Development 126, 671-682 (1999) 671 Printed in Great Britain © The Company of Biologists Limited 1999 DEV3883

Heterologous myb genes distinct from GL1 enhance trichome production when overexpressed in Nicotiana tabacum

Thomas Payne, John Clement, David Arnold and Alan Lloyd* Department of Botany, and Institute for Cellular and Molecular Biology, University of Texas at Austin, Austin, TX 78713, USA *Author for correspondence (e-mail: [email protected])

Accepted 20 November 1998; published on WWW 20 January 1999

SUMMARY

Myb-class transcription factors implicated in cell shape In addition, floral papillae were converted to multicellular regulation were overexpressed in Arabidopsis thaliana and trichomes. CotMYBA, a myb gene which is expressed in Nicotiana tabacum in an attempt to assess the extent to Gossypium hirsutum ovules and has some homology to which cellular differentiation programs might be shared MIXTA, was also overexpressed in the two species. A between these distantly related plants. GLABROUS 1, a similar but distinct syndrome of abnormalities, including myb gene required for trichome development in the production of cotyledonary trichomes, was observed in Arabidopsis, did not alter the trichome phenotype of the 35S:CotMYBA tobacco transformants. However, CotMYBA tobacco plants in which it was overexpressed. MIXTA, did not alter trichome production in Arabidopsis. These which in Antirrhinum majus is reported to regulate certain results suggest that the trichomes of Arabidopsis and aspects of floral papillae development, did not complement Nicotiana are merely analogous structures, and that the the glabrous 1 mutant of Arabidopsis. However, 35S:MIXTA myb genes regulating their differentiation are specific and transformants of N. tabacum displayed various separate. developmental abnormalities, most strikingly production of supernumerary trichomes on cotyledons, leaves and stems. Key words: CotMYBA, MIXTA, Trichomes, Nicotiana tabacum

INTRODUCTION and the order of action in the differentiation pathway of the genes so identified has been deduced by epistatic analysis Trichomes are specialized structures which originate and (Hülskamp et al., 1994). project from the above-ground epidermal tissues of most Wild-type Arabidopsis leaf trichomes are unicellular, plants. A vast array of morphologies occur in addition to the typically have three branches, and lack glands. Progression hair-like shape suggested by the term. Trichomes may be through the stages of leaf trichome morphogenesis in this composed of one or more cells, may be unbranched or species is correlated with overall cell enlargement and branched, and may or may not possess glands which endoreduplicative DNA increase. Three rounds of endomitosis accumulate or secrete alkaloids like nicotine, terpenoids such occur initially, concommitant with extension growth from the as menthol and camphor, or other compounds repellent or toxic epidermal surface. A fourth round of endomitosis occurs after to phytophagous insects (Johnson, 1975; Levin, 1973; primary branching of the developing trichome in a Rodriguez et al., 1984). Different trichome types may occur on proximodistal orientation to the leaf primordium surface has the same plant of a given species, with type and spacing begun. Subsequently the distal branch itself branches, and the determined by the plant organ or organ surface (ie, adaxial vs. nucleus migrates to the branching point from its previous abaxial, leaf vs. stem) from which the trichome extends, and position below it. Trichome cells are regularly spaced in the the developmental phase of the plant at the time of organ mature Arabidopsis leaf. Initiation and maturation of trichomes initiation. proceeds in a generally basipetal direction over the leaf The trichome is a useful cell type for studying regulation of primordium, but additional trichomes may be initiated between cell differentiation in plants. It is morphologically distinct from mature trichome cells as organ growth continues (Hülskamp et surrounding epidermal cells, and its genesis and development al., 1994). from this population are readily observed due to its Mutations at either of two loci, TRANSPARENT TESTA accessibility to visual inspection and its relatively large size. GLABRA 1 (TTG1) or GLABROUS 1 (GL1), result in the In the laboratory, trichomes are non-essential and thus can be virtual absence of leaf trichomes from A. thaliana. TTG1 is genetically manipulated without a reduction in plant viability. thought to have roles in trichome precursor selection, trichome A number of mutants lacking or producing fewer, aberrant, or initiation, and in a nearest-neighbor inhibition of initiation in mis-positioned trichomes have been isolated from Arabidopsis, surrounding protodermal cells. Mutations at this locus have 672 T. Payne and others pleiotropic effects: in addition to the absence of trichomes, but retain other characters normally associated with the homozygous mutants fail to produce anthocyanin pigments and conical cell type (pentagonal symmetry, striations), indicating seed coat mucilage (Koorneef, 1981), and, conversely, produce that the role of the MIXTA gene in this differentiation pathway supernumerary root hairs on root epidermal cells that are is specific and late. Recently, Glover et al. (1998) have normally hairless (Galway et al., 1994). Previously, it was published a characterization of the MIXTA overexpression found that overexpression of the maize R gene (a myc- phenotype in Nicotiana tabacum. The data presented herein homologous transcription factor which regulates anthocyanin are in good agreement with their results which indicate that synthesis in that species) under control of the CaMV 35S the transgene can also regulate trichome differentiation in promoter could suppress the various phenotypes associated tobacco. with the ttg1-1 mutant. In wild-type Arabidopsis Suppression of the pleiotropic Arabidopsis ttg1-1 mutant overexpression of R leads to production of extra trichomes on by overexpression of the R gene from maize, a monocot, leaves and stems, and in some transformants to trichomes on dramatically demonstrated the potential utility of organs which normally lack them, such as petals, stamens and heterologous gene expression as a tool for manipulating and pistils (Lloyd et al., 1992). TTG1 has recently been cloned and studying the regulation of cellular differentiation processes in the gene is reported to encode a protein with WD-40 repeats plants. Such an approach necessarily assumes that the and no myc homology (Walker et al., 1997). Presumably, TTG1 regulatory mechanisms of similar pathways and regulates or acts in concert with the activity of a myc-class developmental programs have been at least partly conserved transcription factor or factors. We have recently identified two over evolutionary time. At the outset of the present study we endogenous Arabidopsis myc-class genes. When constitutively hypothesized that this conservation would be reflected by expressed in transgenic plants both suppress to varying extents heterologous functioning of known epidermal cell shape the trichome and other defects of the ttg1-1 mutation, albeit regulators. Herein we describe an unsuccessful attempt to less dramatically than R (Lloyd et al., unpublished). replace the trichome-initiating function of the Arabidopsis Both GL1 and TTG 1 are necessary for normal Arabidopsis GL1 gene by