Histone Demethylases ELF6 and JMJ13 Antagonistically Regulate Self-Fertility in Arabidopsis

Abstract

The chromatin modification H3K27me3 is involved in almost every developmental stage in Arabidopsis. Much remains unknown about the dynamic regulation of this histone modification in flower development and control of self-fertility. Here we demonstrate that the H3K27me3-specific demethylases ELF6 and JMJ13 antagonistically regulate carpel and stamen growth and thus modulate self-fertility. Transcriptome and epigenome data are used to identify potential targets of ELF6 and JMJ13 responsible for these physiological functions. We find that ELF6 relieves expansin genes of epigenetic silencing to promote cell elongation in the carpel, enhancing carpel growth and therefore encouraging out-crossing. On the other hand, JMJ13 activates genes of the jasmonic acid regulatory network alongside the auxin responsive SAUR26, to inhibit carpel growth, enhance stamen growth, and overall promote self-pollination. Our evidence provides novel mechanisms of self-fertility regulation in A. thaliana demonstrating how chromatin modifying enzymes govern the equilibrium between flower self-pollination and out-crossing.

Keywords: chromatin regulation; epigenetics; flower development; histone demethylases; histone modification; self-fertility.

Figure 1

Fertility phenotypes of H3K27me3 demethylase knock-out mutants. (A) Varying degrees of self-fertility in the demethylase knock-out mutants. The white arrow heads point to aborted siliques caused by failed self-pollination, as seen in the first flower of Col-0 and to the eighth flower of jmj13. Scale bars represent 1 cm. (B) Quantification of self-fertility. Numbers of aborted siliques are plotted with asterisks indicating which data point is statistically significant as compared to that of Col-0 (p < 0.05, t-test). The dashed horizontal line represents the median y-axis value of Col-0.

Figure 2

Quantification of floral organ lengths in H3K27me3 knock-out mutants. (A) Representative images of stage 14 flowers of the indicated genotypes from flower positions 2 and 4 (scale bars represent 1 mm). Black arrow heads indicate the lowest point of the stigma closest to the receptacle, whilst white arrow heads indicate highest point of the tallest stamen, the difference of which correlates with self-fertility. (B) The absolute heights of stamen and stigma from Col-0, elf6, and jmj13 flowers. Measurements were taken from receptacle to the top of the highest stamen and to the bottom of the stigma. n >= 20 for each genotype in each flower position. An asterisk indicates statistical significance compared to the respective Col-0 data of the same floral organ and flower position group (p <= 0.05, t-test). (C) The lengths from receptacle to the tip of the tallest stamen, and receptacle to the lowest point of the stigma were measured. The difference between these two lengths is plotted for every flower measured at flower positions 1–2 and distributed by genotype. Groups marked with an asterisk are statistically significantly different from the Col-0 stamen/stigma height differences (p < 0.05, t-test). The violin graph represents the density of data point distribution displayed symmetrically along the y axis. n >= 20 for each genotype. (D) Stamen-stigma height differences were measured and plotted as in (C) but for flower positions 3–6. n >= 20 for each genotype.

Figure 3

Transcriptome changes in the bud in elf6, ref6 and jmj13 knock-out mutants compared to Col-0. (A) Transcriptomes of elf6, jmj13, and ref6 buds compared after differential expression analysis against the transcriptome of Col-0 buds. The significance thresholds drawn in dashed lines represent a 2-fold change in expression and a probability score > 0.8. Genes over these thresholds are classed as differentially expressed and colored in lime green, forest green and cyan dependent on the genotype. (B) DEGs extracted from the elf6 and jmj13 transcriptomes. They are split into up and down regulated DEGs and represented as individual venn groups, with the shared group representing genes that are either differentially up or down regulated in both elf6 and jmj13 buds compared to Col-0.

Figure 4

Effects of elf6 knock-out on carpel epidermal cell length. (A) Confocal microscopy of stage 14 carpel mid sections using propidium iodide staining to highlight individual cells. The image on the left is taken from a Col-0 carpel, whilst the right hand image is from elf6. White arrow heads indicate the epidermal cell layer. Scale bar depicts 60 μm. (B) Measurements of cell length in carpel epidermis. A significant 15% reduction in mean epidermal cell length was observed in elf6 carpels compared to Col-0 carpels (n >= 3 carpels per genotype, n >= 95 cells measured per genotype, p <= 0.05, t-test). The dashed line represents the mean carpel epidermal cell length of Col-0 carpels. The asterisk denotes a significant change in cell length between the indicated genotypes (p <= 0.05, t-test).