Section of Cell and Developmental Biology, U.C. San Diego, 9500 Gilman Drive, La Jolla, CA 92093-0116, USA
* Author for correspondence (e-mail: lsmith{at}biomail.ucsd.edu)
Accepted 29 October 2002
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SUMMARY |
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Key words: Cell polarity, Cell morphogenesis, Maize leaf development, Mosaic analysis, BRICK1, HSPC300
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INTRODUCTION |
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Drug studies have demonstrated crucial roles for F-actin in tip growth, one
of which is to transport secretory vesicles containing cell wall components
along longitudinal F-actin bundles to the vicinity of the growth site
(Geitmann and Emons, 2000;
Hepler et al., 2001
). A fine
meshwork of cortical F-actin observed at or near the growth site in tip
growing cells is thought to have a function separate from that of the
longitudinal F-actin bundles, which is not well understood
(Geitmann and Emons, 2000
;
Hepler et al., 2001
).
Pharmacological and genetic studies have shown that F-actin also promotes the
expansion of diffusely growing cells
(Smith, 2003
). By analogy to
its well-established role in tip growth, it has been proposed that the primary
role of F-actin in diffusely growing cells is to guide the deposition of
secreted wall components (Baskin and
Bivens, 1995
; Dong et al.,
2001
).
As the principal structural component of the primary cell wall, cellulose
is thought to constrain cell expansion and thereby function as a key
determinant of shape in diffusely growing cells. Drug studies have shown that
normal patterns of diffuse growth also depend on microtubules, which, during
interphase, are arranged in the cell cortex in a pattern mirroring that of
cellulose deposition into the wall (Cyr,
1994). Although the precise nature of the relationship linking
microtubule organization to cellulose deposition pattern remains unknown, it
has been proposed that microtubules help to guide the deposition pattern of
cellulose microfibrils into the wall
(Giddings and Staehelin, 1991
;
Baskin, 2001
).
Some plant cells acquire complex shapes through multidirectional cell
expansion patterns involving both microtubule and actin-dependent mechanisms.
Three-branched trichomes that form on the Arabidopsis epidermis
provide a good example. Drug studies have established that microtubules, but
not actin filaments, play a crucial role in the initial polarized outgrowth of
trichomes and trichome branches. However, subsequent outgrowth of trichome
branches proceeds by means of an actin-dependent, diffuse growth process
(Szymanski et al., 1999;
Mathur et al., 1999
) (M.
Hülskamp, personal communication). Mutations disrupting various aspects
of trichome morphogenesis have identified dozens of genes required for the
proper shaping of this cell type (Bouyer et
al., 2001
). Molecular analysis of some of the corresponding genes
has begun to shed light on microtubule-dependent mechanisms involved in branch
formation (Oppenheimer et al.,
1997
; Burk et al.,
2001
; Folkers et al.,
2002
; Kim et al.,
2002
). Further molecular analysis of these and other gene products
yet to be identified will undoubtedly advance our understanding of both
microtubule- and F-actin-dependent mechanisms governing trichome
morphogenesis.
The lobed shapes of leaf epidermal and mesophyll cells provide another
example of a complex shape resulting from multidirectional cell expansion.
Lobes arise as polarized outgrowths at multiple sites along the margins of
cells whose overall size is increasing via diffuse growth. In lobe-forming
cells from a wide variety of species, emergence of lobes is associated with
reorganization of cortical microtubules into bands focused at lobe sinuses
(Jung and Wernicke, 1990;
Apostolakos et al., 1991
;
Wernicke et al., 1993
;
Panteris et al., 1993a
;
Panteris et al., 1993b
;
Panteris et al., 1994
;
Wasteneys et al., 1997
;
Qiu et al., 2002
;
Frank and Smith, 2002
).
Microtubule bands have been proposed to direct the localized deposition of
cellulose, creating periodic wall thickenings; intervening thinner regions of
the wall are presumed to be more extensible, bulging out under the force of
turgor pressure to form lobes.
