BrrTCP4b interacts with BrrTTG1 to suppress the development of trichomes in Brassica rapa var. rapa

The number of trichomes significantly increased in CRISPR/Cas9-edited BrrTCP4b turnip(Brassica rapa var.rapa)plants.However,the underlying molecular mechanism remains to be uncovered.In this study,we performed the Y2H screen using BrrTCP4b as the bait,which unveiled an interaction between BrrTCP4b and BrrTTG1,a pivotal WD40-repeat protein transcription factor in the MYB-bHLH-WD40(MBW)complex.This physical interaction was further validated through bimolecular luciferase complementation and co-immunoprecipitation.Furthermore,it was found that the interaction between BrrTCP4b and BrrTTG1 could inhibit the activity of MBW complex,resulting in decreased expression of BrrGL2,a positive regulator of trichomes development.In contrast,AtTCP4 is known to regulate trichomes development by interacting with AtGL3 in Arabidopsis thaliana.Overall,this study revealed that BrrTCP4b is involved in trichome development by interacting with BrrTTG1 in turnip,indicating a divergence from the mechanisms observed in model plant A.thaliana.The findings contribute to our understanding of the regulatory mechanisms governing trichome development in the non-model plants turnip.

Less hairy leaf 1,an RNaseH-like protein,regulates trichome formation in rice through auxin

The trichomes of rice leaves are formed by the differentiation and development of epidermal cells.Plant trichomes play an important role in stress resistance and protection against direct ultraviolet irradiation.However,the development of rice trichomes remains poorly understood.In this study,we conducted ethylmethane sulfonate(EMS)-mediated mutagenesis on the wild-type(WT)indica rice‘Xida 1B’.Phenotypic analysis led to the screening of a mutant that is defective in trichome development,designated lhl1(less hairy leaf 1).We performed map-based cloning and localized the mutated gene to the 70-kb interval between the molecular markers V-9 and V-10 on chromosome 2.The locus LOC_Os02g25230 was identified as the candidate gene by sequencing.We constructed RNA interference(LHL1-RNAi)and overexpression lines(LHL1-OE)to verity the candidate gene.The leaves of the LHL1-RNAi lines showed the same trichome developmental defects as the lhl1 mutant,whereas the trichome morphology on the leaf surface of the LHL1-OE lines was similar to that of the WT,although the number of trichomes was significantly higher.Quantitative real-time PCR(RT-qPCR)analysis revealed that the expression levels of auxin-related genes and positive regulators of trichome development in the lhl1 mutant were down-regulated compared with the WT.Hormone response analysis revealed that LHL1 expression was affected by auxin.The results indicate that the influence of LHL1 on trichome development in rice leaves may be associated with an auxin pathway.

OsSPL10 controls trichome development by interacting with OsWOX3B at both transcription and protein levels in rice(Oryza sativa L.)

Plant trichomes are a specialized cellular tissue that functions in resistance to biotic and abiotic stresses.In rice,three transcription-factor genes:OsWOX3B,HL6,and OsSPL10,have been found to control trichome development.Although studies have shown interactions between the three genes,their full relationship in trichome development is unclear.We found that the expression levels of OsWOX3B and HL6 were both reduced in OsSPL10-knockout plants but increased in OsSPL10-overexpression plants,suggesting that OsSPL10 positively regulates their expression.Physical interaction between OsSPL10 and OsWOX3B was found both in vivo and in vitro and attenuated their abilities to bind to the promoter of HL6 to activate its transcription.This mechanism may regulate trichome length by adjusting the expression of HL6.A rice gene network regulating trichome development is proposed.

Protective and defensive roles of non-glandular trichomes against multiple stresses:structure-function coordination

