AGL15 Controls the Embryogenic Reprogramming of Somatic Cells in Arabidopsis through the Histone Acetylation-Mediated Repression of the miRNA Biogenesis Genes

Abstract

The embryogenic transition of somatic cells requires an extensive reprogramming of the cell transcriptome. Relevantly, the extensive modulation of the genes that have a regulatory function, in particular the genes encoding the transcription factors (TFs) and miRNAs, have been indicated as controlling somatic embryogenesis (SE) that is induced in vitro in the somatic cells of plants. Identifying the regulatory relationships between the TFs and miRNAs during SE induction is of central importance for understanding the complex regulatory interplay that fine-tunes a cell transcriptome during the embryogenic transition. Hence, here, we analysed the regulatory relationships between AGL15 (AGAMOUS-LIKE 15) TF and miR156 in an embryogenic culture of Arabidopsis. Both AGL15 and miR156 control SE induction and AGL15 has been reported to target the MIR156 genes in planta. The results showed that AGL15 contributes to the regulation of miR156 in an embryogenic culture at two levels that involve the activation of the MIR156 transcription and the containment of the abundance of mature miR156 by repressing the miRNA biogenesis genes DCL1 (DICER-LIKE1), SERRATE and HEN1 (HUA-ENHANCER1). To repress the miRNA biogenesis genes AGL15 seems to co-operate with the TOPLESS co-repressors (TPL and TPR1-4), which are components of the SIN3/HDAC silencing complex. The impact of TSA (trichostatin A), an inhibitor of the HDAC histone deacetylases, on the expression of the miRNA biogenesis genes together with the ChIP results implies that histone deacetylation is involved in the AGL15-mediated repression of miRNA processing. The results indicate that HDAC6 and HDAC19 histone deacetylases might co-operate with AGL15 in silencing the complex that controls the abundance of miR156 during embryogenic induction. This study provides new evidence about the histone acetylation-mediated control of the miRNA pathways during the embryogenic reprogramming of plant somatic cells and the essential role of AGL15 in this regulatory mechanism.

Keywords: AGL15; DCL1; HDAC; HEN1; SERRATE; TOPLESS co-repressor; acetylation; miR156; miRNA biogenesis; somatic embryogenesis.

Figure 1

Relative expression level of pri-miR156s in the embryogenic cultures of the 35S::AGL15 and agl15 agl18 transgenic lines. The relative miRNA level was normalised to an internal control (At4g27090) and calibrated to a Col-0 culture of the same age. * value significantly different from the Col-0 culture of the same age (p < 0.05; n = 3 ± SD); ^# value significantly different from the 35S::AGL15 culture of the same age (p < 0.05; n = 3 ± SD).

Figure 2

Abundance of mature miR156s in the embryogenic cultures of the 35S::AGL15 and agl15 agl18 transgenic lines. The relative miRNA level was normalised to an internal control (At4g27090) and calibrated to the Col-0 culture of the same age. * value significantly different from the 35S::AGL15 culture of the same age (p < 0.05; n = 3 ± SD).

Figure 3

Relative expression level of the miRNA biogenesis genes (DCL1, HEN1, SERRATE and HYL1) in the embryogenic cultures of the 35S::AGL15 (A) and agl15 agl18 (B) transgenic lines. The relative expression level was normalised to an internal control (At4g27090) and calibrated to the Col-0 culture of the same age. * value significantly different from the Col-0 culture of the same age (p < 0.05; n = 3 ± SD).

Figure 4

Relative expression level of the miRNA biogenesis genes (DCL1, HEN1, SERRATE) in the embryogenic cultures of the 35S::AGL15 (A) and agl15 agl18 (B) transgenic lines that had been treated with a TSA (Trichostatin A). The relative expression level was normalised to an internal control (At4g27090) and calibrated to the untreated culture of the genotypes of the same age. * value significantly different from the culture of the same age untreated with a TSA (p < 0.05; n = 3 ± SD).

Figure 5

Expression analysis of HDAC6 and HDAC19 (A) in the SE culture of the Col-0 genotype. Capacity for SE in the cultures of the hdac6 and hdac6 hdac19 mutants and their parental genotype, Col-0. Percentage of explants producing somatic embryos (B) and SE productivity (C) of the IZE explant culture that was induced on an E5 medium. The relative transcript level was normalised to an internal control (At4g27090) and calibrated to freshly isolated explants (0d). * value significantly different from the freshly isolated explants (0d) (p < 0.05; n = 3 ± SD); ^# values significantly different from the Col-0 (p < 0.05; n = 3 ± SD).

Figure 6

Relative expression of the miRNA biogenesis genes (DCL1, HEN1, SERRATE) in the SE cultures of the hdac6 and hdac6 hdac19 mutant lines. The relative transcript level was normalised to an internal control (At4g27090) and calibrated to the Col-0 culture of the same age. * values significantly different from the Col-0 culture of the same age (p < 0.05; n = 3 ± SD).

Figure 7

H3ac enrichment in a selected fragment of a promoter and TSS + 300 of the DCL1 (A), SERRATE (B) and HEN1 (C) genes in the explants of the Col-0, 35S::AGL15 and agl15 agl18 lines. * value significantly different from the Col-0 (p < 0.05; n = 3 ± SD); ^# value significantly different from the 35S::AGL15 (p < 0.05; n = 3 ± SD).

Figure 8

Relative expression level of the genes encoding the TOPLESS co-repressors (TPL, TPR1-4) in the embryogenic culture of Col-0 (A). Capacity for somatic embryogenesis in the cultures of mutants in the TOPLESS co-repressor genes (tpl, tpr1, 3, 4) and their parental genotype, Col-0. Percentage of explants producing somatic embryos (B) and SE productivity (C) of the IZE explant culture that was induced on an E5 medium. The relative expression level was normalised to an internal control (At4g27090) and calibrated to freshly isolated explants (0d). * value significantly different from the freshly isolated explants (0d) (p < 0.05; n = 3 ± SD); ^# values significantly different from Col-0 (p < 0.05; n = 3 ± SD).

Figure 9

Relative expression of the miRNA biogenesis genes (DCL1, HEN1, SERRATE) in the embryogenic culture of the TOPLESS co-repressor mutants (tpl, tpr1, tpr4). The relative transcript level was normalised to an internal control (At4g27090) and calibrated to the Col-0 culture of the same age. * values significantly different from the Col-0 culture of the same age (p < 0.05; n = 3 ± SD).

Figure 10

A putative model of the histone acetylation-related and AGL15-mediated control of miR156 during embryogenic induction. AGL15 is postulated to fine tune the abundance of miR156 by activating the MIR156 gene (MIR156h) expression and the containment of the mature miR156 molecules as a result of the repression of the miRNA biogenesis genes DCL1, SERRATE and HEN1. To repress the target genes, AGL15 interacts with the TOPLESS co-repressors (TPL, TPR1, TPR4) and recruits the histone deacetylases, HDAC6 and HDAC19, which are components of the SIN3/HDAC gene silencing complex [39,48]. The AGL15-mediated histone deacetylation might also control other miRNAs including miR167 and miR172 in embryogenic culture. AGL15—AGAMOUS-like15, DCL1—DICER-like1, HEN1-HUA-ENHANCER1, TPL—TOPLESS, TPR—TOPLESS-RELATED, HDAC—HISTONE DEACETHYLASE.