Genome-wide analysis of the complex transcriptional networks of rice developing seeds

Abstract

Background: The development of rice (Oryza sativa) seed is closely associated with assimilates storage and plant yield, and is fine controlled by complex regulatory networks. Exhaustive transcriptome analysis of developing rice embryo and endosperm will help to characterize the genes possibly involved in the regulation of seed development and provide clues of yield and quality improvement.

Principal findings: Our analysis showed that genes involved in metabolism regulation, hormone response and cellular organization processes are predominantly expressed during rice development. Interestingly, 191 transcription factor (TF)-encoding genes are predominantly expressed in seed and 59 TFs are regulated during seed development, some of which are homologs of seed-specific TFs or regulators of Arabidopsis seed development. Gene co-expression network analysis showed these TFs associated with multiple cellular and metabolism pathways, indicating a complex regulation of rice seed development. Further, by employing a cold-resistant cultivar Hanfeng (HF), genome-wide analyses of seed transcriptome at normal and low temperature reveal that rice seed is sensitive to low temperature at early stage and many genes associated with seed development are down-regulated by low temperature, indicating that the delayed development of rice seed by low temperature is mainly caused by the inhibition of the development-related genes. The transcriptional response of seed and seedling to low temperature is different, and the differential expressions of genes in signaling and metabolism pathways may contribute to the chilling tolerance of HF during seed development.

Conclusions: These results provide informative clues and will significantly improve the understanding of rice seed development regulation and the mechanism of cold response in rice seed.

Figure 1. Analysis approaches of gene expression patterns in rice developing seeds.

Work flow of data analysis was shown, genes of specific set and regulated set were analyzed respectively (See Materials and Methods section).

Figure 2. Corroboration of expression patterns of genes associated with seed development.

The results by qRT-PCR analysis (box plot) are compared with data obtained from microarray hybridization (horizontal bars) to validate the predominantly expressed genes (A) and regulated genes (B). Expression of genes in root (R), leaf (L), seedling (S), ovary (O), embryo (Em) and endosperm (Endo) was analyzed and the value at 3 DAF in embryo was set as 1.0 for predominantly expressed genes (A). For the time course expression (B), the first time point was set as 1. Blue and yellow in cells reflect low or high expression levels, respectively, as indicated in the scale bar. The numbers of genes with different pattern in Zhonghua 11 (ZH) and Nipponbare (Nip) were calculated in embryo and endosperm (C). “U” or “D” in parenthesis indicates the genes are up-regulated or down-regulated. The data of Nip were obtained from Gene Expression Omnibus (GSE21494).

Figure 3. GO category enrichment of seed development-associated genes.

The enrichment of process categories associated with metabolism, development, hormone signaling and cellular regulation were shown. “Em” or “En” indicates genes that are predominantly expressed in embryo or endosperm, respectively; “Both” indicates genes that are predominantly expressed in both embryo and endosperm; “Em_T” or “En_T” indicates genes that are regulated in time during embryo or endosperm development, respectively. Chi-square test was performed to calculate the p-values, which were log10 transformed. As indicated in the scale bar, the highly enriched GO categories were in red color.

Figure 4. Expression patterns of genes involved in hormone biosynthesis and signaling, which are highly expressed in rice seeds.

The expression data were normalized using Z score and genes involved in ABA, auxin, ethylene and gibberellin pathways were shown. The scale bar was shown at the top the figure.

Figure 5. Expression patterns of genes involved in ABA and gibberellin biosynthesis during rice seed development.

The ABA (A) and GA (B) biosynthesis pathways were from PlantCyc (www.plantcyc.org) and the expressions of relevant genes were log2 transformed. The expression of genes in root (R), leaf (L), seedling (S), ovary (O), embryo and endosperm were shown. The bar represented the scale of relative expression levels.

Figure 6. Expression patterns of transcription factor encoding genes during seed development.

The expressions were normalized and the red or blue lines (words) indicate the up-regulated or down-regulated genes, respectively, during seed development. The scale bar was shown at the top the figure.

Figure 7. Transcriptional co-expression network involved in seed development.

The relationship of transcription factors (circle) and the associated functions (yellow round rectangle) were shown as overview (A) and two modules predominantly expressed in embryo (B) and endosperm (C) respectively, were shown in detail. The circles with red, green and blue colors represent the transcription factors expressed in embryo, endosperm, or both embryo and endosperm.

Figure 8. Regulated genes in chilling response of rice seeds.

A. Low temperature treated rice seed of Zhonghua 11 (ZH) and Hanfeng (HF). Developing seeds of ZH and HF at 4 DAF were treated at 14°C for 2 days (4 D-2 DC) and seeds developed for 6 days (6 D) were used as control. Developing seeds of ZH at 10 DAF were treated at 14°C for 2 days (10 D-2 DC) and ZH seeds developed for 12 days (12 D) were used as control. Appearance of glumes and grains were shown under each treatment. Bar = 1 mm. B. Numbers of genes regulated by low temperature. ZH or HF represents the Zhonghua 11 or Hanfeng variety. “C” indicates the low temperature treatment; “D” indicates days after fertilization. Rice plants at 4 or 10 days after fertilization were treated with low temperature (14°C) for 2 days (ZH 4D-2DC, ZH 10D-2DC and HF 4D-2DC), with corresponding controls growing at normal temperature (ZH6 D, ZH 12D, HF6D). In each comparison, “Up” indicates number of genes higher expressed in later group, whereas “Down” indicates number of genes lower expressed in later group.

Figure 9. Expression patterns of seed development associated genes regulated by low temperature.

The vertical bar indicated the pattern of genes regulated by cold in Zhonghua 11(ZH) seeds. Heat map showed the expression pattern during embryo (Em) and endosperm (Endo) development and in root (R), leaf (L), seedling (S) and ovary (O). The data were normalized using Z score as indicated in the scale bar.

Figure 10. Common genes regulated by low temperature in seed and seedling.

Genes above red line are regulated at 6 DAF and genes under red line are regulated at 12 DAF. The numbers indicate the ratios of gene expressions regulated by low temperature, and the color represents the log2 transformed ratio as indicated in the scale bar.