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3205
Development 127, 3205-3213 (2000)
Printed in Great Britain © The Company of Biologists Limited 2000
DEV0311
The SCARFACE gene is required for cotyledon and leaf vein patterning
Michael K. Deyholos2,*, Glen Cordner2, Dwight Beebe3 and Leslie E. Sieburth1,‡
1Department of Biology, University of Utah, 257 South 1400 East, Salt Lake City, UT 84112, USA
2McGill University, Department of Biology, 1205 Docteur Penfield Avenue, Montreal, Quebec, Canada H3A 1B1
3University de Montreal, Institut de recherche en biologie végétale, 4101 rue Sherbrooke Est, Montréal, QC, Canada,
H1X 2B2
*Present address: Department of Plant Science, University of Arizona1140 E. South Campus Drive, Tuscon, AZ 85721, USA
‡Author for correspondence (e-mail: [email protected])
Accepted 8 May; published on WWW 10 July 2000
SUMMARY
Mechanisms controlling vein patterning are poorly
understood. We describe a recessive Arabidopsis mutant,
scarface (sfc), which maps to chromosome 5. sfc mutants
have vein pattern defects in cotyledons, leaves, sepals and
petals. In contrast to the wild type, in which these organs
all have linear veins that are continuous with at least one
other vein, in sfc mutants these organs' secondary and
tertiary veins are largely replaced by small segments of
discontinuous veins, which we call vascular islands.
Patterning defects are manifest in cotyledon provascular
tissue, suggesting that the patterning defect occurs early in
organogenesis. sfc mutants have exaggerated responses to
exogenous auxin. Analysis of monopteros (mpT370) sfc1 double mutants suggested that SFC has partially
overlapping functions with MP in patterning of both
primary and secondary veins.
INTRODUCTION
streams are established that later specify positions for vein
development (Sachs, 1991). Recently, several studies have
investigated possible roles of polar auxin transport in leaf
development through the use of auxin polar transport inhibitors
(Mattsson et al., 1999, Sieburth, 1999; Tsiantis et al., 1999). In
all three studies, seedling growth in the presence of polar auxin
transport inhibitors resulted in multiple morphological
changes, including altered leaf vein patterns. However, we do
not know whether these changes were the direct result of
reduced transport or an indirect result of increased auxin
concentrations, and we also do not yet know which molecules
effected the changes in vein pattern in response to the altered
auxin conditions.
To investigate vein patterning, we have conducted a screen
for Arabidopsis vein patterning mutants, and in this study, we
present our characterization of plants containing mutations at
the scarface (sfc) locus. sfc mutants produce organs with
defects in the axial continuity of secondary and higher-order
veins. Our investigations suggest that sfc mutants have
heightened auxin responses, and that SFC functions in
processes closely related to those of MONOPTEROS.
In vascular plants, veins form a network that interconnects all
parts of the organism. Veins contain two tissues, xylem and
phloem, that transport water and dissolved mineral nutrients
(xylem), or sugars, amino acids and other compounds
(phloem). Broad organs, such as leaves or cotyledons, have
acute physiological needs for veins to be distributed throughout
the lamina. Large amounts of water are lost due to
transpiration, and this water must be replenished through
the xylem. In addition, leaves are the primary site of
photosynthesis, and so produce large amounts of sugars. Much
of this sugar must be exported from the leaf and transferred to
either storage organs or rapidly growing parts of the plant; this
transport uses phloem.
Strategies used to achieve a distributed pattern of veins in
leaves vary between different groups of plants. For example,
monocots typically show parallel venation, in which major
veins extend parallel along the proximal/distal leaf axis and
largely lack major vein branching. In contrast, dicot leaves
typically contain branched veins, and groups of dicots vary
with respect to numbers and positions of vein branches.
Although leaf vein patterns have been well characterized,
almost nothing is known about the developmental mechanisms
that specify these patterns.
