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Development 126, 671-682 (1999)
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
regulation were overexpressed in Arabidopsis thaliana and
Nicotiana tabacum in an attempt to assess the extent to
which cellular differentiation programs might be shared
between these distantly related plants. GLABROUS 1, a
myb gene required for trichome development in
Arabidopsis, did not alter the trichome phenotype of the
tobacco plants in which it was overexpressed. MIXTA,
which in Antirrhinum majus is reported to regulate certain
aspects of floral papillae development, did not complement
the glabrous 1 mutant of Arabidopsis. However, 35S:MIXTA
transformants of N. tabacum displayed various
developmental abnormalities, most strikingly production of
supernumerary trichomes on cotyledons, leaves and stems.
INTRODUCTION
Trichomes are specialized structures which originate and
project from the above-ground epidermal tissues of most
plants. A vast array of morphologies occur in addition to the
hair-like shape suggested by the term. Trichomes may be
composed of one or more cells, may be unbranched or
branched, and may or may not possess glands which
accumulate or secrete alkaloids like nicotine, terpenoids such
as menthol and camphor, or other compounds repellent or toxic
to phytophagous insects (Johnson, 1975; Levin, 1973;
Rodriguez et al., 1984). Different trichome types may occur on
the same plant of a given species, with type and spacing
determined by the plant organ or organ surface (ie, adaxial vs.
abaxial, leaf vs. stem) from which the trichome extends, and
the developmental phase of the plant at the time of organ
initiation.
The trichome is a useful cell type for studying regulation of
cell differentiation in plants. It is morphologically distinct from
surrounding epidermal cells, and its genesis and development
from this population are readily observed due to its
accessibility to visual inspection and its relatively large size.
In the laboratory, trichomes are non-essential and thus can be
genetically manipulated without a reduction in plant viability.
A number of mutants lacking or producing fewer, aberrant, or
mis-positioned trichomes have been isolated from Arabidopsis,
In addition, floral papillae were converted to multicellular
trichomes. CotMYBA, a myb gene which is expressed in
Gossypium hirsutum ovules and has some homology to
MIXTA, was also overexpressed in the two species. A
similar but distinct syndrome of abnormalities, including
the production of cotyledonary trichomes, was observed in
35S:CotMYBA tobacco transformants. However, CotMYBA
did not alter trichome production in Arabidopsis. These
results suggest that the trichomes of Arabidopsis and
Nicotiana are merely analogous structures, and that the
myb genes regulating their differentiation are specific and
separate.
Key words: CotMYBA, MIXTA, Trichomes, Nicotiana tabacum
and the order of action in the differentiation pathway of the
genes so identified has been deduced by epistatic analysis
(Hülskamp et al., 1994).
Wild-type Arabidopsis leaf trichomes are unicellular,
typically have three branches, and lack glands. Progression
through the stages of leaf trichome morphogenesis in this
species is correlated with overall cell enlargement and
endoreduplicative DNA increase. Three rounds of endomitosis
occur initially, concommitant with extension growth from the
epidermal surface. A fourth round of endomitosis occurs after
primary branching of the developing trichome in a
proximodistal orientation to the leaf primordium surface has
begun. Subsequently the distal branch itself branches, and the
nucleus migrates to the branching point from its previous
position below it. Trichome cells are regularly spaced in the
mature Arabidopsis leaf. Initiation and maturation of trichomes
proceeds in a generally basipetal direction over the leaf
primordium, but additional trichomes may be initiated between
mature trichome cells as organ growth continues (Hülskamp et
al., 1994).
Mutations at either of two loci, TRANSPARENT TESTA
GLABRA 1 (TTG1) or GLABROUS 1 (GL1), result in the
virtual absence of leaf trichomes from A. thaliana. TTG1 is
thought to have roles in trichome precursor selection, trichome
initiation, and in a nearest-neighbor inhibition of initiation in
surrounding protodermal cells. Mutations at this locus have
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T. Payne and others
pleiotropic effects: in addition to the absence of trichomes,
homozygous mutants fail to produce anthocyanin pigments and
seed coat mucilage (Koorneef, 1981), and, conversely, produce
supernumerary root hairs on root epidermal cells that are
normally hairless (Galway et al., 1994). Previously, it was
found that overexpression of the maize R gene (a mychomologous transcription factor which regulates anthocyanin
synthesis in that species) under control of the CaMV 35S
promoter could suppress the various phenotypes associated
with the ttg1-1 mutant. In wild-type Arabidopsis
overexpression of R leads to production of extra trichomes on
leaves and stems, and in some transformants to trichomes on
organs which normally lack them, such as petals, stamens and
pistils (Lloyd et al., 1992). TTG1 has recently been cloned and
the gene is reported to encode a protein with WD-40 repeats
and no myc homology (Walker et al., 1997). Presumably, TTG1
regulates or acts in concert with the activity of a myc-class
transcription factor or factors. We have recently identified two
endogenous Arabidopsis myc-class genes. When constitutively
expressed in transgenic plants both suppress to varying extents
the trichome and other defects of the ttg1-1 mutation, albeit
less dramatically than R (Lloyd et al., unpublished).
Both GL1 and TTG 1 are necessary for normal Arabidopsis
trichome initiation. The GL1 gene was cloned by T-DNA
tagging (Marks and Feldmann, 1989). It encodes a putative
transcription factor with myb-type DNA binding and Cterminal acidic activation domains (Oppenheimer et al., 1991).
By itself GL1 is not sufficient to initiate trichomes in
Arabidopsis. Overexpression from a 35S:GL1 construct does
not suppress the ttg1-1 mutation, but it does lead to the
production of an occasional supernumerary trichome on the
cotyledons of wild-type transformants. In fact, a reduction in
leaf trichome number is observed, and this paradoxical result
has been attributed to a squelching phenomenon (Larkin et al.,
1994) akin to that described by Ptashne and Gann (1990).
