Download Tissue layer and organ specificity of trichome

2283
Development 125, 2283-2289 (1998)
Printed in Great Britain © The Company of Biologists Limited 1998
DEV0160
Tissue layer and organ specificity of trichome formation are regulated by
GLABRA1 and TRIPTYCHON in Arabidopsis
Arp Schnittger, Gerd Jürgens and Martin Hülskamp*
Lehrstuhl für Entwicklungsgenetik, Universität Tübingen, Auf der Morgenstelle 1, D-72076 Tübingen, Germany
*Author for correspondence (e-mail: [email protected])
Accepted 7 April; published on WWW 19 May 1998
SUMMARY
In animal development, cellular diversity is generated
within tissues which in turn are derived from germ layers.
Similar to the germ layers in animals, plants establish three
distinct tissue layers early in development which each give
rise to a distinct set of cell types. To investigate the role of
tissue-layer-specific cues in generating plant cellular
diversity we studied the spatial regulation of an epidermal
cell type, trichomes (hairs), by the two genes, GLABRA1
(GL1) and TRIPTYCHON (TRY). Ubiquitous expression of
the positive regulator GL1 in the absence of the negative
regulator TRY leads to ectopic trichome formation not only
on additional organs but also in subepidermal tissue layers.
Trichomes in inner tissue layers can differentiate the same
morphology and show a spacing pattern comparable to
trichomes in the epidermis. This clearly shows that cell type
specification takes place downstream of tissue-specific cues.
We propose a model of how the tissue and organ specificity
of trichome induction is regulated in normal development.
INTRODUCTION
composed of a few distinct cell types that are specific to this
layer, namely epidermal pavement cells, stomatal guard cells
and trichome cells. We use trichome development in
Arabidopsis as a model system to study epidermal pattern
formation. Trichomes are large single-celled epidermal hairs
that are regularly distributed on leaves, sepals and stems.
Several genes have been identified that act early in trichome
initiation and patterning. Two genes, GLABRA1 (GL1) and
TRIPTYCHON (TRY), appear to be specifically involved in
trichome development (Esch et al., 1994; Hülskamp et al.,
1994; Marks and Feldmann, 1989; Oppenheimer et al., 1991),
whereas TRANSPARENT TESTA GLABRA (TTG) is also
required in other developmental processes, including the
regulation of anthocyanin biosynthesis (Koornneef, 1981), the
production of seed coat mucilage (Koornneef, 1981) and root
hair differentiation (Galway et al., 1994). GL1 and TTG can be
considered positive regulators of trichome initiation since
homozygous mutant plants are nearly devoid of trichomes
(Koornneef et al., 1982). Evidence for the molecular function
of the TTG gene in trichome initiation comes from the analysis
of Arabidopsis plants that constitutively overexpress the maize
R gene. Overexpression of the R gene, which encodes a protein
with sequence similarity to myc-related transcription
activators, is sufficient to rescue the ttg phenotype (Lloyd et
al., 1992). Although the recent cloning of the TTG gene
revealed that TTG is not an R gene homolog (A. Walker and J.
Gray, personal communication), overexpression of the R gene
can still be considered to reflect ectopically provided TTG
function. Plants that constitutively express the R gene are
During the development of multicellular organisms cells
become progressively restricted in their developmental
potential. The final specification of cells can be considered to
be determined by two independent spatial inputs: molecular
circuits that generate patterns of cells or cell groups, and tissuespecific cues that dictate the actual differentiation pathway.
Well-studied cell differentiation processes include the
specification of epidermal sensory organs (Campos-Ortega,
1993; Ghysen et al., 1993; Hinz et al., 1994; Tepass and
Hartenstein, 1995; Yan and Jan, 1993), the development of the
tracheal system (Wilk et al., 1996), eye development (Hafen
and Basler, 1990), and somatic myogenesis (Baylies and Bate,
1996) in Drosophila. In contrast to animal development, little
is known about the organ- and tissue-layer-specific
developmental constraints on cell type specification in plants.
Also plants establish distinct tissue layers that are maintained
as separate layers throughout development: the L1 layer that
produces the epidermis and the two inner tissue layers, the L2
and the L3, that give rise to the mesophyll and the vascular
system (Medford, 1992; Poethig, 1989; Satina et al., 1940;
Smith and Hake, 1992). If cells are displaced from the L1 layer
to the L2 layer they change fate according to their new position
(Dermen, 1953; Stewart and Burk, 1970). This implies that
cell-type specification depends on tissue-layer-specific cues.