One molecule that has been proposed to play a role in vein
patterning is the plant hormone auxin. Auxin is synthesized in
apical parts of the plant, and is moved basipetally by a polar
transport mechanism (reviewed by Jones, 1998). It has been
proposed that early in organogenesis, polar auxin transport
Key words: Arabidopsis thaliana, Vein patterning, Auxin,
SCARFACE, MONOPTEROS
MATERIALS AND METHODS
Mutagenesis and mutant screening
We mutagenized seeds of Arabidopsis thaliana ecotype Landsberg
erecta (Ler) for 3 hours in 130 mM solutions of EMS (ethane methyl
sulfonate; Sigma). The self-fertilized progeny of these seeds were
collected as 5259 individual M2 families, and screened for
morphological abnormalities after 10 days growth on germination
3206 M. K. Deyholos and others
medium (GM; 0.5× Murashige and Skoog basal salts (Sigma), 1%
sucrose, 0.5 g/l MES buffer (2-[N-Morpholino]ethanesulfonic acid;
Sigma), 0.7% phytagar (GIBCO-BRL), pH 5.7). Abnormal seedlings
were fixed in a solution of 3:1 ethanol:glacial acetic acid, cleared in
saturated chloral hydrate (Sigma), and vein patterns were examined
microscopically using dark-field illumination. After identifying lines
that segregated putative mutants, we backcrossed heterozygotes to Ler
plants through four successive cycles before proceeding with detailed
phenotypic analyses. Independent lines with mutant phenotypes
similar to sfc-1 were crossed to sfc-1 and examined in the F1
generation to assess complementation.
Growth conditions
For detailed physiological and anatomical analyses, seeds were
surface-sterilized and plated on GM in Petri plates or magenta boxes.
They were cold-stratified at 4°C for 3 days, and then transferred to a
TC-30 Conviron controlled environment chambers at 24°C, under
constant light at 100-120 µE/m2/second. Seedling age refers to days
after transfer from the cold stratification.
Genetic mapping
We extracted the DNA of more than 100 individual F2 mutant
seedlings from crosses of sfc-1 heterozygotes to ecotype Columbia
(Dellaporta et al., 1983). This DNA was genotyped at several
microsatellite loci that are polymorphic between ecotypes Ler and
Columbia (Col-0) (Bell and Ecker 1994), allowing us to narrow the
map position down to chromosome 5. For more than 100 sfc-1 mutant
plants, we combined pairwise recombination frequencies between the
microsatellite markers nga225, nga249, and nga106 and the sfc-1
locus (using MAPMAKER software (Whitehead Institute, version
2.0) to place the SFC gene approximately 7 cM south of nga249.
Root elongation assays
Root elongation assays were based on the method of Estelle and
Somerville (1987). We grew Ler and segregating populations of sfc1 on vertically oriented GM plates for 4 days, then transferred the
seedlings to fresh GM, or GM supplemented with either indole-3acetic acid (Sigma), or 2,4-dichlorophenoxyacetic acid (Sigma),
(10−6 M to 10−10 M in a 10-fold dilution series). After transfer, the
position of each root tip was marked on the plate, and then marked
again after 3 days of additional growth. Each root's increase in length
was then determined by measuring digitized images of the roots with
NIH Image 1.61 software. We calculated the mean increase in root
length at each concentration of hormone and divided this by the mean
change in root length on hormone-free medium to calculate the
percentage elongation for each genotype at each hormone
concentration.
Histology
Leaves and cotyledons were fixed (2.5% gluteradehyde, 3%
paraformaldehyde, 0.1 M sodium phosphate, pH 7.3) by first vacuum
infiltrating for 5 minutes, followed by microwave fixation (Pelco
model 3420). Following fixation, the tissue was rinsed (0.1 M NaPO4
buffer, pH 7.3), and a secondary fix performed using 1% osmium
tetroxide (in 0.1 M NaPO4 buffer, pH 7.3). Following a buffer rinse,
the tissue was dehydrated in an ethanol series (10%, 30%, 50%, 70%,
95%, 100%), and infiltrated with Spurr resin (steps of 25%, 50%,
75%, and 100%). 1 µm sections were prepared and stained with 1%
toluidine blue (w/v) in 1% sodium borate (w/v), and photographed
using an Olympus BX-50 microscope.
To examine embryonic cotyledon vein patterning, embryos were
dissected from dry seed that had imbibed in 70% ethanol overnight.
They were transferred to saturated chloral hydrate for 24 hours, and
examined as whole-mounts using DIC optics.
