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

Mediation of The Salicylic Acid Pathway by ROS1 in Response to Abiotic Stresses | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research article Mediation of The Salicylic Acid Pathway by ROS1 in Response to Abiotic Stresses Liping Yang, Chenjing Lang, Yanju Wu, Dawei Meng, Tianbo Yang, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-128268/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 10 You are reading this latest preprint version Abstract 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 confirmed 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. Plant Molecular Biology and Genetics Plant Physiology and Morphology DNA methylation salicylic acid ROS1-mediated DNA demethylation abiotic stresses Figures Figure 1 Figure 2 Figure 3 Figure 4 Background DNA methylation is one of the most common forms of DNA covalent modification 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 [2-4]. 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 [13-15]. When plants are under cold stress, ABA can regulate the expression of cold-resistant genes in plants in response to stress [16-18]. 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 profiles of the MYB, b-ZIP, and AP2/DREB transcription factor families significantly 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 [24-29]. 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 pathway-related gene ACO3 , and the stress resistance gene GSTF14 significantly increased after treatment with drought, cold, and salt stresses. Sequencing results confirmed 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 significant 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 confirmed that ACD6 , GSTF14 , and ACO3 were significantly upregulated after drought-stress treatment, and the upregulation of GSTF14 expression was more significant (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). Figure 1 Detection and analyses of the expression of defense genes in Arabidopsis plants treated with drought stress (A) The untreated Arabidopsis Col-0 plants. (B, C) The leaves of Arabidopsis plants treated with drought stress turned slightly yellow and shrunk during days 5-7. (D) Anthocyanin accumulation in the leaves of Arabidopsis plants treated with drought stress clearly increased, and the leaves turned severely yellow and withered on day 14. (E) Transcript levels of related genes in Arabidopsis plants treated with drought stress were analyzed by sqPCR; untreated Col-0 plants served as controls. (F) Transcript levels of related genes in Arabidopsis plants treated with drought stress were analyzed by qPCR. The statistical analysis was performed; asterisks indicate statistically significant differences compared with control plants (P < 0.05). (G) Transcript levels of defense genes in Arabidopsis plants treated with drought stress were analyzed by sqPCR. 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 significantly 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 confirmed that cold stress and salt stress activated the expression of ACD6 , which was significantly 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 significant in the plants treated with cold stress for 24 h (Figure 2C). Figure 2 Detection and analyses of the expression of defense genes and stress resistance genes in Arabidopsis (A, B) The defense genes and ACO3 transcript levels in Arabidopsis plants treated with cold and salt stress were analyzed by sqPCR; untreated Col-0 plants served as controls. (C, D) ACD6 , GSTF14, and ACO3 transcript levels in Arabidopsis plants treated with cold and salt stress were analyzed by qPCR; untreated Col-0 plants served as controls. The statistical analysis was performed; asterisks indicate statistically significant differences compared with control plants (P < 0.05). 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 significantly, while the CNG and CHH methylation of the repeats in the ACO3 promoter decreased significantly, 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 significantly, 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 significantly, while the CNG and CHH methylation of the repeats in the ACO3 promoter decreased significantly, 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. Figure 3 Analyses of DNA methylation of the promoters in plants treated with different stresses (A) Percentage of DNA methylation in the repeat regions of the ACD6 promoter in plants treated with different stresses and untreated Col-0 plants. (B) Percentage of DNA methylation in the repeat regions of the ACO3 promoter in plants treated with different stresses and untreated Col-0 plants. (C) Percentage of DNA methylation in the repeat regions of the GSTF14 promoter in plants treated with different stresses and untreated Col-0 plants. Fifteen individual clones of each genotype were used for sequencing, and the original data are shown in supplement Data S1. The statistical analysis was performed using OriginPro 8 (http://www.originlab.com). Values are means ± SEM, and asterisks indicate statistically significant differences compared with control plants (one-way analysis of variance, P < 0.05). 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 confirmed 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 stress-treated Col-0 plants significantly increased. However, the increase in ACD6 expression in the cold stress-treated ros1 mutants and loss-of-function ros1dml2dml3 ( rdd ) mutants was significantly 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 finding was confirmed 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 confirmed that the DNA methylation levels of the repeats in the ACD6 promoter in cold stress-treated Col-0 plants were significantly 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 significantly 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. Figure 4 Analyses of DNA methylation and the expression levels of genes (A) Analyses of the expression levels of ACD6 and GSTF14 in the mutants ago4 , met1 , drm1/2 and cmt3 by northern blotting; wild-type Col-0 ecotype served as background controls for the mutant genotypes. (B) Analyses of the expression levels of ACD6 , ACO3 , and GSTF14 by RT-qPCR in DNA methylation mutant plants ago4 , with wild-type as background control for the mutant genotypes. (C) The related genes were detected in the Col-0, ros1 and rdd mutants by RT-qPCR. (D) The related genes were detected in the untreated Col-0, the Col-0 treated with cold stress, ros1 , and ros1 dml2 dml3 ( rdd ) mutant plants treated with cold stress by RT-qPCR. (E) Analyses of DNA methylation in the repeat regions of the ACD6 promoter in Col-0, and Col-0 plants and ros1 mutants treated with cold stress. (F) Analyses of DNA methylation in the repeat regions of the ACO3 promoter in Col-0, the Col-0 plants, and ros1 mutants treated with cold stress. The statistical analysis was performed using OriginPro 8 (http://www.originlab.com); asterisks indicate statistically significant differences compared with control plants (P < 0.05). 