Elucidation of Complex Nature of PEG Induced Drought-Stress Response in Rice Root Using Comparative Proteomics Approach

Abstract

Along with many adaptive strategies, dynamic changes in protein abundance seem to be the common strategy to cope up with abiotic stresses which can be best explored through proteomics. Understanding of drought response is the key to decipher regulatory mechanism of better adaptation. Rice (Oryza sativa L.) proteome represents a phenomenal source of proteins that govern traits of agronomic importance, such as drought tolerance. In this study, a comparison of root cytoplasmic proteome was done for a drought tolerant rice (Heena) cultivar in PEG induced drought conditions. A total of 510 protein spots were observed by PDQuest analysis and 125 differentially regulated spots were subjected for MALDI-TOF MS-MS analysis out of which 102 protein spots identified which further led to identification of 78 proteins with a significant score. These 78 differentially expressed proteins appeared to be involved in different biological pathways. The largest percentage of identified proteins was involved in bioenergy and metabolism (29%) and mainly consists of malate dehydrogenase, succinyl-CoA, putative acetyl-CoA synthetase, and pyruvate dehydrogenase etc. This was followed by proteins related to cell defense and rescue (22%) such as monodehydroascorbate reductase and stress-induced protein sti1, then by protein biogenesis and storage class (21%) e.g. putative thiamine biosynthesis protein, putative beta-alanine synthase, and cysteine synthase. Further, cell signaling (9%) proteins like actin and prolyl endopeptidase, and proteins with miscellaneous function (19%) like Sgt1 and some hypothetical proteins were also represented a large contribution toward drought regulatory mechanism in rice. We propose that protein biogenesis, cell defense, and superior homeostasis may render better drought-adaptation. These findings might expedite the functional determination of the drought-responsive proteins and their prioritization as potential molecular targets for perfect adaptation.

Keywords: 2-DE; MALDI-MS/MS; SOTA analysis; drought; proteomics; rice root; tolerant cultivar.

Figure 1

2-DE analysis of root proteome of rice. (A) Rice plant showing different stages of drought. (B) Proteins were extracted from rice root tissue and equal amounts (250 μg) of proteins were separated by 2-DE as described in Materials and Methods Section. (C) Three replicate silver-stained gels for each stage were computationally combined using PDQuest software and one representative master standard gel image was generated.

Figure 2

A higher level master image was created in silico by PDQuest from three replicate gels of each time point. The boxed areas marked with dotted lines represent the zoomed-in gel sections in Figure 3. The numbers correspond with the spot IDs listed in Table 1.

Figure 3

Some of the differentially expressed root proteins represented in the enlarged gel sections (A–H) corresponds to the marked boxed areas in Figure 2.

Figure 4

Distribution of 78 identified differentially expressed rice root proteins in five functional classes based on their putative functions assigned them using protein database.

Figure 5

Expression clustering of 78 differentially expressed proteins showing 8 clusters based on their expression profiles. The gray lines represent expression profile of protein separately and the mean expression profile is indicated by pink line. Total number of proteins with same expression profile is provided in inset within the respective cluster. Detailed information on proteins within each cluster present in Figure S1.

Figure 6

Quantitative validation of relative expression of selected transcripts related to different functional classes of rice root. Heat map of some differentially regulated known and hypothetical protein showing their relative expression by qRT-PCR along with their protein expression data obtained from 2DE. The signal intensity of each transcript was normalized using ubiquitin house keeping gene. The top most bar represents the fold change expression values of selected genes.

Figure 7

Venn diagram showing the distribution of differentially expressed proteins in time-specific and overlapping manner during drought stress. The areas shown in the diagram are not proportional to the number of proteins in the groups.

Figure 8

Illustration of role of differentially regulated proteins involved in different pathways in rice for sustaining during drought stress. Proteins engaged in carbon breakdown, protein synthesis and ROS pathway are displayed on the corresponding metabolic pathways. Graphs are the representatives of expression profile of individual protein and number given below in each graph indicates the protein identification number.