Chitosan suppresses the expression level ofWRKY17 on red chili (Capsicum annuum) plant under drought stress

Chili pepper plays a significant role in the global market. However, the production is often impeded by drought stress involving WRKY genes as the defense regulator. Chitosan is considered as a promising alternative fertilizer and defense elicitor. Hence, this study aimed to determine the role of chitosan in improving plant growth and survival of red chili pepper against drought stress. At the onset of the generative phase, chili plants were subjected to 1 mg/mL chitosan, 50% drought, or chitosan‐drought treatment. Observations were made on several growth parameters, opened stomata, and WRKY gene expression. The results showed that chitosan‐drought treatment decreased plant growth and yielded significantly. The percentage of opened stomata was recorded at 0.56‐fold lower than control. It was followed by the decrease of the relative expression ofWRKY17 andWRKY53 genes up to 0.56 and 0.72‐fold lower than control, respectively. Therefore, we suggested that the double treatment of chitosan‐drought might decrease plant growth performance but increase the defense system by suppressing the expression level of theWRKY17 gene. Interestingly, the drought treatment significantly increased WRKY17 expression level up to 7‐fold higher than control. Hence, it was suggested that WRKY17 has a specific role in response to drought stress.


Introduction
Red chili pepper (Capsicum annuum) is a widely domes ticated and very popular plant throughout the world, rou tinely consumed by about onefourth of the global popu lation (Khan et al. 2014). In Indonesia, this plant is one of the most prioritized vegetables with a high economic value used for daily consumption, food industries, and ex port commodities. In 2014, the total production was about 1.075 million tons or around 9.02% of Indonesia's national vegetable production (KEMENTAN 2015). The average demand for this commodity in the urban area is around 66,000 tons per month increasing up to 20% at a certain period. However, red chili production in Indonesia fluctu ates from time to time and is often unable to meet the mar ket needs. In addition to pathogen infection, the decrease of production is also caused by limited hydration. For in stance, the phenomenon of drought in the Semarang dis trict caused 500 Ha of agricultural land to be affected, and 72 hectares experienced crop failure in 2014 (KEMEN TAN 2016). If this phenomenon occurs in a prolonged pe riod, it might inhibit plant growth or death, resulting in low productivity, increased prices, and decreased export commodities.
Drought is the most frequent abiotic stress experienced by plants due to global climate change in recent years (Khan et al. 2014). In general, the plant will respond to water stress by synthesizing abscisic acid (ABA) as the defense regulator, which leads to closed stomata. It aims to reduce the rate of transpiration, but it also impacts de creasing photosynthesis rate, resulting in decreased plant growth and productivity (Iriti et al. 2009). Under stress conditions, a plant can carry out the various defense mech anisms through molecular, cellular, and biochemical inte gration (Khan et al. 2014). WRKY is a transcription factor known to play a role in the abscisic acid (ABA) and jas monic acid (JA) signaling pathways in response to drought stress. WRKY is characterized by the existence of one or two conserved WRKY domains in the Nterminus and a zinc finger motif in the Cterminus. These two elements are crucial for the affinity of WRKY protein binding with the consensus sequence (C/T) TGAC (C/T) called Wbox. There are 74 kinds of WRKY protein found in Arabidopsis and classified into three groups based on the number and type of WRKY domain. Group III, such as WRKY46 and 53, was characterized by one CCHC zinc finger, group II (for example, WRKY11, 15, 17, and 39) by one CCH H zinc finger, while group I by two CCHH zinc finger (Yan et al. 2014). The previous study reported that the al teration of WRKY genes expression level was in line with plant defense regulation under stress (Yan et al. 2014; Sun andYu 2015).
