Functional Analysis Of Trichomes Development Regulatory Genes PaTCP4、PaTCP15 And PaNAC089 From Platanus Acerifolia

Platanus acerifolia(London plane)belongs to Platanus in Platanaceae,which is widely used and irreplaceable as urban afforestation tree because of its dense canopy rapid growth and good resistance.However,the main defect of P.acerifolia as urban afforestation tree is that its trichomes are serious air pollutants.Multicellular trichomes grow on the surface of P.acerifolia tender organs.These trichomes can be divided into two categories according to their secretory characteristics: glandular trichomes and non-glandular trichomes.The non-glandular trichomes,especially the infructescence trichomes,are the main air pollutants produced by P.acerifolia.Therefore,the main breeding goal of P.acerifolia is to cultivate “hairless London plane tree”.However,current studies show that it is difficult for traditional breeding methods to achieve this objective,genetic engineering methods may be the best way to breeding glabrous cultivars.To date,little is known about the regulation mechanism of trichomes development in P.acerifolia.In this study,we hope to screen out the key genes involved in the development of trichomes in P.acerifolia by RNA-seq and verify the function and regulation mechanism of selected genes by heterologous overexpression of Arabidopsis.Based on these results,the role of the selected genes in the trichome development was speculated in P.acerifolia.The major results are as follows:1.The morphology of fruit hair during the development of infructescence was observed by scanning electron microscope.The fruit of P.acerifolia is syncarp,and each nut on the infructescence is a complete fruit.Two types of non-glandular trichomes were observed on the surface of nut: serrated trichomes and long straight trichomes;The serrated trichomes is mainly distributed in the middle and upper part of nut.The long straight trichomes are mainly distributed in the middle and lower part of nut.Based on the observation results of nut trichomes,we collected the mixed samples of different development stages of long straight trichomes for RNA-seq.The results of RNA-seq showed that many transcription factors and plant hormones were involved in regulating the development of long straight trichomes of P.acerifolia,among which MYB and TCP genes are highly expressed.2.Based on the trichomes transcriptome data of P.acerifolia,we isolated a TCP family gene with high expression in fruit hair of London plane.Multiple sequence alignment and phylogenetic analysis results showed that the gene is a At TCP4 homologous gene and belongs to the Class ⅡTCP subfamily.We named it Pa TCP4.Subcellular localization result showed that Pa TCP4 is localized to the nucleus.The expression of Pa TCP4 is detected in various organs in London plane,especially in trichomes.Overexpression of Pa TCP4 in Arabidopsis reduces the trichome density of the first pair of true leaves,and 5-branched trichomes were also detected on those leaves.Furthermore,the protein product of Pa TCP4 was shown to directly activate At CPC,At TCL2,At GL3,Pa GIS and Pa GL3 in yeast one-hybrid assays and dual-luciferase reporter system assay.Taken together,these results identify a role for Pa TCP4 in trichome initiation and branching in Arabidopsis.Pa TCP4 may be a candidate gene for regulating the trichomes development in London Plane trees.3.We isolated a TCP gene from London plane according to the trichomes transcriptome data.Multiple sequence alignment and phylogenetic analysis results showed that the gene is a At TCP15 homologous gene and belongs to the Class TCP suⅠ bfamily.We named it Pa TCP15.Subcellular localization result showed that Pa TCP15 is localized to the nucleus.The expression of Pa TCP15 is detected in various organs in London plane,including trichomes.In addition,GUS staining(p Pa TCP15::GUS)showed that the p Pa TCP15 was expressed in Arabidopsis trichomes,and 6-BA treatment of transgenic lines(p Pa TCP15::GUS)could deepen the GUS staining.Moreover,promoter sequence analysis revealed that the promoter of Pa TCP15 contained many hormone responsive elements and various cis-acting elements in response to stress response.Pa TCP15 can increase the number of Arabidopsis inflorescence trichomes,especially sepal trichomes.Dualluciferase reporter system assay showed that Pa TCP15 could activates At ZFP6.5.We isolated a gene sequence from trichomes transcriptome data of P.acerifolia,and multiple sequence alignment and phylogenetic analysis results showed that this gene encodes a membrane-tethered transcription factor(MTTF),which belongs to NAC(NAM,ATAF1/2,CUC2)transcription factor family.Subcellular localization showed that Pa NAC089 devoid of the transmembrane domin(?Pa NAC089)was localized in the nucleus,while the full-length Pa NAC089 was localized in the endoplasmic reticulum(ER).In addition,RT-q PCR results showed that Pa NAC089 was expressed in all tissues/organs of P.acerifolia,and the expression level in leaves increased gradually with leaf maturation.Heterogenous overexpression of ?Pa NAC089 decreased the number of Arabidopsis rosette leaves.Consistent with this phenotype,the expression of genes At GL1 and At GL2,which positively regulate the development of Arabidopsis trichomes,was inhibited in the?Pa NAC089 transgenic lines.Ectopic expression of ?Pa NAC089 delays flowering and inhibits chlorophyll breakdown to produce dark green rosette leaves in Arabidopsis.The yeast one-hybrid assays showed that ?Pa NAC089 could directly bind the At CO promoter.In addition,chlorophyll content in the rosette leaves of the transgenic lines was significantly higher than that of CK rosette leaves by chlorophyll content measurement.The yeast onehybrid and luciferase(LUC)transient expression assays showed that ?Pa NAC089 could directly inhibit the activity of CCGs(At NYE1,At NYE2,At NYC1)in Arabidopsis.Taken together,Pa NAC089 is an MTTF that regulates flowering,chlorophyll breakdown,trichome induction and silique development in Arabidopsis.