by tandem repeats of the −116 to −2 region of HRE2 promoter and transformed it into Arabidopsis protoplasts ( Figure 4a). The longest HRE2 promoter, namely the −180 promoter, was used as the positive control ( Figure 4a). Tandem repeats of the −116 to −2 region of HRE2 promoter showed firefly luciferase activity similar to that of the −180 promoter (Figure 4b), demonstrating that the −116 to −2 region of HRE2 promoter includes positive cis-regulatory element(s) responsible for hypoxia response.
poxia, we generated a construct containing the firefly luciferase gene controlled by tandem repeats of the −116 to −2 region of HRE2 promoter and transformed it into Arabidopsis protoplasts ( Figure 4a). The longest HRE2 promoter, namely the −180 promoter, was used as the positive control ( Figure 4a). Tandem repeats of the −116 to −2 region of HRE2 promoter showed firefly luciferase activity similar to that of the −180 promoter (Figure 4b), demonstrating that the −116 to −2 region of HRE2 promoter includes positive cis-regulatory element(s) responsible for hypoxia response.

Isolation of Transcription Factor(s) That Bind to the −116 to −2 Region of HRE2 Promoter Using Yeast One-Hybrid Screening
To isolate the transcription factor(s) that bind to the −116 to −2 region of HRE2 promoter, we performed yeast one-hybrid screening using a cDNA library of Arabidopsis seedlings subjected to hypoxia, in which cDNAs were fused to the GAL4 activation domain (AD). As a result of the screening, a total of 25 positive colonies were obtained from 8.8 × 10 5 yeast transformants by growth assay using HIS3 and ADE2 as reporter genes (Table S1). Plasmid DNAs with AD were isolated from the yeast colonies; we confirmed that the 25 positive plasmid DNAs represented 13 individual genes (Table S2). Interestingly, domain analysis showed that 9 of the 13 genes were homeodomain superfamily genes. Six of these nine genes belonged to the HD-Zip family, while the remaining three belonged to the zinc finger homeodomain (ZF-HD) family (Table S2).
We generated constructs including full-length ORFs of the nine homeodomain superfamily genes fused to GAL4 AD, which were then co-transformed into yeasts, together with AUR1-C or lacZ reporter genes controlled by the tandem repeats of the −116 to −2 region of HRE2 promoter. Based on the yeast growth and β-galactosidase orthonitrophenyl-β-D-galactopyranoside (ONPG) assays, At4g37790 transactivated the reporter genes most strongly ( Figure 5). At4g37790 encodes HAT22/ABIG1, which belongs to class II HD-Zip (HD-Zip II) subfamily. We selected HAT22/ABIG1 for further studies.
We generated constructs including full-length ORFs of the nine homeodomain superfamily genes fused to GAL4 AD, which were then co-transformed into yeasts, together with AUR1-C or lacZ reporter genes controlled by the tandem repeats of the −116 to −2 region of HRE2 promoter. Based on the yeast growth and β-galactosidase orthonitrophenyl-β-D-galactopyranoside (ONPG) assays, At4g37790 transactivated the reporter genes most strongly ( Figure 5). At4g37790 encodes HAT22/ABIG1, which belongs to class II HD-Zip (HD-Zip II) subfamily. We selected HAT22/ABIG1 for further studies.

HAT22/ABIG1 Is Subcellularly Localized in the Nucleus
We investigated the subcellular localization of HAT22/ABIG1 in Arabidopsis protoplasts using an sGFP-HAT22/ABIG1 fusion construct. The GFP signal of the sGFP-HAT22/ABIG1 construct was observed in the nucleus where it overlapped with the 4 ,6diamidino-2-phenylindole signal ( Figure 6), indicating that HAT22/ABIG1 functions in the nucleus.

