Antisense OsPDCD5 Transgenic Rice Enhances Salt Tolerance Of Rice And LRK1 Gene Affects Rice Introgression

●Antisense-OsPDCD5increases rice tolerance to salt stressEnvironmental stressesinfluence the growth of plants and theproductivity of crops.Salinity is one of the most important abiotic stresses for agricultural crops.High salinity causes hyperosmotic and ionic stresses, which in turn mightgenerate secondary stresses such as oxidative stress.Plants have commonmechanisms, not only in terms of the molecular and cellularresponse, but also in terms of the physiologicaland biochemical response, for adaptation to these stresses.Upon exposure to the stresses, many genes are induced andfunction either as cellular protectants of stress-induceddamage or as regulators of gene expression and signal transduction.Programmed cell death (PCD) is a key element during normal plant growth and development.Besides the involvement in normal growth and developmental processes,PCD is also induced by a variety of biotic and abiotic stresses in algae and higher plants, including high salinity treatment. OsPDCD5, an ortholog to the mammalian-programmed cell death5gene, is up-regulated under lowtemperature and salt treatments in rice (Oryza sativa).In this study, we found transgenic rice that constitutively expressed antisense-OsPDCD5increased salt stress tolerance. After the highsalinity treatment, subsequent watering with half-strength MS mediumfor recovery,45-55%of the transgenic rice seedlings survived and recovered, while the growth of salt-stressedNC rice was severely inhibitedand finally died. Measurement of changes in chlorophyll fluorescencefurther verified theenhanced salt tolerance of the transgenic rice.Real-time PCRanalysis showed the endogenous OsPDCDS is repressed under both the normalcondition and salt stress treatment in transgenic rice. Real-time PCRanalysis was also performed on expressionprofiles oftwo PCD markeror PCD-relatedgenes (OsBI-1and Os11g0506800) to confirm the PCD pathways were involved in salttolerance of the transgenic rice.The results demonstrated the PCD pathways are blocked in transgenic rice, when PCD is induced in NC riceby high salinity. Real-time PCRanalysis also showed calcineurin B-like interacting protein kinase23(OsCIPK23), which interacts withOsPDCD5protein, was repressed underboth the normal condition and salt stress treatment in transgenic rice, which was identical to theresponse shown by OsPDCD5. It is likely OsCIPK23acts as a molecular integrator ofthe cross-talk betweenPCD pathwaysand salt stress pathways.Using transcript microarrayanalysis,38stress-related genesregulated in transgenic rice were identified, which appeared to render the transgenic rice adapted for salt stress conditions, consequently conferring the plant with increased tolerance to salt stress.We concluded that antisense-OsPDCD5transgenic riceincreasessalt stress tolerancein unique ways by blocking PCD pathways and regulating specific groups of stress-related genes.PCD pathways are blocked and salt stress signaling pathways areregulated in transgenic rice even under the normal growth conditions withoutstress. PCD is further inhibited and these stress-related genes are further regulated to increase tolerance to high salinity under salt stress conditions, thereby leading to build-up of stress protection mechanisms in transgenic rice. ●LRK1restricts internode elongation in ricePlant height is a critical constituent of plant architecture. Rice (Oryza sativa) plants have the potential to undergo rapid internodal elongation, which determines plant height. A number of physiological studies have proved that gibberellin is involved in internode elongation,and alarge number of genes that encode components of the GA signaling or biosynthetic pathways have been found to be associated with internodal elongation.Leucine-rich repeat receptor-like kinases (LRR-RLKs) are the largest subfamily of transmembrane receptor-like kinases in plants, with more than200members in Arabidopsis, and300members in the rice genome.Plant LRR-RLKs have been found toplay important functions in mediating a variety of cellular processes, including pollen self-incompatibility, meristematic growth, embryogenesisand morphogenesis, and regulating responses to environmental signals such as light, pathogen attack and hormones. LRK1, a PSK receptorhomolog, is a member of the LRR-RLKs family. Transformation experiments have demonstrated that LRK1influences rice plant height and branching, and therefore affects plant architecture and yield.In the present study, we found LRK1transgenic lines showed dwarf phenotypes, the transgenic line060615were5.4%shorter than9311control. Every elongated internode was shortened, especially the third internode, which was shortened by an average of43.8%. Both9311control and transgenic seedlings showed rapid internode elongation after treated with100μM GA3for48h at the three-leaf stage, and LRK1transgenic seedlings did not show negative sensitivity to GA treatment. Together, these results suggested that LRK1negatively regulates theinternode elongation process, but the responsiveness of transgenic seedlingsto GA is not impaired.We used microarray analysis to compare expression of GA biosynthetic genes between9311controland LRK1transgenic060615seedlings at the six-leafstage. The resultsshowed that OsK02expression in LRK1transgenic seedlings was sharply down-regulated, whereas no significant differences were observed between9311controland LRK1transgenic seedlings in expression of OsCPS1,OsKS1,OsKAO and GA20ox2. Real-time PCR analysis confirmed these results. This finding suggested that the GA response was suppressed in the transformantspossibly because of the lack of bioactive GAs.We determined theendogenous GA level at the three-leaf stage in9311controland LRK1transgenic060615and060402plants. The endogenous levels of all GAs testedwere significantly lower in LRK1transgenic plants than in9311control plants. GA levels were decreased together with thesuppression of OsKO2expressionin LRK1transgenic plants.We concluded that LRK1restricts gibberellin responsiveness during the internode elongation process by down-regulation of the gibberellin biosynthetic gene, ent-KAURENE OXIDASE (OsKO2).