Validation of identified genes for water stress in rice (Oryza sativa L.) mediated by Pseudomonas fluorescens

Abstract

Rice is the staple food for an expansive part of the world, particularly Asia. It is grown under diverse water regimes ranging from poorly drained waterlogged situations to well drain or water stressed upland conditions. Drought is one of the major abiotic stresses that adversely affect crop plants limiting their growth and yield potential. It disrupts the ionic and osmotic equilibrium of the cell. Susceptibility to water stress in rice is more pronounced at the reproductive stage and causes the greatest reduction in yield when stress coincides with the irreversible reproductive processes. Transcriptome analysis was carried out in Matta Triveni (PTB 45) to understand the influence of PGPR under water stress tolerance. Matta Triveni is a popular short duration (100-105 days) rice variety of Kerala, recommended for rainfed ecosystem especially in lowlands during the first crop and irrigated conditions during summers (third crop). With growing emphasis on expanding area under upland rice in the state, the need for an early, profusely tillering, drought tolerant variety is on the rise. Matta Triveni though high yielding is found to be adversely affected by drought. Hence, the present study was performed to assess the impact of application of Pseudomonas fluorescens in inducing tolerance to drought in variety Matta Triveni. In the present study the already identified differentially expressed genes such as Cytochrome oxidase subunit I (CO I), Protein kinase domain protein (PKDP), bZIPl (basic Leucine Zipper), AP2-EREBP (APETALA2 and ethylene-responsive element binding protein), Hsp20 (Heat shock protein 20) and COC1 (Circadian oscillator component) which are involved in ABA dependent and independent signal transduction pathways under water stress were validated using real-time PCR. Seedlings of rice variety Matta Triveni were given three treatments. Unstressed plants were maintained as absolute control. Water was withheld in second treatment and in the third treatment water was withheld in plants for which Pseudomonas fluorescens Pf1(KAU strain) was applied. Plants were given two applications of Pseudomonas fluorescens(pf) i.e., seed treatment and foliar spray. Water was withheld continuously for 15 days during reproductive stage (panicle initiation stage) after foliar spray. Plants exhibited leaf rolling which was scored as per IRRI, SES and was used for total RNA isolation. Biometric parameters were recorded after harvest. Total RNA was isolated from 65 days old rice plants after drought induction using TRIzol reagent. The first strand cDNA was synthesized by reverse transcriptase PCR from the above RNA samples. The cDNA was normalized using actin, which is a housekeeping gene. The real-time PCR primers were designed for six differentially expressed genes such as COI, PKDP, bZIPl, AP2-EREBP, Hsp20 and COC1 using IDT PrimerQuest software and the designed primers were validated using IDT OligoAnalyzer 3.1 tool. The target genes were analyzed by keeping actin as endogenous control. Comparative Δ Ct method was used to analyze the expression levels of genes. Results revealed that all these genes were upregulated in water stressed + Pf treated plants. COI,PKDP, bZIPl, AP2-EREBP, Hsp20 and COC1 genes were found to have 2.3, 2, 6, 2.8, 4.5 and 4 fold increases in relative expression levels respectively in the same treatment as compared to control. In-silico analysis was performed to identify the Transcription Factor Binding Sites (TFBSs) and combinatorial cis-regulatory elements for COI, PKDP, bZIP, AP2- EREBP, Hsp20 and MYB genes which are expressed in treatments. TFBSs were identified by AthaMap database, a genome-wide map of TFBSs in Arabidopsis thaliana, and STIFDB2 database which is a comprehensive collection of biotic and abiotic stress responsive genes in Arabidopsis and Oryza sativa. The significant TFBSs were analyzed based on the parameters provided by databases and were cross validated. The results revealed that the WRKY, MYB, HSF, bZIP, ARF, AP2/EREBP, bHLH, Trihelix and ABI3/VP1 TF families and their respective regulatory elements were predicted as functionally significant. The combinatorial cis- regulatory elements for three genes viz, CO 7, AP2-EREBP and Hsp20 were also identified. The transcription factors belonging to MYB, WRKY, bHLH and Trihelix families which are mainly responsible for drought stress signals and were predicted as co-associated. The study had shown that gene expression in plants lacking adequate water can be remarkably influenced by microbial colonization. The activation of the ABA dependent signaling pathway induced by colonization of P. fluorescens might be a key element for induced systemic tolerance. The predicted TFBSs and combinatorial cis-regulatory elements would be responsible for the upregulation of genes under water stress.