Genome-wide identification and comparative analysis of DNA methyltransferase and demethylase gene families in two ploidy Cyclocarya paliurus and their potential function in heterodichogamy
BMC Genomics volume 24, Article number: 287 (2023)
DNA methylation is one of the most abundant epigenetic modifications, which plays important roles in flower development, sex differentiation, and regulation of flowering time. Its pattern is affected by cytosine-5 DNA methyltransferase (C5-MTase) and DNA demethylase (dMTase). At present, there are no reports on C5-MTase and dMTase genes in heterodichogamous Cyclocarya paliurus.
In this study, 6 CpC5-MTase and 3 CpdMTase genes were identified in diploid (2n = 2 × = 32) C. paliurus, while 20 CpC5-MTase and 13 CpdMTase genes were identified in autotetraploid (2n = 4 × = 64). 80% of identified genes maintained relatively fixed positions on chromosomes during polyploidization. In addition, we found that some DRM subfamily members didn’t contain the UBA domain. The transcript abundance of CpC5-MTase and CpdMTase in male and female flowers of two morphs (protandry and protogyny) from diploidy was analyzed. Results showed that all genes were significantly up-regulated at the stage of floral bud break (S2), but significantly down-regulated at the stage of flower maturation (S4). At S2, some CpC5-MTase genes showed higher expression levels in PG-M than in PG-F, whereas some CpdMTase genes showed higher expression levels in PA-M than in PA-F. In addition, these genes were significantly associated with gibberellin synthesis-related genes (e.g. DELLA and GID1), suggesting that DNA methylation may play a role in the asynchronous floral development process through gibberellin signal.
These results broaden our understanding of the CpC5-MTase and CpdMTase genes in diploid and autotetraploid C. paliurus, and provide a novel insight into regulatory mechanisms of DNA methylation in heterodichogamy.
As a conserved epigenetic modification, DNA methylation was widely found in plants and animals, which can alter gene expression and phenotype without altering DNA sequence and maintain genomic stability [1,2,3]. DNA methylation is controlled by a series of cytosine-5 DNA methyltransferase (C5-MTase) and DNA demethylase (dMTase). In plants, DNA methylation commonly occurs at symmetrical CG and CHG sequence context, and asymmetrical CHH context (H = A, T, or C), which can be maintained or established de novo DNA methylation by distinct pathway . Generally, methylation at the CG and CHG contexts is maintained by methyltransferase 1 (MET1) and chromomenthylase 2/3 (CMT2/3), while methylation at the CHH context is maintained by CMT2 and domain rearranged methyltransferase 2 (DRM2) [4, 5]. Among these, DRM catalyzes de novo methylation via the RNA-directed DNA methylation (RdDM) pathway in both symmetric and asymmetric sequence contexts [2, 4, 6]. CMT is a plant-specific C5-MTase, containing bromo-adjacent homology (BAH) and chromo domain bound to histone H3K9 to maintain DNA methylation . DNA methyltransferase 2 (DNMT2) is the smallest methyltransferase in eukaryotes, which can methylate tRNA(Asp)C38 in vitro .
DNA methylation level is dynamically regulated depending not only on the establishment and maintenance of DNA methylation but also on DNA demethylation. DNA methylation can be passively removed by inhibiting C5-MTase activity in DNA replication or actively removed by a series of demethylases [2, 4, 8]. The dMTase proteins can be classified into four subfamilies in Arabidopsis thaliana, including transcriptional activator demeter (DME), repressor of silence 1 (ROS1), demeter-like 2 (DML2), and demeter-like 3 (DML3) . As the necessity for the establishment of genomic imprinting in the endosperm, DME mainly expresses in central cells; whereas ROS1 and DML2/3 express widely in all vegetative tissues [10,11,12]. Furthermore, ROS1 can inhibit the methylation of gene promoters and transposons , and DML2/3 can protect genes from potentially deleterious methylation .
