Download Multicellular trichomes in Arabidopsis - Development

3931
Development 127, 3931-3940 (2000)
Printed in Great Britain © The Company of Biologists Limited 2000
DEV0305
SIAMESE, a gene controlling the endoreduplication cell cycle in Arabidopsis
thaliana trichomes
Jason D. Walker1, David G. Oppenheimer2, Joshua Concienne1 and John C. Larkin1,*
1Department
2Department
*Author
of Biological Sciences, Louisiana State University, Baton Rouge, LA 70803, USA
of Biological Sciences and Coalition for Biomolecular Products, University of Alabama, Tuscaloosa, AL 35487, USA
for correspondence (e-mail: [email protected])
Accepted 3 July; published on WWW 22 August 2000
SUMMARY
Cell differentiation is generally tightly coordinated with the
cell cycle, typically resulting in a nondividing cell with
a unique differentiated morphology. The unicellular
trichomes of Arabidopsis are a well-established model for
the study of plant cell differentiation. Here, we describe a
new genetic locus, SIAMESE (SIM), required for
coordinating cell division and cell differentiation during the
development of Arabidopsis trichomes (epidermal hairs). A
recessive mutation in the sim locus on chromosome 5 results
in clusters of adjacent trichomes that appeared to be
morphologically identical ‘twins’. Upon closer inspection,
the sim mutant was found to produce multicellular
trichomes in contrast to the unicellular trichomes produced
by wild-type (WT) plants. Mutant trichomes consisting of
up to 15 cells have been observed. Scanning electron
microscopy of developing sim trichomes suggests that the
cell divisions occur very early in the development of mutant
trichomes. WT trichome nuclei continue to replicate their
DNA after mitosis and cytokinesis have ceased, and as a
consequence have a DNA content much greater than 2C.
INTRODUCTION
Even a casual inspection reveals that patterns of cell division
in plants are highly organized. Division is concentrated
in meristematic regions exhibiting distinctly nonrandom
division patterns. For example, shoot meristems are organized
into recognizable cell layers maintained by the orientation of
cell division planes (Steeves and Sussex, 1989). Cell
differentiation typically occurs following cessation of the
mitotic cell cycle, and the final morphology is generated
by polarized cell expansion. These observations suggest that
cell division patterns are tightly coupled to the generation of
form.
In contrast, a number of observations have shown that plant
morphogenesis is surprisingly independent of cell division
patterns (Kaplan and Hagemann, 1991). Embryo development
and organogenesis can proceed in the absence of fixed cell
lineages (Poethig and Sussex, 1985; McDaniel and Poethig,
1988; Irish and Sussex, 1992). In a now classic series of
studies, the initial development of leaf primordia and lateral
This phenomenon is known as endoreduplication.
Individual nuclei of sim trichomes have a reduced level
of endoreduplication relative to WT trichome nuclei.
Endoreduplication is also reduced in dark-grown sim
hypocotyls relative to WT, but not in light-grown
hypocotyls. Double mutants of sim with either of two other
mutants affecting endoreduplication, triptychon (try) and
glabra3 (gl3) are consistent with a function for SIM in
endoreduplication. SIM may function as a repressor of
mitosis in the endoreduplication cell cycle. Additionally,
the relatively normal morphology of multicellular sim
trichomes indicates that trichome morphogenesis can occur
relatively normally even when the trichome precursor cell
continues to divide. The sim mutant phenotype also has
implications for the evolution of multicellular trichomes.
Key words: Trichome, Arabidopsis, Endoreduplication,
Endoduplication, Cell cycle, Morphogenesis, Differentiation, Plant
development, Leaf
root primordia was shown to proceed even when mitosis was
blocked (Haber, 1962; Foard et al., 1965; Foard, 1971). In
more-recent genetic studies, mutants in maize (Smith et al.,
1996; Reynolds et al., 1998) and Arabidopsis (Torres-Ruiz and
Jürgens, 1994; Traas et al., 1995) have been identified that
dramatically alter the orientation of cell divisions without
causing major changes in the shape of the resulting tissue. In
addition, experimental manipulation of regulatory components
of the cell cycle has revealed that plants have a remarkable
ability to compensate for altered cell division rates. Hemerly
and co-workers (Hemerly et al., 1995) introduced a dominantnegative mutant of a mitotic cyclin-dependent kinase gene into
transgenic tobacco plants, which resulted in plants with
essentially normal morphology, but fewer and larger cells. In
another study, overexpression of a mitotic B-cyclin in
Arabidopsis resulted in an increase in the number of cells in
the roots (Doerner et al., 1996). Although the roots were
longer, they were otherwise morphologically normal. These
observations suggest that cell division patterns may not play a
direct causal role in morphogenesis.
3932 J. D. Walker and others
The majority of these studies have been at the level of whole
organ or tissue development, and much less is known about
coordination of morphogenesis and cell division at the singlecell level. One of the most thoroughly studied plant cell
differentiation pathways is the development of trichomes (leaf
hairs) in Arabidopsis thaliana (Marks, 1997; Hülskamp et al.,
1999). Arabidopsis trichomes are single cells that extend from
the epidermis of leaves and stems. On leaves, these cells have
an unusual branched shape resulting from a dramatic program
of cellular morphogenesis. On stems, trichomes are generally
unbranched. The single nucleus of a wild-type (WT) trichome
continues to replicate its genomic DNA during differentiation,
reaching nuclear DNA levels of 20C to 32C (Melaragno et
al., 1993; Hülskamp et al., 1994), a process known as
endoreduplication. Many genes involved in trichome
development have been identified and several of these affect
endoreduplication. Mutations at the glabra3 (gl3) locus reduce
endoreduplication, trichome branching and trichome cell size
(Hülskamp et al., 1994). In contrast, mutations at the triptychon
(try) and kaktus (kak) loci increase endoreduplication, trichome
branching and trichome cell size (Hülskamp et al., 1994).