Little is known about the molecular basis of trichome
differentiation in other species, although the inheritance of
indumental characters correlated with pest resistance has been
studied in some crop plants such as cotton (Stephens and Lee,
1961; Wannamaker, 1957), soybean (Broersma et al., 1972),
and tomato (Gentile et al., 1969; Goffreda et al., 1990). In his
1954 monograph, Goodspeed recognized five basic
morphological classes of trichomes variously distributed
within the genus Nicotiana, and assessed their transmission to
F1 interspecific hybrids and naturally occurring amphidiploids.
Such experiments are difficult to interpret, but differences in
the inheritance of trichome types, especially in regard to organ
distribution and degree of structural elaboration, were
documented. Such differences are likely to be indicative of
separate regulatory mechanisms for the various trichome
classes.
Noda et al. (1994) have cloned the gene encoding another
myb-class transcription factor, MIXTA, from a Tam4-tagged
allele of Antirrhinum majus. MIXTA appears to be a positive
regulator of a directional cell wall synthesis which results in
the conical-papillate shape of epidermal cells on wild-type
adaxial petal surfaces. This cell shape is thought to affect the
optical properties of the petal epidermis such that incident
light passes through the pigmented cell before it is reflected,
and thus perceived intensity of pigmentation is enhanced.
Epidermal cells of mixta mutant petals are flat by comparison,
but retain other characters normally associated with the
conical cell type (pentagonal symmetry, striations), indicating
that the role of the MIXTA gene in this differentiation pathway
is specific and late. Recently, Glover et al. (1998) have
published a characterization of the MIXTA overexpression
phenotype in Nicotiana tabacum. The data presented herein
are in good agreement with their results which indicate that
the transgene can also regulate trichome differentiation in
tobacco.
Suppression of the pleiotropic Arabidopsis ttg1-1 mutant
by overexpression of the R gene from maize, a monocot,
dramatically demonstrated the potential utility of
heterologous gene expression as a tool for manipulating and
studying the regulation of cellular differentiation processes in
plants. Such an approach necessarily assumes that the
regulatory mechanisms of similar pathways and
developmental programs have been at least partly conserved
over evolutionary time. At the outset of the present study we
hypothesized that this conservation would be reflected by
heterologous functioning of known epidermal cell shape
regulators. Herein we describe an unsuccessful attempt to
replace the trichome-initiating function of the Arabidopsis
GL1 gene by overexpression of MIXTA, a myb-class cell
shape regulator from another dicotyledonous plant,
Antirrhinum, and CotMYBA, a closely related myb-class gene
from cotton. We further describe the MIXTA overexpression
phenotype in Nicotiana tabacum, which, in contrast to our
results with Arabidopsis, includes the production of
supernumerary trichomes. Overexpression of CotMYBA in
Nicotiana also activates trichome initiation, additionally
amplifying a distinct class of multicellular trichomes. Since
overexpression of either GL1 or R has no effect on trichome
initiation in Nicotiana tabacum, our data indicate that the
trichomes of Arabidopsis and Nicotiana are not homologous
structures and that their differentiation is controlled by
distinct regulatory genes. Furthermore, the development of
papillate cells and certain classes of trichomes in Nicotiana
appears to be controlled by the same regulatory pathway
while separate myb-elements can differentially regulate the
cell-fate decision to produce separate, partially overlapping
subsets of trichomes.
MATERIALS AND METHODS
Plant strains and growth conditions
Arabidopsis thaliana. pMXCD, pCMAX41 and pLBJ4 constructs
were used to transform the wild-type WS ecotype, the ttg1-1 mutant
in Landsberg erecta outcrossed to WS one time, and the gl1-1 mutant
of ecotype Columbia. The R gene was previously overexpressed in the
wild-type WS, wild-type RLD, and in the Landsberg erecta ttg1-1
mutant outcrossed to either WS or RLD one time (Lloyd et al., 1992).
Plants were potted in Sunshine Mix no. 1 (Premier Horticulture Inc.)
and grown under continuous fluorescent illumination at 22°C.
Nicotiana tabacum. All constructs were used to transform tobacco
introduction TI1347 ‘Praecox’ pMXCD was also transformed into
strain NC744. Both were supplied by the US Tobacco Germplasm
Collection. Previously, R and C1 overexpression phenotypes were also
evaluated in the ‘Xanthi’ cultivar. Potting mix consisted of a 2:1:1
mixture of Baccto, Sunshine no. 1, and vermiculite. Plants were grown
in a warm greenhouse with supplemental fluorescent light to achieve
16-hour days.
Myb genes enhance tobacco trichome production
Vector construction
pLBJ4. The oligonucleotide GL1-C (5′-GGGGGGGGCTGCAGATTAAACTAAAGGCAGTACTC-3′) was used to prime reverse
transcription of the GL1 cDNA from Col-0 RNA. The cDNA was
amplified by PCR using the primers GL1-C and GL1-A (5′-GGGGGGGGGAATTCATGAGAATAAGGAGAAGAGATG-3′), and then
cloned into pBluescript II SK− (Stratagene) as an EcoRI-PstI
fragment to create pSRV2. The ends of the insert from pSRV2 were
sequenced and subsequently cloned into the binary plant vector
pKYLX71 (Schardl et al., 1987) as a HindIII-XbaI fragment. The gl11 mutation has been complemented by transformation of this construct
into Arabidopsis (Lloyd et al., unpublished).
pMXCD. The MIXTA cDNA was cloned by ligating a HindIII-SacI
restriction fragment from the pUC-18-derived plasmid pJAM980
(Glover et al., 1998) into pKYLX71.