Far fewer studies have considered the role of F-actin in lobe formation. In
expanding wheat mesophyll cells, bands of cortical F-actin were observed to
co-localize with microtubule bands; cytochalasin treatments suggested an
important role for F-actin in the organization of cortical microtubule bands
(Wernicke and Jung, 1992).
However, two recent studies have provided evidence of a different role for
F-actin in the formation of epidermal cell lobes. These studies showed that,
in expanding epidermal cells of maize and Arabidopsis, localized
enrichment of cortical F-actin is observed at the tips of emerging lobes,
somewhat similar to the F-actin arrangement at the tips of tip-growing cells
(Frank and Smith, 2002
;
Fu et al., 2002
). Expression
of dominant negative and constitutively active forms of ROP2 (a Rho-related
GTPase) disrupted the formation or localization of these cortical F-actin
enrichments and also perturbed the formation of lobes
(Fu et al., 2002
). In maize
brk1 mutants, epidermal lobes completely fail to form, although cells
expand to achieve a normal overall size. Loss of lobes on brk1 mutant
epidermal cells is associated with a failure of localized cortical F-actin
enrichments to form, whereas cortical microtubules bands are still present
(Frank and Smith, 2002
). Thus,
both studies suggest a crucial role for localized F-actin enrichments in
epidermal lobe formation separate from simply promoting the formation of
microtubule bands.
In addition to its effects on epidermal pavement cell shape, brk1
causes 20-40% of stomatal subsidiary cells to form abnormally
(Gallagher and Smith, 2000).
In wild-type leaves, subsidiary cells arise from the asymmetric divisions of
subsidiary mother cells (SMCs), whose premitotic polarization involves the
formation of a localized enrichment of F-actin at a specific site in the cell
cortex. Interestingly, abnormal brk1 subsidiary cells could be
attributed to defects in the polarization of SMCs associated with loss of this
localized enrichment of cortical F-actin
(Gallagher and Smith, 2000
).
Thus, our observations have revealed a mechanistic link between the formation
of polarized outgrowths on the margins of expanding epidermal cells and the
polarization of premitotic SMCs, and suggest a role for Brk1 in an
actin-dependent aspect of both processes.
In this study, we present an analysis of two additional mutants, brk2 and brk3, which have essentially the same phenotype as brk1. Through a combination of phenotypic, double mutant and mosaic analyses, we demonstrate that all three Brk genes have distinct functions in a common pathway promoting lobe formation and polarized cell division in the maize leaf epidermis.
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MATERIALS AND METHODS |
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The brk2 and brk3 mutations were mapped to chromosome
arms using B-A translocations (Beckett,
1994). The brk2 locus is located on chromosome arm 1S,
and brk3 on 10S. Linkage analysis with visible genetic markers on 1S
showed that brk2 is
6 cM from Vp5. More precise mapping
relative to restriction fragment length polymorphism markers showed that
brk2 is
7 cM from asg31 at the distal end of 1S, and
brk3 is less than 4 cM from php20075a near the tip of
10S.
Analysis of brk phenotypes
Epidermal peels were prepared and stained with Toluidine Blue O as
described by Gallagher and Smith
(Gallagher and Smith, 1999).
For cross sections, mature adult leaf pieces were fixed in 4% formaldehyde/50
mM KPO4 pH 7 for at least 2 hours at room temperature, rinsed,
dehydrated through an ethanol series and then infiltrated and embedded in
methacrylate resin as described by Gubler
(Gubler, 1989
). 3 µm
sections were attached to slides coated with Biobond (Electron Microscopy
Sciences), incubated in acetone for 10 minutes to remove the resin,
rehydrated, stained with Toluidine Blue O and examined with a 20x
objective under bright field conditions in a Nikon Eclipse E600 microscope.
Visualization of microtubules and F-actin in fixed cells was performed as
described by Frank and Smith (Frank and
Smith, 2002
). For analysis of F-actin in living cells via
transient expression of green-fluorescent-protein/talin (GFP-talin), strips of
leaf tissue 2-8 cm from the bases of immature adult leaves 20-28 cm long were
excised and cultured as described by Gallagher and Smith
(Gallagher and Smith, 1999
).
