Study Of The Roles Of Arabidopsis Protein Kinase PTI1-5 In Growth Of Pollen Tubes And Root Hairs

Pollon tube and root hair are the plant cells that exhibit tip growth.They play important roles in sexual plant reproduction and uptaking of water and nutrition,respectively,which are closely related to agricultural production.During anthesis,the pollen grains produced in anther of stamen are released and transferred to stigma of pistil.Then they get hydrated and generated a pollen tube by the vegetative cell.The pollen tube invades into the stigmatic cells,extends in the transmitting tract,enters the female gametophyte(embryo sac)and finally discharges the two sperm cells which it carried into the embryo sac for double fertilization.Therefore,growth of pollen tube is essential for sexual plant reproduction.Defect in pollen tube growth may result in male sterility.Root hairs are generated by the epidermal trichoblasts in the root tip region.Formation of root hairs significantly increases surface area of the root epidermal cells.It is helful for the uptaking of water and nutrition and interaction of root cells with soil microbial organisms,which can enhance growth and development of plant cells and effective response to the environmental stresses.In addition,pollen tubes and root hairs are also the good models for studies of cell polar growth.Therefore,studying molecular genetic mechanisms of pollen and root hair growth not only enable us to understand the regulation of cell growth,but also could provide genetic references for improving cultivation and breeding of crops.Studies show that growth of both pollen tubes and root hairs is controlled by many factors,such as Ca2+,small G proteins,cytoskeleton,protein kinases and so on.However,a very limited knowledge has been learnt about molecular genetic mechanisms that regulate growth of polen tubes and root hairs.Especially,little has been known about the roles of protein kinases in growth of pollen tubes and root hairs.Our research group is mainly focusing on the genetic mechanisms of pollen formation and pollen tube growth.We previously isolated a male gametophyte-defective mutant kd640,which is caused by insertion of Ds transposon element.The primary characterization showed that the Ds element was inserted into PTI1-5(At2g41970)that encodes a protein kinase belonging to Arabidopsis Pto-interacting 1(PTI1)protein kinase family.The pti1-5 mutant drastically affected growth of pollen tubes,resulting gametophytic male sterility.This study was to futher characterize the pti1-5 mutant and study the roles of PTI1-5 in pollen tube growth and development.The later research results showed that PTI1-5 is expressed not only in pollen grains and pollen tubes,but also in root hairs.Therefore,the roles of PTI1-5 in root hair growth were investigated as well.The ptil-5 was identified in a screening of the Arabidopsis gene-trap and enhancer-trap Ds insertion lines for male gamephyte-defective mutants.Genetic analysis showed that insertion of Ds element in the PTI1-5 gene caused complete loss of male gametophytic transmission.No homozygous pti1-5 mutant plant was obtained.Phenotypic characterization showed that the pollen tubes had burst after germinated,drastically affecting the normal growth of pollen tubes.These results indicated that PTI1-5 plays important roles in pollen tube growth.The quantative real-time PCR and promoter activity assays showed that PTI1-5 was mainly expressed in pollen grains,pollen tubes,roots and root hairs.The pollen tube-rescue by pollen and pollen tube-specific expresson of PTI1-5 in the ptil-5 mutant could restore normal growth of the mutant pollen tubes and could generate homozygous ptil-5 mutant plants.The pollen tube-rescued ptil-5 mutant plants exhibited strong defect in root hair growth.These results indicated that PTI1-5 not only is essential for normal growth of pollen tubes,but also plays important roles in root hair growth.The PTI1-5 encodes a putative receptor-like cytoplasmic protein kinase(RLCK),one of the 11 members in the PTI1 protein kinase subfamily that belongs to the RLCK VIII family.PTI1-5 was mainly localized in pollen tube cytoplasms and the plasma membrane in the tip regions of growing pollen tubes.Yeast two-hybrid assays,luciferase complementation imaging assays and co-immunoprecipitation experiments showed that PTI1-5 could interact with Oxidative signal-inducible 1(OXI1)and its closest homolog AGC2-2.The kinase assays showed that PTI1-5 could be phosphorylated by OXI1 in vitro.Previous studies have shown that OXI1 is expressed mainly in root hairs.Furthermore,the oxil is mainly defective in growth of root hairs,but does not affected pollen tube growth.Therefore,PTI1-5 might participate in the OXI1-related signal transduction through interaction with OXI1 to control growth of root hairs.In addition,the point mutation assays showed that the predited phosphorylated sites T239 and T244 were important for pollen germination and pollen tube development.Mutations in these two points drastically affected pollen germination and pollen tube growth.However,whether these two points are the actual phosporilation points and related to OXI1 in pollen tube growth remains unclear.In summary,the results from this study indicated that PTI1-5 plays important roles in growth of pollen tubes and root hairs.Furthermore,PTI1-5 may be involved in root hair growth by participating in the OXI1-related signaling through interaction with OXI1.These results could provide reference informations for further studying the roles of PTI1 members in plant cell growth and development.They also could provide a new scue and thought for further studying the molecular mechanisms of pollen tube and root hair growth.