pMMKY-1. Oligonucleotides MXM5 (5′-GGAAGCTTGAATTCATGGTAAAAAAAGGGCCATGGACTG-3′) and MXM3 (5′GGGAGCTCGTCGACCTAACGCTTCTTTAGATGAGTGTTCCAG3′) were used to amplify the first 356 nucleotides of MIXTA from
pJAM980. MXM3 incorporates a stop codon after the arginine residue
which terminates the R3 myb repeat. The PCR product was cloned
into pBluescript II KS+ (Stratagene) as a SacI-HindIII fragment to
create pMXM1-1. The pMXM1-1 insert was completely sequenced
from M13 forward and reverse primers, and then subcloned into
pKYLX71 (Schardl et al., 1987) as a SacI-HindIII fragment.
pCMAX41. The CotMYBA gene was amplified by PCR from
genomic Gossypium hirsutum cv. Texas Marker-1 DNA using the
primers COTMYBA-5 (5′-GCGAATTCCTCGAGCCCATGGGACGATCACGTTGT-3′) and COTMYBA-3 (5′-GCGGATCCTCTAGACCTATGAATCCAAGGGTCTAC-3′). After digestion with XbaI
and XhoI, the PCR product was ligated into pBluescript II KS+
(Stratagene) to create pBSCMAX4. The ends of this insert were
sequenced and then the insert was subcloned into pKYLX71 as an
XhoI to XbaI restriction fragment.
pCMANX2. This overexpression construct contains an intronless
version of CotMYBA amplified by RT-PCR from a pCMAX41transformed TI1347 line using the COTMYBA-5 and -3 primers
described above. The PCR product was cloned into pBluescript II
KS+ (Stratagene) as an XbaI to XhoI fragment to create pCMP2. The
insert was completely sequenced and compared to the GenBank
cDNA accession to verify correct splicing. The cDNA insert from
pCMP2 was then subcloned into pKYLX71 as an XhoI to XbaI
fragment. TI1347 plants transformed with pCMANX2 displayed the
same range of developmental abnormalities seen in pCMAX41
transformants (data not shown).
The construction of pAL144, 35S:R, and pAL71, 35S:C1, is
described elsewhere (Lloyd et al., 1992).
Plant transformation
Binary constructs were introduced into Agrobacterium tumefaciens
strain GV3101 containing pMP90 (Koncz and Schell, 1986) by
electroporation. Arabidopsis was transformed using the protocol of
Valvekens et al. (1988), except in the case of the pCMANX2
construct, which was vacuum infiltrated essentially as described by
Bechtold et al. (1993). Nicotiana tabacum transformation was
accomplished essentially as described by Horsch et al. (1985), except
that explants were derived from hypocotyls of one-week-old
seedlings. With the exception of pMMKY, a minimum of fifteen to
twenty-five independent transformants were produced for each
construct. Where overexpression phenotypes were not observed,
transcription of the transgenes was verified for subsets of
transformants using either northern blot analysis or RT-PCR.
Scanning electron microscopy
Plant materials were fixed overnight in 2% glutaraldehyde and 0.1 M
cacodylate, then taken through an alcohol dehydration series, once in
25, 50, 75, 85, 95% and twice in 100% ethanol, for at least 2 hours
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per step. Specimens were critical point dried in a Tousimis Samdri790 and sputter coated with a gold-palladium alloy using a Ladd
instrument. Specimens were visualized with a Phillips 515 scanning
electron microscope and photographed with Polaroid film.
Plant sectioning and light microscopy
Tissue samples were fixed and stained as described by Mauseth et al.
(1984). Sections were photographed through an Olympus BX60
microscope with an Olympus PM-C35DX camera.
Image processing
Images for figures were scanned with a Sharp JX325 High Resolution
Color Scanner. Figures were constructed using Adobe Photoshop 4.0.
Colors were corrected to match the living specimens and adjustments
in brightness and contrast were made using this program. Images were
printed with a FUJIX Pictrography 3000 printer.
RESULTS
Overexpression of MIXTA in Arabidopsis thaliana
does not complement the gl1-1 mutation
Given the structural similarities between GL1 and MIXTA and
their roles in the control of epidermal cell differentiation, we
hypothesized that MIXTA might be able to substitute for the
function of the Arabidopsis trichome regulator. The MIXTA
cDNA was placed under the transcriptional control of the
CaMV 35S promoter in a binary vector and used to transform
by means of Agrobacterium tumefaciens wild-type Arabidopsis
and the gl1-1 and ttg1-1 mutants. No differences in trichome
production or other epidermal characters could be discerned.
Transcription of the transgene was verified by northern blot
analysis (data not shown).
Since the MIXTA gene was isolated from Antirrhinum majus
based on its effects on floral papillation, we also examined the
petals of pMXCD transformants. Scanning electron
micrographs of petal epidermis (data not shown) revealed no
differences between 35S:MIXTA and untransformed plants of
any of the three genotypes. No published transformation
protocols for Antirrhinum exist, and the reciprocal experiment,
overexpression of GL1 in that species, was not attempted.
Mixta overexpression in Nicotiana tabacum results
in supernumerary trichome production
Like Antirrhinum majus and unlike Arabidopsis thaliana,
Nicotiana tabacum produces flowers pigmented by
anthocyanins. Concurrent with the Arabidopsis experiment, we
transformed N. tabacum with pMXCD to evaluate the effects
of MIXTA overexpression on floral pigmentation. Noda et al.
(1994) have previously shown that in Antirrhinum the gene
exerts its effects on floral phenotype by altering epidermal cell
shape and thus perception of pigmentation rather than quantity
of pigment. One floral phenotype of 35S:MIXTA transformants
is shown in Fig. 1D. Several other transformants produced
mottled pink flowers different from the one shown. The adaxial
floral epidermis of the transformants was examined by
scanning electron microscopy. The SEM shown in Fig. 1H is
from the same transformant that produced the small flower in
Fig. 1D. Remarkably, many of the papillae have elongated to
produce what appear to be multicellular, uniseriate trichomes.
