OsWRKY97, an Abiotic Stress-Induced Gene of Rice, Plays a Key Role in Drought Tolerance

Drought stress is one of the major causes of crop losses. The WRKY families play important roles in the regulation of many plant processes, including drought stress response. However, the function of individual WRKY genes in plants is still under investigation. Here, we identified a new member of the WRKY families, OsWRKY97, and analyzed its role in stress resistance by using a series of transgenic plant lines. OsWRKY97 positively regulates drought tolerance in rice. OsWRKY97 was expressed in all examined tissues and could be induced by various abiotic stresses and abscisic acid (ABA). OsWRKY97-GFP was localized to the nucleus. Various abiotic stress-related cis-acting elements were observed in the promoters of OsWRKY97. The results of OsWRKY97-overexpressing plant analyses revealed that OsWRKY97 plays a positive role in drought stress tolerance. In addition, physiological analyses revealed that OsWRKY97 improves drought stress tolerance by improving the osmotic adjustment ability, oxidative stress tolerance, and water retention capacity of the plant. Furthermore, OsWRKY97-overexpressing plants also showed higher sensitivity to exogenous ABA compared with that of wild-type rice (WT). Overexpression of OsWRKY97 also affected the transcript levels of ABA-responsive genes and the accumulation of ABA. These results indicate that OsWRKY97 plays a crucial role in the response to drought stress and may possess high potential value in improving drought tolerance in rice.


Introduction
Drought is one of the major stresses that seriously affects plant growth and reduces yield [1]. Plants have evolved various strategies to cope with drought stress for survival and development.The response process of plants to drought stress includes stress signal perception, signal transduction and amplification, and adaptation at morphological, physiological, and molecular levels [2].In these processes, many diverse stress-related proteins are expressed that enhance drought resistance via outputs such as osmotic adjustment, stomatal closure, and reactive oxygen species (ROS) scavenging [3].
ABA has been characterized extensively as an important plant hormone, which responds to drought stress by regulating stomatal closure and transpiration rate [4,5].It has been reported that most water stress-inducing genes respond to treatment with exogenous ABA and relate to ABA signal transduction pathways [6].ABA-mediated stomatal closure is usually accompanied by the production of H 2 O 2 [7].It was reported that H 2 O 2 participated in ABA signal transduction in plant guard cells and triggered stomatal closure by Quantitative Polymerase Chain Reaction (qRT-PCR), so that the expression pattern of OsWRKY97 in different tissues at different stages of rice was detected.OsWRKY97 was expressed in various tissues of rice at different stages (Figure S1).We investigated the expression patterns of OsWRKY97 seedlings under various abiotic stresses by qRT-PCR.These results suggested that OsWRKY97 may be involved in responses to various stresses (Figure 1).For drought treatment, the transcription level of OsWRKY97 gradually increased until reaching a maximum at 8 h (Figure 1A).The transcription level of OsWRKY97 reached its maximum after treatment with 50 µM ABA for 12 h, and similar results were obtained after treatment with 250 mM NaCl (Figure 1B,C).The expression of OsWRKY97 increased significantly in 12 h to 24 h under cold stress (Figure 1D).However, the transcription level of OsWRKY97 under high-temperature treatment was not significantly induced (Figure 1E).A-E) OsWRKY97 expression analysis (qRT-PCR) in leaves of 2-week-old rice seedlings subjected to 20% (w/v) PEG6000, 50 µM ABA, 250 mM NaCl, cold (4 °C), and heat (42 °C) treatments, respectively.OsActin gene was used as internal controls.The data are represented as the mean ± SD (n = 3), with three biological experiments.Asterisks indicates a significant difference from the value at 0 h (t-test, * p < 0.05, ** p < 0.01).

Phylogenetic Analysis of OsWRKY97
We searched the homologous amino acid sequence of OsWRKY97 by using the National Center for Biotechnology Information (NCBI, http://www.ncbi.nlm.nih.gov/,accessed on 10 July 2019) BLASTp online tool.The phylogenetic tree of OsWRKY97 protein sequences and other similar sequences was constructed using the N-J method in MEGA-X software.The results showed that OsWRKY97 is closer to TaWRKY13, followed by ZmWRKY46 (Figure 2).However, the identity of OsWRKY97 with other orthologs was lower than that with TaWRKY13; this shows that OsWRKY97 has a wide range of changes with other members.cessed on 10 July 2019) BLASTp online tool.The phylogenetic tree of OsWRKY97 protein sequences and other similar sequences was constructed using the N-J method in MEGA-X software.The results showed that OsWRKY97 is closer to TaWRKY13, followed by ZmWRKY46 (Figure 2).However, the identity of OsWRKY97 with other orthologs was lower than that with TaWRKY13; this shows that OsWRKY97 has a wide range of changes with other members.

