Comparative transcriptomic analysis of Rosa sterilis inflorescence branches with different trichome types reveals an R3-MYB transcription factor that negatively regulates trichome formation

MA Wen-tao ,LU Min ,AN Hua-ming ,Yl Yin

1 Guizhou Engineering Research Center for Fruit Crops,Agricultural College,Guizhou University,Guiyang 550025,P.R.China

2 Key Laboratory of Plant Resource Conservation and Germplasm Innovation in Mountainous Region (Ministry of Education),Institute of Agro-bioengineering,College of Life Science,Guizhou University,Guiyang 550025,P.R.China

3 Key Laboratory of Plant Physiology and Development Regulation,Guizhou Normal University,Guiyang 550025,P.R.China

Abstract Rosa sterilis S.D.Shi is an important economic tree in China that produces fruits with high nutritional and medicinal value.Many of R.sterilis’ organs are covered with different types of trichomes or prickles that directly affect fruit appearance and plant management.This study used RNA sequencing technology to analyze the transcriptomes of two parts of the inflorescence branch,namely inflorescence stems with flagellated trichomes and pedicels with both flagellated and glandular trichomes.Comparative transcriptomic analysis showed that many transcription factors (TFs)are potentially involved in the formation and development of trichomes.The accumulation of RsETC1,a TF of the R3-MYB family,was significantly higher in inflorescence stems than in pedicels;quantitative reverse transcription PCR (qRTPCR) verified that its expression was significantly higher in inflorescence stems than in pedicels during the first three development stages,indicating its inhibitory action on the initiation of glandular trichomes in R.sterilis.The mRNA level of RsETC1 accumulated to significantly higher levels in trichomeless tissues than in tissues with trichromes,suggesting that this gene may inhibit the formation of trichomes in R.sterilis.Over-expression of RsETC1 in Arabidopsis resulted in glabrous phenotypes,and the expression of trichome-related endogenous genes,except for TTG1,was markedly reduced.In addition,the contents of the phytohormones jasmonic acid (JA),gibberellin A3 (GA3),and cytokinins (CKs) in pedicels were significantly higher than those in inflorescence stems,and the expression patterns of the genes related to hormone biosynthesis and signal transduction presented consistent responses,suggesting that the transduction of these hormones might be crucial for trichome initiation and development.These data provide a new perspective for revealing the molecular mechanism of trichome formation in R.sterilis.

Keywords: comparative transcriptome,inflorescence stem,pedicels,R3-MYB transcription factor,trichome

1.lntroduction

The aerial parts of almost all terrestrial plants are covered with trichomes (Huaet al.2022).These trichomes are presented in different forms,such as simple trichomes inArabidopsis,fruit thorns in cucumbers,and prickles in roses.Terrestrial plants mainly depend on trichomes for protection and defense against pathogens and predators(Karabourniotiset al.2020;Wanget al.2020).Many plants in the families Rosaceae,Araliaceae,Fabaceae,and Rutaceae bear prickles on their leaves,stems,and fruits (Zhonget al.2021).However,prickles and trichomes on stems cause issues in fruit harvesting,garden management,and transport process.

RosasterilisS.D.Shi,commonly known as Golden Cili,is a perennial shrub fruit crop that belongs to the Rosaceae family and is endemic to China.Its rapid growth,strong root system,and early and rapid harvest times have madeR.sterilisa preferred tree for the economic and ecological development of karst regions.R.sterilisfruit is nutritious and contains abundant functionally active components.Previous research suggests that components ofR.sterilishave antioxidant,immunity-promoting,anti-cancer,and anti-aging effects(Heet al.2016;Liet al.2016).These features are appreciated by consumers and have madeR.sterilisan important emerging commercial crop in Southwest China.The study of plant prickles has attracted attention in recent years.Previous research has found that the pedicel ofR.sterilishas glandular trichomes in the early stage and that some of the glandular trichomes lignify into prickles during fruit ripening (Maet al.2021).

