Epigenetic aspects of floral homeotic genes in relation to sexual dimorphism in the dioecious plant Mercurialis annua

Abstract

In plants, dioecy characterizes species that carry male and female flowers on separate plants and it occurs in about 6% of angiosperms; however, the molecular mechanisms that underlie dioecy are essentially unknown. The ability for sex-reversal by hormone application raises the hypothesis that the genes required for the expression of both sexes are potentially functional but are regulated by epigenetic means. In this study, proteomic analysis of nuclear proteins isolated from flower buds of females, males, and feminized males of the dioecious plant Mercurialis annua revealed differential expression of nuclear proteins that are implicated in chromatin structure and function, including floral homeotic proteins. Focusing on floral genes, we found that class B genes were mainly expressed in male flowers, while class D genes, as well as SUPERMAN-like genes, were mainly expressed in female flowers. Cytokinin-induced feminization of male plants was associated with down-regulation of male-specific genes concomitantly with up-regulation of female-specific genes. No correlation was found between the expression of class B and D genes and the changes in DNA methylation or chromatin conformation of these genes. Thus, we could not confirm DNA methylation or chromatin conformation of floral genes to be the major determinant regulating sexual dimorphisms. Instead, determination of sex in M. annua might be controlled upstream of floral genes by one or more sex-specific factors that affect hormonal homeostasis. A comprehensive model is proposed for sex-determination in M. annua.

Keywords: Mercurialis annua; Chromatin; DNA methylation; cytokinin; dioecy; epigenetics; feminization; floral homeotic gene; nuclear proteome; sex-determination.

Fig. 1.

Morphology of dioecious Mercurialis annua. (A) Female plant, (B) male inflorescence, and (C) feminized male inflorescence, induced by spraying plants with 6-benzylaminopurine three times daily for 4 weeks. Feminized males produced bi-carpellet flowers, some of which are indicated by arrows.

Fig. 2.

Proteome analysis of nuclei of Mercurialis annua isolated from flower buds of female, male, and feminized male plants (treated with 6-benzylaminopurine, BAP). (A) Down-regulated and (B) up-regulated proteins following male feminization. (C-F) The label-free quantification (LFQ) intensity reflecting the relative amounts of the indicated proteins, which were calculated using peptide intensities normalized between the samples (the corresponding Arabidopsis homolog gene ID is given in brackets). F, female; M, male. (This figure is available in colour at JXB online.)

Fig. 3.

Expression of MADS-box genes in flower buds and open flowers of female and male plants of Mercurialis annua. Relative expression of (A) MaPI, (B) MaAP3, (C) MaAG1, (D) MaAGL1, and (E) MaAGL3 determined using RT-qPCR. The relative transcript levels are normalization to the Actin gene. Data are means (±SE) of three biological replicates. Significant differences between means are indicated by different numbers of asterisks as determined by Tukey’s HSD test (P<0.05). (This figure is available in colour at JXB online.)

Fig. 4.

Expression patterns of floral homeotic genes in different organs of female and male plants of Mercurialis annua. Expression of class B, C, D, and SUPERMAN-like (SUP) genes was determined using semi-quantitative PCR using cDNAs derived from RNA extracted from the various plant organs. Actin was used as a ubiquitously expressed reference gene. M, molecular size markers.

Fig. 5.

Time-course of the expression of floral genes in Mercurialis annua during feminization induced by treatment with 6-benzylaminopurine (BAP). Plants at 25 d old were sprayed three times daily with water (Control) or with BAP and newly emerging inflorescences were collected on the days indicated. RNA was extracted and subjected to cDNA synthesis, which was then used to determine expression using semi-quantitative PCR. Class B, C, D, and SUPERMAN-like (SUP) genes are indicated. Actin was used as the reference gene.

Fig. 6.

Transcriptionally active floral genes are methylated in both females and males of Mercurialis annua. (A) Analysis of DNA methylation at the promoters (-P) of MaAGL1, MaSL1, and MaSL2 by as determined by chop-PCR. A fragment of MaSL1 lacking the CCGG site (no CCGG) was used as an internal control. Left panel is a control of undigested DNA (Ud). Ud, Undigested DNA; H, HpaII; M, MspI; L, molecular size markers in base pairs. (B) Analysis of methylation at the promoter and in the gene-body of MaAP3 and MaSL1 as determined by bisulfite sequencing. The percentage of cytosine methylation for each fragment was determined from at least 10 different clones. (This figure is available in colour at JXB online.)

Fig. 7.

Analysis of chromatin configuration of selected floral genes in Mercurialis annua as determined by micrococcal nuclease assays. (A) Nuclei prepared from male and feminized male flower buds (treated with 6-benzylaminopurine for 14 d, before female flowers were visible) were treated with MNase for the time periods indicated. DNA was extracted from the treated nuclei and resolved on 1.5% agarose gel. M, molecular size markers, in base pairs. (B) Assessment of chromatin configuration of promoters as determined by PCR using DNA recovered from the MNase-treated nuclei shown in (A). Group I refers to male-related identity genes and Group II refers to female-related identity genes. Actin was used as the reference for open chromatin configuration. M, molecular size markers.

Fig. 8.

A proposed model of sex determination in the dioecious plant Mercurialis annua. Differentiation of unisexual flowers is controlled genetically/epigenetically by as yet unknown sex-determining genes that affect hormonal homeostasis. A high cytokinin/auxin ratio activates the transcription of effector genes such as female-identity class D genes (MaAGL1 and MaAGL3). The class D proteins together with class C and class E proteins promote female flower development. Alternatively, a high auxin/cytokinin ratio is presumed to lead to transcriptional activation of male-identity class B genes (MaPI, MaAP3, and MaTM6). The class B proteins together with class C (MaAG1) and class E (MaSEP1–4) promote male flower development. Exogenous application of cytokinin feminizes males by inducing down-regulation of class B genes and up-regulation of class D genes. Exogenous auxin masculinizes females (Delaigue et al., 1984), probably via up-regulation of class B genes concomitantly with down-regulation of class D genes. (This figure is available in colour at JXB online.)