Double mutant analyses
For double mutant analysis with auxin-resistant mutants, we crossed
sfc-1 heterozygotes to plants that were homozygous for one of each
of the following mutations: aux1-7 (Pickett et al., 1990), axr1-3
(Lincoln et al., 1990), axr2-1 (Timpte et al., 1992), and axr4 −2
(Hobbie and Estelle, 1995). From crosses of sfc-1 to aux1-7 and axr13, we identified F3 families in which all individuals (except the sfc-1
homozygotes) were resistant to 2×10−7 M 2,4-D. All seedlings within
these families were presumed to be homozygous for the specific
auxin-resistant mutation, and sfc-1 mutants segregated within these
families at near 3:1 ratios (χ2 P=0.41, 0.46 for double mutants of sfc1 with axr1-3, and aux1-7 respectively). Because no F3 families that
were homozygous for axr4-2 were identified, seedlings from these
families were scored separately for the segregation of sfc-1 and axr42. sfc-1 segregated within F2 families of the dominant axr2-1 mutation
at near-expected ratios (χ2 P=0.07).
To obtain double mutants of sfc-1 and the seedling lethal mutants
mpT370 (Berleth and Jürgens, 1993) and rty-5 (Windsor and Waddell,
pers. comm.), heterozygotes were crossed. Double mutants were
identified within F2 families that segregated for each single mutant,
and were easily distinguished by the presence of the distinct
morphological features characteristic of each parental mutant
phenotypes. For each of the double mutant combinations, we
examined at least 191 seedlings from two or more F2 families. The
segregation ratios were subjected to χ2 analysis, with resulting P
values of 0.2, and 0.003 for expected dihybrid segregation ratios of
sfc-1 with mpT370 and rty-5.
RESULTS
To identify genes required for normal vein patterning in leaves
and cotyledons, we screened for vein patterning mutants
among the progeny of 5259 EMS-mutagenized Arabidopsis
plants. We identified several alleles of one mutant with vein
pattern defects in both the cotyledons and leaves. In contrast
to the wild type, which has continuous veins in cotyledons and
leaves (depicted in Fig. 1, shown in Fig. 2A,C), in these
mutants some vein classes in both leaves and cotyledons were
replaced by isolated clusters of vascular tissue (Fig. 2B,D). We
called these putative mutants scarface (sfc) in reference to this
disfigured vasculature.
We observed segregation of sfc mutants in a ratio consistent
with a single recessive lesion (3.1:1; n=148). Using molecular
markers (SSLPs; simple sequence length polymorphisms), we
mapped the SFC locus to chromosome 5, between markers
nga106 and nga249 (Bell and Ecker, 1994). In total, we
A
B
1
2
1
2
3
IV
mid
30%
Fig. 1. Cotyledon and leaf vein patterns in wild-type plants.
(A) Cotyledon vein pattern. (B) Leaf vein pattern. Both cotyledons
and leaves contain primary veins (1˚) and secondary veins (2˚).
Leaves also contain tertiary (3˚) veins and an intramarginal vein (IV).
The gray box in B indicates the region from which the paradermal
sections shown in Fig. 3 were obtained; dashed lines indicate
positions used for transverse sections shown in Fig. 3.
SCARFACE in plant vein patterning 3207
length of the leaf proximal/distal axis. Between 8
and 12 secondary veins branch from the midvein,
and tertiary veins branch from the secondary
veins.
The leaves produced by sfc mutants resembled
those of the wild type in gross morphology,
adaxial/abaxial epidermal patterning, and color;
however, they were typically only half the size of
the wild type. The vein pattern of the first leaf of
a sfc-1 mutant is shown in Fig. 2D. The leaf
primary vein and the circuit of intramarginal veins
(depicted in Fig. 1B) were typically contiguous in
sfc mutants, but most of the rest of the leaf lamina
contained VIs of varying lengths. All rosette and
cauline leaves produced by sfc-1 plants showed
similar vein patterns (data not shown). These
results indicate that SFC function is required for
the formation of the normal pattern of continuous
veins in both leaves and cotyledons.
Vein patterns of other organs
To determine whether the sfc mutation affected
vein continuity in other organs, we compared
veins of roots, hypocotyls, inflorescence stems,
sepals and petals for sfc-1 and wild type. In wild
type, sepals contained 3 to 5 veins that extended along the
proximal/distal axis (Fig. 2E). In sfc-1 mutants, sepals
contained a reduced number of intact veins, and some VIs (Fig.