Significant progress has been made in trichome initiation and development with unicellular trichomes,especially the leaf trichomes of the model plantArabidopsis(Hülskamp 2004;Schellmann and Hülskamp 2005;Machadoet al.2009).Transcription factors (TFs)such as the MYB,bHLH,WD40,WRKY,and C2H2 zinc finger family proteins are known to play crucial roles in cell fate determination in unicellular trichomes (Pattanaiket al.2014;Chopraet al.2019).Compared with unicellular trichomes,the regulatory networks and development of multicellular trichomes are not clear,and most of the relevant work has been performed in cucumber (Tanet al.2012;Liuet al.2016).Evidence suggests thatCsGL1,TBH,andMICTare alleles of the same gene generated by alternative splicing.TRILandCsGL3are allelic as well,and these two alleles can override the regulation ofCsGL1,TBH,andMICTon the development of multicellular trichomes,thereby affecting their initiation(Chenet al.2014;Panet al.2021).The biosynthesis and signaling of gibberellin (GA),jasmonic acid (JA),salicylic acid (SA),and cytokinins (CKs) are also known to affect trichome initiation (Andradeet al.2017;Chenet al.2017).However,there has been little progress in revealing the molecular cues behind glandular trichome development.In order to explore the molecular signals for different types of trichomes,this study performed the main hormones and high-throughput RNA-sequencing (RNA-Seq) analyses of pedicel and inflorescence stems ofR.steriliswith different types of trichomes on the surface.Differentially expressed genes (DEGs) were identified,and their functions were investigated using Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis and Gene Ontology (GO) annotations.Based on the transcriptome results,an R3-MYB family member was selected as a negative regulator to be heterologously overexpressed inArabidopsis.Overall,this integrated analysis provides insight into the molecular changes in different types ofR.sterilistrichomes.

2.Materials and methods

2.1.Plant materials and sample preparation

Ten-year-oldR.steriliswere planted in an experimental field at the fruit germplasm repository of Guizhou University,Guiyang,China (26°42′N,106°67′E).Samples were collected from a total of 24 independent trees with similar genotypes.The 24 trees were randomly divided into six groups,with four trees in each.Every two groups were combined into a compound sample as one repetition,for three biological repetitions in total.Pedicels with both flagellated and glandular trichomes and inflorescence stems with flagellate trichomes were collected for transcriptomic sequencing and hormone analysis on April 1,2019 (Fig.1),when the pedicel and inflorescence stems could be clearly distinguished;other samples were collected at varying stages,from flower bud germination to flowering (S1-S6) (Fig.2).The Columbia(Col-0) plants were grown at 24°C under 16 h light/8 h dark long-day conditions.

Fig.1 Photos showing different types of trichomes on pedicel and inflorescence stem surface of Rosa sterilis.A,gross morphology of inflorescence branch.B,single inflorescence branch appearance.C,light microscopy images of flagellate trichomes (black arrows) on inflorescence stem and glandular trichomes (white arrows) on the pedicel.The red box and the blue box refer to sampling pedicel and inflorescence stem,respectively.

Fig.2 The macroscopic depiction of six stages of development (S1-S6).

2.2.Scanning electron microscopy

During scanning electron microscopy (SEM),the sample was fixed with 3% glutaraldehyde.Dehydration was performed with 30,50,70,90,and 100% concentrations of tert-butanol and ethanol mixtures (repeated three times),and the samples were then vacuumed and dried at 4°C.Samples were coated with a thin layer of gold and observed with an S-3400 N scanning electron microscope(Hitachi,Tokyo,Japan).

2.3.Hormone analysis

The extraction of hormones from the pedicels and inflorescence stems with different types of trichomes was based on a previously described method (Liuet al.2013) with modifications.Fresh samples weighing 0.2 g were crushed into tubes with extracting solution(methanol/water/formic acid at a ratio of 7.9:2.0:0.1).The homogenates were well mixed in an ultrasonic bath and then kept at 4°C overnight,after which they were centrifuged at 10 000 r min-1for 15 min,and the supernatant was then collected and transferred to a new 10-mL centrifuge tube.A total of 1 mL of extraction solution was added to the residue,and after 1 h of shaking,the mixture was centrifuged at 12 000 r min-1and 4°C for 20 min.Phytohormones were separated into the acid with a reverse-phase-cation exchange solid-phase extraction(SPE) chromatography (Waters,MA,USA).Finally,the combined supernatants were dried under nitrogen,mixed with 0.15 mL of the mobile phase (which contained formic acid/water/acetonitrile in a ratio of 0.9:94.1:5.0),and filtered through a 0.2-μm filter.A total of 100 μL of the filtrate were used to analyze the concentrations of endogenous hormones through liquid chromatography-mass spectrometry (LC/MS) with an ACQUITY UPLC H-Class system (Waters,MA,USA) coupled to a Micromass Quattro II tandem quadrupole mass spectrometer equipped with an electrospray ionization source.The data were normalized based on internal standards (D2-GA3,D1-CKs,D3-BRs,D5-JA,D6-ABA,D2-IAA,D4-SA,and D4-ACC).