Numerous studies have focused on epidermal cell type
specification because the epidermal surface is readily
accessible and therefore easily studied. The epidermis is
Key words: Trichomes, Pattern formation, Tissue layer specificity,
Arabidopsis thaliana, GLABRA1, TRIPTYCHON
2284 A. Schnittger, G. Jürgens and M. Hülskamp
strongly affected in trichome patterning such that trichome
density is increased and trichomes are initiated ectopically on
petals, stamens and carpels (Lloyd et al., 1992). The GL1 gene
encodes a protein with homology to the Myb family of
transcription factors (Oppenheimer et al., 1991). GL1 is
expressed initially at low levels in all cells of the developing
epidermis and then high levels of transcripts accumulate in
developing trichome cells (Larkin et al., 1993). These data
suggest GL1 to be involved in early events of trichome
initiation. However, ectopic expression of GL1 is not sufficient
to trigger trichome development in cells that normally do not
form trichomes (Larkin et al., 1994). TRIPTYCHON (TRY), is
thought to act as a negative regulator of trichome fate. In try
mutants trichomes are often found in clusters, suggesting that
TRY acts to prevent neighboring cells from adopting a
trichome fate (Hülskamp et al., 1994).
We reasoned that the trichome specificity of GL1 and TRY
reflects that both genes function downstream of most other
developmental cues. In this case one would expect that
overexpression of the positive regulator GL1 in the absence of
the negative regulator TRY would bypass other developmental
restrictions of trichome initiation. To test this hypothesis we
studied trichome distribution in a homozygous try mutant that
constitutively expresses GL1 under the control of the
cauliflower mosaic virus 35S RNA promoter (35S::GL1). In
addition, the role of TTG in organ- and tissue-specific trichome
initiation was analyzed in several double and triple mutant
combinations.
MATERIALS AND METHODS
Plants and plant culture
The wild-type strain used in this work was the Landsberg erecta
ecotype. The ttg-1 allele was induced in a Landsberg erecta genetic
background and was obtained from M. Koornneef (Agricultural
University, Wageningen, Netherlands). The try-EM1 allele was
isolated as a recessive mutation in a Landsberg erecta background
(Hülskamp et al., 1994). Although partial dominance was observed
with respect to the branch number in try heterozygotes (Folkers et al.,
1997), none of the three known try alleles show patterning defects in
heterozygous plants (A. Schnittger, unpublished observations).
Therefore, in the following the patterning phenotype of try mutants
will be considered as a recessive trait. The transgenic lines containing
the 35S::GL1 construct (Larkin et al., 1994) and the pGGE4 construct
(Larkin et al., 1993) were obtained from D. Marks. The 35S::R-GR
line was obtained from A. Lloyd (Lloyd et al., 1994). The plants were
grown under constant illumination as described previously (Mayer et
al., 1993). The 35S::GL1 try line was constructed in two steps.
Initially plants were selected among the F2 progeny of a cross between
35S::GL1 homozygotes and try mutants that displayed a new strong
cluster phenotype. Plants of the genotype 35S::GL1 were identified
as F2 plants that did not segregate try plants among their F3 progeny.
The double mutant line was backcrossed with both parentals to verify
the genotype. A similar strategy was used to generate the 35S::R-GR
try line. The +/35S::GL1 try/try +/pGGE4 plants were obtained from
crosses between 35S::GL1 try with pGGE4 try. +/35S::GL1 +/ttg
+/try plants were obtained from a cross between 35S::GL1 try and ttg
mutants. +/35S::GL1 +/ttg try/try plants were generated by crossing
a 35S::GL1 try homozygote with a ttg try double mutant. +/ttg
+/35S::GL1 plants were F1 plants from a cross between homozygous
ttg plants and homozygous 35S::GL1 plants. +/ttg +/try
transheterozygotes were obtained from a cross between homozygous
ttg and try mutants. +/ttg try/try plants were generated by crossing ttg
try double mutants with homozygous try mutants.
Histology
For confocal laser-scanning microscopy, plant material was fixed for
1 hour by vacuum infiltration in 4% paraformaldehyde in MTSBT (50
mM PIPES, 5 mM EGTA, 5 mM MgSO4, 0.1% Triton X-100, pH 7).