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 field, 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 [24-29]. 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 confirmed 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 significantly (Figure 3A, C). However, the CG methylation of the repeats in the ACO3 promoter barely changed, but their CHG and CHH methylation significantly 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 confirmed 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 significant 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 significantly 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 significantly 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 confirm 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], ros1 and 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. Arabidopsis thaliana Col-0 plants were treated with abiotic stresses, such as cold stress (4 °C, 24 h), salt stress (150 mM NaCl, 3 days), and drought stress (not watered, 7 days). The significant experimental details were as follows. Cold stress Salt stress Drought stress 4°C 150 mM NaCl not watered 24 hours 3 days 7 days RT- sqPCR , RT-qPCR and RNA gel blot analysis Total RNA was isolated using TRIzol reagent (Invitrogen) according to the manufacturer's protocols. The total RNA was subsequently used for RT-sqPCR, RT-qPCR, and RNA gel blotting analysis. For RT-sqPCR, total RNA was extracted from the treated plants and subsequently used for reverse transcription and semiquantitative PCR. For RT-qPCR, the complementary DNA synthesis was performed using the Reverse Transcription kit (Takara). Quantitative RT-PCR was performed using SYBR green mix (Qiagen). Each experiment consisted of three biological replicates and was repeated twice. For the high molecular weight RNA gel blot analysis, 10 mg of total RNA was extracted from the treated plants and separated on 1% agarose-formaldehyde gels, transferred to Hybond-Nþ membranes, and hybridized as described previously [4]. ACD6 (AT4G14400) and GSTF14 (AT1G49860) probe primer pairs were as follows: F (ACD6), 5’-TCTCCCTGGTGAAGATGTCG-3’ and R (ACD6), 5’-TTACCGATGCAACAAGAGCC-3’; F (GSTF14), 5’-AGGCGAGTCTCC TTACTTGG-3’ and R (GSTF14), 5’-TTATAGGCAAACGACGCTGC-3’; F(ACO3), 5’-ACGAGTCA ATCACCAAGGGT-3’ and R (ACO3), 5’-GAAGTCCT TACGGTCAACGC-3’. Bisulfite sequencing Total DNA was extracted using cetyl trimethyl ammonium bromide (CTAB) buffer as previously described [23] and purified using a DNA purification kit (Promega). The purified DNA was used for bisulfite treatment using the EpiTect bisulfite kit (Qiagen, http://www.qiagen.com/default.aspx), according to the manufacturer’s instructions. The purified bisulfite-treated DNA was amplified by ACD6 (AT4G14400) and GSTF14 (AT1G49860) promoter-specific primer pairs as follows: F (ACD6), 5′- AAGTTTATTGATGAAAGGAG-3′ and R (ACD6), 5′-CTTACTT (G/A) TCTTCATCAA-3′; F (GSTF14), 5′-TTTGAAAGTTGGTGTATTAAA-3′ and R (GSTF14), 5′-CCCATACCTATC ATATTTCAT-3′; F (ACO3), 5′- GTAATATTAGTAAAGATGTGT-3′ and R (ACO3), 5′- CACTAC TTTC ATTATACTCTTT-3′. PCR cycles include 95℃ 30s，55℃ 30s， 50℃ 30s， 62℃ 2min，repeat 40 cycles. The cytosine methylation analysis was performed as described previously [35]. Abbreviations SA: salicylic acid; ABA: abscisic acid; JA: jasmonic acid; ET: ethylene;ACD6: accelerated cell death6; ACO3: aconitate hydratase 3; GST: glutathione S-transferase; DCL3: Dicer-like 3; RdDM: RNA-directed DNA methylation; siRNAs:small interfering RNAs; AGO4: Argonaute protein 4; ROS1: Repressor of silencing 1; Pol IV: RNA polymerase IV; RDR2: RNA-dependent RNA polymerase; DRM1/2: DNA methyltransferases 1/2; RT-qPCR: Reverse transcription-quantitative PCR; Col-0: Columbia; RT-sqPCR: Reverse transcription- semiquantitative PCR; rdd : ros1 dml2 dml3 ; Declarations Ethics approval and consent to participate Not applicable Consent for publication Not applicable Availability of data and material All data generated or analysed during this study are included in this published article。 Competing interests The authors declare no conflict 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. Acknowledgements We thank Prof. Chengguo Duan, Shanghai Center for Plant Stress Biology, Shanghai Institute of Biological Sciences, Chinese Academy of Sciences (CAS), for providing the ros1 and ros1dml2dml3 mutant seeds and Prof. Xiaofeng Cao, Institute of Genetics and Developmental Biology, CAS, for ago4–1 , met1 , drm1/2 and cmt3 mutant seeds. This work was supported by the Key Laboratory of Jilin Province for Plant Resources Science and Green Production, China. References Baulcombe D. RNA silencing in plants. Nature. 2004; 431:356-363. Ascencio-Ibáñez JT, Sozzani R, Lee TJ, Chu TM, Wolfinger RD, Cella R, et al. Global analysis of Arabidopsis gene expression uncovers a complex array of changes impacting pathogen response and cell cycle during geminivirus infection. Plant Physiol. 2008;148(1):436-454. Raja P, Sanville BC, Buchmann RC, Bisaro DM. Viral genome methylation as an epigenetic defense against geminiviruses. J. Virol. 2008; 82(18):8997-9007. 