2.4.RNA-Seq assembly,annotation,and transcriptome sequence analysis

Total RNA from pedicels and inflorescence stems was extracted using the TRIzol method (TaKaRa,Dalian,China) according to the manufacturer’s instructions.The quality purity was checked using a NanoPhotometer?spectrophotometer (IMPLEN,California,USA) and agarose gel electrophoresis.A total of 1 μg of RNA was used as the input material for the preparation of RNA samples.HISAT2(http://ccb.jhu.edu/software/hisat2/index.shtml) was used to map the clean reads to theRosagenome sequence(http://ftp.ensemblgenomes.org/pub/plants/release-48/gff3/rosa_chinensis/),and the mapped reads were assembled using StringTie based on the reference genome sequence.If assembled reads were mapped to an unannotated transcript and encoded ＞50 amino acids,they were defined as a new gene.The edgeR package was used for the identification and analysis of DEGs between pedicels and inflorescence stems,and unigenes with |log2(fold change)|＞1 and an adjustedP-value＜0.05 were considered DEGs.GO enrichment (http://www.geneontology.org/) and KEGG enrichment (http://www.genome.jp/kegg/) analyses were performed according to Ashburneret al.(2000) and Kanehisaet al.(2004),respectively.The Illumina RNASeq data have been submitted to the Genome Sequence Archive of the National Genomics Data Center with the accession number PRJNA786413.

2.5.ldentification and isolation of the RsETC1 gene

According to the comparative transcriptomic analysis and previous research (Chenet al.2014;Pattanaiket al.2014),this study selected a set of genes that have been implicated in having a role in trichome development.First,the expression levels of these genes were determined in a different stage of trichome development on pedicels and inflorescence stems by quantitative reverse transcription PCR (qRT-PCR).The expression level of RchiOBHm-Chr2g0159921 was significantly different in pedicels and inflorescence stems with different types of trichomes.Then,the expression level of RchiOBHm-Chr2g0159921 was tested in eight tissues (inflorescence stems,inflorescence stems after trichome removal,pedicels,pedicels after trichome removal,floral buds,floral buds after trichome removal,the vein with glandular trichomes,and the mesophyll without any trichomes) at S3 (Fig.2) by qRTPCR.The results showed a similarly negative effect.Finally,RchiOBHm-Chr2g0159921 was identified as a candidate regulatory gene controlling trichome development.Based on the sequence of RchiOBHm-Chr2g0159921,a 225-bp open reading frame (ORF) gene was identified inR.sterilisand namedRsETC1.cDNA or DNA from pooled tissues were used as the template for PCR,and amplicons were inserted into pMD18-T (TaKaRa,Beijing,China) and sequenced.The primer sequences used are shown in Appendix A.

2.6.Subcellular localization

The subcellular localization assays were conducted in tobacco (Nicotianabenthamiana) leaves.To investigate the subcellular localization of the RsETC1 protein,the coding sequence ofRsETC1without the stop codon was amplified and then cloned into pBWA(V) HS.The recombined plasmids were transformed intoAgrobacteriumtumefaciensstrain GV3101 and subsequently transiently co-transformed intoN.benthamianaleaves using a 1-mL needless syringe.The transformed samples were incubated in the dark at 22°C for 24 h and then maintained at 25°C in normal light cycles for 2 days.The subcellular localization of each expressed protein was visualized using a confocal microscope (Olympus,Tokyo,Japan).The primers used are listed in Appendix A.

2.7.Overexpression vector construction and plant transformation

For sequencing,the entireRsETC1coding region was ligated into pMD18-T (TaKaRa,Beijing,China),and accurate fragments were introduced into the pCAMBIA2301 binary vector,which contained the CaMV35S promoter and the nopaline synthase terminator.The GV3101 strain ofA.tumefaciensthat contained this construct was used to transform wild-typeArabidopsisby floral dipping (Clough and Bent 1998).T3 transgenic lines with a wild-typeArabidopsisbackground were chosen for further analysis.

2.8.Semiquantitative RT-PCR and quantitative real-time PCR

RNA was extracted fromR.sterilisand approximately three-week-old wild-type and transgenicArabidopsisplants using the TRIzol RNA Purification Kit (TaKaRa,Beijing,China).gDNA eraser was used to remove genomic DNA,and the first strand cDNA was generated using the PrimeScript RT Reagent Kit (TaKaRa,Kyoto,Japan).Ubiquitin (UBQ) was used as a reference gene to normalize the expression data.Primers used for qRTPCR were designed with Primer Premier 6 (Appendix A).The 20-μL semiquantitative RT-PCR reaction mixture was subjected to 28-35 RT-PCR cycles,depending on the linear range of PCR amplification for each gene.The ABI ViiA 7 DX System (Applied Biosystems,CA,USA)and SYBR PremixExTaq(TaKaRa,Beijing,China) were used for qRT-PCR.The relative transcript levels of genes were determined using the 2-ΔΔCTmethod (Livak and Schmittgen 2001).Three biological replicates and three technical repeats were analyzed for each sample.