overexpression of MIXTA, a myb-class cell trichome initiation. The GL1 gene was cloned by T-DNA shape regulator from another dicotyledonous plant, tagging (Marks and Feldmann, 1989). It encodes a putative Antirrhinum, and CotMYBA, a closely related myb-class gene transcription factor with myb-type DNA binding and C- from cotton. We further describe the MIXTA overexpression terminal acidic activation domains (Oppenheimer et al., 1991). phenotype in Nicotiana tabacum, which, in contrast to our By itself GL1 is not sufficient to initiate trichomes in results with Arabidopsis, includes the production of Arabidopsis. Overexpression from a 35S:GL1 construct does supernumerary trichomes. Overexpression of CotMYBA in not suppress the ttg1-1 mutation, but it does lead to the Nicotiana also activates trichome initiation, additionally production of an occasional supernumerary trichome on the amplifying a distinct class of multicellular trichomes. Since cotyledons of wild-type transformants. In fact, a reduction in overexpression of either GL1 or R has no effect on trichome leaf trichome number is observed, and this paradoxical result initiation in Nicotiana tabacum, our data indicate that the has been attributed to a squelching phenomenon (Larkin et al., trichomes of Arabidopsis and Nicotiana are not homologous 1994) akin to that described by Ptashne and Gann (1990). structures and that their differentiation is controlled by Little is known about the molecular basis of trichome distinct regulatory genes. Furthermore, the development of differentiation in other species, although the inheritance of papillate cells and certain classes of trichomes in Nicotiana indumental characters correlated with pest resistance has been appears to be controlled by the same regulatory pathway studied in some crop plants such as cotton (Stephens and Lee, while separate myb-elements can differentially regulate the 1961; Wannamaker, 1957), soybean (Broersma et al., 1972), cell-fate decision to produce separate, partially overlapping and tomato (Gentile et al., 1969; Goffreda et al., 1990). In his subsets of trichomes. 1954 monograph, Goodspeed recognized five basic morphological classes of trichomes variously distributed within the genus Nicotiana, and assessed their transmission to MATERIALS AND METHODS F1 interspecific hybrids and naturally occurring amphidiploids. Such experiments are difficult to interpret, but differences in Plant strains and growth conditions the inheritance of trichome types, especially in regard to organ Arabidopsis thaliana. pMXCD, pCMAX41 and pLBJ4 constructs distribution and degree of structural elaboration, were were used to transform the wild-type WS ecotype, the ttg1-1 mutant documented. Such differences are likely to be indicative of in Landsberg erecta outcrossed to WS one time, and the gl1-1 mutant separate regulatory mechanisms for the various trichome of ecotype Columbia. The R gene was previously overexpressed in the classes. wild-type WS, wild-type RLD, and in the Landsberg erecta ttg1-1 Noda et al. (1994) have cloned the gene encoding another mutant outcrossed to either WS or RLD one time (Lloyd et al., 1992). myb-class transcription factor, MIXTA, from a Tam4-tagged Plants were potted in Sunshine Mix no. 1 (Premier Horticulture Inc.) allele of Antirrhinum majus. MIXTA appears to be a positive and grown under continuous fluorescent illumination at 22°C. regulator of a directional cell wall synthesis which results in Nicotiana tabacum. All constructs were used to transform tobacco the conical-papillate shape of epidermal cells on wild-type introduction TI1347 ‘Praecox’ pMXCD was also transformed into strain NC744. Both were supplied by the US Tobacco Germplasm adaxial petal surfaces. This cell shape is thought to affect the Collection. Previously, R and C1 overexpression phenotypes were also optical properties of the petal epidermis such that incident evaluated in the ‘Xanthi’ cultivar. Potting mix consisted of a 2:1:1 light passes through the pigmented cell before it is reflected, mixture of Baccto, Sunshine no. 1, and vermiculite. Plants were grown and thus perceived intensity of pigmentation is enhanced. in a warm greenhouse with supplemental fluorescent light to achieve Epidermal cells of mixta mutant petals are flat by comparison, 16-hour days. Myb genes enhance tobacco trichome production 673

Vector construction per step. Specimens were critical point dried in a Tousimis Samdri- pLBJ4. The oligonucleotide GL1-C (5′-GGGGGGGGCTGCAGA- 790 and sputter coated with a gold-palladium alloy using a Ladd TTAAACTAAAGGCAGTACTC-3′) was used to prime reverse instrument. Specimens were visualized with a Phillips 515 scanning transcription of the GL1 cDNA from Col-0 RNA. The cDNA was electron microscope and photographed with Polaroid film. amplified by PCR using the primers GL1-C and GL1-A (5′-GGGG- GGGGGAATTCATGAGAATAAGGAGAAGAGATG-3′), and then Plant sectioning and light microscopy cloned into pBluescript II SK− (Stratagene) as an EcoRI-PstI Tissue samples were fixed and stained as described by Mauseth et al. fragment to create pSRV2. The ends of the insert from pSRV2 were (1984). Sections were photographed through an Olympus BX60 sequenced and subsequently cloned into the binary plant vector microscope with an Olympus PM-C35DX camera. pKYLX71 (Schardl et al., 1987) as a HindIII-XbaI fragment. The gl1- 1 mutation has been complemented by transformation of this construct Image processing into Arabidopsis (Lloyd et al., unpublished). Images for figures were scanned with a Sharp JX325 High Resolution pMXCD. The MIXTA cDNA was cloned by ligating a HindIII-SacI Color Scanner. Figures were constructed using Adobe Photoshop 4.0. restriction fragment from the pUC-18-derived plasmid pJAM980 Colors were corrected to match the living specimens and adjustments (Glover et al., 1998) into pKYLX71. in brightness and contrast were made using this program. Images were pMMKY-1. Oligonucleotides MXM5 (5′-GGAAGCTTGAAT- printed with a FUJIX Pictrography 3000 printer. TCATGGTAAAAAAAGGGCCATGGACTG-3′) and MXM3 (5′- GGGAGCTCGTCGACCTAACGCTTCTTTAGATGAGTGTTCCAG- 3′) were used to amplify the first 356 nucleotides of MIXTA from RESULTS pJAM980. MXM3 incorporates a stop codon after the arginine residue which terminates the R3 myb repeat. The PCR product was cloned Overexpression of MIXTA in Arabidopsis thaliana into pBluescript II KS+ (Stratagene) as a SacI-HindIII fragment to create pMXM1-1. The pMXM1-1 insert was completely sequenced does not complement the gl1-1 mutation from M13 forward and reverse primers, and then subcloned into Given the structural similarities between GL1 and MIXTA and pKYLX71 (Schardl et al., 1987) as a SacI-HindIII fragment. their roles in the control of epidermal cell differentiation, we pCMAX41. The CotMYBA gene was amplified by PCR from hypothesized that MIXTA might be able to substitute for the genomic Gossypium hirsutum cv. Texas Marker-1 DNA using the function of the Arabidopsis trichome regulator. The MIXTA primers COTMYBA-5 (5′-GCGAATTCCTCGAGCCCATGGGAC- cDNA was placed under the transcriptional control of the GATCACGTTGT-3′) and COTMYBA-3 (5′-GCGGATCCTCTAG- ′ CaMV 35S promoter in a binary vector and used to transform ACCTATGAATCCAAGGGTCTAC-3 ). After digestion with XbaI by means of Agrobacterium tumefaciens wild-type Arabidopsis and XhoI, the PCR product was ligated into pBluescript II KS+ (Stratagene) to create pBSCMAX4. The ends