Overexpression of Maize Glutathione S-Transferase ZmGST26 Decreases Drought Resistance of Arabidopsis

: Drought stress critically endangers the growth and development of crops. Glutathione S-transferase plays a vital role in response to abiotic stress. However, there are few studies on the role of glutathione S-transferase in maize drought stress. In this study, the signiﬁcantly downregulated expression of ZmGST26 in roots under drought stress was analyzed by qRT-PCR. Promoter analyses showed that there were several cis-acting elements related to drought stress and that were involved in oxidative response in the promoter region of ZmGST26 . Subcellular localization results showed that ZmGST26 was localized in the nucleus. The transgenic lines of the Arabidopsis over-expressing ZmGST26 were more sensitive to drought stress and ABA in seed germination and inhibited ABA-mediated stomatal closure. Under drought stress, phenotypic analyses showed that the germination rate, root length and survival rate of ZmGST26 overexpressing lines were signiﬁcantly lower than those of wild-type lines. The determination of physiological and biochemical indexes showed that the water loss rate, malondialdehyde, O 2 − and H 2 O 2 of the overexpression lines signiﬁcantly increased compared with wild-type Arabidopsis , but the antioxidant enzyme activities (CAT, SOD and POD), and proline and chlorophyll contents were signiﬁcantly reduced. Subsequently, the qRT-PCR analysis of drought stress-related gene expression showed that, under drought stress conditions, the expression levels of DREB2A , RD29A , RD29B and PP2CA genes in ZmGST26 overexpression lines were signiﬁcantly lower than those in wild-type Arabidopsis . In summary, ZmGST26 reduced the drought resistance of plants by aggravating the accumulation of reactive oxygen species in Arabidopsis .


Introduction
Plants often suffer from some abiotic stresses during growth and development, among which drought and water shortages are the most serious abiotic stresses that threaten plant production [1].The process of plants resisting drought is very complex.When subjected to drought stress, morphological changes, physiological changes and molecular changes will occur to resist the effects of drought and water shortage.Among them, regulating the balance of intracellular reactive oxygen species (ROS) content is essential [2].Under normal growth conditions, ROS is necessary for plant growth and also acts as a signal transduction molecule in order to positively guide plant growth [3].Under drought stress conditions, excessive ROS can cause different degrees of peroxidation with respect to cell membrane lipids, damage membrane stability and change the cell membrane's permeability [4].In order to effectively resist drought stress, the enzymatic antioxidant system in plants is activated, which can effectively help plants to remove excessive ROS and free radicals in vivo, protect cells from damage, reduce the degree of peroxidation of cell membrane and improve their drought resistance [5].The enzymatic active oxygen-scavenging systems mainly include peroxidase (POD), catalase (CAT), superoxide dismutase (SOD) and so on.Among them, superoxide dismutase (SOD) is the first line of defense against ROS and free radical damage to the cell membrane, and it reacts to remove excess O 2 − [6].The antioxidant function of POD is mainly reflected in the early stages of drought stress, scavenging excessive hydrogen peroxide (H 2 O 2 ).When plants suffer from drought stress for too long or severe drought stress, POD will be directly involved in the process of reactive oxygen species production, directly leading to plant senescence [7].
Glutathione S-transferase (GST) is a family of enzymes with a wide range of functions in plants.It has important functions in abiotic stress [8], antioxidant [9], plant detoxification [10] and resistance to pathogen infections [11].At the same time, GSTs, as one of the main members of the plant ROS-scavenging enzymes, catalyze the reaction of electrophilic substrates to remove ROS and exogenous compounds, thereby protecting plants from damage.Plant GST plays an antioxidant role by repairing membrane phospholipid damage caused by free radicals and inhibiting microsomal peroxidation [12].GSTs are classified into eight classes: Phi, Tau, Theta, Zeta, Lambda, EF1G, DHAR and TCHQD, based on protein homology and genome structure [13].In recent years, several plant-specific GSTs, such as Tau and Phi, received extensive attention in regulating the removal of reactive oxygen species in response to abiotic stress.Some studies have shown that, under the osmotic stress of ABA, in Arabidopsis, glutathione S-transferase AtGSTU17 knockout mutants show enhanced drought and salt stress tolerance [14].The overexpression of the Arabidopsis glutathione S-transferase AtGSTU7 gene reduces the germination rate of seeds and reduces the tolerance to droughts [15].Soybean glutathione S-transferase GsGSTU42 enhances the antioxidant capacity of leaves by enhancing ROS detoxification or homeostasis reconstruction [16].The jasmonic acid-responsive glutathione S-transferase gene SlGSTU24 can reduce cold-induced oxidative stress in tomato plants [17].The overexpression of rice Tau-like glutathione S-transferase OsGSTU30 in Arabidopsis enhances its tolerance to heavy metals and drought stress by biochemical and physiological analyses [18].The overexpression of cotton glutathione S-transferase GhGSTF12 gene causes anthocyanin accumulation in cotton [19].
Many glutathione S-transferases have been identified in previous studies.However, there are relatively few studies on the function of glutathione s-transferase in maize [20,21].In this study, the ZmGST26 gene with significantly downregulated expressions under drought stress was screened from the transcriptome sequencing data of maize H8186 [22].We first cloned and characterized the ZmGST26 gene, and we analyzed its expression pattern in roots, stems and leaves under drought stress by real-time PCR and determined its subcellular localization.The overexpression of ZmGST26 in Arabidopsis decreased the antioxidant enzyme activity of transgenic Arabidopsis, inhibited stomatal closure and increased the accumulation of ROS, thereby reducing its tolerance to drought stress.These results suggest that ZmGST26 may be involved in the negative regulation of maize responses to drought stress.

