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Dioecious plants have evolved sex-specific floral development mechanisms. However, the precise gene expression patterns in dioecious plant flower development remain unclear. Here, we used andromonoecious poplar, an exceptional model system, to eliminate the confounding effects of genetic background of dioecious plants. Comparative transcriptome and physiological analysis allowed us to characterize sex-specific development of female and male flowers. Transcriptome analysis identified genes significantly differentially expressed between the sexes, including genes related to floral development, phytohormone synthesis and metabolism, and DNA methylation. Correlation analysis revealed a significant correlation between phytohormone signaling and gene expression, identifying specific phytohormone-responsive genes and their cis-regulatory elements. Two genes related to DNA methylation, METHYLTRANSFERASE1 (MET1) and DECREASED DNA METHYLATION 1 (DDM1), which are located in the sex determination region of Chromosome XIX, have differential expression between female and male flowers. A time-course analysis revealed that MET1 and DDM1 expression may produce different DNA methylation levels in female and male flowers. Understanding the interactions of phytohormone signaling, DNA methylation and target gene expression should lead to a better understanding of sexual differences in floral development. Thus, this study identifies a set of candidate genes for further studies of poplar sexual dimorphism and relates sex-specific floral development to physiological and epigenetic changes.

Background: It is thought that methylcytosine can be inherited through meiosis and mitosis, and that epigenetic variation may be under genetic control or correlation may be caused by neutral drift. However, DNA methylation also varies with tissue, developmental stage, and environmental factors. Eliminating these factors, we analyzed the levels and patterns, diversity and structure of genomic methylcytosine in the xylem of nine natural populations of Chinese white poplar., Principal Findings: On average, the relative total methylation and non-methylation levels were approximately 26.567% and 42.708% (P<0.001), respectively. Also, the relative CNG methylation level was higher than the relative CG methylation level. The relative methylation/non-methylation levels were significantly different among the nine natural populations. Epigenetic diversity ranged from 0.811 (Gansu) to 1.211 (Shaanxi), and the coefficients of epigenetic differentiation (GST  = 0.159) were assessed by Shannon's diversity index. Co-inertia analysis indicated that methylation-sensitive polymorphism (MSP) and genomic methylation pattern (CG-CNG) profiles gave similar distributions. Using a between-group eigen analysis, we found that the Hebei and Shanxi populations were independent of each other, but the Henan population intersected with the other populations, to some degree., Conclusions: Genome methylation in Populus tomentosa presented tissue-specific characteristics and the relative 5'-CCGG methylation level was higher in xylem than in leaves. Meanwhile, the genome methylation in the xylem shows great epigenetic variation and could be fixed and inherited though mitosis. Compared to genetic structure, data suggest that epigenetic and genetic variation do not completely match.

Unlabelled: The andromonoecious poplar is an exceptional model system for studying sex-specific flower development in dioecious plants. There is increasing evidence that epigenetic regulation, particularly DNA methylation, is an important regulatory factor during flower development. Here, methylation-sensitive amplified polymorphism (MSAP) was used to screen for sex-specific DNA methylation alterations in the andromonoecious poplar. The sequences of 27 sex-specific amplified fragments were obtained from DNA prepared from sex-specific flower tissues. PtGT2, PtPAL3, and PtCER4, which are homologous to MF26, MF29, and MF35, respectively, were cloned as candidate genes. Expression analysis and DNA methylation pattern profiling of the three candidate genes revealed that gene expression upregulation was always associated with gene body methylation. The results suggested that DNA methylation sites have the potential to regulate the genes' transcript levels. These three genes were shown to play important roles during different phases of flower development. This study will help to provide candidates for future experiments aimed at understanding the mechanism, whereby DNA methylation regulates gene expression in poplar., Key Messages: We report the first screen for sex-specific DNA methylation alterations in the andromonoecious poplar. 27 sex-specific methylation sites were identified. The gene expression levels and DNA methylation patterns were detected for three candidate genes.