2F). Wild-type petals contained a central primary vein that
extended most of the length of the organ, and three to five
secondary veins (occasionally bifurcated) that branched from
the primary vein (Fig. 2G). Petals of sfc mutants contained an
intact primary vein, but within the petal lamina, secondary
veins were replaced by a small number of VIs (Fig. 2H). For
sfc roots, hypocotyls and inflorescence stems, the veins
resembled those of the wild type in both the radial and
longitudinal axes (data not shown). Taken together, these
observations suggest that sfc-1 vascular defects are limited to
the secondary and higher order veins of flat organs (cotyledons,
leaves, sepals, petals).
Fig. 2. Vein patterns in wild type and sfc mutants. Leaves and cotyledons are from
14-day plants. Wild type (A,C,E,G) and sfc-1 (B,D,F,H). (A,B) Cotyledons;
(C,D) leaves; (E,F) sepals; (G,H) petals. Scale bars, 0.5 mm.
recovered six independent sfc alleles. All six alleles showed
the same seedling phenotype and the same vein pattern defects.
Here we report the characterization of a representative allele,
sfc-1.
Vascular defects in sfc-1 mutants
Cotyledon vein patterns
We compared cotyledon vein patterns of 14-day sfc-1 and wildtype seedlings. In sfc mutants, cotyledons resembled the wild
type in gross morphology, except that they were smaller,
slightly epinastic, and often had anthocyanin pigment
accumulation on their abaxial side. Vein pattern in wild-type
cotyledons features a primary vein that is continuous with
the hypocotyl vasculature and extends the length of the
proximal/distal axis, and typically four secondary veins that
branch from the primary vein (shown in Fig. 2A, depicted in
Fig. 1A). As in the wild type, sfc cotyledons invariably
contained a central primary vein that was continuous with the
hypocotyl vasculature and that extended the length of the
cotyledon proximal/distal axis (Fig. 2B). However, in contrast
to the wild type, the secondary veins were mostly replaced by
short vein-like segments, which we refer to as vascular islands
(VIs). Most of the VIs appeared in positions where secondary
veins might be found in a wild-type cotyledon, however,
approximately 5% of the VIs appeared in positions atypical for
veins (e.g. very close to the cotyledon margin). These
observations indicate that SFC function is required for the
formation of a normal continuous pattern of cotyledon
secondary veins.
Leaf vein patterns
We also compared wild-type and sfc-1 leaf vein patterns.
Arabidopsis wild-type leaf vein pattern, shown in Fig. 2C and
depicted in Fig. 1B, has been described previously (Kinsman
and Pyke, 1998; Candela et al., 1999); a central primary vein
connects with the vascular tissue of the stem, and extends the
Anatomical characterizations
To characterize the anatomy of sfc-1 mutants, we compared
sections prepared from resin-embedded leaves and cotyledons.
Our previous characterizations used dark-field illumination of
cleared intact tissue, which allowed us to observe xylem
tracheary elements. To determine whether other vascular cell
types (e.g. phloem sieve tube elements) formed connections
between VIs, we compared paradermal sections (parallel to
leaf surface) of leaf tissue prepared from the middle of the leaf
adjacent to the primary vein (region depicted by the gray box
in Fig. 1B). Sections from wild type are shown in Fig. 3A,B;
veins were all continuous with other veins on at least one end.
Sections from sfc-1 are shown in Fig. 3C-E. Some veins
matched those observed in the wild type, and were continuous
with other veins on at least one end (data not shown). Other
veins were composed of irregularly shaped vascular cells and
corresponded to VIs. When we compared the serial sections of
the VI regions, there were no vascular cell types that were
continuous with other veins (Fig. 3C-E). These observations
confirmed that the VIs were discontinuous.
3208 M. K. Deyholos and others
Fig. 3. Anatomical characterization of wild type and sfc-1 cotyledons and leaves. (A-E) Paradermal sections of 19-day leaves. (A-B) Wild-type
leaf secondary vein, sections taken midway between apex and base of the leaf, adjacent to the primary vein. A and B show the same vein, A is
further toward the adaxial surface than B; the two sections are separated by approximately 12 µm. (C-E) sfc-1 leaf vascular islands. These three
sections depict the same two vascular islands (indicated by arrows). Each section is separated by approximately 15 µm. (F,G) Transverse
sections of 19-day leaf tissue. (F) wild-type; (G) sfc-1. PP, palisade parenchyma; SP, spongy parenchyma; BS, bundle sheath cells.