Cyclocarya paliurus, also known as “sweet tea”, is a medicinal plant belonging to the family Juglandaceae . C. paliurus is rich in triterpenes, flavonoids, polysaccharides, polyphenols, and other active substances, which have hypoglycemic and hypotensive functions. The attractive ingredients in its leaves have led to massive plantations for leaf-use in China. However, heterodichogamy in C. paliurus seems to slow down its popularization due to low seed fullness, which has been found in half species of Juglandaceae . As a transitional type from dichogamy to dioecy, heterodichogamy possesses two mating types: protogyny (PG) and protandry (PA). Opposite-sex flowers of the intra-mating type are asynchronized in flowering, whereas these of the inter-mating type are synchronized . Supposedly, DNA methylation could regulate flowering by affecting the expression of flowering-related genes. For example, the changes in methylation status of MeGI caused the reversible sex determination in Diospyros kaki . In Arabidopsis thaliana, the reduction of genomic methylation level promoted flowering by suppressing FLC expression; while in cotton, the inhibition of DNA methylation promoted flowering by activating the COL2 expression [19, 20]. Campos-Rivero et al.  showed that epigenetic regulates hormone signaling to inhibit or promote flowering in angiosperms, such as gibberellin, auxin, and ethylene. Moreover, plant hormones can alter DNA methylation status in specific and redundant ways . Previous research has indicated that gibberellin, especially GA3, plays an important role in asynchronous floral development in heterodichogamous C. paliurus . However, the regulatory mechanism of DNA methylation in asynchronous floral differentiation and development in heterodichogamous C. paliurus is still unclear. Additionally, new findings indicated that minority diploid and majority autotetraploidy co-exist in nature . Thus, heterodichogamy coupled with an autotetraploid complicates the issue of seed setting in C. paliurus. Fortunately, whole genomes of two ploidy C. paliurus (PG and PA in diploidy and PA in autotetraploidy) have been published, revealing that the evolving process of autotetraploid experienced three whole-genome duplications (WGD) . It is also well known that DNA methylation plays an important role in the adaptation of polyploid plants to WGD [24, 25]. For instance, autotetraploid rice has been shown to reduce deleterious genomic dosage effects by increasing the methylation of class II transposable elements .
C5-MTases and dMTases gene families with species specificity have been identified in numerous plants [4, 9, 12, 26]. Moreover, gene duplication and loss events constitute the main factors driving the evolution of these two gene families . In C. paliurus, we hypothesized that: 1) C5-MTase and dMTase gene families undergo gene expansion or loss events during polyploidization; and 2) heterodichogamous character is regulated by methylation of related genes. To verify these hypotheses, our work focused on: 1) identifying and analyzing all members of C5-MTase and dMTase gene families by screening whole genomes of autotetraploid and its diploid ancestors; and 2) comparatively analyzing the expression profiles of two gene families in both male and female flowers between two mating types (PA and PG) from diploid plants. This study may lay the foundation for further research on the role of DNA methylation in C. paliurus.
Genome-wide identification and structural analysis of C5-MTase and dMTase genes in C. paliurus
In our study, we identified 26 C5-MTase and 16 dMTase genes from 2 genomes of C. paliurus based on HMM and Blastp analyses. Among them, 6 CpC5-MTase and 3 CpdMTase genes were identified in diploid C. paliurus, while 20 CpC5-MTase and 13 CpdMTase genes were identified in autotetraploid C. paliurus. The CpC5-MTase genes in diploid included 1 MET (CpMET-D1), 2 CMT (CpCMT2-D1 and CpCMT3-D1), 1 DNMT2 (CpDNMT2-D1), and 2 DRM (CpDRM-D1/2), while 7 CMT (CpCMT2-T1/2/3/4 and CpCMT3-T1/2/3), 4 MET (CpMET-T1/2/3/4), 6 DRM (CpDRM-T1/2/3/4/5/6), and 3 DNMT2 (CpDNMT2-T1/2/3) were identified in autotetraploid. The results found that the protein length of 26 CpC5-MTase varied from 393 (CpDNMT2-D1) to 1578 (CpMET-T2) amino acids (aa), and their predicted molecular weights ranged from 44,309.27 to 177,676.64 kDa. The PI values spanned from 5.26 to 9.01; the subfamily DNMT2 was deemed the basic proteins (PI > 7) (Table 1). The grand average of hydropathicity (GRAVY) values ranged from -0.606 to -0.338, indicating that all CpC5-MTase proteins were hydrophilic. Most of the proteins were predicted to be located in the cell nucleus, except for CpDRM-D2, CpCMT2-T2, and CpDRM-T5, which were predicted to be in the cytoplasm.
Sixteen CpdMTase contained 2 DME (CpDME-D1/2) and 1 ROS1 (CpROS1-D1) in diploid, 9 DME (CpDME-T1/2/3/4/5/6/7/8/9) and 4 ROS1 (CpROS1-T1/2/3/4) in autotetraploid, respectively. The sequence characteristic analysis showed that the protein length of CpdMTase varied from 1784 (CpROS1-T4) to 2667 (CpDME-T4) aa, the predicted molecular weights were from 200,250.16 to 301,133.41 kDa, and the PI values ranged from 5.83 to 6.94. The GRAVY values of all proteins were < 0, indicating that CpdMTase proteins were all acidic (PI < 7) and hydrophilic. Putative subcellular localization revealed that all CpdMTase proteins localized to the nucleus (Table 1).
Based on the results of structural domain analysis (Fig. 1), the N-terminus of CpC5-MTase proteins in each subfamily showed different combinations of conserved domains. However, no difference in the structure of C5-MTase proteins was found between the two genomics, except that the average length in autotetraploid (999 aa) was greater than that in diploid (811 aa) (Table 1). CpMETs contained 2 DNMT1-RFD domain and 2 BAH domain (Fig. 1), while CpCMTs contained 1 BAH domain, and CpDNMT2s had no N-terminus. Most CpDRMs contained 1 UBA domain, but both CpDRM-D2 and CpDRM-T5 possessed an extra incomplete DNA methylase domain instead of a UBA domain. The RRM-DME, PERM-CXXC, and ENDO3c domains (including the HhH-GPD domain) appeared in all CpdMTases, which were also reported in AtdMTases (Fig. S1). Similarly, the average length of CpdMTases in autotetraploid (1964 aa) was greater than that in diploid (1910 aa) (Table 1).
