Identification of phytohormone changes and its related genes under abiotic stresses in transgenic rice

Abiotic stresses, such as drought and salinity, adversely affect plant growth and productivity. Comparison between non transgenic and transgenic rice harboring CaMsrB2 gene, which induces tolerance to abiotic stress, is important to observe response of gene under abiotic stress. Phytohormone showed a tendency to increase under the drought stress or salinity stress in the transgenic plant. RT-PCR analysis showed that gene expression and phytohormone levels under abiotic stress, to be closely related. The CaMsrB2 gene is related to the expression of JA and ABA hormones. Therefore, the level of expression of these genes and hormones was observed. The transcription levels of LOX2 and OsWRKY45 were substantially higher in the wild type rice in comparison to the transformants, which suggested that phytohormone are also required for the regulation of leaf and root. Comparison between control and transgenic rice overexpressing a CaMsrB2 gene, resulted in different pattern of ABA, JA levels under different stress condition. In both drought and salinity stresses, the expression of OsWRKY45 gene was similar in both treatments with time. These results suggest that gene involved in the plant physiology response in mechanism to abiotic stress. BIOCELL 2019 43(3): 215-224 Doi: 10.32604/biocell.2019.07549 www.techscience.com/biocell Introduction Abiotic stresses such as extreme temperatures, drought, salinity, chemical, toxicity, and oxidative stress are serious threats to agriculture and have a negative impact on the environment. Abiotic stress is the primary cause of crop loss worldwide, reducing average yields for most major crop plants by more than 50% (Boyer, 1982; Bray et al., 2000). This environmental threat leads to a series of morphological, physiological, biochemical and molecular changes that adversely affect plant growth and productivity (Wang et al., 2001). Drought and soil salinity are among the most damaging abiotic stresses affecting agricultural yield in present times due to the unpredictable rainfall (Jianhua et al., 2006). They are significant plant stressors with major impact on plant development and productivity, which cause serious agricultural yield losses (Flowers, 2004; Godfray et al., 2010; Tester and Langridge, 2010). Plants exposed to abiotic stress may experience limited growth and development. Abiotic stress might cause a worldwide yield reduction of approximately 70% (Acquaah, 2007). The stresses of drought and salinity contribute to water deficit, ionic toxicity, imbalances in ion homeostasis, and the occurrence of *Address Correspondence to: Kyung-Min Kim, kkm@knu.ac.kr oxidative stress (Foolad, 2007). Plants exhibit some adaptive responses to abiotic stresses at the molecular and cellular level, mainly because of changes in the expression patterns of a group of genes (Kumar, 2013). Therefore, to understand the stress response mechanism in plants, it is important to identify the genes that respond to drought and salinity. CaMsrB2, a pepper (Capsicum annuum) gene, which encodes a methionine sulfoxide reductase B2, has been implicated in the reactive oxygen species (ROS)-generating response in plants against pathogens (Oh et al., 2010). Kim et al. (2014) reported that transgenic rice varieties that overexpress CaMsrB2 perform better against drought stress than their wild-type counterparts. They reported that transgenic plants show an effect on reactive oxygen scavenging that results from drought stress (Xie et al., 2011). Differential responses to general abiotic stress and stress tolerance can involve different phytohormone levels, sets of genes, and gene products. One strategy that can be employed is the investigation of the genetic and hormonal changes involved in complex biological pathways and processes (Vleesschauwer et al., 2013). The responses to environmental stresses are integrated by signaling networks that mediate the sensors and receptor signals leading to specific mechanisms of responses to complex stress conditions and thus minimize damage. In the present study, we observed the correlation of CaMsrB2 expression to the tolerance of stress through the evaluation 216 MOhAMMAD UBAIDILLAh et al. of phytohormone levels under the drought stress or salinity stress in rice plants. The experimental advantages associated with exploiting the designated pathways of the mechanism mediated by CaMsrB2 gene when stress exposed of genes that functional with observation of phytohormone level. Materials and Methods Plant materials Oryza sativa L. Japonica-type Ilmi (control), CaMsrB2-8, and CaMsrB2-23 mutant strains of rice were used in this study. CaMsrB2-8 and CaMsrB2-23 are drought-tolerant transgenic lines of Ilmi. The transgenic rice seeds were supplied by the National Academy of Agricultural Science, Rural Development Administration of Korea (Oh et al., 2013). The rice seeds were germinated by soaking in water for 3 days, transferred to