of this insert were and the gl1-1 and ttg1-1 mutants. No differences in trichome sequenced and then the insert was subcloned into pKYLX71 as an production or other epidermal characters could be discerned. XhoI to XbaI restriction fragment. Transcription of the transgene was verified by northern blot pCMANX2. This overexpression construct contains an intronless analysis (data not shown). version of CotMYBA amplified by RT-PCR from a pCMAX41- Since the MIXTA gene was isolated from Antirrhinum majus transformed TI1347 line using the COTMYBA-5 and -3 primers based on its effects on floral papillation, we also examined the described above. The PCR product was cloned into pBluescript II petals of pMXCD transformants. Scanning electron KS+ (Stratagene) as an XbaI to XhoI fragment to create pCMP2. The micrographs of petal epidermis (data not shown) revealed no insert was completely sequenced and compared to the GenBank differences between 35S:MIXTA and untransformed plants of cDNA accession to verify correct splicing. The cDNA insert from any of the three genotypes. No published transformation pCMP2 was then subcloned into pKYLX71 as an XhoI to XbaI fragment. TI1347 plants transformed with pCMANX2 displayed the protocols for Antirrhinum exist, and the reciprocal experiment, same range of developmental abnormalities seen in pCMAX41 overexpression of GL1 in that species, was not attempted. transformants (data not shown). The construction of pAL144, 35S:R, and pAL71, 35S:C1, is Mixta overexpression in Nicotiana tabacum results described elsewhere (Lloyd et al., 1992). in supernumerary trichome production Like Antirrhinum majus and unlike Arabidopsis thaliana, Plant transformation Nicotiana tabacum produces flowers pigmented by Binary constructs were introduced into Agrobacterium tumefaciens anthocyanins. Concurrent with the Arabidopsis experiment, we strain GV3101 containing pMP90 (Koncz and Schell, 1986) by transformed N. tabacum with pMXCD to evaluate the effects electroporation. Arabidopsis was transformed using the protocol of Valvekens et al. (1988), except in the case of the pCMANX2 of MIXTA overexpression on floral pigmentation. Noda et al. construct, which was vacuum infiltrated essentially as described by (1994) have previously shown that in Antirrhinum the gene Bechtold et al. (1993). Nicotiana tabacum transformation was exerts its effects on floral phenotype by altering epidermal cell accomplished essentially as described by Horsch et al. (1985), except shape and thus perception of pigmentation rather than quantity that explants were derived from hypocotyls of one-week-old of pigment. One floral phenotype of 35S:MIXTA transformants seedlings. With the exception of pMMKY, a minimum of fifteen to is shown in Fig. 1D. Several other transformants produced twenty-five independent transformants were produced for each mottled pink flowers different from the one shown. The adaxial construct. Where overexpression phenotypes were not observed, floral epidermis of the transformants was examined by transcription of the transgenes was verified for subsets of scanning electron microscopy. The SEM shown in Fig. 1H is transformants using either northern blot analysis or RT-PCR. from the same transformant that produced the small flower in Scanning electron microscopy Fig. 1D. Remarkably, many of the papillae have elongated to Plant materials were fixed overnight in 2% glutaraldehyde and 0.1 M produce what appear to be multicellular, uniseriate trichomes. cacodylate, then taken through an alcohol dehydration series, once in These results are similar to those presented by Glover et al., 25, 50, 75, 85, 95% and twice in 100% ethanol, for at least 2 hours (1998). For comparison, both a flower and floral epidermis 674 T. Payne and others from untransformed TI1347 are shown in Fig. 1A,E, (Fig. 1L). Hypocotyls and true leaves also produce respectively. The red flower in Fig. 1B is from a plant supernumerary trichomes. These same seedlings were fixed, overexpressing the maize R gene. Note that R overexpression sectioned, and stained for light microscopy as above. up-regulates anthocyanin production in flowers but does not Longitudinal sections through untransformed TI1347 and alter floral papillation (Fig. 1F). 35S:MIXTA seedling cotyledons are compared in Fig. 3A and Effects of MIXTA overexpression in tobacco were, however, B, respectively. Note that the 35S:MIXTA cotyledon is smaller visible prior to flowering. After transfer to soil, rooted shoots than the untransformed cotyledon; only the distal portion of the began to produce abnormal concave leaves with a spoon-like latter is shown. In general, cell expansion appears to be reduced appearance. This leaf curvature was reversed in later leaves, in 35S:MIXTA cotyledons, perhaps due to premature cell wall which were often convex. Leaf blades also showed a thickening. The papillate epidermis (both adaxial and abaxial) characteristic variegation, dark green tissue associated with is visible in Fig. 3B; associated debris is derived from veins against pale green ground tissue, the latter becoming trichomes projecting across the plane of the section. increasingly yellow as the leaves aged. The leaf epidermis in 35S:MIXTA root phenotypes have not been extensively the vein-associated dark green areas possessed a sheen which characterized, but a pronounced tendency toward reduced was absent from the rough-looking epidermis of the lighter elongation has been documented and increased cell wall green areas. This variegation and its corresponding uneven thickening is suggested by Fig. 4B and D, as compared witho pattern of reflectivity are seen in a close-up photograph of the A and C respectively. This is in contrast to the findings of adaxial leaf blade in Fig. 2B. To determine whether this Glover et al. (1998), who note that they were unable to variegation might be due to sub-epidermal cell shape changes, correlate a root phenotype with MIXTA overexpression. thick sections (25 µm) through adult leaves were sputter coated When GL1 was overexpressed in Nicotiana tabacum the and examined by SEM, data not shown. No gross differences phenotype of transgenic plants was indistinguishable from wild in sub-epidermal morphology or wall thickness judged to be type (compare Fig. 1C, G and K, with A, E, and I). sufficient to account for the variegation were seen (data not Transcription of the 35S:GL1 transgene was confirmed by RT- shown). Thinner sections (10 µm) from the same specimens PCR (data not shown). This result is further evidence that GL1 were stained with safranin and counterstained with fast green, and MIXTA have distinct regulatory activities. In addition, R then examined by light microscopy. Adaxial epidermis with a was not observed to affect cotyledonary or leaf trichome pale, rough green appearance was primarily composed of development (Fig. 1,J). papillate cells and trichomes, whereas dark green areas were composed of mostly rounded cells and occasional trichomes The myb gene CotMYBA affects trichome like those seen in the leaf epidermis of untransformed controls. differentiation when overexpressed in Nicotiana This differential pattern of trichome occurrence and papillation tabacum was subsequently confirmed in SEMs of adaxial leaf epidermis A Blast search (Altschul et al., 1990) of the National Center for (Fig. 2C-G). Leaves and petals are presumed to be Biotechnology Information database using the complete