(H-N) Transverse sections through veins, xylem tissue has been circled in red, phloem tissue in yellow. (H) Higher magnification of wild-type
leaf primary vein shown in F. (I) Higher magnification of sfc-1 leaf primary vein shown in G. (J-K) Transverse sections through 11-day
cotyledon primary veins. (J) Wild-type cotyledon primary vein. (K) sfc-1 cotyledon primary vein. (L-N) Transverse sections of secondary vein
regions. (L) Wild-type cotyledon secondary vein. (M,N) two sfc-1 vascular islands. Scale bars (A,B,F,G) 100 µm; (C-E, H-N) 50 µm.
To determine whether sfc defects were restricted to vascular
tissue, we compared transverse sections of leaves, taken at their
midpoint (region of section depicted in Fig. 1B). In the wild
type, leaf mesophyll cells are differentiated into palisade
parenchyma on the adaxial (upper) side and spongy
parenchyma on the abaxial (lower) side, and the primary vein
is surrounded by a ring of bundle sheath cells (Fig. 3F). These
pattern elements were also present in the sfc mutant (Fig. 3G).
Cotyledon transverse sections were also compared; pattern
elements of the two genotypes were similar (data not shown).
These data suggest that sfc patterning defects in these organs
are limited to the vascular tissue.
To determine whether the sfc vascular defects extended to
the abaxial/adaxial patterning of vascular cell types, we
compared transverse sections of vascular bundles of wild type
and sfc mutants. Wild-type and sfc leaf primary veins are
shown in Fig. 3H,I, and cotyledon veins in Fig. 3J,K. In both
genotypes, vascular bundles were organized in the normal
pattern, with xylem tissues toward the adaxial surface and
phloem tissues oriented toward the abaxial surface, although in
sfc mutants, the adaxial/abaxial alignment was occasionally
rotated by approximately 20˚.
To determine whether the abaxial/adaxial vascular cell type
patterning was intact in VIs, we prepared transverse sections
from cotyledons at a position 30% the distance from the lamina
base to the distal end (position depicted in Fig. 1A). In the wild
type, normal secondary veins were typically present in these
positions, while in sfc mutants, this region invariably contained
VIs. Radial organization of wild-type secondary veins
resembled the primary vein (compare Fig. 3J to L). Two
different VI cross sections are shown (Fig. 3M,N). The
adaxial/abaxial orientations of xylem and phloem were
preserved in the sfc VIs. Taken together, these anatomical
analyses indicated that the sfc mutation disrupts normal axial
(longitudinal) patterning of secondary and higher-order veins.
Provascular patterning is disrupted in sfc mutants
The sfc vein pattern defect could, in theory, result from either
an initial patterning defect, or from a normal pattern that
failed to differentiate uniformly. To distinguish between these
possibilities, we compared provascular patterning in sfc1 mutants and the wild type. Provascular tissue is the first
morphological indication of vein differentiation, and is
distinguishable because the cells are elongated. In Arabidopsis,
cotyledon vein pattern is established during embryogenesis,
and can be detected in cotyledons of dry seed embryos in the
same pattern as the veins of fully differentiated cotyledons
(Sieburth, 1999). Therefore, to assess sfc provascular
SCARFACE in plant vein patterning 3209
Fig. 5. IAA-induced inhibition of primary root elongation. Wild-type
(open circle) and sfc-1 (solid square) elongation is expressed as a
proportion (%) of the root elongation on hormone-free medium. Each
point represents the mean of at least 15 independent measurements.
Error bars are the standard error of the mean.
Fig. 4. Provascular tissue patterning in embryonic cotyledons.
(A) Wild-type embryonic cotyledon, cleared, and viewed with DIC
optics. B and D are the same images as A and C respectively but the
provascular tissue is colored. The elongated provascular cells form a
pattern that matches the normal differentiated cotyledon vein; note
provascular cells are not in the focal plane at the lower left of this
image. (C) sfc embryonic cotyledon, cleared, viewed with DIC
optics. Normal primary vein provascular tissue is present, however
some irregular islands of provascular-like cells, not continuous with
other veins, are also present. Scale bars, 50 µm.
patterning, we compared cotyledons of dry seed embryos
obtained from a plant that segregated for sfc-1 mutants to dry
seed embryos of wild-type plants.