The improvement of plant growth and resistance us ing environmentally friendly technologies is continuously developed since the excessive use of chemical substances is often destructive such as the accumulation of pesticide residues, pathogen resistance, eradication of natural preda tors, and environmental pollution (Duriat et al. 2007). Chi tosan is one of the most promising candidates of natural fertilizer that is widely studied due to its various charac teristics such as environmentally friendly, edible, having antifungal and antitranspirant activity, and also can boost plant defense system. The application of foliar chitosan spraying on coffee increased chlorophyll and carotenoid content of leaves around 46.38 to 73.51%, mineral ab sorption which includes 9.49% N, 11.76% P, 18.75% Mg, and 3.77% Ca and plant height up to 33.51%, and also in creased resistance to drought (Dzung et al. 2011). In ad dition, the application of 100 and 125 ppm chitosan was proven to increase the growth and productivity of okra (Mondal et al. 2013). The previous study demonstrated that the antitranspirant activity of chitosan was related to the synthesis of ABA as a regulator of resistance mecha nisms by inducing closed stomata (Iriti et al. 2009). Thus, chitosan is coincided as the potential agent to promote plant growth and survival under drought conditions. However, the role of chitosan on physiological and molecular mechanisms during drought stress has not been studied thoroughly, particularly in the plant defense mech anisms. Thus, in this study, we used red chili pep per LADO cultivar, which was given a combination of 1 mg/mL chitosan and 50% of drought treatment to de termine the plant physiological (growth performance and stomatal behavior) and molecular (WRKY genes expres sion) responses. This study aimed to determine the role of chitosan in improving plant growth and survival of red chili pepper against drought stress.

Plants Materials and Treatments
The cultivation and treatments of red chili pepper (C. an nuum) cultivar LADO (EastWest Seed Indonesia Ltd.) was conducted at the greenhouse at Institut Teknologi Ban dung, Indonesia. This experiment was conducted for ± 112 d with the average of photoperiod was 12 h, relative humidity of 78.96%, a light intensity of 10240 lux, and at temperature ± 27.78°C (Supplementary 1). Individual red chili pepper was grown in a plastic polybag with 3 kg planting medium containing soil, husks, and organic fertil izer (4: 3: 2). The plantlets were watered up to 100% field water capacity as long as 7 of the weeks. At the onset of generative phase, as indicated by the emergence of flower buds, the plantlets were subjected to the treatments of chi tosan (Chi), chitosan & drought (ChiD), drought (D), and control (C) up to sixteenth weeks of planting (n = 3 bio logical replications of each treatment).
Chitosan solution was prepared as described in Esyanti et al. (2019) by diluting chitosan powder into a glacial acetic acid solution (0,07%) to a concentration of 1 mg/mL and sprayed weekly by foliar feeding technique to Chi and ChiD treatments. It suggested that the 1 mg/mL chitosan solution was able to maintain the cultivar's growth rate (Esyanti et al. 2019). Drought treatment was applied to reach 50% of field water capacity by using 500 mL of wa ter in 2 d subjected to D and ChiD treatments. It sug gested that 50% of deficit irrigation is a feasible irrigation strategy to maintain plant growth performance (Dorji et al. 2005). Also, the control group (C) was watered by 100% field water capacity but not sprayed with any solutions. Fresh leaves from 3 plants were collected at 50 60 d af ter flowering (harvesting period) (Sung et al. 2005) into one sample and used for RNA extraction to analyze WRKY genes expression level (n = 3 technical replications of each treatment).

Measurement of Physiological Parameters
The physiological parameters, including plant height, the number of leaves, flowers, and fruits were recorded weekly starting from week 8 to 16. Plant height was mea sured using a ruler, determining the length of the main stem from the base line up to the tip, while the firmly at tached organs, including leaf, flower, and fruit, were hand counted. It determines the remaining plant organ num ber from abscission, fallen organs. Stomatal behavior was evaluated by making the mold on the lower part of chili leaves during the day and then observed under a light mi croscope (NIKON, SM2445). Furthermore, the percent age of the opened stomata was calculated using the corre sponding method described by Fibriyanti (2008).