HAT22/ABIG1 Is Subcellularly Localized in the Nucleus
We investigated the subcellular localization of HAT22/ABIG1 in Arabidopsis protoplasts using an sGFP-HAT22/ABIG1 fusion construct. The GFP signal of the sGFP-HAT22/ABIG1 construct was observed in the nucleus where it overlapped with the 4′,6diamidino-2-phenylindole signal ( Figure 6), indicating that HAT22/ABIG1 functions in the nucleus. It has been reported that HD-Zip II proteins, such as AtHB2, HAT1, HAT2, and AtHB4, function as transcriptional repressors by means of the EAR motif located in their N-terminal regions [25][26][27][28]. HAT22/ABIG1 also contains an EAR motif at its N-terminus  It has been reported that HD-Zip II proteins, such as AtHB2, HAT1, HAT2, and AtHB4, function as transcriptional repressors by means of the EAR motif located in their N-terminal regions [25][26][27][28]. HAT22/ABIG1 also contains an EAR motif at its N-terminus [33], indicating that HAT22/ABIG1 might function as a transcriptional repressor in the regulation of downstream genes. To check the transcriptional repression of HRE2 by HAT22/ABIG1, firefly luciferase gene controlled by the tandem repeats of the −116 to −2 region of HRE2 promoter was co-transformed with the HAT22/ABIG1 OX construct into Arabidopsis protoplasts (Figure 7a). The firefly luciferase activity with HAT22/ABIG1 was almost one-third of that without HAT22/ABIG1 (Figure 7b), demonstrating that HAT22/ABIG1 represses HRE2 transcription via the −116 to −2 region of HRE2 promoter in Arabidopsis plants. The relative firefly luciferase activity in Arabidopsis protoplasts. The transformation efficiency was normalized using Nano luciferase activity. The normalized firefly luciferase activity of the negative control was set as 1. The empty effector plasmid was used for the negative control. Data are shown as means ± S.D. (n = 5). Different letters display significant differences (p < 0.05).
We also tested whether the EAR motif in the N-terminal region of HAT22/ABIG1 is important for its transcriptional repression activity. We generated the HAT22/ABIG1 OX construct (ΔN52 HAT22/ABIG1), in which 52 aa of the N-terminus of HAT22/ABIG1, including the EAR motif, were deleted. We then analyzed the effect of ΔN52 HAT22/ABIG1 on the firefly luciferase activity controlled by the tandem repeats of the −116 to −2 region of HRE2 promoter (Figure 7a). The firefly luciferase activity with ΔN52 HAT22/ABIG1 was recovered to the level observed for that without HAT22/ABIG1 (Figure 7b). It was previously reported that HD-Zip II proteins bind to the promoters of downstream genes through homeodomain [13]. Predicted nuclear localization sequences (NLS) of AtHB4, a HD-Zip II protein, is in the homeodomain and the EAR motif-deleted AtHB4 is subcellularly localized in the nucleus [28]. We found that predicted NLS of HAT22/ABIG1 is also in the homeodomain (125-179 aa region) using NLS Mapper (https://nls-mapper.iab.keio.ac.jp/cgi-bin/NLS_Mapper_form.cgi, accessed on 29 April 2022) (data not shown), suggesting that ΔN52 HAT22/ABIG1 is translocated to the nucleus and binds to the −116 to −2 region of HRE2 promoter. Indeed, GFP signal of the sGFP-ΔN52 HAT22/ABIG1 construct was observed in the nucleus ( Figure S2), demonstrating that ΔN52 HAT22/ABIG1 is translocated to the nucleus. Our results together with the predictions indicate that the EAR motif in the N-terminus of HAT22/ABIG1 is important for its repression of HRE2 transcription.
We further tested the transactivation activity of HAT22/ABIG1 to check whether HAT22/ABIG1 acts as a transcriptional activator. HAT22/ABIG1 was fused to the GAL4 DNAbinding domain (BD) and transformed into yeast. As expected, HAT22/ABIG1 did not show transactivation activity in the yeast growth and β-galactosidase ONPG assays ( Figure S3). The relative firefly luciferase activity in Arabidopsis protoplasts. The transformation efficiency was normalized using Nano luciferase activity. The normalized firefly luciferase activity of the negative control was set as 1. The empty effector plasmid was used for the negative control. Data are shown as means ± S.D. (n = 5). Different letters display significant differences (p < 0.05).