In the wild-type dry seed embryos, the cotyledon
provascular tissue pattern matched the mature cotyledon vein
positions (Fig. 4A,B). However, among embryos from
heterozygous sfc-1 parents, approximately one quarter had
cotyledons with normal primary vein provascular tissue, but in
secondary vein positions, the provascular tissue was
interrupted and often also disorganized (Fig. 4C,D). These
observations suggest that sfc vein axial discontinuities resulted
from defects in provascular patterning.
sfc mutants have altered auxin responses
Because aspects of vascular cell type differentiation and
vascular patterning have been linked to the hormone auxin
(reviewed by Aloni, 1995), it is possible that the sfc phenotype
resulted from defects in auxin perception. To test whether
auxin perception was altered in sfc-1, we compared the efficacy
of auxin to inhibit root elongation for sfc and wild-type
seedlings (Fig. 5). In the wild type, exogenous auxin inhibits
root elongation and the ID50 (dose conferring 50% inhibition
of root elongation; Maher and Martindale, 1980) for IAA was
2.7×10−8 mol/l. In sfc-1 mutants, exogenous auxin also
inhibited root elongation (Fig. 5). However, in contrast to the
wild type, for sfc-1 mutants, the ID50 was 5.8×10−9 mol/l, about
a 20-fold increase in sensitivity. We obtained similar root
elongation responses in replicates and in experiments using the
synthetic auxin 2,4-D in the place of IAA. These results
indicate that, at least in roots, sfc-1 mutants showed increased
auxin sensitivity.
We next wanted to determine whether SFC function is
related to that of the auxin-response factor gene,
MONOPTEROS (MP). Molecular characterization of MP has
shown that it encodes a transcription factor that binds to auxin
response elements in the promoters of auxin-regulated genes
(Hardtke and Berleth, 1998) and phenotypic characterizations
have shown that mp mutants have no root, a highly reduced
hypocotyl, and cotyledons with greatly reduced vein patterns
(Berleth and Jürgens, 1993). We constructed double mutants
using mpT370. This is a strong allele, and cotyledons contained
a primary vein that typically extended only half the length of
the cotyledon and no secondary veins (Berleth and Jürgens,
1993) (Figs 6, 7A). We used DIC optics to assess the presence
of provascular tissue in mpT370 cotyledons. We did not observe
provascular tissue in the lamina where secondary veins
typically appear, however for mpT370 primary veins, we
invariably observed provascular tissue that extended from the
distal end of the mp primary vein (averaging close to 50% the
length of the cotyledon) into the distal-most 2/3 of the organ
(n=54/54, Fig. 7G,H). These observations agree with, and
extend those of Przemeck et al. (1996), and suggest that mpT370
vascular defects include both vein patterning and
differentiation of vascular cell types from provascular tissue.
We identified the sfc-1 mpT370 double mutants among the
seedlings that were missing basal seedling parts, like the mp
single mutant. The sfc-1 mpT370 double mutant had cotyledons
that contained a short primary vein, no secondary veins and a
variable number of small VIs (Fig. 7D). The differentiated
primary vein of the double mutant tended to be shorter than
that of the mp single mutant (Fig. 6). More strikingly, in the
double mutant the primary vein provascular tissue extended
distally no more than 2 elongated cells beyond the
differentiated primary vein (n=39/39, Fig. 7I). This loss of
3210 M. K. Deyholos and others
percent of cotyledon primary veins
in each length class
100
75 - 100%
50 - 75%
25 - 50%
0 - 25%
90
80
70
60
50
40
30
20
10
scf-1 (124)
mpT370 (140)
mpT370 scf-1 (33)
primary vein provascular tissue was not observed in either
single mutant. In addition, sfc-1 mpT370 double mutants also
had a unique VI phenotype in which small VIs were scattered
in an apparently random pattern in the cotyledon lamina. The
observation of a unique and enhanced phenotype (the loss of
primary vein provascular tissue and the complete
randomization of VI positions) in the sfc-1 mpT370 double
mutant suggests that MP and SFC have partially overlapping
roles in patterning of cotyledon primary and secondary veins.
