Mediation of The Salicylic Acid Pathway by ROS1 in Response to Abiotic Stresses

Background: DNA methylation plays an important role in the growth and development of plants in response to various abiotic stresses. Salicylic acid (SA) is an important signaling molecule that is synthesized by plants and induces the expression of defense genes. Results: In this paper, we investigated the molecular mechanisms by which an upstream regulator (ACD6) in the SA pathway, an ABA pathway-related gene (ACO3), and a stress resistance gene (GSTF14) were induced by various abiotic stresses. The results demonstrated that abiotic stresses, including drought, cold, and salt stresses, induced the demethylation of the repeats in the promoters of ACD6, ACO3, and GSTF14 and transcriptionally activated their expression. Furthermore, our results revealed that ROS1-mediated DNA demethylation plays an important role in the process of transcriptional activation of ACD6 and GSTF14 when Arabidopsis plants were under cold stress. Conclusions: Our results conrmed that ROS1 plays an important role in the process of defense genes in the SA pathway and stress resistance gene GSTF14 in response to abiotic stresses. Our study reveals the molecular mechanism that plant defense genes in the SA pathway and the stress resistance genes are involved in response to various abiotic stresses. The results show that the RdDM pathway has an important role in maintaining the low transcription levels of ACD6, GSTF14, and ACO3 in wild-type Col-0 plants. Further studies reveal that abiotic stresses induced DNA demethylation of the ACD6, ACO3, and GSTF14 promoters and transcriptionally activated the expression of defense and stress resistance genes. Moreover, ROS1-mediated DNA demethylation plays an important role in this process.

Background DNA methylation is one of the most common forms of DNA covalent modi cation in the genome of eukaryotes. It plays an important role in the growth and development of plants and in response to various abiotic stresses. RNA silencing is a conserved pathway that results in the blockage of gene expression in both the cytoplasm and nucleus of eukaryotic organisms [1]. In plants, small interfering RNAs (siRNAs) target homologous sequences for DNA methylation, a process known as RNA-directed DNA methylation (RdDM); this process plays an important role in regulating gene expression, controlling the activity of transposable elements, and defending against foreign DNAs, such as DNA viruses [2][3][4]. This type of small interfering RNA (siRNA) is synthesized by RNA polymerase IV (Pol IV), RNA-dependent RNA polymerase (RDR2), and Dicer-like 3 (DCL3) together [5]. The Argonaute protein 4 (AGO4) and the DNA methyltransferases DRM1/2, MET1, and CMT3 perform de novo methylation and maintain methylation of the target DNA [6]. DNA methylation can be removed by DNA glycosylases/lyases in Arabidopsis, and this process is known as active demethylation [7]. Repressor of silencing 1 (ROS1) can negatively regulate the RdDM pathway [8,9]. ROS1-mediated DNA demethylation helps determine genomic DNA methylation patterns and protects active genes from being silenced [10].
Abiotic stresses mainly include drought, cold, and salt stresses, which severely threaten plant growth or crop yield [11,12]. Abiotic stresses can induce accumulation of endogenous abscisic acid (ABA), triggering ABA signal transduction to cope with adverse environmental factors [13][14][15]. When plants are under cold stress, ABA can regulate the expression of cold-resistant genes in plants in response to stress [16][17][18]. Abiotic stress also affects the dynamic changes in DNA methylation in plants. Changes in methylation levels and patterns regulate the expression of stress-responsive genes, thereby improving the resistance of plants to stress [19]. Aluminum, salt, and cold stresses induce the demethylation of the coding sequence of the NtGPDL gene in tobacco, thereby promoting the expression of this gene [20].
Soybean has been found to show abnormal expression of approximately 49 transcription factors under salt stress, with expression pro les of the MYB, b-ZIP, and AP2/DREB transcription factor families signi cantly correlated with the DNA methylation of their gene sequences [21]. Variation in DNA methylation of four potato cultivars before and after cryopreservation has indicated that DNA methylation patterns can change in cryopreserved materials [12]. Abiotic stress can regulate the expression of stress-responsive genes by inducing dynamic changes in DNA methylation, thereby improving the adaptability of plants to the environment. Changes in methylation status caused by stress can be passed on to offspring, namely, stress memory [22].
Salicylic acid (SA) is an important signaling molecule in plant defense responses and can induce the expression of defense genes and acquisition of systemic resistance [23]. There are at least three upstream regulators of SA, and accelerated cell death 6 (ACD6) belongs to the second class of SA upstream regulators. The gain-of-function mutant of ACD6, acd6-1, can increase the expression of the genes ACD6-1, EDS1, PAD4, and NPR1 and induce an increase in SA accumulation [24][25][26][27][28][29]. Plants respond to pathogens via the salicylic acid (SA), jasmonic acid (JA), and ethylene (ET) pathways [2]. Our previous study revealed the molecular mechanisms underlying the induction of defense genes in the SA pathway by biotic stresses [4], but the regulatory mechanism of the SA defense pathway in response to abiotic stresses remains unclear.
In this study, we determined the molecular mechanisms underlying functioning of the upstream regulator ACD6 of the SA pathway, the stress resistance gene GSTF14 in the glutathione S-transferase (GST) superfamily and aconitate hydratase 3 (ACO3) in response to abiotic stresses. The results showed that the expression levels of defense genes (ACD6, NPR1, and PR5) in the SA pathway, the ABA pathwayrelated gene ACO3, and the stress resistance gene GSTF14 signi cantly increased after treatment with drought, cold, and salt stresses. Sequencing results con rmed that abiotic stresses induced the demethylation of the repeats in the promoters of ACD6, ACO3, and GSTF14 and transcriptionally activated their expression. Further experiments revealed that ROS1-mediated DNA demethylation plays an important role in the process of the SA pathway in response to abiotic stresses.