These results are similar to those presented by Glover et al.,
(1998). For comparison, both a flower and floral epidermis
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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 RTshown). 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
The myb gene CotMYBA affects trichome
composed of mostly rounded cells and occasional trichomes
differentiation when overexpressed in Nicotiana
like those seen in the leaf epidermis of untransformed controls.
tabacum
This differential pattern of trichome occurrence and papillation
A Blast search (Altschul et al., 1990) of the National Center for
was subsequently confirmed in SEMs of adaxial leaf epidermis
Biotechnology Information database using the complete
(Fig. 2C-G). Leaves and petals are presumed to be
Antirrhinum MIXTA cDNA sequence as a query, calls up a long
homologous structures (Esau, 1977), so it is perhaps not
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 mybmany
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-weekeither
unicellular
or
old seedlings showing adaxial epidermis of cotyledons (to the left and right) and first 2-3 true leaves. Scale
bar, 1 mm. (A,E,I) Untransformed TI1347; (B,F,J) 35S:R. (C,G,K), 35S:GL1; (D,H,L), 35S:MIXTA
multicelluar and uniseriate
Myb genes enhance tobacco trichome production
Fig. 2. Textural variegation of Nicotiana tabacum adaxial leaf
epidermis. (A) Photograph of untransformed adaxial leaf epidermis.
The midvein is vertical. (B) 35S:MIXTA leaf. (C-G) SEMs of adaxial
leaf epidermis. (C) Untransformed. (D) 35S:MIXTA, from region
adjacent to vein, corresponding to reflective areas in (B).
(E) 35S:MIXTA, from interveinal, rough-looking regions as seen in B.
Scale bar, 0.1 mm. (F) Detail of non-papillate cells seen in D.
(G) Detail of papillate epidermis as in E. Area shown in F and G is
1 mm2.
list of myb-homologous plant genes. One of the highest scoring
but presumably non-orthologous sequences (Clement, Payne,
and Lloyd, unpublished) is a GenBank accession, CotMYBA
(L04497), isolated from Gossypium hirsutum 3-day pre-anthesis
ovules. Cotton fibers are trichomes which are initiated and begin
elongation from the epidermis of the cotton ovule at about the
time of anthesis (Basra and Malik, 1984). Cotton fiber-specific
promoters have previously been shown to direct transcription of
reporter genes in trichomes of N. tabacum (Dang et al., 1996),
suggesting that the two trichome types share some ontogenetic
similarities. To explore the possibility that CotMYBA is a
positive regulator of trichome differentiation, we overexpressed
the genomic sequence (see Materials and Methods) in tobacco.
SEMs of tobacco seedlings overexpressing the CotMYBA
transgene are shown in Fig. 4. Whole 10-day-old T2 seedling
shoots seen in A-D demonstrate the variation in overall
severity of the phenotype observed in progeny of independent
transformants. In all cases the cotyledons are distorted in
comparison to untransformed plants (see Fig. 1I), with many
surface irregularities. Cotyledonary margins are wavy and
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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
transformant cotyledons looks more random, at least partly as
a consequence of the aforementioned epidermal irregularities.
To date, only T2 plants with the mildest phenotypes (Fig.
4A) have survived transplantation to soil. This is not surprising
given our observations of seedlings allowed to continue growth
on nutrient medium without hormones for up to one month
(Fig. 4J-L). The cotyledons of such seedlings may become
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
not be green at emergence, but often pale as they age.
Invariably they are distorted to some degree. In the most
severely affected seedlings the epidermis coarsens and
becomes increasingly disorganized as it ages, even to the point
of being callus-like. Bulbous projections from the epidermis
occur, and these are distinct from observed trichomes (compare
Fig. 4I, with J and L). The “shoulder” regions of the cotyledons
may become pronounced, flattened projections distinguishable
from young first true leaves by their positions relative to the
rest of the cotyledon (Fig. 4K).
A section through a one-month-old 35S:CotMYBA
cotyledon is seen in Fig. 3D. Extreme variation in cell size and
shape is characteristic. Large cells lack chloroplasts. (The
overall appearance of tissue prior to fixation was chlorotic.)
Whereas some tissue is collapsed and apparently necrotic,
there are also isolated pockets of adventitious mitotic activity
(Fig. 3F compare with Fig 3C and E, untransformed).
A distinctive root phenotype is also associated with
overexpression of CotMYBA in tobacco. As seen in Fig. 5E,
35S:CotMYBA seedling roots produce lollipop-like root hairs
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T. Payne and others
with bulbous tips. Similar structures are occasionally seen on
untransformed TI1347 seedling roots, but on 35S:CotMYBA
seedling roots they are much more numerous, and their
frequency is directly correlated with severity of the phenotype
of the aerial organs. A tendency toward disorganization and
uncoordinated cell expansion has also been observed as
35S:CotMYBA roots age (data not shown).
Arabidopsis thaliana was also transformed with the
pCMAX41 construct. Neither of the trichome mutants (ttg1-1,
gl1-1) were suppressed. Initial experiments using the
transformation protocol of Valvekens et al. (1988) resulted in
a small number of regenerants which were stunted and
produced aberrant flowers. Attempts to secure transgenic seed
or correlate floral phenotypes with degree of papillation on
petal epidermis were likewise unsuccessful.
In a recent experiment, vacuum infiltration was used to
transform wild-type Arabidopsis with the pCMANX2 construct,
which would allow us to evaluate transgenic cotyledon
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 kanamycinresistant 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
We have shown that the independent
overexpression
of
two
myb-class
transcription factors, MIXTA and
CotMYBA,
significantly
perturb
development of transgenic tobacco plants,
engendering complex phenotypes which
include the production of supernumerary trichomes. These
same transgenes failed to alter the trichome phenotype of
Arabidopsis. A known regulator of trichome initiation from
Arabidopsis, GL1, likewise had no effect on the trichome
complement of Nicotiana tabacum when overexpressed in that
species. Previous experiments in which the myc-homologous
R gene was overexpressed in the two species suggested that
whereas the regulation of the anthocyanin pathway was
conserved even between monocots and dicots and in both
Arabidopsis and N. tabacum, the development of trichomes in
these dicots was regulated differently. Present results
corroborate this hypothesis by identifying two genes which can
control trichome cell-fate in Nicotiana but not in Arabidopsis.