Samples were washed 3× in MTSBT for 5 minutes, 1× in dH2O,
stained for 1 hour in YO-PRO-1 solution (1:1000 in dH2O, Molecular
Probes, Y-3603) by vacuum infiltration, washed in dH2O and mounted
in Citifluor. For GUS detection, plant material was stained and cleared
as described previously (Vroemen et al., 1996).
Microscopy and graphics work
DAPI staining and cytophotometry was done as previously described
(Hülskamp et al., 1994). For histological analysis leaves were fixed
and processed as described previously (Hülskamp et al., 1994).
Confocal laser-scanning microscopy was done with an inverse
fluorescence microscope (Leitz), using the Leica TCS NT program.
Scanning electron microscopy was done as previously described
(Laux et al., 1996). Images were processed using Adobe Photoshop
3.0 and Aldus Freehand 7.0 software.
Statistical comparison of epidermal and subepidermal
trichome distribution
A map of the relative position of epidermal and subepidermal
trichomes in 35S::GL1 try plants was constructed with the help of
camera lucida drawings of GUS-stained 35S::GL1 try pGGE4 leaves.
All leaves were approximately equal in age, size and the number of
epidermal trichomes. To facilitate a standardization of data, a square
of 2×2 mm was defined for each leaf. For further statistical analysis
data from 12 leaves with a total of 397 subepidermal and 131
epidermal trichomes were pooled. In order to test whether the
subepidermal trichome distribution is linked to the epidermal
trichome distribution the distances between all subepidermal
trichomes and their nearest epidermal trichomes were compared to the
distances of 10 000 randomly selected positions and epidermal
trichomes. The two sets of data define two distribution functions that
were compared by a χ2 test to determine whether they are statistically
significantly different. The program used for this analysis was written
in C programming language and can be obtained on request.
RESULTS
Overexpression of GL1 in the absence of TRY
causes ectopic trichome formation on various
organs
In wild-type plants trichome initiation is restricted to leaves,
sepals and stems. This organ-specific initiation of trichomes is
not affected in try plants. 35S::GL1 plants occasionally have
trichomes on cotyledons. 35S::GL1 try plants display a high
number of trichomes on the cotyledons (Fig. 1A). In addition,
35S::GL1 try plants display trichomes ectopically on several
organs. We found large clusters of trichomes at the base of
flower stalks at a position characteristic for the development of
bracts in other plant species (Fig. 1B). The pistil is covered
densely with branched trichomes which are normally present
only on leaves (Fig. 1C). Occasionally we found trichomes on
the stamen (Fig. 1D). 35S::GL1 try plants show a marked
increase in the size of trichome clusters. While try mutants
usually have small clusters of no more than four trichomes,
35S::GL1 try mutants have clusters of up to 8 trichomes. A
closer inspection revealed that most of these supernumerary
Subepidermal trichomes in 35S::GL1 try plants 2285
trichomes are derived from trichome accessory cells. In young
leaves most trichomes are found as single or twin trichomes
that are surrounded by a ring of eight accessory cells. Slightly
later a variable number of accessory cells start to emerge from
the leaf surface and develop into trichomes (Fig. 1E,F).
Subepidermal trichome formation in 35S::GL1 try
plants
In wild-type plants trichomes are exclusively initiated in the
epidermal cell layer. This cell-layer specificity is lost in
35S::GL1 try mutants which produce trichomes also in
subepidermal tissue layers. In mature leaves we observed
subepidermal cells that were unusually large and contained a
much larger nucleus than the surrounding cells. These cells
lacked chloroplasts which are a typical feature of mesophyll
cells. Two observations suggest that these large cells have
adopted a trichome fate. Many acquire a polarization
characteristic for trichome cells. Some cells start to expand
towards the leaf surface (Fig. 2A-C,G). Others grow through
the epidermal layer and initiate branching (Fig. 2D-F,H,I).
Additional evidence was obtained from studying the
expression of a trichome-specific GUS marker construct
(pGGE4) in a 35S::GL1 try mutant background. The pGGE4
construct contains a short promoter fragment of the GL1 gene
that drives the expression of the GUS gene specifically in
trichomes and stipules (Larkin et al., 1993). The GUS
histochemical staining in 35S::GL1 try pGGE4 plants revealed
abnormally large GUS-positive subepidermal cells in leaves
(Fig. 2J,K). Subepidermal trichomes were not only produced
on the adaxial side of the leaf but also on the abaxial side (Fig.
