Characterization Of Rice Homologues Of The Arabidopsis Core Trichome Initiation Regulating Transcription Factors

The control of cell-fate determination has been a centralquestion in developmental biology.Arabidopsis trichome and root hair initiation has emerged as an importantmodel system for cell-fate determination studies in plants.Trichomesare special haircellsproducedbytheoutwardgrowthofepidermalcells in aerial parts of plants.Trichomescan increase the skin thickness,reduce moisture and heatloss,protectplantsfrominsect and pathogen,alleviatemechanical damage.In Arabidopsis,trichomes are specialized cells,which distributed onthesurfaceof rosette leaves,stem leaves,stemsandflowerorgans.There are three types of trichome in rice:macro hair,micro hair and glandular hair.The glabrous feature of rice(Oryzasativa)is considered as a favoriteagronomic trait because it has greaterpacking capability of rice grains,dries and produces less dustthat causes itching effect on farmers.Therefore,glabrous feature of rice is widely used in rice cultivation.Root hairs are specialized long tubular outgrowth on the surface of the epidermal cells.Root hairs canincrease the root and soil contact area.They are important for water and nutrient uptake fromthe rhizosphere,and serve as a site of interaction with soilmicro-organisms.In Arabidopsis,the R2R3MYBtranscriptionfactorGLABRA1(GL1),thebHLHtranscriptionfactorGLABRA3(GL3)orENHANCEROFGLABRA3(EGL3),and the WDR proteinTRANSPARENTTESTAGLABRA1(TTG1)form MBWtranscriptional activator complex activates thehomeodomain protein gene GLABRA2(GL2),leading to the promotion of trichome formation.The same MBW complex also activates single-repeat R3 MYB genes includingTRICHOMELESS1(TCL1),TCL2,TRYPTICHON(TRY),CAPRICE(CPC),ENHANCE ROFTRYANDCPC1(ETC1),ETC2andETC3.These R3 MYB transcriptional factors move to their neighboring cells,and compete with GL1 for binding GL3,inhibiting the formation of the MBW complex,thus inhibiting the trichome formation.The mechanism of regulating root hair formation is similar with trichome formation.But in root epidermis cells,GL1 is replaced by another R2R3 MYB transcriptionfactorWEREWOLF(WER).WER,GL3/EGL3 and TTG1 form a transcriptional activator complex promote the expression of GL2 in downstream,thus makingepidermiscellsdifferentiateintohairlesscells.When WER is replaced by R3 MYB transcriptionfactor,this kind of complex will not activate the expression of GL2,thus the corticalcells will differentiateinto root hair cells.Currently,little is known about whether MBW transcriptional complex regulates trichome and root hair formation in rice.In this study,we identified the homologous proteins of GL1,GL3,TTG1 and TCL1 by BLASTing the riceprotein database using their entire amino acid sequence.Defined and named these homologs as OsGL1(OsGL1A,OsGL1 B,OsGL1C,OsGL1 D,OsGL1E),OsGL3(OsGL3A,OsGL3 B,OsGL3C),Os TTG1(OsTTG1A,OsTTG1B),OsTCL1 and OsTCL2.Protein subcellular localization results showed that OsGL1 E,OsGL3B,OsTTG1 A and OsTCL1 are all nuclear protein,butOsTTG1 A and OsTCL1 are also associated with cytoplasm and plasmamembranerespectively.The result showed that OsGL3 B was a transcriptional activator by testing in protoplasts.OsTTG1 A and OsTCL1 had very weak transcriptional activation activity.And OsGL1 E didn't havetranscriptional activation activity.In protoplast,GL1,TTG1 and TCL1 interacted with OsGL3 B.OsGL1E,OsTTG1 A and OsTCL1 interacted with GL3 B,and also interacted with OsGL3.These results suggested that the corresponding transcription factors in rice can also form MBW complex.We took OsTCL1 as an example to study how it regulates the trichome and root hair initiationin Arabidopsis.When expressed under the control of the double 35 S promoter,OsTCL1 inhibited trichomeformation and promoted root hair formationin transgenic Arabidopsis plants.On the other hand,phenotypic complementation experiment in tcl1,try and cpc mutants indicate that OsTCL1 is functional similar,although it may not be equivalent to TCL1.Quantitative RT-PCR results showed that the expression of GL1 was also dramatically reduced in thetransgenic Arabidopsis plants expressing OsTCL1.These results suggest that OsTCL1 regulates trichome and root hair formation in Arabidopsisby a manner similar to that of the Arabidopsis R3 MYB transcription factors.Inttg1 mutants background,overexpressing OsTTG1 A could rescue the non trichome phenotype,indicating that OsTTG1 A is functional similar to TTG1 in regulating Arabidopsis trichome formation.In rice,we overexpressed OsTCL1 to study the regulation of rice trichome and root hair formation.We found the phenotype of trichome and root hair formation in transgenic plants remained largely unaffected.Rather than decreased expression of OsGL2 was observed in transgenic rice overexpressing OsTCL1,and elevated expression of OsGL3 C was also observedin transgenic rice overexpressing OsTCL1,whereas expression of GL3 in transgenic Arabidopsis overexpressingTCL1 remained largely unchanged.These results suggest that rice may use different mechanisms toregulate trichome and root hair formation.