HAT22/ABIG1 Represses HRE2 Transcription via 7 bp Conserved Negative Cis-Regulatory
We also tested whether the EAR motif in the N-terminal region of HAT22/ABIG1 is important for its transcriptional repression activity. We generated the HAT22/ABIG1 OX construct (∆N52 HAT22/ABIG1), in which 52 aa of the N-terminus of HAT22/ABIG1, including the EAR motif, were deleted. We then analyzed the effect of ∆N52 HAT22/ABIG1 on the firefly luciferase activity controlled by the tandem repeats of the −116 to −2 region of HRE2 promoter (Figure 7a). The firefly luciferase activity with ∆N52 HAT22/ABIG1 was recovered to the level observed for that without HAT22/ABIG1 (Figure 7b). It was previously reported that HD-Zip II proteins bind to the promoters of downstream genes through homeodomain [13]. Predicted nuclear localization sequences (NLS) of AtHB4, a HD-Zip II protein, is in the homeodomain and the EAR motif-deleted AtHB4 is subcellularly localized in the nucleus [28]. We found that predicted NLS of HAT22/ABIG1 is also in the homeodomain (125-179 aa region) using NLS Mapper (https://nls-mapper.iab.keio.ac.jp/cgi-bin/NLS_Mapper_form.cgi, accessed on 29 April 2022) (data not shown), suggesting that ∆N52 HAT22/ABIG1 is translocated to the nucleus and binds to the −116 to −2 region of HRE2 promoter. Indeed, GFP signal of the sGFP-∆N52 HAT22/ABIG1 construct was observed in the nucleus ( Figure S2), demonstrating that ∆N52 HAT22/ABIG1 is translocated to the nucleus. Our results together with the predictions indicate that the EAR motif in the N-terminus of HAT22/ABIG1 is important for its repression of HRE2 transcription.
We further tested the transactivation activity of HAT22/ABIG1 to check whether HAT22/ABIG1 acts as a transcriptional activator. HAT22/ABIG1 was fused to the GAL4 DNA-binding domain (BD) and transformed into yeast. As expected, HAT22/ABIG1 did not show transactivation activity in the yeast growth and β-galactosidase ONPG assays ( Figure S3).

HAT22/ABIG1 Is Responsive to Both Hypoxia and Salt Stresses
Previously, HAT22/ABIG1 was found to be responsive to drought stress and ABA [32] (Figure 10). However, responses of HAT22/ABIG1 to hypoxia and/or salt stress have not yet been reported. To determine the expression of HAT22/ABIG1 under hypoxic and salt stress conditions, the transcript abundance of HAT22/ABIG1 was examined under these conditions. Quantitative RT-PCR (RT-qPCR) results showed that the expression of HAT22/ABIG1 increased at 1 h after being subjected to hypoxia and then gradually decreased until 8 h after hypoxia treatment ( Figure 10). In addition, the expression of HAT22/ABIG1 also increased at 1 h after the treatment with NaCl, and the expression level was maintained up to 4 h after the treatment (Figure 10). Increased expression of ADH1 and RD29A, hypoxia and osmotic-stress marker genes, respectively, confirmed that the hypoxia, NaCl, and mannitol stresses were properly treated ( Figure 10). These results indicated that HAT22/ABIG1 is involved in the response to hypoxia and salt stress.  The relative firefly luciferase activity in Arabidopsis protoplasts. The transformation efficiency was normalized using Nano luciferase activity. The normalized firefly luciferase activity of negative control was set as 1. The empty effector plasmid was used for the negative control. Data are shown as means ± S.D. (n = 5). Different letters display significant differences (p < 0.05).