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Supplementary Files Supplementaldata2GSTF14.jpg Supplementaldata3ACO3.jpg Supplementaldata4thebisulfiteconversioncontrol.doc Supplementaldata1ACD6.jpg Cite Share Download PDF Status: Under Review Version 1 posted Reviewer # 4 agreed at journal 20 May, 2021 Reviewer # 3 agreed at journal 19 May, 2021 Review # 2 received at journal 15 Dec, 2020 Review # 1 received at journal 15 Dec, 2020 Reviewer # 2 agreed at journal 14 Dec, 2020 Reviewer # 1 agreed at journal 13 Dec, 2020 Editor assigned by journal 10 Dec, 2020 Reviewers invited by journal 10 Dec, 2020 Submission checks completed at journal 10 Dec, 2020 Editor invited by journal 10 Dec, 2020 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-128268","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research article","associatedPublications":[],"authors":[{"id":6411178,"identity":"7ab46878-7691-4d61-b3a4-17080350ffb2","order_by":0,"name":"Liping Yang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABEklEQVRIie2RMUsDMRTHXwmcS2zWHAr2IzwIRIUDP4jLg8JNp/QDFDwo2NHV0e/gF7jyQJfKrTd0iIs4dDg3h4KmJwUH724Vmt+SEP6/PP4JQCDwX3EIEkB9OMJEKCHY9SrUKGDQTdKDeB6l2K/8LDZ2NSss5Uh3pfH5hd9psjo+VQ9jJBRHhiUgTJPLVmV5nZ4Rvsnze7fwXSJj+bBw8JRe5W1KkVl/OUusFjO/kWPLQ8JBzu1Kud4pHGlCffM4k6g7lSozrlHK262CAkWPEldrC9suWEnhx5HQ7B+ZOroMy8zU9WZ1geVy8Pq5+RLqjtnV06RVGRUQ+V8oADT9Oqa/0w0nOYi6UVTREQsEAoG95huYKmIfA6C2jgAAAABJRU5ErkJggg==","orcid":"","institution":"Institute of Life Sciences","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Liping","middleName":"","lastName":"Yang","suffix":""},{"id":6411179,"identity":"8cc7ddde-c468-4c90-bc5b-4df4e8d4a7ce","order_by":1,"name":"Chenjing Lang","email":"","orcid":"","institution":"life science","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Chenjing","middleName":"","lastName":"Lang","suffix":""},{"id":6411180,"identity":"4cc174f0-8882-4305-a5e0-d288a4711086","order_by":2,"name":"Yanju Wu","email":"","orcid":"","institution":"Institute of Life Sciences","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Yanju","middleName":"","lastName":"Wu","suffix":""},{"id":6411181,"identity":"1ceaad49-a572-4748-b6bc-4a6ad93c382e","order_by":3,"name":"Dawei Meng","email":"","orcid":"","institution":"life csience","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Dawei","middleName":"","lastName":"Meng","suffix":""},{"id":6411182,"identity":"8e8a0424-c919-4dfd-8cdc-cc322ec32f93","order_by":4,"name":"Tianbo Yang","email":"","orcid":"","institution":"life science","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Tianbo","middleName":"","lastName":"Yang","suffix":""},{"id":6411183,"identity":"8349db83-f5e3-45f7-9264-54ea77629a29","order_by":5,"name":"Taicheng Jin","email":"","orcid":"","institution":"Life science","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Taicheng","middleName":"","lastName":"Jin","suffix":""},{"id":6411184,"identity":"de6d6044-a689-42a6-9825-47a1a70fc1df","order_by":6,"name":"Xiaofu Zhou","email":"","orcid":"","institution":"Institute of Life Sciences","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Xiaofu","middleName":"","lastName":"Zhou","suffix":""}],"badges":[],"createdAt":"2020-12-14 11:51:05","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-128268/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-128268/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":4315934,"identity":"d1950e6b-863f-47ba-9926-4ae5a45449a0","added_by":"auto","created_at":"2020-12-16 20:49:41","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":318493,"visible":true,"origin":"","legend":"Detection and analyses of the expression of defense genes in Arabidopsis plants treated with drought stress\n(A) The untreated Arabidopsis Col-0 plants. (B, C) The leaves of Arabidopsis plants treated with drought stress turned slightly yellow and shrunk during days 5-7. (D) Anthocyanin accumulation in the leaves of Arabidopsis plants treated with drought stress clearly increased, and the leaves turned severely yellow and withered on day 14. (E) Transcript levels of related genes in Arabidopsis plants treated with drought stress were analyzed by sqPCR; untreated Col-0 plants served as controls. (F) Transcript levels of related genes in Arabidopsis plants treated with drought stress were analyzed by qPCR. The statistical analysis was performed; asterisks indicate statistically significant differences compared with control plants (P \u003c 0.05). (G) Transcript levels of defense genes in Arabidopsis plants treated with drought stress were analyzed by sqPCR. ","description":"","filename":"Fig01.PNG","url":"https://assets-eu.researchsquare.com/files/rs-128268/v1/4d360a91dddae95a18ef1662.PNG"},{"id":4315935,"identity":"4e830e25-86d5-4dd4-9a9b-272268d08248","added_by":"auto","created_at":"2020-12-16 20:49:41","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":52005,"visible":true,"origin":"","legend":"Detection and analyses of the expression of defense genes and stress resistance genes in Arabidopsis \n(A, B) The defense genes and ACO3 transcript levels in Arabidopsis plants treated with cold and salt stress were analyzed by sqPCR; untreated Col-0 plants served as controls. (C, D) ACD6, GSTF14, and ACO3 transcript levels in Arabidopsis plants treated with cold and salt stress were analyzed by qPCR; untreated Col-0 plants served as controls. The statistical analysis was performed; asterisks indicate statistically significant differences compared with control plants (P \u003c 0.05).","description":"","filename":"Fig02.PNG","url":"https://assets-eu.researchsquare.com/files/rs-128268/v1/97b7ac531468cff60452f7a6.PNG"},{"id":4315936,"identity":"00c54388-3cb2-44bf-82c9-aa1060d8b8d1","added_by":"auto","created_at":"2020-12-16 20:49:41","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":26511,"visible":true,"origin":"","legend":"Analyses of DNA methylation of the promoters in plants treated with different stresses\n(A) Percentage of DNA methylation in the repeat regions of the ACD6 promoter in plants treated with different stresses and untreated Col-0 plants. (B) Percentage of DNA methylation in the repeat regions of the ACO3 promoter in plants treated with different stresses and untreated Col-0 plants. (C) Percentage of DNA methylation in the repeat regions of the GSTF14 promoter in plants treated with different stresses and untreated Col-0 plants. Fifteen individual clones of each genotype were used for sequencing, and the original data are shown in supplement Data S1. The statistical analysis was performed using OriginPro 8 (http://www.originlab.com). Values are means ± SEM, and asterisks indicate statistically significant differences compared with control plants (one-way analysis of variance, P \u003c 0.05). ","description":"","filename":"Fig03.PNG","url":"https://assets-eu.researchsquare.com/files/rs-128268/v1/8193fc9d9c003423908bf1c7.PNG"},{"id":4315937,"identity":"91314f80-04f2-4248-a3e5-71bc89dbbf13","added_by":"auto","created_at":"2020-12-16 20:49:41","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":74001,"visible":true,"origin":"","legend":"Analyses of DNA methylation and the expression levels of genes (A) Analyses of the expression levels of ACD6 and GSTF14 in the mutants ago4, met1, drm1/2 and cmt3 by northern blotting; wild-type Col-0 ecotype served as background controls for the mutant genotypes. (B) Analyses of the expression levels of ACD6, ACO3, and GSTF14 by RT-qPCR in DNA methylation mutant plants ago4, with wild-type as