homologous structures (Esau, 1977), so it is perhaps not Antirrhinum MIXTA cDNA sequence as a query, calls up a long surprising that MIXTA overexpression alters the epidermal characters of both, but our results are particularly interesting in that they suggest these alterations can affect perception of pigments with absorption spectra as diverse as anthocyanins and chlorophylls. Tobacco plants transformed with the 35S:MIXTA construct were selfed and the resulting T2 seedlings examined by SEM. The cotyledons of N. tabacum are normally devoid of trichomes except at the very base of the petiole (Fig. 1I), but in plants overexpressing MIXTA most of the cells of the cotyledonary epidermis have a papillate appearance, and Fig. 1. Floral and seedling phenotypes of Nicotiana tabacum overexpressing heterologous myc- and myb- many differentiate into class transcription factors. First row: flowers, all the same magnification, flower in A is approximately 2.25 glandular trichomes which are cm wide. Second row: SEMs of adaxial petal epidermis. Scale bar, 0.1 mm. Third row: SEMs of 2-week- either unicellular or old seedlings showing adaxial epidermis of cotyledons (to the left and right) and first 2-3 true leaves. Scale multicelluar and uniseriate bar, 1 mm. (A,E,I) Untransformed TI1347; (B,F,J) 35S:R. (C,G,K), 35S:GL1; (D,H,L), 35S:MIXTA Myb genes enhance tobacco trichome production 675

bordered by elongated, turgid-looking cells. The exaggerated appearance of these border cells is emphasized in Fig. 4E,F. In untransformed seedlings they are similarly discernible as a double layer of transluscent cells at the cotyledonary margin, but lack the turgid appearance of the transformants. The angle of attachment of cotyledons to hypocotyl varies; some seedlings are Y-shaped as in Fig. 4B, with cotyledons remaining at less than 45o relative to the long axis of the hypocotyl, while others assume a normal perpendicular orientation. Interestingly, transformant cotyledon emergence from the seed coat is delayed relative to untransformed seedlings, whereas radicle emergence is not. One of the most striking characteristics of tobacco seedlings overexpressing CotMYBA is “loss” of the cotyledonary petiole. The cotyledonary petioles of untransformed seedlings are narrower than the cotyledonary blade, and epidermal cells are elongate and occur in longitudinally aligned files (see Fig. 4G). In the most severe 35S:CotMYBA phenotypes (Fig. 4D,H) the cotyledonary petiole becomes a wider shoulder region in which tissue bulges out from the plane of the blade, epidermal cells have a rounded appearance, and cell files are difficult to recognize. Like the plants overexpressing Mixta, 35S:CotMYBA plants produce supernumerary epidermal trichomes on cotyledons and other organs. In many of the transformants a preponderance of short-stalked, multicellular “jingle bell” trichomes (corresponding in Goodspeed’s classification scheme to E1, also referred to as hydathodes) like those shown in Fig. 4I, occur. Supernumerary trichomes of this type were not observed in plants overexpressing MIXTA. Whereas 35S:MIXTA cotyledonary trichomes in general appear to be regularly spaced, trichome initiation in 35S:CotMYBA Fig. 2. Textural variegation of Nicotiana tabacum adaxial leaf transformant cotyledons looks more random, at least partly as epidermis. (A) Photograph of untransformed adaxial leaf epidermis. a consequence of the aforementioned epidermal irregularities. The midvein is vertical. (B) 35S:MIXTA leaf. (C-G) SEMs of adaxial To date, only T2 plants with the mildest phenotypes (Fig. leaf epidermis. (C) Untransformed. (D) 35S:MIXTA, from region 4A) have survived transplantation to soil. This is not surprising adjacent to vein, corresponding to reflective areas in (B). given our observations of seedlings allowed to continue growth (E) 35S:MIXTA, from interveinal, rough-looking regions as seen in B. on nutrient medium without hormones for up to one month Scale bar, 0.1 mm. (F) Detail of non-papillate cells seen in D. (Fig. 4J-L). The cotyledons of such seedlings may become (G) Detail of papillate epidermis as in E. Area shown in F and G is 1mm2. extremely chlorotic, whereas cotyledons of untransformed seedlings of the same age maintained on the same medium remain green and healthy. Subsequent true leaves may or may list of myb-homologous plant genes. One of the highest scoring not be green at emergence, but often pale as they age. but presumably non-orthologous sequences (Clement, Payne, Invariably they are distorted to some degree. In the most and Lloyd, unpublished) is a GenBank accession, CotMYBA severely affected seedlings the epidermis coarsens and (L04497), isolated from Gossypium hirsutum 3-day pre-anthesis becomes increasingly disorganized as it ages, even to the point ovules. Cotton fibers are trichomes which are initiated and begin of being callus-like. Bulbous projections from the epidermis elongation from the epidermis of the cotton ovule at about the occur, and these are distinct from observed trichomes (compare time of anthesis (Basra and Malik, 1984). Cotton fiber-specific Fig. 4I, with J and L). The “shoulder” regions of the cotyledons promoters have previously been shown to direct transcription of may become pronounced, flattened projections distinguishable reporter genes in trichomes of N. tabacum (Dang et al., 1996), from young first true leaves by their positions relative to the suggesting that the two trichome types share some ontogenetic rest of the cotyledon (Fig. 4K). similarities. To explore the possibility that CotMYBA is a A section through a one-month-old 35S:CotMYBA positive regulator of trichome differentiation, we overexpressed cotyledon is seen in Fig. 3D. Extreme variation in cell size and the genomic sequence (see Materials and Methods) in tobacco. shape is characteristic. Large cells lack chloroplasts. (The SEMs of tobacco seedlings overexpressing the CotMYBA overall appearance of tissue prior to fixation was chlorotic.) transgene are shown in Fig. 4. Whole 10-day-old T2 seedling Whereas some tissue is collapsed and apparently necrotic, shoots seen in A-D demonstrate the variation in overall there are also isolated pockets of adventitious mitotic activity severity of the phenotype observed in progeny of independent (Fig. 3F compare with Fig 3C and E, untransformed). transformants. In all cases the cotyledons are distorted in A distinctive root phenotype is also associated with comparison to untransformed plants (see Fig. 1I), with many overexpression of CotMYBA in tobacco. As seen in Fig. 5E, surface irregularities. Cotyledonary margins are wavy and 35S:CotMYBA seedling roots produce lollipop-like root hairs 676 T. Payne and others with bulbous tips. Similar structures are occasionally seen on include the production of supernumerary trichomes. These untransformed TI1347 seedling roots, but on 35S:CotMYBA same transgenes failed to alter the trichome phenotype of seedling roots they are much more numerous, and their Arabidopsis. A known regulator of trichome initiation from frequency is directly correlated with severity of the phenotype Arabidopsis, GL1, likewise had no effect on the trichome of the aerial organs. A tendency toward disorganization and complement of Nicotiana tabacum when overexpressed in that uncoordinated cell expansion has also been observed as species. Previous experiments in which the myc-homologous 35S:CotMYBA roots age (data not