Global warming-associated increases in temperature, particularly during the night, are detrimental to rice production and yield, as over-warm nighttime temperatures increase the rate of grain growth and decrease grain growth duration [26, 27]. The molecular mechanisms associated with the response of rice to HNT have been widely investigated in the past decade, and several of the genes, proteins, metabolites, and probable gene-regulatory networks involved in the rice HNT response have been explored [21–23]. However, the primary genetic elements and specific molecular mechanisms conferring rice heat tolerance remain unclear. Cytosine methylation in genomic DNA is an important epigenetic mechanism that may regulate the expression of coding and non-coding genetic elements at the transcriptional and post-transcriptional levels in response to adverse environmental conditions, without changing the DNA sequence [16, 18]. In Arabidopsis, cytosine methylation is involved in the regulation of gene expression and participates in the response to temperature stress [19]. In rice, it was previously shown that the DNA methylation status of the gene OsFIE1 was sensitive to temperature change and affected endosperm development [20]. Here, we demonstrated that HNT induced the demethylation of successive cytosines in the promoter regions of genes involved in ABA-related oxidation and ROS scavenging, thus contributing to rice heat tolerance.
In plants, cytosine methylation may occur in CG, CHG, or CHH contexts. CHH is the primary context, while CG is the primary context of mC [18, 28]. However, variations in methylation status in the response of rice to stress and during growth are most common in the CHH context [29, 30]. Consistent with this, we identified over 64.8% of all cytosines in the CHH context across the heat-tolerant and heat-sensitive coisogenic rice strains, and identified 48.8–51.0% of all mCs in the CG context. In contrast, most of the HNT-induced DMCs between the heat-tolerant and heat-sensitive coisogenic rice strains were identified in the CHH context. The results suggested that the methylation or demethylation of cytosine in the rice genome mostly occurs in the CG context, while the cytosine in CHH was the main context associated with HNT stress.
In the rice genome, most cytosines in the CHH context are methylated by O. sativa domains rearranged methyltransferases (OsDRMs), and the OsDRMs are negatively regulated by micro RNA (miR) 820, which is located in the CACTA transposon element (TE) primarily on chromosome 7. MiR820 transcription is regulated not by copy dosage but by the epigenetic state of each copy [31]. Our results showed that 18 successive cytosines in the downstream region of CACTA TE (LOC_Os07g09540) were consistently HNT-induced demethylated in the heat-tolerant rice strain (Fig. 4 and Table S4). This suggested that the DNA methylation also regulated the genome cytosine methylation by modifying the methylation status of transposons or miRNA, and demethylation of the successive cytosines downstream of the gene LOC_Os07g09540 caused fewer CHH methylation and contributes to rice heat tolerance.
During the initiative transcription of nearly all mRNAs, RNA polymerase II assembles with basal TFs, including TFIID and the mediator of RNA polymerase II, to form the transcription preinitiation complex (PIC) [32]. TFIID is a large, multi-subunit complex comprised of the TATA-binding protein (TBP) and TBP-associated factors (TAFs), which contain subunits capable of promoter DNA recognition. Mediator of RNA polymerase II acts as a checkpoint for gene activation and TFIID activity, and functions by relaying signals from TFs directly to RNA polymerase II, thereby facilitating the TF-dependent regulation of gene expression. Mediator of RNA polymerase II is also targeted by sequence-specific, DNA-binding TFs that work to control gene expression in response to developmental or environmental cues [33]. In the present study, we discovered 16 successive cytosines located 358–399 bp downstream from the transcription termination site (TTS) of the transcription initiation factor TFIID subunit 11 (LOC_Os01g48810) that were consistently HNT-induced demethylated in the heat-tolerant strain (Fig. 4 and Table S4). In addition, 18 successive cytosines located 2–60 bp downstream from the TTS of the mediator of RNA polymerase II transcription subunit 31 (LOC_Os07g07020) were also consistently HNT-induced demethylated in the heat-tolerant rice strain (Fig. 4 and Table S4). These results indicated that the mediator of RNA polymerase II regulates gene expression in response to developmental or environmental cues not only when targeted by sequence-specific DNA-binding TFs, but also due to the demethylation of the successive cytosines in the promoter region of TFIID and the mediator of RNA polymerase II itself [33].