background control for the mutant genotypes. (C) The related genes were detected in the Col-0, ros1 and rdd mutants by RT-qPCR. (D) The related genes were detected in the untreated Col-0, the Col-0 treated with cold stress, ros1, and ros1 dml2 dml3 (rdd) mutant plants treated with cold stress by RT-qPCR. (E) Analyses of DNA methylation in the repeat regions of the ACD6 promoter in Col-0, and Col-0 plants and ros1 mutants treated with cold stress. (F) Analyses of DNA methylation in the repeat regions of the ACO3 promoter in Col-0, the Col-0 plants, and ros1 mutants treated with cold stress. The statistical analysis was performed using OriginPro 8 (http://www.originlab.com); asterisks indicate statistically significant differences compared with control plants (P \u003c 0.05). ","description":"","filename":"Fig04.png","url":"https://assets-eu.researchsquare.com/files/rs-128268/v1/7ba30b70d66b27b7d99c3623.png"},{"id":13634936,"identity":"b41bdc08-4d3b-4587-b5c3-e4993400031a","added_by":"auto","created_at":"2021-09-17 08:34:23","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":771900,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-128268/v1/0c3a608f-b32b-41f4-8878-4a42f66c49f9.pdf"},{"id":4315938,"identity":"271a96f3-16cf-45e9-8f45-1690c3f3f4eb","added_by":"auto","created_at":"2020-12-16 20:49:42","extension":"jpg","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":3431156,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementaldata2GSTF14.jpg","url":"https://assets-eu.researchsquare.com/files/rs-128268/v1/bf8ffe421c0019ceffa9030a.jpg"},{"id":4315939,"identity":"a20bbdc9-f492-4e8b-970c-a2f10ac59dc7","added_by":"auto","created_at":"2020-12-16 20:49:42","extension":"jpg","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":2477195,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementaldata3ACO3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-128268/v1/714d892c75455f9d7f916415.jpg"},{"id":4315940,"identity":"73c77c4c-f8a4-437c-b778-ab9bb74e1798","added_by":"auto","created_at":"2020-12-16 20:49:42","extension":"doc","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":24064,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementaldata4thebisulfiteconversioncontrol.doc","url":"https://assets-eu.researchsquare.com/files/rs-128268/v1/1c83dbb0ec27a9b3bde93938.doc"},{"id":4315941,"identity":"65ef488d-d211-46c5-96a7-dfbe374d3494","added_by":"auto","created_at":"2020-12-16 20:49:42","extension":"jpg","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":3019827,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementaldata1ACD6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-128268/v1/333e7a6020c8208bcde4e852.jpg"}],"financialInterests":"","formattedTitle":"\u003cp\u003eMediation of The Salicylic Acid Pathway by ROS1 in Response to Abiotic Stresses\u003c/p\u003e","fulltext":[{"header":"Background","content":"\u003cp\u003eDNA methylation is one of the most common forms of DNA covalent modification 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 [2-4]. 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].\u003c/p\u003e\n\u003cp\u003eAbiotic 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 [13-15]. When plants are under cold stress, ABA can regulate the expression of cold-resistant genes in plants in response to stress [16-18]. 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 profiles of the MYB, b-ZIP, and AP2/DREB transcription factor families significantly 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].\u003c/p\u003e\n\u003cp\u003eSalicylic 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, \u003cem\u003eacd6-1\u003c/em\u003e, can increase the expression of the genes \u003cem\u003eACD6-1\u003c/em\u003e, \u003cem\u003eEDS1\u003c/em\u003e, \u003cem\u003ePAD4\u003c/em\u003e, and \u003cem\u003eNPR1\u003c/em\u003e and induce an increase in SA accumulation [24-29]. 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.\u003c/p\u003e\n\u003cp\u003eIn this study, we determined the molecular mechanisms underlying functioning of the upstream regulator ACD6 of the SA pathway, the stress resistance gene \u003cem\u003eGSTF14\u003c/em\u003e 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 (\u003cem\u003eACD6\u003c/em\u003e, \u003cem\u003eNPR1\u003c/em\u003e, and \u003cem\u003ePR5\u003c/em\u003e) in the SA pathway, the ABA pathway-related gene \u003cem\u003eACO3\u003c/em\u003e, and the stress resistance gene GSTF14 significantly increased after treatment with drought, cold, and salt stresses. Sequencing results confirmed that abiotic stresses induced the demethylation of the repeats in the promoters of \u003cem\u003eACD6\u003c/em\u003e, \u003cem\u003eACO3\u003c/em\u003e, and \u003cem\u003eGSTF14 \u003c/em\u003eand 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.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003e\u003cem\u003eActivation of the expression of the upstream regulator ACD6 of the SA pathway by drought stress\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eOur 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 \u003cem\u003eACD6 \u003c/em\u003ein the SA pathway and stress resistance genes \u003cem\u003eGSTF14\u003c/em\u003e and \u003cem\u003eACO3\u003c/em\u003e, the wild-type Columbia (Col-0) line of \u003cem\u003eArabidopsis\u003c/em\u003e \u003cem\u003ethaliana\u003c/em\u003e was selected for drought-stress treatment, cold-stress treatment, and salt-stress treatment. There were no significant phenotypic changes in plants treated with cold stress (4 \u0026deg;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).\u003c/p\u003e\n\u003cp\u003eWe 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\u0026ndash;semiquantitative polymerase chain reaction (RT-sqPCR) assay showed higher expression levels of the regulator ACD6 of the SA pathway, the stress resistance gene \u003cem\u003eGSTF14\u003c/em\u003e, and \u003cem\u003eACO3\u003c/em\u003e in the plants after drought-stress treatment than in untreated Col-0 plants; \u003cem\u003eGAPDH\u003c/em\u003e 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 confirmed that \u003cem\u003eACD6\u003c/em\u003e, \u003cem\u003eGSTF14\u003c/em\u003e, and \u003cem\u003eACO3\u003c/em\u003e were significantly upregulated after drought-stress treatment, and the upregulation of GSTF14 expression was more significant (Figure 1F). Since ACD6 is an upstream regulator of the SA pathway, the increase in \u003cem\u003eACD6\u003c/em\u003e expression could upregulate the expression of the defense genes \u003cem\u003eNPR1\u003c/em\u003e and \u003cem\u003ePR5 \u003c/em\u003e(Figure 1G).