3A). No indication of a general transformation of subepidermal
tissue layers into epidermal tissue layers was found, e.g. no
stomata or other epidermal cell types were found. Except for
the subepidermal trichome cells all other cells showed a
morphology and organization characteristic of subepidermal
leaf tissues (Fig. 2G-I).
Subepidermal trichome formation was also found in other
plant organs including the stem and cauline leaves (Fig. 3B).
Also cotyledons produced unusually large subepidermal cells.
These cells, however, did not show GUS-staining in a pGGE4
background. No subepidermal trichomes were found in flower
tissues or in the hypocotyl. Also root hair patterning and
differentiation was indistinguishable from wild type.
Subepidermal trichome distribution in 35S::GL1 try
plants
Subepidermal trichomes appear to be distributed regularly on
the leaf. No correlation of their spatial distribution and other
morphological landmarks such as the vascular system was
found. The subepidermal trichome pattern appears to be
random with respect to the distribution of epidermal trichomes
(Fig. 4A). Subepidermal trichomes were found in direct contact
with epidermal trichomes as well as at some distance from
epidermal trichomes. In order to test whether subepidermal
trichome patterning is coupled to the epidermal trichome
pattern we compared all distances of subepidermal trichomes
to their nearest epidermal trichome and the distances of
randomly chosen positions and epidermal trichomes. If
subepidermal trichome patterning were independent of the
epidermal trichome pattern one would expect a similar
distribution of the subepidermal-epidermal distances and the
distances between random positions and epidermal trichomes.
However, if epidermal and subepidermal trichome patterning
depend on each other, one would expect that the two
distributions are different. As shown in Fig. 4B the distances
between subepidermal and epidermal trichomes show a
distribution with a broad peak between 125 µm and 500 µm.
A similar distribution was observed for the values for distances
between 10 000 randomly chosen positions and epidermal
trichomes. The maximum of this distribution, however, was
much narrower, with a peak at 250 µm. A difference between
the two distributions was found to be statistically significant
(χ2=34,37; α=0.01). These results clearly rule out a preference
of subepidermal trichome initiation in the proximity of
epidermal trichomes. The larger fraction of more distant
subepidermal trichomes may suggests a lateral inhibition of
subepidermal and epidermal trichomes. The biological
significance of this observation is not clear and remains to be
studied in more detail.
Another aspect of trichome patterning, cluster formation as
found in try mutants, was also observed for subepidermal
trichomes in 35S::GL1 try plants. Approximately 31% of all
subepidermal trichome sites contained clusters (n=219). The
formation of trichome clusters appears to depend on the gene
dosage of TRY. This is suggested by the finding that
subepidermal trichomes in +/35S::GL1 +/try plants were never
found in clusters (n=100).
The finding that trichome patterning in subepidermal tissue
layers in 35S::GL1 try plants is comparable to that in the
epidermis is remarkable. It suggests that trichome patterning
takes place among cell types as different as mesophyll cells and
epidermal pavement cells. This suggests that the underlying
patterning mechanisms are inherent to the activation of
trichome-specific genes.
Subepidermal trichomes lack accessory cells
During trichome maturation all epidermal cells adjacent to the
base of a trichome differentiate as accessory cells which are
characterized by a rectangular shape. The cell fate of accessory
cells appears to be determined by lateral signaling of the
central trichome cell rather than by cell lineage since the
accessory cells are not clonally related to each other or to the
trichome cell (Larkin et al., 1996). In order to assess whether
subepidermal trichomes recruit surrounding mesophyll cells to
aquire accessory cell fate we inspected optical sections of
subepidermal tissue layers by confocal microscopy (Fig. 5). By
morphological criteria, subepidermal cells that are in
immediate contact with a subepidermal trichome cell are
indistinguishable from other mesophyll cells. All surrounding
cells contain chloroplasts suggesting that these cells have not
changed their normal identity. Also cells in contact with large,
branched subepidermal trichomes showed no transformation of
mesophyll cells into accessory cells (Fig. 2I). This observation
renders it unlikely that differentiation of accessory cells is not
initiated simply because trichome development does not reach
a critical stage.