Drought Stress Induced the Downregulated Expression of ZmGST26 Gene
In maize, we obtained a ZmGST26 gene, which is a member of the glutathione S-transferase family and belongs to the Tau class.To further understand the evolutionary relationship between ZmGST26 and other reported tau proteins, we performed a phylogenetic analysis of the amino acid sequences of 20 Tau genes, and the results showed that ZmGST26 was relatively close to these proteins as the highest homology to OsGSTU7 protein; meanwhile, because the expression level of the gene was regulated by the upstream promoter, when analyzed by a 2000 bp promoter element from the upstream promoter of the ZmGST26 gene, it showed that the upstream promoter of the ZmGST26 gene contained several active oxygen-, drought-, ABA and light-responsive elements (Table S1).Therefore, the expression of the ZmGST26 gene in different tissues of maize plants under drought stress was verified by qRT-PCR.The results showed that the expression level of the ZmGST26 gene was significantly downregulated in roots, stems and leaves under drought stress (Figure 1B), and in roots, it reached the lowest level at 8 h and then stabilized.In leaves and stems, the lowest levels were reached at 4 h and 8 h, and subsequently, the expression increased again, but it was lower than the control level.These results suggest that ZmGST26 is likely involved in the drought response in maize.
gene contained several active oxygen-, drought-, ABA and light-responsive elements (TableS1).Therefore, the expression of the ZmGST26 gene in different tissues of maize plants under drought stress was verified by qRT-PCR.The results showed that the expression level of the ZmGST26 gene was significantly downregulated in roots, stems and leaves under drought stress (Figure 1B), and in roots, it reached the lowest level at 8 h and then stabilized.In leaves and stems, the lowest levels were reached at 4 h and 8 h, and subsequently, the expression increased again, but it was lower than the control level.These results suggest that ZmGST26 is likely involved in the drought response in maize.

Subcellular Localization
Understanding the subcellular localization of gene expression products is of great significance for the functional analyses of genes.To determine the subcellular localization of ZmGST26, p35S::GFP and p35S::-ZmGST26-GFP were transiently expressed in tobacco leaves.As shown in Figure 2, the unfused GFP green fluorescence signal in tobacco leaves was distributed in the nucleus and cytoplasm, while the green fluorescence signal

Subcellular Localization
Understanding the subcellular localization of gene expression products is of great significance for the functional analyses of genes.To determine the subcellular localization of ZmGST26, p35S::GFP and p35S::-ZmGST26-GFP were transiently expressed in tobacco leaves.As shown in Figure 2, the unfused GFP green fluorescence signal in tobacco leaves was distributed in the nucleus and cytoplasm, while the green fluorescence signal of ZmGST26-GFP was only distributed in the nucleus.It was further confirmed that ZmGST26 acts in the nucleus.

