Functional Characterization Of OsELF3-1 Gene For Heading Date In Rice(Oryza Sativa L.) And Applications Of Osa-miR393a In Perennial Turfgrass

Chapter I:Functional Characterization of OsELF3-1 Gene for Heading Date in Rice(Oryza sativa L.)Rice(Oryza sativa L.)is one of the most important food crops which the mankind depends on for living.Rice flowering,referred to heading date in rice is an important agronomic trait for the reproductive development of rice.It affects the distribution,introduction,adaption and production of rice cultivars.The regulation of plant reproductive growth is a complicated process,involving many genes.EARLY FLOWERING 3(ELF3)is a positive regulator of circadian clock in Arabidopsis,which works between photoreceptor and core oscillator,and maintains the viability of the circadian rhythm to obtain the accurate amplitude and period.Mutations in ELF3 locus result in the loss of both photoperiod sensitivity and circadian regulation,and early flowering in both LD and SD light conditions.Through the study on a reproductive development-delayed T-DNA insertion mutant,we found the mutation was located in rice chromosome 6.The mutation caused from the loss of function gene OsELF3-1,which is the orthologs of ELF3 in Arabidopsis.In this study,we elucidated the function and regulation mechanisms of OsELF3-1,which will contribute to further study the role of OsELF3-1 in rice heading date regulation,explore the complex network of rice flowering pathways and enrich mechanism in rice photoperiodic flowering.The main results are summarized as follows:1?By blast in The Rice Genome Annotation Database Release 6.0(http://rice.plantbiology.msu.edu)shows that there are two putative orthologs of Arabidopsis ELF3 in rice,which are OsELF3-1 and OsELF3-2.OsELF3-1 and OsELF3-2 share 35%and 30%identity with ELF3 in amino acid sequence,respectively.Phylogenetic analysis showed that two homologs exist in most examined species.The two homologs in plants might have occurred during genome duplication and performed important roles respectively during plant genome evolution.2?Gene expression during a 24-hour period of OsELF3-1 was examined in OsELF3-1 RNAi and Nip under SD and LD conditions.We found that in OsELF3-1 RNAi,OsELF3-1 was constitutively repressed regardless of the conditions.All the homozygous T2 progenies of OsELF3-1 RNAi transgenic lines consistently showed delayed flowering compared with Nip,Which resulted from the repression of OsELF3-1 gene.3?OsELF3-1 is expressed in various tissues or organs,and the transcript levels were highest in leaves and leaf sheaths.There is a circadian rhythm of OsELF3-1 transcript levels,which accumulates in darkness,reaches a peak at dawn,and decreases after sunrise.4?OsELF3-1 can affect rice circadian systems via the positive regulation of OsLHY and the negative regulation of OsPRR1,OsPRR37,OsPRR73 and OsPRR95.In addition,OsLHY and OsPRRs were not affected under DD in mutants,indicating that OsELF3-1 might be responsible for mediating light input to the core circadian oscillator.5.OsELF3-lis involved in regulating the rice photoperiodic flowering pathway.Under short-day(SD)conditions,OsELF3-1 directly activates the flowering activator Ehdl and Hd3a to accelerate flowering.Under long-day(LD)conditions,OsELF3-1 suppresses the flowering repressor Ghd7 to indirectly accelerate flowering.6?OsELF3-1 is essential for Ehdl activation in response to blue light in the morning under SD conditions.OsELF3-1 is involved in blue light signaling by activating Ehdl to promote rice flowering under SD conditions.Chapter II:Applications of Osa-miR393a in Perennial TurfgrassAs an essential part of the world landscape ecosystem,turfgrass is a precious natural resource.Turfgrass is important in human's manufacture and life.It plays a good role in beautifying,protecting and improving our environments.Environmental stresses significantly influence turfgrass quality and production and lead to economic loss.Therefore,improvement of abiotic tolerance in turfgrass will greatly reduce water usage and turfgrass management cost,benefiting the environment and the turfgrass industry.Plant miRNAs regulate gene expression at post-transcriptional levels,which are involved in many biological processes,including plant growth and development,signal transduction and response to environmental stresses.The miR393 family is one of the first characterized and conserved miRNA families in plants.In rice,the miR393 family is encoded by two loci,Osa-miR393a and Osa-miR393b.Osa-miR393 could target two rice auxin receptor genes(OsTIR1and OsAFB2).An increase in tillers and early flowering were caused by overexpression of OsmiR393.OsmiR393 plays an important role in plant response to salt stress and auxin.In this study,we generated transgenic creeping bentgrass(Agrostis stolonifera L.)overexpressing rice microRNA393a gene(Osa-miR393a)to investigate the roles and mechanism