Limited correlations between myb gene sequence
homology and function are seen
Fig. 7 shows an alignment of the myb DNA binding domains
of the three plant myb-element transcription factors used in the
Fig. 3. Nicotiana tabacum seedling sections. (A) Untransformed TI1347 cotyledon.
(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
of 35S:CotMYBA seedling showing abnormal cotyledon. (E) Longitudinal section through
untransformed TI1347 cotyledon. (F) Detail of 35S:CotMYBA cotyledon seen in D,
showing internal “islands” of small, darkly staining cells surrounded by larger, apparently
quiescent, highly vacuolated cells. Scale bars (A-C,F), 20 µm; (D and E) 50 µm and 10 µm
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) Closeup 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
maize C1 protein (Cone et al., 1986; Paz-Ares et al. 1987),
AtMIXTA, a presumed MIXTA ortholog from Arabidopsis
(Rabinowicz et al., 1996), and PhMYB1 from Petunia hybrida,
which has now been shown to regulate papillae development
in that species (Avila et al., 1993; van Houwelingen et al.,
1998). Overexpression of C1 in concert with R results in the
up-regulation of anthocyanin synthesis in Arabidopsis, but
overexpression of C1 either alone or together with R does not
alter trichome production in tobacco, nor does C1 enhance the
exaggerated trichome phenotype of 35S:R Arabidopsis plants
(Lloyd et al. 1992). The similarities calculated for the myb
DNA binding domains of the putative orthologs MIXTA,
AtMIXTA, and PhMYB1 are over 90% while the similarity for
any other pair is from 60-68%. Our results suggest a role for
the GIDPXXH motif (Avila et al., 1993) in cell shape
regulation. Of the five myb genes implicated in plant cell shape
control, four contain this motif.
Most of the plant myb genes so far described possess two
myb repeats. Based on amino acid sequence similarities, it is
thought that these have been derived from the ancestral
sequences which gave rise to the vertebrate myb protooncogene second (R2) and third (R3) myb repeats (Lipsick,
1996; Martin and Paz-Ares, 1997; Rosinski and Atchley,
1998). A comparative sequence analysis of 50 plant R2R3-type
myb genes recently conducted in this lab (Clement, Payne and
Lloyd, unpublished) revealed the existence of many previously
unrecognized conserved motifs outside the myb DNA binding
domain, some conserved between monocots and dicots. Since
a majority of known plant myb genes have been cloned by
DNA binding domain sequence homology (for example, Li and
Parrish, 1995), gene function is often unknown, making the
assignment of function to the motifs largely conjectural.
Heterologous overexpression experiments such as those
described herein, together with sequence analysis, may enable
such assignments to be made with greater confidence,
especially when the gene in question is derived from a species
in which complementable or revertable mutants and
transformation/regeneration technology is lacking.
A short N-terminal motif occurs in 31 of 50 R2R3 plant myb
genes examined. Immediately C-terminal to the myb DNA
binding domain, 20 genes examined encode a GIDPXXH
amino acid sequence, as described in Avila et al. (1993); of
these, 18 also encode the small amino-terminal motif,
including MIXTA and CotMYBA. These motifs are boxed in
Fig. 7. None of the flavonoid biosynthesis regulating mybs
possess the GIDPXXH sequence, whereas it is present in all
mybs directly implicated in plant cell shape regulation except
GL1. A role in plant cell shape regulation for another myb from
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
seedling root. (B) Longitudinal section through 35S:MIXTA root. (CE) Light micrographs of living 10-day-old seedling primary root tips
growing on MS agar. (C) Untransformed TI1347 seedling root. (D)
35S:MIXTA root. (E) 35S:CotMYBA root. Since both 35S:MIXTA and
35S:CotMYBA roots elongate less than untransformed, D and E show
proportionally more of the primary seedling root than C. Scale bars
(A,B) 10 µm; (C-E) 1 mm.
in which Arabidopsis genes were expressed under an inducible
promoter in Schizosaccharomyces pombe, and isolated based
on their apparent ability to cause cell division anomalies in the
yeast (Xia et al., 1996). However, Moyano et al. (1996) have
reported that its most similar counterpart in Antirrhinum majus
is a regulator of phenylalanine ammonia lyase and other
phenylpropanoid biosynthetic genes, suggesting that the result
in S. pombe is due to a coincidence in gene product structure
not correlated with gene function in plants. AmMYB308, which
also contains the GIDPXXH motif, may affect pallisade cell
shape in Antirrhinum (Noda et al., 1994) and it is reported to
inhibit lignin biosynthesis when overexpressed in tobacco
(Tamagnone et al., 1998).
As previously mentioned (see Introduction), the
morphogenesis of unicellular Arabidopsis trichomes is
associated with endoreduplicative DNA increase. Melaragno et
al. (1993) have suggested that once a plant cell undergoes a
round of endoreduplication it becomes incompetent to undergo
further cell division. Endoreduplication may thus constitute
cellular commitment to a terminal differentiation program.
Although no attempt to quantify DNA content has been made
in the present study, the tobacco trichomes affected by
overexpression of MIXTA and CotMYBA are frequently
The phenotypes of tobacco plants overexpressing
MIXTA or CotMYBA are distinct
The most parsimonious explanation for our results would be
that in Nicotiana tabacum, a myb-class transcription factor
with extensive homology to MIXTA or CotMYBA regulates
trichome differentiation, and that overexpression of the
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
complexity of the observed phenotypes and the superficiality
of their resemblance.
A hypothetical gene-for-gene relationship is a better fit for
the MIXTA data than for CotMYBA, since the effects of its
overexpression appear to be specific to the epidermis. As
previously stated (see Results), overexpression of MIXTA in
tobacco results in hyperpapillate leaf and petal epidermis,
which in turn alters perception of plant pigments consistent
with the reported function of the gene in Antirrhinum.