Within 6 hours of the initiation of tissue cultures, plates were bombarded
with 0.3-0.8 µg of pYSC14 (Kost et al.,
1998
) as described by Ivanchenko et al. (Ivanchenko et al., 2000),
except that 1 µm gold particles were used as the microcarrier, and tissue
was cultured under continuous light conditions at 22°C for 22-26 hours
prior to examining by confocal microscopy as described by Frank and Smith
(Frank and Smith, 2002
).
Double mutant analysis
To generate double mutants, homozygous brk mutants in a B73
background were crossed in each pairwise combination. F1 progeny
were selfed to generate an F2 generation. Each mutant in the
F2 generation was test-crossed to both of the appropriate single
mutants, and test-cross progeny were analyzed to determine the genotypes of
the F2 mutants. Cyanoacrylate glue impressions of leaf surfaces
were examined under DIC conditions in a Nikon E600 microscope using a
10x objective, captured with a DAGE MTI CCD72 camera coupled to a Scion
LG-3 framegrabber, and processed with Adobe Photoshop 4.0. To collect data for
Table 1, at least four images
from each impression were captured from randomly chosen locations.
|
Mosaic analysis
For mosaic analysis, brk3 Oy1-700/brk3 oyl+, brk2
vp5/brk2 Vp5+ and brk1 lw2/brk1 Lw2+
plants were crossed to wild-type plants to generate seeds of the following
genotypes: brk1 lw2/Brk1+ Lw2+
(n=3500), brk2 vp5/Brk2+ Vp5+
(n=1500), and brk3 Oy1-700/Brk3 oyl+
(n=1750). This crossing scheme ensured that each brk mutant
allele would always be linked in cis with the marker mutation in the
doubly heterozygous progeny. Seeds were germinated, irradiated and grown as
described previously (Walker and Smith,
2002). As discussed below, vp5-marked brk2
sectors were also generated by means of Ac/Ds-induced chromosome
breakage. Glue impressions of the leaf surface spanning the entire width of
each sector were inspected for the brk phenotype. Sectors showing the
brk phenotype were then fixed and processed for epidermal peels as
described by Gallagher and Smith
(Gallagher and Smith, 1999
).
Epidermal peels and hand-cut cross sections from these sectors were examined
in a Nikon E600 microscope under epifluorescence conditions using a rhodamine
filter set, and under DIC conditions. Images were acquired and processed as
described earlier for double mutant analysis.
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RESULTS |
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To determine whether brk mutations affect cell morphology in other
leaf layers in addition to the epidermis, internal tissue layers were
examined. As found previously for brk1
(Frank and Smith, 2002),
transverse sections revealed no differences in the sizes, shapes or
organization of cells in internal tissue layers of brk2 or
brk3 leaves compared with the wild type
(Fig. 1C,D). Furthermore,
direct examination of isolated mesophyll cells confirmed that their lobed
shapes are normal in both brk2 and brk3 mutant leaves (data
not shown). However, epidermal cells of brk2 and brk3 mutant
leaves appear more rounded in cross section than those of wild-type leaves.
This appears to be due mainly to a lack of outer epidermal cell wall material
filling the crevices between adjacent epidermal cells in wild type leaves
(Fig. 1C,D, arrows).
As discussed in the Introduction, several previous studies have attributed
lobe formation in expanding epidermal and mesophyll cells to the organization
of cortical microtubules into bands that direct a non-uniform pattern of
cellulose deposition. As predicted from these earlier studies, cortical
microtubules in expanding wild-type epidermal pavement cells of maize are
organized into bands focused at lobe sinuses
(Fig. 2A, white arrows). In
expanding brk1 pavement cells, cortical microtubules also form bands,
although they are less distinct than those in wild-type cells
(Frank and Smith, 2002).
Similarly, in expanding brk2 and brk3 pavement cells,
cortical microtubule bands are observed that are less distinct than those in
wild-type cells (Fig. 2B,C compared with
2A) but more pronounced than in those in brk1 cells.