Phylogenetic analysis of C5-MTase and dMTase in C. paliurus and other plant species
To further explore the evolutionary relationship between CpC5-MTase and CpdMTase, a phylogenetic tree was constructed based on the protein sequences of C5-MTase and dMTase from typical monocots (Oryza sativa), dicots (A. thaliana), and woody species (Populus trichocarpa) (Fig. 2). Obviously, the phylogenetic tree showed that C5-MTase families from the 4 species were naturally grouped into 4 categories, namely CMT, MET, DRM, and DNMT2, respectively (Fig. 2A). The DNMT2 category, with 6 genes, was the smallest in the phylogenetic tree, including AtDNMT2, PtDNMT2, CpDNMT-D1, and CpDNMT-T1/2/3. Twenty-nine dMTase proteins were divided into 3 categories, namely DME, ROS1, and DML, respectively (Fig. 2B), in which ROS1s and DMLs showed a closer relationship. In addition, CpC5-MTase and CpdMTase proteins sequences showed high conservation in diploid C. paliurus, autotetraploid C. paliurus, A. thaliana, and P. trichocarpa, indicating their similar functions in different species.
Chromosomal location and synteny analysis
For diploid C. paliurus (2n = 2 × = 32), 6 CpC5-MTase genes were located on 6 chromosomes (Chr2, Chr4, Chr5, Chr6, Chr12, and Chr16) and 3 CpdMTase genes on 3 chromosomes (Chr7, Chr12, and Chr14) (Fig. 3A). For autotetraploid (2n = 4 × = 64), 33 identified genes were distributed unevenly in 25 chromosomes, among them, 20 CpC5-MTase genes were located on Chr2B/C/D, Chr4C/D, Chr5A/D, Chr6A/C/D, Chr12A/C/D, and Chr16A/C/D; 13 CpdMTase genes were located on Chr7A/B/C/D, Chr12A/B/C, and Chr14A/B/C/D (Fig. 3B). Comparatively, Chr4C contained more genes (CpCMT2-T1/2/3) than others (only 1 or 2 genes). Noticeably, CpC5-MTase and CpdMTase families were not found in the following 8 pairs of homologous chromosomes 1, 3, 8, 9, 10, 11, 13, and 15. Most genes of CpC5-MTase and CpdMTase were found to situate near two ends of chromosomes with relatively stable positions both in diploid and autotetraploid C. paliurus.
Synteny analysis revealed the locus relationship of homologous genes between diploid and autotetraploid C. paliurus. As shown in Fig. 4, gene pairs with a collinear relationship were connected with gray lines, while CpC5-MTase and CpdMTase syntenic gene pairs were linked with red lines. Many chromosomes in both genomes were joined together by lines, indicating that they were evolutionarily related. Also, most genes were conserved during polyploidization, such as CpC5-MTase and CpdMTase. The duplication types of CpC5-MTase and CpdMTase genes were mostly WGD or segmental in autotetraploid (Table S1), resulting in the expansion of two families. Furthermore, Ka/Ks ratios were used to estimate the selection pressure among duplicated gene pairs in the autotetraploid genome. The Ka/Ks values of all duplicated CpC5-MTase and CpdMTase genes pairs were lower than 1.0 (Fig. S2), except for two pairs (CpCMT2-T2 & CpCMT2-T4, CpDRM-T4 & CpDRM-T6), suggesting that most of them may undergo purifying selection .
Gene structure and conserved motif distribution analysis
As shown in Fig. 5A, different exon–intron distribution patterns were observed in the structure of CpC5-MTase in diploid C. paliurus. Among them, CpCMT-Dn contained the largest number of introns, while CpCMT2-D1 and CpCMT3-D1 had 21 and 20 introns, respectively. In other subfamily genes, the number of introns varied from 3 to 11. However, in autotetraploid C. paliurus, the ranges of introns in CpCMT-Tn, CpDRM-Tn, CpMET-Tn, and CpDNMT2-Tn were 11–23, 3–20, 11, and 12–16, respectively. Comparatively, the length of intron in CpC5-MTases exhibited expansion in autotetraploid, such as CpCMT2-T3/4, and CpDRM-T1. Furthermore, a significant increment in the number of introns CpDNMT2-Tn from autotetraploid was found in comparison with that from diploid. Interestingly, all members of the MET subfamily from both ploidies contain 11 introns, demonstrating their stability during polyploidization. For CpdMTase genes (Fig. 5B), the number of introns ranged from 13 to 19 regardless of ploidy. Among them, the least number of introns (13) occurred in CpDME-T2. Moreover, CpROS1s contained 17–19 introns, indicating their relative conservation in gene structure during polyploidization.