Stress leads to high levels of ROS accumulation in plant cells, which may result in metabolic disorders, cellular damage, and premature senescence [34]. During cellular respiration, most ROS are generated by oxidative phosphorylation (OXPHOS) processes, which are performed by a set of membrane-associated enzymes and complexes, including complex I (NADH dehydrogenase ubiquinone oxidoreductase), complex II (succinate ubiquinone oxidoreductase), complex III (cytochrome bc1), complex IV (cytochrome c oxidase), and complex V (ATP synthase) [35]. Complex IV catalyzes water synthesis from hydrogen ions (H+) and oxygen (O−) to remove ROS, while complex V modulates ROS formation [35]. The inhibition of complex V leads to ROS accumulation during the OXPHOS process [36]. The activity levels of complex IV may be influenced by the DNA methylation of its components; for example, complex IV activity is inhibited by COX7A1 methylation [37]. In addition, the expression levels of the components of complex V are influenced by the methylation of mitochondrial DNA [38]. Here, 18 successive cytosines 209–260 bp upstream from the TSS of cytochrome C oxidase assembly protein COX15 (LOC_Os08g38720), one component of complex IV, were unchanged in methylation status in the heat-tolerant rice strain, but the corresponding cytosines in the heat-sensitive strain were consistently HNT-induced methylated. We also identified 19 successive cytosine located 538–616 bp upstream from the TSS of the plasma membrane H+-ATPase (LOC_Os06g08310) that were consistently HNT-induced demethylated in the heat-sensitive strain but unchanged in the heat-tolerant rice strain (Fig. 4 and Table S4). These results indicated that, in rice, OXPHOS processes and ROS production could be disrupted by cytosine methylation in the promoter region of COX15 in complex IV and of H+-ATPase in complex V. These methylation events are detrimental to rice heat tolerance.
ABA is an important messenger that acts as the signaling mediator during oxidation and stress adaption. ABA reduces ROS damage in two ways: by inhibiting ROS production during OXPHOS and by regulating the expression of genes related to ROS scavenging [39]. During OXPHOS, ABA binds to the receptor Mg-chelatase H subunit/putative ABA receptor (CHLH/ABAR) to induce TF PPR. PPR then regulates components of complex I and complex V post-transcription [40]. To influence the ROS scavenging pathway, ABA binds to the receptor pyrabactin resistance/pyrabactin resistance-Like (PYR/PYL) to inhibit downstream protein phosphatases type 2C (PP2C) [41]. PP2C inhibition indirectly activates the sucrose nonfermenting 1-related kinase2 (SnRK2), which then induces several downstream TFs, including MYB, cysteine 2/histidine 2 -type zinc finger protein (C2H2), and bZIP [41–44]. Previous studies have shown that, in response to environmental stress or during development, DNA methylation in rice and /or Arabidopsis regulates PPR expression during OXPHOS, as well as MYB, C2H2, and bZIP, in the ROS scavenging pathway [45–48]. In the present study, we detected that 21 successive cytosines located 1495–1577 bp upstream from the TSS of the TF PPR repeat domain containing protein (LOC_Os07g28900) were consistently HNT-induced demethylated in the heat-tolerant rice strain, whereas the corresponding cytosines in the heat-sensitive strain were unchanged or methylated. Eighteen successive type III cytosines 1748–1799 bp downstream from the TTS of MYB family TF (LOC_Os08g33150), and 18 successive cytosines 1669–1737 bp downstream from the TTS of the TF ZOS3-15-C2H2 zinc finger protein (LOC_Os03g41390), were unchanged in the heat-tolerant rice strain, whereas the cytosines in the heat-sensitive strain were either HNT-induced methylated or demethylated. Twenty successive cytosines 651–728 bp downstream from the TTS of the TF bZIP (LOC_Os02g52780) were consistently HNT-induced methylated in the heat-tolerant strain, while the cytosines were unchanged in the heat-sensitive strain. Thus, consistent methylation of the successive cytosines in the promoter region of the TFs might regulate ABA cellular oxidation pathways during rice in response to HNT (Fig. 4 and Table S4). The consistent demethylation of the cytosines in the promoter region of PPR, the consistent methylation of the cytosines in the downstream region of bZIP, as well as the stable methylation state in the downstream regions of MYB and C2H2 contribute to rice heat tolerance [46].