Results
Activation of the expression of the upstream regulator ACD6 of the SA pathway by drought stress Our previous studies have shown the molecular mechanism underlying the induction of defense gene expression in the SA pathway by biotic stresses [4,30]. To investigate whether abiotic stress could induce the expression of defense gene ACD6 in the SA pathway and stress resistance genes GSTF14 and ACO3, the wild-type Columbia (Col-0) line of Arabidopsis thaliana was selected for drought-stress treatment, cold-stress treatment, and salt-stress treatment. There were no signi cant phenotypic changes in plants treated with cold stress (4 °C) for 24 h or salt stress (150 mM) for 3 days. On days 5-7, the leaves of Col-0 plants treated with drought stress turned slightly yellow and shrunk ( Figure 1B, C) in comparison to untreated Col-0 plants ( Figure 1A). On day 14, anthocyanin accumulation in the leaves of Col-0 plants treated with drought stress clearly increased, and the leaves turned severely yellow and withered (Figure 1  D).
We extracted the total RNA from Col-0 plants on the 7th day of drought-stress treatment for comparative analysis of gene expression. The results of the reverse transcription-semiquantitative polymerase chain reaction (RT-sqPCR) assay showed higher expression levels of the regulator ACD6 of the SA pathway, the stress resistance gene GSTF14, and ACO3 in the plants after drought-stress treatment than in untreated Col-0 plants; GAPDH acted as a reference gene in this study ( Figure 1E). Consistent with the RT-sqPCR results, the quantitative reverse transcription-polymerase chain reaction (RT-qPCR) analysis con rmed that ACD6, GSTF14, and ACO3 were signi cantly upregulated after drought-stress treatment, and the upregulation of GSTF14 expression was more signi cant ( Figure 1F). Since ACD6 is an upstream regulator of the SA pathway, the increase in ACD6 expression could upregulate the expression of the defense genes NPR1 and PR5 ( Figure 1G). Induction of SA pathway-related defense genes by cold and salt stress To further investigate whether cold stress could also induce the expression of defense genes in the SA pathway, we extracted total RNA from wild-type Arabidopsis Col-0 plants treated under different conditions and detected the related defense genes. RT-sqPCR results showed that compared with controls, A. thaliana plants treated with cold or salt stress had signi cantly higher expression levels of defense genes ACD6, NPR1, and PR5 and ABA pathway-related gene ACO3 (Figure 2A, B). Consistent with the RT-sqPCR results, the RT-qPCR results further con rmed that cold stress and salt stress activated the expression of ACD6, which was signi cantly increased after 24 h of cold-stress treatment ( Figure 2C, D). We also compared the expression of the stress resistance gene GSTF14. The results showed that the upregulation of GSTF14 was the most signi cant in the plants treated with cold stress for 24 h ( Figure  2C). Direct correlation between the increased expression of defense and stress resistance genes and the reduction in promoter DNA methylation To investigate whether the increase in the expression of these defense and stress resistance genes was related to the changes in their promoter DNA methylation, the DNA methylation of the plants under stress treatments was detected and compared. Untreated Arabidopsis Col-0 plants were used as the controls. After drought-stress treatment, the CG, CNG, and CHH methylation of the repeats in the ACD6 promoter decreased from 78.30% to 62.03%, from 21.67% to 8.11%, and from 13.51% to 5.80%, respectively. After cold-stress treatment, the CG, CNG, and CHH methylation of the repeats in the ACD6 promoter decreased from 78.32% to 57.77%, from 21.67% to 7.56%, and from 13.51% to 5.36%, respectively. After salt-stress treatment, the CG, CNG, and CHH methylation of the repeats in the ACD6 promoter decreased from 78.32% to 63.46%, from 21.67% to 8.26, and from 13.51% to 5.25%, respectively ( Figure 3A).