of miR393 in controlling plant development and plant response to abiotic stress for improved quality and enhanced stress tolerance in perennial turfgrass.The main results are summarized as follows:1?Based on Osa-miR393a cDNA sequence on Gramene website,we designed primers to amplify Osa-miR393a gene fragment.PCR products were cloned into the binary vector pZH01,generating an Osa-miR393a overexpression gene construct pZH01-Osa-miR393a,which contains a CaMV35S promoter driving Osa-miR393a and a Hyg gene for hygromycin resistance as a selectable marker.2?The over-expression vector pZH01-Osa-miR393a was transferred into embryogenic callus of creeping bentgrass,a perennial turfgrass species,through Agrobactetium-mediated transformation.After tissue culture,antibiotic resistance gene screening and callus differentiation,the regenerated plants were gained.Through screening these regenerated plants by amplifying Hyg gene with semi-quantitative PCR and real-time PCR,five positive transgenic plants are obtained.3?Osa-miR393a transgenics had fewer and longer tillers than WT control plants.However,no significant difference in shoot biomass between WT control and Osa-miR393a plants was observed,suggesting that the increased tiller length in Osa-miR393a plants might have compensated for the loss in biomass caused by decreased tillering.In contrast,the root biomass of Osa-miR393a plants was significantly lower than that of the WT controls.In addition,Osa-miR393a plants displayed wider leaves and larger stems than WT controls.Microscopic analysis indicates that it might be associated with the increased vein number of stem and leaf in transgenics in comparison with WT controls.4?Overexpression of miR393 improves salt tolerance in creeping bentgrass,which is associated with enhanced water retention,cell membrane integrity,and photosynthetic efficiency.No significant difference in root and shoot Na+ content was observed between Osa-miR393a transgenics and control plants before salt stress.After salt stress,transgenic plants showed significantly higher Na+ contents than wild-type controls,whereas their Na+ contents were similar in roots.During salt treatment,K+contents were decreasing in wild-type and transgenic plants in comparison with the normal growth conditions.Under both normal and salt stress conditions,transgenic plants showed significantly higher K+ contents than wild-type controls in shoots and roots.5?After heat stress,most wild-type leaves were yellow and wilted,while the leaves of TG plants kept green with minor heat damage and less degree of reduced biomass compared to wild-type controls,suggesting an enhanced heat tolerance in Osa-miR393a transgenics,which is associated with enhanced water retention,cell membrane integrity and photosynthetic efficiency,and the induced expression of chloroplast-localized small heat shock proteins.6?Fifteen days after water withholding,wild-type plants started to display dehydration symptoms,whereas Osa-miR393a transgenics remained turgid without obvious damage.Twenty days after water withholding,wild-type plants showed serious tissue damage while most Osa-miR393a transgenics remained green.Water loss and drought-elicited cell membrane damage in Osa-miR393a plants were less severe than in wild-type plants.When subjected to limited water supply treatment,wild-type plants displayed many morphological changes with shortened leaves and stems,while Osa-miR393a transgenics were kept growing with less impaction.Although the tillers of wild-type plants were still more than that of Osa-miR393a transgenics,the shoot biomass of control plants was significantly reduced.No significant difference in root biomass was observed.The results suggest that overexpression of miR393 improves drought tolerance in creeping bentgrass,which is associated with reduced stomata density on leaf surface and dense cuticle.7.Based on the sequence of OsTIR1 and OsAFB2 in rice,we blasted the corresponding creeping bentgrass homologs AsTIR1 and AsAFB2 based on our RNA-seq data,and identified the putative target sites which were highly complementary to miR393.The target genes AsTIR1 and AsAFB2 were all down-regulated in three Osa-miR393a transgenic lines,and they responded to various abiotic stresses,including salt,heat and drought,which might contribute to the enhanced abiotic tolerance of transgenics.In addition,other stress-related genes,AsNAC60 and AsDREB2B were down-regulated in Osa-miR393a transgenic plants,indicating that miR393 indirectly regulates AsNAC60 and As DREB2B.In summary,overexpression of miR393 impacts plant development and enhances plant drought,heat and salt tolerance.The miR393-mediated down-regulation of target genes in transgenic plants may have caused changes in various biological processes.Therefore,the manipulation of miR393 target genes provides novel molecular strategies to crop species for enhanced resistance to environmental stress.