Subcellular Localization of OsWRKY97
To determine the subcellular localization of OsWRKY97, the full-length cDNA sequence of OsWRKY97 was fused to green fluorescent protein (GFP) and driven by CaMV35S.The fusion protein and GFP control were transiently expressed in tobacco cells via Agrobacterium infiltration, and meanwhile, the carrier of the red fluorescent protein (RFP) NLS-RFP connected with the nuclear localization signal is used as the nuclear localization control.The fluorescence signal of the fusion protein was located in the nucleus (Figure 3A), while the fluorescence signal of the GFP control was located in the nucleus and cytoplasm (Figure 3B).OsWRKY97-GFP fusion protein and NLS-RFP were co-located in the nucleus, indicating that OsWRKY97 is a nuclear protein.

Subcellular Localization of OsWRKY97
To determine the subcellular localization of OsWRKY97, the full-length cDNA sequence of OsWRKY97 was fused to green fluorescent protein (GFP) and driven by CaMV35S.The fusion protein and GFP control were transiently expressed in tobacco cells via Agrobacterium infiltration, and meanwhile, the carrier of the red fluorescent protein (RFP) NLS-RFP connected with the nuclear localization signal is used as the nuclear localization control.The fluorescence signal of the fusion protein was located in the nucleus (Figure 3A), while the fluorescence signal of the GFP control was located in the nucleus and cytoplasm (Figure 3B).OsWRKY97-GFP fusion protein and NLS-RFP were co-located in the nucleus, indicating that OsWRKY97 is a nuclear protein.
sequences and other similar sequences was constructed using the N-J method in MEGA-X software.The results showed that OsWRKY97 is closer to TaWRKY13, followed by ZmWRKY46 (Figure 2).However, the identity of OsWRKY97 with other orthologs was lower than that with TaWRKY13; this shows that OsWRKY97 has a wide range of changes with other members.

Subcellular Localization of OsWRKY97
To determine the subcellular localization of OsWRKY97, the full-length cDNA sequence of OsWRKY97 was fused to green fluorescent protein (GFP) and driven by CaMV35S.The fusion protein and GFP control were transiently expressed in tobacco cells via Agrobacterium infiltration, and meanwhile, the carrier of the red fluorescent protein (RFP) NLS-RFP connected with the nuclear localization signal is used as the nuclear localization control.The fluorescence signal of the fusion protein was located in the nucleus (Figure 3A), while the fluorescence signal of the GFP control was located in the nucleus and cytoplasm (Figure 3B).OsWRKY97-GFP fusion protein and NLS-RFP were co-located in the nucleus, indicating that OsWRKY97 is a nuclear protein.

Analysis of OsWRKY97 Promoter Domain
The promoter region contains many cis-acting elements related to stress response.In order to further understand the regulatory mechanism of OsWRKY97, we analyzed the promoter region upstream of OsWRKY97 ATG initiation codon.The results showed that the promoter region contained the W-box elements, and MYB and MYC binding sequences.In addition, gibberellin-responsive elements (GAREs) and SA-responsive element (W-Box) were identified (Table 1).In this study, we found that OsWRKY97 was strongly induced by osmotic stress (Figure 1A).To further verify whether OsWRKY97 was involved in regulating the sensitivity of rice to stress, we constructed an OsWRKY97 overexpression vector and transferred it into wild-type rice.The expression level of OsWRKY97 in overexpressed plants (OE) was analyzed by qRT-PCR.The results showed that the expression of OsWRKY97 was significantly enhanced in transgenic lines (Figure 4A).The screened T2 transgenic plants all showed significant response to drought stress, but the OE-1 and OE-23 lines showed the most significant performance.Therefore, these two independent transgenic rice lines, OE-1 and OE-23, were selected for future testing.Under normal conditions, the germination rates of WT and overexpression lines were not significantly different.However, after 5 days of absorption under osmotic stress (20% (w/v) PEG6000), the germination rate of overexpressed lines OE-1 and OE-23 (76% and 80%, respectively) were higher than that of WT (50%) (Figure 4B,C).To learn about the sensitivity of overexpression lines to osmotic stress at the post-germination stage, seedings of WT and overexpression lines that were growing under normal conditions for 4 days were selected and transferred to the nutrient solution under normal and osmotic stress conditions.After 12 days, each material was photographed, and its plant height was measured.Under normal conditions, the seedlings of WT and overexpressing lines were similar in growth.Under osmotic stress, the plant height of WT seedlings was 8.6 cm, while the plant height of overexpression seeding OE-1 was 10.8 cm, and that of OE-23 was 10.6 cm (Figure 4D,E).These results showed that overexpression of OsWRKY97 in rice did not affect seed germination and seedling growth under normal growth conditions, but significantly attenuated the inhibitory effects on seed germination and seedling under osmotic stress induced by 20% (w/v) PEG6000.

