Functional Analysis Of OsWRKYX And G1/ELE Genes In Rice

As one of the most important food crop in the world,rice is often subjected to biotic and abiotic stresses by the external environment because of its fixed property.It is an important strategy to improve and enhance the resistance in rice by screening and cloning of stress-resistant genes which are involved in plant biotic and abiotic stress responses.WRKY transcription factors bind to the W-box through their WRKY domains and regulate the expression of genes related to disease resistance,drought,heat,cold,high salt,oxidative damage and senescence;participating the response to biotic and abiotic stress,hormone signaling pathways,developmental and metabolic process.In this study,a T-DNA insertion line of OsWRKYX was screened and analyzed.Meanwhile,a series of experiments were performed.Main results are below: 1.OsWRKYX is extensively expressed in roots,stems,leaves and flowers,while the expression level in roots and leaves are higher.The expression is induced by PEG,ABA and SA,but not induced by high temperature(40?),low temperature(4?)or Nacl.2.The result of molecular detection showed the T-DNA was inserted in the first intron of OsWRKYX and resulted in the obvious decrease of the expression level.Furthermore,under PEG stress,the concentration of proline is higher and the stomatal aperture is smaller in oswrkyx-1 than wild type.These results suggest the ability to resist to drought stress of oswrkyx-1 is stronger than wild type plants,and OsWRKYX is a negative regulator for response to drought stress.3.After treatment by ABA,the ratio of germination of oswrkyx-1 seeds is much lower than wild type seeds and more sensitive to ABA than wild type seeds.The expression of key genes associated with ABA signaling pathway was analyzed by quantitative RT-PCR.As predicted,the expression of OsEM1 and OsRD29 B were down-regulated,while the expression of OsABI5,Os RAV1 and OsMYC2 were up-regulated in mutant,further indicating that function of OsWRKYX is associated with the ABA pathway.4.The over expression vector was constructed and transformed into rice and Arabidopsis thaliana,and the transgenic plants were obtained.Rice empty glumes,also named sterile lemmas or rudimentary lemmas according to different interpretations,are distinct from lemmas in morphology and cellular pattern.Consistently,the molecular mechanism to control the development of lemmas is different from that of empty glumes.Rice LEAFY HULL STERILE1(OsLHS1)and DROOPING LEAF(DL)regulate the cellular pattern and the number of vascular bundles of lemmas respectively,while LONG STERILE LEMMA1(G1)/ELONGATED EMPTY GLUME(ELE)and PANICLE PHYTOMER2(PAP2)/Os MADS34 determine identities of empty glumes.Though some progress has been made,but identities of glumes,genetic interaction between lemma genes and glume genes have been less elucidated.1.In this research,a new G1/ELE mutant g1-6 was identified and the phenotype was analyzed.Similar to previously reported mutant lines of G1/ELE,empty glumes of g1-6 plants transform into lemma-like organs.Furthermore,the genetic interaction between G1/ELE and OsLHS1 was analyzed.Essentially,the phenotype of empty glumes in double mutant plants is similar to that of g1-6plants,while phenotypes of inner floral organs mimic those of osmads1-z plants.These additive phenotypes imply these two genes function independently.2.Meanwhile,expression patterns of G1/ELE in osmads1-z flowers and OsLHS1 in g1-6 flowers indicate they do not regulate each other at the level of transcription.Detailed analyses showed abnormal empty glumes and lemmas in double mutant plants form more vascular bundles than those of g1-6 or osmads1-z plants,respectively.Additionally,the thickness of these organs in double mutant plants is thinner than that of g1-6 or osmads1-z plants because of smaller cell volumes and decreased layers of cells.These findings suggest G1/ELE play a role in the development of lemmas redundant with OsLHS1.3.Finally,down-regulation of the empty glume gene OsMADS34/PAP2,and ectopic expression of the lemma gene DL,in the g1-6 background provide further evidence that empty glumes are sterile lemmas.Furthermore,G1/ELE appears to regulate the expression of OsMADS34/PAP2.In general,by adapting the genetic strategy,molecular and physiological technology,preliminary results suggest OsWRKYX might regulate the reponse to drought stress negatively via ABA pathway.In addition,findings on the function of G1/ELE also provided valuable information for better understanding functions of G1,OsLHS1 in flower development and identities of empty glumes.Our findings lay a foundation for elucidating the biological function of OsWRKYX and G1/ELE.