As superficial structures,non-glandular trichomes,protect plant organs against multiple biotic and abiotic stresses.The protective and defensive roles of these epidermal appendages are crucial to developing organs and can be attributed to the excellent combination of suitable structural traits and chemical reinforcement in the form of phenolic compounds,primarily fl avonoids.Both the formation of trichomes and the accumulation of phenolics are interrelated at the molecular level.During the early stages of development,non-glandular trichomes show strong morphological similarities to glandular ones such as the balloon-like apical cells with numerous phenolics.At later developmental stages,and during secondary wall thickening,phenolics are transferred to the cell walls of the trichomes.Due to the diff use deposition of phenolics in the cell walls,trichomes provide protection against UV-B radiation by behaving as optical fi lters,screening out wavelengths that could damage sensitive tissues.Protection from strong visible radiation is also aff orded by increased surface light refl ectance.Moreover,the mixtures of trichome phenolics represent a superfi-cial chemical barrier that provides protection against biotic stress factors such as herbivores and pathogens.Although the cells of some trichomes die at maturity,they can modulate their quantitative and qualitative characteristics during development,depending on the prevailing conditions of the external biotic or abiotic environment.In fact,the structure and chemical constituents of trichomes may change due to the particular light regime,herbivore damage,wounding,water stress,salinity and the presence of heavy metals.Hence,trichomes represent dynamic protective structures that may greatly aff ect the outcome of many plant–environment interactions.

Up-regulation of a homeodomain-leucine zipper gene HD-1 contributes to trichome initiation and development in cotton

Plant trichomes originate from epidermal cells.In this work,we demonstrated that a homeodomain-leucine zipper(HD-Zip)gene,Gh_A06G1283(Gh HD-1A),was related to the leaf trichome trait in allotetraploid cotton and could be a candidate gene for the T_1 locus.The ortholog of GhHD-1A in the hairless accession Gossypium barbadense cv.Hai7124 was interrupted by a long terminal repeat(LTR)retrotransposon,while GhHD-1A worked well in the hairy accession Gossypium hirsutum acc.T586.Sequence and phylogenetic analysis showed that GhHD-1A belonged to the HD-Zip IV gene family,which mainly regulated epidermis hair development in plants.Silencing of GhHD-1A and its homoeologs GhHD-1D in allotetraploid T586and Hai7124 could significantly reduce the density of leaf hairs and affect the expression levels of other genes related to leaf trichome formation.Further analysis found that GhHD-1A mainly regulated trichome initiation on the upper epidermal hairs of leaves in cotton,while the up-regulated expression of GhHD-1A in different organs/tissues also altered epidermal trichome development.This study not only helps to unravel the important roles of GhHD-1A in regulating trichome initiation in cotton,but also provides a reference for exploring the different forms of trichome development in plants.

Identifying candidate genes involved in trichome formation on carrot stems by transcriptome profiling and resequencing

Trichomes are specialized structures developed from epidermal cells and can protect plants against biotic and abiotic stresses.Trichomes cover carrots during the generative phase.However,the morphology of the carrot trichomes and candidate genes controlling the formation of trichomes are still unclear.This study found that carrot trichomes were nonglandular and unbranched hairs distributed on the stem,leaf,petiole,pedicel,and seed of carrot.Resequencing analysis of a trichome mutant with sparse and short trichomes(sst)and a wild type(wt)with long and dense trichomes on carrot stems was conducted.A total of 15396 genes containing nonsynonymous mutations in sst were obtained,including 42 trichomerelated genes.We also analyzed the transcriptome of the trichomes on secondary branches when these secondary branches were 10 cm long between wt and sst and obtained 6576 differentially expressed genes(DEGs),including 24 trichome-related genes.qRT-PCR validation exhibited three significantly up-regulated DEGs,20 significantly downregulated,and one with no difference.We considered both the resequencing and transcriptome sequencing analyses and found that 12 trichome-related genes that were grouped into five transcription factor families containing nonsynonymous mutations and significantly down-regulated in sst.Therefore,these genes are potentially promising candidate genes whose nonsynonymous mutations and down-regulation may result in scarce and short trichomes mutation on carrot stems in sst.

Pollen morphology and trichome types of Betula spp. in the Hyrcanian forests of northern Iran

Pollen and trichome morphology of the genus Betula were evaluated from three main habitats in Iran using light and electron microscopy.Pollen types were identical and tri-porate;however,in terms of quantitative data,some differences were observed in exine(outside)wall thickness,length of polar axis and length of equatorial axis,possibly due to the environment.Pollen alone is insufficient to separate species of Iranian birch.Investigation of the trichome trait(small hairs or other outgrowths)revealed that birch species in Iran belong to:(1)Betula litwinowii Doluch.according to the low density of trichomes on the adaxial surface of the leaves and the lack of trichomes on the abaxial surface and many trichomes on the petioles;(2)Betula pendula Roth.due to the absence of trichomes on the adaxial surface and the low density of trichomes on the abaxial surface;and,(3)in the adaxial and abaxial surfaces of leaves and petioles,the trichomes had different types of pilose(long,soft hairs),and this strengthens the possibility of a different species or more likely a hybrid of B.pendula.