RNA Extraction and qPCR Analysis
The total RNA was extracted from the frozen pepper leaves (50 mg) using the PureLinkTM RNA mini kit (In vitrogen). The quality of RNA was evaluated in a 1.5% (w/v) agarose gel with gel red as a coloring agent using an electrophoresis instrument. Meanwhile, the quantity of RNA was evaluated using a nanodrop spectrophotometer (Eppendorf, biospectrometer) to know the purity of RNA and also to quantify RNA concentration. DNAse treat ment was performed using DNAseI (Thermo Scientific), while cDNA synthesis was carried out using iScript cDNA Synthesis Kit (BioRad) containing reverse transcriptase enzyme. The quality of cDNA was determined in a 2% agarose using CaUbi3 as the housekeeping gene with the specific primer, as explained in Table 1. The Polymerase Chain Reaction (thermal cycler) condition was performed as follow: 95°C 3 min, 95°C 30 s, 58°C 30 s, 72°C 1 min, 72°C 5 min (35 cycles). The experiment was re peated three times (n = 3 technical replications). Amplification of WRKY genes was performed by us ing specific primers, as mentioned in Table 1. After con firmed by using agarose electrophoreses, sequencing was performed through the Macrogen service, Korea. And then, the quantitative PCR was performed by using instru ments and software from MyGo Pro with the dye from Toyobo (Thunderbirds Sybr qPCR Mix QPS201). The quantification cycle (Cq) score was then used for quan tifying the expression level through a relative method by using the corresponding formula, as described by Livak and Schmittgen (2001).

Statistical analysis
The significant differences among the treatments were determined using the Tukey HSD test at P<0.05 using R studio 3.5.2 (32/64 bit) application. The principal componentbiplot analysis (PCA), the heatmap clustering analysis (HCA), and the correlation analysis were per formed using the MetaboAnalysed 4.0 application. Those analyses were performed to cluster among the treatments and also to determine the correlation among the parame ters. In addition, the effect of chitosan, drought, and the in teraction of both treatments towards the increase in height, the percentage of opened stomata, and WRKY genes ex pression level were analyzed by Twoway ANOVA per formed using IBM SPSS statistics 22 software.

Chitosan Application under Drought Stress Decreased Plants Growth Performance
The effect of chitosan application and/or drought treatment to red chili pepper on growth performance parameters in cluding height increment, the number of leaves, flowers, and fruits in the generative phase was indicated in Figure 1. The initial plant growth condition at the vegetative phase was determined at the similar performance indicated by the similar growth parameters at week 8 for the number of leaves and flower and also at week 10 for the number of fruits. According to the investigation, the minimum height increment was recorded on the double treatment of chi tosan and drought (ChiD) at 30.67 cm. The maximum increase in height was showed by Chi treatment at 49.67 cm, whereas that of D and C treatments was at 44 cm and 47.67 cm, respectively. In addition, the drought treatment showed an average decrease of ± 5 cm in plants compared to the control. The effect of chitosan application during drought stress condition was also determined based on the number of leaves from week 8 to 16. Overall, plants under ChiD treatment possessed the minimum number of leaves, with a total of 114 in week 16 ( Figure 1B). Chi treatment showed the maximum number of leaves in weeks 9 and 10. In general, D and ChiD treatments suppressed the number of leaves since week 10 when compared to that of C and Chi. Hence, the application of 1 mg/mL chitosan under 50% of drought stress condition inhibited growth in red chili pepper showed by the lowest height increment and leaves number.
The yield was evaluated as the number of flowers and fruits. The results showed that chitosan application dur ing drought conditions (ChiD) resulted in the minimum total number of flowers. The maximum number of flow ers was observed in week 11 on the control group (Fig  ure 1C). A steep decrease was detected after week 11 in all treatments that could occur due to the abscission and fruit development. An exponential fruit emergence was recorded in weeks 11 to 12 ( Figure 1D) in almost all treat ments. However, the immature fruits did not entirely reach the mature stage, with fruit abscised as early as in week 12. ChiD treatment was only able to produce the least number of ripe fruits. However, ChiD treatment showed inferior values in almost all measured parameters of growth per formance and yield.

