Transcriptome analysis of proliferating Arabidopsis endosperm reveals biological implications for the control of syncytial division, cytokinin signaling, and gene expression regulation

Abstract

During the early stages of seed development, Arabidopsis (Arabidopsis thaliana) endosperm is syncytial and proliferates rapidly through repeated rounds of mitosis without cytokinesis. This stage of endosperm development is important in determining final seed size and is a model for studying aspects of cellular and molecular biology, such as the cell cycle and genomic imprinting. However, the small size of the Arabidopsis seed makes high-throughput molecular analysis of the early endosperm technically difficult. Laser capture microdissection enabled high-resolution transcript analysis of the syncytial stage of Arabidopsis endosperm development at 4 d after pollination. Analysis of Gene Ontology representation revealed a developmental program dominated by the expression of genes associated with cell cycle, DNA processing, chromatin assembly, protein synthesis, cytoskeleton- and microtubule-related processes, and cell/organelle biogenesis and organization. Analysis of core cell cycle genes implicates particular gene family members as playing important roles in controlling syncytial cell division. Hormone marker analysis indicates predominance for cytokinin signaling during early endosperm development. Comparisons with publicly available microarray data revealed that approximately 800 putative early seed-specific genes were preferentially expressed in the endosperm. Early seed expression was confirmed for 71 genes using quantitative reverse transcription-polymerase chain reaction, with 27 transcription factors being confirmed as early seed specific. Promoter-reporter lines confirmed endosperm-preferred expression at 4 d after pollination for five transcription factors, which validates the approach and suggests important roles for these genes during early endosperm development. In summary, the data generated provide a useful resource providing novel insight into early seed development and identify new target genes for further characterization.

Figure 1.

Heat map showing endosperm-preferred genes in the AtGenExpress seed data. This heat map includes genes with endosperm-preferred expression from our arrays that correlate with SUC5 expression (based on an r value cutoff of 0.75) across the AtGenExpress expression library using online tools provided at the University of Toronto BAR Web site. The DataMetaFormatter tool created a heat map of probe intensities for genes in AtGenExpress seed development series and also calculated the median expression intensities in all other wild-type (WT) tissues. This heat map only includes genes that had a median expression level in AtGenExpress of <100 units in non-seed-containing tissues and a median expression of >100 across the seed series. Stages 3, 4, 5, 6, 7, 8, 9, and 10 correspond to siliques containing seeds at 48 to 66 h after flowering (HAF), siliques containing seeds at 66 to 84 HAF, siliques containing seeds at 84 to 90 HAF, isolated seeds at 90 to 96 HAF, isolated seeds at 96 to 108 HAF, isolated seeds at 108 to 120 HAF, isolated seeds at 120 to 144 HAF, and isolated seeds at 144 to 192 HAF, respectively. [See online article for color version of this figure.]

Figure 2.

Interaction summary of endosperm-preferred MADS box transcription factors. Interaction plot of endosperm-preferred MADS box transcription factors in the array data adapted from the output of the Arabidopsis Interactions Viewer at http://bar.utoronto.ca/. Black outline corresponds to genes differentially expressed in our array data. Dark and pale green fill corresponds to >2.0-fold endosperm-enriched and 1.5- to 2-fold endosperm-enriched transcripts, respectively. Gray outline represents genes not differentially expressed in the array data. Connecting lines indicate that a protein-protein interaction is evident in the literature. The more red the line, the better the correlation for transcript expression across the AtGenExpress expression series, as assessed by Pearson correlation. [See online article for color version of this figure.]

Figure 3.

Heat map showing qRT-PCR validation of early seed and/or early seed-specific expression. The expression level of each transcript was calculated relative to ACTIN2. R, ST, L, FB, SIL, and DS represents roots, stems, leaves, flower buds, siliques, and dissected seeds, respectively. Context relates to the section of the article in which a particular gene is discussed. [See online article for color version of this figure.]

Figure 4.

Promoter-GUS expression during early seed development. GUS reporter constructs were made for a selection of transcription factors to assess their use as markers for early endosperm development. A to E, Expression of the At1g65300 (AGL38/PHE2) construct was seen in mature pollen (B) and subsequently in the central cell after fertilization (A). Expression then persisted in the very early embryo and endosperm (C) but became restricted to the endosperm around the late globular stage (D). Expression was then specific to the chalazal region during transition (E). F, The At1g49190 (ARR19) promoter was expressed in the chalazal endosperm during globular and early heart stages of seed development. G, The At5g60440 (AGL62) promoter was expressed throughout the syncytial endosperm, albeit at low levels, and is shown here during the globular stage. H, The At4g18870 (HSF15) promoter had very strong expression in the chalazal endosperm plus strong expression in the peripheral endosperm at the globular stage of development. Staining was not apparent in the micropylar domain. I and J, The At4g21080 (DOF4.5) promoter showed very strong chalaza-specific expression at the globular stage (I), with expression becoming more widespread in the endosperm at the heart stage (J). The labels on E are as follows: cze, chalazal endosperm; em, embryo; mpe, micropylar endosperm; pe, peripheral endosperm.