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

Rosa sterilis S.D.Shi is an important economic tree in China that produces fruits with high nutritional and medicinal value.Many of R.sterills’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(GA_(3)),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.

New Allele of HL6 Regulates Trichome Elongation in Rice

Trichomes are specialized epidermal cells that plav multiple roles in plant development.However,knowledge about the molecular mechanism of trichome development in rice is limited.In this study,a hairy leaf locus HL6SWWR of rice variety Suwangwanger(SWWR)was identified by map-based cloning.Transgenic complementation and knock-out mutation demonstrated that HL6SWWR regulated trichomes on leaves and expression levels of HL6SWWR affected trichome elongation.Transgenic experiments of PROswwr:HL6nipand PROnip:HL6swwr indicated that the promoter and coding sequences of HL6swwr were indispensable for trichome regulation.Sequencing alignment of the promoter and coding regions of HL6 in 22 varieties showed that some c/s-binding elements in the promoter region may be related to trichome development,while no decisive variation was found in the coding sequence.Furthermore,RNA-sequencing analysis revealed that 1415 differential expression genes(DEGs)were detected in hl6SWWR mutant vs wild type and that 1010 DEGs were found in HL6SWWR complementary transgenic line vs wild type.Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway analysis showed that most of the DEGs were involved in metabolite pathway,secondary metabolite biosynthesis,plant pathogen interaction and phytohormone signal transduction in both the two groups.The results indicated that these enhanced pathways are critical during trichome development in rice.Taken together,our results provided new views into the regulatory mechanism of trichome formation in rice.

Localization of a defensive volatile 4-hydroxy-4-methylpentan-2-one in the capitate glandular trichomes of Oenothera glazioviana

Glandular trichomes of plants produce a wide variety of secondary metabolites which are considered as major defensive chemicals. The capitate glandular trichomes of Oenothera glazioviana(Onagraceae) were collected with laser microdissection and analyzed by gas chromatography-mass spectrometry. The volatile compound 4-hydroxy-4-methylpentan-2-one(1) was identified. We found that compound 1 displays antimicrobial, insecticidal, and phytotoxic activities. These results suggest that compound 1 might function as a defensive compound in the capitate glandular trichomes of O. glazioviana against pathogens, insect herbivores, and presumably competitive plants as well.

Cloning and Expression Analysis of <i>TTG</i>1 Gene Related to <i>Rosa rugosa</i>Trichomes Formation

The TTG1 transcription factor plays an important role in the formation of plant trichomes. Based on the R. rugosa transcriptome data, this study cloned a R. rugosa TTG1 gene, named RrTTG1, and carried out bioinformatics analysis and fluorescence quantitative analysis to explore the relationship between TTG1 gene and R. rugosa trichomes formation, in order to lay a good foundation to cultivate a thornless plant in the family Rosaceae. In this experiment, six hybrid cultivars of R. rugosa “Zizhi”, R. rugosa “Xizi”, R. rugosa “Tang fen”, R. rugosa “Hun chun”, R. rugosa “Zi long wo chi” and R. rugosa “Tian e huang” were used as experimental materials, and the cDNA full length of this gene was obtained by RT-PCR and RACE, and the full length of the cDNA was 1348 bp. After bioinformatics analysis, it is predicted that its molecular formula is C1723H2661N465O529S12, the molecular weight is 38.71 KB, and the isoelectric point is 5.00. Its instability index is 54.30, which belongs to unstable protein;and its hydrophilic amino acid distribution is relatively uniform, and the amount is larger than hydrophobic amino acid, which belongs to hydrophilic protein. Phylogenetic tree was constructed for the TTG1 gene. Evolutionary analysis indicated that RrTTG1 is closely related to the TTG1 protein of Rosaceae family, and has a close relationship with other families. The expression analysis showed that the expression of RrTTG1 protein was negatively correlated with the trichome content of R. rugosa stems and leaves. The expression levels of the three spiny varieties of R. rugosa “Hun chun”, R. rugosa “Xizi” and R. rugosa “Zi long wo chi” were lower, and the expressions of the three less thorn varieties of R. rugosa “Zizhi”, R. rugosa “Tian e huang” and R. rugosa “Tang fen” were higher. According to the above results, it was speculated that RrTTG1 is involved in the synthesis of R. rugosa trichomes and belongs to the negative regulation mechanism.