Overexpression of ZmGST26 Reduced the Drought Tolerance of Transgenic Arabidopsis
To better define the role of ZmGST26 under abiotic stress, we employed an overexpression strategy.Transgenic Arabidopsis plants were obtained by the overexpression of ZmGST26 under the control of the 35S promoter.We verified and screened 15 lines of ZmGST26 overexpression in T3 generation.Finally, three lines OE-4, OE-9 and OE-13 with obvious overexpression effects were selected for subsequent phenotypic experiments (Figure 3A).The seeds of the overexpression lines and wild-type Arabidopsis thaliana were inoculated on a 1/2 MS medium containing different concentrations of mannitol (0, 100 and 200 mM) and different concentrations of ABA (0.100 and 200 μM), as shown in the figure (Figure 3B-E).Under normal conditions, there were no noticeable changes during germination among the transgenic and WT plants, and germination was about 95% in all cases.However, seed germination was inhibited under mannitol and ABA stress conditions to a more pronounced extent for the overexpression lines relative to the WT.Under 100 mM mannitol stress, the germination rate of the WT seeds reached 72.3%; however, the overexpression lines showed 53.4%, 51.2% and 49.3% germination.This gap was more significant at 200 mm mannitol stress, with 50.2% germination in the wild-type, but only 29.6%, 25.3% and 30.2% in the overexpression lines.At 100 μM ABA stress, the germination rate of wild-type Arabidopsis was 75.7%, whereas that of the overexpression line Arabidopsis was 37.6%, 36.9% and 37.2%.At 200 μM ABA stress, the germination rates of the overexpression line for Arabidopsis were 24.6%, 22.8% and 15.9% in the overexpression lines, whereas it was 38.9% in wild-type Arabidopsis, indicating that the overexpression of ZmGST26 decreased the germination rate of Arabidopsis.

Overexpression of ZmGST26 Reduced the Drought Tolerance of Transgenic Arabidopsis
To better define the role of ZmGST26 under abiotic stress, we employed an overexpression strategy.Transgenic Arabidopsis plants were obtained by the overexpression of ZmGST26 under the control of the 35S promoter.We verified and screened 15 lines of ZmGST26 overexpression in T 3 generation.Finally, three lines OE-4, OE-9 and OE-13 with obvious overexpression effects were selected for subsequent phenotypic experiments (Figure 3A).The seeds of the overexpression lines and wild-type Arabidopsis thaliana were inoculated on a 1/2 MS medium containing different concentrations of mannitol (0, 100 and 200 mM) and different concentrations of ABA (0.100 and 200 µM), as shown in the figure (Figure 3B-E).Under normal conditions, there were no noticeable changes during germination among the transgenic and WT plants, and germination was about 95% in all cases.However, seed germination was inhibited under mannitol and ABA stress conditions to a more pronounced extent for the overexpression lines relative to the WT.Under 100 mM mannitol stress, the germination rate of the WT seeds reached 72.3%; however, the overexpression lines showed 53.4%, 51.2% and 49.3% germination.This gap was more significant at 200 mm mannitol stress, with 50.2% germination in the wild-type, but only 29.6%, 25.3% and 30.2% in the overexpression lines.At 100 µM ABA stress, the germination rate of wild-type Arabidopsis was 75.7%, whereas that of the overexpression line Arabidopsis was 37.6%, 36.9% and 37.2%.At 200 µM ABA stress, the germination rates of the overexpression line for Arabidopsis were 24.6%, 22.8% and 15.9% in the overexpression lines, whereas it was 38.9% in wild-type Arabidopsis, indicating that the overexpression of ZmGST26 decreased the germination rate of Arabidopsis.This gap was more significant at 200 mm mannitol stress, with 50.2% germination i wild-type, but only 29.6%, 25.3% and 30.2% in the overexpression lines.At 100 μM stress, the germination rate of wild-type Arabidopsis was 75.7%, whereas that o overexpression line Arabidopsis was 37.6%, 36.9% and 37.2%.At 200 μM ABA stres germination rates of the overexpression line for Arabidopsis were 24.6%, 22.8% and 1 in the overexpression lines, whereas it was 38.9% in wild-type Arabidopsis, indic that the overexpression of ZmGST26 decreased the germination rate of Arabidopsis.Similarly, the seeds of the overexpression lines and wild-type Arabidopsis were sown on a 1/2 MS medium with different concentrations of mannitol and cultured in light and vertical conditions to observe the root's length.After 10 days of culture, as shown in the figure (Figure 4A,E), there were no significant changes in the root length of the overexpression lines and the wild-type line under the control treatment without mannitol.Under the treatment of 100 mM and 200 mM mannitol, the root length of the three overexpression lines was significantly shorter than that of the wild-type Arabidopsis thaliana.In order to further verify the role of the ZmGST26 gene in drought stress, the overexpression lines grown for 21 days and wild-type Arabidopsis plants were treated with drought stress for 10 days and then re-watered for 3 days.The results showed that the survival rate of the overexpression lines was significantly lower than that of wild-type Arabidopsis.The survival rate after rehydration was 67.6% for the wild-type, but only 27.3%, 29.2% and 25% for the overexpression lines (Figure 4B,C).Similarly, the seeds of the overexpression lines and wild-type Arabidopsis were sown on a 1/2 MS medium with different concentrations of mannitol and cultured in light and vertical conditions to observe the root's length.After 10 days of culture, as shown in the figure (Figure 4A,E), there were no significant changes in the root length of the overexpression lines and the wild-type line under the control treatment without mannitol.Under the treatment of 100 mM and 200 mM mannitol, the root length of the three overexpression lines was significantly shorter than that of the wild-type Arabidopsis thaliana.In order to further verify the role of the ZmGST26 gene in drought stress, the overexpression lines grown for 21 days and wild-type Arabidopsis plants were treated with drought stress for 10 days and then re-watered for 3 days.The results showed that the survival rate of the overexpression lines was significantly lower than that of wild-type Arabidopsis.The survival rate after rehydration was 67.6% for the wild-type, but only 27.3%, 29.2% and 25% for the overexpression lines (Figure 4B,C).
pression lines was significantly shorter than that of the wild-type Arabidopsis thaliana.In order to further verify the role of the ZmGST26 gene in drought stress, the overexpression lines grown for 21 days and wild-type Arabidopsis plants were treated with drought stress for 10 days and then re-watered for 3 days.The results showed that the survival rate of the overexpression lines was significantly lower than that of wild-type Arabidopsis.The survival rate after rehydration was 67.6% for the wild-type, but only 27.3%, 29.2% and 25% for the overexpression lines (Figure 4B,C).The determination of physiological and biochemical parameters such as water loss rate, proline, malondialdehyde and chlorophyll content can well reflect the physiological changes exhibited by plants subjected to drought stress and thus demonstrate whether plants are tolerant to drought stress.After drought stress treatments, compared to the wild-type line, all three overexpression lines showed significantly lower levels of proline (Figure 4F) and chlorophyll (Figure 4H).However, the water loss rate (Figure 4D) and malondialdehyde content (Figure 4G) were significantly elevated.These results indicated that the overexpression of ZmGST26 reduced the drought tolerance of transgenic Arabidopsis.