3.Results

3.1.Morphology of trichomes on pedicels and inflorescence stems

SEM imaging revealed the differences between pedicels and inflorescence stems (Fig.3).There were two types of trichomes on pedicels,and both were multicellular.Flagellate trichomes were abundant on the surfaces of the inflorescence stems and pedicels.The capitate glandular trichomes were comprised of glandular heads with multiple layers of epidermal cells and long stalks (Fig.3-B).In addition,the capitate glandular trichomes were only readily observed on the surfaces of the pedicel.

Fig.3 Scanning electron micrographs (SEM) micrographs showing pedicel and inflorescence stem.A,flagellate trichomes (black arrow) on inflorescence stem.B,flagellate trichomes (black arrow),and glandular (white arrow) trichomes on a pedicel.

3.2.De novo assembly and comparative analysis of RNA-Seq data

To investigate the genes associated with different types of trichomes,six cDNA libraries were constructed from the total RNA extracted from pedicels and inflorescence stems with or without glandular trichomes.An overview of sequence assembly after Illumina sequencing is shown in Table 1.In total,151 160 554 and 151 903 248 raw reads were generated from the three replicates of pedicels and inflorescence stems,respectively.After the adapter sequence and low-quality tags had been removed,145 635 274 and 147 956 022 clean reads were obtained from pedicels and inflorescence stems,respectively.A total of 44.03 Gb of clean bases were generated for the six libraries,with an average of 7.3 Gb of clean bases for each library.The average GC content was 46.85%,and average Q30 and Q20 values were 88.33 and 96.38%,respectively.Clean reads were assembled into unique tags and mapped toRosagenome sequences and annotated transcripts(Table 1).Expression levels were represented as the total tag count mapped to the sense strand of each annotatedRosagene.A total of 2 868 DEGs (fold-change＞1 with aP-value＜0.05) were identified,of which 1 128 were upregulated and 1 740 were downregulated in the samples from pedicels with glandular trichomes (Fig.4).

Fig.4 Differential gene expression analysis.A,M-vs.-A (MA) plot with log2(fold change) plotted versus base mean fold change.B,volcano with log2(fold change) plotted vs.-log10P-adj.,horizontal line represents P=0.05.

Twenty DEGs were randomly selected for qRT-PCR validation,and the results were consistent with the RNASeq data (Pearson correlation coefficient 0.989) (Table 2),suggesting that the transcriptomic data were reliable and valid.

Table 1 Summary statistics relating to the RNA-Seq libraries derived from pedicel and inflorescence stem

Table 2 qRT-PCR confirmation of differentially expressed Rosa sterilis genes identified by different expression gene (DEG)

3.3.GO enrichment classification and KEGG pathway analysis

GO analysis was used to investigate the functions of the DEGs identified in pedicel and inflorescence stem samples (Fig.5).The DEGs were clustered into three main categories according to GO classification: biological process,cellular component,and molecular function.In both comparisons,the most highly enriched terms in the biological process category were single-organism process,cellular process,and metabolic process;the dominant terms in the cellular component category were membrane,membrane part,cell part,and cell.Most molecular function terms were found in the catalytic activity category,followed by binding.The DEGs were also subjected to a KEGG enrichment analysis (Fig.6).A total of 784 genes were annotated and assigned to 108 KEGG pathways (Appendix B).The upregulated genes that differed between pedicel and inflorescence stem tissues were significantly enriched in phenylpropanoid biosynthesis (ko00940,29,8.3%),and the downregulated genes were enriched in the plant hormone signal transduction pathway (ko0475,49,11.2%).

Fig.5 Classification of Gene Ontology (GO) terms concerning analysis of differentially expressed genes between pedicel and inflorescence stem.

Fig.6 Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis of differentially expressed genes (DEGs) between pedicel and inflorescence stem.A,upregulated DEGs in pedicel.B,downregulated DEGs in pedicel.

3.4.Differential hormonal composition in pedicel and inflorescence stem tissues

The transcription data revealed that many genes were enriched by endogenous hormones in the KEGG pathway.To identify the endogenous hormones regulating trichomes,eight kinds of endogenous hormones related to trichome development were determined in inflorescence stems and pedicels (Table 3).The contents of CKs,JA,GA3,auxin (IAA),and 1-aminocyclopropane carboxylic acid (ACC) in pedicels were higher than in inflorescence stems.However,only the contents of CKs,JA,and GA3differed significantly between inflorescence stems and pedicels,while the contents of SA,abscisic acid (ABA),brassinosteroids (BRs) in pedicels and inflorescence stems showed the opposite trend as the other hormones.In particular,the content of SA was significantly higher than pedicels in inflorescence stems.