Overexpression of OsWRKY97 in Rice Enhanced Drought Stress Tolerance
To further validate the biological function of OsWRKY97, we tested the drought stress tolerance of OsWRKY97 overexpression lines by water deficit.OsWRKY97-overexpressing lines and WT seedlings with similar vigor were sown in the same pots, watering was

Overexpression of OsWRKY97 in Rice Enhanced Drought Stress Tolerance
To further validate the biological function of OsWRKY97, we tested the drought stress tolerance of OsWRKY97 overexpression lines by water deficit.OsWRKY97-overexpressing lines and WT seedlings with similar vigor were sown in the same pots, watering was stopped until the leaves curled, and then resumed.Growth conditions were similar for all plants before stress was applied.After drought treatment, WT plants showed more severe leaf curling than OsWRKY97-overexpressing plants.After re-watering, the survival rate of overexpressed plant OE-1 was 53%, and that of OE-23 was 55%; however, the survival rate of WT was only 14-16% (Figure 5A,B).These results indicated that overexpression of OsWRKY97 enhances the drought tolerance of rice.The relative water loss rate of detached leaves is an important characteristic reflecting drought tolerance [24].Leaves of 2-week-old transgenic plants and WT seedlings were removed and exposed to water-free air for dehydration.Compared with WT, the leaf water loss of overexpression lines was significantly slower (Figure 5C), which means that OsWRKY97 played an active role in improving the water retention capacity of plants under dehydration conditions.
Plants 2023, 12, x FOR PEER REVIEW 7 of 15 stopped until the leaves curled, and then resumed.Growth conditions were similar for all plants before stress was applied.After drought treatment, WT plants showed more severe leaf curling than OsWRKY97-overexpressing plants.After re-watering, the survival rate of overexpressed plant OE-1 was 53%, and that of OE-23 was 55%; however, the survival rate of WT was only 14-16% (Figure 5A,B).These results indicated that overexpression of OsWRKY97 enhances the drought tolerance of rice.The relative water loss rate of detached leaves is an important characteristic reflecting drought tolerance [24].Leaves of 2-weekold transgenic plants and WT seedlings were removed and exposed to water-free air for dehydration.Compared with WT, the leaf water loss of overexpression lines was significantly slower (Figure 5C), which means that OsWRKY97 played an active role in improving the water retention capacity of plants under dehydration conditions.

Effects of OsWRKY97 Overexpression on Related Physiological Indexes under Drought Stress
To further clarify the physiological mechanism by which OsWRKY97 confers tolerance to drought, we investigated the possible physiological basis related to the enhance-

