Screening And Verification Of Epidermal Hair Characteristics And Regulatory Genes For Stalk Thorn Formation In Seedless Roxburghi

Rosa sterilis S.D.Shi(R.sterilis),a perennial deciduous shrub from the Rosaceae family,is seedless due to the stamen abortion.The nutrients and active substances contents of R.sterilis fruit are particularly rich and exhibit considerable processing suitability for high sugar content and low astringency.Therefore,R.sterilis has become an important horticultural crop in Guizhou Province,with the economic cultivation area of at least 26,000 hectares.Trichomes are present on the buds,stems,pedicels and fruits of R.sterilis,especially the acicular prickles on the stems and pedicels,which affects the plant management and harvesting.No systematic study has been conducted on the types,anatomical structures,development and chemical compositions of trichomes in R.sterilis,and little known about the formation or regulation of trichomes.In this paper,we systematically observe the types and morphology of trichomes,and focus on the pedicels(with glandular trichome and flagellate trichome)and inflorescence stem(with flagellate trichome only)with different trichome types,using transcriptomic and proteomics to screen genes that could potentially be regulated formation formation.At the same time,we analyzed the spatiotemporal expression pattern of the screened genes and heterologous transformation verification.The data presented here provide a basis for creating new Rosa varieties with desired trichome growth and density through breeding and genetic engineering.The main results are as follows:1.To characterize the morphology of R.sterilis trichomes in details,we examined the trichomes with SEM(scanning electron microscopy)and TEM(transmission electron microscopy),we found that all R.sterilis trichomes were multicellular.Two main forms of trichomes were present in R.sterilis,namely glandular and non-glandular trichomes,and their cell ultrastructures are significantly different.There are three types of non-glandular trichomes(flagellate,acicular,and branched)and two types of glandular trichomes(capitate and bowl-shaped).The distribution of trichome in different organs is quite different.The trichomes on the receptacle/fruit were mainly flagelliform trichomes,acicular trichomes and capitate glandular trichomes with long stalk in the early stage.The flagelliform trichomes gradually fell off,while the other two kinds of trichomes were lignified to prickles,and the fruit prickles are easy to fall off when fruits ripening.On the surfaces of sepals,there are four types of trichomes,including flagellate trichomes,acicular trichomes,branched trichomes and capitate glandular trichomes.No trichomes were observed on the leaf blade adaxially and abaxially,but a few capitate glandular trichomes and acicular trichome on the vains.Capitate glandular and bowl-shaped glandular trichomes and prickles were abundant on the surface of young stems.Prickles of fruit and stem mainly consisted of lignin,suberin,cellulose and hemicelluloses,whose content in upper tissue was higher than that in beneath tissue,consistent with the results of lignin staining.We measured the DNA content of acicular trichomes on fruit and prickles on stem with flow cytometry,no difference in nuclear ploidy levels was observed between two types trichome and leaf tissue.It shows that the ploidy change of cell chromosomes is not involved in trichome formation mechanism.The above observations show that the trichome of R.sterilis exhibited polymorphism in organ distribution,morphology and type.2.The inflorescence stem developed after the germination of mixed flower buds had only flagellate trichome distributed,and gradually fell off and became smooth with the development process.The pedicel developed later has both flagellate trichomes and long-stalked glandular trichomes,and some of the head cells at the top of the glandular hairs fall off to acicular trichomes,which are then lignified into acicular prickles.Therefore,transcriptome and TMT proteome quantification were performed on these two parts during early development.Base on our RNA-seq data,a total of 2485 statistically differentially expressed genes were identified,956up-regulated and 1529 down-regulated genes were found in the pedicel with glandular trichomes,which includes a total of 122 transcription factors belonging to 18 families.The DEGs were largely enriched in phenylpropanoid biosynthesis(ko00940),MAPK signaling pathway-plant(ko04016)and plant hormone signal transduction(ko04075).A total of 277 differentially expressed proteins were identified in the proteome,of which 161 were up-regulated and 116 were down-regulated in the pedicel;12 were related to hormone biosynthesis and signal transduction,including 2 GASA in the GA pathway,1 AHP in CKs,1 LOX in JA,and 1 PAL in SA.Further hormone analysis found that the contents contents of phytohormone JA,GA3,and CKs in pedicels were significantly higher than those in inflorescence stems,but the opposite was true in SA.There are 35 genes with the same transcription level,including 13 JA-related genes,12 GA-related genes,8 CKs-related genes,and 2SA-related genes,suggesting that these hormones transduction might be crucial for trichome initiation and development in R.sterilis.3.Taking Arabidopsis,cucumber and other plants as references,10 homologous genes that may be involved in the initiation and development of trichomes were screened from transcriptome data based on family conserved domains and their expression levels and spatiotemporal expression pattern were detected by q RT-PCR.It suggested that GL1,GL2 and PDF2 were positively regulated the formation and development of acicular trichomes and long-stalked glandular trichomes,ETC1 and MYB-like were more prominent in negative effect on trichome formation.The expression of these genes peaked in the B5 stage of flower bud development(the trichome can be observed in the later B7 stage).This trend of simultaneous up-regulation of positive and negative regulated genes is consistent with the "activator-inhibitor" pattern of trichome,and the B5-B6 stage may be the key phases trichome morphogenesis.4.Base on rose genome and trichome regulatory mechanism,and through the transcriptome-proteome association analysis of pedicel and inflorescence stem,Rchi OBHm-Chr2g0159921 was identified as a candidate negative regulatory gene controlling prickle of pedicel formation.After cloning the full-length sequence,we found that the ORF of this gene is 225 bp,encoding 75 aa;bioinformatics analysis showed that this gene was identical to the amino acid sequence of rose Rc ETC1(R3MYB family),so it was named Rs ETC1.In view of the expression characteristics and important role of another R3 MYB family member,MYB-like(novel.29299.1),we also cloned the full-length sequence of this gene.Its ORF is 222 bp,encoding 74 aa.No homologous sequence was found in the rose genome,it may be a specific gene of R.sterilis,named Rs MYB-like.Subcellular localization experiments showed that Rs ETC1 and Rs MYB-like were located both in cytoplasmic and nuclear.Over-expression of Rs ETC1 and Rs MYB-like in Arabidopsis resulted in glabrous phenotypes.In addition,expression of endogenous GL1,GL2,GL3,EGL3,MYB23,TTG2 and a set of R3 MYB-encoding genes was markedly reduced in Rs ETC1 transgenic plants,thus verifying its effect on trichome.The above results show that the R3 MYB family plays an important role in regulation trichome formation in R.sterilis.