Trichomes of try mutants also occur in clusters in the
epidermis, suggesting that the TRY gene plays a role in
determining trichome cell fate in addition to its role in trichome
differentiation. The GLABRA1 (GL1) gene is another cell fate
determination gene that may also play a role in the control
of endoreduplication, although published evidence is
contradictory (Schnittger et al., 1998; Szymanski and Marks,
1998).
Endoreduplication is an alternate version of the cell cycle
that occurs in a wide variety of organisms (Nagl, 1976; Barlow,
1978; Traas et al., 1998). During endoreduplication cycles
(endo cycles), nuclear DNA is replicated without mitosis or
cytokinesis, resulting in cells with a DNA content much greater
than 2C. In angiosperms, endoreduplication is particularly
common and occurs in a wide variety of tissues and cell types
(Barlow, 1978; Galbraith et al., 1991; Gendreau et al., 1997),
including agriculturally important tissues such as maize
endosperm (Kowles and Phillips, 1985). Animal cell types that
undergo endoreduplication include many Drosophila larval
tissues (Orr-Weaver, 1994), some molluscan neurons (Chase
and Tolloczko, 1987), mammalian megakaryocytes
(Angchaisuksiri et al., 1994) and placental trophoblast cells
(Sarto et al., 1982). The function of endoreduplication is
unknown, although proposed roles include gene amplification,
radiation resistance and cell differentiation (Nagl, 1976;
Barlow, 1978). In plants, there is often a correlation between
the final volume of a differentiated cell and its DNA content
(Barlow, 1978; Melaragno et al., 1993), but evidence of a
functional link between DNA content and cell volume is
lacking. The control of endoreduplication, and its relationship
to cell differentiation and morphogenesis, is unclear, and until
recently, has been little studied.
In this study, we describe a mutation in a newly identified
gene, SIAMESE (SIM), that results in multicellular trichomes,
in contrast to the unicellular trichomes of WT plants. Nuclei
in the multicellular trichomes have reduced levels of
endoreduplication. Double mutants between sim and either gl3
or try are consistent with a role for sim in endoreduplication.
SIM appears to encode a repressor of mitosis required for
normal trichome endo cycles.
MATERIALS AND METHODS
Plant growth and mutant isolation
All plants were grown as described previously (Larkin et al., 1999),
except that for studies of hypocotyl endoreduplication, surfacesterilized seeds were sown on agar containing Murashige and Skoog
salts, and the plates were incubated vertically for eight days, either in
continuous light or in darkness. Dark-grown conditions were obtained
by wrapping the plates in aluminum foil and placing them in a drawer.
The sim mutant was identified in a screen of ethyl-methanesulfonate
(EMS) mutagenized wild-type Columbia M2 seeds purchased from
Lehle Seeds (Roundrock, TX). The initial mutant was crossed to
Columbia (Col-1) wild-type once, backcrossed once and sim mutant
plants were selected from the self-progeny of the backcrossed plants.
No phenotypes other than the sim phenotype were observed
segregating in any families derived from the original sim mutant.
These plants were used for the initial studies. An additional backcross
has now been performed, and the sim phenotype in the self-progeny
of this backcross is indistinguishable from the phenotype of first
backcross sim plants. Other Arabidopsis strains used in this study
were gl3-1 Landsberg erecta (Ler) background, Koornneef et al.,
1982), a gl3-1 line that had been crossed three times to Col before
selfing (J. L. and J. W., unpublished), try-JC (Col background, Larkin
et al., 1999) and a 35SGUS::Ac line (Lawson et al., 1994; Larkin et
al., 1996).
Complementation and genetic mapping
For the complementation test, sim was crossed to try-JC plants, and
the resulting F1 plants were examined. sim fully complemented the
recessive trichome clustering phenotype of try. The increased
trichome branching phenotype of try is incompletely dominant (J. C.
L., unpublished). sim/+ try/+ heterozygotes exhibited a slight increase
in branching, indistinguishable from that observed in try/+
heterozygotes. In the F2 derived from the double heterozygotes, 39
WT, 12 sim, 12 try, and 8 sim try plants were observed (χ2=1.9, P>0.5
for 9:3:3:1 segregation ratio), demonstrating that sim and try were
unlinked.
Plants for mapping experiments were produced from crosses
between homozygous sim plants and WT plants of the either the RLD
or Ler ecotypes. Total nucleic acids were prepared from F2 plants that
had the sim phenotype, and PCR amplification with primer-pairs for
various molecular markers was performed essentially by standard
methods (Bell and Ecker, 1994). Molecular markers tested for linkage
were nga63, nga128, nga168, nga6, nga8, nga172, nga1139, AG,
DET1, AtS0191, nga249, and nga158 (Konieczny and Ausubel, 1993;
Bell and Ecker, 1994). Significant linkage was found between sim and
nga158 (8 of 128 chromosomes recombinant, χ2=98.0, P<0.005).