Effects of OsWRKY97 Overexpression on Related Physiological Indexes under Drought Stress
To further clarify the physiological mechanism by which OsWRKY97 confers tolerance to drought, we investigated the possible physiological basis related to the enhancement of drought resistance in OsWRKY97-overexpressing plants.ROS were generated in plants when they were subjected to abiotic stresses such as drought, salinity, heat, and cold [25].H 2 O 2 , which is an important second messenger, is one of the most significant of these ROS [26].Therefore, we measured the accumulation of H 2 O 2 in plants after drought treatment.We found that the accumulation of H 2 O 2 in OsWRKY97-overexpressing plants after drought treatment was much lower than that of WT (Figure 6A).To explore the potential mechanism of the reduction of active oxygen level in transgenic plants, ROS scavenging enzyme activity was also measured [11].As shown in Figure 6, there was no significant difference in superoxide dismutase (SOD), catalase (CAT), and peroxidase (POD) activities between transgenic lines and WT under normal conditions, while the enzyme activities of transgenic lines were higher than those of WT under drought stress (Figure 6B,D).Abiotic stresses to plants lead to enhanced membrane peroxidation and accumulation of osmotic substances in plants to maintain osmotic potential tissues of leaves [27].As shown in Figure 6E, under drought conditions, the contents of malondialdehyde (MDA) in the OsWRKY97-overexpressing plants were significantly lower than that in the WT, which indicated that overexpression of OsWRKY97 in rice could reduce membrane lipid peroxidation under drought stress.Under drought stress, the proline content of transgenic plants was significantly higher than that of control plants (Figure 6F).This result clearly shows that overexpression of OsWRKY97 can increase proline synthesis and protect rice plants to better cope with drought stress.
Plants 2023, 12, x FOR PEER REVIEW 8 of 15 cold [25].H2O2, which is an important second messenger, is one of the most significant of these ROS [26].Therefore, we measured the accumulation of H2O2 in plants after drought treatment.We found that the accumulation of H2O2 in OsWRKY97-overexpressing plants after drought treatment was much lower than that of WT (Figure 6A).To explore the potential mechanism of the reduction of active oxygen level in transgenic plants, ROS scavenging enzyme activity was also measured [11].As shown in Figure 6, there was no significant difference in superoxide dismutase (SOD), catalase (CAT), and peroxidase (POD) activities between transgenic lines and WT under normal conditions, while the enzyme activities of transgenic lines were higher than those of WT under drought stress (Figure 6B,D).Abiotic stresses to plants lead to enhanced membrane peroxidation and accumulation of osmotic substances in plants to maintain osmotic potential tissues of leaves [27].
As shown in Figure 6E, under drought conditions, the contents of malondialdehyde (MDA) in the OsWRKY97-overexpressing plants were significantly lower than that in the WT, which indicated that overexpression of OsWRKY97 in rice could reduce membrane lipid peroxidation under drought stress.Under drought stress, the proline content of transgenic plants was significantly higher than that of control plants (Figure 6F).This result clearly shows that overexpression of OsWRKY97 can increase proline synthesis and protect rice plants to better cope with drought stress.

OsWRKY97 Is a Positive Regulator in ABA Signaling under Drought Stress
In this study, we found that OsWRKY97 was also strongly induced by ABA (Figure 1B), to verify this expression pattern, the sensitivities of OsWRKY97-overexpressing plants to exogenous ABA were examined.The results showed that the germination rate of WT plants was higher than OsWRKY97-overexpressing plants under the treatment of exogenous ABA (Figure 7A,B).Therefore, we suspect that the drought tolerance of OsWRKY97overexpressing transgenic lines may be related to ABA.In order to verify this hypothesis, we measured the ABA content in OsWRKY97-overexpressing lines and WT, respectively, under drought stress.The results showed that the endogenous ABA level was not significantly different between WT plants and OsWRKY97-overexpressing plants under normal conditions.However, under drought stress, the endogenous ABA level in OsWRKY97overexpressing plants was significantly higher than WT plants (Figure 7C).In addition, we also analyzed the transcription level of response genes in the ABA signaling pathway in WT plants and OsWRKY97-overexpressing plants, including OsRAB21, OsRD22, OsRAB16A, and OsNCED3 [28,29].As shown in Figure 7D, the transcription level of these genes in OsWRKY97-overexpressing plants was significantly higher than WT plants under drought stress.These results indicate that OsWRKY97 may improve drought tolerance via the ABA signaling pathway.In this study, we found that OsWRKY97 was also strongly induced by ABA (Figure 1B), to verify this expression pattern, the sensitivities of OsWRKY97-overexpressing plants to exogenous ABA were examined.The results showed that the germination rate of WT plants was higher than OsWRKY97-overexpressing plants under the treatment of exogenous ABA (Figure 7A,B).Therefore, we suspect that the drought tolerance of OsWRKY97overexpressing transgenic lines may be related to ABA.In order to verify this hypothesis, we measured the ABA content in OsWRKY97-overexpressing lines and WT, respectively, under drought stress.The results showed that the endogenous ABA level was not significantly different between WT plants and OsWRKY97-overexpressing plants under normal conditions.However, under drought stress, the endogenous ABA level in OsWRKY97overexpressing plants was significantly higher than WT plants (Figure 7C).In addition, we also analyzed the transcription level of response genes in the ABA signaling pathway in WT plants and OsWRKY97-overexpressing plants, including OsRAB21, OsRD22, OsRAB16A, and OsNCED3 [28,29].As shown in Figure 7D, the transcription level of these genes in OsWRKY97-overexpressing plants was significantly higher than WT plants under drought stress.These results indicate that OsWRKY97 may improve drought tolerance via the ABA signaling pathway.overexpression lines under exogenous ABA stress (t-test, * p < 0.05, ** p < 0.01).(C) ABA contents of OsWRKY97 overexpression and WT plants under normal and drought stress conditions.Results are means ± SD from three independent biological experiments.(D-G) Real-time PCR analysis of the expression of ABA biosynthesis and responsive genes under normal and drought stress conditions.The data are represented as the mean ± SD, with three biological experiments.The asterisk represents the statistically significant difference between the WT and OsWRKY97 overexpression lines under osmotic stress (t-test, * p < 0.05, ** p < 0.01).