soil, and cultivated in a greenhouse at Kyungpook National University, Daegu, Korea. Drought tolerance assay The drought experiment was performed during the vegetative stages of growth (until the 45-day-old stage). The sterilized seeds from non-transgenic rice plants (wild type) and T8 lines of CaMsrB2 transgenic rice (by PCR analysis, Supplementary Fig. 1) were planted with two germinated seeds per 5 cm in a plastic tray with 15 holes and supplied with adequate water for growth. The growing plantlets were maintained in the greenhouse under natural light conditions (16-h light/8-hdark cycle) at an ambient temperature of 30 ± 2°C. Drought stress was imposed upon 5-week-old plants by decanting all water from the tray and stopping irrigation for 4 days. The survival rate was measured in 12 plants among 30 plants of each genotype. Salinity tolerance assay To analyze the salinity tolerance, the plants were grown as described above. Salinity stress was induced by the irrigation of the plants with 200 mM sodium chloride (NaCl) solution for 7 and 14 days. Transgenic and wild-type plants were treated for salinity by decanting all the resting waster into the plastic box and irrigating the solution twice after 1 day. The survival rate and chlorophyll content of the transgenic plants were observed and compared with those of the control plants, which served as an indication of salinity tolerance. The chlorophyll content in different parts of the leaves was analyzed in triplicate by using a chlorophyll meter, SPAD-502 (Minolta, Japan). The experiment was conducted in five replicates. Sample collection The leaf and root samples were collected after drought treatment for 3 and 4 days after salt treatment for 0, 1, and 2 weeks. The samples were used immediately after collection or frozen in liquid nitrogen and stored at -70°C for later use in gene expression and hormone analysis. Extraction of total RNA from leaf and roots tissues Total RNA was isolated from leaf and root tissues by using RNeasy Plant Mini Kit (QIAGEN, Germany) in accordance with the manufacturer’s protocol. cDNA was synthesized from total RNA using a qPCRBIO cDNA synthesis kit (PCR Biosystem, USA) in accordance with the manufacturer’s protocol. Reverse transcriptase PCR analysis The expression of the transgene CaMsrB2 was analyzed with reverse transcriptase (RT)-PCR by using the SuperScript III One-step RT-PCR system with Platinum Taq DNA Polymerase (Invitrogen, USA). RT-PCR was performed by using 2× reaction mixture (25 μL), forward and reverse primer (1 μL at 20 pmol/μL), SuperScript III RT/ Platinum TaqMix (25 μL), and total RNA (500 ng). Each gene investigated was amplified from three independent biological samples using specific primers (Supplementary Tab. 1). The cycling conditions for the RT-PCR were: 94°C for 2 min, 94°C for 15 s, 60°C for 30 s, and 72°C for 1 min. Forty cycles of the amplification reaction were performed with a final extension at 72°C for 10 min. The 210 bp product was amplified from CaMsrB2 by using the genespecific primers 5’-GTCAGGAGTGCGATTAATAGC-3’and 5’-CCTGAGCAGTTCCGTATTCT-3’ for the analysis of confirmed transgenic plants (data not shown). Quantitative real-time PCR The quantitative (q) RT-PCR analysis was conducted by using real-time PCR Pre-mix qPCRBIO SyGreen Mix LoROX (PCR BIOSYSTEMS, UK) in accordance with the manufacturer’s protocol. cDNA concentration was checked by NanoDrop 2000 (Thermo Scientific, USA). After amplification, the experiment was converted to comparative quantification (calibrator) experiment type and the data were analyzed with the Eco software (Illumina, USA). The rice actin gene Os11g0247300 was used as an endogenous control. Accession numbers Sequence data from this article can be found in the GenBank/ EMBL databases under accession numbers AY026332 ( JAMyb), AY838897 (NCED1), AK241395 (LOX2), AK068993 (PAL1), AK120715 (OsNPR1), DQ298181 (OsWRKY45), and AK060893 (OsACTIN). Quantitative analysis of major phytohormones The quantitative analysis of the major phytohormones abscisic acid (ABA), jasmonic acid (JA), and GA3 was performed as described by Ubaidillah et al. (2016). Statistical analysis We sowed seeds in three replicates of 30 each for each experiment and then used 12 plants to test the two abiotic stresses and phytohormone changes in the plants. The results were expressed as means ± standard deviations (SD) of at least three independent experiments performed in duplicate. P < 0.05 was considered statistically significant and was determined using SPSS software (version 14.0, SPSS Science, Chicago, IL, USA). Statistical differences were determined by one-way analysis of variance (ANOVA) and Tukey’s post-test. ABIOTIC STRESS IN TRANSGENIC RICE 217 RT-PCR. Expression patterns of the JsMyb gene related to jasmonic acid were not expressed on the 0th day of the drying treatment and were expressed the same from the 3rd day to the 4th day. The expression of NCED1 gene associated with ABA was very small on days 0 to 3 and strong on day 4 (Supplementary Fi


