The Cloning And Functional Analysis Of Two Genes LPL2 And DSP1 That Regulate The Epidermal Cell Development In Rice

The developmental process of epidermal cell was a classical model for studying cell division,differentiation and morphogenesis in plant.It has been a hot area of research for the past few years.Rice(Oryza sativa)is not only a classic model of monocotyledons but also an important crop.Therefore,the study on epidermal cells in rice has great significance for theoretical study and agriculture application.At present,the study on the stomata and epidermal cells mainly focuses on Arabidopsis,however,corresponding advancement in rice was seldom reported.This article mainly illustrates the molecular mechanism of the two novel genes,LPL2 and DSP1,which are involved in epidermal cells and stomatal development in rice.Three mutants of epidermal pavement cells with smooth marginal lobes,lpll-1(less pronounced lobe epidermal cell 1-1),lpl2-1 and lpl3-1 were screened.Our research focuses on lpl2-1.lpl2-1 shows decreased papilla and smooth epidermal cell.Meanwhile,the stomatal density of lpl2-1 is higher than that in wild type,and some abnormal stomata are observed in lpl2-1.The plant height and root or root cells length of lpl2-1 seedlings are reduced;at mature period,both the height of plant and the length of spikes are shorter than Zhonghua11.lpl2-1 was sensitive to drought and salt stress.Genetic statistical analysis indicated that lpl2-1 is controlled by the recessive heredity of single gene of autosomes.Through map-based cloning,we confirmed that the mutant gene is 0s03g05020,encoding a puptive protein homologous to PIR in Arabidopsis and BRK2 in maize,and we named it as LPL2 in rice.lpl2 lacks 26 bp in the 13th exon and the 594th Glycine turned into stop codon,leading to premature transcription termination.lpl2-1 was hybridized with two T-DNA lines of LPL2,lpl2-2 and lpl2-3,and the phenotypes of F1 generation were the same as their parents.What's more,overexpression of LPL2 can completely rescue the lpl2-1 phenotype,while overexpression of LPL2 in wild type ZH11 shows no abnormal phenotypes.In summary,the genetic and transgenic evidence demonstrated that losing function of LPL2 caused the mutant phenotype.qRT-PCR analysis indicates that LPL2 is widely expressed in all tissues.Subcellular localization suggests that LPL2 protein is localized in the cytoplasm and cell membrane.F-actin-labeling indicates that distribution of micro filament in epidermal cell was disturbed,leading to the changed cytoskeleton.LPL2 is the homologous protein of PIR,a subunit of SCAR/WAVE complex in Arabidopsis.Overexpression of LPL2 in pir mutant could partly rescue its distorted trichomes of leaves and stems.By bioinformatics analysis,we further found two rice homologues to Arabidopsis and maize HSPC300(BRK1)and NAP1(BRK3),which are named as LPL1(Os02g58320)and LPL3(Os08g43130),respectively.lpll-1 was screened from offspring of RNAi-LPL1/ZH11 and lpl3-1 was ordered from T-DNA insertion mutant library,both of which are similar to lpl2-1 with dwarf phenotype and abnormal epidermal pavement cells lacking marginal lobes.In addition,lpll-1 epidermis has shortened,blunt prickle hairs.Yeast two hybrid assay suggests that LPL2 directly interact with other two subunits,LPL1 and LPL3,and also interact with OsROP2.These results indicate that LPL2 may involve in the OsROPs-SCAR/WAVE-OsARP2/3 signaling pathway in rice.The stomata mutant dsp-1(disturbed stomatal pattern-1)shows stomata clustering,abnormal stomata and decreased stomatal density.Homozygous dsp-1 is dwarf with abnormal flower and sterile pollen.We found the T-DNA is located between two genes Os07g03730 and Os07g03740,by hiTAIL-PCR.qRT-PCR analysis showed that Os07g03730 expression is increased more than 200 times.However,overexpression of Os07g03730 in Zhonghua11 failed to recapitulate the disturbed stomatal phenotype of dsp1.RNAi-DSP1/dsp-1 could not rescue the phenotype of dsp-1 yet.In accord with these results,we finally found that the phenotype of dsp-1 did not link with the T-DNA insertion.Then the candidate gene DSP1 was cloned by map-based cloning,and we found the loss-of-function of DSP1 results in abnormal stomatal development in the first and the second step,and form various abnormal stomata.