Map order was determined by the pattern of recombination with the
linked marker nga249. None of the other markers showed significant
linkage. sim has been further mapped to two BAC clones distal to
nga158 that total approximately 200kb. We currently have 20 sim
recombinants mapping within this region, and all of them retain all
aspects of the sim phenotype. It is thus likely that the sim phenotype
is due to a mutation in a single locus.
Phenotypic analysis
Cryo-scanning electron microscopy (see Figs 1C, 2, 5) was performed
as described by Luo and Oppenheimer (1999). The scanning electron
microscopy in Fig. 1A,B was performed as previously described
(Larkin et al., 1999). All images were electronically processed in
Adobe Photoshop, adjusting only for brightness and contrast. Samples
for anatomical sectioning were fixed, embedded, sectioned and
stained essentially as described by Fernandez and co-workers
(Fernandez et al., 2000), using standard methods (Ruzin, 1999).
For 4′,6-diamidino-2-phenylindole (DAPI) staining, leaves were
fixed in FAA (50% ethanol, 5% glacial acetic acid, 10% formalin) and
stained for 15 minutes to 2 hours with 20 µg/ml DAPI in McIlvaine’s
Multicellular trichomes in Arabidopsis 3933
buffer (60 mM citric acid, 80 mM Na2HPO4, pH 4.1), then washed
three times for 15 minutes each with McIlvaine’s buffer. Samples were
mounted in 50% glycerol in McIlvaine’s buffer and observed via
epifluorescence on a Nikon Microphot FXA microscope.
Fluorescence of individual nuclei was measured with a Nikon spot
photometer attachment. Background readings from an area in the
specimen without a nucleus were subtracted from the fluorescence of
each nucleus prior to further analysis. In some experiments,
fluorescence from guard cell nuclei in epidermal peels mounted on
the same slide were obtained.
For deconvolution microscopy, samples stained with DAPI and
mounted as described above were observed with a 63× oil immersion
objective on a Leica DMRXA Research Microscope. Data were
collected and analyzed using the Slidebook software from Intelligent
Imaging Innovations (Denver, CO). Nuclear volumes were estimated
from nuclear diameters assuming that the nucleus was roughly
spherical, which appears to be true for trichome and epidermal cell
nuclei in the early developmental stages examined here.
Guard cell nuclei have been reported to have a 2C DNA content
(Melaragno et al., 1993), and have often been used to standardize
trichome DNA values. However, we have obtained variable results
when normalizing to guard cell relative fluorescence units. In our
hands, guard cell nuclei have a fluorescence level only slightly above
background, which results in a small number that includes a relatively
large amount of variation. Much more consistent results were obtained
when samples were normalized to the mean fluorescence intensity of
a sample of Col trichome nuclei included in the same experiment. For
the trichome experiments reported here, measured fluorescence levels
for individual nuclei were divided by the mean value for Col trichome
nuclei, and the resulting number was multiplied by 32. Although our
data are expressed as relative fluorescence units (RFU) normalized to
Col, we have artificially set Col at 32 RFU. Our RFU values should
thus be roughly comparable with the C values reported by others. A
Col trichome nuclear DNA content of 32C is consistent with the
results we have obtained in experiments where guard cell DNA
content was measured. Data for epidermal pavement cells and
hypocotyl nuclei were normalized similarly, using mean C values of
pavement cells from Fig. 2A of Melaragno et al. (1993) and of lightgrown hypocotyls from Fig. 3A of Gendreau et al. (1998).
Because the DNA content of endoreplicated nuclei is unlikely to
follow a normal distribution, pairwise nonparametric Mann-Whitney
U-tests were performed for the genetically relevant comparisons using
the Statistica software package (StatSoft, Inc., Tulsa, OK). The
standard Bonferroni technique was used to obtain P values corrected
for the multiple comparisons conducted (Rice, 1989). The results of
one set of experiments are reported here; all results on trichome
nuclear DNA content have been replicated in at least one independent
experiment.
Analysis of L1 clonal sectors
For the production of clonal sectors, a sim 35SGUS::Ac strain was
constructed by crossing sim plants with a previously described
35SGUS::Ac transgenic line (Lawson et al., 1994). Leaf tissue was
histochemically stained for β-glucuronidase (GUS) activity as
described previously (Larkin et al., 1996), and sectors on first leaves
were examined with DIC optics at 200× magnification. Sectors were
chosen for analysis if they extended at least 50% of the length of the
leaf. This type of sector results from transposon excision prior to the
initiation of trichome development (Larkin et al., 1996). Trichome
clusters were included in the analysis if at least one trichome in the
cluster was stained, and at least one trichome in the cluster was
immediately adjacent to unstained cells (i.e., was at the edge of the
stained sector).
To estimate the probability that a clonal sector boundary would be
expected to include only one trichome in a cluster of two trichomes,
we took advantage of the observation that at the time of trichome
initiation, cells in the epidermis approximate a hexagonally packed
array, i.e., each cell is surrounded by an average of six neighbors
(Larkin et al., 1996). In this case, a pair of adjacent trichomes (the
cluster) would be surrounded by eight other cells. It is straightforward
to calculate that a total of 262 combinations occur (Boas, 1966) in
which both trichomes are labeled, and one, two, three, four, five, six
or seven of the surrounding cells are labeled. These combinations
correspond to the number of ways that a sector boundary can pass
adjacent to a cluster of two trichomes and include both trichomes
within the GUS-expressing sector. It is more difficult to calculate the
number of combinations in which only one of the trichomes is
included in the sector, owing to the complication that the stained cells
must be contiguous. However, direct counting revealed a minimum of
at least 224 ways that a sector could include only one trichome of the
cluster. Thus, there are a total of at least 486 ways that a developing
trichome cluster could contact the edge of a clonal sector. Thus we
would expect that in a minimum of 224/486, or 46%, of trichome
clusters at a sector border, the sector boundary would pass between
the two trichomes in the absence of any constraint on the clonal
relationship of the two trichomes.