Introduction
Abiotic stresses such as extreme temperatures, drought, salinity, chemical, toxicity, and oxidative stress are serious threats to agriculture and have a negative impact on the environment. Abiotic stress is the primary cause of crop loss worldwide, reducing average yields for most major crop plants by more than 50% (Boyer, 1982;Bray et al., 2000). This environmental threat leads to a series of morphological, physiological, biochemical and molecular changes that adversely affect plant growth and productivity (Wang et al., 2001). Drought and soil salinity are among the most damaging abiotic stresses affecting agricultural yield in present times due to the unpredictable rainfall (Jianhua et al., 2006). They are significant plant stressors with major impact on plant development and productivity, which cause serious agricultural yield losses (Flowers, 2004;Godfray et al., 2010;Tester and Langridge, 2010). Plants exposed to abiotic stress may experience limited growth and development. Abiotic stress might cause a worldwide yield reduction of approximately 70% (Acquaah, 2007). The stresses of drought and salinity contribute to water deficit, ionic toxicity, imbalances in ion homeostasis, and the occurrence of *Address Correspondence to: Kyung-Min Kim, kkm@knu.ac.kr oxidative stress (Foolad, 2007). Plants exhibit some adaptive responses to abiotic stresses at the molecular and cellular level, mainly because of changes in the expression patterns of a group of genes (Kumar, 2013). Therefore, to understand the stress response mechanism in plants, it is important to identify the genes that respond to drought and salinity. CaMsrB2, a pepper (Capsicum annuum) gene, which encodes a methionine sulfoxide reductase B2, has been implicated in the reactive oxygen species (ROS)-generating response in plants against pathogens (Oh et al., 2010). Kim et al. (2014) reported that transgenic rice varieties that overexpress CaMsrB2 perform better against drought stress than their wild-type counterparts. They reported that transgenic plants show an effect on reactive oxygen scavenging that results from drought stress (Xie et al., 2011). Differential responses to general abiotic stress and stress tolerance can involve different phytohormone levels, sets of genes, and gene products. One strategy that can be employed is the investigation of the genetic and hormonal changes involved in complex biological pathways and processes (Vleesschauwer et al., 2013). The responses to environmental stresses are integrated by signaling networks that mediate the sensors and receptor signals leading to specific mechanisms of responses to complex stress conditions and thus minimize damage. In the present study, we observed the correlation of CaMsrB2 expression to the tolerance of stress through the evaluation of phytohormone levels under the drought stress or salinity stress in rice plants. The experimental advantages associated with exploiting the designated pathways of the mechanism mediated by CaMsrB2 gene when stress exposed of genes that functional with observation of phytohormone level.

Plant materials
Oryza sativa L. Japonica-type Ilmi (control), CaMsrB2-8, and CaMsrB2-23 mutant strains of rice were used in this study. CaMsrB2-8 and CaMsrB2-23 are drought-tolerant transgenic lines of Ilmi. The transgenic rice seeds were supplied by the National Academy of Agricultural Science, Rural Development Administration of Korea (Oh et al., 2013). The rice seeds were germinated by soaking in water for 3 days, transferred to soil, and cultivated in a greenhouse at Kyungpook National University, Daegu, Korea.

