MADS-Box and bHLH Transcription Factors Coordinate Transmitting Tract Development in Arabidopsis thaliana

Abstract

The MADS-domain transcription factor SEEDSTICK (STK) controls several aspects of plant reproduction. STK is co-expressed with CESTA (CES), a basic Helix-Loop-Helix (bHLH) transcription factor-encoding gene. CES was reported to control redundantly with the brassinosteroid positive signaling factors BRASSINOSTEROID ENHANCED EXPRESSION1 (BEE1) and BEE3 the development of the transmitting tract. Combining the stk ces-4 mutants led to a reduction in ovule fertilization due to a defect in carpel fusion which, caused the formation of holes at the center of the septum where the transmitting tract differentiates. Combining the stk mutant with the bee1 bee3 ces-4 triple mutant showed an increased number of unfertilized ovules and septum defects. The transcriptome profile of this quadruple mutant revealed a small subset of differentially expressed genes which are mainly involved in cell death, extracellular matrix and cell wall development. Our data evidence a regulatory gene network controlling transmitting tract development regulated directly or indirectly by a STK-CES containing complex and reveal new insights in the regulation of transmitting tract development by bHLH and MADS-domain transcription factors.

Keywords: MADS-box; bHLH; cell death; cell wall; extracellular matrix; transmitting tract.

FIGURE 1

Analysis of CES expression. In situ hybridization (A–F) and GUS activity of the pCES::GUS marker line (G–I) showed CES expression in ovules (A–C,G–I), transmitting tract (B), and in the embryos of the developing seeds (D–F). Em, embryo; fu, funiculus; ov, ovule; tt, transmitting tract. Scale bars: 50 μm.

FIGURE 2

Unfertilized ovules analysis in multiple mutant combination of stk, ces-4, and shp1 shp2. (A) Stereomicroscope images of opened siliques of the stk and ces-4 single mutants, the stk ces-4 and the shp1 shp2 double mutants, the shp1 shp2 ces-4 triple mutant and the wild type Col-0; Scale bars: 1 mm. (B) Box plots of all the mutants analyzed in comparison to wild type Col-0. Statistical analysis was performed using Anova followed by Tukey HSD test (**p < 0.01). The experiment was repeated four times. For each experiment 5 siliques were collected from 4 independent plant lines of the same genotype to obtain a total of 20 analyzed siliques.

FIGURE 3

Unfertilized ovules analysis in multiple mutant combination of bee1, bee2, bee3, stk, and ces-4. (A) Stereomicroscope images of opened siliques of the bee1 and bee3 single mutants, the bee1 bee3 double mutant, the bee1 bee2 bee3 triple mutant, the bee1 bee2 bee3 stk, the bee1 bee2 bee3 ces-4, and bee1 bee3 stk ces-4 quadruple mutants, the bee1 bee2 bee3 stk ces-4 quintuple mutants and the wild type Col-0; Scale bars: 1 mm. (B) Box plots of all the mutants analyzed in comparison to wild type Col-0. Statistical analysis was performed using Anova followed by Tukey HSD test (**p < 0.01). The experiment was repeated four times. For each experiment 5 siliques were collected from 4 independent plant lines of the same genotype to obtain a total of 20 analyzed siliques.

FIGURE 4

Septum analysis and pollen tube growth. (A) Scanning Electron Microscope pictures of the septum of opened siliques of stk, ces-4, stk ces-4, bee1 bee2 bee3 stk, bee1 bee2 bee3 ces-4, bee1 bee3 stk ces-4 and bee1 bee2 bee3 stk ces-4 and wild type Col-0; Scale bars: 100 μm. (B) Aniline blue staining of stk ces-4, bee1 bee3 stk ces-4 and bee1 bee2 bee3 stk ces-4 and wild type Col-0; Scale bar: 50 μm.

FIGURE 5

STK, CES and BEE1 interaction assays in yeast and in planta. (A) BiFC assays showing the interaction of STK with CES. STK-N or CES-N: STK or CES fusion with the N-terminal part of the split YFP; STK-C or CES-C: STK or CES fusion with the C-terminal part of the split YFP. CES-N CES-C interaction was used as positive control. STK-N EMPTY-C and CES-N EMPTY-C were used as negative control. (B) Yeast two-hybrid assays testing interactions between STK-AD, BEE1-AD, and CES-BD; interactions were considered positive when growth on -W-L-H +15 mM of 3-AT selective media was observed. The experiments were performed at 22°C. The CES-BEE1 interaction was chosen as a positive control (Poppenberger et al., 2011), the growth of transformants carrying empty AD or BD vectors were used as negative controls of the experiments.

FIGURE 6

Heatmap of differentially expressed genes described in Table 1. Expression levels are shown for each of the 6 independent biological replicates analyzed in this study. Genes are represented in the columns and the biological replicates in the rows. To facilitate comparison, the expression values were standardized with respect to the average expression of each gene. Dark green indicates low expression, light brown indicates high expression. In the columns, red is used to mark biological replicates for the quadruple mutant, blue is used to indicate biological replicates for wild type plants.

FIGURE 7

Proposed model of transmitting tract development controlled by STK, CES, BEE1, and BEE3. The blue box symbolizes the interaction (STK-CES) that we found in this research project; The black box represents the interaction (CES-BEE1-BEE3) that has been previously described (Crawford and Yanofsky, 2011; Poppenberger et al., 2011). ECM, extracellular matrix; PCD, programmed cell death; BR, brassinosteroid.