Overexpression of ZmGST26 Increased the Sensitivity of Transgenic Arabidopsis to Drought
Plants produce more ROS in adversity.In order to avoid the damage of excess ROS, plants initiate feedback mechanisms to increase the enzymatic scavenging system and non-enzymatic scavenging system activity; this eliminates excess ROS in vivo.The activity of ROS-scavenging enzymes in plants is significantly different due to the different species.The activity of ROS-scavenging enzymes is reflected by the effects of O 2 − production rates, H 2 O 2 content, membrane lipid peroxidation and antioxidant enzyme activity in plants.
Firstly, NBT and DAB staining were used to observe the color of leaves to identify the changes in the ROS content in leaves.The results (Figure 5A,B) showed that, with the increase in drought stress time, the color of overexpressed ZmGST26 lines was significantly darker than that of wild-type lines, indicating that the ROS content in overexpressed Arabidopsis leaves increased.Then, we measured the content of H 2 O 2 and O 2 − , and the results are shown in Figure 5C,D.Under normal conditions, there is no significant difference between the two; after drought stress treatments, the content of O 2 − and H 2 O 2 in overexpression lines was significantly higher than that in wild-type Arabidopsis, resulting in a decrease in drought resistance.
increase in drought stress time, the color of overexpressed ZmGST26 lines was significantly darker than that of wild-type lines, indicating that the ROS content in overexpressed Arabidopsis leaves increased.Then, we measured the content of H2O2 and O2 − , and the results are shown in Figure 5C,D.Under normal conditions, there is no significant difference between the two; after drought stress treatments, the content of O2 -and H2O2 in overexpression lines was significantly higher than that in wild-type Arabidopsis, resulting in a decrease in drought resistance.The high and low activities of antioxidant enzymes can well reflect the amount of ROS in plants; the antioxidant enzyme activities, such as SOD, POD and CAT, were measured in the overexpression lines of Arabidopsis and the wild-type Arabidopsis.The results showed that after drought stress treatments, the activities of SOD, POD and CAT antioxidant enzymes (Figure 5E-G) were significantly lower in all three overexpression lines compared with the wild-type Arabidopsis.Thus, this further illustrated that overexpressing the ZmGST26 gene after drought stresses decreased antioxidant enzyme contents and elevated O2 − and H2O2 contents in the body of the overexpression lines, which led to the accumulation of ROS content in vivo; this then caused the overexpression lines to cope with reduced drought-stress response tolerance.
Under a normal culture environment, the stomatal aperture of the leaves of each line was almost the same.However, under 100 μM ABA stress, the stomatal apertures in overexpressing Arabidopsis lines were 0.358 μm, 0.299 μm and 0.375 μm.However, wild-type Arabidopsis stomata tended to close completely, thus indicating that stomatal pore sizes differ significantly between the overexpression lines and wild-type Arabidopsis (Figure 5H,I).We speculated that overexpressing ZmGST26 reduced plant tolerances to drought stress by regulating the stomatal opening and thus causing water loss.