\u003c/p\u003e\n\u003cp\u003eFigure 1 Detection and analyses of the expression of defense genes in \u003cem\u003eArabidopsis\u003c/em\u003e plants treated with drought stress\u003c/p\u003e\n\u003cp\u003e(A) The untreated \u003cem\u003eArabidopsis\u003c/em\u003e Col-0 plants. (B, C) The leaves of \u003cem\u003eArabidopsis\u003c/em\u003e plants treated with drought stress turned slightly yellow and shrunk during days 5-7. (D) Anthocyanin accumulation in the leaves of \u003cem\u003eArabidopsis\u003c/em\u003e plants treated with drought stress clearly increased, and the leaves turned severely yellow and withered on day 14. (E) Transcript levels of related genes in \u003cem\u003eArabidopsis\u003c/em\u003e plants treated with drought stress were analyzed by sqPCR; untreated Col-0 plants served as controls. (F) Transcript levels of related genes in \u003cem\u003eArabidopsis\u003c/em\u003e plants treated with drought stress were analyzed by qPCR. The statistical analysis was performed; asterisks indicate statistically significant differences compared with control plants (P \u0026lt; 0.05). \u0026nbsp;(G) Transcript levels of defense genes in \u003cem\u003eArabidopsis\u003c/em\u003e plants treated with drought stress were analyzed by sqPCR.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eInduction of SA pathway-related defense genes by cold and salt stress\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo 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 significantly higher expression levels of defense genes \u003cem\u003eACD6\u003c/em\u003e, \u003cem\u003eNPR1\u003c/em\u003e, and \u003cem\u003ePR5\u003c/em\u003e and ABA pathway-related gene \u003cem\u003eACO3\u003c/em\u003e (Figure 2A, B). Consistent with the RT-sqPCR results, the RT-qPCR results further confirmed that cold stress and salt stress activated the expression of \u003cem\u003eACD6\u003c/em\u003e, which was significantly increased after 24 h of cold-stress treatment (Figure 2C, D). We also compared the expression of the stress resistance gene \u003cem\u003eGSTF14\u003c/em\u003e. The results showed that the upregulation of \u003cem\u003eGSTF14\u003c/em\u003e was the most significant in the plants treated with cold stress for 24 h (Figure 2C).\u003c/p\u003e\n\u003cp\u003eFigure 2 Detection and analyses of the expression of defense genes and stress resistance genes in \u003cem\u003eArabidopsis\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e(A, B) The defense genes and \u003cem\u003eACO3\u003c/em\u003e transcript levels in \u003cem\u003eArabidopsis\u003c/em\u003e plants treated with cold and salt stress were analyzed by sqPCR; untreated Col-0 plants served as controls. (C, D) \u003cem\u003eACD6\u003c/em\u003e, \u003cem\u003eGSTF14, \u003c/em\u003eand\u003cem\u003e ACO3 \u003c/em\u003etranscript levels in \u003cem\u003eArabidopsis\u003c/em\u003e plants treated with cold and salt stress were analyzed by qPCR; untreated Col-0 plants served as controls. The statistical analysis was performed; asterisks indicate statistically significant differences compared with control plants (P \u0026lt; 0.05).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eDirect correlation between the increased expression of defense and stress resistance genes and the reduction in promoter DNA methylation\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo 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).\u003c/p\u003e\n\u003cp\u003eSimilarly, 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 significantly, while the CNG and CHH methylation of the repeats in the ACO3 promoter decreased significantly, 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 significantly, 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 significantly, while the CNG and CHH methylation of the repeats in the ACO3 promoter decreased significantly, from 65.89% to 21.43% and from 42.22% to 9.19%, respectively (Figure 3B).\u003c/p\u003e\n\u003cp\u003eDNA 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 \u003cem\u003eACD6\u003c/em\u003e and \u003cem\u003eGSTF14\u003c/em\u003e promoters was different from that of the \u003cem\u003eACO3 \u003c/em\u003epromoter.\u003c/p\u003e\n\u003cp\u003eFigure 3 Analyses of DNA methylation of the promoters in plants treated with different stresses\u003c/p\u003e\n\u003cp\u003e(A) Percentage of DNA methylation in the repeat regions of the \u003cem\u003eACD6\u003c/em\u003e promoter in plants treated with different stresses and untreated Col-0 plants. (B) Percentage of DNA methylation in the repeat regions of the \u003cem\u003eACO3\u003c/em\u003e promoter in plants treated with different stresses and untreated Col-0 plants. (C) Percentage of DNA methylation in the repeat regions of the \u003cem\u003eGSTF14\u003c/em\u003e promoter in plants treated with different stresses and untreated Col-0 plants. Fifteen individual clones of each genotype were used for sequencing, and the original data are shown in supplement Data S1. The statistical analysis was performed using OriginPro 8 (http://www.originlab.com). Values are means \u0026plusmn; SEM, and asterisks indicate statistically significant differences compared with control plants (one-way analysis of variance, P \u0026lt; 0.05).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eRole of ROS1 in the regulation of the SA pathway in response to abiotic stresses\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo 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 \u003cem\u003eACD6\u003c/em\u003e and \u003cem\u003eGSTF14\u003c/em\u003e clearly increased in the mutant \u003cem\u003eago4\u003c/em\u003e and DNA methyltransferase mutants \u003cem\u003emet1\u003c/em\u003e, \u003cem\u003edrm1/2\u003c/em\u003e and\u003cem\u003e cmt3\u003c/em\u003e with ecotypes Col-0 as controls (Figure 4A). RT-qPCR results further confirmed that \u003cem\u003eACD6\u003c/em\u003e, \u003cem\u003eGSTF14\u003c/em\u003e, and \u003cem\u003eACO3\u003c/em\u003e were upregulated in the \u003cem\u003eago4\u003c/em\u003e mutant (Figure 4B), indicating that RdDM has an important role in maintaining the low transcription levels of \u003cem\u003eACD6\u003c/em\u003e, \u003cem\u003eGSTF14\u003c/em\u003e, and \u003cem\u003eACO3\u003c/em\u003e 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 \u003cem\u003eros1\u003c/em\u003e and \u003cem\u003erdd \u003c/em\u003emutants, when the Col-0 plants were used as the control (Figure 4C).