Drought tolerance assay
The drought experiment was performed during the vegetative stages of growth (until the 45-day-old stage). The sterilized seeds from non-transgenic rice plants (wild type) and T 8 lines of CaMsrB2 transgenic rice (by PCR analysis, Supplementary  Fig. 1) were planted with two germinated seeds per 5 cm 3 in a plastic tray with 15 holes and supplied with adequate water for growth. The growing plantlets were maintained in the greenhouse under natural light conditions (16-h light/8-hdark cycle) at an ambient temperature of 30 ± 2°C. Drought stress was imposed upon 5-week-old plants by decanting all water from the tray and stopping irrigation for 4 days. The survival rate was measured in 12 plants among 30 plants of each genotype.

Salinity tolerance assay
To analyze the salinity tolerance, the plants were grown as described above. Salinity stress was induced by the irrigation of the plants with 200 mM sodium chloride (NaCl) solution for 7 and 14 days. Transgenic and wild-type plants were treated for salinity by decanting all the resting waster into the plastic box and irrigating the solution twice after 1 day. The survival rate and chlorophyll content of the transgenic plants were observed and compared with those of the control plants, which served as an indication of salinity tolerance. The chlorophyll content in different parts of the leaves was analyzed in triplicate by using a chlorophyll meter, SPAD-502 (Minolta, Japan). The experiment was conducted in five replicates.

Sample collection
The leaf and root samples were collected after drought treatment for 3 and 4 days after salt treatment for 0, 1, and 2 weeks. The samples were used immediately after collection or frozen in liquid nitrogen and stored at -70°C for later use in gene expression and hormone analysis.

Extraction of total RNA from leaf and roots tissues
Total RNA was isolated from leaf and root tissues by using RNeasy Plant Mini Kit (QIAGEN, Germany) in accordance with the manufacturer's protocol. cDNA was synthesized from total RNA using a qPCRBIO cDNA synthesis kit (PCR Biosystem, USA) in accordance with the manufacturer's protocol.

Reverse transcriptase PCR analysis
The expression of the transgene CaMsrB2 was analyzed with reverse transcriptase (RT)-PCR by using the SuperScript III One-step RT-PCR system with Platinum Taq DNA Polymerase (Invitrogen, USA). RT-PCR was performed by using 2× reaction mixture (25 µL), for ward and reverse primer (1 µL at 20 pmol/µL), SuperScript III RT/ Platinum TaqMix (25 µL), and total RNA (500 ng). Each gene investigated was amplified from three independent biological samples using specific primers (Supplementary Tab. 1). The cycling conditions for the RT-PCR were: 94°C for 2 min, 94°C for 15 s, 60°C for 30 s, and 72°C for 1 min. Forty cycles of the amplification reaction were performed with a final extension at 72°C for 10 min. The 210 bp product was amplified from CaMsrB2 by using the genespecific primers 5'-GTCAGGAGTGCGATTAATAGC-3'and 5'-CCTGAGCAGTTCCGTATTCT-3' for the analysis of confirmed transgenic plants (data not shown).

Quantitative real-time PCR
The quantitative (q) RT-PCR analysis was conducted by using real-time PCR Pre-mix qPCRBIO SyGreen Mix Lo-ROX (PCR BIOSYSTEMS, UK) in accordance with the manufacturer's protocol. cDNA concentration was checked by NanoDrop 2000 (Thermo Scientific, USA). After amplification, the experiment was converted to comparative quantification (calibrator) experiment type and the data were analyzed with the Eco software (Illumina, USA). The rice actin gene Os11g0247300 was used as an endogenous control.

Quantitative analysis of major phytohormones
The quantitative analysis of the major phytohormones abscisic acid (ABA), jasmonic acid (JA), and GA 3 was performed as described by Ubaidillah et al. (2016).