Chitosan Application During Drought Condition Promoted Stomatal Closure
The closed stomata state of ChiD treatment and the opened stomata of control are indicated in Figure 2A and B. Chitosan application under drought stress (Chi D) significantly decreased the number of opened stom ata. The result showed that there was only a 30.67% of opened stomata in the ChiD treatment, whereas that in D treatment was recorded at 32.44%. Chitosan treat ment also decreased the percentage of opened stomata by 42.75%, while the highest of opened stomata, 54.97%, was recorded in control ( Figure 2C).

Transcription Factor WRKY genes Regulated Growth and Defense Mechanism During Chitosan Application Under Drought Condition
To conduct the relative gene expression analysis, the vali dation in every former step needs to be performed. Based on the total RNA visualization, there are two bands with the sizes of 28s and 18s (Supplementary 2) by means that the total RNA was successfully extracted. Spectropho tometry analysis showed that the purity of RNA (ratios λ260 and λ280) ranged from 2.12 to 2.14, while the ob tained concentrations ranged from 353.1 to 410.9 µg/mL. Based on the sequencing result for a ±150 bp of the PCR product, after being analyzed using BLAST, a 100% iden tity value was confirmed with the WRKY17 gene (acces sion number: XM_016699929.1) and WRKY53 gene (ac cession number: NM_001324692.1) (Supplementary 3). WRKY17 and WRKY53 were used to analyze the influence of chitosan on plant growth and defense response against drought stress.
In this study, we found that the relative expres sion level of WRKY17 and WRKY53 gene was signif icantly downregulated in almost all treatments. The result showed that chitosan application under drought stress (ChiD) decreased the relative expression level of WRKY17 and WRKY53 up to 0.56 and 0.72fold lower than control, respectively ( Figure 3). Individual chitosan application (Chi) showed the relative expression level of WRKY17 and WRKY53 up to 0.67 and 0.47fold lower than control, respectively. Interestingly, in addition to almost all genes expression level of WRKY were recorded lower in all treatments, the only expression level of WRKY17 in D treatment was upregulated up to 7fold higher than control. Different from WRKY17, the expression level of WRKY53 in D treatment was only 0.12fold lower than control. We also found that chitosan application under drought stress resulted in the different responses between WRKY17 and WRKY53 gene expression.

The Correlation of Physiological and Molecular Responses after Coupled Treatment of Chitosan and Drought in Chili Plants
Principal Componentbiplot Analysis (PCA) was carried out to understand the grouping pattern of the measured parameter with the treatment groups. In Figure 4A, the PCA result showed a 91.005% score of PC1 followed by an 8.816% of PC2. This indicates a classification pattern of the treatments defining that chitosan application un der drought condition (ChiD) is distinct from the drought treatment (D) group. In contrast chitosan treatment (Chi) and the normal condition (C) are classified into one group. The number of leaves indicates a powerful parameter clus tering Chi and C groups into the one quadrant, while the expression of the WRKY17 gene only separates the D group to the other quadrant. Furthermore, to generate a deeper understanding of the correlation among parameters, the heatmap clustering analysis (HCA) was generated. The result ( Figure 4B) showed that Chi and C groups are clustered together, as shown in the PCA score plot result. Interestingly, Chi D and D groups are in another cluster. The clustering among parameters, it is forms two major groups defin ing the WRKY17 gene as the outgroup. In another group, there is a physiological parameter clustered in one group, while the WRKY53 as the other outgroup. This is sup ported by the correlation analysis ( Figure 4C) stated that almost all physiological parameters showed a strong pos itive correlation with each other (r = 0.61 -0.89). How ever, the expression of the WRKY53 gene is positively cor related with the stomatal behavior (r = 0.64), contrasting the negative correlation (r = 0.78) between WRKY17 and WRKY53.