\u003c/p\u003e\n\u003cp\u003eTo determine whether ROS1 plays a role in the responses of these genes to abiotic stress, we performed cold-stress treatment on loss-of-function \u003cem\u003eros1\u003c/em\u003e mutants and compared the expression of the \u003cem\u003eACD6\u003c/em\u003e gene between the cold stress-treated \u003cem\u003eros1\u003c/em\u003e 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 \u003cem\u003eACD6\u003c/em\u003e in the cold stress-treated Col-0 plants significantly increased. However, the increase in \u003cem\u003eACD6\u003c/em\u003e expression in the cold stress-treated \u003cem\u003eros1\u003c/em\u003e mutants and loss-of-function\u003cem\u003e ros1dml2dml3 \u003c/em\u003e(\u003cem\u003erdd\u003c/em\u003e) mutants was significantly 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 finding was confirmed by the expression levels of \u003cem\u003eGSTF14\u003c/em\u003e and \u003cem\u003eACO3\u003c/em\u003e. When the cold stress-treated Col-0 plants were used as the control, the increase in \u003cem\u003eGSTF14\u003c/em\u003e and \u003cem\u003eACO3 \u003c/em\u003eexpression was inhibited in the cold stress-treated \u003cem\u003eros1\u003c/em\u003e mutants (Figure 4D).\u003c/p\u003e\n\u003cp\u003eSequencing analysis confirmed that the DNA methylation levels of the repeats in the \u003cem\u003eACD6\u003c/em\u003e promoter in cold stress-treated Col-0 plants were significantly reduced, including the CG, CNG and CHH sites, while the decrease in DNA methylation levels of the repeats in the \u003cem\u003eACD6\u003c/em\u003e promoter in cold stress-treated \u003cem\u003eros1\u003c/em\u003e mutants was obviously inhibited (Figure 4E). The results further demonstrated that the DNA methylation at CNG and CHH sites in the \u003cem\u003eACO3\u003c/em\u003e promoter in cold stress-treated Col-0 plants was significantly decreased, while the decrease in DNA methylation at CNG and CHH sites in the\u003cem\u003e ACO3\u003c/em\u003e promoter in cold stress-treated \u003cem\u003eros1\u003c/em\u003e mutants was obviously inhibited (Figure 4F).\u003c/p\u003e\n\u003cp\u003eOur results revealed that the activation of the expression of the regulator ACD6 in the SA defense pathway, the stress resistance gene \u003cem\u003eGSTF14\u003c/em\u003e and ABA pathway-related gene \u003cem\u003eACO3\u003c/em\u003e by abiotic stresses was related to ROS1-mediated DNA demethylation.\u003c/p\u003e\n\u003cp\u003eFigure 4 Analyses of DNA methylation and the expression levels of genes\u003c/p\u003e\n\u003cp\u003e(A) Analyses of the expression levels of \u003cem\u003eACD6\u003c/em\u003e and \u003cem\u003eGSTF14\u003c/em\u003e in the mutants \u003cem\u003eago4\u003c/em\u003e, \u003cem\u003emet1\u003c/em\u003e,\u003cem\u003e drm1/2\u003c/em\u003e and\u003cem\u003e cmt3 \u003c/em\u003eby northern blotting; wild-type Col-0 ecotype served as background controls for the mutant genotypes. (B) Analyses of the expression levels of \u003cem\u003eACD6\u003c/em\u003e, \u003cem\u003eACO3\u003c/em\u003e, and \u003cem\u003eGSTF14\u003c/em\u003e by RT-qPCR in DNA methylation mutant plants\u003cem\u003e ago4\u003c/em\u003e, with wild-type as background control for the mutant genotypes. (C) The related genes were detected in the Col-0, \u003cem\u003eros1\u003c/em\u003e and\u003cem\u003e rdd\u003c/em\u003e mutants by RT-qPCR. (D) The related genes were detected in the untreated Col-0, the Col-0 treated with cold stress, \u003cem\u003eros1\u003c/em\u003e, and \u003cem\u003eros1 dml2 dml3\u003c/em\u003e (\u003cem\u003erdd\u003c/em\u003e) mutant plants treated with cold stress by RT-qPCR. (E) Analyses of DNA methylation in the repeat regions of the \u003cem\u003eACD6\u003c/em\u003e promoter in Col-0, and Col-0 plants and\u003cem\u003e ros1\u003c/em\u003e mutants treated with cold stress. (F) Analyses of DNA methylation in the repeat regions of the \u003cem\u003eACO3\u003c/em\u003e promoter in Col-0, the Col-0 plants, and\u003cem\u003e ros1\u003c/em\u003e mutants treated with cold stress. The statistical analysis was performed using OriginPro 8 (http://www.originlab.com); asterisks indicate statistically significant differences compared with control plants (P \u0026lt; 0.05).\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn 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 field, 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.\u003c/p\u003e\n\u003cp\u003eThis 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 \u003cem\u003eACO3\u003c/em\u003e in \u003cem\u003eArabidopsis\u003c/em\u003e plants (Figure 1E, F). The gain-of-function mutant of ACD6, \u003cem\u003eacd6-1\u003c/em\u003e, can increase the expression of the genes \u003cem\u003eACD6-1\u003c/em\u003e, \u003cem\u003eEDS1\u003c/em\u003e, \u003cem\u003ePAD4\u003c/em\u003e, and \u003cem\u003eNPR1\u003c/em\u003e and induce an increase in SA accumulation [24-29]. Therefore, we hypothesized that the increase in \u003cem\u003eACD6\u003c/em\u003e expression would further activate the expression of defense genes \u003cem\u003eNPR1\u003c/em\u003e and \u003cem\u003ePR5\u003c/em\u003e (Figure 1G) in the SA pathway.\u003c/p\u003e\n\u003cp\u003eUnder 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 confirmed that the increase in the expression of \u003cem\u003eACD6\u003c/em\u003e, \u003cem\u003eGSTF14\u003c/em\u003e, and\u003cem\u003e ACO3 \u003c/em\u003ewas related to the reduction in DNA methylation levels of the promoters of these genes. The CG, CNG, and CHH methylation in the \u003cem\u003eACD6\u003c/em\u003e and \u003cem\u003eGSTF14 \u003c/em\u003epromoters decreased to varying degrees, and the CG methylation decreased significantly (Figure 3A, C). However, the CG methylation of the repeats in the\u003cem\u003e ACO3 \u003c/em\u003epromoter barely changed, but their CHG and CHH methylation significantly decreased (Figure 3B). Our results reveal that abiotic stresses (cold stress, drought, and salt stress) induced DNA demethylation of the \u003cem\u003eACD6\u003c/em\u003e, \u003cem\u003eACO3\u003c/em\u003e, and \u003cem\u003eGSTF14\u003c/em\u003e promoters and transcriptionally activated the expression of defense and stress resistance genes, thereby enhancing the adaptability of plants to abiotic stresses.