Similarly, we used untreated Col-0 as a control to perform DNA methylation sequencing of the repeats in the ACO3 promoter in plants under drought-, cold-, and salt-stress treatments. After drought-stress treatment, the CG methylation of the repeats in the ACO3 promoter did not change signi cantly, while the CNG and CHH methylation of the repeats in the ACO3 promoter decreased signi cantly, from 65.89% to 33.33% and from 42.22% to 8.89%, respectively. After the cold-stress treatment, the CG methylation of the repeats in the ACO3 promoter did not change, while the CNG and CHH methylation of the repeats in the ACO3 promoter decreased signi cantly, from 65.89% to 20% and from 42.22% to 8.16%, respectively. After salt-stress treatment, the CG methylation of the repeats in the ACO3 promoter did not change signi cantly, while the CNG and CHH methylation of the repeats in the ACO3 promoter decreased signi cantly, from 65.89% to 21.43% and from 42.22% to 9.19%, respectively ( Figure 3B). DNA methylation of the GSTF14 promoter was analyzed next. After drought-stress treatment, the CG, CNG, and CHH methylation of the repeats in the GSTF14 promoter decreased from 90.30% to 75.49%, from 64.04% to 48.61%, and from 20.78% to 8.72%, respectively. After cold-stress treatment, the CG methylation of the repeats in the GSTF14 promoter decreased, from 90.30% to 73.03%, the CNG and CHH methylation decreased from 64.04% to 51.46% and from 20.78% to 9.63%, respectively. After salt-stress treatment, the CG methylation of the repeats in the GSTF14 promoter decreased, from 90.30% to 75.50%, the CNG and CHH methylation decreased, from 60.60% to 52.75% and from 20.78% to 8.65%, respectively ( Figure 3C). Our results revealed that drought, cold, and salt stresses could induce DNA demethylation of the repeats in the gene promoters and increase the expression of these defense and stress resistance genes. Moreover, under drought, cold, and salt stresses, the pattern of DNA methylation variation of the ACD6 and GSTF14 promoters was different from that of the ACO3 promoter. Role of ROS1 in the regulation of the SA pathway in response to abiotic stresses To further study the molecular mechanisms underlying the functioning of defense genes of the SA pathway in response to abiotic stresses, we used RNA gel blotting to detect the expression of related genes in plants mutated at key functional elements of the RdDM pathway. The results showed that the expression of ACD6 and GSTF14 clearly increased in the mutant ago4 and DNA methyltransferase mutants met1, drm1/2 and cmt3 with ecotypes Col-0 as controls ( Figure 4A). RT-qPCR results further con rmed that ACD6, GSTF14, and ACO3 were upregulated in the ago4 mutant ( Figure 4B), indicating that RdDM has an important role in maintaining the low transcription levels of ACD6, GSTF14, and ACO3 in wild-type plants; however, these mutants showed increased transcript levels for those genes. Repressor of silencing 1 (ROS1) can negatively regulate the RdDM pathway [8,9]. The results further showed that the expression levels of these genes were lower in the ros1 and rdd mutants, when the Col-0 plants were used as the control ( Figure 4C).
To determine whether ROS1 plays a role in the responses of these genes to abiotic stress, we performed cold-stress treatment on loss-of-function ros1 mutants and compared the expression of the ACD6 gene between the cold stress-treated ros1 mutants (ros1+cold) and the cold stress-treated Col-0 (Col-0+cold). The results showed that when Col-0 was used as the control, the expression of ACD6 in the cold stresstreated Col-0 plants signi cantly increased. However, the increase in ACD6 expression in the cold stresstreated ros1 mutants and loss-of-function ros1dml2dml3 (rdd) mutants was signi cantly inhibited when compared with the cold stress-treated Col-0 plants ( Figure 4D). ROS1 plays an important role in the activation of defense and stress resistance genes in response to abiotic stress, and this nding was con rmed by the expression levels of GSTF14 and ACO3. When the cold stress-treated Col-0 plants were used as the control, the increase in GSTF14 and ACO3 expression was inhibited in the cold stress-treated ros1 mutants ( Figure 4D). Sequencing analysis con rmed that the DNA methylation levels of the repeats in the ACD6 promoter in cold stress-treated Col-0 plants were signi cantly reduced, including the CG, CNG and CHH sites, while the decrease in DNA methylation levels of the repeats in the ACD6 promoter in cold stress-treated ros1 mutants was obviously inhibited ( Figure 4E). The results further demonstrated that the DNA methylation at CNG and CHH sites in the ACO3 promoter in cold stress-treated Col-0 plants was signi cantly decreased, while the decrease in DNA methylation at CNG and CHH sites in the ACO3 promoter in cold stress-treated ros1 mutants was obviously inhibited ( Figure 4F).
Our results revealed that the activation of the expression of the regulator ACD6 in the SA defense pathway, the stress resistance gene GSTF14 and ABA pathway-related gene ACO3 by abiotic stresses was related to ROS1-mediated DNA demethylation.