Discussion
Plants will suffer from various abiotic stresses in their natural environment, which will affect the normal development of plants and even affect their yield [30,31].At present, many studies have shown that WRKY genes are negatively or positively involved in the integration of signaling pathways in abiotic stress responses [32].However, the functions of many WRKY genes in plants, especially stress responses, are still unclear.In this study, we examined whether OsWRKY97 participates in the regulation of the response to drought stress and its effect on rice.In addition, we found that OsWRKY97-GFP subcellular localization was in the nucleus of tobacco epidermal cells (Figure 3), which is consistent with previous studies on other WRKY families and may be related to its function [33].
Increasing evidence shows that WRKY families play an important role in the drought stress response, as ectopic expression of OsWRKY11 enhanced tolerance to drought stress and induced constitutive expression of drought-responsive genes [34].Overexpression of GsWRKY20 from Glycine soja L.G07256 in Arabidopsis resulted in increased sensitivity to ABA when stomata were closed and stronger drought tolerance compared with the WT [35].Similarly, our results showed that OsWRKY97 expression was rapidly activated under drought stress, suggesting that OsWRKY97 may play an important role in drought stress.Therefore, we constructed OsWRKY97-overexpressing transgenic plants and tested their resistance to drought stress.The results showed that the germination rate of OsWRKY97overexpressing plants at the germination stage was significantly higher, and the growth inhibition of OsWRKY97 at the seedling stage was also weakened compared with that of WT plants under osmotic stress (Figure 4).These results suggest that OsWRKY97 may positively regulate drought stress tolerance.In addition, this conclusion was supported by the result that the survival rate of OsWRKY97-overexpressing plants was higher than that of WT plants under drought conditions (Figure 5A,B).
Plants respond to water loss at physiological, cellular, and molecular levels [36].ABA is an important plant hormone involved in the plant developmental process, which is widely considered as the main regulatory factor of plant response to drought.ABA reduces water loss by inducing stomatal closure and induces a number of stress response genes [37,38].It has been reported that ABA-independent and ABA-dependent regulatory systems both exist in response to drought stress [39].In this study, first, we found that OsWRKY97 was strongly induced by exogenous ABA (Figure 1B).Second, OsWRKY97 overexpression enhances the sensitivity of plants to exogenous ABA (Figure 7C).Third, the expression level of OsWRKY97 induces ABA accumulation and the expression level of ABA-responsive genes under drought stress (Figure 7A,D); this result was consistent with the capability of OsWRKY97 to reduce the water loss rate of plants under drought conditions (Figure 5C).These results intimate that OsWRKY97 improves drought tolerance by enhancing water retention of rice through the ABA-dependent pathway.Meanwhile, the up-regulation of OsWRKY97 expression under drought stress will lead to the up-regulation of ABA biosynthesis and response genes, resulting in ABA accumulation and increased sensitivity to exogenous ABA.
H 2 O 2 is not only an important ROS but also the pivot for the mutual conversion of ROS, which is also an important signal molecule at normal levels [26].The ABA signal interacts with H 2 O 2 in plant tissues.There is evidence that H 2 O 2 acts upstream of the ABA signaling pathway.Exogenous H 2 O 2 increases ABA catabolism during seed germination by enhancing the expression of CYP707A genes [40,41].It has also been reported that H 2 O 2 plays an important role as a second messenger in ABA-induced stomatal closure in guard cells [8,9].It is reported that H 2 O 2 can be induced by ABA, which is mediated by inducing plant gene expression encoding NADPH oxidase to respond to ABA [42,43].However, the increase in H 2 O 2 content induced by drought stress is more obvious than that caused by exogenous ABA; this shows that the water deficit signal enhances the production of ROS to a greater extent, which will pose a threat to plants [44].As expected, the level of H 2 O 2 in plants increased under drought stress.However, the accumulation of H 2 O 2 in WT plants was significantly higher than that in OsWRKY97-overexpressing plants (Figure 6A), and the activities of major oxygen scavenging enzymes, including SOD, POD, and CAT, were also observably higher than those in OsWRKY97-overexpressed plants (Figure 6B,D).SOD, POD, and CAT, which maintain the ROS homeostasis, were activated in OsWRKY97-overexpressing plants.
As a product of ROS-stimulated lipid peroxidation, MDA contents can be used to evaluate the extent of ROS-mediated injuries in plants.In our research, we found that the accumulation of ROS and MDA in WT plants under drought stress were higher than that in OsWRKY97-overexpressing plants (Figure 6E).In addition, we also found that the accumulation of proline in OsWRKY97-overexpressing plants was greater than that in WT plants under drought stress (Figure 6F).Under osmotic conditions, proline, as an important osmotic protective agent, can maintain low water potential of cells so as not to be damaged by active enzymes [45].These results showed that WT was more seriously damaged than OsWRKY97-overexpressing plants under osmotic conditions, thus increasing the probability of WT death.