Several additional TFs, including flowering control locus A (FCA), bZIP, tryptophan-arginine-lysine-tyrosine (WRKY), APETALA2 (AP2), cysteine 8-cysteine 5 or cysteine 3-histidine-type zinc finger protein (C8C5C3H), and DNA binding with one finger protein (Dof), were also reported to be regulated by DNA methylation in plants [49–51]. The post-transcriptional regulator FCA, which has two RNA-binding motifs (RRMs), upregulates the gene mitogen-activated protein kinase 5 lacking an intact kinase domain (OstMAPKKK5). The upregulation of OstMAPKKK5 then upregulates the endogenous gibberellin (GA) content to affect the size of rice grain cells [52]. The ABA-induced bZIP directly induces amylase- and sugar-transporter genes to increase the accumulation of soluble sugars in plants [53]. WRKY is involved in carbohydrate anabolism during starch synthesis in the endosperms of rice and wheat, while AP2 regulates both embryo cell number and cell size by controlling sugar metabolism [54, 55]. C8C5C3H is associated with the regulation of the gene Glub-1 to control glutelin accumulation in rice grains, and Dof influences the expression of seed-storage genes, including sugar transporter genes, starch synthesis genes, and glutelin genes [56, 57]. Here, we identified that the successive cytosines in the promoter or downstream regions from the TTS of FCA (LOC_Os09g03610), bZIP (LOC_Os02g52780), WRKY80 (LOC_Os03g63810), Dof (LOC_Os12g39990), AP2 (LOC_Os12g41030), and C8C5C3H (LOC_Os01g68860) were HNT-induced differentially methylated or demethylated between the heat-tolerant and heat-sensitive coisogenic rice strains (Fig. 4 and Table S4). The results indicated that the methylation status of the cytosines in the promoter or downstream regions of these TFs is associated with grain size, energy synthesis, and energy transport, ultimately affecting rice heat tolerance.
Based on the DMG-associated pathways identified in KEGG and the literature, we constructed a composite DMG regulatory network (Fig. 5). In the constructed network, the successive cytosines in the region upstream of the gene encoding the TF PPR, which is activated when ABA binds to CHLH/ABAR and participates in the regulation of complex I and complex V components [40], were consistently HNT-induced demethylated and also triggered cellular oxidation and ROS production in response to HNT stress. In contrast, the successive cytosines in the gene body of the TF Homeobox were consistently HNT-induced demethylated, which regulates the binding of ABA to PYR/PYL and activates PP2C. SnRK2, a key regulator of ROS scavengers (e.g., MYB, C2H2, and bZIP), is negatively regulated by PP2C [41–44]. However, the cytosines in the promoter or downstream region of the TFs MYB and C2H2 could be HNT-induced demethylated in the heat-sensitive rice strain but were unchanged in the heat-sensitive strain, which might allow for the removal of excessive ROS and reduction of cellular damage in the heat-tolerant strain. SnRK2 also plays important roles in sugar transport and starch synthesis by regulating bZIP, WRKY80, AP2, and C8C5C3H [48–50, 58]. The cytosines in the promoter or downstream regions of bZIP and WRKY80 were HNT-induced methylated, while the cytosines in the promoter or downstream regions of AP2 and C8C5C3H were unchanged in the heat-tolerant rice strain. The constructed network thus showed that the methylation or demethylation of certain regions in genes contributes to rice heat tolerance.