Field transcriptome revealed critical developmental and physiological transitions involved in the expression of growth potential in japonica rice

Abstract

Background: Plant growth depends on synergistic interactions between internal and external signals, and yield potential of crops is a manifestation of how these complex factors interact, particularly at critical stages of development. As an initial step towards developing a systems-level understanding of the biological processes underlying the expression of overall agronomic potential in cereal crops, a high-resolution transcriptome analysis of rice was conducted throughout life cycle of rice grown under natural field conditions.

Results: A wide range of gene expression profiles based on 48 organs and tissues at various developmental stages identified 731 organ/tissue specific genes as well as 215 growth stage-specific expressed genes universally in leaf blade, leaf sheath, and root. Continuous transcriptome profiling of leaf from transplanting until harvesting further elucidated the growth-stage specificity of gene expression and uncovered two major drastic changes in the leaf transcriptional program. The first major change occurred before the panicle differentiation, accompanied by the expression of RFT1, a putative florigen gene in long day conditions, and the downregulation of the precursors of two microRNAs. This transcriptome change was also associated with physiological alterations including phosphate-homeostasis state as evident from the behavior of several key regulators such as miR399. The second major transcriptome change occurred just after flowering, and based on analysis of sterile mutant lines, we further revealed that the formation of strong sink, i.e., a developing grain, is not the major cause but is rather a promoter of this change.

Conclusions: Our study provides not only the genetic basis for functional genomics in rice but also new insight into understanding the critical physiological processes involved in flowering and seed development, that could lead to novel strategies for optimizing crop productivity.

Figure 1

Overview of gene expression profile of organs and tissues at various stages of growth . (A) Number of expressed genes in each organ and tissue across the entire spatiotemporal developmental cycle. The genes with normalized signal intensities above -5 were extracted as 'expressed' genes. (B) PCA applied to the expression profiles of 48 samples identified organ/tissue-specific gene expression signatures. The average normalized signal intensities for each sample were used in this analysis.

Figure 2

Heat map of organ and tissue-specific expressed genes . A total of 731 organ/tissue-specific genes identified by the Shannon entropy based method were analyzed by hierarchical clustering. A heat map was constructed using the relative expression values of the genes based on correlation distance and average linkage method. As a result, the 731 genes were grouped into 7 clusters based on organ/tissue-specificity of gene expression. High expression values are shown in red. Details of the samples used for each organ and tissue are described in Table 1.

Figure 3

Diurnal and growth stage-specificity of gene expression . (A) Frequency of diurnally expressed genes in the vegetative organs. Genes differentially expressed between daytime (12:00) and nighttime (24:00) were extracted based on the t-test and fold change criteria (FDR < 0.05 and fold change, FC > 3) in each organ/tissue. Red and blue bars represent highly expressed genes at daytime and nighttime, respectively. (B) Venn diagram of differentially expressed genes from the vegetative to reproductive phases in leaf blade, leaf sheath, and root during daytime. The differentially expressed genes were statistically extracted based on the t-test and fold change criteria (FDR < 0.05 and FC > 3).

Figure 4

Correlation of expression profiles of the leaf from 13 to 125 DAT . Pearson's correlation coefficients (PCCs) were calculated using the normalized signal intensities of the 29,119 genes. Samples were clustered based on Euclidian distance and complete linkage. Transcriptome profiles were apparently grouped into phase 1 (13-41 DAT), phase 2 (48-90 DAT), and phase 3 (97-125 DAT) corresponding approximately to vegetative, reproductive, and ripening stages of growth, respectively. The color scale represents the PCC scores. DAT: days after transplanting.

Figure 5

Change in transcriptome associated with the transition to reproductive stage . (A) Expression pattern of 29,119 genes from 20 to 76 DAT based on relative expression values indicate drastic change between 41-48 DAT. Blue, yellow, and red lines indicate high, middle, and low expression values, respectively, at 20 DAT. (B) Expression pattern of rice florigen genes, Hd3a (blue) and RFT1 (red), from 20 to 76 DAT. Error bars indicate s.e.m. (n = 3). (C) Expression pattern of seven miR169 precursors from 20 to 76 DAT. Each miRNA precursor was represented in the microarray as two probes corresponding to the 3' and 5' sequence, respectively. Error bars indicate s.e.m. (n = 3). (D) GO analysis of the 357 genes upregulated from 41 to 48 DAT. The colored circles represent enriched categories based on the p-values corrected for multiple testing (FDR) ranging from 0.05 (yellow) or below (orange). The size of the circle is proportional to the number of genes annotated to that node. (E) Expression pattern of five miR399 precursors from 20 to 76 DAT. Error bars indicate s.e.m. (n = 3).

Figure 6

Change in transcriptome associated with flowering and grain filling . (A) Expression pattern of 29,119 genes from 62 to 125 DAT based on relative expression values. Transient expression of pollen specific genes at 90 DAT (indicated by asterisk), which also coincided with the peak of flowering, was due to pollen contamination of leaf samples (See Additional file 15). (B) PCA of the expression profile of flag leaf at 83, 90, 97 and 104 DAT. (C) Cluster analysis of fertile (F) and sterile (S) plants at 0, 1, 2 and 3 weeks after heading (WAH) based on correlation distance and complete linkage. (D) Differentially expressed genes in flag leaf of fertile (blue) and sterile (red) lines after heading were statistically extracted based on t-test and fold change criteria (FDR < 0.05 and FC > 2).