ZmGST26 Negatively Regulates the Expression of Drought-Related Genes in Overexpressed Arabidopsis
The response of plants to drought stress can be evaluated by the expression level of known drought-related genes.In this study, the genes such as DREB2A, RD29A, RD29B and PP2CA, which were significantly upregulated under drought stress, were selected.In this experiment, the relative expression levels of these four genes in wild-type and overexpressed Arabidopsis were detected.Via an analysis of qRT-PCR results (Figure 6), the expression levels of DREB2A, RD29A, RD29B and PP2CA were upregulated in wild-type and overexpression lines under drought stress treatments, but the expression The high and low activities of antioxidant enzymes can well reflect the amount of ROS in plants; the antioxidant enzyme activities, such as SOD, POD and CAT, were measured in the overexpression lines of Arabidopsis and the wild-type Arabidopsis.The results showed that after drought stress treatments, the activities of SOD, POD and CAT antioxidant enzymes (Figure 5E-G) were significantly lower in all three overexpression lines compared with the wild-type Arabidopsis.Thus, this further illustrated that overexpressing the ZmGST26 gene after drought stresses decreased antioxidant enzyme contents and elevated O 2 − and H 2 O 2 contents in the body of the overexpression lines, which led to the accumulation of ROS content in vivo; this then caused the overexpression lines to cope with reduced drought-stress response tolerance.
Under a normal culture environment, the stomatal aperture of the leaves of each line was almost the same.However, under 100 µM ABA stress, the stomatal apertures in overexpressing Arabidopsis lines were 0.358 µm, 0.299 µm and 0.375 µm.However, wild-type Arabidopsis stomata tended to close completely, thus indicating that stomatal pore sizes differ significantly between the overexpression lines and wild-type Arabidopsis (Figure 5H,I).We speculated that overexpressing ZmGST26 reduced plant tolerances to drought stress by regulating the stomatal opening and thus causing water loss.

ZmGST26 Negatively Regulates the Expression of Drought-Related Genes in Overexpressed Arabidopsis
The response of plants to drought stress can be evaluated by the expression level of known drought-related genes.In this study, the genes such as DREB2A, RD29A, RD29B and PP2CA, which were significantly upregulated under drought stress, were selected.In this experiment, the relative expression levels of these four genes in wild-type and overexpressed Arabidopsis were detected.Via an analysis of qRT-PCR results (Figure 6), the expression levels of DREB2A, RD29A, RD29B and PP2CA were upregulated in wild-type and overexpression lines under drought stress treatments, but the expression levels of DREB2A, RD29A, RD29B and PP2CA in the ZmGST26 overexpression lines were significantly lower than those in wild-type plants.Therefore, ZmGST26 may regulate plant drought stressrelated genes, thereby negatively regulating plant responses to drought stress.

Plant Materials and Growth Conditions
The H8186 maize inbred line was used as the material.Maize seeds with full and well-developed kernels were sown in soil and cultivated in an artificial culture room (25 °C, 16 h light/ 8 h dark).
Arabidopsis thaliana takes the Colombian ecotype as the wild-type material.The seeds were first disinfected with 75% ethanol for 1 min and then sterilized with 1% sodium hypochlorite for 10 min; finally, they were rinsed with sterile water 3-5 times.Sterilized Arabidopsis seeds were cultured in a 1/2 MS medium at 4 °C in darkness for 72 h and then transferred to the tissue culture room (22 °C, 16 h light/8 h dark) for germination and growth.After 7 days of incubation, the seedlings were transplanted into soil (22 °C, 16 h light /8 h dark).