HAT22/ABIG1 Is Responsive to Both Hypoxia and Salt Stresses
Previously, HAT22/ABIG1 was found to be responsive to drought stress and ABA [32] ( Figure 10). However, responses of HAT22/ABIG1 to hypoxia and/or salt stress have not yet been reported. To determine the expression of HAT22/ABIG1 under hypoxic and salt stress conditions, the transcript abundance of HAT22/ABIG1 was examined under these conditions. Quantitative RT-PCR (RT-qPCR) results showed that the expression of HAT22/ABIG1 increased at 1 h after being subjected to hypoxia and then gradually decreased until 8 h after hypoxia treatment ( Figure 10). In addition, the expression of HAT22/ABIG1 also increased at 1 h after the treatment with NaCl, and the expression level was maintained up to 4 h after the treatment (Figure 10). Increased expression of ADH1 and RD29A, hypoxia and osmotic-stress marker genes, respectively, confirmed that the hypoxia, NaCl, and mannitol stresses were properly treated ( Figure 10). These results indicated that HAT22/ABIG1 is involved in the response to hypoxia and salt stress. HAT22/ABIG1 increased at 1 h after being subjected to hypoxia and then gradually decreased until 8 h after hypoxia treatment ( Figure 10). In addition, the expression of HAT22/ABIG1 also increased at 1 h after the treatment with NaCl, and the expression level was maintained up to 4 h after the treatment (Figure 10). Increased expression of ADH1 and RD29A, hypoxia and osmotic-stress marker genes, respectively, confirmed that the hypoxia, NaCl, and mannitol stresses were properly treated ( Figure 10). These results indicated that HAT22/ABIG1 is involved in the response to hypoxia and salt stress.