Statistical analysis
We sowed seeds in three replicates of 30 each for each experiment and then used 12 plants to test the two abiotic stresses and phytohormone changes in the plants. The results were expressed as means ± standard deviations (SD) of at least three independent experiments performed in duplicate. P < 0.05 was considered statistically significant and was determined using SPSS software (version 14.0, SPSS Science, Chicago, IL, USA). Statistical differences were determined by one-way analysis of variance (ANOVA) and Tukey's post-test.
RT-PCR. Expression patterns of the JsMyb gene related to jasmonic acid were not expressed on the 0th day of the drying treatment and were expressed the same from the 3rd day to the 4th day. The expression of NCED1 gene associated with ABA was very small on days 0 to 3 and strong on day 4 ( Supplementary Fig. 2). The transcript levels in CaMsrB2-8 and CaMsrB2-23 had similar profiles, but with varying timing during drought and salinity stress conditions (Fig. 2). The levels of gene expression in the transgenic species were evaluated by qRT-PCR. We found that CaMsrB2 was highly expressed with similar patterns under the drought stress or salinity stress (Fig. 2). In transgenic rice overexpressing CaMsrB2, the CaMsrB2 transcript level was strongly detected during the drought stress period. The transcripts in CaMsrB2-8 and CaMsrB2-23 were high in all parts of the root and leaf. The length of the drought stress period did not affect the transcript level. The transcript level profiles in CaMsrB2-8 and CaMsrB2-23 were similar, although they occurred at different times during 0, 3, and 4 days of drought condition. CaMsrB2 transgenic rice showed high abundance from the beginning throughout the prolonged salinity stress treatment. The transcripts were strongly detected in both the root and leaf in the CaMsrB2-8 and CaMsrB2-23 lines. The length of stress treatment did not affect the transcript levels. The transcript levels in the CaMsrB2-8 and CaMsrB2-23 plants had similar profiles, although at different time points, of 0, 1, and 2 weeks of salinity stress. We determined the expression level of CaMsrB2 when plants were exposed to stress conditions such as drought and salinity and results showed its high level of expression.

Plant response during drought and salinity stress
We evaluated the performance of CaMsrB2-overexpressing transgenic rice under drought stress and observed it to be better than the control plants. Both lines of transgenic rice were observed to tolerate drought and salinity better than the control plants. The survival rates of all rice lines under both conditions are shown in Fig. 1(A); the survival rates of the CaMsrB2-8 and CaMsrB2-23 lines were almost 40% higher than those of the control. Under the salinity stress, the chlorophyll content was decreased in all plants by approximately 5 units after 1-2 weeks of treatment ( Fig.  1(B)). The two transgenic lines that overexpress CaMsrB2 demonstrated similar responses, which suggested similarities in the stress tolerance mechanisms of these plants.

Gene expression analysis using RT-PCR and qRT-PCR
In the transgenic rice, the CaMsrB2 transcript level was high during the drought stress period; expression in CaMsrB2-8 and CaMsrB2-23 was strongly detected in both of the root and leaf. The length of the drought stress period did not affect the transcript level. The transcript levels were high in conditions of salinity stress. The CaMsrB2 transcript in transgenic rice was highly abundant from the beginning and throughout the prolonged salinity stress. The transcripts were strongly detected in both root and leaf in both CaMsrB2-8 and CaMsrB2-23 lines. The length of the salinity stress treatment did not affect the transcript levels ( Supplementary  Fig. 2). Jsmyb (JA) and NCED1 (ABA) were confirmed by

Alteration of hormone levels under drought and salinity stress
To investigate the early effects of drought stress on hormone levels, the hormones abscisic acid (ABA) and jasmonic acid (JA) were examined in leaf and root tissues after 0, 3, and 4 days of drought stress (Fig. 3). The ABA levels in control rice increased after 3 days of drought stress but decreased after 4 days of drought stress. In contrast, the ABA levels in CaMsrB2-8 and CaMsrB2-23 leaf tissue did not change before 3 days but increased after an additional day of stress. The ABA levels in the root tissue of CaMsrB2-8 and CaMsrB2-23 showed no significant changes after 4 days of drought stress compared with the control.  The level of JA in the control rice increased after 3 days of drought in both leaf and root tissues, but it decreased after an additional day of drought stress treatment. JA levels in the transgenic rice CaMsrB2 were evaluated. In the leaf tissues of CaMsrB2-8 and CaMsrB2-23, the JA level increased after 3 days of drought stress, but it decreased slightly after the fourth day in both leaf and root tissues. The JA levels in control plant and transgenic CaMsrB2 rice were the same after 3 and 4 days of drought stress. however, the JA levels in leaf tissue were slightly higher in transgenic plants than in control plants after 4 days of treatment.
The evaluation GA 3 showed that under drought stress, the accumulation of this hormone in plant tissues was low. The levels were observed in both transgenic and control plants. Under normal conditions, GA 3 accumulates at low levels in leaf and root tissues, but no GA 3 was detected during drought stress (data not shown).

