Comprehensive expression analysis of rice phospholipase D gene family during abiotic stresses and development

Abstract

Phospholipase D is one of the crucial enzymes involved in lipid mediated signaling, triggered during various developmental and physiological processes. Different members of PLD gene family have been known to be induced under different abiotic stresses and during developmental processes in various plant species. In this report, we are presenting a detailed microarray based expression analysis and expression profiles of entire set of PLD genes in rice genome, under three abiotic stresses (salt, cold and drought) and different developmental stages (3-vegetative stages and 11-reproductive stages). Seven and nine PLD genes were identified, which were expressed differentially under abiotic stresses and during reproductive developmental stages, respectively. PLD genes, which were expressed significantly under abiotic stresses exhibited an overlapping expression pattern and were also differentially expressed during developmental stages. Moreover, expression pattern for a set of stress induced genes was validated by real time PCR and it supported the microarray expression data. These findings emphasize the role of PLDs in abiotic stress signaling and development in rice. In addition, expression profiling for duplicated PLD genes revealed a functional divergence between the duplicated genes and signify the role of gene duplication in the evolution of this gene family in rice. This expressional study will provide an important platform in future for the functional characterization of PLDs in crop plants.

Figure 1. Expression of rice PLD gene family has been represented by a heat map. Developmental stages comprising three vegetative stages (L-leaf, R-root and SL-7­d­old seedling), six stages of panicle [P1 (0–3 cm), P2 (3–5 cm), P3 (5–10 cm), P4 (10–15 cm), P5 (15–22 cm), and P6 (22–30 cm)] and five stages of seed [S1 (0–2 DAP), S2 (3–4 DAP), S3 (4–10 DAP), S4 (11–20 DAP) and S5 (21–29 DAP)]. Clustering of the expression profile was done with log transformed average values taking mature leaf as base line. Three stress conditions are denoted as C, Cold Stress; D, Drought Stress; S, Salt Stress and SL, control, seven day old unstressed seedling. The scale at the bottom of each heat map is given in log₂ intensity value.

Figure 3. Venn diagram for differentially expressed PLDs. PLD genes up- and downregulated (A) under different abiotic stress conditions and (B) under stresses and developmental stages. Different compartments showing genes specific to either a particular stress/developmental stage or common to more than one stress and/or developmental stage.

Figure 2. Expression profiles of differentially expressed PLD genes during developmental stages. Average signal intensity value of three replicates from microarray for all the developmental stages has been plotted to show the differential expression. Standard error bars have been shown. Y-axis represents signal values from microarray and X-axis shows different developmental stages.

Figure 4. Expression profiles of duplicated PLD genes. Expression profiles of segmentally and tandemly duplicated gene pairs/clusters from microarray data were compared in various developmental stages including leaf (L), root (R) and seven day old seedling (S) tissue, in various stages of panicle development (P1-P6), seed development (S1-S5) and under cold stress (CS), dehydration stress (DS) and salt stress (SS). Each area graph represents compilation of the mean normalized signal intensity values.

Figure 5. Validation of microarray expression profiles for differentially expressing PLD genes by quantitative RT-PCR. Two and three biological replicates were analyzed by real time PCR and microarray, respectively. Standard error bars have been shown for both microarray and real time PCR data. Y-axis represents raw expression values from microarray and normalized expression value from real time PCR and X-axis shows different experimental conditions.