Silicon Deposition in Leaf Trichomes of Cucurbitaceae Horticultural Plants: A Short Report

Silicon deposition in leaf trichome of six horticultural Cucurbitaceae species, cucumber (Cucumis sativus), pumpkin (Cucurbita maxima), melon (Cucumis melo), watermelon (Citrullus lanatus), sponge gourd (Luffa cylindrica) and bottle gourd (Lagenaria siceraria var. hispida) was observed by an X-ray microanalyzer coupled with an environmental scanning electron microscope. The elements that presented in the surface of three or four leaves of the individual species were detected and mapped by the X-ray microanalyzer. In leaves of cucumber, pumpkin, and melon, high accumulation of silicon was detected in cells surrounding the bases of the trichome hair and the hair itself deposited calcium. On the other hand, in sponge gourd and bottle gourd, high accumulation of silicon was detected only in the hair. In watermelon leaves, silicon deposited both in the hair and in cells surrounding the bases of the hair. Thus, horticultural Cucurbitaceae plants have interspecific variation in the pattern of silicon deposition in leaf trichomes.

Leaf Traits and Histochemistry of Trichomes of Conocarpus lancifolius a Combretaceae in Semi-Arid Conditions

Leaf traits, structure and water status of Conocarpus lancifolius, a Combretaceae were investigated under semi-arid conditions. The leaf traits examined included leaf area and thickness, stomatal distribution, sclerophylly, succulence and relative water content. Additionally, the types of secretory structures, histochemistry of trichomes, and chemical nature of the cuticlular waxes were evaluated. Leaves showed xerophytic characteristics including a high degree of sclerophylly, thick cuticle and outer epidermal cell wall, low relative water content and high trichome density on younger leaves. The species has two types of trichomes;a secretory, short-stalked capitate trichome and a non-secretory trichome with a bulbous base and a pointed tip. The leaves also have a pair of extrafloral nectaries on both sides of the distal end of the petiole, 3-4 pairs near the leaf apex and two secretory ducts or cavities on mature leaves that secreted polysaccharides, epicuticlar waxes and polyphenols. Compared to young leaves mature leaves had almost 3 times total cuticular wax deposit or load. The most abundant fatty acids were palmitic, stearic, nondecanoic, behenic and arachidic acids. The leaf traits and structures are discussed in relation to semi-arid habitat.

A Systematic Revision of the Genus Plectranthus L. (Lamiaceae) in Saudi Arabia Based on Morphological, Palynological, and Micromorphological Characters of Trichomes

This study aims to investigate the morphology and ultrastructure characters of pollen grains and trichomes in order to evaluate their systematic value of these characters in specific and intraspecific separation of the Saudi Arabian Plectranthus species. A critical systematic revision of 7 species of Plectranthus (Lamiaceae) in Saudi Arabia was conducted by means of numerical analyses based on thirty-one morphological characters, including vegetative parts, seeds, pollen grains, and trichomes. Macro- and micro-morphological characters, including seed and pollen shape, size, coat sculpture, trichome structure, were studied. It reveals the presence of seven species, including two endemic species. The pollen grains were zonocolpate, hexacolpate, prolate to subprolate. Three types of exine ornamentation were recognized. Also, two trichome types could be distinguished and classified into glandular and non-glandular. The glandular trichomes could be distinguished as peltate, capitate and digitiform. The eglandular trichomes were single, uniseriate, multicellular. Pollen and trichome characters were found to be valuable, while seed characters presented only minor taxonomic value. On the basis of UPGMA clustering analysis four branches and clusters were distinguished. The results offer useful data for evaluating the taxonomy of Plectranthus both at subgeneric and sectional levels. Our results indicated some degree of similarity among the species of subgenus Burnatastrum. Plectranthus arabicus is considered as a separate group and may be treated as separate subgenus. Furthermore, the endemic species Plectranthus asirensis and Plectranthus hijazensis constitute a monophyletic group and there are close relationships between this group and Plectranthus tenuiflorus. A key for the identification of the investigated taxa based on studied characters is provided.

