Protein phosphatase complement in rice: genome-wide identification and transcriptional analysis under abiotic stress conditions and reproductive development

Abstract

Background: Protein phosphatases are the key components of a number of signaling pathways where they modulate various cellular responses. In plants, protein phosphatases constitute a large gene family and are reportedly involved in the regulation of abiotic stress responses and plant development. Recently, the whole complement of protein phosphatases has been identified in Arabidopsis genome. While PP2C class of serine/threonine phosphatases has been explored in rice, the whole complement of this gene family is yet to be reported.

Results: In silico investigation revealed the presence of 132-protein phosphatase-coding genes in rice genome. Domain analysis and phylogenetic studies of evolutionary relationship categorized these genes into PP2A, PP2C, PTP, DSP and LMWP classes. PP2C class represents a major proportion of this gene family with 90 members. Chromosomal localization revealed their distribution on all the 12 chromosomes, with 42 genes being present on segmentally duplicated regions and 10 genes on tandemly duplicated regions of chromosomes. The expression profiles of 128 genes under salinity, cold and drought stress conditions, 11 reproductive developmental (panicle and seed) stages along with three stages of vegetative development were analyzed using microarray expression data. 46 genes were found to be differentially expressing in 3 abiotic stresses out of which 31 were up-regulated and 15 exhibited down-regulation. A total of 82 genes were found to be differentially expressing in different developmental stages. An overlapping expression pattern was found for abiotic stresses and reproductive development, wherein 8 genes were up-regulated and 7 down-regulated. Expression pattern of the 13 selected genes was validated employing real time PCR, and it was found to be in accordance with the microarray expression data for most of the genes.

Conclusions: Exploration of protein phosphatase gene family in rice has resulted in the identification of 132 members, which can be further divided into different classes phylogenetically. Expression profiling and analysis indicate the involvement of this large gene family in a number of signaling pathways triggered by abiotic stresses and their possible role in plant development. Our study will provide the platform from where; the expression pattern information can be transformed into molecular, cellular and biochemical characterization of members belonging to this gene family.

Figure 1

Phylogenetic relationship among various phosphatase classes of rice. An un-rooted NJ tree is made from the domains sequences of rice phosphatases. Tree was made using ClustalX 1.81 and viewed in Treeview 1.6.6 software. The whole protein phosphatase gene family is divided into different classes, PP2A, PP2C, DSP, PTP and LMWP, each represented by a clade. PP2C class is further subdivided into different classes (A-K) each represented by a subclade as described by Xue et al [26]. Scale bar represents 0.1 amino acid substitutions per site.

Figure 2

Phylogenetic analysis of rice and Arabidopsis protein phosphatase genes. An Un-rooted NJ tree made from the domain sequences of rice and Arabidopsis protein phosphatases. Tree was made using ClustalX 1.81 and viewed using treeview 1.6.6. software. PPs from rice and Arabidopsis belong to same class falling in the same clades are based on the bootstrap support value ≥ 50%. Scale bar represents 0.1 amino acid substitutions per site.

Figure 3

Chromosomal localization of OsPP genes on 12 chromosomes of rice. Respective chromosome numbers are written at the top. Genes belonging to five classes have been marked by different colors. Corresponding numbers as described in Additionl file 5 indicate gene names. Dashed lines join the genes, lying on duplicated segments of the genome. Tandemly duplicated genes are joined with vertical lines. Chromosomes are grouped randomly to show the duplication with clarity.

Figure 4

Expression profiles of OsPPs under abiotic stress conditions. Three experimental stress conditions are denoted as CS: Cold Stress, DS: Drought Stress, SS: Salt Stress and S: control, 7 days old unstressed seedling. Color bar at the base represents log2 expression values, thereby green color representing low level expression, black shows medium level expression and red signifies high level expression. A gene is considered differentially expressed under abiotic stress conditions if it is up- or down-regulated at least two-fold, at P-value ≤ 0.05, with respect to the 7-days-old unstressed seedling.

Figure 5

Venn diagram for differentially expressed OsPPs. Protein phosphatase genes up-regulated (A), down-regulated (B) under different abiotic stress conditions. Different compartments showing the genes specific to either one particular stress (salt or drought or cold), involved in two stresses, or involved in all the three stresses. Protein phosphatase genes up-regulated (C), down-regulated (D) in stress and reproductive development showing overlapping expression pattern. Different compartments showing the genes specific to stress, panicle or seed stage or involved in stress-panicle, stress-seed or seed-panicle or involved in all the three conditions.

Figure 6

Expression profiles of OsPPs during reproductive development. Reproductive development comprising 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)] development. Genes are considered as up- or down-regulated w.r.t. all the vegetative controls, (L-mature leaf, R-root, and S-7-days-old seedling). Clustering of the expression profile was done with log transformed average values taking mature leaf as base line. The color scale at the bottom of the heat map is given in log2 intensity value. A gene is considered differentially expressed during reproductive development if it is up- or down-regulated at least two-fold, at P-value ≤ 0.05, with respect to the three vegetative controls (mature leaf, root and 7-days-old seedling).

Figure 7

Validation of expression profiles for selected OsPPs by Q-PCR. Two and three biological replicates were taken for Q-PCR and microarray analysis respectively. Standard error bars have been shown for data obtained using both the techniques. Y-axis represents raw expression values obtained using microarray and Q-PCR expression values normalized with the maximum average value obtained by microarray data and X-axis shows different experimental conditions; red bars represent the expression from microarrays, while blue bars represent the real-time PCR values.

Figure 8

Expression pattern of duplicated OsPP genes. The expression values of duplicated genes obtained from microarray data were compared in leaf (L), root (R) and 7-day-old seedling (SDL) tissue, and in various stages of panicle development (P1-P6), seed development (S1-S5) and cold stress (CS), dehydration stress (DS) and salt stress (SS). Each area graph represents compilation of the mean normalized signal intensity values from 17 stages of development/stress conditions. Gene pairs have been grouped into retention of expression, neo-functionalization and pseudo-functionalization based on their respective profile (A), expression pattern of OsPP genes in segmentally duplicated region of rice genome and (B), expression pattern of OsPP genes in tandem duplication.