Sequence Analysis
The cDNA sequence of the ZmGST26 gene was derived from NCBI (https://www.ncbi.nlm.nih.gov/(accessed on 11 November 2022.))The phylogenetic tree was constructed by the neighbor-joining method using MEGA7 software.The parameter of boot strap repetitions was set to 1000 [23].The 2000 bp promoter upstream of the ZmGST26 gene was analyzed by Plant CARE (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/(accessed on 11 November

Plant Materials and Growth Conditions
The H8186 maize inbred line was used as the material.Maize seeds with full and well-developed kernels were sown in soil and cultivated in an artificial culture room (25 • C, 16 h light/8 h dark).
Arabidopsis thaliana takes the Colombian ecotype as the wild-type material.The seeds were first disinfected with 75% ethanol for 1 min and then sterilized with 1% sodium hypochlorite for 10 min; finally, they were rinsed with sterile water 3-5 times.Sterilized Arabidopsis seeds were cultured in a 1/2 MS medium at 4 • C in darkness for 72 h and then transferred to the tissue culture room (22 • C, 16 h light/8 h dark) for germination and growth.After 7 days of incubation, the seedlings were transplanted into soil (22 • C, 16 h light/8 h dark).

RNA Extraction and qRT-PCR Analysis
Total RNA was isolated from maize tissues under drought stress by the TRIzol method.The seedlings at the three-leaf stage were treated with 200 mmol mannitol.The tissues were collected at 0, 2, 4, 8, 12 and 24 h after treatment.The harvested samples were immediately frozen in liquid nitrogen for subsequent RNA extraction, and then the first strand of cDNA was generated by using the M-MLV reverse transcription kit (Takara, Kusatsu, Japan).qRT-PCR was applied using SYBR green and the Light Cycler ® 96 PCR detection system (Roche, Switzerland).The reaction procedure was 95 • C 30 s, 95 • C 10 s and 60 • C 30 s at 40 The melting point curve and data analysis were performed using the 2 −∆∆CT method.ZmActin1 was used as an internal reference gene for calculating the Ct value.
The rosette leaves of Arabidopsis thaliana with normal growth and drought stress for 10 days were selected.The total RNA was extracted and the first strand cDNA was synthesized for qRT-PCR detection.The marker gene-specific primers DREB2A-F/R, RD29A-F/R, RD29B-F/R and PP2CA-F/R were used for qRT-PCR analyses.AtActin2 was used as an internal reference gene to calculate the expression of four stress-related marker genes.
Each sample was repeated three times.The primers used in this study are shown in Supplementary Materials Table S2.

Subcellular Localization
The full-length coding sequence of the ZmGST26 gene without a stop codon was amplified by PCR using specific primers.Then, the NcoI and SpeI sites of pCAMBIA1302-35S-GFP were selected to generate the pCAMBIA1302-35S-ZmGST26-GFP recombinant vector.The recombinant vector was transferred into the Agrobacterium tumefaciens EHA105 strain.It was infiltrated and co-injected into the tobacco leaf and cultured in an incubator at 25 • C for 48 h.It was observed using an FV1000 laser scanning confocal microscope (LSCM IX83-FV1200, Olympus, Hamburg, Germany).

Acquisition and Phenotypic Analysis of Overexpressed Arabidopsis
The full-length cDNA sequence of the ZmGST26 gene was cloned into the pCAMBIA3301-35S-Bar vector.Subsequently, the pCAMBIA3301-35S-ZmGST26-Bar vector was introduced into the Agrobacterium strain EHA105 and transformed into Arabidopsis plants by the floral dip method.Positive plants were screened by Basta until homozygous Arabidopsis plants were obtained in T 3 generation.Three overexpression lines were selected for subsequent research.
After disinfection, the seeds were sown on a 1/2 MS medium containing 0 mM, 100 mM, 200 mM mannitol, 100 µM ABA and 200 µM ABA; these seeds grew in a greenhouse.After 8 days of culture, the germination rate of the seedlings was recorded: germination rate = germinated seeds/total seeds × 100% [25].Each sample contains three biological replicates.A one-way analysis of variance was used for statistical analyses, and the average value of each overexpression line was compared with the wild-type line.
The method of root length determination was first performed by sanitizing the seeds sown on a 1/2 MS medium containing 0 mm, 100 mm and 200 mm mannitol, and then growing them vertically for 10 days.The main root lengths of Arabidopsis thaliana under different mannitol concentrations were measured.All the experiments were repeated three times.Each biological replicate contained at least 12 seedlings [26].
The Arabidopsis thaliana that grew normally during the four-leaf stage was subjected to drought stress, with watering stopped for 10 days, before it was watered for 3 days to investigate its survival rate.Following the rewatering treatment, plants were considered survivors if they had green and healthy new leaves.All the experiments were repeated three times.