shown). R gene was overexpressed in the two species suggested that Arabidopsis thaliana was also transformed with the whereas the regulation of the anthocyanin pathway was pCMAX41 construct. Neither of the trichome mutants (ttg1-1, conserved even between monocots and dicots and in both gl1-1) were suppressed. Initial experiments using the Arabidopsis and N. tabacum, the development of trichomes in transformation protocol of Valvekens et al. (1988) resulted in these dicots was regulated differently. Present results a small number of regenerants which were stunted and corroborate this hypothesis by identifying two genes which can produced aberrant flowers. Attempts to secure transgenic seed control trichome cell-fate in Nicotiana but not in Arabidopsis. or correlate floral phenotypes with degree of papillation on petal epidermis were likewise unsuccessful. Limited correlations between myb gene sequence In a recent experiment, vacuum infiltration was used to homology and function are seen transform wild-type Arabidopsis with the pCMANX2 construct, Fig. 7 shows an alignment of the myb DNA binding domains which would allow us to evaluate transgenic cotyledon of the three plant myb-element transcription factors used in the phenotypes even in the absence of fertile transgenics. Cotyledonary abnormalities somewhat comparable to those seen in 35S:CotMYBA tobacco seedlings were observed in approximately one quarter of the V1 transformants, but no cotyledonary trichomes were seen. Of 56 kanamycin- resistant V1 seedlings transplanted to soil, twenty-five survived to flowering. Survival on soil was inversely correlated with severity of the cotyledon phenotype. Among the survivors were six plants (derived from three different infiltration seed lots) which shared a syndrome of developmental abnormalities. These included small concave or convex rosette leaves (Fig. 6A) as well as floral defects like those seen in the earlier transformation experiment. In these plants first- and second-whorl floral organs fail to expand properly, or do so in an uncoordinated fashion. As a consequence, fourth- and (to a lesser extent) third-whorl organs are exposed prematurely (Fig. 6B). To date neither pollen dehiscence nor self-set siliques have been observed, although the plants appear to be fertile when pollinated with wild-type pollen. Further characterization of the 35S:CotMYBA Arabidopsis phenotype is planned for these out-crossed progeny of the surviving abnormal V1 seedlings once sufficient seed stocks have been accumulated.

DISCUSSION Fig. 3. Nicotiana tabacum seedling sections. (A) Untransformed TI1347 cotyledon. We have shown that the independent (B) 35S:MIXTA cotyledon, same age as in A. (C) Longitudinal section of TI1347 seedling shoot apex showing parts of one cotyledon and two young leaves. (D) Longitudinal section overexpression of two myb-class of 35S:CotMYBA seedling showing abnormal cotyledon. (E) Longitudinal section through transcription factors, MIXTA and untransformed TI1347 cotyledon. (F) Detail of 35S:CotMYBA cotyledon seen in D, CotMYBA, significantly perturb showing internal “islands” of small, darkly staining cells surrounded by larger, apparently development of transgenic tobacco plants, quiescent, highly vacuolated cells. Scale bars (A-C,F), 20 µm; (D and E) 50 µm and 10 µm engendering complex phenotypes which respectively. Myb genes enhance tobacco trichome production 677

Fig. 4. SEMs of untransformed and 35S:CotMYBA tobacco seedlings. (A-D) T1 35S:CotMYBA seedlings 10-14 days post-germination, demonstrating variation in severity of phenotype. (Compare with Fig.1I.) (E) Cotyledonary margin of untransformed TI1347 seedling. (F) Cotyledonary margin of 35S:CotMYBA seedling seen in A. (G) Cotyledonary petiole of untransformed TI1347 seedling. (H) Cotyledonary petiole region of 35S:CotMYBA seedling seen in C. (I) Detail of 35S:CotMYBA seedling first true leaf adaxial epidermis showing preponderance of trichomes of Goodspeed types A and E. (J) Detail of one-month-old 35S:CotMYBA seedling showing progressive epidermal distortion; a cotyledon is seen far right. (K) Detail of petiolar/“shoulder” region of another one-month-old 35S:CotMYBA seedling. (L) Close- up of rounded-out epidermal cell from cotyledon of seedling shown in K. Scale bars (A-D,G,H,J,K) 1 mm; (E,F,I,L) 0.1 mm. present overexpression experiments, as well as those of the myb genes recently conducted in this lab (Clement, Payne and maize C1 protein (Cone et al., 1986; Paz-Ares et al. 1987), Lloyd, unpublished) revealed the existence of many previously AtMIXTA, a presumed MIXTA ortholog from Arabidopsis unrecognized conserved motifs outside the myb DNA binding (Rabinowicz et al., 1996), and PhMYB1 from Petunia hybrida, domain, some conserved between monocots and dicots. Since which has now been shown to regulate papillae development a majority of known plant myb genes have been cloned by in that species (Avila et al., 1993; van Houwelingen et al., DNA binding domain sequence homology (for example, Li and 1998). Overexpression of C1 in concert with R results in the Parrish, 1995), gene function is often unknown, making the up-regulation of anthocyanin synthesis in Arabidopsis, but assignment of function to the motifs largely conjectural. overexpression of C1 either alone or together with R does not Heterologous overexpression experiments such as those alter trichome production in tobacco, nor does C1 enhance the described herein, together with sequence analysis, may enable exaggerated trichome phenotype of 35S:R Arabidopsis plants such assignments to be made with greater confidence, (Lloyd et al. 1992). The similarities calculated for the myb especially when the gene in question is derived from a species DNA binding domains of the putative orthologs MIXTA, in which complementable or revertable mutants and AtMIXTA, and PhMYB1 are over 90% while the similarity for transformation/regeneration technology is lacking. any other pair is from 60-68%. Our results suggest a role for A short N-terminal motif occurs in 31 of 50 R2R3 plant myb the GIDPXXH motif (Avila et al., 1993) in cell shape genes examined. Immediately C-terminal to the myb DNA regulation. Of the five myb genes implicated in plant cell shape binding domain, 20 genes examined encode a GIDPXXH control, four contain this motif. amino acid sequence, as described in Avila et al. (1993); of Most of the plant myb genes so far described possess two these, 18 also encode the small amino-terminal motif, myb repeats. Based on amino acid sequence similarities, it is including MIXTA and CotMYBA. These motifs are boxed in thought that these have been derived from the ancestral Fig. 7. None of the flavonoid biosynthesis regulating mybs sequences which gave rise to the vertebrate myb proto- possess the GIDPXXH sequence, whereas it is present in all oncogene second (R2) and third (R3) myb repeats (Lipsick, mybs directly implicated in plant cell shape regulation except 1996; Martin and Paz-Ares, 1997; Rosinski and Atchley, GL1. A role in plant cell shape regulation for another myb from 1998). A comparative sequence analysis of 50 plant R2R3-type Arabidopsis, AtMYB305, has been inferred from experiments 678 T. Payne and others

Fig. 6. Leaf and floral phenotypes of Arabidopsis overexpressing CotMYBA. (A) Concave rosette leaf of V1 35S:CotMYBA A. thaliana seedling, WS ecotype. (B) Flower of same, showing uncoordinated expansion of floral organs.

