Gene Cloning And Functional Analysis Of OsPLT2,an AP2/ERF Transcription Factor Regulate Trichome Development In Rice

Trichome refers to a hair-like structure that extends from the epidermis of the aerial parts of the plant and is distributed on the epidermis of most land plants.Rice(Oryza sativa spp.)is one of the most important cereal crops in the world.For the most of rice cultivars,the plants and grains are covered by epidermal hairs or trichomes;while for glabrous cultivars,the plant and grain are glabrous due to the loss of trichomes on epidermis.In fact,the absence of trichomes in rice which leads to glabrous leaf and hulls is a favorite agronomic trait in rice production,and the glabrous rice is also regarded as important germplasm resource in rice breeding.Because trichomes originate from pluripotent epidermal cells(promeristem)and are easily accessible,their development has become an excellent model system to study all aspects of cell differentiation and growth in Arabidopsis.However,their molecular mechanism in rice and other monocots remains unclear.In the present study,a rice gene,OsPLT2,which encodes a member of the AP2/EREBP transcription factor,was cloned and characterized by a series of molecular analyses.The main results are as follow:(1)Gene cloning revealed that the full-length cDNA sequence of OsPLT2 is 1744 bp,which encodes a protein of 469 amino acids;Sequence analysis indicated that the OsPLT2 protein is a putative AP2/ERF transcription factor containing two AP2/ERF domains.Furthermore,phygenetic analysis showed that OsPLT2 is homologous to AIL5(At5g57390)and PLT2(At1g51190)in Arabidopsis thaliana.So it is named OsPLT2.(2)Gene expression analysis using qRT-PCR revealed that OsPLT2 was expressed in 19 tissues including callus,seedling stage,tillering stage,booting stage,and heading stage.However,the expression in callus was higher,and the expression in other tissues was low.Transgenic analysis of OsSPLT2 promoter driven GUS reporter gene also confirmed the expression pattern of OsPLT2.(3)It was showed that the OsPLT2 promoter region contains several functional elements such as CGTCA-motif,MBS and ABRE.Gene expression analysis revealed that the OsPLT2 was sensitive to Me JA(methyl jasmonate),MV(methyl viologen,oxidants)and low temperature treatments,which can significantly induce the expression of OsPLT2.(4)By transient expression the OsPLT2-eGFP fusion protein,it was showed that theGFP fluorescence signal was clearly localized in the nucleus of rice protoplasts.Moreover,the yeast experiment showed that the OsPLT2 protein had transcriptional activation activity,further indicating that the OsPLT2 is a transcription factor.(5)The trangentic plants were obtained by overexpression of the OsPLT2 under the control of the maize Ubi promoter.Compared with the control,overexpression of the OsPLT2 leads to hairy phenotype in trangenic plants.Scanning electron microscopic observtation further revealed that the hairy phenotype is due to a significant increase of the trichome length in the overexpression lines.(6)Using CRISPR/Cas9 mediated gene modification,the knock-out mutants of OsPLT2 was obtained in the present study.However,no significant phenotypic changes was observed in the osplt2 mutants as compaired with WT.Moreover,the scanning electron microscopic observtation further revealed that knock-out of OsPLT2 had no effect on trichome development in the osplt2 mutants.(7)Previous studies have shown that loss-of-function of GL1/OsWOX3 b,a gene encoding WUS-related homeobox transcription factor,leads to loss of trichome in rice plants.In the study,yeast two hybrid(Y2H)experiment indicated that the OsPLT2 protein directly interact with GL1/OsWOX3 b protein.The interaction between OsPLT2 and GL1/OsWOX3 b protein was further confirmed by the bimolecular fluorescence complementation(BiFc)experiment in rice protoplasts.In summary,the present study demonstrated that the OsPLT2 encodes an AP2/ERF transcription factor and controls trichome development in rice.The OsPLT2 protein interacts with GL1/OsWOX3 b,indicating that the OsPLT2 might regulate trichome development by interacting with theGL1/OsWOX3 b.Takgen together,our results imply that the OsPLT2 play an important role in rice trichome development.The elucidation of OsPLT2 function has theoretical values for the improvement of trichome traits in rice.