A total of 10 motifs were identified in the CpC5-MTase proteins (Fig. 5A; Table S2). Motifs 1, 2, 4, 5, and 8 located at the C-terminus were highly conserved in CpCMTs and CpMETs. All CpDRMs consisted of motifs 1, 4, 6, 7, 8, 9, and 10. Compared to the other three subfamilies, the DNMT2 subfamily had a unique structure, with only motifs 1, 2, 3, and 4 located at the N-terminus. These results may explain the division of CpC5-MTase proteins into three branches, CpCMTs and CpMETs proteins were clustered into a small one, while CpDRMs and CpDNMT2s belonged to separate branches. Similarly, 10 motifs were identified in the CpdMTase proteins, which were grouped into 3 branches (Fig. 5, Table S2). Among these, the structure of the ROS1 subfamily was relatively stable, with the consistent distribution of 10 motifs; all CpDMEs contained all 10 motifs, except for CpDMTE-T3 and CpDMTE-T4 (missing motif 7), CpDME-D1 (missing motif 8).
Cis-acting element analysis of CpC5-MTase and CpdMTase
In C. paliurus, the cis-acting elements in promoter regions of CpC5-MTase and CpdMTase genes were classified into four categories: light response, phytohormone response, stress response, and plant growth and development (Fig. 6). Notably, each gene was enriched with multiple light-responsive elements. Phytohormone response elements, including ABA, auxin, GA, MeJA, and salicylic acid were also widely observed. Moreover, multiple stress-related elements were also predicted in CpC5-MTase and CpdMTase genes, implying their role in responding to various stresses, such as anaerobic induction, drought, low temperature, and defense response. In addition, some tissue-specific elements related to endosperm-specific, root, and stem meristem specific were identified. In a word, most of the cis-acting elements found in CpC5-MTase and CpdMTase were associated with physiological processes, indicating their involvement in plant growth and development, as well as the stress response.
Expression patterns of CpC5-MTase and CpdMTase genes in various tissues of diploid C. paliurus
The flower buds of C. paliurus undergo physiological differentiation (S0), dormancy (S1), bud break (S2), inflorescence elongation (S3), and flower maturation stages (S4) (Fig. 7A, cited from ). The gene expression patterns of floral buds at the S2 and S4 were analyzed, including female and male buds in protandry (PA) and protogyny (PG) individuals, respectively . The results showed that all CpC5-MTase and CpdMTase genes were expressed in floral buds. According to heatmap-clustering analysis (Fig. 7B), the expression of all genes was significantly up-regulated at S2 and significantly down-regulated at S4. At S2, the expression levels of CpCMT3-D1, CpDNMT2-D1, CpMET-D1, and CpDRM-D1 in male floral buds were higher than those in female ones regardless of mating type, indicating that CpC5-MTases plays an important role in the morphological development of male flowers. The expression levels of CpDNMT2-D1, CpCMT3-D1, and CpMET-1 were higher in PG-M than those in PG-F. In addition, some CpdMTases (CpROS1-D1 and CpDME-D1) showed higher expression levels in PA-M than those in PA-F. However, some genes (e.g. CpDRM-D1, CpCMT3-D1 and CpMET-D1) displayed the opposite expression pattern in the same sexual floral bud, showing higher expression levels in PG-M versus lower levels in PA-M; whereas CpDRM-D1 and CpDME-D1 showed higher expression levels in PG-F than in PA-F. At S4, CpC5-MTases showed lower expression levels in male flowers than in female ones regardless of mating type, except that CpDNMT2-D1 and CpDRM-D2 expressed higher levels in PA-M than in PA-F. Similarly, the expression levels of CpdMTase in male flowers were lower than those in female ones. Moreover, the expression levels of these genes in the same sexual floral bud were also different, such as CpCMT2-D1, CpDRM-D1, and CpDME-D1/2 showed higher expression levels in PA-M than in PG-M, CpROS1-D1 and CpDME-D2 were expressed higher in PA-F than in PG-F. Thus, we speculate that these genes may fulfill important functions in heterodichogamous characteristics of C. paliurus.
Expression correlation analysis between candidate genes and flowering-related genes
To investigate the potential role of CpC5-MTases and CpdMTases in heterodichogamy, based on previous research results [17, 23, 30], correlation analysis was performed on the differential genes in the lowering process of PA and PG. Figure 8 illustrated some genes that may play roles in heterodichogamy, such as ERFs, Trihelix-1, and GA synthesis genes. CpC5-MTases and CpdMTases were significantly correlated with several genes involved in GA biosyntheses (Fig. 8), including KO, GA20ox, GID1, and DELLA. Notably, CpC5-MTases showed significant positive correlations with GA20ox. Both CpC5-MTases and CpdMTases were significantly positively correlated with KO. With the exception of CpDRM-D2 and CpROS-D1, most of the CpC5MTases and CpdMTases showed significant negative correlations with GID1, but remarkable positive correlation with DELLA. Additionally, except for CpDRM-D2, most of the CpC5MTases and CpdMTases were significantly positively correlated with WRKY55 and ERF090, but negatively correlated with Trihelix-1.
