Harmful ROS content usually increases dramatically under drought stress and attacks membrane lipids, accompanied by peroxidative damage to DNA, proteins, and membrane lipids . To resist oxidative damage, plants have gradually evolved many protectives scavenging or antioxidant defense mechanisms, such as SOD and POD antioxidant enzymes . In this paper, the contents of malondialdehyde (MDA) and relative electrical conductivity (REC), which are indicators of oxidative damage , were significantly increased at T8 (P < 0.05), and continue to increase sharply at T12 (P < 0.01) (Fig. 1A, B), indicating that F. nilgerrensis were suffering from severe oxidative damage from T8. Correspondingly, the activities of POD and SOD were also displayed an increasing tread from T8 to T12, thereby alleviating the ROS damages. Besides these enzymes, we also found a significant rise in proline (Pro) content from T8 to T12 in F. nilgerrensis, which can modulate intracellular and extracellular osmotic potential to improve plant water retention . The smallest increase of all the five parameters (Pro, MDA, SOD, POD, REC) were detected at T4. These drought inducible physiological changes indicated that multiple physiological processes, which were regulated by molecular alterations, were initialed near or at T8.
Changes of differentially expressed genes (DEGs) under drought stress
To explore the drought resistance mechanism of F. nilgerrensis, we sequenced the transcriptome and whole genome bisulfite sequencing of leaves at four time points (T0, T4, T8, and T12). Among them, transcriptome sequencing was completed in 11 samples with an average of ~ 6.4Gb per sample, which were subsequently mapped to the F. nilgerrensis genome. The mapping rate was as high as about 95%, and the unique mapping rate was greater than 92% on average (Supplementary Table S1). The Pearson correlation coefficients (R2) among replicates mostly exceeded 0.9, indicating the high reproducibility between replicates of each time point (Supplementary Fig. S1A).
Compared with T0, the most DEGs (5,308) were detected at T8, of which significantly up-regulated and down-regulated genes were 2225 and 3083, respectively (Fig. 2A). Notably, the lowest number of DEGs was found between T12 vs. T8 (100 DEGs), suggesting the gene expression patterns of T8 and T12 were similar. Consistently, Venn plot showed that T8 and T12 shared most DEGs (2,593) (Supplementary Fig. S1B) as well as the hierarchical cluster analysis of DEGs, which showed that T8 and T12 were clustered together (Supplementary Fig. S1C). These results further indicated that dramatic molecular and metabolic alterations of F. nilgerrensis occurred at T8. Then we searched common temporal expression patterns using the Short Time-series Expression Miner (STEM) program  and found 11 significant profiles (Supplementary Fig. S1D), among which two broad profiles exhibited up (1,549 genes) or down (1,274 genes) regulation at T8 (Fig. 2B). Notably, the Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis revealed that up-regulated genes were enriched in glycerophospholipid metabolic pathway, MAPK signaling and ribosome biosynthesis etc., while down-regulated genes were enriched in carbon fixation and photosynthesis (Fig. 2B). Previous studies have shown that the MAPK cascade is a key signal mechanism in response to various external stimuli, including drought stress . The Glycerophospholipid metabolic pathway has also been reported in drought tolerance studies on the tolerant diploid common wheat ancestor Aegilops tauschii . In contrast, stomatal closure under water-deficient conditions allows plants to minimize water loss by transpiration, and with the inaccessibility of CO2, the rate of photosynthesis decreases . Thus, these genes maybe play crucial role in respondse to drought in F. nilgerrensis.
Consistently, if we focused on DEGs at T8 vs. T0, a larger number of pathways were enriched in drought resistance according to GO (Gene Ontology) enrichment analysis. The up-regulated genes were mostly enriched in response to water deprivation, oxidative stress, abscisic acid-activated signaling pathway, UDP-glucosyltransferase activity, calmodulin binding, and active transmembrane transporter activity (Fig. 2C). Whereas the down-regulated DEGs were enriched in photosynthesis, cell wall organization or biogenesis, transmembrane receptor protein kinase activity and water channel activity. The expression patterns of DEGs at T8 roughly delineated the processes involved in drought resistance of F. nilgerrensis.
Methylation landscape of F. nilgerrensis under drought stress
To explore the regulatory mechanisms of methylation level after drought, we simultaneously performed WGBS analysis. A total of ~ 126Gb clean data were produced, with an average of ~ 10.5Gb per sample, a sequencing depth of over 30x (305.9Mb for the genome), and a unique mapping rate of 67.75%-73.13%. In each library, the lowest BS conversion rate was 99.499%, while the lowest conversion rates were 96.78% and 90.18% for Q20 and Q30, respectively (Supplementary Table S2).