Thus, while trichome patterning appears to be normal in
subepidermal tissue layers the induction of morphologically
recognizable accessory cells is not observed. This suggests that
the initiation of accessory cells does not take place because
tissue-layer-specific developmental constraints cannot be
bypassed. Alternatively, the cell fate of mesophyll cells may
2286 A. Schnittger, G. Jürgens and M. Hülskamp
have been irreversibly determined by the time the trichome cell
starts lateral signaling.
The role of the TTG/R gene in subepidermal
trichome formation
In addition to GL1 and TRY, the TTG gene is thought to be a
key factor in the regulation of trichome patterning. The lack of
trichome phenotype suggests that the TTG gene acts similarly
to the GL1 gene as a positive regulator of trichome initiation.
We therefore tested whether overexpression of the R gene alone
or in the absence of TRY activity results in subepidermal
trichome initiation. For these studies we used transgenic lines
overexpressing an inducable form of the R gene that includes
a N-terminal fusion of the vertebrate glucocorticoid receptor
(35S::R-GR; Lloyd et al., 1994). Neither induced 35S::R-GR
nor 35S::R-GR try plants exhibited subepidermal trichomes.
Thus, activation of the TTG pathway via overexpression of the
R gene is not sufficient to trigger trichome initiation in
subepidermal tissue layers. The phenotypic difference between
the 35S::GL1 try and 35S::R-GR try plants could be explained
by assuming that TTG is normally present in subepidermal
tissues while GL1 is specifically expressed in the epidermis and
hence is limiting in 35S::R-GR try plants (see Discussion).
A second aspect of TTG function during trichome patterning
is its apparent negative role during lateral signaling between
trichome precursor cells. A reduction of the TTG dosage in
35S::GL1 plants results in a high frequency of clustered
trichomes (Larkin et al., 1994). A similar phenotype is
produced by weak ttg alleles (Larkin et al., 1994). Both sets of
data suggest that the relative concentration of TTG is important
Fig. 1. Ectopic trichome formation in 35S::GL1 try plants.
(A) Cotyledons. (B) Trichomes at the base of a flower stalk.
(C) Pistil with branched trichomes. (D) Anther with a trichome.
(E) One accessory cell forming a trichome. (F) Mature, branched
accessory cell.
during trichome selection. In order to test whether the gene
dosage of TTG might play a role in subepidermal trichome
formation in try, 35S::GL1 or 35S::GL1 try plants we
constructed various double mutants that are heterozygous for
ttg. Plants of the genotype +/ttg try/try, +/ttg +/try or
35S::GL1 +/ttg did not produce subepidermal trichomes. We
also found no increased cluster frequency in +/35S::GL1 +/ttg
try/try plants as compared to 35S::GL1 try plants. Both lines
show approximately the same cluster frequency of 35.7%
(n=213) and 31.5% (n=219) respectively; no significant
difference was found in a Welch test (t′=−0,273, α=0.01). Also
the total number of subepidermal trichomes in +/35S::GL1
Fig. 2. Trichome formation in subepidermal tissue layers of
35S::GL1 try plants. (A) Confocal image of a polarized
subepidermal trichome cell, optical section perpendicular to the leaf
surface. (B) Schematic drawing of the confocal image in (A);
epidermal cells are shaded blue, subepidermal cells are green, and
the trichome cell is shown in red. (C) SEM image, epidermal view of
a putative subepidermal trichome causing a bulge of the epidermis.
(D) Confocal image, optical section perpendicular to the leaf surface:
a subepidermal trichome cell which has grown through the epidermis
and started branching. Note surrounding epidermal cells reach up to
the branches. (E) Schematic drawing of the confocal image in (D);
color code as in B. (F) SEM image: epidermal view of a putative
subepidermal trichome breaking through the epidermis.
(G-I) Histological sections of subepidermal trichomes. (G) Polarized
subepidermal trichome cell. (H) Subepidermal trichome cell growing
through the epidermis. (I) Subepidermal trichome similar in size and
branching to epidermal trichomes. (J,K) DIC images of GUS-stained
35S::GL1 try pGGE4 plants, focused on the leaf epidermis (J) and
the leaf ground tissue (K).
Subepidermal trichomes in 35S::GL1 try plants 2287
Fig. 3. Subepidermal trichome formation in different organs of
35S::GL1 try plants. (A) Histological section of a mature leaf. Note,
subepidermal trichomes are formed on the adaxial and the abaxial
side of the leaf (arrows). (B) DIC image of a GUS-stained 35S::GL1
try pGGE4 plant, focused on the ground tissue of the stem.
