Molecular Cloning And Functional Analysis Of Leaf Trichome Related Genes From Platanus Acerifolia

Trichomes are defined as hair-like structures that originate from the epidermis of most terrestrial plants.According to their cell numbers,trichomes are constitutied of unicellular and multicellular trichomes.Morphologically,trichomes are typically grouped into two general categories namely,branched and unbranched trichomes.They may also be divided into two classes according to their secretory characteristics namely,glandular and non-glandular trichomes.The hair-like structures have widely served as an excellent experimental system for the study of all aspects of cell differentiation and development.Significant progress has been obtained about molecular regulation of trichome in Arabidopsis,however,little was known about London plane(Platanus acerifolia).London plane is cultivated widely as an ornamental tree in parks and gardens and is commonly used for the formation of avenues in cities due to its innately desirable characteristics such as a rapid growth habit,dense crown and good resistance.However,the numerous stiff hairs released limit the universality of P.acerifolia.Therefore,understanding of the molecular mechanisms of trichome development in London plane is helpful for breeding glabrous cultivars.In this study,we document the critical genes related trichome regulation which provide scientific basis for breeding.The major results as following:1.Four R3 MYB transcription factors were cloned and identified from London plane on the basis of homologous cloning and transcriptome data.The results of the phylogenetic analysis showed that one of the four London plane sequences corresponded to the clade of group I,whereas,the other three sequences were clustered within the group II clade.The group I and group II Platanus genes were most closely related to the TRY and CPC-like sequences,respectively,from Nelumbo nucifera.The q RT-PCR results showed that Pa CPC-like1 was expressed in all tissues tested,the tissue-specific pattern of Pa TRY、Pa CPC-like2 and Pa CPC-like3 were similar which highest in stems but was undetectable in leaves.Four R3 MYB transcription factors were both cytoplasmic and nuclear-localized transcription factors.Overexpressing of Pa TRY and Pa CPC-like genes in Arabidopsis resulted in glabrous phenotypes.The R3 MYB transcription factors from London plane inhibit trichome formation in Arabidopsis by interact with the b HLH transcription factors to regulate the expression of At CPC、At TRY、At ETC1、At GL1、At MYB23、At TTG2 and At GL2 thereby regulating the trichome formation.2.Pa MYB82 was isolated and characterized from London plane.Phylogenetic tree analysis placed Pa MYB82 and At MYB82 together in the same clade which was closest to R2R3-MYB subgroup-15 and is a nuclear-localized transcription factor although does not actually belong to the R2R3-MYB subgroup-15.Pa MYB82 was found to be expressed in all of the examined tissues,with transcript levels highest in the stems,which implies that it may play a role in the stem development of P.acerifolia.Expression of the Pa MYB82 gene under the control of the viral Ca MV35 S promoter caused a nearly glabrous phenotype in wild type Arabidopsis and can partially rescue the gl1 mutant phenotype.Protein-protein interactions occurred between Pa MYB82 and Pa GL3、At GL3、At EGL3 as well as At MYC1 and the expression levels of At CPC、At ETC1、At TRY、At GL2 and At TTG2 were markedly increased in transgenic lines overexpressing Pa MYB82.These results indicating that Pa MYB82 gene regulates trichome formation in London plane through a similar set of regulatory mechanisms as found in Arabidopsis.3.We isolated and characterized Pa GL1-like1 and Pa GL1-like2 genes from London plane(Platanus acerifolia).Sequence alignment and phylogenetic analysis indicated that these Pa GL1-like genes are homologous to At GL1.Quantitative real-time PCR(q RT-PCR)analysis showed that Pa GL1-like1 was expressed in all of the tested organs taken from adult London plane trees,including trichomes、petioles after trichome removal、stems after trichome removal、leaves after trichome removal and also in the roots、cotyledons、hypocotyls and true leaves of seedlings.By contrast,the Pa GL1-like2 gene was expressed only in trichomes、leaves after trichome removal from adult trees and in the cotyledons and true leaves of seedlings.Overexpression of Pa GL1-like genes caused trichome abortion when transferred into wild type Arabidopsis and promoted trichome formation in the gl1 mutant.The expression profiles of some trichome-related genes were changed in transgenic Arabidopsis lines,and yeast twohybrid analysis indicated that Pa GL1-like proteins can directly interact with b HLH proteins from both P.acerifolia and Arabidopsis.These results suggest that Pa GL1-like genes are functional in Arabidopsis and may play an important role in the regulation of trichome development in London plane.Pa GL1-like2 including Pa GL1-like2.1 and Pa GL1-like2.2,sequence alignment indicated that two amino acids changed in the R2 repeat between them(Pa GL1-like2 indicated Pa GL1-like2.1 in previous description).To our surprise,overexpressing Pa GL1-like2.1 also caused dwarfish phenotype in wild type Arabidopsis.In order to further investigate the putative function of two Pa GL1-like2 genes,we overexpress them in tobacco,respectively.The results indicated that overexpress Pa GL1-like2.1increased axillary buds growth and developed narrower leaves.In addition,ectopic expression Pa GL1-like2.1 advanced the accumulation of anthocyanins in stamens、immature seeds and tubes in transgenic tobacco.The results of yeast two hybrid assay and analysis of gene expression affirmed that Pa GL1-like2.1 interact with anthocyanins related b HLH transcription factors to up-regulate the expression of Nt ANS、Nt DFR、Nt An1 a and Nt An1 b to increase the accumulation of anthocyanins in tobacco.