Rice plants are constantly exposed to various adverse abiotic and biotic stress factors that alter the molecular and physiological pathways leading to differential gene expression. Roots are the primary plant organs to perceive abiotic stress (drought and salinity) signals, which later induce specific signal transduction to shoot regions. Drought responsive genes along with novel transcripts were identified from QTL introgressed line rice (NIL of IR64, with QTL introgression from Azucena, a japonica rice variety) induced to moisture stress. Differential Display Reverse Transcription Polymerase Chain Reaction (DDRTPCR) strategy (Liang and Pardee 1992) was employed to identify genes, selectively expressed due to drought stress induction.

Rice plants were grown for one month in plastic pots at 100% of the water capacity of the field and at 31°C. Drought stress was imposed withholding water for one week from the stress pots so as to reach 50% field capacity leading to the appearance of drought while the control plants were maintained at 100% field capacity. Total RNA was extracted from root samples of both controlled and stressed plants for gene expression analysis. Different poly (A) tailed mRNA sub-populations from the total RNA extract were pooled by combinations of single penultimate based primer H-T11G (2μM/μL) and H-T11C (2μM/μL) The second strand synthesis and PCR was performed using arbitrary primers H-AP9 (2μM/μL), H-AP10 (2μM/μL) (GenHunter Corporation, USA).Reaction temperatures and number of cycles were followed as per the company indications. The amplified cDNA products were resolved on a 6% sequencing gel, eluted and purified. Automated sequencing analysis (Bangalore Genei Pvt Ltd, India) of the purified samples and their subsequent sequence homology search against the NCBI non-redundant database using the BLAST program indicated the candidacy of genes correlating to Diacyl-glycerol kinase (DAGK), Calcium Dependent Protein Kinase (CDPK), and hypothetical sequences. The hypothetical transcripts were translated using Swissprot ExPasy Translation Tool. Both 5'-3' and 3'-5'Aminoacid sequence reading frames were analyzed for heir amino-acid contents, protein instability index, and also for Grand average of hydropathicity (GRAVY) using ProtParam Program of ExPasy tools. Table 1 summarizes the sequencing results for the cDNAs obtained by differential display.

The transcript-1 (DQ632738) exhibited 96% homology to Diacyl-Glycerol Kinase (DAGK) from Escherichia coli K-12 MG1655 (Gen Bank accession no: U00096 AE000111-AE000510). Automated sequencing analysis (Bangalore Genei, India) of transcript-1 gave the sequence pattern as depicted in the (Fig. 2).

Various lipid compounds are known to be involved in signal transduction of drought stress as second messengers in plants. Three major categories of Glycerolipids found in plant membranes are Acylglycerols, Phospholipids and Glycolipids (Ohlrogge and Jawroski 1997, Lea and Leegood 1993). Diacyl-glycerols (DAG) in plants are the basic components of all Glycerolipids (Harwood 1980). The Diacyl-Glycerol Kinase (DAGK) enzyme catalyzes the following reaction: ATP + 1, 2-diacyl-glycerol = ADP + 1, 2-diacyl-glycerol 3-phosphate. An increase in the accumulation of Phosphatidic Acid (PA) and Diacyl-Glycerol Phosphate (DAGP) is common during water deficit conditions (Munnik T et al. 2000) ). Diacyl-glycerol kinase (DAGK) phosphorylates DAG to generate PA. The increase in the level of PA in plant cells is partly due to DAGK activity, while most of it is synthesized due to Transphosphatidylation of 1-butanol. Further conversion of PA to DAGP by Pyrophosphorylation is indicative of drought signaling. DAGKs are known to be involved in membrane trafficking as well as defense responses (Munnik T et al. 2000). Transcript- 3 (DQ632737) showed homology to putative Calcium Dependent Protein Kinase (CDPK) of Nipponbare. CDPKs are a family of Ser/Thr protein kinases found abundantly in plants. When plants are exposed to any type of stress they first respond with an early set of events that include a shift in membrane fluidity and drought induced calcium flux, followed by a secondary response in which calcium-regulated protein kinases and phospshatases are believed to be involved. Calcium flux, first characterized in plants as the secondary messenger in response to cold acclimation processes, is also known to take part in drought signal transduction. Various membrane-bound, drought and cold-responsive CDPKs have been identified by biochemical analysis. The activation of CDPK is detected after 12-18 hrs of stress indicating that the kinase does not participate in the initial response to stress but rather in the adaptive process to adverse conditions. The involvement of CDPKs in osmotic signaling has been suggested by the transcriptional induction of CDPK genes in response to salinity, cold, or drought in rice (Oryza sativa), since over expression of OsCDPK7 conferred cold, drought and salt tolerance by inducing the expression of stress responsive genes (Mariana Laura Martin et al. 2001).

The novel, hypothetical transcripts (DQ632736, DQ632739, DQ632740, and DQ632741) identified were translated using Swissprot Translation Tool. Aminoacid sequence reading frames were analyzed for their aminoacid contents, Protein Instability Index and Grand Average Hydropathicity, results interpreted as in table 2. Leucine rich repeats (LRR) and Proline rich repeats (PRR) were identified. Proline accumulation is a common metabolic response of higher plants to water deficit and salinity stress, and has been the subject of numerous reviews over the past 20 years. Solubel-Prolines protect membranes and proteins against dehydration and temperature extremes (Paleg et al. 1981, Paleg et al. 1985, Santoro et al. 1992). A decrease in the Proline oxidation rate can contribute to net proline accumulation during drought and salinity stress (Kiyosue et al. 1996). The sequence data of all the transcripts is being used for EST marker designing and rice genome mapping.

Acknowledgements

The authors gratefully acknowledge the financial support by the Rockefeller Foundation (RF-FS031), USA to carry out this work.

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