Construction of double mutants
Double mutants were produced by crossing the individual mutant
homozygotes and examining trichome phenotypes in the F2.
Suspected double mutants were crossed individually to each parental
mutant and confirmed by the absence of complementation in the
resulting progeny. The double mutants were also allowed to self, and
the self-progeny scored to show that the double mutant phenotype
breeds true.
RESULTS
sim mutants produce multicellular trichomes
On WT leaves, virtually all trichomes occur singly (Fig. 1A).
Instances where two or more adjacent trichomes occur with
no intervening cells have been termed trichome clusters
(Larkin et al., 1994). A mutant producing frequent clusters of
trichomes was identified in a screen of EMS-mutagenized
plants (Fig. 1B). Because many of the mutant trichome
clusters looked like ‘twins’ with trichomes of nearly identical
morphology joined at the base, the gene identified by this
mutation was named SIAMESE (SIM). Approximately 65% of
the trichomes on sim first leaves were adjacent to another
trichome (Table 1); however, the number of sites on the leaf
Table 1. Number of trichomes, trichome initiation sites
(TIS) and percentage of trichomes in clusters
Genotype
Trichomes per leaf
(mean±s.d.)
TIS per leaf
(mean±s.d.)
% in clusters
Col
sim
gl3*
try
sim gl3‡
sim try
28.4±4.8
47.3±9.8
23.3±5.3
30.7±5.2
16.9±5.31
n.c.§
28.3±4.7
31.9±6.06
21.9±5.1
27.4±4.8
3.5±5.2
26.2±2.3
0.7
5.3
10.7
27.4
41.4
>99
Data are based on counts of all of the trichomes on the adaxial surface of
ten first leaves per genotype. Trichomes were considered to be in a cluster if
they were immediately adjacent to another trichome with no intervening cells.
*gl3-1 allele crossed three times to Col before selfing.
‡gl3-1 allele in Ler background was used in construction of the double
mutant.
§n.c., not counted. The trichomes associated with each sim try cluster were
too crowded to count (Fig. 5D). Most TIS of this genotype appeared to
contain four to ten trichomes that were in contact with other epidermal cell.
One instance of a solitary trichome was observed in this sample.
3934 J. D. Walker and others
Fig. 2. Cross sections through sim multicellular trichomes.
(A) Multicellular trichome with relatively normal morphology.
(B) Multicellular trichome with altered morphology and several
division planes. Arrow indicates a cell junction similar to those
indicated in Fig. 1B. Bar, 75 µm.
Fig. 1. siamese (sim) trichomes are multicellular. (A) A WT
unicellular trichome. (B) A cluster of two sim trichomes, each of
which consists of two cells. Cell junctions are indicated by white
arrows. (C) Close-up of basal junction of two sim trichomes.
(D) DAPI-stained multicellular stem trichome with six nuclei,
separated by cell walls. Bars, 100 µm (A); 100 µm (B); 15 µm (C).
containing at least one trichome (trichome initiation sites, TIS)
was approximately the same as was seen on WT leaves. No
other aspects of plant growth and development appeared to
be affected. In particular, fertility, leaf expansion, and
development of other epidermal cell types, root hairs and root
cortical cells all appeared to be unaffected by the mutation.
The sim phenotype superficially resembled the trichome
clustering phenotype of try mutants. However, unlike try, the
individual trichomes in each cluster were typically joined well
above the epidermal surface (Fig. 1C). Additionally, try
trichomes had an increased number of trichome branches,
whereas sim trichomes had a WT branching pattern.
Complementation tests and segregation analysis demonstrated
that sim was not allelic to try (see Materials and Methods). The
sim gene was mapped to a position approximately 6 cM distal
to nga158 on chromosome 5.
Closer inspection revealed that many of the sim trichomes
were multicellular (Fig. 1B,D; Fig. 2A,B – arrows in Fig. 1B
indicate cell junctions). Fig. 2A shows a cross section of a
multicellular trichome of relatively normal morphology similar
to the trichomes in the cluster shown in Fig. 1B, while Fig. 2B
shows a cross section of a trichome with distorted morphology
and relatively disorganized planes of division. On stems, as
many as 15 cells per trichome have been observed, although
most leaf trichomes are composed of only two or three cells.
Staining of trichomes with the fluorescent DNA stain DAPI
demonstrated that each of the cells in a multicellular trichome
has its own nucleus, and each cell in a multicellular sim
trichome has only a single nucleus (Fig. 1D).
Development of sim trichomes
Cell division has never been observed in hundreds of
developing WT trichomes that have been examined (J. C. L.
and D. G. O., unpublished). In contrast, cell division was often
observed early in the development of sim trichomes (Fig. 3A).
Divisions occurring shortly after initiation of trichome
development appeared to result in two fully formed adjacent
trichomes, producing the trichome clusters that were initially
observed (Figs 1B,C, 3B). Divisions occurring later resulted
either in cells that reiterated the trichome branching pattern and
other aspects of the morphogenic program while attached to
another trichome cell (Fig. 3C), or in multicellular trichomes
with relatively normal morphology (Figs 1B, 3D). These
divisions did not appear to affect later stages of trichome
differentiation such as branching and secondary cell wall
thickening, although some cell boundaries within a mutant
trichome did appear to be correlated with branch junctions
Fig. 3. Development of sim trichomes. (A) Early stage of
development, showing evidence of recent cell divisions in a
developing trichome. (B) Slightly later stage in the development of a
sim trichome cluster. (C) Same stage as B, single trichome composed
of two cells. Upper cell is reiterating trichome branching pattern.