Discussion
Most of the time, plants live in a habitat composed of a complex set of stresses. Understanding crop plant re sponses towards these stresses are fundamental to develop strategies that retain yield. Drought is considered as one of the most threatening conditions in the face of climate change, impeding the production of crop plants. Plant's response to drought is also complex, in which some are general across cultivars or even species, while others are distinctive in a certain genotype only. LADO is a com mercial cultivar of red chili pepper (C. annuum) from East West Seed Ltd. LADO is widely cultivated due to its adap tive properties to a wide range of altitudes and the ability to produce 1820 tons/Ha. It was shown that plants response to drought involves jasmonic acid (JA), but suppresses the activity of growth hormones such as gibberellic acid (GA). In this presented study, the low increase in height indicated a growth restraint during drought (Wasternack 2014). The similar results were showed that 50% of drought stresses decreased 1315% of plant height (Him and Radhouane 2015), while a 34.7% decrease of fresh weight under the same stress was evaluated by Dorji et al. (2005).
The increase in the height of chitosan treatment (Chi) was more significant than control, suggesting this poly mer capability to improve the plant's growth. The pre vious study showed that chitosan application under nor mal conditions by the foliar feeding technique resulted in an increase in the height of coffee plants up to 33.51% (Dzung et al. 2011). Besides, it was also demonstrated that chitosan application increased plants height significantly about 10 cm compared to the control accompanied by an increase in chlorophyll levels (Salachna and Zawadzińska 2014). These phenomena suggested due to chitosan that contains the abundance of amine (N) groups. Thus it was thought to play a role in plant growth, such as forming new cells and cell elongation (Ohta et al. 2004; Dzung et al. 2011. However, the double treatment of chitosan and drought (ChiD) showed the lowest increase in height. This is following the assumption of Iriti et al. (2009) that the combination of chitosan application and drought in plants could cause a synergistic response in improving de fense mechanisms but resulted in inhibited growth.
In each treatment, the number of leaves, flowers, and fruits peaked in weeks 11 and 12, followed by a steep de crease in week 13, which could occur due to the organ abscission. Further, the abscission of main organs such as leaves and flowers leads to a decline in plant growth and productivity (Sumarni and Muharam 2005). Natu rally, abscission is also a strategy to face unfavorable en vironmental conditions. The previous study described that microclimate conditions such as temperature, humidity, and light intensity during cultivation highly influence the plant's growth (Taylor and Whitelaw 2001). We recorded the highest temperature in week 13, with an average of 31.48ºC, which was higher than the suggested temperature for chili plant cultivation at 2527ºC during the day and 1820ºC at night. Thus, this warmer microclimate might promote higher transpiration and reduce turgor in various organs that lead to abscission, as stated by Sumarni and Muharam (2005). Similarly, it was reported that drought stress in C. annuum decreased the number of leaves by 45%, flowers by 79%, and fruits by 88% (Showemimo and Olarewaju 2007).
The previous study suggested that the chitosan appli cation was able to alleviate drought stress. It has been re ported that the application of 0.4 g/L chitosan improved the growth of Ocimum basilicum plants, either in drought treatment or control (Malekpoor et al. 2016). Interestingly, 1 mg/mL chitosan application and 50% drought treatment combination (ChiD) showed the most mediocre growth performances, as observed in the increase in height rate, the number of leaves, flowers, and fruits. However, in the previous study, chitosan was only applied three times dur ing vegetative until generative phases. While in this study, chitosan was applied once a week (eight times) during the generative phase with a concentration of 1 mg/mL, which is higher than the previous study. Thus, there was a dif ferent intensity and concentration of chitosan application, which thought to cause a different response in different species.
Under drought conditions, C3 plants such as chili pep per will respond to close stomata, so that the photosyn thesis rate will decrease. The closure of stomata alters gas exchange in plants, which can induce organ abscis sion (Sumarni and Muharam 2005). The assumption of the negative effects of intensive chitosan application on growth performance was supported by stomatal behavior observation as displayed in Figure 2. Photosynthesis is the primary process of supplying energy for plant growth, while the alteration of stomatal movement might indirectly hinder growth progression. We found that the number of opened stomata decreased in all treatments compared to the control group. However, we recorded that the number of opened stomata in ChiD plants was the least among all treatments. This suggested that both water deficit and elicitation from foliar application of chitosan were able to induce irregular stomatal state.