Plant Material and Growth Conditions
The plant material Oryza sativa L. subsp.japonica cv.Nipponbare was used in this experiment as the wild-type rice.Rice seeds were sterilized with 0.1% NaClO for 30 min before being soaked in distilled water for 2 days in the dark and were then transferred to a culture dish containing Hoagland nutrient solution and grown in a climate chamber (Southeast instrument, Ningbo, China) with a temperature of 28 • C, a relative humidity of 70%, and a 14 h light/10 h dark photoperiod [46].

Abiotic Treatments
To determine the expression pattern of OsWRKY97 under different stress conditions, two-week-old seedlings of Nipponbare rice were subjected to various stress treatments.For drought treatment, seedlings were grown in culture solution containing 20% (w/v) PEG6000.For salt treatment, NaCl solution was added to achieve a final concentration of 250 mM.ABA treatment was carried out by adding 50 µM ABA to the culture solution.The seedlings were transferred to a 4 • C climate chamber for cold treatment.For heat stress, the seedlings were subjected to 42 • C heat shock treatment [47,48].Samples were collected at 0, 1, 2, 4, 8, 12, 16 and 24 h after treatment.Two-week-old seedings of OsWRKY97 overexpression lines were subjected to drought treatment, and samples were collected at 0 h and 10 h after treatment, respectively.

RNA Extraction and Real-Time PCR
The leaves of 14-day-old plants were sampled, and total RNA was extracted with TRIzol reagent (Invitrogen, Nanjing, China) according to the manufacturer's instructions.One microgram of DNase-treated RNA was reverse-transcribed using a RevertAid RT Reverse Transcription Kit (TaKaRa, Beijing, China) according to the manufacturer's protocol.Real-Time Quantitative Polymerase Chain Reaction was performed on a Bio-Rad CFX96 real-time PCR system.Each reaction was performed in triplicate, and the reaction procedure was as follows: 95 • C for 10 min and, then 39 cycles of 95 • C for 10 s and, 60 • C for 30 s.The data of relative expression level were analyzed by the 2 −∆∆Ct method [49].The OsActin rice gene was used as an internal control gene, and relevant primer pairs are listed in Table S1.similar growth potential were selected and transferred to nutrient solution containing 20% (w/v) PEG6000, and their growth conditions were observed and recorded.To simulate an arid environment, the two-week-old seedlings were transferred to the sand, and watering was stopped after two days of normal irrigation.The mortality rates were determined.In order to test the sensitivity of ABA sensitivity at germination stage, seeds of OsWRKY97overexpressing plants and WT plants were germinated on culture solution containing 5 µM ABA, and the germination rate was calculated on the 6th day after germination.