Table 3 Hormonal content in pedicel and inflorescence stem (ng g-1)1)

3.5.Expression levels of genes related to endogenous hormones

Endogenous hormones play an important role in trichome formation and development.Based on the expression analyses and the hormone levels in the pedicel and inflorescence stem tissues,a heat map was constructed referring to CKs,GA,JA,and SA (Fig.7),which were significantly different in inflorescence stems and pedicels.A total of 35 DEGs (Appendix C) involved in hormone metabolism and signal transduction were found.Those related to JA (13 genes) and GA (12 genes) were the most abundant,followed by those related to CKs (eight genes) and SA (two genes).In the case of CKs,the mRNA forZOX,which is associated with CK biosynthesis,was exclusively upregulated in pedicel tissue,and the hormone signal transduction gene ofHKwas more highly expressed in pedicel tissue as well.The gene profiling data also showed thatGASAandDELLAtranscripts,which are markers for the GA response,were upregulated in pedicels.However,the degradation geneGA2oxwas upregulated in the inflorescence stem tissue.AOC,AOS,andJMTwere involved in regulating JA metabolism,and these genes were more highly expressed in the inflorescence stems.The other two DEGs involved in JA signal transduction showed increased transcript abundance in inflorescence stem tissue compared to the pedicels.The SA-responsive transcripts NPR1 and NPR5 exhibited higher expression levels than pedicels in the inflorescence stems.

Fig.7 Heat map of the expression of genes related to endogenous hormone biosynthesis and signal transduction in comparison of inflorescence stem and pedicel of the pedicel.A,related to cytokinins (CKs) biosynthesis and signal transduction.B,related to jasmonic acid (JA) biosynthesis and signal transduction.C,related to gibberellin A (GA) biosynthesis and signal transduction.D,related to salicylic acid (SA) biosynthesis and signal transduction.Red or blue represents up-or down-regulation,respectively.The bars represent the log2(fold changes) in expression.Numbers in brackets indicate gene ID.

3.6.ldentification of TFs involved in the formation of different types of trichomes

Numerous TFs have been reported to modulate trichome development.In this study,a total of 152 DEGs belonging to 36 TF families were identified.Based on previous research,116 candidate genes were pinpointed whose encoded TFs likely regulated trichome development(Appendix D).These TFs were subdivided into 17 gene families (Fig.8-A),including the homeodomain,MYBrelated,WRKY,bHLH,C2H2,MADS,AP2/ERF,TCP,HSF,NAC,YABBY,TIFY,DOF,SBP,bZIP,GRF,and Trihelix families.Among these DEGs,six genes (two MYBrelated,one MADS,two AP2/ERF,and one TCP family genes) were expressed only in pedicel tissue,whereas the transcription of four genes (two MYB-related,one MADS,and one C2H2 family genes) was not detected in pedicel tissue.The expression of all TCP (four),YABBY(one),and SBP (one) family members was higher in the pedicels,whereas the expression of all TIFY (one) and GRF (two) family members was lower in the pedicels.

This study then focused on the mechanistic basis underlying the differences in trichome development between the pedicel and inflorescence stem tissues and identified key genes in the MYB-bHLHWD40 (MBW) trimer and the downstreamGL2TF that controlArabidopsistrichome initiation and development.The information from nine TFs,such as the bHLH,C2H2 zinc finger,MYB,WD40 repeat,and WRKY families,are presented in Appendix E.OnlyETC1was significantly downregulated in pedicels compared with inflorescence stems (Fig.8-B).

Fig.8 Identification and validation of transcription factors (TFs) associated with trichome development,and their expression levels at pedicel and inflorescence stem.A,numbers upregulated or downregulated in pedicel and inflorescence stem.B,transcript expression analysis for TFs associated with trichome development in Arabidopsis.Red or blue represents up-regulation or downregulation,respectively,the bars represent the log2(fold changes) in expression.Numbers in brackets indicate gene ID.

3.7.ldentification and isolation of RchiOBHm-Chr2g0159921

To further explore the role of trichome-related genes in the development of trichromes,the expression levels of nine TFs were analyzed in pedicel and inflorescence stem tissues from flower bud germination to six flowering stages (Fig.2)by qRT-PCR analysis (Fig.9).Trichome-related genes have been found to be related to plant growth and development.Positively regulated genes and negatively regulated genes show different tendencies.Positively regulated genes reach their maximum at S3 in pedicels,while negatively regulated genes reach their maximum at S2 in inflorescence stems.Among the negative regulatory genes,only the expression level of RchiOBHm-Chr2g0159921 in inflorescence stems was significantly higher from S1 to S3 compared to that in pedicels.

Fig.9 qRT-PCR expression assays of trichome-related genes in six stages (S1-S6) pedicel and inflorescence stem.RsUBQ was used as an internal control.The data points represent the means of three biological replicates±SD.＊,P＜0.05 (Student’s t-test).