Furthermore, the previous study reported that chi tosan application could reduce opened stomata by trig gering reactive oxygen species (ROS) production such as H 2 O 2 . At the same time drought stress also leads to highly synthesized ABA, which also induces ROS production (Pichyangkura and Chadchawan 2015). ABA is highly synthesized when the plants experienced water deficiency, which will further induce the ROS production as the main signaling molecule. Following this, ROS will trigger nitric oxides (NO) production to inhibit membrane proton pump and the influx of Ca 2+ between the plasma membrane and vacuole. Increased Ca 2+ concentration causes anion ef flux, so the membrane is depolarized, then the pressure of turgor and cell volume decreases, and the stomata are closed (Arve et al. 2011).
Plant develops an integrated mechanism to maintain FIGURE 4 The correlation between physiological and molecular responses after chitosan and drought treatment combination in red chili plants including (A) score plot of the principal component-biplot analysis (PCA), the longer and the more clear the initial line, the stronger the certain parameters classify each treatment group, (B) the heatmap clustering analysis (HCA), the higher similarity score is defined by the red color, while the lower score is by the green, and (C) the correlation analysis, the positive correlation is defined by the blue color, while the negative correlation is by the red. HEIGHT, height increment; LEAVES, the number of leaves; FLOWER, the number of flowers; FRUIT, the number of fruit; STOMATA, the percentage of opened stomata; WRKY17, the expression level of WRKY17 gene; WRKY53, the expression level of WRKY53 gene. Chi, 1 mg/mL chitosan; Chi-D, 1 mg/mL chitosan and 50% drought; D, 50% drought; C, control.
its growth and survival under stress. Hence, the analy sis of plant signaling molecules such as transcription fac tor WRKY will shed a light on how the mechanism is carried out. WRKY is a group of transcription factors characterized by the presence of one or two conserved WRKY domains on the Nterminus and a zinc finger mo tif on the Cterminus, involved in growth, development, and stress response regulation through ABA and ROS sig naling mechanisms. For instance, WRKY41, WRKY70, and WRKY1 were reported involved in increasing drought stress tolerance in transgenic tobacco plants by regulat ing stomatal closure and ROS level. They also docu mented the wider opened stomata in the overexpressed Gh WRKY17 mutant line compared to wild type when treated with exogenous ABA (Yan et al. 2014). In addition, it was also documented that AtWRKY53 was thought to regulate opened stomata by increasing starch degradation for keep ing stomatal opened. However, the opened stomata under drought stress conditions could promote water loss, result ing in plant dehydration. Therefore, the higher expression of WRKY17 and WRKY53 gene could decrease the plant's sensitivity against stress (Sun and Yu 2015). The WRKY transcription factor was reported as a pos itive or negative regulator in many biological processes in cluding, plant development, defense regulation, and stress response (Cai et al. 2017). The superior expression level of WRKY17 under stress conditions might decrease the sen sitivity of ABA signaling. The decreased ABA sensitiv ity to guard cells and the lower ABA content is thought to play a role in increasing water loss and decreasing drought tolerance. Based on the previous study, during drought stress the expression of genes related to ROS degrading en zymes including catalase (CAT) and superoxide dismutase (SOD) decreases in GhWRKY17 transgenic plants group, while the expression of genes related to ROS (RbohA and RbohB) production increases significantly compared to wild type plants. This causes an increase in ROS accumu lation such as hydrogen peroxide (H 2 O 2 ) and anion diox ide (O 2 ). Therefore, Gossypium hirsutum WRKY17 was thought to reduce the plant's tolerance to osmotic stress (Yan et al. 2014). Meanwhile, the role of Arabidopsis WRKY53 in the regulation of stomatal state during drought was thought to be not directly related to ABA signaling, this was characterized by the expression of genes related to ABA biosynthesis (ABA1, ABA2, and ABA3) and ABA levels that do not differ significantly either AtWRKY53 transgenic plants or control group. Based on the previous investigation, AtWRKY53 was suggested to regulate stom atal movement by increasing the degradation of starch to malate for keeping stomata open. In brief, an AtWRKY53 can directly bind to the QQS promoter sequence and then leads to an increase in starch metabolism (Sun and Yu 2015).