multicellular, which might imply that endoreduplication is unnecessary for Nicotiana trichome morphogenesis. If trichome genesis in the two species is as dissimilar as our experiments indicate, one would not expect to encounter homology between the implicated myb genes other than that which was minimally required for DNA binding domain function. Besides the common N-terminal motif, homology between CotMYBA and MIXTA outside the myb DNA binding domain is confined to an identically positioned GIDPXTH amino acid sequence, which suggests a role for the latter motif in cell shape regulation in tobacco. Fig. 5. Root phenotypes of tobacco seedlings overexpressing MIXTA and CotMYBA. (A) Longitudinal section of untransformed TI1347 The phenotypes of tobacco plants overexpressing seedling root. (B) Longitudinal section through 35S:MIXTA root. (C- MIXTA or CotMYBA are distinct E) Light micrographs of living 10-day-old seedling primary root tips growing on MS agar. (C) Untransformed TI1347 seedling root. (D) The most parsimonious explanation for our results would be 35S:MIXTA root. (E) 35S:CotMYBA root. Since both 35S:MIXTA and that in Nicotiana tabacum, a myb-class transcription factor 35S:CotMYBA roots elongate less than untransformed, D and E show with extensive homology to MIXTA or CotMYBA regulates proportionally more of the primary seedling root than C. Scale bars trichome differentiation, and that overexpression of the (A,B) 10 µm; (C-E) 1 mm. homologous genes from other species can substitute for or augment its activity to initiate ectopic trichome production. But such a facile interpretation fails to take into account the in which Arabidopsis genes were expressed under an inducible complexity of the observed phenotypes and the superficiality promoter in Schizosaccharomyces pombe, and isolated based of their resemblance. on their apparent ability to cause cell division anomalies in the A hypothetical gene-for-gene relationship is a better fit for yeast (Xia et al., 1996). However, Moyano et al. (1996) have the MIXTA data than for CotMYBA, since the effects of its reported that its most similar counterpart in Antirrhinum majus overexpression appear to be specific to the epidermis. As is a regulator of phenylalanine ammonia lyase and other previously stated (see Results), overexpression of MIXTA in phenylpropanoid biosynthetic genes, suggesting that the result tobacco results in hyperpapillate leaf and petal epidermis, in S. pombe is due to a coincidence in gene product structure which in turn alters perception of plant pigments consistent not correlated with gene function in plants. AmMYB308, which with the reported function of the gene in Antirrhinum. also contains the GIDPXXH motif, may affect pallisade cell However, in tobacco plants overexpressing MIXTA many of shape in Antirrhinum (Noda et al., 1994) and it is reported to these papillate cells become what appear to be multicellular inhibit lignin biosynthesis when overexpressed in tobacco trichomes. One would imagine that natural selection by (Tamagnone et al., 1998). pollinators has optimized pigment perception in the As previously mentioned (see Introduction), the Antirrhinum floral epidermis and thus the duration of the morphogenesis of unicellular Arabidopsis trichomes is directional cell wall deposition signal presumably directed by associated with endoreduplicative DNA increase. Melaragno et MIXTA. In the 35S:MIXTA transgenic tobacco plants an al. (1993) have suggested that once a plant cell undergoes a unregulated exogenous signal has been applied and some round of endoreduplication it becomes incompetent to undergo epidermal cells have demonstrated a prolonged capacity to further cell division. Endoreduplication may thus constitute respond. The multicellularity achieved by some of these cellular commitment to a terminal differentiation program. trichomes may reflect a general cellular mechanism set to Although no attempt to quantify DNA content has been made maintain the nuclear DNA/cytoplasmic volume ratio of cells in the present study, the tobacco trichomes affected by within viable parameters. overexpression of MIXTA and CotMYBA are frequently In the case of CotMYBA, the effects of overexpression in Myb genes enhance tobacco trichome production 679 tobacco are global, implying that some fundamental cellular The results of overexpression studies are potentially process is disrupted or exaggerated, one result being the artifactual. Because the transgenes are constitutively production of supernumerary trichomes. These trichomes are transcribed from the strong CaMV 35S promoter, the results of several distinct morphological classes, again indicating that must be interpreted carefully. It is possible that the the process regulated by CotMYBA is less specific. In severely overexpression phenotypes of MIXTA and CotMYBA in affected 35S:CotMYBA transformants, non-trichome cells tobacco are the result of non-productive interactions with gene maintain the ability to elongate and expand long after normal promoters or endogenous regulatory proteins. The maize C1-I cotyledon development has ceased. Pattern maintenance as mutant allele of C1 is an endogenous dominant repressor of well as pattern formation is defective. It may be that in cotton anthocyanin structural gene transcription. The C1-I gene ovules CotMYBA positively regulates cell wall extensibility to product is truncated relative to C1, resulting in loss of the C- allow for the considerable unidirectional fiber growth which terminal amphipathic alpha helix, which functions in the full- occurs during development of the cotton boll. In Nicotiana length allele as an activator of transcription (Paz-Ares et al., tabacum, increased cell wall extensibility might trigger a 1990; Goff et al., 1992). C1-I retains the myb DNA binding certain subset of competent epidermal cells (perhaps those domain, and apparently represses anthocyanin synthesis by having conducively oriented cytoskeletal elements) to competing with the full-length C1 gene product for binding to differentiate into trichomes. structural gene promoters. C1-I may also titrate out In a preliminary