Structure and evolutionary features of C5-MTase and dMTase gene families during polyploidization
Homozygous comparisons revealed no differences in the C5-MTase and dMTase gene families between PA-dip and PG-dip genomes (Table S4, Fig. S1-B). Therefore, we further analyzed the genomic data from PA-dip and PA-tetra, identifying 6 CpC5-MTase and 3 CpdMTase genes in the diploid C. paliurus, and 20 CpC5-MTase and 13 CpdMTase in the autotetraploidy. Compared with reported plants, the members of C5-MTase and dMTase gene families in diploid C. paliurus were lower than those in Arabidopsis (11 and 4), rice (7 and 6), and tea plant (8 and 4) [12, 26, 31]. The larger genome in diploid C. paliurus (587 Mb) than in rice (466 Mb) and A. thaliana (125 Mb) [12, 17], this suggests that gene absence events may have occurred in CpC5-MTase and CpdMTase genes during the evolution of C. paliurus. The phylogenetic tree showed that CMT1 and DML subfamilies were absent in C. paliurus compared with other species (Fig. 2). Previous research showed that 73 out of 77 species have experienced gene absence events, such as CMT1 in sugarcane and DML in peanut [32,33,34]. In Arabidopsis, AtCMT1 has been proven unessential, as the allotetraploid A. suecica contained an Evelknievel retroelement insertion or inactivation of the truncated proteins . Presumably, CMT1 and DML in C. paliurus were lost during the evolutionary process due to functional redundancy. However, the effects of gene absence on epigenetic modifications need to be further studied.
Additionally, gene absence commonly occurs during genome sequence rearrangement after chromosome doubling. Qu et al.  found a large deletion in Chr8 compared with other chromosomes, and an inversion between Chr4 and Chr16 in the synteny analysis of C. paliurus genomes. Similarly, we observed that some genes’ locations changed in chromosomes (Fig. 4), indicating that chromosome rearrangement may exist in the target gene family. Theoretically, based on the ploidy level, the number of CpC5-MTase and CpdMTase genes in autotetraploid C. paliurus should be 36 (9 × 4). Actually, CpC5-MTase experienced gene loss during polyploidization. For example, there are 6 DRM genes in autotetraploid C. paliurus, compared with 8 DRM genes in diploidy, suggesting that the DRM subfamily may have undergone copy loss during the evolution of autotetraploid C. paliurus. Nonetheless, the number of CpC5-MTase and CpdMTase gene families in autotetraploid (13) was significantly higher than that in diploid C. paliurus (3), allotetraploid Brassica napus (6) and peanut (10) [12, 34]. The quantitative advantage demonstrated that the DME subfamily may experience significant expansion during three WGD events, and the autotetraploid speciated in the most recent WGD (~ 11.2–10.5 Mya) . In this study, we found that CpC5-MTase and CpdMTase genes were mostly involved in WGD and segmental replication (Table S1), which is consistent with the expansion of C5-MTase gene family in autopolyploid sugarcane (Saccharum spontaneum) and allopolyploid wheat (Triticum aestivum) [33, 36]. Segmented replication often occurs in plants, which experienced polyploidy events resulting in a large number of chromosome blocks in the genome. Therefore, the results confirmed our conjectures that these two gene families experienced gene loss and expansion events during polyploidization, while WGD and segmental replication bring about the increment number of CpdMTase genes in autotetraploid C. paliurus.
Although the numbers of C5-MTase and dMTase gene members varied greatly across species, their protein sequences were highly similar. In C. paliurus, the C-terminus of CpC5-MTase proteins contained a conserved structural domain of DNA-methylase, but they were equipped with different N-terminal structural domains (Fig. 1), which is similar to the homologous genes in Arabidopsis . The BAH structural domain was presented in all CpMETs and CpCMTs, and it interacted with chromatin to be related to gene silencing and replication. The UBA structural domain, usually as a protein–protein interaction structural domain, was presented in members of the DRM subfamily . In Arabidopsis, normal RNA-directed DNA methylation required both the UBA and DNA-methylase domain . However, both CpDRM-D2 and CpDRM-T5 owned an additional incomplete DNA-methylase domain instead of the UBA domain, which was also reported in peanuts . It is speculated that DRM without a UBA domain may direct DNA de novo methylation upon forming heterodimer with another DRM with a UBA domain . However, the specific function of CpDRM-D2 and CpDRM-T5 genes in C. paliurus needs to be further explored.
