Extensive modulation of the transcription factor transcriptome during somatic embryogenesis in Arabidopsis thaliana

Abstract

Molecular mechanisms controlling plant totipotency are largely unknown and studies on somatic embryogenesis (SE), the process through which already differentiated cells reverse their developmental program and become embryogenic, provide a unique means for deciphering molecular mechanisms controlling developmental plasticity of somatic cells. Among various factors essential for embryogenic transition of somatic cells transcription factors (TFs), crucial regulators of genetic programs, are believed to play a central role. Herein, we used quantitative real-time polymerase chain reaction (qRT-PCR) to identify TF genes affected during SE induced by in vitro culture in Arabidopsis thaliana. Expression profiles of 1,880 TFs were evaluated in the highly embryogenic Col-0 accession and the non-embryogenic tanmei/emb2757 mutant. Our study revealed 729 TFs whose expression changes during the 10-days incubation period of SE; 141 TFs displayed distinct differences in expression patterns in embryogenic versus non-embryogenic cultures. The embryo-induction stage of SE occurring during the first 5 days of culture was associated with a robust and dramatic change of the TF transcriptome characterized by the drastic up-regulation of the expression of a great majority (over 80%) of the TFs active during embryogenic culture. In contrast to SE induction, the advanced stage of embryo formation showed attenuation and stabilization of transcript levels of many TFs. In total, 519 of the SE-modulated TFs were functionally annotated and transcripts related with plant development, phytohormones and stress responses were found to be most abundant. The involvement of selected TFs in SE was verified using T-DNA insertion lines and a significantly reduced embryogenic response was found for the majority of them. This study provides comprehensive data focused on the expression of TF genes during SE and suggests directions for further research on functional genomics of SE.

Figure 1. Developmental changes in Arabidopsis Col-0 and tanmei IZE explants induced on auxin-containing medium.

A–D) Col-0 accession. E–H) tanmei mutant. Explants were induced on auxin-containing medium (E5) and monitored at days 0 (A, E), 5 (B, F), 10 (C, G) and 15 (D, H) of in vitro culture. A, E) Freshly isolated IZE 12 days after pollination (DAP). B) Straightening, enlargement and swelling of IZE cotyledons. C) Tissue proliferation and somatic embryo-like protuberances formed at adaxial side (arrow). D) Numerous somatic embryos at the adaxial side of IZE cotyledons. F) Anthocyanin accumulation in IZE cotyledons and tissue proliferation from IZE hypocotyl. G) Non-embryogenic watery callus. H) Progression of non-embryogenic callus production. Bars: 0.2 mm (A, B, E, F); 0.3 mm (C, G); 0.6 mm (H) and 1.0 mm (D).

Figure 2. Principal Component Analysis (PCA).

The analysis demonstrates a clear separation of TF expression in Col-0 and tanmei (tan1–2), both in explants (0 d) and during embryogenic culture (5 d and 10 d). Expression data from three independent biological replicates were analysed each. Samples: C0, Col-0, day 0; C5, Col-0, day 5; C10, Col-0, day 10; T0, tan mutant, day 0; T5, tan mutant, day 5; T10, tan mutant, day 10. Numbers 1 to 3 denote replicates 1 to 3. Approximately 67.6% of the variation is captured by the first two components.

Figure 3. Venn diagram demonstrating the number of genes expressed during SE in the Col-0 accession.

Numbers in intersections represent TFs commonly expressed at the different culture time points: 0 d, explant; 5 d, induction phase of SE; 10 d, advanced SE culture.

Figure 4. Numbers of TF genes expressed in explants of the highly embryogenic Col-0 genotype and the non-embryogenic tanmei mutant (A) and in embryogenic Col-0 culture (B).

´+ ´, genes for which expression was observed; ´- ´, genes for which no expression was observed. Differentially expressed and highly-regulated genes show at least 2- (x≥2) and 10-fold (x≥10) change, respectively, in expression level in any of the compared culture points.

Figure 5. Cluster analysis.

K-means clustering revealed four main expression patterns of TF genes in Col-0 embryogenic cultures. The levels of expression changes are given as 40-ddCt. The cluster analysis shows up-regulation of the great majority of TFs (B, C, D), and down-regulation of a small group of TFs (A). Increased TF expression was either restricted to the early stage of SE (C), or was observed during both SE stages, early and advanced (B, D).

Figure 6. Expression profiles of SE-associated genes.

The graph shows contrasting expression levels of TFs in embryogenic (Col-0) and non-embryogenic (tanmei) cultures. The relative transcripts levels of the genes are shown as ddCt.

Figure 7. Number of TF genes of modulated expression in embryogenic cultures.

A) TFs expressed in Col-0 culture. B) TFs of SE-specific expression pattern, i.e. those displaying distinctly different expression profiles in Col-0 and tanmei cultures. Numbers of TFs of steady (fold change<2) and modulated (fold change≥2) expression in embryogenic cultures referenced to the indicated culture time points (i.e. 5 d–0 d; 10 d–0 d, and 5 d–10 d) are given. Genes with up- and down-regulated expression are indicated.

Figure 8. Functional categories of differentially expressed genes.

A) TFs differentially expressed during SE. B) SE-associated TFs. TFs were annotated to four major categories (plant development, phytohormones, stress and others) and various subcategories. Given are the numbers of TFs in the different functional categories.

Figure 9. Hormone-related TFs.

The graph shows the percentages of hormone-related TFs up- or downregulated in embryogenic Col-0 culture. A great majority of the hormone-related TFs is up-regulated including those related to brassinosteroids, auxin, SA, cytokinins, GA, ethylene, JA and ABA.

Figure 10. Functional test of SE-modulated transcription factors.

Embryogenic capacity of TF T-DNA insertion mutants (A, B) and transgenic lines expressing the indicated TFs under the control of a ß-estradiol-inducible promoter (C, D) was analysed and SE efficiency (A, C) and SE productivity (B, D) were evaluated. Values significantly different from the parental Col-0 genotype are marked by asterisks (n  = 3; means ± SD are given; Mann-Whitney’s U test; p<0.05).

Figure 11. Expression profiles of AUX/IAA genes.

Shown are expression levels of AUX/IAA genes (IAA16, IAA29, IAA30 and IAA31) in explants induced towards alternative morphogenic pathways, i.e. somatic embryogenesis (SE) and seedling development (E0). Values significantly different from E0 are labeled by asterisks (n  = 3; means ± SD are given; Mann-Whitney’s U test; p<0.05).