Cloning And Functional Characterization Of Trichome Regulating Transcription Factor HD-Zip ? In Tomato

Plant trichomes develop from the epidermis of nearly all terrestrial plants and provide an excellent model for studying the molecular mechanisms underlying cell fate determination.Trichomes exist in various shapes and sizes,including unicellular and multicellular.Regulation of unicellular trichome in Arabidopsis has been well characterized.A typical MYB/bHLH/WD complex regulated the expression of downstream gene and trigger the trichome formation.However,little is known about the initial and regulatory mechanism of the multicellular trichome formation in tobacco and tomato.Wo(an HD-Zip ? gene)has been cloned and functionally charecertized in this lab,regulating the formation of tomato trichome type-I and homozygous embryo lethality.HD-Zip IV proteins are closely related to critical biological processes in plants.For example,trichome formation,anthocyanin accumulation,stomatal development,etc.Only two members of tomato HD-Zip ? genes(Wo and CD2)have been reported.The function of the other tomato HD-Zip ? genes remains unknown.Identification and function researches of HD-Zip ? genes in tomato were performed in this study.The main results are as follows:1.In this study,15 members of HD-ZIP ? genes in tomato were identified with the conserved domain of Wo blast against SGN database.The genes were asymmetrically distributed on chromosomes,except on chromosome 4 and 5.Gene structure analysis revealed that a module of 11 exons and 10 introns existed.2.The tissue expression profiles of tomato HD-ZIP ? genes revealed their broad expression pattern.The majority of the genes were abundanced in young leaves and flowers.Each gene responded to more than one of phytohormones or abiotic stress treatments.3.13 novel tomato HD-ZIP ? genes were constructed over-expression and RNA interference vector.The vectors were transformed to Alisa Craig mediated by Agrobacterium.Transgenic plants were obtained.4.Compared to the control,the trichome on aerial parts of RNAi-SlHDZIV8 plants decreased significantly.Additionally,the epidermis and trichome cell morphology of RNAi-SlHDZIV8 plants changed significantly through scanning electron microscope observation.However,there is no change in over-expression SlHDZIV8 plants than the control.5.Subcellular location showed that SlHDZIV8 localized in the nucleus.Promoter analysis by GUS histochemical staining was detected in tissues including shoot apexes,axillary meristems,leaf epidermis,root and hypocotyledonary axis.6.We performed yeast two-hybridization to screen the interacting proteins using SIHDZIV8-PGBKT7 fusion protein as bait.Hl/SRAl which regulated tomato trichome mophogenesis was screen out.We demonstrated that SlHDZIV8 protein interact directly with H1 protein by one for one verification in yeast.7.We identified two point mutations in a novel allele(Wov)at Wo locus.A missense mutation(T2075 to G)results in one amino acid change of Ile to Arg.The other one(G2083 to T)results in one amino acid change of Asp to Tyr.The functional complementation analysis was carried out to confirm the mutant caused by Wov.8.Ectopic expression of Wov in tobacco and potato also induces much more trichome formation.It suggested that solanaceae species maybe shared the common network controling the trichome formation.9.The distorted flower and enhancement of insect-resistance also present in Wov transgenic tobacco.To gain new insights into the underlying mechanisms during the processes of these trichomes formation,we compared the gene expression profiles between Wov transgenic and wild-type tobacco by RNA-Seq analysis.A total of 544 Co-DEGs were detected between transgenic and wild-type tobacco.Functional assignments of the Co-DEGs indicated that 33 reliable pathways are altered in transgenic tobacco plants.The most noticeable pathways are fatty acid metabolism,amino acid biosynthesis and metabolism,and plant hormone signal transduction.The results suggest that these enhanced processes are critical for the cell proliferation during multicellular trichome formation in transgenic plants.10.In addition,the transcriptional levels of homologues of multicellular trichome regulators(Wo and SlCycB2)were significantly upregulated,whereas homologues of unicellular trichome regulators were not significantly changed in Wov transgenic tobacco plants.