Furthermore, cortical microtubules in expanding brk2 and
brk3 pavement cells generally appear more aligned with each other
than those in brk1 or wild-type pavement cells
(Fig. 2B,C compared with 2A) and are sometimes aligned at an oblique angle to the cell's long axis
(Fig. 2C for brk3).
Thus, as in brk1 mutants, failure of pavement cell lobes to form in
brk2 and brk3 mutants could not be readily explained in
terms of a lack of cortical microtubule bands during cell expansion.
|
Recent studies of actin organization in expanding epidermal pavement cells
of wild-type maize and Arabidopsis revealed local accumulations of
F-actin at the tips of emerging and elongating lobes
(Fig. 2D, white arrows)
(Frank and Smith, 2002;
Fu et al., 2002
). However, in
brk1 mutants, localized accumulations or `patches' of cortical
F-actin were not observed at any stage of pavement cell expansion
(Frank and Smith, 2002
).
F-actin organization is similarly altered in brk2 and brk3
expanding pavement cells: no cortical F-actin patches were observed when fixed
tissues were stained with FITC-phalloidin
(Fig. 2E,F).
To confirm that these observations were not due to an artefact of chemical
fixation, F-actin organization was also examined in living cells transiently
expressing a GFP-talin fusion protein
(Kost et al., 1998) introduced
via particle bombardment. As in fixed, FITC-phalloidin-stained cells,
expanding wild-type pavement cells expressing GFP-talin showed F-actin patches
in elongating lobe tips (Fig.
2G, white arrows). In expanding brk2 and brk3
pavement cells expressing GFP-talin, no cortical F-actin patches were observed
that were comparable to those seen in the wild type
(Fig. 2H,I). Although small
puncta of GFP-talin fluorescence were occasionally seen at the cell margins at
early stages of mutant pavement cell expansion, they were not localized
periodically and were generally restricted to small areas of the cell (data
not shown). Thus, all three Brk genes are necessary for the formation
of cortical F-actin patches, suggesting that they play an important role in
pavement cell lobe formation.
The resemblance of F-actin organization at the tips of elongating pavement
cell lobes to that in tip-growing cells led us to consider how brk
mutations might affect cells known to expand by means of tip growth. No
obvious alterations in the morphogenesis of root hairs were present in
brk2 or brk3 mutants (data not shown). Moreover, when
crosses are performed with brk2 or brk3 heterozygous pollen,
mutant pollen tubes achieve fertilization at approximately the same frequency
as competing wild-type pollen tubes. Thus, as in brk1
(Frank and Smith, 2002), tip
growth in brk2 and brk3 mutant root hairs and pollen tubes
appears to be normal.
Brk1, Brk2 and Brk3 act in a common pathway
Double mutant analysis was used to investigate the relationships between
the functions of Brk1, Brk2 and Brk3. In populations
segregating both single and double mutants derived from self-pollination of
double heterozygotes, no phenotype distinct from the brk single
mutant phenotype was observed. The genotype of each plant showing a mutant
phenotype was determined by test-crossing to the appropriate single mutants.
Pavement cell shapes in brk1;brk2, brk2;brk3 and brk1;brk3
double mutants are the same as those in brk single mutants
(Fig. 3). The frequency of
abnormal stomatal subsidiaries provides a more quantitative measure of
phenotypic severity. In this respect, brk1 (20-40% abnormal
subsidiaries) is more severe than either brk2 or brk3 (5-10%
abnormal subsidiaries; Table
1). Abnormal subsidiary frequencies in brk1;brk3 and
brk1;brk2 double mutants are similar to those in brk1 single
mutants, whereas abnormal subsidiary frequencies in brk2;brk3 double
mutants are similar to those in both brk2 and brk3 single
mutants (Fig. 3; Table 1). Thus, all three
classes of brk double mutants have the same phenotypes as
brk single mutants, indicating that all three Brk genes
function in a common pathway.
Construction and analysis of genetic mosaics
To gain a better understanding of the function of each Brk gene,
clonal sectors of brk mutant cells in otherwise wild-type leaves were
examined to determine whether each of the Brk genes acts
cell-autonomously or non cell-autonomously. For any one of the brk
mutations, if brk cells neighboring wild-type cells always show the
mutant phenotype then that Brk gene acts cell-autonomously. However,
if brk mutant cells next to wild-type cells show a wild-type
phenotype then the corresponding gene acts non cell-autonomously.