DNA methylation is a common epigenetic modification involved in regulating many biological processes. However, the epigenetic mechanisms involved in the formation of floral scent have rarely been reported within a famous traditional ornamental plant Prunus mume emitting pleasant fragrance in China. By combining whole-genome bisulfite sequencing and RNA-seq, we determined the global change in DNA methylation and expression levels of genes involved in the biosynthesis of floral scent in four different flowering stages of P. mume. During flowering, the methylation status in the "CHH" sequence context (with H representing A, T, or C) in the promoter regions of genes showed the most significant change. Enrichment analysis showed that the differentially methylated genes (DMGs) were widely involved in eight pathways known to be related to floral scent biosynthesis. As the key biosynthesis pathway of the dominant volatile fragrance of P. mume , the phenylpropane biosynthesis pathway contained the most differentially expressed genes (DEGs) and DMGs. We detected 97 DMGs participated in the most biosynthetic steps of the phenylpropane biosynthesis pathway. Furthermore, among the previously identified genes encoding key enzymes in the biosynthesis of the floral scent of P. mume , 47 candidate genes showed an expression pattern matching the release of floral fragrances and 22 of them were differentially methylated during flowering. Some of these DMGs may or have already been proven to play an important role in biosynthesis of the key floral scent components of P. mume , such as PmCFAT1a / 1c , PmBEAT36 / 37 , PmPAL2 , PmPAAS3 , PmBAR8 / 9 / 10 , and PmCNL1 / 3 / 5 / 6 / 14 / 17 / 20. In conclusion, our results for the first time revealed that DNA methylation is widely involved in the biosynthesis of floral scent and may play critical roles in regulating the floral scent biosynthesis of P. mume. This study provided insights into floral scent metabolism for molecular breeding. [ABSTRACT FROM AUTHOR]

Increasing evidence shows that epigenetics plays an important role in phenotypic variance. However, little is known about epigenetic variation in the important ornamental tree Prunus mume. We used amplified fragment length polymorphism (AFLP) and methylation-sensitive amplified polymorphism (MSAP) techniques, and association analysis and sequencing to investigate epigenetic variation and its relationships with genetic variance, environment factors, and traits. By performing leaf sampling, the relative total methylation level (29.80%) was detected in 96 accessions of P. mume. And the relative hemi-methylation level (15.77%) was higher than the relative full methylation level (14.03%). The epigenetic diversity (I* = 0.575, h* = 0.393) was higher than the genetic diversity (I =0.484, h = 0.319). The cultivated population displayed greater epigenetic diversity than the wild populations in both southwest and southeast China. We found that epigenetic variance and genetic variance, and environmental factors performed cooperative structures, respectively. In particular, leaf length, width and area were positively correlated with relative full methylation level and total methylation level, indicating that the DNA methylation level played a role in trait variation. In total, 203 AFLP and 423 MSAP associated markers were detected and 68 of them were sequenced. Homologous analysis and functional prediction suggested that the candidate marker- linked genes were essential for leaf morphology development and metabolism, implying that these markers play critical roles in the establishment of leaf length, width, area, and ratio of length to width. [ABSTRACT FROM AUTHOR]

Dioecious plants have evolved sex-specific floral development mechanisms. However, the precise gene expression patterns in dioecious plant flower development remain unclear. Here, we used andromonoecious poplar, an exceptional model system, to eliminate the confounding effects of genetic background of dioecious plants. Comparative transcriptome and physiological analysis allowed us to characterize sex-specific development of female and male flowers. Transcriptome analysis identified genes significantly differentially expressed between the sexes, including genes related to floral development, phytohormone synthesis and metabolism, and DNA methylation. Correlation analysis revealed a significant correlation between phytohormone signaling and gene expression, identifying specific phytohormone-responsive genes and their cis-regulatory elements. Two genes related to DNA methylation, METHYLTRANSFERASE1 ( MET1) and DECREASED DNA METHYLATION 1 ( DDM1), which are located in the sex determination region of Chromosome XIX, have differential expression between female and male flowers. A time-course analysis revealed that MET1 and DDM1 expression may produce different DNA methylation levels in female and male flowers. Understanding the interactions of phytohormone signaling, DNA methylation and target gene expression should lead to a better understanding of sexual differences in floral development. Thus, this study identifies a set of candidate genes for further studies of poplar sexual dimorphism and relates sex-specific floral development to physiological and epigenetic changes. [ABSTRACT FROM AUTHOR]

Growth and wood formation are crucial and complex biological processes during tree development. These biological regulatory processes are presumed to be controlled by DNA methylation. However, there is little direct evidence to show that genes taking part in wood regulation are affected by cytosine methylation, resulting in phenotypic variations. Here, we detected epimarkers using a methylation-sensitive amplification polymorphism (MSAP) method and performed epimarker–trait association analysis on the basis of nine growth and wood property traits within populations of 432 genotypes of Populus tomentosa. Tree height was positively correlated with relative full-methylation level, and 1101 out of 2393 polymorphic epimarkers were associated with phenotypic traits, explaining 1.1–7.8% of the phenotypic variation. In total, 116 epimarkers were successfully sequenced, and 96 out of these sequences were linked to putative genes. Among them, 13 candidate genes were randomly selected for verification using quantitative real-time PCR (qRT-PCR), and it also showed the expression of nine putative genes of PtCYP450, PtCpn60, PtPME, PtSCP, PtGH, PtMYB, PtWRKY, PtSTP, and PtABC were negatively correlated with DNA methylation level. Therefore, it suggested that changes in DNA methylation might contribute to regulating tree growth and wood property traits. [ABSTRACT FROM AUTHOR]