The DME demethylase regulates sporophyte gene expression, cell proliferation, differentiation, and meristem resurrection

Abstract

The flowering plant life cycle consists of alternating haploid (gametophyte) and diploid (sporophyte) generations, where the sporophytic generation begins with fertilization of haploid gametes. In Arabidopsis, genome-wide DNA demethylation is required for normal development, catalyzed by the DEMETER (DME) DNA demethylase in the gamete companion cells of male and female gametophytes. In the sporophyte, postembryonic growth and development are largely dependent on the activity of numerous stem cell niches, or meristems. Analyzing Arabidopsis plants homozygous for a loss-of-function dme-2 allele, we show that DME influences many aspects of sporophytic growth and development. dme-2 mutants exhibited delayed seed germination, variable root hair growth, aberrant cellular proliferation and differentiation followed by enhanced de novo shoot formation, dysregulation of root quiescence and stomatal precursor cells, and inflorescence meristem (IM) resurrection. We also show that sporophytic DME activity exerts a profound effect on the transcriptome of developing Arabidopsis plants, including discrete groups of regulatory genes that are misregulated in dme-2 mutant tissues, allowing us to potentially link phenotypes to changes in specific gene expression pathways. These results show that DME plays a key role in sporophytic development and suggest that DME-mediated active DNA demethylation may be involved in the maintenance of stem cell activities during the sporophytic life cycle in Arabidopsis.

Keywords: DNA demethylation; cell proliferation; pluripotency; sprorophytic development.

Fig. 1.

dme-2 homozygous seeds are large and dme-2 showed a higher abortion ratio than dme-1 plants. (A) dme-2 homozygous mutant silique with aborted seeds. (B) Comparison of the seed abortion ratio of F3 progenies from dme-1/dme-2 F1 progenitors. The total number of seeds counted for dme-1/dme-1, dme-1/dme-2, and dme-2/dme-2 was 1,306, 1,370, and 1,284, respectively, as shown in SI Appendix, Table S1. (C) Comparison of dme-2 aborting seed with wild-type Ler from the same silique (200x magnification). Black arrowhead, embryo; white arrowhead, endosperm. (Scale bar, 100 µm.) (D) Viable seeds from wild type and dme-2 homozygous mutants. (Scale bar, 400 µm.) (E) Average length of mature seeds from wild type and dme-2 mutants. Individual seed length of the major axis is shown in SI Appendix, Table S7. Data represent means ± SD in B and E.

Fig. 2.

RNA-seq analysis of Ler and dme-2 mutant seedlings. (A) Volcano plot visualizing differentially expressed genes between dme-2 mutant and Ler wild type. The comparison was made using the DESeq2 R package with untransformed RNA-seq read counts. Data from three replicates per sample were used. Totals of 3,874 genes and 1,448 genes are significantly up-regulated and down-regulated, respectively, in dme-2 mutants (log2 fold change >1, FDR <0.05). (B) Venn diagram of genes regulated by DME and ROS1. Genes up-regulated and down-regulated in dme-2 seedlings compared to wild-type Ler are in blue and yellow, respectively. Genes up-regulated and down-regulated in ros1-4 seedlings compared to wild-type Columbia are in green and magenta, respectively. ros1-4 data are from ref. . (C) Venn diagram of genes regulated by DME and RDD. Genes up-regulated and down-regulated in dme-2 seedlings compared to wild-type Ler are in blue and yellow, respectively. Genes up-regulated and down-regulated in rdd immature floral buds compared to wild-type Columbia are in green and magenta, respectively. rdd data are from ref. . All genes shown in the Venn diagram are twofold changed and FDR <0.05.

Fig. 3.

Seed germination comparison between wild type and dme-2 mutant in continuous cold conditions. (A) Sequential stages of wild-type seed germination. (Scale bar, 400 µm.) (B) Distribution ratios of germination stages of wild-type and dme-2 seeds in continuous light with cold condition. Brown, nongerminated seed; yellow, seed-coat rupture; green, endosperm rupture. The counted numbers are shown in SI Appendix, Table S8. (C) Comparison of endosperm rupture. X = DAG, Y = (endosperm ruptured seeds/all seeds) x 100. (D) Comparison of overall germination ratio. X = DAG, Y = (ruptured seeds/all seeds) x 100.