Profiles of the Headspace Volatile Organic and Essential Oil Compounds from the Tunisian Cardaria draba (L.) Desv. and Its Leaf and Stem Epidermal Micromorphology

In this work, we investigated aroma volatiles emanated by dry roots, stems, leaves, flowers, and fruits of Cardariadraba (L.) Desv. growing wild in Tunisia and its aerial part essential oils (EOs) composition. A total of 37 volatileorganic compounds (96.7%–98.9%) were identified;4 esters, 4 alcohols, 7 hydrocarbons, 12 aldehydes, 5 ketones,1 lactone, 1 organosulfur compound, 2 organonitrogen compounds, and 1 acid. The hydrocarbons form the maingroup, representing 49.5%–84.6% of the total detected volatiles. The main constituent was 2,2,4,6,6-pentamethylheptane(44.5%–76.2%) reaching the highest relative percentages. Forty-two compounds were determined in thetwo fractions of EOs, representing 98.8% and 97.2% of the total oil composition, respectively. The principal componentswere hexadecanoic acid (34.6%), 6-methyl-5-hepten-2-one (18.3%), decanal (15.0%), 6,10,14-trimethyl-2-pentadecanone (13.2%), and n-pentacosane (13%). Micromorphological details of the leaf and stem epidermisusing light microscopy revealed polygonal cells with sinuous walls in the adaxial and abaxial leaf surfaces andnearly rectangular and long ones with linear and thick walls for the stem epidermis. The stomata complexes wereanisocytic in the leaf epidermis and mainly anisocytic and rarely paracytic in the stem epidermis. Non-glandulartrichomes were unbranched and long with an acute apex or short with a convex apex. The glandular ones wereidentified for the first time in this species. They were short-stalked with a large secretory head. The highest stomatalindex (17.02%) was recorded in the abaxial leaf surface. The identification of headspace volatiles and essentialoil compounds can be used to characterize this species, and the various epidermis micromorphologicalfeatures are very useful for biosystematics taxonomic studies within Brassicaceae.

TRICHOMEAND ARTEMISININ REGULATOR 1 Is Required for Trichome Development and Artemisinin Biosynthesis in Artemisia annua

Trichomes, small protrusions on the surface of many plant species, can produce and store various secondary metabolic products. Artemisinin, the most famous and potent medicine for malaria, is synthesized, stored, and secreted by Artemisia annua trichomes. However, the molecular basis regulating the biosynthesis of artemisinin and the development of trichomes in A. annua remains poorly understood. Here, we report that an AP2 transcription factor, TRICHOME AND ARTEMISININ REGULATOR 1 （TAR1）, plays crucial roles in regulating the development of trichomes and the biosynthesis of artemisinin in A. annua. TAR1, which encodes a protein specially located in the nucleus, is mainly expressed in young leaves, flower buds, and some trichomes. In TAR1-RNAi lines, the morphology of trichomes and the composition of cuticular wax were altered, and the artemisinin content was dramatically reduced, which could be significantly increased by TAR1 oeverexpression. Expression levels of several key genes that are involved in artemisinin biosynthesis were altered when TAR1 was silenced or overexpressed. By the electrophoretic mobility shift, yeast one-hybrid and transient transformation β-glucuronidase assays, we showed that ADS and CYP71AV1, two key genes in the biosynthesis pathway of artemisinin, are likely the direct targets of TAR1. Taken together, our results indicate that TAR1 is a key component of the molecular network regulating trichome development and artemisinin biosynthesis in A. annua.

Hairy Leaf 6, an AP2/ERF Transcription Factor, Interacts with OsWOX3B and Regulates Trichome Formation in Rice

Trichome formation has been extensively studied as a mechanistic model for epidermal cell differentiation and cell morphogenesis in plants. However, the genetic and molecular mechanisms underlying trichome formation （i.e., initiation and elongation） in rice remain largely unclear. Here, we report an AP2/ERF transcription factor, Hairy Leaf 6 （HL6）, which controls trichome formation in rice. Functional analyses revealed that HL6 transcriptionally regulates trichome elongation in rice, which is dependent on functional OsWOX3B, a homeodomain-containing protein that acts as a key regulator in trichome initiation. Biochemical and molecular genetic analyses demonstrated that HL6 physically interacts with OsWOX3B, and both of them regulate the expression of some auxin-related genes during trichome formation, in which OsWOX3B likely enhances the binding ability of HL6 with one of its direct target gene, OsYUCCA5. Popu- lation genetic analysis indicated that HL6 was under negative selection during rice domestication. Taken together, our findings provide new insights into the molecular regulatory network of trichome formation in rice.