Statistical Analysis
Results are reported as the mean ± standard deviation (SD) values of the three independent biological experiments; all experiments were repeated at least three times, Statistical analysis was performed using the Student's t-test by SPSS 27.0 (IBM SPSS, Chicago, IL, USA) software package, * p < 0.05, ** p < 0.01.

Conclusions
In conclusion, we identified a nuclear gene OsWRKY97, which affects the sensitivity of rice to exogenous ABA and the accumulation of ABA content.In addition, OsWRKY97 affects the redox balance and drought resistance of rice.Redox-related mechanisms might be involved in OsWRKY97-mediated drought tolerance, which might affect the content of proline and MDA in rice.All these results indicated that the OsWRKY97 gene has high potential for improving rice drought resistance.

Figure 2 .
Figure 2. Phylogenetic tree of the protein sequences of OsWRKY97 and other similar sequences.

Figure 3 .
Figure 3. Subcellular localization of OsWRKY97-GFP.OsWRKY97 driven by the CaMV35S promoter was transiently expressed in tobacco leaf epidermal cells and viewed with confocal microscopy.

Figure 2 .
Figure 2. Phylogenetic tree of the protein sequences of OsWRKY97 and other similar sequences.

Figure 2 .
Figure 2. Phylogenetic tree of the protein sequences of OsWRKY97 and other similar sequences.

Figure 3 .
Figure 3. Subcellular localization of OsWRKY97-GFP.OsWRKY97 driven by the CaMV35S promoter was transiently expressed in tobacco leaf epidermal cells and viewed with confocal microscopy.

Figure 3 .
Figure 3. Subcellular localization of OsWRKY97-GFP.OsWRKY97 driven by the CaMV35S promoter was transiently expressed in tobacco leaf epidermal cells and viewed with confocal microscopy.Nuclear and cytosolic localization of GFP protein was shown as a control.(A) 35S: OsWRKY97-GFP; (B) 35S: GFP.Bar = 50 µm.

Figure 4 .
Figure 4. Phenotype of OsWRKY97-overexpressing plants under osmotic stress.(A) Two independent transgenic lines (OE-1, OE-23) of OsWRKY97 were verified by qRT-PCR.OsActin was analyzed as an internal control.Data were means ± SD with at least three biological replicates.Asterisks represent statistically significant differences between WT and OsWRKY97 overexpression lines (t-test, * p < 0.05, ** p < 0.01).The growth performance (B) and germination rate (C) of OsWRKY97 overexpression lines and wild-type at 5 days after germination in nutrient solution under normal and osmotic stress conditions.The data are represented as the mean ± SD with at least three biological replicates, and every replicate contains 20 individual plants.The growth phenotype (D) and plant height © of overexpression lines and WT, which were growing under normal conditions for 4 days, were transferred to nutrient solution under normal and osmotic stress conditions for 12 days.(E) Data are shown as the mean ± SD (n = 4) with four biological replicates, and every replicate contains 40 individual plants.The asterisk represents the statistically significant difference between the WT and OsWRKY97 overexpression lines under osmotic stress (t-test, ** p < 0.01).

Figure 4 .
Figure 4. Phenotype of OsWRKY97-overexpressing plants under osmotic stress.(A) Two independent transgenic lines (OE-1, OE-23) of OsWRKY97 were verified by qRT-PCR.OsActin was analyzed as an internal control.Data were means ± SD with at least three biological replicates.Asterisks represent statistically significant differences between WT and OsWRKY97 overexpression lines (t-test, ** p < 0.01).The growth performance (B) and germination rate (C) of OsWRKY97 overexpression lines and wild-type at 5 days after germination in nutrient solution under normal and osmotic stress conditions.The data are represented as the mean ± SD with at least three biological replicates, and every replicate contains 20 individual plants.The growth phenotype (D) and plant height © of overexpression lines and WT, which were growing under normal conditions for 4 days, were transferred to nutrient solution under normal and osmotic stress conditions for 12 days.(E) Data are shown as the mean ± SD (n = 4) with four biological replicates, and every replicate contains 40 individual plants.The asterisk represents the statistically significant difference between the WT and OsWRKY97 overexpression lines under osmotic stress (t-test, ** p < 0.01).

