Function Analysis Of Receptor-like Kinase PSRK2 Involved In Gibberellin Response In Rice(Oryza Sativa L.)

Rice(Oryza sativa)is a most important food crop,accounted for about one-third of the total area of food crops.More than half of the world population live on rice.Plant height is not only an important determinant factor of rice morphological structure,but also an important agronomic trait for improving rice yield.Rice height is mainly determined by the elongation of stems,and gibberellin is one of the important factors affecting the elongation of stems in rice.Therefore,gibberellin plays a crucial role in the development of plant height in rice.Gibberellins(GAs)is a class of tetracyclic diterpenoids that plays important regulatory roles in the growth and development of higher plants,including seed germination,hypocotyl elongation,stem elongation,stretch of leaves,maturation of pollen and induction of flowering period.Gibberellin-mediated dwarf or semi-dwarf mutants have been studied in rice,maize,wheat and Arabidopsis,which the heights of these dwarf or semi-dwarf mutants are due to defect of stem elongation regulated by gibberellin.In this paper,a dominant negative transgenic population of receptor-like kinase was constructed using reverse genetic tools in rice.Combined with high-throughput gene chip,RNA-seq and plant hormone-induced expression pattern,a receptor-like kinase,Plant Stature Related receptor-like Kinase2(PSRK2),with negative regulation of plant height was obtained.Through bioinformatics analysis,cytological observation,expression pattern,physiological and biochemical analysis,the main results obtained are as follows:(1)Cloned a new receptor-like kinase PSRK2(Plant Stature Related receptor-like Kinase2).PSRK2 encodes a receptor-like protein kinase containing 536 amino acids.The PSRK2 protein sequence contains two core domains,the S-site glycoprotein domain(S_domain_glycoprotein)and the kinase domain(S_TKs domain).A typical receptor-like protein kinase contains three domains,including the signaling domain that senses external signals,the transmembrane domain that transmits signals from extracellularly to intracellular and the kinase domain that transmits signals in intracellular.However,it was found that PSRK2 protein sequence does not contain the transmembrane domain,suggesting that PSRK2 could not anchor the plasma membrane.Therefore PSRK2 may be involved in intracellular signaling through interaction with itself or in extracellular signaling through interaction with a receptor-like kinase containing transmembrane domain.(2)High specificity of PSRK2 amino acid sequence between species(subspecies in rice).Gene databases from different species were used to search for homolog genes and constructing phylogenetic trees with the PSRK2 signaling domain and the PSRK2 kinase domain.Its obtained candidate sequences of 10 species(subspecies)include indica rice,maize and potato using the signaling domain;the obtained candidate sequences of 11 species(subspecies)include indica rice,wheat,and physcomitrella using the kinase domain.Using analyses of phylogenetic trees,we found higher specificity of PSRK2 amino acid sequence in japonica rice,and lower support in other species(including indica rice).The PSRK2 homologous gene was not found in indica rice.(3)Generating and analyzing the interfering expression line psrk2-RNAi and the dominant negative line psrk2-DN,which affects on the elongation of young root and stem internodes in PSRK2 low-expression lines(psrk2-RNAi lines)and PSRK2 lack-of-function lines(psrk2-DN lines).The interference expression vector pHB-PSRK2-RNAi was constructed and transformed into rice using genetic engineering and cell engineering,and the genetically stable T1 interfering expression lines psrk2-RNAi-2 and psrk2-RNAi-6 were obtained.Meanwhile,the dominant negative vector pCAMBIA1301-PSRK2-DN was constructed and transformed into rice,and the genetically stable T1 dominant negative lines psrk2-DN-21 and psrk2-DN-24 were obtained.Further studies on the above two types of transgenic materials revealed that the low level of PSRK2 expression(psrk2-RNAi lines)and the lack of PSRK2 kinase function(psrk2-DN lines)affected the expression of key cyclin genes OsRAN,CycD2 and CycD4 in rice at seedling stage,resulting in a shortened cell cycle,accelerated cell division,and accelerated elongation of the young roots.The developmental transition from the vegetative to the reproductive stage induced internode elongation,especially the first internode obviously elongation.In the psrk2-RNAi and psrk2-DN,low level of PSRK2 expression and the lack of PSRK2 kinase function affected the division of stem node meristem cells,resulting in increased number of cells in the joints of stem internodes and stem nodes.(4)PSRK2 is mainly expressed in the root during the whole growth period and in the stem in the reproductive stage.The expression pattern of PSRK2 was analyzed by qRT-PCR and tissue localization.It was found that the expression of PSRK2 was relatively strong in the root of seedling stage,vegetative stage and reproductive stage;and it was relatively strong in the stem and stem nodes after the transition vegetative to reproductive stage.Additionally,PSRK2 transcripts could be detected in the coleoptile in seed germination stage,young inflorescence after the transition vegetative to reproductive stage,and leaves in mature.(5)PSRK2 participates in the gibberellin response.The ?-amylase degradation starch test showed the psrk2-RNAi and psrk2-DN were more senstive to the degradation of starch compared wild-type,and the ?-amylase encoded gene RAmy1 a had stronger expression.Meanwhile,the second leaf sheath elongation test showed the psrk2-RNAi and psrk2-DN were more sensitive to exogenous GA,and the second leaf sheath had higher elongation compared wild-type.In summary,these results showed that it was sensitive to gibberellin in psrk2-RNAi and psrk2-DN.Moreover,analysis of the key genes of GA biosynthetic metabolic pathways,OsGA20 ox and OsGA2 ox,revealed that the synthetic gene OsGA20 ox and the metabolic gene OsGA2 ox were up-regulated in psrk2-RNAi and psrk2-DN;analysis of key gene of GA signal transduction pathway,GID1 and SLR1,revealed that the expression of GID1 encoding GA receptor protein was significantly up-regulated,while the expression of SLR1 encoding DELLA protein was not significantly changed.In summary,the low level of PSRK2 expression and the lack of PSRK2 kinase function affected the expression of key gene in GA synthesis and transduction pathways,thus making more actively signal transduction pathway,indicating that PSRK2 regulates rice height by participating in the gibberellin response.Based on the above results,the PSRK2 is involved in the gibberellin response pathway.However,the low level of PSRK2 expression(psrk2-RNAi lines)and the lack of PSRK2 kinase function(psrk2-DN lines)resulted in obviously higher stems compared with wild-type,indicating that PSRK2 may be a negative regulator in gibberellin response.There are essential biological implications for the in-depth studies of the molecular mechanisms and regulatory networks of PSRK2 in the gibberellin pathway.