(D) Various stages of sim trichome development, including later
stages. Arrow indicates cell junction. Bars, 10 µm (A); 15 µm (B);
15 µm (C); 50 µm (D).
Multicellular trichomes in Arabidopsis 3935
Fig. 4. sim trichome clusters are clonal in origin. A GUS-stained
clonal sector in the epidermis resulting from Ac transposon excision
is outlined on the surface of the leaf. This sector ran from the base of
the leaf to a point on the leaf margin about 60% of the way to the tip.
All four trichomes in the cluster are stained. The dotted line indicates
where the sector boundary passes behind the four clustered
trichomes. Guard cells are particularly useful in following sector
boundaries, because guard cells and trichomes stain more intensely
than other cell types. White arrows show unstained guard cells
outside the sector. Black arrows show stained guard cells within the
sector. An independent stained vascular sector passes below the
epidermal sector, and staining is also visible in the mesophyll in the
upper right. Note that the guard cells above the stained mesophyll
cells are not stained, indicating that this staining is subepidermal.
These two subepidermal sectors are not relevant to the analysis of the
epidermal sector.
(Fig. 1B). On leaves, there was no obvious pattern to the cell
division planes. On stems, where no branching occurs, adjacent
trichomes were observed, presumably resulting from early cell
divisions, but the later planes of division were always oriented
periclinally (parallel to the surface), resulting in linear chains
of cells (Fig. 1D).
Trichome clusters in sim mutants are clonal lineages
Two different models can be invoked to explain the clusters of
adjacent trichomes observed on sim mutants. If they result from
mitotic cell divisions after the initiation of trichome
development, then all of the trichome cells in a cluster should
be immediate clonal siblings. Alternatively, trichome clusters
may result from a failure of cell signaling during the selection
of trichome precursor cells. The latter model appears to explain
the trichome clusters observed in try mutants (Schnittger et al.,
1999). Although observations of developing sim trichomes
(Fig. 3) would favor the first model, many regulatory factors
Fig. 5. Relative DNA content of DAPI-stained trichome nuclei of
single and double mutants. (A) Col (WT), (B) sim, (C) gl3, (D) sim
gl3, (E) try, (F) sim try. The degree of fluorescence is expressed in
terms of relative fluorescence units (RFU) normalized to the mean
fluorescence of Col. These RFU values have been adjusted to be
roughly comparable to published C values (see Materials and
Methods), but they should only be interpreted as relative
comparisons, not as measurements of absolute DNA contents.
are known to play a role in multiple processes in a
developmental pathway, and we considered the possibility that
SIM plays a role in the selection of trichome precursor cells in
addition to its role in regulating mitosis.
Genetically marked clonal sectors resulting from the
excision of an Ac transposon from a 35SGUS transgene were
used to test whether all adjacent trichomes in a cluster were
clonally related, as described previously (Larkin et al., 1996).
Thirty-eight instances were observed in which a trichome
cluster was located at the border of a GUS-stained sector and
at least one trichome was stained. If there were no constraints
on cell lineage of the cluster, a simple model (see Materials
and Methods) suggests that we would expect 46%, (17.5) of
the clusters to include trichome cells from the adjacent
unstained cell lineage. None of the 38 clusters included any
unstained trichomes, a significant difference from the
3936 J. D. Walker and others
Fig. 6. Nuclear size of WT and sim trichome nuclei during early
trichome development in the leaf protoderm. Arrows indicate
trichome nuclei; arrowheads show undifferentiated protodermal
nuclei. (A) sim protoderm containing a cluster of three developing
trichomes. The trichome closest to the bottom edge has begun to
expand out from the plane of the epidermis (stage 2 of Szymanski et
al., 1998), while the remaining two trichomes in the cluster are
dome-shaped (stage 1 of Szymanski et al., 1998). (B) WT protoderm
containing a stage 1 trichome. In both (A) and (B), the trichome
nuclei are larger than surrounding undifferentiated protodermal cells.
Bar, 10 µm.
expectation for the null hypothesis of no clonal constraint
(χ2=32.4, P<0.001).
It is important to recognize that the model of clonal
boundaries used to generate the expected value used here is an
idealization. For example, real sectors are not oriented at
random on the leaf, and this could bias the frequency with
which all of the trichomes in a cluster would be included within
the sector. However, we have noted that the individual
trichomes in sim clusters appear to be randomly oriented
with respect to the leaf axis (J. W. and J. L., unpublished
observations), which would tend to counteract bias of this type.
Also, some of the observed trichome clusters included more
than two trichomes, which would tend to increase the chance
that one or more trichomes would not be included in the sector.
A particularly striking example of a cluster of four trichomes
at a sector boundary, all stained, is shown in Fig. 4. In this case,
and in several others, the sector boundary appears to make a
substantial detour to include all of the trichomes in the cluster.