Discussion
In recent years, scientists have begun to pay attention to the important role of hormones in the regulation of plant growth and development and resistance to abiotic stresses. In this eld, the ABA pathway has been well studied. ABA is a key hormone regulating the response of plants to abiotic stresses, such as drought. A total of 40 stress-inducible transcription factor genes have been found in Arabidopsis [31]. For example, the MYB transcription factors are indispensable to the adaptation of plants to cold stress and can affect plant resistance to drought by controlling stress-induced ABA synthesis [32]. We know less about the role of the SA defense pathway in the response of plants to abiotic stresses and the related molecular mechanisms.
This study investigated the role of the SA pathway and related defense genes in the response of plants to abiotic stresses. The results showed that drought (Figure 1), cold and salt stresses (Figure 2) induced the expression of the upstream regulator ACD6 of the SA pathway, the stress resistance gene GSTF14, and the ABA pathway-related gene ACO3 in Arabidopsis plants ( Figure 1E, F). The gain-of-function mutant of ACD6, acd6-1, can increase the expression of the genes ACD6-1, EDS1, PAD4, and NPR1 and induce an increase in SA accumulation [24][25][26][27][28][29]. Therefore, we hypothesized that the increase in ACD6 expression would further activate the expression of defense genes NPR1 and PR5 ( Figure 1G) in the SA pathway.
Under the same stress conditions, different genes differ in the levels and patterns of DNA methylation (Figure 3), suggesting complex molecular mechanisms regulate the expression of these genes. Sequencing results con rmed that the increase in the expression of ACD6, GSTF14, and ACO3 was related to the reduction in DNA methylation levels of the promoters of these genes. The CG, CNG, and CHH methylation in the ACD6 and GSTF14 promoters decreased to varying degrees, and the CG methylation decreased signi cantly ( Figure 3A, C). However, the CG methylation of the repeats in the ACO3 promoter barely changed, but their CHG and CHH methylation signi cantly decreased ( Figure 3B). Our results reveal that abiotic stresses (cold stress, drought, and salt stress) induced DNA demethylation of the ACD6, ACO3, and GSTF14 promoters and transcriptionally activated the expression of defense and stress resistance genes, thereby enhancing the adaptability of plants to abiotic stresses.
Further studies revealed that the expression of ACD6 and GSTF14 in the mutants ago4, drm1/2, cmt3 and met1 was higher than that in Col-0 ( Figure 4A). RT-qPCR results con rmed that ACD6, ACO3, and GSTF14 in the mutant ago4 were upregulated ( Figure 4B), indicating that the RdDM pathway has an important role in maintaining the low transcription levels of ACD6, GSTF14, and ACO3 in wild-type plants. DNA methylation can be removed by DNA glycosylases/lyases in Arabidopsis, in which ROS1 can negatively regulate the RdDM pathway [7,8]. To demonstrate that ROS1 also targets these genes, we performed the detection of the expression levels of these genes in the ros1 and rdd mutants. The results showed that the expression levels of these genes were lower in the ros1 and rdd mutants, when the Col-0 plants were used as the control ( Figure 4C). ROS1-mediated DNA demethylation can act on the three DNA methylation sites, CG, CHG, and CHH [33]. DNA methylation sequencing of ros1 mutants has revealed that ROS1 generally targets genes that contain CG, CNG, and CNN methylation in transposable elements and repeats but does not target genes that contain only CG methylation [34]. Our results further reveal that ROS1 also plays an important role in the responses of the defense genes in the SA pathway and stress resistance genes to abiotic stresses. When the Col-0 plants were used as the control, the upregulation of ACD6 and GSTF14 was signi cant in Col-0 plants treated with cold stress for 24 h ( Figure 4D). When the cold stress-treated Col-0 plants were used as the control, the increase in the expression of ACD6 and GSTF14 in ros1 mutants treated with cold stress for 24 h was signi cantly inhibited ( Figure 4D). Furthermore, after 24 h of the cold-stress treatment of Col-0, DNA methylation levels in the repeats in the ACD6 and ACO3 promoters were signi cantly reduced, while the decrease in DNA methylation levels in the repeats in the ACD6 and ACO3 promoters in cold stress-treated ros1 mutants was obviously inhibited ( Figure 4E, F).
These results further con rm that ROS1-mediated DNA demethylation played an important role in the transcriptional activation of the upstream regulator ACD6 of the SA pathway and the stress resistance genes in response to various abiotic stresses. Due to the complexity of the dynamic regulation of DNA methylation, the molecular mechanisms by which plants adapt to various adverse environmental factors and the ways different signaling pathways interact still require in-depth study.