However, in tobacco plants overexpressing MIXTA many of
these papillate cells become what appear to be multicellular
trichomes. One would imagine that natural selection by
pollinators has optimized pigment perception in the
Antirrhinum floral epidermis and thus the duration of the
directional cell wall deposition signal presumably directed by
MIXTA. In the 35S:MIXTA transgenic tobacco plants an
unregulated exogenous signal has been applied and some
epidermal cells have demonstrated a prolonged capacity to
respond. The multicellularity achieved by some of these
trichomes may reflect a general cellular mechanism set to
maintain the nuclear DNA/cytoplasmic volume ratio of cells
within viable parameters.
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 Callow for the considerable unidirectional fiber growth which
terminal amphipathic alpha helix, which functions in the fulloccurs 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 . . . .2 0 . . . . 30 . . . . 40 . . . .5 0 . . . .6 0
unpublished) indicating that the
A mM I X TA M V R S P C C D K V G V-- K K GPW TV DE DQ K LL AY IE E HG HG S WRS LP L KAG LQ R CG K SC RL RW A
A tM I X TA M G R S P C C D K L G L-- K K GPW TP EE DQ K LL AY IE E HG HG S WRS LP E KAG LH R CG K SC RL RW T
MIXTA transgene is epistatic to
P hM Y B 1 M G R S P C C D K V G L-- K K GPW TP EE DQ K LL AY IE E HG HG S WRA LP A KAG LQ R CG K SC RL RW T
the CotMYBA transgene. MIXTA
C ot M Y BA M G R S P C C E K AH T-- N K GAW TK EE DQ R LI NY IR V HG EG C WRS LP K AAG LL R CG K SC RL RW I
Z mC 1
M G R R A C C A K E G V-- K R GAW TS KE DD A LA AY VK A HG EG K WRE VP Q KAG LR R CG K SC RL RW L
may function earlier than
A tG L 1
M R IR R R DE K EN QEY K K GLW TV EE DN I LM DY VL N HG TG Q WNR IV R KTG LK R CG K SC RL RW M
CotMYBA in cell morphogenesis
or
the
expansion-promoting
. . . . 70 . . . .8 0 . . . . 90 . . . 1 00 . . . 11 0 . . . 12 0
activities regulated by the two
A mM I X TA N Y LR P D IK RGP FSL QE EQT II QL HA L LG NR WS A IA SH L PKR TD N EIK NY W NT H LK KR LT R
transgenes may in fact be
A tM I X TA N Y LR P D IK RGK FNL QE EQT II QL HA L LG NR WS A IA TH L PKR TD N EIK NY W NT H LK KR LV K
P hM Y B 1 N Y LR P D IK RGK FTL QE EQT II QL HA L LG NR WS A IA TH L PKR TD N EIK NY W NT H LK KR LV K
antagonistic.
C ot M Y BA N Y LR P D LK RGN FTE EE DEL II KL HS L LG NK WS L IA GR L PGR TD N EIK NY W NT H IK RK LI S
In Antirrhinum majus floral
Z mC 1
N Y LR P N IR RGN ISY DE EDL II RL HR L LG NR WS L IA GR L PGR TD N EIK NY W NS T LG RR AG A
A tG L 1
N Y LS P N VN KGN FTE QE EDL II RL HK L LG NR WS L IA KR V PGR TD N QVK NY W NT H LS KK LV G
papillae, MIXTA is thought to
regulate directional deposition of
. . . 1 30 . . . 14 0 . . . 1 50 . . . 1 60 . . . 17 0 . . . 18 0
cell wall material (Noda et al.,
A mM I X T A M G I D P V T H K P H T HN IL GH- GQ PK DV A NL N H IA Q WE S A R LQ A ER R LVR E S R LA Q NN NK IG T
1994).
Random,
excessive
A tM I X T A M G I D P V T H K P K N ET PL SSL GL SK NA A IL S H TA Q WE S A R LE A EA R LAR E S K LL H LQ HY QT K
P hM Y B 1 M G I D P V T H K P K N DA LL SHD GQ SK NA A NL S H MA Q WE S A R LE A EA R LVR Q S K LR S NS FQ NP L
deposition of cell wall material
C ot M Y B A R G I D P Q T H R P L N QT AN TNT VT AP TE L DF RN SP T SV S K S SSI KN P SLD FN Y NE F QF KS NT D
coupled with an epidermal cell’s
Z mC 1
G A GA G G SW VVV A PD TG SHA TP AA TS G AC ET GQ N SA AH R ADP DS A GTT TT S AA A VW AP KA V
normal tendency to expand might
A tG L 1
D Y SS A V KT TGE DDD SP PSL FI TA AT P SS C H HQ Q EN IY E NI A KS F NGV V S A SY E DK PK QE L
result in the genesis of a trichome,
since the integrity and shape of the
. . . 1 90 . . . 20 0 . . . 2 10 . . . 2 20 . . . 23 0 . . . 24 0
A mM I X TA I Q RR L T WP LCL DNE QS NHY HS AL LN S TS AV GL N QD NS F TNY SA R PDN NN I YD D YE VN NI M
wall facing outward would not be
A tM I X TA T S SQ P H HH HGF THK SL LPN WT TK PH E DQ QQ LE S PT ST V SFS EM K ESI PA K IE F VG SS TG V
reinforced or constricted by the
P hM Y B 1 A S HE L F TS PTP SSP LH KPI VT PT KA P GS PR CL D VL KA W NGV WT K PMN DV L HA D GS TS AS A
C ot M Y BA S L EE P N CT ASS GMT TD EEQ QE QL HK K QQ YG PS N GQ DI N LEL SI G IVS AD S SR V SN AN SA E
walls of surrounding cells. The
Z mC 1
R C TG G L FF FHR DTT PA HAG ET AT PM A GG GG GG G EA GS S DDC SS A ASV SL R VG S HD EP CR S
supernumerary
trichomes
A tG L 1
A Q KD V L MA TTN DPS HY YGN NA -- -- - -- -- -- - -- -- - --- -- - --- -- - -- - -- -- -- 35S:MIXTA plants produce are
uniseriate vectors essentially
. . . 2 50 . . . 26 0 . . . 2 70 . . . 2 80 . . . 29 0 . . . 30 0
A mM I X TA G M IE F N NN SNY FAD SL RLP GF VE GI T DI SS SN I VL GA G VLP SN S DNV VG Y FE E NW SS VL N
perpendicular to the epidermis.