Phytohormone signaling regulates the expression of defenserelated genes to JA signaling pathways in affecting age-related transcript levels of four genes
We selected LOX2, PAL, OsNPR1, and OsWRKY45 genes, which are most closely related to abiotic stress in rice plants. Among these genes, OsWRKY45 showed that the transformant was treated with two stresses clearly expressed in the leaves and roots (Fig. 4, Supplementary Fig. 3). In untreated plants, we observed that the basal levels of LOX2 and OsWRKY45 were higher in control rice than in the CaMsrB2 mutant. To assess the molecular mechanisms by which phytohormone affect OsWRKY45 gene expression in rice, we compared the expression patterns of four genes known to function in JA-dependent defense signaling pathways in old and new leaves (roots) of the control rice and the CaMsrB2 mutant. PAL was expressed with lower levels in roots and leaves. LOX2 and OsWRKY45 were expressed at higher levels in the wild-type plants than in the CaMsrB2 mutant. sNPR1 was expressed at slightly lower levels in the wild-type plants than in the CaMsrB2 mutant at 4 days and 1-2 weeks, respectively.

Discussion
To evaluate their responses to various abiotic stresses, the CaMsrB2 transgenic plants were subjected to the drought stress or salinity stress. The survival rates of transgenic plants after drought stress treatment were higher than those of the control rice plants (Dhungana et al., 2015). Kim et al. (2014) reported that several tests after drought and re-watering indicated that the transgenic lines showed fewer oxidative stress symptoms and a strengthened PSII quantum yield under stress conditions, along with an increased survival rate and chlorophyll index. here, a salt tolerance analysis of CaMsrB2 transgenic rice was performed through the estimation of the level of chlorophyll breakdown. The estimation of the transcript levels in transgenic plants demonstrated that the transcript levels were high and stable throughout prolonged drought stress. The level of gene expression was observed during the salinity stress. In CaMsrB2 transgenic rice, the transcript level was high throughout prolonged drought stress. These transcript levels provide evidence of the role of this gene in the induction of tolerance toward the given stresses.
In this study, the changes in hormone levels in CaMsrB2 transgenic rice, as well as non-transformed control rice, were examined in the drought stress and salinity stress in leaf and root tissues. The changes in hormone levels in rice represent distinct plant responses to abiotic stress that result from different defense mechanisms. CaMsrB2 has been reported as a novel defense regulator against oxidative stress and pathogen attacks (Acquaah et al., 2007). Plants have several physiological and biochemical mechanisms to survive in soils with a deficiency of water and an excess of salt. Under the drought stress and salinity stress, the first organ to experience the stress is the root system. The roots send various signals to the leaves through the xylem sap; phytohormone are considered to be one of the major root-to-shoot stress signals. In addition, drought stress and salinity stress induce calcium signaling (Knight et al., 1998;Laohavisit et al., 2013), which is involved in the activation of the downstream response mechanisms of the involved genes. Steinhorst and Kudla (Steinhorst and Kudla, 2013) observed that Ca 2+ and the reactive oxygen species (ROS) signaling pathway directly enable cell-to-cell communication and thereby long-distance transmission of signals in plants. Under drought stress, ROS production is enhanced owing to stomatal closure and the concomitant limitation on CO 2 fixation (Carvalho, 2008). Osmotic stress during salinity stress impairs the ability of a plant to detoxify ROS (Gupta and huang, 2014). The synthesis of several phytohormone can be induced by ROS (Bogatek and Gniazdowska, 2007). Phytohormone participate prominently in the important regulatory roles in plant physiology that affect developmental processes during abiotic stresses (Chen et al., 2002;Browse, 2009). Three hormones (ABA, JA, and GA 3 ) that are related to the defense mechanisms during abiotic stress, especially against the drought stress and salinity, were investigated. The roles of ABA, JA, and SA as primary signals in the regulation of plant defenses is well established (Bari and Jones, 2009;Pieterse et al., 2009). These hormones generate a signal transduction network that leads to a cascade of events responsible for the physiological adaptation of plants to stress.