Mosaic analysis of each brk mutation involved a similar scheme for generation of marked, clonal sectors, which is summarized in Fig. 4A. Seedlings heterozygous for a brk mutation linked in cis with a recessive or semi-dominant cell-autonomous marker mutation affecting chlorophyll pigmentation were irradiated to produce random chromosome breaks. Cells in which breaks have occurred between the centromere and the wild-type allele for the marker gene divide to produce white (albino) sectors of brk mutant cells in otherwise Brk+, green plants. White sectors of brk1, brk2 and brk3 mutant tissue were analyzed to determine the locations of sector boundaries. Wild-type chloroplasts autofluoresce brightly when examined by fluorescence microscopy using a rhodamine filter set. Sector boundaries in internal tissue layers were located by examining leaf cross sections and in the epidermis by examining epidermal peels. In the epidermis, only stomatal guard cells contain mature chloroplasts and can therefore be scored as mutant or wild type for the albino mutation. Data collected through examination of cross sections and epidermal peels for each sector were compiled to reconstruct the composition of each sector (Fig. 4B).
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Brk2 and Brk3 act cell-autonomously to
promote pavement cell lobe formation
Brk3 is located near the end of chromosome 10S, distal to the
Oil Yellow 1 (Oy1) locus
(Fig. 4A). Plants heterozygous
for the semi-dominant Oy1-700 allele are yellow-green, whereas
homozygous or hemizygous Oy1-700 plants completely lack pigmentation
and are therefore white. Brk2 is located near the end of chromosome
1S 6 cM from Viviparous5 (Vp5). The vp5
mutation is recessive; homozygous mutant plants are white. Albino-marked
brk2 and brk3 sectors were generated by irradiating brk3
Oy1-700 and brk2 vp5 heterozygous plants
(Fig. 4A) (described in
Materials and Methods). A second method was also used to create
vp5-marked brk2 sectors. Plants homozygous for brk2
and heterozygous for vp5 were crossed to wild-type plants homozygous
for a chromosome-breaking Ds transposon on chromosome 1S between the
centromere and Vp5 (Neuffer,
1995
). In the progeny, Ac-induced chromosome breakage at
the site of this Ds insertion resulted in the loss of wild-type
Vp5 and Brk2 alleles, simultaneously uncovering vp5
and brk2. Because Ac/Ds-induced chromosome breaks occur
relatively late in development, the resulting sectors are small, sometimes
only a few cell files in width, whereas sectors induced by irradiation are
much larger (Fig. 4A).
We analyzed 72 brk2 and 64 brk3 lateral epidermal sector boundaries (Table 2; Fig. 4B, boundary types A, C and D). At all boundaries of this type, there was a clear difference between the shapes of brk and wild-type cells. As explained earlier, lateral sector boundaries in the epidermis can be located only to the interval between wild-type and albino stomatal files. Although stomatal files are usually separated by at least one non-stomatal file, we occasionally observed areas where sector boundaries split directly adjacent stomatal files such that one file was wild type and the neighboring file was mutant (Fig. 5A,B for brk2, Fig. 6A,B for brk3). In these areas, we could determine with certainty that wild-type cells were lobed, whereas directly adjacent brk2 or brk3 cells were unlobed. Glue impressions of lateral sector boundaries in the epidermis revealed that, at the junctions between wild-type and brk2 or brk3 pavement cells, lobes of wild-type cells appear to grow over unlobed, brk cells (Fig. 5C, black arrows, for brk2; Fig. 6C, black arrows, for brk3). These results demonstrate that both Brk2 and Brk3 act cell-autonomously in the lateral dimension to promote lobe formation in epidermal cells. We also analyzed transverse sector boundaries, where mutant epidermis lies over wild-type mesophyll (Fig. 4B, boundary type B). For brk2 and brk3, respectively, 30 and 37 such boundaries were analyzed (Table 2). In each case, brk mutant epidermal cells overlying wild-type mesophyll cells were unlobed (Fig. 5A,B for brk2; Fig. 6A,B for brk3), indicating that Brk2 and Brk3 also act cell-autonomously in the transverse dimension.