This study found that chitosan application under drought stress conditions might suppress the expression level of the WRKY17 gene compared to drought treatment. However, the lower expression level was also recorded in WRKY53 in all treatments. This finding suggested that both genes were involved in the stomatal opening regula tion after the treatments. The previous study that charac terized the functional analysis of WRKY17 genes in cotton and Arabidopsis sp. showed a similar result to the over ex pression of GhWRKY17 and AtWRKY53 which could also regulate the wider opened stomata (Yan et al. 2014; Sun andYu 2015). As explained in the correlation analysis ( Figure 4C), the lower expression level of the WRKY53 gene is positively correlated with the lower opened stom ata (r = 0.64). Therefore, it is suggested that the regulation of the stomatal state under the treatments is influenced by the WRKY53 gene, leading to the growth alteration.
WRKY genes involve in ABA and ROS production, this is suggested that the lower percentage of opened stom ata leading to lower plant growth after chitosan applica tion under drought stress condition (ChiD) might be in fluenced by the suppression of the WRKY17 and WRKY53 genes. In addition to impeded plant growth, the lower opened stomata also promote the plant's defense system through maintaining water capacity and/ or signal trans duction (Yan et al. 2014). Under drought conditions, the plant's water capacity becomes more critical to maintain the plant's life, so that the water loss through stomata might need to be regulated. Thus, it is not surprising to find that stomata in the ChiD group are only slightly opened. This further supports our suggestion that the in tensive chitosan application could reduce the number of opened stomata, and more pronounced when combined with drought stress. The previous study indicated that the silencing of GhWRKY27a enhanced drought tolerance in cotton marked by less water loss, higher survival rate, and less H 2 O 2 accumulation (Yan et al. 2015).
Although WRKY17 and WRKY53 in almost all treat ments showed lower gene expression, the only WRKY17 in drought treatment was expressed 7fold higher than con trol. This is supported by the previous study that WRKY17 in maize was upregulated after 12 h of drought stress treat ment (Cai et al. 2017). Although a low percentage of the opened stomata between ChiD and D treatments is quite similar, the superior expression of WRKY17 in drought treatment (D) might associate with the plant's responses under drought stress conditions. This is suggested that WRKY17 has a specific role in response to drought stress. We also suggested that WRKY17 and WRKY53 have dif ferent roles in plant defense mechanisms after chitosan ap plication and drought treatment. This is supported by the statistical analysis that showed a negative correlation (r = 0.78) between the genes. A more comprehensive analysis of WRKY17 and WRKY53 is crucial in developing strate gies to define the plant's molecular response to drought. The determination of WRKY17 and WRKY53 target genes will allow us to manipulate plants response under drought stress.

Conclusions
The double treatment of 1 mg/mL chitosan application and 50% drought stress (ChiD) in red chili plants signifi cantly decreased plant growth performance, including the height increment, followed by the decrease in the num ber of leaves, flower, and fruit. The percentage of opened stomata on chitosandrought treatment significantly de creased by up to 30.67% and followed by the decrease in WRKY17 and WRKY53 genes expression level. There fore, chitosan application was suggested could not allevi ate plants grown under drought stress, but enhance plant's resistance against drought stress. However, WRKY17 ex pression level on drought treatment was significantly up regulated up to 7fold higher than control. Furthermore, we suggested that WRKY17 has a specific role in response to drought stress.