All CpdMTase proteins contained the same conserved structural domains: RRM-DME, Perm-CXXC, and ENDO3c (Fig. 1), which is similar to the homologous genes in Arabidopsis and rapeseed . The ENDO3c domain is the core domain of dMTase, and contains a conserved DNA glycosidase motif that directly removes 5-mC bases . Genes with the same domain, such as CpDMEs and CpROS1, may exert similar functions in the process of demethylation in C. paliurus. Additionally, synteny analysis showed that 80% of analyzed genes situated at the relatively stable positions on the chromosomes (Fig. 4), indicating that the CpC5-MTase and CpdMTase genes are highly conserved at the DNA level during polyploidization in C. paliurus.
Collectively, in both diploid and autotetraploid C. paliurus, the CpC5-MTase and CpdMTase genes were highly conserved not only at the protein level but also at the DNA level, including gene structure, conserved structural domains, as well as the type of motif.
C5-MTase and dMTase gene families play important roles in regulating the heterodichogamous characteristics in C. paliurus
C. paliurus is a typical heterodichogamous species, in which the flowering of female flowers in PG synchronizing with male flowers in PA effectively avoids self-pollination [16, 29, 30]. As reported in many plants, C5-MTase and dMTase genes play important roles in flower development, sex differentiation, and regulation of flowering time [4, 5, 18]. It was documented that the reduction of DNA methylation promoted flowering in Arabidopsis and trifoliate orange (Poncirus trifoliata) [39, 40]. However, this is the first report that C5-MTase and dMTase genes involve in heterodichogamous species. In this study, the expression patterns of 9 candidate genes in counterpart sexual flowers varied across developmental stages (Fig. 7B), presenting their transcript abundances of male and female flowers from two morphs were all higher at S2 than at S4. During the process of floral development in C. paliurus, S2 is defined as the pivotal stage affecting further asynchrony . As presented in Fig. 7B, CpC5-MTases at S2, including CpDNMT2-D1, CpCMT3-D1, and CpMET-D1, displayed a higher expression level in PG-M, while less or no expression level in PG-F. This suggests that the decrease of DNA methylation in PG-F promotes the development of female prior to male buds at S2, which is in accord with the morphological characters of C. paliurus (Fig. 7A). In contrast, CpdMTases (CpROS1-D1 and CpDME-D1) were expressed higher in early flowering PA-M than in PA-F at S2 (Fig. 7B), implying that active demethylation in PA-M could promote the development of male prior to female buds at S2, which is in accord with DNA demethylation induces flowering in plants . DNA methylation expresses dynamic changes during flower development , and the transcript levels of CpC5-MTases and CpdMTases decreased with the maturation of male and female flowers in C. paliurus (Fig. 7B). At S4, except CpDNMT2-D1 and CpDRM-D2, the expression levels of CpC5-MTases and CpdMTases were higher in female than in male flowers regardless of the mating type (Fig. 7B). Moreover, the expression levels of these genes in the same sexual floral bud were also different. Divergent gene expression levels in PA-F/M and PG-F/M at S4 implied that DNA methylation exerts different functions in the morphological development and maturation of male and female flowers. Song et al.  identified higher levels of methylation in male than in female flowers in Populus tomentosa. Xing et al.  argued that the DNA methylation pattern affected gene expression levels in apple buds with different flowering capabilities, to influence the flowering phenotype. Based on the analyzed data from C. paliurus, we speculate that the mechanisms of DNA methylation reduction in early flowering between two morphs are probably induced by: a) DNMT2, CMT3, and MET (responsible for establishing and maintaining DNA methylation) being down-regulated in PG-F; b) DNA demethylase ROS1 being up-regulated in PA-M.
In C. paliurus, Qu et al.  found that GA3 positively regulated the physiological differentiation and germination of floral buds (S2), and GA-related DEGs play central roles in regulating flower development. In addition, Meijón et al.  detected that global DNA methylation in azalea treated with a GA biosynthesis inhibitor during the floral transition could promote flowering. While we found that CpC5-MTases and CpdMTases were significantly associated with several genes related to GA biosynthesis (Fig. 8). It was demonstrated that GA20ox, KO, GID1, and DELLA are the key enzymes in the biosynthesis pathway of gibberellin, and their expression patterns can affect the content of gibberellins [21, 44]. As shown in Fig. 8, DELLA and GID1 were significantly correlated with most CpC5-MTases and CpdMTases. DELLA proteins are negative regulators of GA signaling, and DELLA binding to GA receptor GID1 could lead to the degradation of DELLA proteins and the activation of GA function [45, 46]. These key genes in GA biosynthesis and signaling pathway are regulated by epigenetic mechanisms. It has been demonstrated that DELLA protein regulates GA, brassinosteroid and jasmonic acid pathways by histone deacetylation to adjust plant growth and development [47, 48]. DELLA displayed a higher expression level in PA-F than that in PA-M at S2, indicating that the DELLA proteins suppress nutritional and reproductive growth [23, 45]. Meanwhile, we identified GA-response CREs in the promoters of these two gene families. Our results suggested that GA may affect the level of DNA methylation by regulating the promoter activities of CpC5-MTase and CpdMTase genes. Further, Zhang et al.  found that GA3 participated in the regulation of CiLFY (a central regulator of flowering), while demethylation induced CiLFY gene expression in trifoliate orange . Accordingly, locus-specific methylation analysis of CiLFY showed that the level of DNA methylation decreased during the phase change between juvenile and adult stages in precocious trifoliate orange . Thus, we suggest that the interaction between DNA methylation modifications and gibberellins levels could regulate the asynchronous flowering in heterodichogamous C. paliurus. However, the specific regulatory mechanisms need to be further verified. We will further identify the functions of CpC5MTases and CpdMTases by transient transformation, and the role of DNA methylation modification on the asynchronous floral development process in two morphs will be further revealed by using genome-editing tools.
