Functional Characterization Of Flavone-specific Glucosyltransferase(OsUGT707A2) From Rice And Dof Transcription Factor(SlDof17) From Tomato

This dissertation is divided into two parts and studies are conducted in two economically important crop plants,rice and tomato.In first part of our study,we have identified flavonoids specific glycosyltransferases(UGTs)from rice using metabolitebased genome wide analysis and further characterized their in vitro and in vivo functions.In second part of our study,we have focused on drought responsive tomato Dof transcription factor,Sl Dof17,which was previously up-regulated in our microarray analysis.For further functional characterization,we have generated Sl Dof17-overexpression transgenic plants and elucidate its important roles in abiotic stress tolerance.PART ?The plant kingdom is capable of producing a wide range of secondary metabolites,such as terpenoids,alkaloids,phenylpropanoids as well as flavonoids.In particular,flavonoids are low molecular weight secondary metabolites that are essential for plant survival.Additionally,with over 10,000 structural variants already known,they are bioactive across all major plant lineages.Flavonoids synthesis appears to be ubiquitous in plants and evolved early during land plant evolution,aiding in plant protection and signalling.In plants,various biological processes depend on the formation of myriad of structurally diverse flavonoids and their glycosides and therefore glycosylation is a key modification step to this glycodiversity.Research on rice metabolomics especially secondary metabolites plays a very important role in understanding plant metabolic pathways and improving plant response to environmental stresses.The study of secondary metabolites in rice,especially flavonoids is of great significance for the analysis of rice growth and development,stress response and nutritional quality.At the same time,dissecting the flavonoids variation in rice which is a model organism for gramineous species,will also promote the development of research on other important crops.In this study,using widely targeted metabolite profiling combined with genome-wide association analysis;we were able to identify a single locus strongly associated with flavone-5-O glycosides in rice.Based on gene-based analysis,we identified four putative UGT encoding genes(Os UGT707A2,Os UGT707A3,Os UGT707A4 and Os UGT707A5)within confidence interval of this locus.Phylogenetic analysis was performed with previously reported flavonoid UGTs and the candidate UGTs of rice,identified in this study.Consequently,the candidate UGTs were clustered together in a separate group,which strongly point out their ability to use different substrate to exhibit their novel functions.In vitro biochemical screening showed that Os UGT707A2 enzyme could utilize both flavonols and flavones,however,substrate specificity results showed high preference for apigenin(flavone)compared to other flavonoids.Further,apigenin was the best substrate catalyzed by Os UGT707A2 enzyme as revealed by comparing the rate of glycosylation for different flavonoid substrates.Moreover,UV absorbance spectra revealed that Os UGT707A2 reaction product have single peak with similar retention time to apigenin and tricin 5-O glucosides.These results are also consistent with our prediction from association mapping,therefore we concluded that Os UGT707A2 is a major flavone UGT responsible for glucosylation at 5-OH position of flavones in rice,thus it was later termed as flavone 5-O glucosyltransferase(F5Glc T).To further confirm in vivo functions of Os UGT707A2,we generated overexpression transgenic plants.Instead of its in vitro ability to utilize both flavonols and flavones,interestingly,Os UGT707A2 showed rather strict in vivo substrate activity and we found that only flavone glycosides were dramatically accumulated in transgenic plants.Further,we searched for functional polymorphisms within Os UGT707A2 using Rice Var Map database.As a result,we found that an SNP(sf0719059405,400 bp upstream of coding region)and an In Del(vf0719060607,a C deletion at position 773 bp),represents the functional polymorphisms responsible for variation in content of apigenin 5-O-glucoside in rice.In order to understand the evolutionary context of the identified UGTs at locus 1(corresponds to chromosome 7),which were clustered together and form a distinct clade unique to the previously reported UGTs,an extensive BLAST search was performed.Unlike flavonoid 3-O glycosyltransferases(F3GTs)and flavone 7-O glucosyltransferases(F7Glc Ts),we found that F5 Glc T clade was unique to commelinids and thus validating the exclusive identification of F5 Glc T orthologs.Moreover,apparently the absence of F5 Glc T in Gymnosperms as well as in core eudicots,suggesting an early appearance in common monocot ancestors,which is in accordance with the taxonomic relationship defined by APGIII taxonomic system.Most intriguingly,the presence of a single clade of three tandem UGTs in Elaeis guineensis separated from Gramineae