To further verify the function of RchiOBHm-Chr2g0159921,the expression patterns of different tissues at S3 were determined by qRT-PCR.It was found that RchiOBHm-Chr2g0159921 was significantly more highly expressed in trichomeless tissues than in corresponding trichome tissues (Fig.10).Therefore,it can be speculated that RchiOBHm-Chr2g0159921 participates in the regulation of trichome initiation.

Fig.10 qRT-PCR analysis expression of RsETC1 in different tissues at S3 of inflorescence branch.IS,inflorescence stem;ISa,inflorescence stem after trichome removal;P,pedicel;Pa,pedicel after trichome removal;Ve,vein;Me,mesophyll;Fb,floral bud;Fba,floral bud after trichome removal.The data points represent the means of three biological replicates±SD.＊,P＜0.05 (Student’s t-test).

3.8.Phylogenetic analysis and structural features of RsETC1

Based on the sequence of RchiOBHm-Chr2g0159921,RsETC1,containing a 225-bp open reading frame (ORFs)and encoding 75 amino acids,was identified inR.sterilis(Fig.11-A).RsETC1contained two introns and three exons (48,90,and 87 bp).The alignment of the amino acid sequence of theR.sterilisgene with the R3-MYB proteins ofArabidopsisand Rosaceae revealed that,among them,RsETC1was closest toRcETC1with a 100% sequence similarity,compared with 39.19% toAtETC1and no more than 42% to other R3-MYB family members ofArabidopsis.Therefore,the sequence fromR.steriliswas namedRsETC1(Fig.11-B).Previous studies have confirmed that the conserved amino acid signature [D/E]L×2[R/K]×3L×6L×3R belongs to R/B-like bHLH TFs (Duboset al.2010).InRsETC1,the first conserved D amino acid in the signature [D/E]L×2[R/K]×3L×6L×3R motif is substituted by F.

Fig.11 RsETC1 in Rosa sterilis.A,genomic structures of RsETC1.Boxes indicate exons,black lines indicate introns;numbers show sizes in base pairs.B,sequence alignment of RsETC1,Rosaceae,and Arabidopsis R3-MYB transcription factors using ClustalX.The lines above the sequence indicate the conserved R3-MYB repeats.The blue arrows indicate the conserved R3-MYB motif in R3-MYB([D/E]L×2[R/K]×3L×6L×3R).

To understand the evolutionary relationship betweenRsETC1and other R3-MYB proteins,a phylogenetic tree was constructed using the neighbor-joining method(Fig.12;Appendix F).Cluster analysis demonstrated that theRsETC1and other R3-MYB proteins clustered into two groups.The seven R3-MYB genes ofArabidopsisclustered within the group I clade,and the Rosaceae were contained within the group II clade.Except for inRosa chinensis,RsETC1was most similar toFvCPC-like.

Fig.12 Phylogenetic tree of R3-MYB transcription factors in Rosa sterilis and other plant species based on amino acid sequences.The tree was generated by the neighbor-joining (NJ) method and 500 bootstrap replicates using MEGA7.0 Software.The R3-MYB genes from R.sterilis are marked with blue star.

3.9.Subcellular localization of RsETC1

To determine the subcellular localization ofRsETC1,a green fluorescent protein (GFP) was fused toRsETC1and transiently expressed inN.benthamianaleaves.The GFP fluorescence ofRsETC1-GFP was observed in both cell nuclei and the cytoplasm (Fig.13).This demonstrated thatRsETC1was located in both the cytoplasm and nucleus,consistent with its role as a TF.

Fig.13 Subcellular localization analysis of RsETC1 protein in tobacco (Nicotiana benthamiana) leaves.Bright,bright field image;GFP,GFP fluorescence;Chloropla,chloroplast fluorescence;Merged,merged images of bright,GFP and chloroplast fluorescence images.Bar=20 μm.

3.10.Overexpression of RsETC1 inhibits trichome development in Arabidopsis

To characterize the function ofRsETC1,its cDNA was ectopically expressed in wild-typeArabidopsiswith the CaMV 35S promoter.As reported previously for the R3-MYB TFs of London plane tree (Platanusacerifolia)(Zhanget al.2019) andArabidopsis(Tominaga-Wada and Wada 2017),overexpressingRsETC1genes resulted in glabrous phenotypes,with no trichome formation on the early rosette leaves (Fig.14-F),the adaxial and abaxial surfaces of the later rosette and cauline leaves (Fig.14-E and L),or the surfaces of inflorescences (Fig.14-H and N) or stems (Fig.14-G and M).Semiquantitative RT-PCR was performed to confirm the presence and assess the expression levels of the exogenousR.sterilisgenes.Twelve independent transgenic lines were obtained,among which four displayed a completely glabrous phenotype.Homozygous T3 lines of these four lines were used for further characterization.