Determination of O 2
− , H 2 O 2 and Physiological and Biochemical Indexes in Overexpressed Arabidopsis The content of ROS in vivo was identified by 3,3 -diaminobenzidine (DAB) and p-nitroblue tetrazolium (NBT) histochemical staining, and the specific method was referred to as Fryer's method [27].The content of H 2 O 2 and O 2 − was calculated by using the acetone method, and the specific method was referred to as Wang's method [28].The determination of water loss rates was referred to as Negi's method [29].The content of malondialdehyde was detected by using the thiobarbituric acid method; fresh young leaves were soaked in 95% ethanol for 24 h under dark conditions.The absorbance of solution was measured at 649 nm and 665 nm, and the chlorophyll content was measured by Fitter's method [30]; the proline content was analyzed by the acid ninhydrin method [31].The antioxidant enzyme activities of SOD, POD and CAT were measured according to Xiang's method [32].

Guard Cell Stomatal Aperture Observations
For the stomatal opening test, the leaf epidermis of 3-week-old Arabidopsis seedlings was incubated in a buffer (10 mM KCl, 200 µM CaCl 2 and 10 mM MES-KOH, pH 6.2) for 2 h.Then, the epidermis was immediately transferred to a dish containing 100 µM ABA in the stomatal opening solution for 2 h, and the ABA free epidermis was used as a control.Photographs were taken using a microscope and at least 30 stomata were observed for each treatment.The lengths and widths of stomata were measured and analyzed using the ImageJ software.

Discussion
Glutathione S-transferase is an important enzyme and a superfamily of biotic and abiotic stress responses.Glutathione S-transferase plays a key role in the antioxidant enzyme system.The expression of GST genes plays an important role in response to plant oxidative stress, metal ions, drought, salinity and other abiotic stresses [33][34][35][36].In this study, the differentially expressed ZmGST26 under drought stress was screened by previous laboratory transcriptome sequencing data.On this basis, we performed drought stress on this gene in different tissues of maize.The results showed that ZmGST26 was significantly sensitive to drought stress in roots.A previous study also found that CsWRKY48 directly binds to the W-box element (TTGACC) in the CsGSTU8 promoter and activates its expression by improving the clearance of excess ROS under drought conditions to indicate enhanced drought tolerances [37].The promoter element analysis of 2000 bp upstream of ZmGST26 showed that there were W-box response elements and a large number of elements involved in drought and oxidative responses.Therefore, we speculate that the W-box element (TTGACC) in the ZmGST26 promoter may be regulated by WRKY transcription factors to cope with drought stress.Subsequently, the subcellular localization of ZmGST26 was performed to verify its localization in the nucleus, and this conjecture will be further verified by experiments.
Many studies have determined the function of genes by phenotypic and physiological changes in transgenic Arabidopsis lines.It has been reported that the germination rate and root length of transgenic Arabidopsis overexpressing TaSADR1 under drought stress were significantly lower than those of wild-type Arabidopsis, which confirmed that TaSADR1 reduced the tolerance of plants relative to drought stress [38].PtGSTF1 increases biomass production and salt tolerance by regulating xylem cell proliferation, ion homeostasis and ROS-scavenging in poplar [39]; the overexpression of CsGSH2 reduces phytotoxicity in cucumbers by enhancing antioxidant and glutathione detoxification properties [40]; in this study, after drought stress, the root length, germination rate, chlorophyll content, proline content and survival rate of transgenic lines overexpressing ZmGST26 were significantly lower than those of wild-type Arabidopsis, but the water loss rate and MDA content increased significantly.It showed that ZmGST26 reduced the drought tolerance of transgenic Arabidopsis.
Drought stress leads to the production of ROS, and the excessive accumulation of ROS will cause damage to plant cells.Therefore, ROS is required to remove excessive ROS free radicals and O 2 − ions by activating the enzymatic antioxidant system in vivo.In previous studies, it was reported that MruGSTU39 could detoxify ROS by upregulating the activities of antioxidant enzymes and GST to reduce its damage to membranes and thus enhance the drought tolerance of alfalfa [41].OsGSTU6 enhances rice abiotic stress tolerance by participating in intracellular ROS homeostasis [42].ThGSTZ1 transgenic Arabidopsis enhanced drought tolerances by scavenging excess H 2 O 2 and other studies have shown that ROS can regulate plant drought tolerance [43].In this study, NBT and DAB staining results, H 2 O 2 , O 2 − content and antioxidant enzyme activities were determined.The results showed that under drought conditions, the antioxidant enzyme activity of transgenic lines was significantly lower than that of wild-type plants, and the contents of ROS, H 2 O 2 and O 2 − increased.In recent years, many studies elaborated that stomatal regulation plays an important role in abiotic stress, and the overexpression of AtPUB46 decreased the number of rosette leaves, stomatal density and water loss rate in Arabidopsis, thereby improving tolerance to drought and oxidative stress [44]; the overexpression of GmPUB21 significantly increased stomatal density and water loss, thereby reducing plant resistance to drought and salt tolerance [45].In this study, Arabidopsis overexpressing the ZmGST26 transgenic line showed a significantly higher stomatal aperture and water loss rate than wild-type Arabidopsis under ABA stress conditions; thus, we speculated that overexpressing ZmGST26 reduced plant tolerance to drought stress by regulating the stomatal opening and thus water loss.Subsequently, we performed qRT-PCR on drought-stress-related genes (DREB2A, RD29A, RD29B and PP2CA).The results showed that drought-stress-related genes were significantly downregulated.Therefore, we determined that ZmGST26 increased plant sensitivities to droughts by increasing the accumulation of reactive oxygen species, thereby reducing plant drought tolerances.