ABA is known for its regulatory role of the integration of environmental adversity with the plant development (Christmann et al., 2005). ABA positively contributes to the adaptations of osmotic stress, a major component of several abiotic stresses (Kissoudis et al., 2014). Its involvement in the regulation of the defense responses has been a topic of recent comprehensive reviews (Asselbergh et al., 2008;Ton et al., 209). The synthesis, degradation, and transport processes dynamically maintain ABA levels in plant cells. Therefore, the plants maintain their developmental programs and stress responses through the modulation of endogenous ABA levels (Schwartz et al., 2003). It has been reported that ABA can stimulate the production of anti-apoptotic proteins and reduce the expression of a number of pro-apoptotic proteins (Scarfì et al., 2009). There was more ABA accumulated in the leaves than the roots of plants in the present study, because the roots are not able to combat stress-induced ABA accumulation. JA is a well-known signaling molecule in plant defense and stress response (hoeberichts and Woltering, 2003). The participation of JA in response to abiotic stresses, such as drought and salinity, has been reported in several species. Water stress increases endogenous JA content, which is followed by the synthesis of jasmonate-induced proteins (Lehmann et al., 1995). It has been reported that tomato cultivars differ in salt tolerance owing to different basal JA content. Steady-state amounts of JA and related compounds are higher in plants with salt and water stress tolerance (Pedranzani et al., 2003). It has been suggested that JArelated responses are directly associated with a downstream reset of gene expression in the biosynthesis pathway (Thines et al., 2007). Collectively, these studies indicated that JA was an important component of a pathway that positively improves plant defense in CaMsrB2 transgenic rice. however, the precise mechanisms by which JA signaling improves this defense remain unknown. Plants have developed complex mechanisms to perceive external signals under drought and salinity stresses and allow for an optimal response to the environment. In this study, the accumulation of JA in transgenic lines was higher than that in the non-transgenic plants under both stresses. Based on RT-PCR data, it appeared that the transgene is expressed even before the onset of stress. If it is involved in the regulation of hormonal metabolism, then the transgenic plants should have elevated levels of hormones when compared to non-transgenic plants before onset of stress. But this is not reflected in the data. however, significant increases in JA were present under the salinity stress. JA and ABA might be involved in different stages of the response, which drive an acclimation process during growth through extensive genetic reprogramming to reach a new homeostasis (harb et al., 2010). In Arabidopsis, both JA and ABA act in response to moderate drought (30% of field capacity) (Aimar et al., 2011). The overexpression of CaMsrB2 can improve the endogenous levels of ABA and JA during the early stages of moderate drought. We have suggested that CaMsrB2 is involved in an increase in the endogenous ABA and JA levels and can sufficiently stimulate the preparation of the response needed for drought acclimation.
Rice PAL transcripts have a higher level of accumulation in stems, and observed in lower levels in roots and leaves (Zhu et al., 1995). Nh1 (Arabidopsis NPR1 homolog 1) has been reported to play an important role in the SA signaling pathway downstream of SA (Chern et al., 2005) and to regulate the expression of PR1a and PR1b genes in rice (Shimono et al., 2007). Xie et al. (2011) suggested that these genes are upregulated by phytochrome-mediated signals, which may account for the elevated basal levels of PR1a and PR1b in wild-type plant compared with the phyAphyBphyC mutant. These same genes were developmentally upregulated, as indicated by the higher level of transcripts in the old leaves than in the new leaves. We have proposed a defense model for rice that does not support a dichotomy between the pathogen lifestyle and the effectiveness of the archetypal defense hormones salicylic acid (SA) and jasmonic acid (JA) (Vleesschauwer et al., 2013).
The determination of the changes in the levels of endogenous hormones in transgenic rice subjected to the drought stress and salinity stress will allow a better understanding of the salinity response and assist in the formulation of improved strategies for the generation of salinity-tolerant crops.

Conclusions
This study examined the relationship between two types of abiotic stress and four genes involved in salinity and drought stress. Especially, salinity and drought resistance of the transgenic plant showed similar patterns of plant hormones. Among the four genes, OsWRKY45 was most related to abiotic stress. We demonstrated that the phytohormone in rice required for age-related resistance to abiotic stress may indirectly increase OsWRKY45 gene expression through the regulation of JA-dependent defense pathways.

Conflicts of Interest
The authors declare that there are no conflicts of interest regarding the publication of the paper.

Financial Support
This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.