We have identified members of C5-MTase and dMTase gene families in diploid and autotetraploid Cyclocarya paliurus. WGD or segmental duplication was the main impetus for the expansion of CpdMTase gene families during polyploidization. Furthermore, gene loss events occurred during the speciation of autotetraploid C. paliurus, and the DML and CMT1 were missed in comparison with other species. The CpC5-MTase and CpdMTase gene families were highly conserved at the DNA and protein levels during polyploidization. Combined with transcriptome data, we observed differential transcript abundance of CpC5-MTase and CpdMTase genes in female/male flowers of two morphs (PA and PG) at different stages. Collectively, we speculated that the early flowering of PG-F and PA-M may be caused by the down-regulating expression of CpC5-MTases and the up-regulating expression of CpdMTases, respectively. In addition, CpC5-MTase and CpdMTase genes were significantly associated with GA synthesis-related genes during flowering. Our results provide novel insights into the molecular mechanisms of heterodichogamy.
Materials and methods
Plant materials and transcriptome sequencing
The plants in this study were the diploid C. paliurus growing in the Baima experimental base of Nanjing Forestry University, Nanjing, Jiangsu Province, China (31°35′N, 119°09′E). According to the characteristics of floral developmental phase of floral buds , female and male floral buds in PA and PG individuals were collected at two stages: 1) S2, bud break stage, flower buds began to protrude; 2) S4, mature stage, the feathery stigma of PG-F opened and the mature pollen of PA-M was released; while the stigma of PA-F was not fully formed, and the anther of PG-M was gradual expansion (Fig. 7A). Each sample contained three biological replicates, the collected samples were frozen immediately in liquid nitrogen and stored at -80 °C before use.
The total RNA was extracted using the E.Z.N.A. plant RNA Kit (Omega, Atlanta, Georgia, USA), and converted into cDNA using the PrimeScript™ II 1st Strand cDNA Synthesis Kit (Takara, Dalian, Liaoning, China). All cDNA libraries were loaded onto an Illumina HiSeq™2000 system (2 × 100 bp read length).
Identification of the cytosine-5 DNA methyltransferase and demethylase genes
The Hidden Markov Model (HMM) of the DNA methylase domain (PF00145) was downloaded from the Pfam database (http://pfam.xfam.org) to identify the C5-MTase proteins in 3 genomes (PA-dip, PG-dip, and PA-tetra) of C. paliurus. Similarly, we downloaded the HMMs of HHH-GPD domain (PF00730) and RRM-DME domain (PF15628) from the Pfam database as the probes to search the dMTase proteins. These HMM profiles were used to perform HMM searches against proteins from C. paliurus genome via a local HMMER search program (E-value ≤ 1e−10) . The whole protein sequence of C. paliurus was obtained based on whole-genome sequencing results . To ensure the accuracy of the predicted genes, the sequences of 11 C5-MTase and 4 dMTase proteins in Arabidopsis thaliana were downloaded from the TAIR (https://www.arabidopsis.org) (Table S3) to be used as queries against C. paliurus genome database using the local BLAST program (E-value ≤ 0.001) , and further compared with conserved domains of the predicted genes using NCBI-CDD (http://ncbi.nih.gov/Structure/cdd/cdd.shtml). After the removal of redundant and incomplete sequences, we obtained the putative C5-MTase and dMTase sequences. The new-identified genes were named according to both A. thaliana homologs gene and their chromosomal location in C. paliurus. Genes from the diploid genome were labeled as “-D” and those from autotetraploid as “-T”.
The ExPASy software (http://web.expasy.org/protparam) was used to analyze the grand average of hydrophobicity (GRAVY), molecular weight (MW), and isoelectric point (pI) of CpC5-MTase and CpdMTase proteins. The subcellular localization of CpC5-MTase and CpdMTase was further predicted using the WoLF PSORT server (https://wolfpsort.hgc.jp/).
Phylogenetic tree construction
The MEGA 11  was used to investigate the phylogenetic interactions of C5-MTase and dMTase proteins between C. paliurus and other three species (A. thaliana, O. sativa, and P. trichocarpa) (Table S3). The protein sequences of the other three species downloaded from both UniProt and NCBI Protein databases, were utilized to construct a phylogenetic tree using MEGA 11 software according to the Maximum Likelihood (ML) method with 1000 bootstrap replicates. Visualization of the phylogenetic tree was accomplished by the Interactive Tree of Life (iTOL) online phylogeny tool .