Discussion
HRE2 is a member of the ERF-VII transcription factor group in Arabidopsis, and the ERF-VII group is well known to be involved in the hypoxia response in plants [6]. The ERF-VII group members in Arabidopsis, namely RAP2.12, RAP2.2, RAP2.3, HRE1, and HRE2, are post-transcriptionally regulated by the N-degron pathway; however, their transcriptional regulation is not well understood [6]. Moreover, signal transduction pathways involving ERF-VII group genes, including upstream transcriptional regulators and downstream genes, have not been well studied. In this study, we identified the HRE2 promoter region containing hypoxia-and salt stress-responsive positive cis-regulatory element(s). In addition, we isolated HAT22/ABIG1 as a transcriptional repressor of HRE2 transcription in responses to hypoxia and salt stress, and identified a negative cis-regulatory element bound by HAT22/ABIG1 in HRE2 promoter.
We have previously reported that HRE2 is involved in responses to both hypoxia and salt stress and that the 180 bp promoter of HRE2 includes positive cis-regulatory element(s) responsible for these responses [35]. To elucidate the signal transduction pathway of hypoxia and salt stress responses via HRE2, we first analyzed the region of HRE2 promoter responsible for hypoxia and salt stress responses. The analysis using firefly luciferase and GUS as reporter genes controlled by deletion series of the 180 bp HRE2 promoter showed that the −116 to −2 region of HRE2 promoter includes positive cis-regulatory element(s) responsible for both hypoxia and salt stress responses (Figures 1-4). We analyzed potential cis-regulatory elements in the −116 to −2 region of HRE2 promoter using PLACE (https: //www.dna.affrc.go.jp/PLACE/?action=newplace, accessed on 2 April 2022), software for the analysis of plant cis-regulatory element(s) in the promoter. However, we could not find candidate(s) for hypoxia-responsive positive cis-regulatory element(s) (data not shown).
Using yeast one-hybrid screening, we isolated HAT22/ABIG1, a member of the HD-Zip II subfamily, which binds to the −116 to −2 region of HRE2 promoter (Table S2 and Figure 5). It has been well known that HD-Zip II proteins contain LxLxL-type EAR motif in their N-terminus and repress downstream genes by binding to 7 bp conserved regulatory sequences, 5 -AAT(G/C)ATT-3 , in the promoters of the downstream genes [13]. For example, HAT1 directly binds to the target genes of brassinosteroids and functions as a co-repressor together with BES1 [27]. AtHB2 acts as a negative regulator and induces hypocotyl elongation by inhibiting auxin transport inhibitors [36]. Interestingly, the −116 to −2 region of HRE2 promoter contains 5 -AATGATA-3 sequence, which is similar to the HD-Zip II-binding 7 bp element ( Figure S4). The yeast one-hybrid assay and transrepression assay in Arabidopsis protoplasts showed that HAT22/ABIG1 binds to the 7 bp conserved regulatory sequence and represses the transcription of HRE2 (Figures 8 and 9). Our results demonstrated that HAT22/ABIG1 represses the transcription of HRE2 via the 7 bp negative cis-regulatory element, 5 -AATGATA-3 , in the −116 to −2 region of HRE2 promoter in responses to hypoxia and/or salt stress, and that the EAR motif in the N-terminus of HAT22/ABIG1 plays an important role in this transcriptional repression. This is the first report to clarify that the 7 bp negative cis-regulatory element is involved in hypoxia and salt stress signal transduction via the HD-Zip II protein HAT22/ABIG1. As the transcriptional regulator(s) that activate HRE2 transcription remain unidentified in this study, further studies using the −116 to −2 region of HRE2 promoter are needed to isolate and characterize the transcriptional activators.
Gene expression is tightly regulated by transcriptional activators and repressors. Regulation of the balance between activators and repressors is important for proper gene expression and responses to abiotic stresses [11]. DREB1/CBF proteins transactivate RD29A and COR15A to lead tolerance to freezing temperature, whereas DEAR1 protein represses RD29A and COR15A to tightly control during normal growth and development [37]. NAC016 and AtNAP negatively regulate AREB1 under drought stress, whereas SnRK2.2 positively regulates AREB1, resulting in fine-tuning of the spatiotemporal control of drought stress-responsive signaling [38,39]. Our results showed that the −116 to −2 region of HRE2 promoter contains both positive and negative cis-regulatory elements involved in responses to hypoxia and salt stress and that the negative cis-regulatory element is bound by HAT22/ABIG1, indicating that the transcription of HRE2 might be properly regulated by both transcriptional activator(s) and repressor(s).
The ERF-VII group of the AP2/ERF family can be divided into two types, namely, the RAP-type, which includes RAP2.12, RAP2.2, and RAP2.3, and the HRE-type, which includes HRE1 and HRE2 [6]. Recently, it was reported that RAP2.2 is transactivated by WRKY33 and WRKY12 in the hypoxia response via the W-box, 5 -AGTCAA-3 , in RAP2.2 promoter. However, HRE2 and HRE1 are not regulated by WRKY33 and WRKY12 [11] and our analysis revealed that the HRE2 promoter does not contain the W-box (data not shown). On the other hand, RAP2.12 and RAP2.2 transactivate downstream genes via HRPE, a hypoxia-responsive cis-regulatory element, whereas HRE1 and HRE2 transactivate downstream genes via the GCC box [7][8][9][10]. These results suggest that the RAP-type and HRE-type ERF-VII groups might be involved in separate signal transduction pathways in the hypoxia response.
Taken together, our results demonstrate that the −116 to −2 region of HRE2 promoter contains both positive and negative cis-regulatory elements involved in hypoxia and salt stress responses and that HAT22/ABIG1 transcriptionally represses HRE2 via 5 -AATGATA-3 sequence, which is a negative cis-regulatory element present in the −116 to −2 region.

Plant Materials and Growth Conditions
All Arabidopsis thaliana plants used in this study were of the Columbia (Col-0) ecotype. Arabidopsis seeds preparation, germination, and growth were performed according to previous study [35].

Plasmid Construction
To generate deletion series of HRE2 promoter, −180 to +212, −116 to +212, −2 to +212, and +52 to +212 regions from the transcriptional start site of HRE2 were amplified by PCR and cloned into pFGL1495 or pFGL539 fused with firefly luciferase or GUS, respectively. Two tandem repeats of −116 to −2 region of HRE2 promoter were cloned into pFGL1437 fused with firefly luciferase.
To construct plasmids for the yeast one-hybrid assay, the promoter regions of HRE2 were amplified by PCR and cloned into pAbAi or pLacZi fused with AUR1-C or lacZ, respectively. The full-length ORF of HAT22/ABIG1 was amplified by PCR and cloned into pGADT7 in-frame with GAL4 AD.
To generate plasmids for the transrepression assay in Arabidopsis protoplasts, the promoter regions of HRE2 were amplified by PCR and cloned into pFGL1437 fused with firefly luciferase.
The primers for cloning are listed in Table S3.