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Brk2 and Brk3 act non cell-autonomously to promote
polarized SMC divisions
In all brk mutants, stomatal subsidiaries occasionally form
abnormally. Normal stomatal development in maize is illustrated in
Fig. 7. An asymmetric division
gives rise to a small guard mother cell (GMC), which is thought to signal its
neighbors to the left and right (SMCs) to become polarized with respect to the
GMC and to divide asymmetrically to form subsidiary cells. The GMC later
divides longitudinally to give rise to a guard cell pair. Analysis of stomatal
development in brk1 mutants showed that abnormal subsidiaries arise
from SMCs that failed to polarize properly before dividing
(Gallagher and Smith, 2000).
The presumed role of GMCs in stimulating the polarization of adjacent SMCs
raises the interesting question of whether SMC polarization depends on
Brk gene function in the GMC or in the SMC itself. We addressed this
question by examining mosaic stomata in which mutant guard cells were flanked
by a wild-type subsidiary, or vice versa.
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In contrast to its cell-autonomous action in promotion of pavement cell lobe formation, analysis of mosaic stomata showed that Brk3 acts non cell-autonomously to promote polarized SMC divisions. Wild-type subsidiary cells in sectored leaves were never abnormal, whether flanking wild-type or brk3 guard cells, demonstrating that the presence of a wild-type Brk3 allele in the SMC alone is sufficient for its normal division. Whereas 15% of brk3 subsidiaries flanking brk3 guard cells were abnormal, only 1.5% of brk3 subsidiaries flanking wild-type guard cells were abnormal (Table 3; Fig. 6A, arrowheads), demonstrating that wild-type GMCs rescued the abnormal divisions of adjacent, mutant SMCs. Notably, however, the abnormal divisions of brk3 SMCs were not rescued by adjacent wild-type pavement cells (n=117), so the adjacent GMC appears to be the only cell that can supply Brk3 function to a brk3 mutant SMC.
|
Similar analysis of brk2 mosaic stomata showed that Brk2 acts partially non cell-autonomously to promote the polarized divisions of SMCs. As for Brk3, the presence of a wild-type Brk2 allele in the SMC is sufficient for its normal division, because 3.3% of wild-type subsidiaries in sectored leaves were abnormal whether flanking wild-type or brk2 guard cells. Whereas 18.4% of brk2 subsidiaries flanking brk2 guard cells were abnormal, 10.5% of brk2 subsidiaries flanking wild-type guard cells were abnormal (Table 3; Fig. 5D, arrowheads). Thus, brk2 SMC divisions were partially rescued by an adjacent, wild-type GMC.
Brk1 acts non cell-autonomously
Brk1 is located near the end of chromosome 5S. As illustrated in
Fig. 4A, the recessive
lemon white 2 (lw2) mutation, located between the centromere
and brk1, was used to mark brk1 mutant sectors. To determine
whether Brk1 acts cell-autonomously or non cell-autonomously within
the epidermis, we analyzed 63 lateral sector boundaries
(Fig. 4B, types A, C and D). In
areas where these boundaries split adjacent stomatal cell files, brk1
cells in direct contact with wild-type cells appeared to be wild type, with
pavement cell lobes and normal stomata
(Fig. 8A,B). Moreover, weakly
lobed pavement cells were observed in brk1 mutant cells two to three
cell files away from the nearest wild-type epidermal cell
(Fig. 8A, black arrows). Thus,
direct contact with a wild-type cell is not necessary to rescue the phenotype
of a brk1 cell. From these observations, we conclude that
Brk1 acts non cell-autonomously in the lateral dimension.