+/ttg +/try mutants was not increased as compared to
+/35S::GL1 +/try mutants. Both lines show subepidermal
trichomes at a low frequency with 1-5 subepidermal trichomes
per mature leaf. These results show that the TTG dosage does
not affect subepidermal trichome initiation in 35S::GL1 try
plants.
Regulation of the number of endoreplication cycles
by GL1 and TRY
35S::GL1 try plants are also affected at the cellular level in the
regulation of endoreplication. During wild-type development
trichomes undergo four rounds of endoreplication. TRY has
been described to act as a negative regulator of endoreplication
Fig. 5. Neighboring cells of subepidermal trichomes. (A) Confocal
image of a subepidermal trichome cell in a mature leaf. Chloroplasts
are recognized by their auto-fluorescence. (B) Schematic drawing of
the relative position of cells of the confocal image in A. TR,
trichome, CHL, chloroplast, MC, mesophyll cell.
since try trichomes show twice the amount of DNA as wildtype trichomes (Hülskamp et al., 1994). We found that
35S::GL1 trichomes show a similar increase in nuclear DNA
content (Fig. 6). This indicates that GL1 is not only required
for trichome initiation but also involved in the control of
endoreplication. In 35S::GL1 try plants most trichomes had a
DNA content equivalent to two or even three additional rounds
of endoreplication compared to the two single lines. This result
could be explained by considering the 35S::GL1 try phenotype
as the addition of two separate effects such that TRY and GL1
function in two independent pathways. Alternatively, TRY
could negatively regulate the action of overexpressed GL1
directly.
DISCUSSION
The increased DNA content and the ectopic initiation of
trichomes in 35S::GL1 try plants support the idea that GL1 and
TRY act together downstream of other developmental controls
of trichome development. In particular, the initiation of
subepidermal trichomes is unexpected. The layered
organization and the tissue- layer-specific differentiation of cell
types in diverse plant organs (Medford, 1992; Poethig, 1989;
Satina et al., 1940; Smith et al., 1992) implies that, similar to
germ layers in animals, each tissue layer confers
developmental constraints in which context subordinate
differentiation processes take place. Our data show that tissuelayer-specific cues can be overriden by activating cell-typespecific pathways. Whether this finding reflects a general
difference in the developmental plasticity of plants versus
animals is not known.
Fig. 4. Trichome distribution in subepidermal tissue layers of
35S::GL1 try plants. (A) Schematic drawing of a 35S::GL1 try leaf.
Positions of epidermal trichomes are indicated as blue dots,
subepidermal trichomes as red dots. (B) The frequency of
subepidermal trichomes (____) or random positions (.......) is plotted
against their distance to epidermal trichomes. The two graphs
comprise 15 classes with each class summarizing data of 62.5 µm
intervals. Note, the last class contains all values above 937 µm which
results in a superficial increase of large values.
Regulation of organ specificity of trichome
formation
A model to explain the organ and tissue layer specific
regulation of trichome formation is presented in Figure 7. In
this model, trichome formation depends on two pathways that
receive different environmental and developmental inputs and
both GL1 and TTG functions, are required to initiate trichome
differentiation. Differences in the presence and density of
trichomes on different organs reflect the regulation of trichome
formation by organ-specific developmental programs and by
2288 A. Schnittger, G. Jürgens and M. Hülskamp
these organs (Larkin et al., 1993), TTG function is limiting for
trichome formation. Our finding that 35S::GL1 try plants show
ectopic trichome formation reminiscent of R transgenic plants
is unexpected. One possible explanation is that the TTG
pathway is activated in 35S::GL1 try plants.
134 44
35S::GL1 try
73 20
try
69 26
35S::GL1
45 21
COL
41 17
LER
32
64
96
128
160
DNA
content (C)
Fig. 6. Regulation of endoreplication in 35S::GL1 try plants. Ler and
Col are the corresponding ecotypes of try and 35S::GL1 respectively.
Average DNA value (thick vertical bars) and standard deviations
(horizontal bars) are indicated. Values were normalized relative to
the 2C DNA content of guard cells (Melaragno et al., 1993).
the physiological state or environmental cues. The involvement
of organ- specific cues is evident in mutants that have homeotic
transformations of cotyledons or flower organs into trichome
bearing leaves (Bowman et al., 1991; Meinke et al., 1994).