Figure 5 .
Figure 5. Drought stress analysis of wild-type plants and transgenic plants with OsWRKY97.(A) Performance of 2-week-old seedlings from OsWRKY97 transgenic and wild-type plants subjected to drought stress without water for 15 days and then recovered for 3 days.The experiment contained three biological replicates.(B) Survival rates of transgenic and WT plants under drought stress.Values represent the mean ± SD (n = 3) from three independent biological experiments, and every replicate contains 12 individual plants.Statistically significant differences between WT and OsWRKY97 overexpression lines were indicated by asterisks (t-test, ** p < 0.01).(C) Rate of water loss by detached leaves from control and transgenic plants.It is expressed as percentage of initial fresh weight.Values are the mean ± SD (n = 3) from three independent biological experiments, and every replicate contains 5 individual plants.The asterisk represents the statistically significant difference at the same time between the WT and OsWRKY97 overexpression lines (t-test, * p < 0.05, ** p < 0.01).

Figure 5 .
Figure 5. Drought stress analysis of wild-type plants and transgenic plants with OsWRKY97.(A) Performance of 2-week-old seedlings from OsWRKY97 transgenic and wild-type plants subjected to drought stress without water for 15 days and then recovered for 3 days.The experiment contained three biological replicates.(B) Survival rates of transgenic and WT plants under drought stress.Values represent the mean ± SD (n = 3) from three independent biological experiments, and every replicate contains 12 individual plants.Statistically significant differences between WT and OsWRKY97 overexpression lines were indicated by asterisks (t-test, ** p < 0.01).(C) Rate of water loss by detached leaves from control and transgenic plants.It is expressed as percentage of initial fresh weight.Values are the mean ± SD (n = 3) from three independent biological experiments, and every replicate contains 5 individual plants.The asterisk represents the statistically significant difference at the same time between the WT and OsWRKY97 overexpression lines (t-test, ** p < 0.01).

Figure 6 .
Figure 6.Physiological parameters of OsWRKY97 overexpressed plants under drought stress.Twoweek-old rice seedlings were transferred to nutrient solution under normal and drought stress conditions (20% (w/v) PEG6000).(A) H2O2 content, (B) CAT activity, (C) POD activity, (D) SOD activity, (E) MDA content, (F) proline content.Values are shown as the mean ± SD (n = 3) and the experiments were performed with at least three biological repetitions.The asterisk represents the statistically significant difference between the WT and OsWRKY97 overexpression lines under osmotic stress (ttest, * p < 0.05, ** p < 0.01).

Figure 6 .of 15 (
Figure 6.Physiological parameters of OsWRKY97 overexpressed plants under drought stress.Twoweek-old rice seedlings were transferred to nutrient solution under normal and drought stress conditions

Figure 7 .
Figure 7. ABA accumulation and sensitivity of OsWRKY97-overexpressing plants.The growth performance (A) and the germination rates (B) of OsWRKY97 overexpression and WT seeds under ABA treatment.Values are shown as the mean ± SD from three biological experiments, with 20 plants in each repeat.The asterisk represents the statistically significant difference between the WT and OsWRKY97 overexpression lines under exogenous ABA stress (t-test, * p < 0.05, ** p < 0.01).(C) ABA contents of OsWRKY97 overexpression and WT plants under normal and drought stress conditions.Results are means ± SD from three independent biological experiments.(D-G) Real-time PCR analysis of the expression of ABA biosynthesis and responsive genes under normal and drought stress conditions.The data are represented as the mean ± SD, with three biological experiments.The asterisk represents the statistically significant difference between the WT and OsWRKY97 overexpression lines under osmotic stress (t-test, * p < 0.05, ** p < 0.01).

Figure 7 .
Figure 7. ABA accumulation and sensitivity of OsWRKY97-overexpressing plants.The growth performance (A) and the germination rates (B) of OsWRKY97 overexpression and WT seeds under ABA treatment.Values are shown as the mean ± SD from three biological experiments, with 20 plants in each repeat.The asterisk represents the statistically significant difference between the WT and OsWRKY97

Table 1 .
Putative cis-elements in the OsWRKY97 promoter.