Conclusions
Our study reveals the molecular mechanism that plant defense genes in the SA pathway and the stress resistance genes are involved in response to various abiotic stresses. The results show that the RdDM pathway has an important role in maintaining the low transcription levels of ACD6, GSTF14, and ACO3 in wild-type Col-0 plants. Further studies reveal that abiotic stresses induced DNA demethylation of the ACD6, ACO3, and GSTF14 promoters and transcriptionally activated the expression of defense and stress resistance genes. Moreover, ROS1-mediated DNA demethylation plays an important role in this process.

Methods
Plant growth and abiotic stress treatments Arabidopsis thaliana ecotype Columbia (Col-0) and the mutant plants were used for this work. The ago4 mutant seeds (original source) [36], ros1and ros1dml2 dml3 mutant seeds (original source) [37] were provided by Chengguo Duan Shanghai Center for Plant Stress Biology, Shanghai Institute of Biological Sciences, Chinese Academy of Sciences (CAS). The Col-0, met1, drm1/2 and cmt3 mutant seeds were provided by Institute of Genetics and Developmental Biology, CAS. Seeds were surface-sterilized with 30% bleach, washed three times with sterile water, and sown on Murashige and Skoog (MS) plates. The seedlings were grown for approximately 2 weeks before they were transplanted to soil.

Competing interests
The authors declare no con ict of interest.

Funding
This study was supported by grants from the National Natural Science Foundation of China (Grant Number 31301043), which provides the research fundings used for sequencing and the analysis of data and the Department of Finance of Jilin Province (Grant Number JJKH20191013KJ), which provides the research fundings used to purchase for chemical reagents.

Authors' contributions
Liping Yang had the idea and performed the design of the study, sequencing, the analysis of data and manuscript writing, Chenjing Lang and Yanju Wu performed RNA extraction and the detection of related gene expression, Dawei Meng and Tianbo Yang performed DNA extraction and sequencing, Taicheng Jin participated the analysis of data, Xiaofu Zhou provided help for the analysis of data and the revision of manuscript. The authors have read and approved the manuscript.