The average methylation level across the genome is methylated mC divided by the sum of mC and unmethylated umC at certain cytosine sites. Among three sequence contexts of F. nilgerrensis, the CG context exhibited highest methylation level, followed by the CHG and CHH contexts, with an average of 47.69%, 30.87% and 10.56%, respectively (Fig. 3A, Supplementary Table S3). In addition, we also calculated the percentage of three contexts (mCG, mCHG and mCHH) in the total mC sites and found they exhibited dynamic changes at various time points, of which mCHH not only account for the highest percentage but also showed biggest changes (Fig. 3B) (Supplementary Table S3). This was consistent with previous findings in Morus alba and Gossypium hirsutum that overall methylation levels increased after drought stress and CG methylation has the highest levels [34, 35].
The sum of DMRs between each time point and the control (T0) were also determined and most hypo- and hypermethylated DMRs were detected in T4 and T8 respectively (Fig. 3D). These DMRs mainly occur in CHH context, and about 80% of them were in repeat elements (Supplementary Fig. S2A). Accordingly, the methylation levels for each element in different genomic regions exhibited that repeat elements showed highest methylation level in all the three contexts and all the time points, followed by promoters and introns (Fig. 3C). For the methylation of promoter and gene body usually affect transcriptional regulation differently, we classified differentially methylated genes (DMGs) between each time point and T0 as promoter methylated and gene body methylated genes. The results showed that promoter DMGs were more than gene body DMGs in F. nilgerrenis at all the time points of drought stress (Fig. 3D). Among the promoter DMGs, 317 hypo- and 21 hyper-methylated genes were shared across all the time points respectively (Fig. 3E), this suggests that these genes were may be constantly regulated by DNA methylation and may play a key role in drought resistance. KEGG enrichment showed that these genes were involved in plant hormone signaling transduction, MAPK signaling pathway and ubiquitin mediated proteolysis pathways (Supplementary Fig. S2B). These results were roughly consistent with KEGG enrichment of DEGs.
The relationship between methylation level and gene expression in response to drought
Dynamic changes in DNA methylation is crucial in regulating gene expression of plants under abiotic stresses [14, 36]. Take T8 (containing most DMEGs) for example we illustrated the relationship of DNA methylation and expression in different genomic regions and different contexts. The genes detected in transcriptome were divided into four categories according to their expression levels: no expression (FPKM < 1), low expression, medium expression and high expression level, of which the latter three corresponded to the lower, middle and upper quartiles of FPKM. We found that the unexpressed genes exhibited expectable high methylation levels and highly expressed genes showed low methylation levels correspondingly in gene body except for the CG methylation, which exhibited opposite trend (Fig. 4A). Conversely, genes with medium to high expression levels in all three contexts showed high methylation levels 2 kb upstream of the transcription start site (TSS), indicating that DNA methylation was positively correlated with gene expression in the promoter region (Fig. 4A). It is notable that all three contexts exhibited the lowest methylation levels near TSS and transcription termination sites (TES). This was consistent with previous report that the lack of methylation around transcription initiation and termination sites appears to be important for gene expression regulation [37, 38]. Further, we also divided the methylation levels of these genes into five groups by quintile, with the first group being the lowest and the fifth group the highest (Supplementary Fig. S3). This reverse verification, and analysis gave the same results as above.
To further characterize the relationship between DNA methylation and gene expression, we merged DMGs and DEGs of all the time points and identified 835 genes showed both differential methylation and alteration in gene expression, named as DMEGs (Fig. 4B, Supplementary Table S4). Analysis of the expression levels of DMEGs at different time points showed that the T8 time point showed significantly higher expression levels of hypomethylated DMGs than hypermethylated DMGs and all DEGs (P < 0.001) (Fig. 4C). This was consistent with the fact that most recognized hypomethylation usually promotes gene expression [14, 39]. On the contrary, DMEGs at T4 and T12 showed a slight increase in gene expression after hypermethylation, but it was not significant. In order to further explore the relationship of methylation and expression, these genes were systematically classified into four different clusters (C1, C2, C3 and C4, respectively) based on hyper- and hypo-methylation in promoter and gene body as well as gene expression patterns (Fig. 4D). Among promoter and gene body DMEGs, both positive and negative correlation were found in methylation and gene expression. In promoter DMEGs, the genes of C1 and C2 show typical methylation regulation patterns, i.e., hypermethylation represses the gene expression while hypomethylation promotes expression; in contrast, the genes of C3 and C4 showed positive correlation in methylation and gene expression (Fig. 4D). These gene clusters were found with different functions, such as many genes in C2 (Hypomethylation and high expression) and C3 (Hypermethylation and high expression) were related to hormone signaling, kinases, transcription factors, detydrin, and detoxificants etc., while C1 (Hypermethylation and low expression) and C4 (Hypomethylation and low expression) contained a large number of photosynthesis-related genes (Fig. 4E). Similar pattern was found in gene body DMEGs that genes with increased expression were involved in drought reaction, while the genes with decreased expression were mainly related to the normal physiologic metabolism no matter hypo- or hypermethylated. In rice, promoter DNA methylation has been reported to be associated with transcriptional repression, while gene body methylation usually upregulates gene expression . Whereas, positive associations of DNA methylation to gene expression were found in both promoter and gene body in apple . This may imply a deep relationship between DNA methylation and gene expression, and is not just satisfied with previous reports of maintaining genome stability and suppressing gene expression [35, 40].
