DNA methylation analysis of floral parts revealed dynamic changes during the development of homostylous Fagopyrum tataricum and heterostylous F. esculentum flowers

Abstract

Background: Proper flower development is essential for plant reproduction, a crucial aspect of the plant life cycle. This process involves precisely coordinating transcription factors, enzymes, and epigenetic modifications. DNA methylation, a ubiquitous and heritable epigenetic mechanism, is pivotal in regulating gene expression and shaping chromatin structure. Fagopyrum esculentum demonstrates anti-hypertensive, anti-diabetic, anti-inflammatory, cardio-protective, hepato-protective, and neuroprotective properties. However, the heteromorphic heterostyly observed in F. esculentum poses a significant challenge in breeding efforts. F. tataricum has better resistance to high altitudes and harsh weather conditions such as drought, frost, UV-B radiation damage, and pests. Moreover, F. tataricum contains significantly higher levels of rutin and other phenolics, more flavonoids, and a balanced amino acid profile compared to common buckwheat, being recognised as functional food, rendering it an excellent candidate for functional food applications.

Results: This study aimed to compare the DNA methylation profiles between the Pin and Thrum flower components of F. esculentum, with those of self-fertile species of F. tataricum, to understand the potential role of this epigenetic mechanism in Fagopyrum floral development. Notably, F. tataricum flowers are smaller than those of F. esculentum (Pin and Thrum morphs). The decline in DNA methylation levels in the developed open flower components, such as petals, stigmas and ovules, was consistent across both species, except for the ovule in the Thrum morph. Conversely, Pin and Tartary ovules exhibited a minor decrease in DNA methylation levels. The highest DNA methylation level was observed in Pin stigma from closed flowers, and the most significant decrease was in Pin stigma from open flowers. In opposition, the nectaries of open flowers exhibited higher levels of DNA methylation than those of closed flowers. The decrease in DNA methylation might correspond with the downregulation of genes encoding methyltransferases.

Conclusions: Reduced overall DNA methylation and the expression of genes associated with these epigenetic markers in fully opened flowers of both species may indicate that demethylation is necessary to activate the expression of genes involved in floral development.

Keywords: Fagopyrum esculentum; Fagopyrum tataricum; DNA methylation; Epigenetics; Flowers; Gene expression; Heterostyly.

Fig. 1

Morphology of analysed flowers. F. esculentum (a, d) Pin, (b, e) Thrum, and (c, f) F. tataricum. (a – c) closed flowers were tightly coiled in petals. (d – f) In open flowers, the anatomy is easily visible. Arrows – stigmas, double arrows – nectaries, open arrows – anthers, o – ovaries, p – petals. Scale bars: (a–f) = 300 μm

Fig. 2

Histology of closed flower components (F. esculentum Pin, Thrum; F. tataricum). Schematic diagrams represent analysed flower components: I petals, II stigma, III ovary with ovule, IV nectary, marked in pink on the diagram. (a - c) petals, abundant polyphenols occurrence in epidermis (arrowheads) and mesophyll cells (arrows). (d - f) stigmas, papillae and subpapillae cells rich in polyphenols (arrowheads) but with visible nuclei (arrows). (g - l) ovaries and ovules, polyphenols present in basal part of ovule and in outer integument cells (arrowheads), arrows – nucellus parenchyma cells, embryo sacs (asterisks); double arrow stands for the extent of cells that were taken into account in the measurement of Alexa488 fluorescence intensity. (m - o) nectaries, epidermis rich in polyphenols (arrowheads), secretory trichomes (arrows), np nectary parenchyma. Scale bars: a, d, e, j, k = 10 μm; b, f, g, h, l, m – o = 20 μm; c, i = 50 μm

Fig. 3

Histology of open flower components (F. esculentum Pin, Thrum; F. tataricum). Schematic diagrams represent analysed flower components: I petals, II stigma, III ovary with ovule, IV nectary, marked in pink on the diagram. (a – c) petals, abundant occurrence of polyphenols in the epidermis (arrowheads) and mesophyll cells (arrows). (d – f) stigmas, papillae and subpapillae cells containing polyphenols (arrowheads) and hardly visible nuclei (arrows). (g – i) ovaries and ovules, high polyphenolic content in the basal part of the ovule and in outer integument cells (arrowheads), arrows – nucellus parenchyma cells, embryo sacs (asterisks); double arrow stands for the extent of cells that were taken into account in the measurement of Alexa488 fluorescence intensity. (j – l) nectaries, epidermis rich in polyphenols (arrowheads), secretory trichomes (arrows), np nectary parenchyma. Scale bars: b = 10 μm; a, d – f, i, j = 20 μm; c, k, l = 50 μm; g, h = 100 μm

Fig. 4

Fluorescence intensity measurements of DNA methylation levels. (a) petal; n = 500, (b) stigma, n = 1000, (c) ovule, n = 1500, and (d) nectary, n = 1000 parts of the closed and the open flowers of F. esculentum and F. tataricum. Bars represent standard error; results presented in relative units, letters indicate statistically significant differences between the groups

Fig. 5

Expression level of MET1, MET2, CMET3, DME1, DME3, and ROS1 in closed and open flowers, (a, d) Thrum and (b, d) Pin of F. esculentum and (c, d) F. tataricum. The expression level of genes in open flowers was calibrated to expression in closed flowers of the same type and species (a, b, c). * - values significantly different from closed flowers of the same type and species (a, b,c) (p < 0.05; n = 3; means ± SD are given). The expression level of genes in all types and species was calibrated to expression in closed flowers of the F. tataricum (d). Different letters indicate a significant difference between flower type and species according to Tukey’s HSD test (p < 0.05; n = 3; means ± SD are given)