\u003c/p\u003e\n\u003cp\u003eFurther studies revealed that the expression of \u003cem\u003eACD6\u003c/em\u003e and \u003cem\u003eGSTF14\u003c/em\u003e in the mutants \u003cem\u003eago4\u003c/em\u003e, \u003cem\u003edrm1/2\u003c/em\u003e, \u003cem\u003ecmt3 \u003c/em\u003eand \u003cem\u003emet1\u003c/em\u003e was higher than that in Col-0 (Figure 4A). RT-qPCR results confirmed that \u003cem\u003eACD6\u003c/em\u003e, \u003cem\u003eACO3\u003c/em\u003e, and \u003cem\u003eGSTF14\u003c/em\u003e in the mutant \u003cem\u003eago4\u003c/em\u003e were upregulated (Figure 4B), indicating that the RdDM pathway has an important role in maintaining the low transcription levels of \u003cem\u003eACD6\u003c/em\u003e, \u003cem\u003eGSTF14\u003c/em\u003e, and \u003cem\u003eACO3\u003c/em\u003e in wild-type plants. DNA methylation can be removed by DNA glycosylases/lyases in \u003cem\u003eArabidopsis\u003c/em\u003e, 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 \u003cem\u003eros1\u003c/em\u003e and \u003cem\u003erdd \u003c/em\u003emutants. The results showed that the expression levels of these genes were lower in the \u003cem\u003eros1\u003c/em\u003e and \u003cem\u003erdd \u003c/em\u003emutants, when the Col-0 plants were used as the control (Figure 4C).\u003c/p\u003e\n\u003cp\u003eROS1-mediated DNA demethylation can act on the three DNA methylation sites, CG, CHG, and CHH [33]. DNA methylation sequencing of \u003cem\u003eros1\u003c/em\u003e 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 \u003cem\u003eACD6\u003c/em\u003e and \u003cem\u003eGSTF14\u003c/em\u003e was significant 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 \u003cem\u003eACD6\u003c/em\u003e and \u003cem\u003eGSTF14\u003c/em\u003e in \u003cem\u003eros1\u003c/em\u003e mutants treated with cold stress for 24 h was significantly inhibited (Figure 4D). Furthermore, after 24 h of the cold-stress treatment of Col-0, DNA methylation levels in the repeats in the \u003cem\u003eACD6\u003c/em\u003e and \u003cem\u003eACO3\u003c/em\u003e promoters were significantly reduced, while the decrease in DNA methylation levels in the repeats in the \u003cem\u003eACD6\u003c/em\u003e and \u003cem\u003eACO3\u003c/em\u003e promoters in cold stress-treated \u003cem\u003eros1\u003c/em\u003e mutants was obviously inhibited (Figure 4E, F).\u003c/p\u003e\n\u003cp\u003eThese results further confirm 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.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eOur 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 \u003cem\u003eACD6\u003c/em\u003e, \u003cem\u003eGSTF14\u003c/em\u003e, and \u003cem\u003eACO3\u003c/em\u003e in wild-type Col-0 plants. Further studies reveal that abiotic stresses induced DNA demethylation of the \u003cem\u003eACD6\u003c/em\u003e, \u003cem\u003eACO3\u003c/em\u003e, and \u003cem\u003eGSTF14\u003c/em\u003e promoters and transcriptionally activated the expression of defense and stress resistance genes. Moreover, ROS1-mediated DNA demethylation plays an important role in this process.\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003e\u003cstrong\u003e\u003cem\u003ePlant growth and abiotic stress treatments\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eArabidopsis thaliana ecotype Columbia (Col-0) and the mutant plants were used for this work. The \u003cem\u003eago4\u003c/em\u003e mutant seeds (original source) [36], \u003cem\u003eros1\u003c/em\u003eand \u003cem\u003eros1dml2 dml3 \u003c/em\u003emutant 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, \u003cem\u003emet1\u003c/em\u003e, \u003cem\u003edrm1/2\u003c/em\u003e and \u003cem\u003ecmt3 \u003c/em\u003emutant 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.\u003c/p\u003e\n\u003cp\u003eArabidopsis thaliana Col-0 plants were treated with abiotic stresses, such as cold stress (4 \u0026deg;C, 24 h), salt stress (150 mM NaCl, 3 days), and drought stress (not watered, 7 days). The significant experimental details were as follows.\u003c/p\u003e\n\u003ctable border=\"1\"\u003e\n\u003ctbody\u003e\n\u003ctr\u003e\n\u003ctd width=\"163\"\u003e\n\u003cp\u003eCold stress\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"189\"\u003e\n\u003cp\u003eSalt stress\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"139\"\u003e\n\u003cp\u003eDrought stress\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"163\"\u003e\n\u003cp\u003e4\u0026deg;C\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"189\"\u003e\n\u003cp\u003e150 mM NaCl\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"139\"\u003e\n\u003cp\u003enot watered\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"163\"\u003e\n\u003cp\u003e24 hours\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"189\"\u003e\n\u003cp\u003e3 days\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"139\"\u003e\n\u003cp\u003e7 days\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eRT-\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u003cem\u003esqPCR\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e,\u003cem\u003e RT-qPCR and RNA gel blot analysis\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTotal RNA was isolated using TRIzol reagent (Invitrogen) according to the manufacturer's protocols. The total RNA was subsequently used for RT-sqPCR, RT-qPCR, and RNA gel blotting analysis. For RT-sqPCR, total RNA was extracted from the treated plants and subsequently used for reverse transcription and semiquantitative PCR. For RT-qPCR, the complementary DNA synthesis was performed using the Reverse Transcription kit (Takara). Quantitative RT-PCR was performed using SYBR green mix (Qiagen). Each experiment consisted of three biological replicates and was repeated twice. For the high molecular weight RNA gel blot analysis, 10 mg of total RNA was extracted from the treated plants and separated on 1% agarose-formaldehyde gels, transferred to Hybond-N\u0026thorn; membranes, and hybridized as described previously [4]. ACD6 (AT4G14400) and GSTF14 (AT1G49860) probe primer pairs were as follows: F (ACD6), 5\u0026rsquo;-TCTCCCTGGTGAAGATGTCG-3\u0026rsquo; and R (ACD6), 5\u0026rsquo;-TTACCGATGCAACAAGAGCC-3\u0026rsquo;; F (GSTF14), 5\u0026rsquo;-AGGCGAGTCTCC TTACTTGG-3\u0026rsquo; and R (GSTF14), 5\u0026rsquo;-TTATAGGCAAACGACGCTGC-3\u0026rsquo;; F(ACO3), 5\u0026rsquo;-ACGAGTCA ATCACCAAGGGT-3\u0026rsquo; and R (ACO3), 5\u0026rsquo;-GAAGTCCT TACGGTCAACGC-3\u0026rsquo;.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eBisulfite sequencing\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTotal DNA was extracted using cetyl trimethyl ammonium bromide (CTAB) buffer as previously described [23] and purified using a DNA purification kit (Promega). The purified DNA was used for bisulfite treatment using the EpiTect bisulfite kit (Qiagen, http://www.qiagen.com/default.aspx), according to the manufacturer\u0026rsquo;s instructions. The purified bisulfite-treated DNA was amplified by ACD6 (AT4G14400) and GSTF14 (AT1G49860) promoter-specific primer pairs as follows: F (ACD6), 5\u0026prime;- AAGTTTATTGATGAAAGGAG-3\u0026prime; and R (ACD6), 5\u0026prime;-CTTACTT (G/A) TCTTCATCAA-3\u0026prime;; F (GSTF14), 5\u0026prime;-TTTGAAAGTTGGTGTATTAAA-3\u0026prime; and R (GSTF14), 5\u0026prime;-CCCATACCTATC ATATTTCAT-3\u0026prime;; F (ACO3), 5\u0026prime;- GTAATATTAGTAAAGATGTGT-3\u0026prime; and R (ACO3), 5\u0026prime;- CACTAC TTTC ATTATACTCTTT-3\u0026prime;. PCR cycles include 95℃ 30s，55℃ 30s， 50℃ 30s， 62℃ 2min，repeat 40 cycles. The cytosine methylation analysis was performed as described previously [35].