Chromosome localization and synteny analysis
The distribution information of CpC5-MTase and CpdMTase genes on the chromosomes of C. paliurus was analyzed using TBtools software (GitHub, San Francisco, CA, USA) , then named according to their chromosome orders. The synteny relationships of CpC5-MTase and CpdMTase genes between diploidy and autotetraploidy were exhibited using the TBtools and Multiple Collinearity Scan toolkit (MCScanX) . The nonsynonymous (Ka) and synonymous (Ks) of each duplicated gene pair of two gene families were calculated by the TBtools [28, 53].
Conserved motif and gene structure analysis
Conserved motifs of all CpC5-MTase and CpdMTase proteins were analyzed using Multiple Expectation Maximization for the Motif Elicitation (MEME, http://meme-suite.org/) . Gene structure analysis was conducted with the Gene Structure Display Server tool (GSDS, http://gsds.cbi.-pku.edu.cn/index.php) . Then, the 2000-bp upstream sequence of C5-MTase and dMTase genes was extracted by TBtools, and the cis-acting regulatory element was predicted on the PlantCARE website (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/) [53, 57].
Gene expression analysis
To gain insight into the expression patterns of CpC5-MTase and CpdMTase in C. paliurus, we analyzed transcriptomic data obtained by our research group earlier, including male and female flower buds of both mating types in diploid C. paliurus at S2 and S4, respectively . RNA-seq reads were obtained from the Genome Sequence Archive (SRA) database under accession numbers CRA002980 and CRA016788. The expression levels of all transcripts were obtained by calculating fragments per kilobase per million (FPKM) with the StringTie software .
Identification and correlation analysis of genes related to heterodichogamy
MADS14 (CpaM1st25222) and MADS09 (CpaM1st23860) were highly expressed in PA-M and PG-F, respectively . Trihelix-1 (CpaM1st06815), ERF066 (CpaM1st16083), ERF090 (CpaM1st01501), WRKY55 (CpaM1st00114), and some genes related to GA synthesis were found that they have functions in the heterodichogamy characteristics, including CPS (CpaM1st44543), KO (CpaM1st25482), GA3ox (CpaM1st52632), GA2ox (CpaM1st19769), GA20ox (CpaM1st23650), GID1 (CpaM1st32729), and DELLA (CpaM1st34996) [17, 28]. Pearson correlation coefficient between genes related to heterodichogamy and CpC5-MTase and CpdMTase gene families were analyzed and plotted with Origin software.
Availability of data and materials
The datasets supporting the conclusions of this article are included within the article and its additional files. The flower buds transcriptome raw datasets were deposited in the Genome Sequence Archive (SRA) database (https://ngdc.cncb.ac.cn) under the accession number CRA002980 and CRA016788. The three genome sequences of C. paliurus (PA-dip, PG-dip, and PA-tetra) were from the Genome Warehouse at the NGDC, BIG, CAS / CNCB (GWH: GWHBKKW00000000, GWHBKKX00000000, and GWHBKKY00000000), and are publicly accessible at https://ngdc.cncb.ac.cn/gwh/.
Cytosine-5 DNA methyltransferase
Domain rearranged methyltransferase 2
RNA-directed DNA methylation
DNA methyltransferase 2
Repressor of silence 1
Stage of floral bud break
Stage of flower maturation
Protogynous of diploid plant
Protandrous of diploid plant
Protandrous of autotetraploid plant
Female floral buds from a protandrous plant
Male floral buds from a protandrous plant
Male floral buds from a protogynous plant
Female floral buds from a protogynous plant
Open reading frame; pI: Isoelectric point
Grand average of hydrophobicity
Hidden Markov Model
Multiple expectation maximization for motif elicitation
Non-synonymous substitution rate
Synonymous substitution rate
Fragments per kilobase per million
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We thank all participants of the present study and Qiang Lu for revising the manuscript.
This research was funded by the National Natural Science Foundation of China (project number: 32271859) and the Postgraduate Research and Practice Innovation Program of Jiangsu Province (No. KYCX22_1109). The funding bodies had no role in the design of the study and collection, analysis, and interpretation of data in writing the manuscript.
Ethics approval and consent to participate
The materials of Cyclocarya paliurus in this experiment were collected from the C. paliurus germplasm bank of the College of Forestry, Nanjing Forestry University, located in the Baima experimental base of Nanjing Forestry University, Nanjing, Jiangsu Province, China. The plant materials don’t include any wild species at risk of extinction, no special licenses were needed. We comply with relevant institutional, national, and international guidelines and legislation for plant study.
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Wang, Q., Qu, Y., Yu, Y. et al. Genome-wide identification and comparative analysis of DNA methyltransferase and demethylase gene families in two ploidy Cyclocarya paliurus and their potential function in heterodichogamy. BMC Genomics 24, 287 (2023). https://doi.org/10.1186/s12864-023-09383-5
- DNA methylase
- DNA demethylase