Fig.14 Phenotypes of 35S::RsETC1-overexpression in Arabidopsis.A-D and I-K are wild-type olumbia,E-H and L-N are 35S::RsETC1 transgenic plant.A and E,trichomes on rosette leaves.B and F,stereomicroscopic images of young leaves from 10-day-old seedlings.C and G,main stems.D and H,sepals.I and L,scanning electron micrographs (SEM) of fresh true leaves.J and M,SEM of stems.K and N,SEM of sepals.

3.11.Expression changes of trichome-related genes in Arabidopsis overexpressing RsETC1

To further study howRsETC1regulates trichome development,the expression of several trichomedevelopment-related genes in the transgenic lines was assessed (Fig.15).HighRsETC1expression was detected in the transgenic lines;in contrast,RsETC1expression was not detected in the wild type.Consistent with previous reports on seven R3-MYB negative regulatory families,the overexpression ofRsETC1also strongly inhibited the expression of trichome-related genes inArabidopsis(Fig.15).The endogenousArabidopsisR3-MYB-encoding genesAtTC1,AtETC2,AtETC3,AtTCL1,AtTCL2,AtTRY,andAtCPCwere strongly repressed;in particular,the expression levels ofAtETC1andAtTCL1were extremely low in transgenic plants.Some of these,notablyAtGL1,AtGL21,AtGL3,AtEGL3,AtMYB23,andAtTTG2,appeared to be significantly downregulated relative to the levels in wild-type plants.In contrast,the expression ofAtTTG1was slightly higher in some transgenic lines.

Fig.15 Relative expression of genes related to trichome developmental control in transgenic Arabidopsis lines overexpressing RsETC1.The AtACT2 (AT3G18780) gene was used as an internal control.The expression level of each gene in the wild type was set to 1 and error bars indicated the standard deviation (SD) of three biological replicates.

4.Discussion

Prickles onR.sterilisseriously increase production costs,making the cultivation of prickless plants an urgent need.R.sterilisproduces five types of multicellular trichomes,namely,flagellate trichomes,acicular trichomes,branched trichomes,capitate glandular trichromes,and bowl-shaped glandular trichomes (Maet al.2021).This provides convenience for studying the formation of different types of multicellular trichomes,but the limited available genomic information forR.sterilishas constrained previous genetic studies.Pedicels and inflorescence stems are closely connected parts ofR.sterilis,but their trichome types are not the same.Inflorescence stems contain flagellate trichomes,while pedicels contain both flagellate and glandular trichomes.

Unlike the glandular trichomes on grapes (Maet al.2016),not all glandular trichomes will develop into prickles inR.sterilis.According to a previous study (Maet al.2021),some heads of the glandular trichomes in pedicels will fall off,and they increase in both height and basal width,thus developing into acicular trichomes.Based on these differences,two high-quality RNA-Seq datasets of pedicel and inflorescence stem tissues were generated and analyzed to study the molecular mechanism underlyingR.sterilistrichome formation.Based on these transcriptomic data,2 868 candidate genes obtained were significantly differentially expressed in pedicel and inflorescence stem tissues.

The main difference between glandular trichomes and non-glandular trichomes is the presence or absence of glandular heads (Kellogget al.2011).The main function of glandular head cells is the synthesis and secretion of large amounts of several metabolites,mainly terpenoids,but also flavonoids and phenylpropanoids (Burns 2014).The results of the KEGG pathway enrichment analysis in this study indicated that the biosynthesis of secondary metabolites was a very important pathway in pedicels covered with glandular trichomes.With the exception of phenylpropanoids,DEGs involved in the biosynthetic pathways of other bioactive substances were also enriched,such as those involved in flavonoid biosynthesis,glucosinolate biosynthesis,and stilbenoid,diarylheptanoid and gingerol biosynthesis.

Trichome differentiation is also regulated by phytohormones in plants.However,little is known about the underlying mechanism of phytohormone signaling in the induction of trichomes.Several studies have shown that the metabolic processes of GA,SA,JA,and CKs are involved in trichome density,length,and branching on leaves and stems (Maes and Goossens 2010;Huaet al.2021).In the present study,the hormone content in pedicels and inflorescence stems was analyzed because the KEGG enrichment of the transcriptome was more enriched in endogenous hormone pathways.It was found that the contents of CKs,GA,JA,and SA were significantly different between inflorescence stem and pedicel tissues.Combined with the transcriptomic analysis,the candidate genes involved in hormone metabolism were identified.These genes may indirectly participate in the formation of trichomes inR.sterilisby regulating hormone metabolism.

