Functional Analysis Of OsPP2C30,aType 2C Protein Phosphatase Gene In Rice

Clade A type 2C protein phosphatases(PP2CAs)are important components in the abscisic acid(ABA)signaling pathway and play widely varying roles in ABA-mediated stress responses.There are ten genes in the rice PP2CA family,and some of them have been functionally characterized.However,the roles of OsPP2C30,encoding a PP2CA in rice,remains unknown.In this study,expression analysis,subcellular localization and transgenic assays were employed to reveal the function of OsPP2C30 in plant growth and ABA signaling in rice.The main results are as follows:1.Real-time PCR and promoter-GUS staining analyses were used to investigate the expression pattern of OsPP2C30.OsPP2C30 was abundantly expressed in all the tested tissues,and showed the highest transcript accumulation in young panicles.In addition,the expression decreased during panicle and spikelet development,which suggested that OsPP2C30may play an essential role in panicle and spikelet development.2.Subcellular localization analysis in tobacco leaf epidermal cells showed that OsPP2C30 protein was localized to the nucleus.3.Yeast two-hybrid assays were performed to investigate the interactions between OsPP2C30 and the components of the ABA signaling pathway,including OsPYLs和 OsSAPKs.We observed that OsPP2C30 interacted with OsPYL1,OsPYL4,OsPYL5,OsPYL6,OsPYL8,OsPYL9,OsPYL10 and OsPYL11 regardless of ABA’s presence.In contrast,the interactions between OsPP2C30 and OsPYL2,OsPYL3,as well as OsPYL6,occurred in an ABA-dependent manner.Moreover,we observed that OsPP2C30 interacted with OsSAPK1,OsSAPK2,OsSAPK8,OsSAPK9,and OsSAPK10 in yeast.These data suggest that OsPP2C30 is a component in rice ABA signaling pathway.4.We developed the overexpression lines and CRISPR/Cas9 mutants of OsPP2C30 in the Nipponbare(Oryza sativa L.ssp.japonica)background.Agronomic characterization showed that overexpression of OsPP2C30 promoted plant growth,and positively regulated plant height,grain weight and grain yield per plant.By contrast,knockout of OsPP2C30 decreased plant height,grain weight and grain yield per plant.5.Expression analysis indicated that exogenous ABA significantly induced the transcripts of OsPP2C30.This induction of OsPP2C30 expression became more obvious with the increase of processing time.We also compared the seed germination rates and shoot growth between the transgenic lines and wild-type under various ABA concentrations.We observed that overexpression of OsPP2C30 reduced the sensitivity to ABA treatments,while knockout of OsPP2C30 enhanced the sensitivity to ABA treatments.These data suggest that OsPP2C30 is a negative regulator in ABA signaling pathway in rice.