Figure 1 .
Figure 1.Phylogenetic analysis and expression pattern analysis of ZmGST26.(A) Phylogenetic analysis of the Tau class of Glutathione S-transferases.(B) Expression of ZmGST26 in roots, leaves and stem of maize at different time under drought stress.(ZmActin1 was used as an internal reference gene).* p < 0.05; ** p < 0.01.All bars represent means ± SD, (n ≥ 3).

Figure 1 .
Figure 1.Phylogenetic analysis and expression pattern analysis of ZmGST26.(A) Phylogenetic analysis of the Tau class of Glutathione S-transferases.(B) Expression of ZmGST26 in roots, leaves and stem of maize at different time under drought stress.(ZmActin1 was used as an internal reference gene).* p < 0.05; ** p < 0.01.All bars represent means ± SD, (n ≥ 3).
of ZmGST26-GFP was only distributed in the nucleus.It was further confirmed that ZmGST26 acts in the nucleus.

y 13 Figure 3 .
Figure 3. Drought resistance of wild-type and ZmGST26-overexpressing Arabidopsis plants.(A) ZmGST26 expression in leaves of wild-type and overexpression lines Arabidopsis.(B,C) Seedling growth and germination rate in transgenic and wild-type Arabidopsis under mannitol treatment.(D,E) Seedling growth and germination rate in transgenic and wild-type Arabidopsis under ABA treatment.** p < 0.01.All bars represent means ± SD, (n ≥ 3).

Figure 3 .
Figure 3. Drought resistance of wild-type and ZmGST26-overexpressing Arabidopsis plants.(A) ZmGST26 expression in leaves of wild-type and overexpression lines Arabidopsis.(B,C) Seedling growth and germination rate in transgenic and wild-type Arabidopsis under mannitol treatment.(D,E) Seedling growth and germination rate in transgenic and wild-type Arabidopsis under ABA treatment.** p < 0.01.All bars represent means ± SD, (n ≥ 3).

Figure 4 .
Figure 4. Drought resistance of wild-type and ZmGST26-overexpressing Arabidopsis plants.(A,E) Roots length in transgenic and wild-type Arabidopsis treated with mannitol.(B) Phenotypes of transgenic and wild-type Arabidopsis plants under drought conditions.(C) Survival rates of

Agronomy 2022 , 13 Figure 6 .
Figure 6.Expression of drought stress response genes in wild-type and ZmGST26-overexpressing transgenic Arabidopsis leaves.Quantitative real-time PCR analysis was conducted to detect the relative expression levels of RD29A, RD29B, DREB2A and PP2CA in three-week-old plants after not watering for 10 days.The AtActin2 gene was used as an internal reference.** p < 0.01.All bars represent means ± SD, (n ≥ 3).

Figure 6 .
Figure 6.Expression of drought stress response genes in wild-type and ZmGST26-overexpressing transgenic Arabidopsis leaves.Quantitative real-time PCR analysis was conducted to detect the relative expression levels of (A) RD29A, (B) RD29B, (C) DREB2A and (D) PP2CA in three-week-old plants after not watering for 10 days.The AtActin2 gene was used as an internal reference.** p < 0.01.All bars represent means ± SD, (n ≥ 3).