TFs of the MYB family are known to regulate responses to biotic and abiotic stresses,cell fate and identity,as well as primary and secondary metabolism (Duboset al.2010).In this study,the RNA sequencing results revealed that the members of the MYB family were the most abundant,of which 10 were significantly increased in pedicels,and 17 were decreased.The expression of MYB members was analyzed,and it was found that the expression levels ofMYB61,MYB86,andMYB4were downregulated in inflorescence stems (Appendix B),whereas upregulated in the transcription ofETC1,RL6,andMYR2.It is worth noting that the expression of all the TCPs was upregulated in pedicels compared with their expression in inflorescence stems.The TCP TFs are known to regulate diverse processes of plant trichome initiation and density (Wang and Chen 2014).Collectively,these findings indicate that TCP TFs act as positive regulators and play important roles inR.sterilispedicel glandular trichome development.

InArabidopsis,the key genes involved in trichome initiation encode a bHLH,an MYB,and a WD-40 repeatcontaining protein,which together form an MYB-bHLHWD40 complex that promotes non-glandular trichome initiation.In contrast,TRIPTYCHONandCAPRICEsuppress this process (Yuanet al.2021).The initiation of most glandular trichomes is regulated by MYB TFs,such as tomato type I glandular trichome and tobacco glandular trichome (Fenget al.2021).However,the molecular mechanism of trichome development inRosaremains largely unknown.Transcriptome data showed thatETC1expression displayed significant differences between pedicels and inflorescence stems.qRT-PCR analysis of trichome-related genes at six developmental stages and without trichome tissue indicated that RchiOBHm-Chr2g0159921,encoding an MYB gene,may regulate trichome formation inR.sterilis.

On the basis of the sequence of RchiOBHm-Chr2g0159921,the 225-bp full-length cDNA of the R3-MYB geneRsETC1was isolated fromR.sterilis.Surprisingly,theRsETC1gene identified in this study shared a very high similarity to theR.chinensisRcETC1gene,with 99% nucleotide sequence identity and 100%amino acid sequence identity.TheRcETC1gene has not been assigned biological functions to date.The results from phylogenetic and gene structure analyses support the conclusion thatRsETC1is closely related to thePaCPC-like1,PaCPC-like2,andPaCPC-like3genes,which regulate trichome development in the London plane tree (Zhanget al.2019).According to previous research onArabidopsis and other plants,GL1,GL2,GL3,TTG1,PDF2,PDF2-like,ETC1,ETC3,andCPCare the key TFs involved in regulating trichome development (Hülskamp 2004;Schellmann and Hülskamp 2005).It could thus be speculated thatRsETC1may play a negative regulatory role in trichome development inR.sterilis.

The overexpression ofRsETC1by CMV35S produced glabrous phenotypes inArabidopsis(Fig.14).It was concluded thatRsETC1,an R3-MYB TF fromR.sterilis,negatively controlledArabidopsistrichome formation.The role ofRsETC1inR.steriliswill need to be determined through the transformation ofR.sterilisitself.R3-MYBs promote trichome formation but inhibit root hair development (Wang and Chen 2014).In previous research,TRY(R3-type MYB) has been found to compete withGL1(R2R3-type MYB) for aGL3binding site;they form different MYB-bHLH complexes that regulate the differentiation ofArabidopsistrichomes (Tominaga-Wadaet al.2013;Zhanget al.2019).CPC-likeR3-MYB may be a key common regulator of plant trichome and roothair development in tomatoes (Tominaga-Wada and Wada 2017).The results of gene expression analysis showed that all endogenous genes related to trichome development inArabidopsisexcept forAtTTG1were significantly suppressed in 35S:RsETC1transgenic lines(Fig.15).These data indicate that theRsETC1protein may also disrupt theTTG1-GL3/EGL3-GL1complex,thereby repressingGL2expression.

5.Conclusion

For the first time,this study compared trichome types in two connected organs (pedicels and inflorescence stems)and generated transcriptome profiles to analyze trichome formation inR.sterilis.A member of the R3-MYB family,RsETC1,was isolated and characterized fromR.sterilis,inhibiting trichome development inArabidopsis.In addition,according to the expression levels of DEGs and the content of endogenous hormones in samples,GA,JA,CK,and SA hormone metabolism were analyzed,and some genes that may be related to prickle formation were screened out.The findings have advanced our understanding of the molecular regulatory mechanism of trichome formation inR.sterilis.However,althoughRsETC1can effectively inhibit the initiation of trichomes inArabidopsis,the function ofRsETC1needs further verification because of the lack of genetic transformation available forR.sterilis.It will be worthwhile to study whetherRsETC1directly regulates the trichome types inR.sterilisin the future.

Acknowledgements

This work was supported by grants from the Joint Fund of the National Natural Science Foundation of China and the Karst Science Research Center of Guizhou Province,China (U1812401),and the Talent Project of Guizhou Province,China (20164016).

Declaration of competing interest

The authors declare that they have no conflict of interest.

Appendicesassociated with this paper are available on http://www.ChinaAgriSci.com/V2/En/appendix.htm