MENON, TARA G.

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                                                                                                                                                            ABSTRACTSalinity and drought are the two major environmental constraints thataffect plant growth and in turn crop productivity, a problem that has beenincreased by improper land usage and irrigation practices. Thoroughunderstanding of the salt tolerance adaptations of plants in terms of biochemicaland molecular levels can provide tremendous knowledge about their tolerancestrategies. Salinity stress in plants induces the formation of reactive oxygenspecies (ROS) and plants have formulated an array of ROS scavenging system(enzymatic and nonenzymatic antioxidants) to mitigate the cellular componentsfrom oxidative burst. Mangroves are intertidal plants inhabiting the coastal zones of tropicaland subtropical regions and possess unique characteristics of tolerating high-salinity of seawater and also tidal currents. Recent progresses in understandingthe physiological mechanism of salinity adaptation in mangroves on molecularlevel indicate that the phenomenon is indeed tightly linked to the regulation of gene expression. Recent studies on salinity tolerance in mangroves likeAvicennia marina, Aegiceras corniculatum, Bruguiera gymnorrhiza, Bruguieracylindrica, Ceriops tagal, Acanthus species have identified number of differentially expressed stress regulated genes. Rhizophora apiculata Blume. isa highly salt tolerant mangrove species inhabiting the coastal belts of Kerala.The present work proposes to examine the ROS cycle and also geneexpression in Rhizophora apiculata in response to NaCl stress by suppressionsubtractive hybridization and gene cloning analysis.Salt stress was imposed on R. apiculata seedlings with five differentconcentrations of NaCl (0, 100, 200, 300, 400 and 500 mM) and also with timeslots such as 3, 6, 12 and 24 h durations. NaCl induced remarkable changes intotal protein levels and is visualized with protein banding by SDS-PAGE. NaClix                                                                                                                                                                                            stress induced the profile of superoxide anion and hydrogen peroxide content.Further, the activities of NADPH oxidase, superoxide dismutase (SOD),catalase (CAT) and peroxidae (GPOX) showed varied levels of expression withconcentrations and durations. Different isoenzyme of SOD, CAT and GPOXwere detected with the NaCl treatment. Enhancement in antioxidant enzymeactivities could be an induced response against cellular damage due to NaClstress. Significant ascorbate-glutathione cycle (in terms of activities of ascorbate peroxidase (APX), regenerating enzymes and isozymes of APX)supports the efficiency of the mangrove to detoxify the ROS hydrogen peroxidefrom cells. Ascorbate, reduced glutathione and α-tocopherol increase thecapacity of the species to neutralize the toxic effects of NaCl stress.The molecular part includes Subtractive hybridization using PCR SelectTM Subtraction kit (Clontech, CA, USA). Several stress genes were isolated,which were then classified to nine functional categories viz., Metabolism,Protein degradation and folding, Secondary metabolism, Cell rescue anddefense, Transport facilitation, Signal transduction, Transcription andtranslation, Photosynthesis and Unclassified genes of unknown function. Theexpression patterns of twelve of the genes thus obtained were studied at 6 h,12 h and 24 h time points of salinity stress using Real time PCR. Most geneswere found to be upregulated under salt stress and showed maximumupregulation at the 6 h time point. Two of the genes studied were downregulated after 6 h, implying that the pattern of gene expression varies with timeof application of stress.The most important gene identified by SSH and upregulated in Real timeRT PCR is Cytochrome P450 monooxygenase (S)-N-methylcoclaurine 3′-hydrolase (CYP80B3), which acts in the hydroxylation of (S)-N-Methylcoclaurine to yield (S)-3'-Hydroxy-N-methylcoclaurine.The full lengthgene (1.5 Kb) was isolated and its expression in planta was attempted. Thex                                                                                                   promoter sequence of the gene was also isolated by genome walking approach.The size of the amplified product was ~0.5Kb. Several domains were identifiedin the promoter region such as CAAT box, TATA box, Myb Binding Site (MBS)etc.Another gene that was highly upregulated in the real time PCR analysiswas that of Hevamine A precursor.The gene (1.1Kb) was isolated, cloned into abinary vector, pMDC 139 and expressed in onion epidermal cells. Syringeinfiltration of the binary vector construct into onion peels showed blue colourdue to gus activity.The present results suggest that the synergistic effect of different ROS-scavenging enzymes and compounds effectively improve the biochemicalcaliber of plants, thereby reducing the degree of oxidative injuries. Similarly, thegenes and the gene elements thus isolated may be used to confer the trait of salt tolerance to non – tolerant genotypes, which can ultimately prove beneficialfor crop improvement programmes.Key words: Antioxidant enzymes, Mangrove, NaCl, Oxidative stress, Reactiveoxygen species, Rhizophora apiculata, Salinity, Stress genesxi

MOLECULAR SCREENING FOR SALT TOLERANCE GENES IN Rhizophora apiculata Blume.

http://eprints.csirexplorations.com/18/1/MENON%2C%20TARA%20G.%20%282015%29.pdf

MOLECULAR SCREENING FOR SALT TOLERANCE GENES IN Rhizophora apiculata Blume.

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