A tM I X TA T L MK E P EH DWI NST MH EFE TT QM GE G IE EG FT G LL LG G DSI DR S FSG DK N ET A GE SS GG D
The intriguing possibility exists
P hM Y B 1 T V SV N A LG LDL ESP TS TLS YF EN AQ H IS TG MI Q EN ST S LFE FV G NSS GS S EG G IM NE ES E
that both MIXTA and CotMYBA
C ot M Y BA S K PK V D NN NFQ FLE QA MVA KA VC LC W QL GF GT S EI CR N CQN SN S NGF YS Y CR P LD S- -- Z mC 1
G D GD G D WM DDV RAL AS FLE SD ED WL R CQ TA GQ L A- -- - --- -- - --- -- - -- - -- -- -- perturb cellular processes such that
A tG L 1
- - -- - - -- --- --- -- --- -- -- -- - -- -- -- - -- -- - --- -- - --- -- - -- - -- -- -- the physical properties of cell walls
or cytoskeletal elements are
. . . 3 10 . . . 32 0 . . . 3 30 . . . 3 40 . . . 35 0 . . . 36 0
changed, and that these physical
A mM I X TA N V AS S S SM DSP DVL VN ASS SY KM Y- - -- -- -- - -- -- - --- -- - --- -- - -- - -- -- -- A tM I X TA C N YY E D NK NYL DSI FN FVD PS PS DS P MF -- -- - -- -- - --- -- - --- -- - -- - -- -- -- manifestations in turn trigger the
P hM Y B 1 E D WK G F GN SST GHL PE YKD GI NE NS M SL TS TL Q DL TM P MDT TW T AES LR S NA E DI SH GN N
expression of trichome genes
C ot M Y BA - - -- - - -- --- --- -- --- -- -- -- - -- -- -- - -- -- - --- -- - --- -- - -- - -- -- -- Z mC 1
- - -- - - -- --- --- -- --- -- -- -- - -- -- -- - -- -- - --- -- - --- -- - -- - -- -- -- downstream
of
endogenous
A tG L 1
- - -- - - -- --- --- -- --- -- -- -- - -- -- -- - -- -- - --- -- - --- -- - -- - -- -- -- trichome initiation regulators, bypassing 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;
AtGL1, GL1 protein from Arabidopsis thaliana, ZmC1, functional C1 allele from Zea mays.
(Hernandez and Green, 1993).
680
T. Payne and others
overexpressed in wild-type A. thaliana, an overall reduction in
trichomes and an increase in root hair production are seen. It
has been proposed that CPC, which encodes a single R2homologous myb motif and no obvious transcriptional
activation domain, competes with GL1 in the regulation of the
GLABROUS 2 gene since the phenotypes of gl2 mutants and
CPC overexpressing wild-type plants are similar (Wada et al.,
1997). It is worthwhile to note that like GL1, C1 does not alter
the development of tobacco plants in which it is overexpressed
(Lloyd, unpublished). MIXTA or CotMYBA might bind to
heterologous negative regulatory elements in gene promoters
or titrate regulatory proteins such that trichome structural gene
transcription would be derepressed. Such a competition
hypothesis was tested by overexpressing MIXTA from which
the putative activation domain had been deleted.
A preliminary characterization of N. tabacum plants
transformed with pMMKY, a construct encoding a MIXTA
truncation from which everything C-terminal of the myb
repeats is deleted, has revealed no developmental
abnormalities,
indicating
that
putative
C-terminal
transcriptional activation domains are necessary for production
of the 35S:MIXTA phenotype (data not shown). This result
suggests that the overexpression phenotype of the full-length
MIXTA transgene is unlikely to be an artifact caused by titration
of a MIXTA partner or nonproductive occupation of
downstream gene promoters.
It is unlikely that MIXTA or CotMYBA alter the normal
course or timing of overall plant development
Changes in epidermal characters such as trichome production
have been correlated with phase change and the transition of
the shoot from vegetative to inflorescence development
programs. In maize, glossy-15 mutants result in the precocious
acquisition of adult epidermal characteristics, including the
elaboration of hairs (Moose and Sisco, 1994). Arabidopsis leaf
trichome distribution changes gradually from exclusively
adaxial (on early rosette leaves) to exclusively abaxial (on
bracts) as the shoot develops (Telfer et al., 1997). Plants
overexpressing R do not violate this trend, implying that in
Arabidopsis competence to respond to trichome-specific
differentiation regulators is governed by higher-order
regulatory factors which are themselves modulated by such
environmental stimuli as photoperiod. Increases in stem
pubescence are correlated with inflorescence development in
Antirrhinum majus (Bradley et al., 1996). None of the genes
known to alter the timing of such developmental changes in
trichome distribution (including genes involved in floral
induction) are myb-class transcription factors. Time to
flowering of plants overexpressing MIXTA is similar to that of
untransformed plants. Slow growth of surviving
35S:COTMYBA T2 seedlings makes evaluation of flowering
time problematical, but delays are likely due to pleiotropic
effects of overexpression not associated with transition to
flowering per se.