Fig. 4.

dme mutants show an abnormal RAM with a disorganized quiescent center. (A) Wild-type Ler root development from DAG2 to DAG5, showing natural QC division onset. (B) Major phenotype observed in dme mutant roots. (C) Frequency of abnormal roots. The numbers of primary roots displayed abnormally distorted QC versus normal roots are in SI Appendix, Table S5. (D) Reduced expression of CCS52A gene in dme-2 mutants. Data represent means ± SD.

Fig. 5.

DME suppresses root hair growth. (A and B) Comparison of root hair length in Ler and dme-2 mutants. Data represent mean ± SEM, n = 517 root hairs for Ler and n = 355 root hairs for dme-2 from approximately 20 ∼ 29 roots. The values are from two biological replicates and are significantly different (*P < 0.0001; Student’s t test). (Scale bar, 100 μm.) (C) Relative expression levels of the RHD6 gene from RNA-seq, plotted as TPM value (transcripts per kilobase million, number of transcripts scaled using the average transcript length over samples and then the library size). (D) Relative expression levels of RSL4 from the RNA-seq plotted as TPM value. ** in C and D, FDR-adjusted P value < 0.001 by DESeq2. Data represent means ± SD.

Fig. 6.

DME affects the determination of stomatal precursor cells. (A) Stomata (green) and stomatal precursor cell (light blue) of adaxial leaf and overall SEM visualization (white spots show stomata). (Scale bar, 20 µm.) (B) Normalized counted cell number of stomata and precursor cell in DAG3 seedlings. Pictures in A were used for analysis, n = 4 (*P < 0.02, Student’s t test). (C) qRT-PCR of stomatal regulatory genes, EPF2 and EPF1 (*P < 0.02, **P < 0.001, Student’s t test). Data represent means ± SD.

Fig. 7.

Earlier bolting and resurrection after termination in dme mutants. (A) Rosette leaf counting on flowering (n = 25 for both Ler and dme-2 plants). (B) Flowering time and growth differences between Ler and dme-2 mutants. (C) Growth plot of main stem (n = 16) of each genotype. Resurrection (bloom after the first termination) makes a difference in the final height of the main stem at DAG65. (D) Number of siliques generated from the resurrection (n = 16, *P < 0.001, Student’s t test). (E) Resurrected green siliques are shown on top of the dried terminated IM in dme mutants. Data in A and D represent means ± SD.

Fig. 8.

Increased callus formation and de novo shoot regeneration in dme-2. (A) Callus formation. Leaf explants from the third leaves of 2‐wk‐old plants were used to induce callus on callus‐inducing medium (CIM) (n > 30). Plates were incubated for 2 wk under continuous dark conditions and photographed (Left). Thirty calli dissected from leaf explants were collected to measure fresh weight (Right). (B) De novo shoot regeneration. Calli preincubated for 6 d on CIM were used to induce shoot regeneration on SIM (n > 30). Plates were incubated for 3 wk under continuous light conditions and photographed (Left). The number of regenerated shoots from calli was measured (Right). In A and B, three independent measurements were averaged. Statistically significant differences between wild‐type and mutant calli are indicated by asterisks (Student’s t test, *P < 0.05, ***P < 0.001). Bars indicate ± SEM. (C) Some key regulatory genes involved in pluripotency and shoot formation are overexpressed in dme-2 mutant compared to wild type. RNA-seq analyses were performed as described in Materials and Methods and differentially expressed genes analyzed with DESeq2. The bars represent the average fold expression changes with SEs. (D) A heatmap of genes involved in callus regeneration, pluripotency, and de novo shoot formation in dme-2 mutants and Ler wild type. TPM (transcripts per kilobase million) counts were log2(x + 1) transformed. The Far Left is Min(logTPM + 1) and the Far Right is Max(logTPM + 1). Min and Max mean the minimum and maximum value of log TPM(normalized read count) of the total genes in the figure. Blue color corresponds to a decreasing transcript abundance, while red color corresponds to an increasing transcript abundance. (E) A heatmap of significant gene expression (DESeq2, padj of 0.05) that are up-regulated in dme-2 mutants involved in cytokinin biosynthesis and signaling. Transcriptome reads were log2(x + 1) transformed.