Characterization of key genes for drought response in F. nilgerrensis
The GO enrichment analysis of DEGs indicated the ABA signaling pathway was significantly involved in the drought resistance of F. nilgerrensis (Fig. 2C), which was reported responding to drought stress by regulating stomatal closure and the expression of genes [41, 42]. Notably, the expression of gene NCED1 encoding 9-cis-epoxycarotenoid dioxygenase (a very important enzyme in the ABA biosynthetic pathway) and gene CYP707A2 encoding ABA 8’-hydroxylase (a key enzyme in the oxidative catabolism of ABA) were significantly upregulated by more than 10-fold (Fig. 5A), in which CYP707A2 was found to be accompanied by hypomethylation in the promoter region throughout drought stress. Significant upregulated expression of NCED1 and CYP707A2 and an increased ABA content was also detected in cultivated strawberries under drought stress (F. × ananassa) . It suggested that drought stress on F. nilgerrensis promote ABA biosynthesis and activated ABA-dependent signaling pathways. It was known that under stress conditions, ABA levels increased and the PYRABACTIN RESISTANCE/PYR-LIKE/REGULATORY COMPONENTS OF ABA RECEPTORS (PYR/PYL/RCAR) bounded to ABA to interact with and inhibit downstream target, the clade A type 2 C protein phosphatase (PP2Cs), thereby releasing the SNF1-related protein kinase 2 (SnRK2) . The released SnRK2 (especially encoded by subclasses III and II of SnRK2) eventually leads to the phosphorylation of the downstream ABA-dependent signaling gene ABF/AREB to activate the stress response . Our results showed that expression of five ABA receptor (PYL6, PYR1, PYL8, two PYL2s) genes were down-regulated, while expression of PP2Cs, genes in subclass II of SnRK2 and ABF2 were significantly up-regulated in F. nilgerrensis under drought stress (Fig. 5A, Fig. 6, Supplementary Fig. S4). In contrast to the common expectation that ABA reduces the expression of PYR/PYLs receptors and induces the expression of PP2Cs, no common trend between ABA content and PYR/PYLs expression was detected in some species, which appears to be the mechanism for reducing persistent ABA damage . This pattern was also observed in Arabidopsis that the expression levels of PYR/PYLs were down-regulated, while PP2Cs and ABFs were up-regulated under stress . Consistently, most PYLs were down-regulated except for ZmPYL4, ZmPYL7 and ZmPYL8 in polyethylene glycol (PEG)-treated maize leaves, and PP2Cs and SnRK2s were either up-regulated or unchanged . Furthermore, the lack of differential expression of SnRK2 subclass III genes in F. nilgerrensis may be caused by a missed right time point, and the activation of the downstream target gene ABF2 also supported this speculation. It was reported that subclass III genes of SnRK2 were rapidly activated within minutes after exogenous ABA administration in Arabidopsis .
Through the hierarchical clustering method, we performed weighted gene co-expression network analysis (WGCNA) of physiological traits and RNA-seq data to characterize similar expression patterns, and 29 valid modules (grey is an invalid module) were obtained (Supplementary Fig. S5A, 5B). We found that the orange, lightcyan and turquoise modules were significantly positively correlated with the five physiological traits, with an average correlation of 0.76 (P < 0.05) (Fig. 5B). On the contrary, the blue module showed a significantly negative correlation with these physiological traits. The KEGG enrichment analysis showed that the positively correlated three modules were mainly enriched in ubiquitin mediated proteolysis, amino acid biosynthesis and MAPK signaling pathway, etc. While the negatively correlated blue module was mainly enriched in photosynthesis, phytohormone signal transduction and biosynthesis of secondary metabolites (Supplementary Fig. S5B). More importantly, a large number of important genes in the ABA signaling pathway were also identified in the turquoise color module, including NCED1, CYP707A2, and PP2Cs, which indicated ABA pathway play an important role in regulating physiological response in F. nilgerrensis under drought stress (Fig. 5C, 5D). It’s notable that the FaNCED gene was reported to be expressed only in roots, but not in leaves in cultivated strawberry (Figaro and Flair) . While in our study we detected the high expression of NCED in leaves of F. nilgerrensis under drought. Although the root-derived ABA theory has been widely accepted previously, several recent studies have shown that leaves are considered to be the initial source of ABA biosynthesis during water stress due to the presence of large amounts of endogenous carotenoid precursors required for ABA biosynthesis . Besides, the hub genes of orange module, such as WRKY28, Zinc finger protein 7 ZFP7, and Peroxiredoxin-2B PRXIIB also has been shown to be highly related to drought resistance (Supplementary Fig. S5C). Among them, ZEP7 was reported to regulate ABA signaling in Arabidopsis, and WRKY28, which co-expressed with AtbHLH17, were known to be up-regulated under drought and oxidative stress, improving resistance to abiotic stress in Arabidopsis . Meanwhile, PRXIIBs have been reported to play an significant role in cytoprotection against oxidative stress by eliminating peroxides and acting as sensors of hydrogen peroxide-mediated signaling events .