The leafy cotyledon (lec) mutants of A. thaliana bear a
superficial resemblance to N. tabacum plants overexpressing
MIXTA or CotMYBA, in that they produce cotyledonary
trichomes (Meinke et al., 1994; West et al., 1994). However, the
precocious germination phenotype characteristic of lec1-1 seeds
is not seen in tobacco plants overexpressing either MIXTA or
CotMYBA, and supernumerary trichomes are not confined to
embryonic epidermis. A number of Nicotiana species produce
cotyledonary trichomes as a matter of course, and a change in
the pattern of expression of a trichome regulatory gene seems a
simpler evolutionary explanation than homeotic conversion or
heterochrony. Although the cotyledons of plants overexpressing
CotMYBA are distorted, their progressive loss of photosynthetic
capacity makes them decidedly unlike normal true leaves. Since
radicle emergence is simultaneous, slight delays in cotyledonary
escape from the seed coat relative to wild-type are probably due
to these distortions rather than to abnormalities in GA or ABA
metabolism or response, which one would expect to have an
effect on germination.
Apparent contradictions between evolutionary
distance and heterologous function of transgenes
probably reflect parallel conservation of posttranscriptional regulatory mechanisms
Paradoxically, overexpression of the Antirrhinum majus
MIXTA gene was not observed to perturb development of floral
papillae in Arabidopsis thaliana, even though the latter genome
contains a putative orthologue which is 91.3% similar through
the myb DNA binding domain. A likely explanation might be
that the two genes are insufficiently diverged to overcome posttranscriptional checks operating in A. thaliana. Sequencespecific DNA binding by the vertebrate MYB oncoprotein has
been shown to be regulated by a variety of post-transcriptional
means, including phosphorylation and redox state (Guehmann
et al., 1992; Lüscher et al., 1990; Myrset et al., 1993). Capacity
for interactions with other regulatory proteins may also be vital
to myb gene function. In maize, the C1 (a myb) and B (one of
several R orthologues in this species, a myc) gene products
interact to regulate anthocyanin synthesis (Goff et al., 1992).
Myb- and myc-class transcription factors also co-regulate
anthocyanin synthesis in Petunia hybrida (Quattrocchio,
1994); recently, another anthocyanin regulatory locus, An 11,
has been cloned (de Vetten et al., 1997). It encodes a cytosolic
protein with WD-40 repeats which acts upstream of the myb
gene An 2. The TTG1 locus of Arabidopsis is reported to
encode a WD-40 protein as well (Walker et al., 1997).
Preliminary results of experiments conducted in this lab
indicate that while overexpression of the maize R gene can
strongly suppress the phenotype of the ttg1-1 mutant, two
endogenous myc-class homologues do so more weakly.
Overexpression of these same genes in a non-mutant
background results in both excess trichome and anthocyanin
production equivalent to wild-type plants overexpressing R,
indicating that TTG1 influences the functioning of the
endogenous myc gene products. The monocot gene may be
sufficiently diverged to circumvent restraints imposed by
TTG1 on its activity. MIXTA from A. majus may be too similar
to its Arabidopsis orthologue to overcome equivalently similar
regulatory mechanisms. It is interesting to note in this regard
that overexpression of DELILA, the R orthologue from A.
majus, results in less anthocyanin accumulation in tobacco than
does overexpression of its monocot counterpart, and will not
suppress the ttg1-1 mutant phenotype in A. thaliana (Mooney
et al., 1995).
The term “trichome” has been used as a catch-all designation
for plant epidermal protuberances that in fact vary widely in
morphology and function (Mauseth, 1988). Arabidopsis
trichomes are exclusively unicellular, and probably function as
Myb genes enhance tobacco trichome production
physical and chemical barriers to insect feeding (Mauricio,
1998). Of the five distinct morphological classes of trichomes
produced by N. tabacum (Goodspeed, 1954), two (types C and
E) are glandular. Type D trichomes are defined by the presence
of specialized stalk cells which may also accumulate secretory
products. The fact that these do not occur together in every
species of the genus Nicotiana implies that development of each
type proceeds from the activity of specific and in some cases
separate sets of regulatory genes, which would not preclude
sharing of structural genes or trichome building mechanisms
between types. MIXTA appears to exert its effects primarily on
trichome types A and C. Overexpression of CotMYBA also
promotes the development of Type E trichomes, which may
indicate that it regulates or perturbs some growth process or fatedetermining mechanism fundamental to all three. At least 23
myb gene sequences have already been cloned from Arabidopsis
(Wang et al., 1997), and according to one estimate up to a
hundred may be present in the genome (Martin and Paz-Ares,
1997). It has been suggested that proliferation of the myb
regulatory gene family was correlated with the evolution in
plants of specialized physiological functions (Martin and PazAres, 1997). Indeed, the generalization can be made that those
mybs to which functions have been assigned regulate late or
downstream events in plant organ development, such as the
epidermal characteristics of petals or leaves. Of course this bias
in recovery might stem from the lethality of mutations in more
fundamental developmental pathways. Given the large size of
the myb gene family and observed regulatory association of
these genes with downstream developmental events, it is perhaps
not surprising that myb genes have been implicated in the
differentiation of trichomes as different morphologically as those
of Arabidopsis and N. tabacum. Our results imply that these myb
genes (GL1 vs. MIXTA or CotMYBA) are as distinct as the
structures their activities give rise to. This is in sharp contrast to
the well-documented conservation of regulatory genes which
exists in flower development (Coen and Meyerowitz, 1991;
Mandel et al., 1992). From a hierarchical standpoint this
disparity is readily explained by the non-essential character of
the trichome relative to the fundamental importance of the flower
in the plant life-cycle. And since trichomes are simpler organs,
one would suppose that fewer structural genes need be subjected
to coordinate regulation in order to produce them.
We thank Dr Cathie Martin of the John Innes Institute for pJAM
980, Barbara Goettgens and John Mendenhall of the University of
Texas Cell Research Institute for assistance with SEMs and image
processing, and Dr Verne Sisson of the Oxford Tobacco Research
Station for supplying Nicotiana tabacum germplasm. This work was
partially supported by the Institute for Cellular and Molecular
Biology, UT-Austin, and the Texas Higher Education Coordinating
Board.
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