Generation of Arabidopsis Transgenic Plants
The constructs for expression in Arabidopsis were transformed into Agrobacterium tumefaciens strain GV3101 (pMP90) using the freeze-thaw method [40] and then introduced into WT Arabidopsis using the floral-dipping method [41]. Transgenic plants were selected by 50 mg/L of kanamycin in MS plates.

Stress Treatment
For the hypoxia treatment, 10-day-old WT seedlings grown on MS plates were transferred to MS medium-saturated filter paper and then were treated with 99.99% N 2 gas under dark conditions for 0, 1, 2, 4, and 8 h.
For NaCl and mannitol treatments, 10-day-old WT seedlings grown on MS plates were transferred to filter papers saturated with MS medium containing 150 mM NaCl or 300 mM mannitol and kept for 0, 1, 2, and 4 h.

Histochemical GUS Assay
GUS activity was detected histochemically following a previously described protocol [35].

Protoplast Transformation
Arabidopsis protoplast isolation and transformation were conducted according to Yoo et al. [42].

cDNA Library Generation and Yeast One-Hybrid Screening
To generate a hypoxia cDNA library, 7-and 14-day-old seedlings grown under shortday conditions were subjected to hypoxia for 1 and 3 h. Total RNA was isolated using RNAqueous Kit (Invitrogen, Carlsbad, CA, USA) and Plant RNA Isolation Aid (Invitrogen, Carlsbad, CA, USA). Subsequently, cDNA library was generated using Make Your Own "Mate & Plate" Library System (Clontech Laboratories, Inc., Mountain View, CA, USA). The cfu value of the cDNA library was 1.43 × 10 7 . Yeast one-hybrid screening was performed using Matchmaker ® Gold Yeast One-Hybrid Library Screening System. pADE2i harboring two tandem repeats of the −116 to −2 region of HRE2 promoter was used as the bait in yeast one-hybrid screening. cDNA library generation and yeast one-hybrid screening were performed by PanBioNet (http://www.panbionet.com, accessed on 19 February 2019).

Yeast Transformation and Assay
The constructs for the yeast one-hybrid assay were transformed into Y1HGOLD or YM4271. Yeast transformation was performed by the Frozen-EZ Yeast Transformation II TM Kit (Zymo Research Corp., Irvine, CA, USA), in accordance with the manufacturer's instructions. A quantitative β-galactosidase assay was performed using ONPG as a substrate. The unit of β-galactosidase activity was calculated using the formula 1000 × OD 420 /(OD 600 × assay time in min × assay volume in mL). Transformants were analyzed using 5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside as a substrate for the β-galactosidase filter assay. The reaction was carried out for 6 h. For the yeast growth assay, transformants were streaked onto synthetic minimal media lacking leucine and uracil containing 150 ng/mL Aureobasidin A (AbA) and incubated for 3-5 days at 30 • C.
4.10. RNA Isolation, cDNA Synthesis, and RT-qPCR Total RNA was isolated by RNAqueous Kit (Invitrogen, Carlsbad, CA, USA) and Plant RNA Isolation Aid (Invitrogen, Carlsbad, CA, USA) in accordance with the manufacturer's protocol. Two micrograms of total RNA was reverse-transcribed using Moloney murine leukemia virus reverse transcriptase (Promega Corp., Madison, WI, USA). RT-qPCR was performed and analyzed using Power SYBR Green PCR Master mix (Applied Biosystems, Foster, CA, USA), QuantStudio TM 3 real-time PCR system (Applied Biosystems, Foster, CA, USA), and QuantStudio TM Design and Analysis software v.1.4.3 (Applied Biosystems, Foster, CA, USA) in accordance with the manufacturer's manual. Three independent reactions were conducted for each technical replicate. Two technical replicates were conducted for each biological replicate. The primers for RT-qPCR are listed in Table S4.