characterization of tobacco plants transcriptional activators by formation of non-functional overexpressing both MIXTA and CotMYBA, F1 progeny heterodimers with C1 (Franken et al., 1994) or other regulatory seedlings closely resemble seedlings overexpressing MIXTA proteins. When the Arabidopsis CAPRICE (CPC) gene is alone (Payne and Lloyd, . . . .10 . . . .20 . . . .30 . . . .40 . . . .50 . . . .60 unpublished) indicating that the AmMIXTA MV RSPCCD KV GV--KKGPWTVDEDQKLLAYIEEHGHGSWRSLPLKAGLQRCGKSCRLRWA MIXTA transgene is epistatic to AtMIXTA MGRSPCCD KL GL--KKGPWTPEEDQKLLAYIEEHGHGSWRSLPEKAGLHRCGKSCRLRWT PhMYB1 MGRSPCCD KV GL--KKGPWTPEEDQKLLAYIEEHGHGSWRALPAKAGLQRCGKSCRLRWT the CotMYBA transgene. MIXTA CotMYBA MGRSPCCE KAHT--NKGAWTKEEDQRLINYIRVHGEGCWRSLPKAAGLLRCGKSCRLRWI may function earlier than ZmC1 MGRRA CCA KE GV--KRGAWTSKEDDALAAYVKAHGEGKWREVPQKAGLRRCGKSCRLRWL CotMYBA in cell morphogenesis AtGL1 MKRIRRRDE ENQEYKKGLWTVEEDNILMDYVLNHGTGQWNRIVRKTGLKRCGKSCRLRWM or the expansion-promoting . . . .70 . . . .80 . . . .90 . . . 100 . . . 110 . . . 120 activities regulated by the two AmMIXTA NYLRPDIKRGPFSLQEEQTIIQLHALLGNRWSAIASHLPKRTDNEIKNYWNTHLKKRLTR transgenes may in fact be AtMIXTA NYLRPDIKRGKFNLQEEQTIIQLHALLGNRWSAIATHLPKRTDNEIKNYWNTHLKKRLVK antagonistic. PhMYB1 NYLRPDIKRGKFTLQEEQTIIQLHALLGNRWSAIATHLPKRTDNEIKNYWNTHLKKRLVK CotMYBA NYLRPDLKRGNFTEEEDELIIKLHSLLGNKWSLIAGRLPGRTDNEIKNYWNTHIKRKLIS In Antirrhinum majus floral ZmC1 NYLRPNIRRGNISYDEEDLIIRLHRLLGNRWSLIAGRLPGRTDNEIKNYWNSTLGRRAGA papillae, MIXTA is thought to AtGL1 NYLSPNVNKGNFTEQEEDLIIRLHKLLGNRWSLIAKRVPGRTDNQVKNYWNTHLSKKLVG regulate directional deposition of cell wall material (Noda et al., . . . 130 . . . 140 . . . 150 . . . 160 . . . 170 . . . 180 AmMIXTA MGIDPVTHKPHTHNILGH-GQPKDVANLNHQSRAIA WE A LQ ERRLVRE SRLAQNNNKIGT 1994). Random, excessive AtMIXTA MGIDPVT HKPKNETPLSSLGLSKNAAILSHQSRATA WE A LE EARLAREK SLLHLQHYQTK deposition of cell wall material PhMYB1 MGIDPVTHKPKNDALLSHDGQSKNAANLSHQSRAMA WE A LE EARLVRQ SKLRSNSFQNPL CotMYBA RGIDPQ THRPLNQTANTNTVTAPTELDFRNSPTSVSKSSSIKNPSLDFNYNEFQFKSNTD coupled with an epidermal cell’s ZmC1 GAGAGGSWVVVAPDTGSHATPAATSGACETGQNSAAHRADPDSAGTTTTSAAAVWAPKAV normal tendency to expand might AtGL1 DYSSAVKTTGEDDDSPPSLFITAATPSSCHQHQ ENIYENI AKSFNGVV SASYEDKPKQEL result in the genesis of a trichome, since the integrity and shape of the . . . 190 . . . 200 . . . 210 . . . 220 . . . 230 . . . 240 wall facing outward would not be AmMIXTA IQRRLTWPLCLDNEQSNHYHSALLNSTSAVGLNQDNSFTNYSARPDNNNIYDDYEVNNIM AtMIXTA TSSQPHHHHGFTHKSLLPNWTTKPHEDQQQLESPTSTVSFSEMKESIPAKIEFVGSSTGV reinforced or constricted by the PhMYB1 ASHELFTSPTPSSPLHKPIVTPTKAPGSPRCLDVLKAWNGVWTKPMNDVLHADGSTSASA walls of surrounding cells. The CotMYBA SLEEPNCTASSGMTTDEEQQEQLHKKQQYGPSNGQDINLELSIGIVSADSSRVSNANSAE ZmC1 RCTGGLFFFHRDTTPAHAGETATPMAGGGGGGGEAGSSDDCSSAASVSLRVGSHDEPCRS supernumerary trichomes AtGL1 AQKDVLMATTNDPSHYYGNNA------35S:MIXTA plants produce are uniseriate vectors essentially . . . 250 . . . 260 . . . 270 . . . 280 . . . 290 . . . 300 perpendicular to the epidermis. AmMIXTA GMIEFNNNSNYFADSLRLPGFVEGITDISSSNIVLGAGVLPSNSDNVVGYFEENWSSVLN AtMIXTA TLMKEPEHDWINSTMHEFETTQMGEGIEEGFTGLLLGGDSIDRSFSGDKNETAGESSGGD The intriguing possibility exists PhMYB1 TVSVNALGLDLESPTSTLSYFENAQHISTGMIQENSTSLFEFVGNSSGSSEGGIMNEESE that both MIXTA and CotMYBA CotMYBA SKPKVDNNNFQFLEQAMVAKAVCLCWQLGFGTSEICRNCQNSNSNGFYSYCRPLDS---- ZmC1 GDGDGDWMDDVRALASFLESDEDWLRCQTAGQLA------perturb cellular processes such that AtGL1 ------the physical properties of cell walls or cytoskeletal elements are . . . 310 . . . 320 . . . 330 . . . 340 . . . 350 . . . 360 changed, and that these physical AmMIXTA NVASSSSMDSPDVLVNASSSYKMY------manifestations in turn trigger the AtMIXTA CNYYEDNKNYLDSIFNFVDPSPSDSPMF------PhMYB1 EDWKGFGNSSTGHLPEYKDGINENSMSLTSTLQDLTMPMDTTWTAESLRSNAEDISHGNN expression of trichome genes CotMYBA ------downstream of endogenous ZmC1 ------trichome initiation regulators, by- AtGL1 ------passing them. A possible role for Fig. 7. Alignment of five plant myb proteins. The homology within the DNA binding domain is physical constraints in determining indicated by shading. The amino- and carboxy-terminal conserved regions are boxed. AmMixta, organ differentiation has been MIXTA protein from Antirrhinum majus; AtMIXTA, MIXTA homolog from Arabidopsis thaliana; demonstrated in Helianthus PhMYB1, myb protiein from Petunia hybrida, CotMYBA, myb protein from Gossypium hirsutum; (Hernandez and Green, 1993). AtGL1, GL1 protein from Arabidopsis thaliana, ZmC1, functional C1 allele from Zea mays. 680 T. Payne and others overexpressed in wild-type A. thaliana, an overall reduction in embryonic epidermis. A number of Nicotiana species produce trichomes and an increase in root hair production are seen. It cotyledonary trichomes as a matter of course, and a change in has been proposed that CPC, which encodes a single R2- the pattern of expression of a trichome regulatory gene seems a homologous myb motif and no obvious transcriptional simpler evolutionary explanation than homeotic conversion or activation domain, competes with GL1 in the regulation of the heterochrony. Although the cotyledons of plants overexpressing GLABROUS 2 gene since the phenotypes of gl2 mutants and CotMYBA are distorted, their progressive loss of photosynthetic CPC overexpressing wild-type plants are similar (Wada et al., capacity makes them decidedly unlike normal true leaves. Since 1997). It is worthwhile to note that like GL1, C1 does not alter radicle emergence is simultaneous, slight delays in cotyledonary the development of tobacco plants in which it is overexpressed escape from the seed coat relative to wild-type are probably due (Lloyd, unpublished). MIXTA or CotMYBA might bind to to these distortions rather than to abnormalities in GA or ABA heterologous negative regulatory elements in gene promoters metabolism or response, which one would expect to have an or titrate regulatory proteins such that trichome structural gene effect on germination. transcription would be derepressed. Such a competition hypothesis was tested by overexpressing MIXTA from which Apparent contradictions between evolutionary the