Taken together, these results indicate that sim trichome clusters
arise through divisions of a trichome precursor cell, and
provide no evidence in favor of a role for sim in signaling
pathways involved in the selection of precursor cells.
sim mutants have altered endoreduplication
WT trichomes have been reported to have nuclear DNA levels
of approximately 20C to 32C (Melaragno et al., 1993;
Hülskamp et al., 1994). In three independent experiments, we
found that sim nuclei had significantly less DNA than WT
(P<0.001). The results of one such experiment are shown in
Fig. 5. These data indicate that sim trichome nuclei have about
one-third the DNA content of WT trichome nuclei (Fig. 5A,B).
Nuclei of multicellular trichomes had slightly less DNA than
nuclei of unicellular sim trichomes (Table 2). Within the
multicellular trichomes, there did not appear to be a difference
in nuclear DNA content along the proximo-distal axis of the
trichome (Table 2). Nuclei of the cells attached directly to the
epidermis (basal cells) did not have a significantly different
DNA content than nuclei of other cells within the multicellular
trichomes (P=0.755).
Fig. 7. Relative DNA content of DAPI-stained hypocotyl nuclei of
WT and sim grown in the light or dark. (A) A WT DAPI-stained
dark-grown hypocotyl. (B) A sim DAPI-stained dark-grown
hypocotyl. (C-F) represent the pooled measurements of nuclei of the
hypocotyl epidermis and cortex of the following genotypes and
treatments: (C) WT grown in light, (D) sim grown in light,
(E) WT grown in dark and (F) sim grown in dark. RFU, relative
fluorescence units.
In trichomes of morphological stage 1 and early stage 2, as
described by Szymanski et al. (1998: stage 1, radial expansion
within epidermis; stage 2, expansion from epidermis prior to
branching), both sim (Fig. 6A) and WT (Fig. 6B) developing
trichomes had nuclear volumes of approximately 100-150 µm3,
in contrast to ordinary protodermal cells showing no signs of
trichome development (Fig. 6A,B), which had nuclear volumes
of approximately 12 to 36 µm3. Several of the sim developing
trichomes observed were in clusters of adjacent trichomes (Fig.
6A), and their nuclei had similar nuclear volumes to isolated
sim trichomes and WT trichomes. We did not observed a
Multicellular trichomes in Arabidopsis 3937
Fig. 9. Model for role of SIM in
endoreduplication. SIM is
hypothesized to encode a repressor
of mitosis that is necessary to
establish the endo cycle. GL3 is
hypothesized to function in the endo
cycle by activating and inhibiting S
phase, respectively. Pointed arrows
indicate activation, and blunt arrows
indicate inhibition.
Fig. 8. Double mutant phenotypes. (A) gl3 trichome, (B) sim gl3
trichome cluster with a multicellular trichome (arrow),
(C) try trichome cluster and (D) sim try trichome cluster. Bars, 100
µm (A); 70 µm (B); 100 µm (C); 100 µm (D).
mutant trichome just after a division that generates a true
multicellular trichome in this analysis (i.e., not just a cluster of
trichomes, but rather multicellular in the aerial part of the
trichome). Thus, although it is clear that sim trichomes can
undergo some degree of endoreduplication early in trichome
development, we do not yet understand the timing of the
divisions generating the aerial multicellular class of trichomes
relative to the timing of endoreduplication.
We have also examined endoreduplication in other tissues.
Epidermal pavement cell nuclei undergo endoreduplication,
although to a lesser degree than trichome nuclei (Melaragno et
al., 1993). In contrast to the situation for trichome nuclei, sim
epidermal pavement cell nuclei did not have a decreased
amount of DNA relative to WT (Table 2, P=0.21).
Another tissue with a well-documented pattern of
endoreduplication is the Arabidopsis hypocotyl (Gendreau et
al., 1997). When grown in the dark, WT hypocotyl cells
undergo two to three rounds of endoreduplication, while in the
Table 2. Endoreduplication in multicellular and
unicellular sim trichomes and epidermal pavement cells
Cell type
sim trichomes
unicellular
multicellular (all)*
basal cell
other cells
WT trichomes (unicellular)
sim pavement cells
WT pavement cells
Mean RFU±s.d.
5.5±11.11
11.1±8.1
11.3±8.9
11.0±7.4
32.0±15.8
3.9±9.6
3.4±2.0
Median RFU
2.2‡
9.1‡
8.2
9.9
30.1
2.81
3.1
light, they undergo approximately one less round of
endoreduplication. A phytochrome-dependent pathway acts to
repress one round of endoreduplication in the light (Gendreau
et al., 1998). WT and sim hypocotyl epidermal and cortical cell
nuclei had similar levels of DNA when grown in the light
(Fig. 7C,D; Table 3). However, when grown in the dark,
sim hypocotyl nuclei failed to undergo the additional
endoreduplication seen in WT hypocotyl nulcei grown in the
dark (Fig. 7A,B,E,F; Table 3). The sim mutant thus affects at
least one endoreduplication pathway in addition to trichomes,
but some endoreduplication pathways remain unaffected by the
mutant.
Genetic interactions of sim with two mutations that
alter endoreduplication
Double mutants were constructed between sim and two
mutations that affect endoreduplication, gl3 and try. Trichomes
on gl3 plants were smaller, had reduced branching, and reduced
nuclear DNA content relative to WT (Figs 5C, 8A). Trichomes
on gl3 plants also exhibited more clustering than WT trichomes
(Table 1). Trichomes on sim gl3 plants resembled gl3
trichomes, except that some multicellular trichomes occurred
(Fig. 8A,B, arrow indicates a cell junction). The frequency of
trichome clusters on leaves of sim gl3 plants was 41.4%, a
value intermediate between that of the gl3 and sim single
mutants (Table 1). The degree of endoreduplication in sim gl3
trichome nuclei was significantly less than that of the gl3 single
mutant (Fig. 5C,D; P<0.01), and was greater than that of
the sim single mutant although not significantly (Fig. 5B,D;
P=0.185). The primarily additive nature of the sim gl3 double
n
33
80
38
42
42
68
102
n, number of nuclei; RFU, relative fluorescence units (normalized to the
WT mean as described in Materials and Methods). The basal cell in a sim
multicellular trichome are the cell that is directly attached to the epidermis.
