The Arabidopsis BET bromodomain factor GTE4 is involved in maintenance of the mitotic cell cycle during plant development

Abstract

Bromodomain and Extra Terminal domain (BET) proteins are characterized by the presence of two types of domains, the bromodomain and the extra terminal domain. They bind to acetylated lysines present on histone tails and control gene transcription. They are also well known to play an important role in cell cycle regulation. In Arabidopsis (Arabidopsis thaliana), there are 12 BET genes; however, only two of them, IMBIBITION INDUCIBLE1 and GENERAL TRANSCRIPTION FACTOR GROUP E6 (GTE6), were functionally analyzed. We characterized GTE4 and show that gte4 mutant plants have some characteristic features of cell cycle mutants. Their size is reduced, and they have jagged leaves and a reduced number of cells in most organs. Moreover, cell size is considerably increased in the root, and, interestingly, the root quiescent center identity seems to be partially lost. Cell cycle analyses revealed that there is a delay in activation of the cell cycle during germination and a premature arrest of cell proliferation, with a switch from mitosis to endocycling, leading to a statistically significant increase in ploidy levels in the differentiated organs of gte4 plants. Our results point to a role of GTE4 in cell cycle regulation and specifically in the maintenance of the mitotic cell cycle.

Figure 1.

Phylogenetic analysis of the Arabidopsis BET bromodomain family. The rooted tree was obtained with a ClustalX alignment and subsequent analysis with Phylyp protdist neighbor joining; the alignment was made considering only the bromodomain (of human BRD4, only the second bromodomain was considered). The numbers represent the branching reproducibility over 100 bootstraps.

Figure 2.

Analysis of the gte4 mutant. A, Schematic representation of the T-DNA insertion in GTE4. B, Differences in inflorescence height in the wild type (WT) and the gte4 mutant. C, Rosettes of the gte4 mutant are smaller than those of wild-type plants. D, Comparison of leaf size and shape between the gte4 mutant and the wild type. gte4 leaves are visibly more jagged than those of the wild type. E, Transections of wild-type and gte4 stem internodes showing the reduced stem thickness in gte4 plants at 55 d after germination with unstained sections under light microscopy. Bars = 50 μ m. F, Wild-type and gte4 flowers at anthesis. The stamen number (arrows) in the gte4 flower is reduced in comparison with the wild type. Bars = 0.5 mm. [See online article for color version of this figure.]

Figure 3.

Effects of the gte4 mutation on root development in young seedlings. A and B, Young seedlings. C and D, Primary roots. E to L, Lateral root apices. A and B, Wild-type (WT; A) and gte4 (B) unstained sections under light microscopy of 4-d-old seedlings. The gte4 primary root is shorter than that of the wild type due to a reduction in length in the hairy region (h.r.) and the elongation region (e.r.). Bars = 100 μ m. C and D, Wild-type (C) and gte4 (D) apices of the primary root of seedlings at day 4 after germination showing the different lengths of the apical region (brackets). Bars = 10 μ m. Magnifications of the QC cells and surrounding initial cells are shown in the insets. The arrows in the inset of D show anomalous anticlinal divisions in the procambial cells. Bars = 10 μ m. E and F, Unstained sections under light microscopy showing details of the primary root of wild-type (E) and gte4 (F) 9-d-old seedlings revealing a lower density, and a reduced development, of lateral roots (arrows) in the mutant. Bars = 200 μ m. G and H, Wild-type (G) and gte4 (H) lateral root apices at early emergence from the primary root in 12-d-old seedlings showing apical initials and QC cells equally defined in both genotypes. Bars = 10 μ m. I and J, Details of wild-type (I) and gte4 (J) lateral root apices during the expansion phase of the emerged organ in 14-d-old seedlings showing that expansion in the QC cells and columella initials is higher in gte4 roots than in the wild type (squares). Bars = 10 μ m. K and L, Apices of protruded lateral roots in wild-type (K) and gte4 (L) plants. Rectangles show QC cells, columella, and procambial initials in the gte4 mutant in comparison with the wild type. A magnification of hardly visible QC cells and of irregularly sized surrounding cells is shown in the inset. Bars = 10 μ m. In C, D, and G to L, images are by Nomarski microscopy. QC cells are marked with asterisks.

Figure 4.

Effects of the gte4 mutation on embryo development. Images on the left are from the wild type (WT) and those on the right are from the gte4 mutant. A to D, In the gte4 mutant, there is no visible effect on embryo development at the octant stage (A and B) and the heart stage (C and D), whereas the suspensor cells are enlarged. Bars = 10 μ m. E and F, Wild-type (E) and gte4 (F) embryos at the cotyledonary stage showing no significant alteration in the cotyledons. Bars = 50 μ m. G and H, Details of the root pole of the embryo at the cotyledonary stage showing irregularly shaped QC cells (arrows) and irregularly divided columella initials (brackets) in the mutant. Bars = 10 μ m. I and J, Details of wild-type and gte4 apices of mature embryos showing a different shape in the mutant. Bars = 10 μ m. In A to H, longitudinal toluidine blue-stained sections of developing seeds are observed under light microscopy; in I and J, images by Nomarski microscopy are shown after 5 h of imbibition on filter paper. [See online article for color version of this figure.]

Figure 5.

Expression of QC markers in the root apex of wild-type (A, C, and E) and gte4 (B, D, and F) seedlings. A and B, Expression of AGL42:GFP in the apex of the primary root. A, Wild-type (WT) root showing a strong GFP signal in the QC cells (higher magnification in the inset). B, Absence of GFP signal in the root apex of a gte4 mutant containing the same construct. Bars = 20 μ m. C to F, Expression of the QC46 (C and D) and QC25 (E and F) GUS reporter in the QC cells of wild-type and gte4 mutant roots. Bars = 10 μ m.

Figure 6.

Quantitative RT-PCR analysis of genes that are involved in the E2F pathway. The graphs show the relative expression of each gene in the gte4 mutant compared with the wild type (WT). Error bars indicate se. RNA was extracted from 4-d-old Arabidopsis plantlets.