demonstrate that F5 Glc T genes evolved independently after speciation between Palmaceae and Gramineae.Further,co-linear genomic analyses and the recombinant protein assay for F5 Glc T also supported the idea of their independent duplication.Taken together,this data provide biochemical insight and genetic regulation of flavone biosynthesis in rice.In addition,considering the collective roles of different classes of flavonoids and their positive roles in various stresses,this study suggests that adoption of the positive allele of this gene into breeding programs will further pave the way to produce stress-tolerant plants.PART ?Transcription factors are those proteins that contain a conserved domain through which they influence the transcription of other genes via binding to specific sites in their promoter.Dof(DNA binding with one finger)are specialized family of transcription factors and are involved in multiple aspects of plants growth and development.In this study a tomato Dof,annotated as Sl Dof17,was cloned and over expressed in tomato to generate transgenic lines for functional characterization.Our phylogenetic analysis demonstrate that Sl Dof17 clade together with cyclic Dofs from other plant species,suggesting its involvement in circadian regulation.In addition,presence of number of stress-responsive and circadian regulatory cis-elements in the promoter region strongly suggests that Sl Dof17 is actively involved in circadian-regulated plant growth and development under stress conditions.Expression analysis revealed that Sl Dof17 transcripts oscillate during diurnal cycles and is responsive to subjective light/dark(L/D)and continuous light(L/L)conditions,suggesting that Sl Dof17 follows the circadian clock irrespective of light conditions.Moreover,early flowering was observed in Sl Dof17-OE lines compared to WT under neutral day(ND)photoperiod.Further,we found the strong up-regulation of flower promoting gene,SFT,while flowering repressor,SP,was down-regulated in Sl Dof17-OE lines under ND conditions.Our study also showed that chlorophyll contents were enhanced in OE plants than in WT growing under ND conditions.Surprisingly,most of the chlorophyll biosynthetic genes were strongly induced in transgenic lines under ND conditions,whereas no significant difference was found under long day(LD)photoperiod,hence explaining the Sl Dof17 regulated molecular mechanism of distinct chlorophyll accumulation under ND and LD conditions.Our previous microarray screening showed that Sl Dof17 expression was upregulated during drought stress.We therefore extend our study and analyzed the tolerance level of Sl Dof17-overexpression plants subjected to drought and salt stress,the two major abiotic stress factors severely affecting economically important crops including tomato.Our preliminary test showed that Sl Dof17 was responsive to number of different abiotic stresses as well as plant hormones,suggesting that its transcriptional activation participates in broad-spectrum signaling pathways.Interestingly,seedlings from OE transgenic plants overall showed better performance in terms of seed germination rate and seedling growth in MS media supplemented with mannitol and Na Cl stress as compared to control.In addition,when soil-grown mature plants were subjected to drought and salinity stress,we found that Sl Dof17-OE plants showed enhanced resistance compared to WT plants.Under drought stress,WT plants have shown severe wilting symptoms after 14 days whereas OE plants remained turgid except yellowing of the basal leaves.In case of salt stress,WT plants could not survive after 14 days while Sl Dof17-OE plants showed enhanced tolerance,however,we lost few basal leaves at this stage.Moreover,Sl Dof17 is involved in ABA biosynthesis/signal transduction pathways as indicated through modulation of ABA biosynthesis and receptors related genes expression during stress response in transgenic tomato.Consequently,number of physiological and biochemical changes were noticed under drought and salt stresses in OE transgenic plants.At the molecular level,expression of six drought stress related genes were up-regulated in OE plants.Most importantly,reduced levels of MDA were observed both under drought and salt stress in OE transgenic as compared to WT,suggesting that OE plants encountered less cell membrane injury.Under drought stress,leaf chlorophyll,photosynthesis and soluble sugars all were well maintained and thus enhanced plant biomass was recorded in Sl Dof17-OE lines than in WT.Most intriguingly,our results provide evidence that overexpression of Sl Dof17 helps mitigate the oxidative stress and strengthen the antioxidant system,as indicated by higher accumulation of polyamines(Put,Spd,Spm)and increased antioxidant enzyme activities(SOD,POD and CAT)in OE plants,thus demonstrating a valid reason of enhanced tolerance in transgenic tomato.Overall,these results insinuate that Sl Dof17 is involved in circadian clock mediated physiological responses and play important roles in abiotic stress tolerance.