*In this analysis, trichomes were considered multicellular only if the aerial
parts of the trichome were multicellular, i.e., trichomes in clusters joined at
the base were included in the unicellular class.
‡These two samples differ significantly (P=0.045).
Table 3. Hypocotyl nuclear DNA content and hypocotyl
length in sim and WT plants grown in the light or in the
dark
Genotype
WT
sim
Treatment
Light
Dark
Light
Dark
Mean nuclear RFU
±s.d. (median)
5.6±2.9 (5.0)*
8.2±3.1 (8.1)*,‡
5.9±3.3 (5.8)
4.9±3.1 (4.2)‡
n
56
49
92
56
Mean hypocotyl
length (mm)±s.d.
2.2±0.4
22.1±2.8
2.4±0.3
24.4±2.2
Fluorescence of epidermal and cortical nuclei of hypocotyls was measured
as described in Materials and Methods. RFU, relative fluorescence units,
normalized to the WT mean, as described in Materials and Methods.
*These two samples differ significantly (P<0.001).
‡These two samples differ significantly (P<0.001).
3938 J. D. Walker and others
mutant phenotype suggests that these two genes reduce
endoreduplication via different mechanisms.
The phenotype of sim try trichomes was particularly
striking. try single mutants produce some clusters of adjacent
trichomes, apparently by affecting cell signaling during
trichome initiation (Schnittger et al., 1999). In addition, try
had effects on later aspects of trichome development,
resulting in increased cell size, increased branching and
increased nuclear DNA content (Figs 5E, 8C). The sim try
double mutants produced large clusters of highly
multicellular trichomes (Fig. 8D). Virtually all of the leaf
trichomes on these plants were in clusters, a higher frequency
than was observed in either individual mutant (Table 1). The
sim try double mutants exhibited a relative DNA content
similar to that found in the sim single mutant (Fig. 5B,F;
P=0.56), and much less than the increased DNA content of
try mutants (Fig. 5E,F; P<0.001). This indicates that sim is
epistatic to try with regard to the degree of endoreduplication.
These results are consistent with the hypothesis that sim and
try function in the same pathway.
DISCUSSION
The presence of numerous multicellular trichomes on sim
plants (Figs 1, 2, 3) demonstrates that trichome differentiation
is not dependent upon the cessation of cell division.
Furthermore, the relatively normal morphology of some
multicellular sim trichomes indicates that cellular
morphogenesis can occur surprisingly normally in the
presence of continued cell division. In unbranched stem
trichomes, division planes are periclinal once the trichome
has begun to expand. Trichome clusters do occur on the stem,
perhaps because anticlinal division planes were already
established in some cells prior to commitment to the trichome
pathway. In multicellular branched leaf trichomes, division
planes are much more variable, perhaps because the
branching mechanism interferes with the regular
establishment of division planes. These results are consistent
with the view that cell division is not essential for the
generation of plant form, even at the cellular level (Kaplan
and Hagemann, 1991).
A simple model to explain the sim mutant phenotype is
shown in Fig. 9. SIM is proposed to encode a repressor of
mitosis that functions in the establishment of the
endoreduplication cell cycle. In addition to the observation
that sim trichomes continue to divide, several lines of
evidence support this model. Trichome clusters in sim
are clonal, consistent with the possibility that they are
generated by division of a committed trichome precursor
cell. The DNA content of sim trichome nuclei is reduced
relative to WT, consistent with a role in the endo cycle.
Although we have not measured C values of nuclei directly
(see Materials and Methods), comparison of our data with the
data of others suggests that most sim trichome nuclei are
arrested with DNA contents of 4-8C. The sim mutation is also
epistatic to try with regard to endoreduplication, consistent
with the hypothesis that these two genes function in the same
pathway.
Although sim affects endoreduplication in trichomes, not all
endoreduplication pathways are affected. Endoreduplication in
epidermal pavement cells is unaltered, as is endoreduplication
in light-grown hypocotyls. However, the additonal level of
endoreduplication seen in dark-grown hypocotyls is absent in
sim mutants. We do not yet know if sim is interfering with a
specific phytochrome-repressed pathway in hyptocotyl
development (Gendreau et al., 1998), or whether it is disrupting
endoreduplication by a more general mechanism. This question
is currently under investigation.
The endoreduplication cell cycle has been most thoroughly
studied in Drosophila melanogaster (Orr-Weaver, 1994). The
first larval endo cycles begin after mitosis 16 during embryo
development. Endoreduplication occurs in tissue-specific
domains, and S phases alternate with a single G phase,
suggesting that endo cycles are highly regulated (Smith and
Orr-Weaver, 1991). Cyclin E appears to play an important role
in promoting endo cycles (Knoblich et al., 1994; Sauer et al.,
1995), while escargot, cdc2, Cyclin A and Myb appear to act
as inhibitors of endoreduplication (Sauer et al., 1995; Hayashi,
1996; Katzen et al., 1998). In addition, fizzy-related functions
as an inhibitor of mitotic cyclins during endo cycles, and has
been proposed to be involved in the degradation of these
cyclins (Sigrist and Lehner, 1997).