Role of trichome of Pteris vittata L. in arsenic hyperaccu-mulation

Environmental scanning electron microscope (ESEM) fitted with an energy disper- sive X-ray microanalyzer (EDX) was used to investigate the surface micromorphology and arse- nic (As) micro-distribution in Chinese brake (Pteris vittata L.). It was found that amounts of trichome, which possessed multicellular structure with the average length of 160 μm and with an average diameter of 28 μm, existed in the frond of P. vittata, and the density of trichome on the pinnate axial surface was higher than that on the petiole. Visible X-ray peak of As was recorded in the epidermal cell and trichome. The relative weight of As in the pinnate trichome, which con- tained the highest concentration of As among all tissues of the plant, was 2.4 and 3.9 times as much as that in the epidermal and mesophyllous cells, respectively. The As concentrations in the basal and stalk cells of the same trichome were higher than that in its cap cell. This is the first time to report that the trichome of P. vittata plays an important role in arsenic hyperaccumulation. The finding from the present study implies that much attention should be paid to the role of the trichome in understanding the hyperaccumulation and detoxicity of As in the hyperaccumulator and improving the ability of As accumulation.

Micro-trichome as a class I homeodomain-leucine zipper gene regulates multicellular trichome development in Cucumis sativus

Plant trichomes serve as a highly suitable model for investigating cell differentiation at the single-cell level. The regulatory genes involved in unicellular trichome develop- ment in Arabidopsis thaliana have been intensively studied, but genes regulating multicellular trichome development in plants remain unclear. Here, we characterized Cucumis sativus （cucumber） trichomes as representative multicellular and unbranched structures, and identified Micro-trichome （Mict）, using map-based cloning in an F2 segregating population of 7,936 individuals generated from a spontaneous mict mutant. In mitt plants, trichomes in both leaves and fruits, are small, poorly developed, and denser than in the wild type. Sequence analysis revealed that a 2,649-bp genomic deletion, spanning the first and second exons, occurred in a plant-specific class I homeodomain-leucine zipper gene. Tissue-specific expression analysis indicated that Mict is strongly expressed in the trichome cells. Transcriptome profiling identified potential targets of Mict including putative homologs of genes known in other systems to regulate trichome development, meristem determinacy, and hormone responsiveness. Phylo- genic analysis charted the relationships among putative homologs in angiosperms. Our paper represents initial steps toward understanding the development of multicellular trichomes.

SAD2 in Arabidopsis Functions in Trichome Initiation through Mediating GL3 Function and Regulating GL1,TTG1 and GL2 Expression

Most genes identified that control Arabidopsis trichome initiation and formation are transcription factors or regulatory components in transcriptional networks and include GLABROUS1 （GL1）, GLABRA2 （GL2）, GLABRA3 （GL3） and TRANSPARENT TESTA GLABRA1 （TTG1）. Herein, we report that an importin β-like protein, SENSITIVE TO ABA AND DROUGHT2 （SAD2）, is required for trichome initiation. Mutations in SAD2 disrupted trichome initiation resulting in reduced trichome number, but had no effect on trichome development or root hair number and development. Expression levels of GL1, MYB23, GL2 and TTG1 were reduced in shoots of sad2 mutants while expression levels of GL3 and ENHANCER OF GLABRA3 （EGL3） were enhanced. Overexpression of GL3 increased trichome numbers in wild type but not in sad2 mutants, indicating that the function of the GL3 protein is altered in the sad2 mutants. In contrast, overexpression of GFP-GL1 decreased trichome number in both wild type and sad2. Double mutant analysis of gll sad2 and g13 sad2 indicated that SAD2 functions genetically, at least in part, in the same pathway with these two genes. Co-immunoprecipitation indicated that the sad2 mutation does not disrupt formation of the TTG1-GL3-GL1 complex. Analysis of GFP fusions of GL1, GL2, GL3 and TTG1 suggested that these proteins are most likely not direct cargo of SAD2. Our data suggest that SAD2 is involved in trichome initiation by regulating these nuclear genes.