\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eSA: salicylic acid; ABA: abscisic acid; JA: jasmonic acid; ET: ethylene;ACD6: accelerated cell death6; ACO3: aconitate hydratase 3; GST: glutathione S-transferase; DCL3: Dicer-like 3; RdDM: RNA-directed DNA methylation; siRNAs:small interfering RNAs; AGO4: Argonaute protein 4; ROS1: Repressor of silencing 1; Pol IV: RNA polymerase IV; RDR2: RNA-dependent RNA polymerase; DRM1/2: DNA methyltransferases 1/2; RT-qPCR: Reverse transcription-quantitative PCR; Col-0: Columbia; RT-sqPCR: Reverse transcription- semiquantitative PCR; \u003cem\u003erdd\u003c/em\u003e: \u003cem\u003eros1 \u003c/em\u003e\u003cem\u003edml2 dml3\u003c/em\u003e;\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and material\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data generated or analysed during this study are included in this published article。\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no conflict of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis 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.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors' contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eLiping 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.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank Prof. Chengguo Duan, Shanghai Center for Plant Stress Biology, Shanghai Institute of Biological Sciences, Chinese Academy of Sciences (CAS), for providing the \u003cem\u003eros1\u003c/em\u003e and \u003cem\u003eros1dml2dml3 \u003c/em\u003emutant seeds and Prof. Xiaofeng Cao, Institute of Genetics and Developmental Biology, CAS, for \u003cem\u003eago4\u0026ndash;1\u003c/em\u003e, \u003cem\u003emet1\u003c/em\u003e, \u003cem\u003edrm1/2\u003c/em\u003e and \u003cem\u003ecmt3\u003c/em\u003e mutant seeds. This work was supported by the Key Laboratory of Jilin Province for Plant Resources Science and Green Production, China.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eBaulcombe D. RNA silencing in plants. 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Cell Res. 2017;27:226\u0026ndash;240.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bmc-plant-biology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"pbio","sideBox":"Learn more about [BMC Plant Biology](http://bmcplantbiol.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/pbio/default.aspx","title":"BMC Plant Biology","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"DNA methylation, salicylic acid, ROS1-mediated DNA demethylation, abiotic stresses","lastPublishedDoi":"10.21203/rs.3.rs-128268/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-128268/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground:\u003c/strong\u003e 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. \u003c/p\u003e\u003cp\u003e\u003cstrong\u003eResults:\u003c/strong\u003e In this paper, we investigated the molecular mechanisms by which an upstream regulator (ACD6) in the SA pathway, an ABA pathway-related gene (\u003cem\u003eACO3)\u003c/em\u003e, and a stress resistance gene (\u003cem\u003eGSTF14\u003c/em\u003e) 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\u003cem\u003e ACD6\u003c/em\u003e, \u003cem\u003eACO3\u003c/em\u003e, and \u003cem\u003eGSTF14\u003c/em\u003e 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 \u003cem\u003eACD6 \u003c/em\u003eand \u003cem\u003eGSTF14\u003c/em\u003e when \u003cem\u003eArabidopsis\u003c/em\u003e plants were under cold stress.\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eConclusions:\u003c/strong\u003e Our results confirmed that ROS1 plays an important role in the process of defense genes in the SA pathway and stress resistance gene \u003cem\u003eGSTF14 \u003c/em\u003ein response to abiotic stresses.\u0026nbsp;\u003c/p\u003e","manuscriptTitle":"Mediation of The Salicylic Acid Pathway by ROS1 in Response to Abiotic Stresses","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2020-12-16 20:49:39","doi":"10.21203/rs.3.rs-128268/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewerAgreed","content":"","date":"2021-05-21T00:00:00+00:00","index":4,"fulltext":""},{"type":"reviewerAgreed","content":"","date":"2021-05-20T00:00:00+00:00","index":3,"fulltext":""},{"type":"editorInvitedReview","content":"","date":"2020-12-16T00:00:00+00:00","index":2,"fulltext":"Recommendation: Reviewer's comments unavailable pending editorial decision\n"},{"type":"editorInvitedReview","content":"","date":"2020-12-16T00:00:00+00:00","index":1,"fulltext":"Recommendation: Reviewer's comments unavailable pending editorial decision\n"},{"type":"reviewerAgreed","content":"","date":"2020-12-15T00:00:00+00:00","index":2,"fulltext":""},{"type":"reviewerAgreed","content":"","date":"2020-12-14T00:00:00+00:00","index":1,"fulltext":""},{"type":"editorAssigned","content":"","date":"2020-12-11T00:00:00+00:00","index":"","fulltext":""},{"type":"reviewersInvited","content":"","date":"2020-12-11T00:00:00+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2020-12-10T23:00:00+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2020-12-10T23:00:00+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-plant-biology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"pbio","sideBox":"Learn more about [BMC Plant Biology](http://bmcplantbiol.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/pbio/default.aspx","title":"BMC Plant Biology","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"18e606e3-cab8-485c-9083-2d2f14f20128","owner":[],"postedDate":"December 16th, 2020","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":1506018,"name":"Plant Molecular Biology and Genetics"},{"id":1506019,"name":"Plant Physiology and Morphology"}],"tags":[],"updatedAt":"2022-02-15T15:45:33+00:00","versionOfRecord":[],"versionCreatedAt":"2020-12-16 20:49:39","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-128268","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-128268","identity":"rs-128268","version":["v1"]},"buildId":"rHA-KDH7Qsr4HCuvH75dn","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}