|
To investigate whether Brk1 also acts non cell-autonomously in the transverse dimension, 53 sectors were identified in which brk1 mutant epidermis was overlying wild-type internal tissue layers. At 28 boundaries with wild-type mesophyll directly underlying brk1 epidermis (Fig. 4B, boundary type B), the mutant epidermis was indistinguishable from wild-type epidermis, with lobed pavement cells and normal stomata (Fig. 8C,D, right-hand side). By contrast, at 25 boundaries where brk1 epidermis was separated from a wild-type cell layer by one or more layers of brk1 mesophyll (Fig. 4B, boundary type E), the epidermis appeared to be mutant, lacking marginal lobes and having some abnormal subsidiary cells. These results show that Brk1 is expressed and functions in internal tissue layers even though no mutant phenotype was observed in these layers of brk1 leaves. Furthermore, Brk1 expressed in these layers rescues the phenotype of directly overlying brk1 mutant epidermal cells. Therefore, Brk1 acts non cell-autonomously in the transverse dimension but its influence does not travel as far in this dimension as it does within the epidermis.
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DISCUSSION |
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Consistent with many previous studies of lobe formation, cortical
microtubules are organized into bands focused at lobe sinuses of expanding
wild-type pavement cells in the maize leaf epidermis. In spite of the absence
of lobe formation, these microtubule bands are also present in all three
brk mutants, although they are somewhat less distinct. Visualization
of F-actin in expanding wild-type pavement cells by means of phalloidin
staining of fixed cells and GFP-talin expression in living cells showed
distinct enrichments of cortical F-actin at lobe tips. These observations are
similar to those reported in a recent study in which F-actin organization was
visualized in living, expanding Arabidopsis epidermal pavement cells
expressing GFP-talin (Fu et al.,
2002). Another recent study of lobe formation in
Arabidopsis pavement cells revealed relatively little enrichment of
cortical F-actin in lobe tips (Qiu et al.,
2002
). In this study, however, antibody staining of fixed cells
was used to visualize F-actin, so some cortical F-actin might have been lost
during the fixation process. Our observation of cortical F-actin enrichments
at lobe tips in both fixed and living maize cells indicates that this feature
of cytoskeletal organization is not an artefact of the F-actin visualization
method used. In both living and fixed expanding epidermal pavement cells of
all three brk mutants, cortical F-actin enrichments were not
observed, supporting the conclusion that these F-actin enrichments are
important for lobe formation, and indicating that the Brk genes are
required for their formation. By analogy to the proposed function of F-actin
in both tip-growing and diffusely growing cells, the F-actin enriched in lobe
tips might function to guide or promote localized secretion.
Analysis of brk1;brk2, brk1;brk3 and brk2;brk3 double
mutants showed that they have the same phenotypes as the corresponding single
mutants. These results indicate that all three Brk genes act in a
common pathway. Although this might mean that the genes act in a linear
sequence, it is also possible that the products of these genes simply act
together in a common process, possibly directly interacting with each other.
Although the Brk1 gene product has been identified
(Frank and Smith, 2002),
investigation of its functional relationship to Brk2 and
Brk3 gene products awaits the cloning of these other two genes.
Mosaic analyses were carried out to determine whether each Brk
gene acts cell-autonomously or non cell-autonomously. Brk1 acts non
cell-autonomously in both lateral and transverse leaf dimensions over a short
distance. Although the non cell-autonomous influence of Brk1 does not
depend on direct cell-cell contact, it travels farther within the epidermal
layer (2-3 cell diameters) than it does between leaf layers (1 cell diameter).
The non cell-autonomous action of Brk1 suggests that this gene
encodes or controls the production of a diffusible or transported molecule.
The Brk1 gene was shown to encode a novel protein of 8 kDa that
is highly conserved in both plants and animals
(Frank and Smith, 2002
).
Neither BRK1 nor BRK1-related proteins in other organisms have the predicted
signal peptides expected for secreted proteins. Moreover, recent work has
implicated the mammalian BRK1 homolog HSPC300 in the regulation of actin
polymerization (Eden et al.,
2002
), clearly pointing to an intracellular function for BRK1.