Little was known about the control of endoreduplication in
plants until recently (Traas et al., 1998). In maize endosperm,
biochemical evidence has been obtained for two separable
endoreduplication-promoting factors: an M phase inhibitor
and an increase in S phase-related protein kinase activity
(Grafi and Larkins, 1995). In addition, altered
phosphorylation of a retinoblastoma homolog correlates with
the onset of endoreduplication (Grafi et al., 1996). In animals,
the phosphorylation state of retinoblastoma protein controls
entry into S phase. The Arabidopsis thaliana cyclindependent kinase cdc2b is expressed in endoreplicating cells,
and may play a role in this process (Segers et al., 1996). These
studies suggest that the control of endoreduplication in plants
is similar to the situation in animals, with different factors
acting to stimulate S phase and inhibit M phase of the cell
cycle.
SIM is a good candidate for a gene encoding a component
of this mitosis-inhibiting pathway during the endo cycle,
similar to the role of fizzy-related in Drosophila (Sigrist and
Lehner, 1997). GL3 encodes a basic-helix-loop-helix protein
similar to the maize R gene, and probably functions in the
initiation of trichome development (M. Brown and J. C. L.,
unpublished). GL3 is a good candidate to function as a
transcription factor promoting S-phase during trichome
development (Fig. 9). If GL3 were necessary for
endoreduplication to occur, one might expect that GL3 function
would be required for the multicellularity resulting from the
sim mutation, i.e., gl3 would be epistatic to sim. This is in
contrast to the additive phenotype seen here (Figs 5B,C,D,
8A,B). However, at least one other R-like gene exists in the
Arabidopsis genome whose function probably overlaps in
function with that of GL3 (Payne et al., 2000), consistent with
the presence of some endoreduplication in gl3 trichomes.
Functional redundancy can result in additive phenotypes when
mutations acting in the same pathway are combined
(Martiensen and Irish, 1999).
The phenotype of the sim try double mutant is particularly
striking (Figs 5B,E,F, 8C,D). Although the nature of the TRY
gene product is unknown, we speculate that TRY may encode
Multicellular trichomes in Arabidopsis 3939
a factor that regulates the level of endoreduplication by acting
to inhibit endo S phase (Fig. 9). If SIM acts to inhibit mitosis
during each endocycle, then the sim mutation would convert
the extra rounds of endoreduplication due to the try mutation
into additional cells. One problem with this interpretation is
that try mutants produce only one additional round of
endoreduplication in trichomes, but sim try double mutants
appear to produce more than twice as many cells as sim
trichomes per TIS (Figs 1B, 3D, 8C,D; Table 1). It is possible
that in this double mutant, feedback mechanisms that normally
control the degree of endoreduplication are further disrupted.
We cannot rule out the possibility that the sim try phenotype
results from an interaction with the trichome branching aspect
of the try phenotype, or some unknown function of try other
than endoreduplication. Given the results of our clonal analysis
of sim trichome clusters (Fig. 4), however, it seems unlikely
that sim interacts with the proposed cell signaling function of
try during trichome initiation.
Of course, we do not yet know the molecular or biochemical
nature of the SIM gene and its mutation, and thus these genetic
interactions must be interpreted with some caution (Martiensen
and Irish, 1999). In addition, we only have a single sim allele.
The sim phenotype is very recognizable, but despite long-term
screening for trichome mutants involving the progeny of tens
of thousands of M1 plants, no additional alleles have been
recovered. During these screens, multiple alleles of most
known trichome genes have been identified. Thus, it is quite
possible that this allele is a partial loss-of-function allele, or
mutation in very specific domain of the gene product.
Nevertheless, the evidence presented here supports a model for
the regulation of trichome endoreduplication that is quite
consistent with the evidence from other systems indicating that
the endocycle is regulated at both the G1/S and G2/M
transitions. This dual control of the endo cycle may explain
why the sim mutation does not result in trichome ‘tumors’
exhibiting uncontrolled growth.
In addition, the sim mutant phenotype may have implications
for the evolution of trichome multicellularity. One other
example of induction of multicellular trichomes in a plant that
normally has unicellular trichomes occurs in cotton fibers,
where tissue culture in the presence of auxin and GA can cause
developing cotton fibers to divide (Van’t Hof and Saha, 1997).
Cotton fiber cells undergo endoreduplication during normal
development, similar to the case of Arabidopsis trichomes
(Van’t Hof, 1999). Both unicellular and multicellular trichomes
are widespread among angiosperms. The sim mutant
phenotype suggests that it may be surprisingly easy to switch
between unicellular and multicellular trichomes during
evolution, and that this switch could be accomplished relatively
independent of changes in trichome shape.
We wish to thank Ginger Brininstool, Mohamed Noor and Jim
Moroney for critical reading of the manuscript; Jolanta Nunley and
Olga Borkhsenious for expertise with scanning electron microscopy;
Margaret C. Henk and Ron Bouchard for additional technical
assistance with microscopy and image production; and Bill Platt,
Mohamed Noor and Charles Ramcharan for advice on probability and
statistical methods. We also wish to thank two anonymous reviewers
for helpful and constructive suggestions. J. C. L.’s research is
supported by Grant IBN 9728047 from the National Science
Foundation. D. G. O.’s research is supported by Grant GM 5-30753
from the National Institutes of Health.
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