R2R3 MYB-dependent Auxin Signaling Regulates Trichome Formation In Tomato

Plant trichomes,are differentiated epidermal cells,functioning as mechanical and chemical barrier against herbivores.Typically,Arabidopsis trichomes are unicellular,branched and non-glandular.Trichome formation in Arabidopsis is controlled by a transcription factor(TF)complex including R2R3 MYB,WD40 repeat(WDR)and basic helix–loop–helix(b HLH)proteins.Studies indicated that the regulatory pathways of trichome formation are different between multicellular trichomes in tomato and unicellular trichomes in Arabidopsis.However,the mechanism of formation as well as development of glandular trichome in tomato remains unclear.The auxin,as the most important phytohormone,controls many physiological processes of plant growth and development.Auxin treatment obviously increased formation of type ?,? and ? trichomes in tomato leaves.The short-lived Aux/IAAs proteins and auxin response factors(ARFs)are involved in auxin-dependent transcriptional regulation.ARFs act as either transcriptional repressors or activators of auxin-responsive genes.Our research focused on functional characterization of SlARF4 and systematical construction of its downstream regulatory network.The important experimental results and conclusions are listed below.(1)q PCR showed that SlARF4 is expressed in all tissue tested,with highest expression level in leaf trichomes.GUS staining was detected in type ?,?,and ? trichomes of Pro SlARF4: GUS tomato plants.Next,we functionally analyzed SlARF4 in tomato.Fully expanded and expanding leaves were used in this study.Over-expression of SlARF4 increased type ?,type ? and type ? density in both stages and down-regulation of SlARF4 led to decrease trichome density.The OE-SlARF4 lines also displayed increase in the density of epidermal pavement cells in the leaves,whereas RNAi lines exhibited decreased density compared with WT plants.Subsequently,an apparent increase in the number of trichomes per epidermal cell in the overexpressed lines and decrease in that of the RNAi lines were observed.Auxin treatment showed that the downregulation of SlARF4 decreases the effect of IAA treatment on the formation of type ?,?,and ? trichomes in tomato leaves and that the inducement of auxin on the formation of type ?,?,and ? trichomes is dependent on SlARF4.Overexpression of SlARF4 increased excretion of y-elemene,?-caryophyllene and?-humulene from leaves and tolerance to spider mites in tomato.(2)RNA sequencing(RNA-seq)was used to identify the differentially expressed genes(DEGs)between the WT and RNAi-SlARF4 lines.Among these,two MYB genes,SlTHM1 and SlMYB52 contained the Aux RE(TGTCTC)motif and auxin response element(TGA motif,AACGAC),respectively,which are the binding motifs of ARF proteins.The binding of SlARF4 protein to SlTHM1 and SlMYB52 genes was verified by gene-protein interaction experiment.Dual-luciferase assay,EMSA assay and ChIP-q PCR showed that the SlARF4 bound to the Aux RE and TGA motifs of the SlTHM1 and SlMYB52 promoters,respectively,and inhibited their expression.Hybridization experiments showed that SlARF4 depended on SlTHM1 to regulate type ? and ? trichome formation and SlARF4 depended on SlMYB52 to regulate type ? trichome formation.(3)SlTHM1 is expressed in all tissue tested,with highest expression level in leaf trichomes.GUS staining was detected in type ? and ? trichomes of p SlTHM1-GUS tomato plants.SlTHM1 is located in cell nucleus and has transcriptional activation activity.SlTHM1 negatively regulated the formation of type ? and ? trichome in tomato leaves.SlTHM1 also negatively regulated the terpenoids excretion and tolerance to the spider mites in tomato.SlMYB52 was specifically expressed in type ? trichomes and negatively regulated the formation of type ? trichome in tomato.Down-regulation of SlMYB52 doesn‘t influence tolerance to the spider mites in tomato.(4)SlCycB2,a B-type cyclin gene,plays a key role in trichome initiation.SlTHM1 and SlMYB52 directly targeted cyclin gene SlCycB2 and activated its expressions.SlIAA15 negatively regulated the formation of type ?,? and ? trichomes in leaves.SlTHM1 and SlMYB52 directly targeted cyclin gene SlIAA15 and activated its expressions.We proposed a model of how auxin induces the formation of type ?,?,and ? trichomes in tomato leaves.Auxin induces the expression of SlARF4,and SlARF4 protein inhibits the expressions of SlTHM1 and SlMYB52.Decreased SlTHM1 levels reduce SlCycB2 and SlIAA15 expression,which promotes the formation of type ? and ? trichomes.Decreased SlMYB52 levels inhibit SlCycB2 and SlIAA15 expression,resulting in the promotion of the formation of type ? trichomes.The SlTHM1/SlMYB52-dependent auxin signaling pathway modulates the formation of unicellular and multicellular trichomes in tomato,and increasing trichome density is an effective method to improve the tolerance of tomato to spider mites.