However, the non cell-autonomous action of Brk1 might reflect
movement of BRK1 from cell to cell through plasmodesmata. Particularly at
early developmental stages, a variety of proteins considerably larger than
BRK1 appear to be transported from cell to cell via plasmodesmata in maize and
several other plant species (Jackson,
2000
). Whether the BRK1 protein moves intercellularly and the
functional significance of any such movement remain to be determined.
Unlike Brk1, mosaic analyses showed that Brk2 and Brk3 act cell-autonomously to promote the formation of epidermal pavement cell lobes, indicating that both gene products act in or on each pavement cell to direct its own morphogenesis. Surprisingly, however, Brk3 acts non cell-autonomously, and Brk2 partially non cell-autonomously, to promote the polarized divisions of SMCs. Although polarization of SMCs appears to depend on a signal from the adjacent guard mother cell (GMC), it is unlikely that Brk3 or Brk2 function directly in this signaling process. If they did, we would expect that a subsidiary cell's phenotype would depend solely on the genotype of its adjacent GMC, but we found instead that the presence of a wild-type Brk3 allele in either the SMC or the adjacent GMC is sufficient for a normal SMC division. The same is true for Brk2, except that a wild-type Brk2 allele in the GMC only partially rescues the polarized divisions of adjacent brk2 SMCs. In summary, the Brk2 and Brk3 function needed for polarized SMC divisions can be provided by the SMC itself, or by the adjacent GMC, but not by surrounding pavement cells. The specificity observed in the non cell-autonomous action of Brk3 and Brk2 might be due to diffusion or active transport of BRK3 protein (and to a lesser extent, BRK2 protein) from GMCs to SMCs, but not between other leaf cell types. Alternatively, presence of BRK3 and BRK2 proteins in either the SMC or the GMC might in some way facilitate intercellular communication between these two cell types leading to proper polarization of SMCs.
In summary, genetic and phenotypic analyses of brk mutations show
that they define three genes that act in a common pathway promoting specific
aspects of maize leaf epidermal morphogenesis and division that involve local
actin polymerization. Moreover, mosaic analyses yielding different results for
each Brk gene show that the product of each gene has a distinct
function in that pathway. The mammalian homolog of BRK1, HSPC300, has recently
been directly implicated in regulation of Arp2/3-dependent actin
polymerization (Eden et al.,
2002). The Arp2/3 complex (putative components of which are
encoded by various plant genomes) nucleates polymerization of new actin
filaments at specific sites in the cell when activated by proteins that
respond to localized intracellular or extracellular cues
(Higgs and Pollard, 2001
).
HSPC300 was identified as a component of a multiprotein complex isolated from
bovine brain extracts that also includes WAVE, an Arp2/3 activator whose
activity is regulated by Rac. The intact WAVE complex is inactive but, in the
presence of GTP-Rac (or Nck, another regulatory protein), WAVE and HSPC300
dissociate from the rest of the complex to form a two-protein subcomplex that
stimulates Arp2/3-dependent actin polymerization
(Eden et al., 2002
). Thus, BRK1
probably functions as part of a complex that also contains an Arp2/3
activator. Brk2 and Brk3 might encode other components of
such a BRK1-containing complex, or factors that positively regulate its
activity. Indeed, either Brk2 or Brk3 might encode a WAVE
ortholog, a particularly exciting possibility given that plant genomes are not
predicted by sequence homology to encode WAVE-like proteins or other Arp2/3
activators. Interestingly, the Arabidopsis SPK1 gene is also required
for the formation of epidermal pavement cell lobes; this encodes a protein
containing a motif that has previously been shown to interact with Rac GTPases
(Qiu et al., 2002
). Moreover,
expression of both dominant negative and constitutively active forms of the
Rac/Rho-related Arabidopsis GTPase ROP2 also perturbs the formation
of epidermal pavement cell lobes (Fu et
al., 2002
). In combination with recent findings on the role of
HSPC300 in regulation of local actin polymerization by Rac, these findings
suggest that BRK proteins might function in the same pathway (probably
downstream of) maize SPK1 and ROP2 orthologs. To investigate these
possibilities, future work will be aimed at the identification and analysis of
Brk2 and Brk3 gene products.
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ACKNOWLEDGMENTS |
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