Environmental parameters that modulate trichome distribution
include light, day length and the plant hormone gibberellin
(Chien and Sussex, 1996; Misera et al., 1994). Two lines of
evidence suggest that trichome formation on cotyledons,
carpels and stamens depends on the TTG pathway. First,
constitutive expression of the R gene is sufficient to induce
ectopic trichome formation on these organs (Lloyd et al.,
1992). Second, cop1 (fus1) mutant sectors in the epidermis of
carpels produce ectopic trichomes (Misera et al., 1994). The
COP1 gene is involved in light-induced signal transduction and
has been proposed to function as a negative regulator of TTG
with respect to the regulation of anthocyanin biosynthesis in
subepidermal cells and trichome formation in epidermal cells
(Misera et al., 1994). Since GL1 is expressed at low levels in
Organ-Specific
Factor(s)
Epidermis-Specific
Factor(s)
TTG
GL1
TRY
Trichome
Formation
Fig. 7. Model of the organ and tissue layer specificity of trichome
formation. Evidence for this model is summarized in the text. Arrows
indicate positive action, blunted bars indicate negative regulation.
Regulation of tissue layer specificity of trichome
formation
By contrast, tissue layer specificity, the restriction of trichome
formation to the epidermal cell layer, appears to be regulated
by the tissue-layer specific transcriptional activation of the GL1
gene (Fig. 7). TTG is required as a prerequisite for trichome
formation but does not provide spatial information. Activation
of the TTG pathway in subepidermal tissue layers is not
sufficient to trigger trichome formation. Plants expressing the
R gene constitutively under the control of the 35S promoter do
not show subepidermal trichomes. Also fus mutants or
subepidermal fus sectors in adult plants do not produce
subepidermal trichomes (Misera et al., 1994). Although TTG
is active in these tissue layers, trichome formation is not
promoted in subepidermal tissue layers, presumably because
the GL1 gene is not activated. Consistent with this idea, the
activation of the GL1 TRY pathway results in subepidermal
trichome initiation.
GL1 and TRY play a dual role in trichome patterning
and cell differentiation
Pattern formation and cell fate determination are, in animal
development, usually separate processes. A well-studied
example is the regulation of cell type specification by
neurogenic and proneural genes in Drosophila. The neurogenic
genes together with the proneural genes form a gene cassette
(Yan et al., 1993) that acts in ectodermal and endodermal
patterning. Different cell fates are selected by this system in
the respective tissue layers: in the ectoderm, epidermal versus
neural precursor cells (Campos-Ortega, 1993; Ghysen et al.,
1993), in the endoderm, the segregation of midgut epithelial
cells, the midgut precursor cells, and interstitial cell precursors
(Tepass and Hartenstein, 1995). By contrast, trichome pattern
formation and trichome differentiation are intimately coupled.
This is suggested by two lines of evidence. First, 35S::GL1 try
plants initiate an apparently normal trichome pattern in a new
developmental context, the subepidermal tissue layer. Hence,
in this situation not only patterning is initiated but also cell
identity is imposed on the selected cells. Second, both genes,
GL1 and TRY, play a dual role in cell differentiation
(endoreplication) as well as in the spatial control of trichome
initiation. Although this could reflect that both genes are
involved in two independent regulation events, the same
regulatory interactions between GL1 and TRY in both
processes, i.e. the suppression of GL1 by TRY, suggests that
both processes share the same molecular function of the two
genes. This appears to be a paradox since the regulation of cell
differentiation processes typically utilize intracellular
functions while patterning processes usually involve cell-cell
communication processes. Further clarification of the
molecular and cell biological function of GL1 and TRY is
needed to resolve this paradox.
We thank Birgitt Schönfisch for help with the statistic analysis of
the distribution pattern and for writing the simulation program. We
Subepidermal trichomes in 35S::GL1 try plants 2289
thank U. Folkers, P. Grini, H. Ilgenfritz, S. Schellmann, K. Schrick,
T. Steinmann, B. Schwab for helpful suggestions and critical reading
of the manuscript. We thank Heinz Schwarz and Jürgen Berger for
help with the scanning electron microscopy and the confocal
microscope. We would like to thank Charles N. David (Department
of Zoology, University of Munich) for making available the
microscope photometer for cytophotometry. This work was supported
by the SFB 446 grant to M.H. and a Leibniz award to G.J. from the
Deutsche Forschungsgemeinschaft.
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