putative activation domain had been deleted. distance and heterologous function of transgenes A preliminary characterization of N. tabacum plants probably reflect parallel conservation of post- transformed with pMMKY, a construct encoding a MIXTA transcriptional regulatory mechanisms truncation from which everything C-terminal of the myb Paradoxically, overexpression of the Antirrhinum majus repeats is deleted, has revealed no developmental MIXTA gene was not observed to perturb development of floral abnormalities, indicating that putative C-terminal papillae in Arabidopsis thaliana, even though the latter genome transcriptional activation domains are necessary for production contains a putative orthologue which is 91.3% similar through of the 35S:MIXTA phenotype (data not shown). This result the myb DNA binding domain. A likely explanation might be suggests that the overexpression phenotype of the full-length that the two genes are insufficiently diverged to overcome post- MIXTA transgene is unlikely to be an artifact caused by titration transcriptional checks operating in A. thaliana. Sequence- of a MIXTA partner or nonproductive occupation of specific DNA binding by the vertebrate MYB oncoprotein has downstream gene promoters. been shown to be regulated by a variety of post-transcriptional means, including phosphorylation and redox state (Guehmann It is unlikely that MIXTA or CotMYBA alter the normal et al., 1992; Lüscher et al., 1990; Myrset et al., 1993). Capacity course or timing of overall plant development for interactions with other regulatory proteins may also be vital Changes in epidermal characters such as trichome production to myb gene function. In maize, the C1 (a myb) and B (one of have been correlated with phase change and the transition of several R orthologues in this species, a myc) gene products the shoot from vegetative to inflorescence development interact to regulate anthocyanin synthesis (Goff et al., 1992). programs. In maize, glossy-15 mutants result in the precocious Myb- and myc-class transcription factors also co-regulate acquisition of adult epidermal characteristics, including the anthocyanin synthesis in Petunia hybrida (Quattrocchio, elaboration of hairs (Moose and Sisco, 1994). Arabidopsis leaf 1994); recently, another anthocyanin regulatory locus, An 11, trichome distribution changes gradually from exclusively has been cloned (de Vetten et al., 1997). It encodes a cytosolic adaxial (on early rosette leaves) to exclusively abaxial (on protein with WD-40 repeats which acts upstream of the myb bracts) as the shoot develops (Telfer et al., 1997). Plants gene An 2. The TTG1 locus of Arabidopsis is reported to overexpressing R do not violate this trend, implying that in encode a WD-40 protein as well (Walker et al., 1997). Arabidopsis competence to respond to trichome-specific Preliminary results of experiments conducted in this lab differentiation regulators is governed by higher-order indicate that while overexpression of the maize R gene can regulatory factors which are themselves modulated by such strongly suppress the phenotype of the ttg1-1 mutant, two environmental stimuli as photoperiod. Increases in stem endogenous myc-class homologues do so more weakly. pubescence are correlated with inflorescence development in Overexpression of these same genes in a non-mutant Antirrhinum majus (Bradley et al., 1996). None of the genes background results in both excess trichome and anthocyanin known to alter the timing of such developmental changes in production equivalent to wild-type plants overexpressing R, trichome distribution (including genes involved in floral indicating that TTG1 influences the functioning of the induction) are myb-class transcription factors. Time to endogenous myc gene products. The monocot gene may be flowering of plants overexpressing MIXTA is similar to that of sufficiently diverged to circumvent restraints imposed by untransformed plants. Slow growth of surviving TTG1 on its activity. MIXTA from A. majus may be too similar 35S:COTMYBA T2 seedlings makes evaluation of flowering to its Arabidopsis orthologue to overcome equivalently similar time problematical, but delays are likely due to pleiotropic regulatory mechanisms. It is interesting to note in this regard effects of overexpression not associated with transition to that overexpression of DELILA, the R orthologue from A. flowering per se. majus, results in less anthocyanin accumulation in tobacco than The leafy cotyledon (lec) mutants of A. thaliana bear a does overexpression of its monocot counterpart, and will not superficial resemblance to N. tabacum plants overexpressing suppress the ttg1-1 mutant phenotype in A. thaliana (Mooney MIXTA or CotMYBA, in that they produce cotyledonary et al., 1995). trichomes (Meinke et al., 1994; West et al., 1994). However, the The term “trichome” has been used as a catch-all designation precocious germination phenotype characteristic of lec1-1 seeds for plant epidermal protuberances that in fact vary widely in is not seen in tobacco plants overexpressing either MIXTA or morphology and function (Mauseth, 1988). Arabidopsis CotMYBA, and supernumerary trichomes are not confined to trichomes are exclusively unicellular, and probably function as Myb genes enhance tobacco trichome production 681 physical and chemical barriers to insect feeding (Mauricio, mediated gene transfer by infiltration of adult Arabidopsis thaliana plants. 1998). Of the five distinct morphological classes of trichomes C. R. Acad. Sci. Ser. III Sci. Vie 316, 1194-1199. produced by N. tabacum (Goodspeed, 1954), two (types C and Bradley, D., Vincent, C., Carpenter, R. and Coen, E. (1996). Pathways for inflorescence and floral induction in Antirrhinum. Development 122, 1535- E) are glandular. Type D trichomes are defined by the presence 1544. of specialized stalk cells which may also accumulate secretory Broersma, D. B., Bernard, R. L. and Luckmann, W. H. (1972). Some effects products. The fact that these do not occur together in every of soybean pubescence on populations of the potato leafhopper. J. 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