In addition, drought usually prevent the entering of CO2 into leaves and finally result in the decrease of photosynthetic rate . Consistent with this point, a large number of key photosynthesis related genes showed significantly down regulated expression and differentially methylation in our results, such as the genes encoding fructose 1,6-bisphosphatase (FBPase), chlorophyll-binding protein (CBP) and ribulose-1,5-bisphosphate carboxylase (RuBisco) (Fig. 6), which were important regulators of photosynthesis . Furthermore, the drought-induced stomatal closure, reduced carbon dioxide uptake, reduced photosynthetic rate, and changes in chloroplast photochemical reactions also could cause overproduction of ROS . Consistently, we observed that the expression of respiratory burst oxidase homologs (RBOH) was significantly unregulated with changed methylation (Fig. 6), which were reported to be the key genes for ROS synthesis and play a crucial role in their responses to biotic and abiotic stresses [57, 58]. Excessive ROS would cause oxidative stress to impair DNA, proteins and lipids, resulting in cell dysfunction and death . Thus, ROS levels under stress conditions are associated with ROS production and ROS clearance maintenance, which also represents the ability of the stress response . Our results showed that in response to ROS damage, ROS scavenging systems were activated and expression of genes such as POD, glutathione transferase (GST), glutathione reductase (GR) and glutathione peroxidase (GPX) were significantly increased, regulated by different methylation (Fig. 6). It indicated that rapid and effective antioxidant defense system involved in ROS detoxification in F. nilgerrensis under drought stress may provide them strong resistance.
Transcription factors (TFs) are inevitably activated in response to transduction of stress signals and subsequently trigger the expression of a large number of stress-responsive genes [61, 62]. To illustrate the changes in expression and methylation of TFs under drought stress, 1285 TFs in F. nilgerrensis genomes were identified according to the transcription factor database PlantTFDB (http://planttfdb.gao-lab.org), among which, 189 TFs differentially expressed under drought stress (Supplementary Table S5). These differentially expressed TFs were from 34 gene families, including NAC (19 members), WRKY (15 members), MYB (23 members), bZIP (14 members), ERF (13 members) and DREB (7 members). As the heat map showed that a large number of TFs showed significantly increased expression under drought stress, including NAC, WRKY, HSF and ERF ect., and some of them were accompanied by differential methylation (Fig. 6). Interestingly, we identified a significant upregulation of dehydration response element binding (DREB), which is a key stress TF in the regulation of the ABA-independent pathway. This may seem to imply that there are both ABA-dependent and -independent signals in F. nilgerrensis for drought response.
Among these differentially expressed TFs, 25 TFs were also showed methylation changes in the promoter region, including the heat stress transcription factor A-6b, the AP2/ERF family transcription factor ERF4, and the possible WRKY transcription factor 46 (Supplementary Table S6). Notably, we found that ERF1, WRKY46, B3 domain-containing and Dof3.6 showed continuously increased expression and hypomethylation across all the time points. Among them, ERF1 was reported to be able to integrate stress-specific gene regulation of multiple hormonal signals to play an active role in drought and heat stress tolerance in Arabidopsis . Previous studies have shown that WRKY64 is involved in stress osmotic and stomatal regulation . Besides, B3 domain-containing  and Dof  also have been reported to be involved in drought stress. These results suggested that these TFs should play a central role in drought stress tolerance in F. nilgerrensis. Finally, we randomly selected the above 12 genes (NCED1, CYP707A2, two each of PYLs and PP2Cs, ABF2, WRKY46, WRKY28, ERF1, ERF4 and B3) for qRT-PCR verification, and obtained results consistent with the transcriptome trend (Fig. 5D).