We also characterized double mutants between sfc-1 and the
auxin-resistant mutants axr1-3, axr2, axr4-2 and aux1-7
(Estelle and Somerville, 1987; Hobbie and Estelle, 1995;
Fig. 7. Cotyledon vein patterns of 11day wild type, sfc-1, mpT370, and
sfc-1 mpT370 double mutants.
(A-D) Dark-field images of cleared
intact cotyledons. (A) Wild-type
cotyledon. (B) sfc-1. (C) mpT370;
arrows correspond to the positions of
arrows in G,H. (D) sfc-1 mpT370
double mutant, arrows indicate some
of the vascular islands. (E-L) DIC
images of cleared intact cotyledons.
(E-I) Cotyledon primary vein or
primary vein provascular tissue.
(E) Wild-type primary vein. (F) sfc-1
primary vein. (G-H) mp primary vein
and provascular tissue from the
cotyledon shown in C, the white
arrow indicates distal end of
differentiated tracheary elements, and
the black arrows indicate provascular
tissue that extends distally from the
vein, H overlaps G by approximately
10%. (I) sfc-1 mpT370 double mutant
does not contain provascular tissue
distal from the midvein.
(J-L) Secondary vein area.
(J) Secondary vein from a wild-type
cotyledon. (K) Secondary vein region
of a sfc-1 mutant showing vascular
islands. (L) Vascular islands from a
sfc-1 mpT370 double mutant. Scale
bars, (A-D) 1 mm; (E-L) 100 µm.
Fig. 6. Primary vein lengths in mp, sfc and mp sfc double mutants.
Bars represent the percentage of cotyledon primary veins that
extended and terminated in each quarter of the cotyledon; white bars
indicate the proximal-most 25% of the cotyledon, light gray bars the
next 25%, dark gray bars the next 25%, and back bars the distal-most
25%. Numbers in parenthesis following each genotype indicate the
number of cotyledon primary veins scored.
Maher and Martindale, 1980; Pickett et al., 1990; Wilson et al.,
1990). We did not observe significant changes in cotyledon
vein pattern for any of these double mutants (data not shown).
These results suggest that the sfc vein pattern defects were not
affected by the reduced auxin responses resulting from auxinresistant mutations. We also analyzed double mutants between
sfc-1 and the auxin-overaccumulating mutant rty-5 (Boerjan et
al., 1995; King et al., 1995; Windsor and Waddell, personal
communication). The vein pattern of these double mutants also
matched that of the sfc single mutant (data not shown),
suggesting that interrupted sfc secondary veins could not be
ameliorated simply by increasing auxin levels.
sfc-1 seedling phenotype
To determine what other phenotypes accompanied the
interrupted vein pattern of sfc mutants, we characterized the
sfc-1 seedling phenotype. sfc-1 mutants could first be
distinguished from the wild type after 3 days by their smaller
size. At 14 days, the wild-type cotyledons and first leaf pair
were fully expanded; sfc mutants at this stage had organs that
were approximately 50% the size of the wild type (Fig. 8A,B).
The morphology of sfc-1 mutants matched wild-type plants
SCARFACE in plant vein patterning 3211
suggest that SFC function is required for normal activity of
both axillary meristems and the shoot apical meristem.
DISCUSSION
We have described the isolation and characterization of
mutants at the SCARFACE (SFC) locus. sfc mutants have
cotyledons, leaves, sepals and petals with discontinuous
secondary and higher order veins. That all four of these organ
types show similar defects indicates that a common mechanism
is used in secondary vein patterning in these organ types.
Fig. 8. Gross morphology of wild type and sfc-1 mutants. (A) 14-day
sfc-1. (B) 14-day wild type. (C) 63-day sfc-1, shown from the top,
has exaggerated growth from axillary meristems. (D) 63-day wild
type, which produced an inflorescence from the shoot apical
meristem, and for which axillary meristems were not activated. Scale
bars, (A-D) 2 mm.
in terms of organ number and organ position for the first 3
weeks, but soon after, activation of sfc-1 axillary meristems
resulted in very different-looking plants. In wild-type plants,
axillary meristems were largely inactive, and activation
resulted in production of secondary inflorescences (Fig. 8D).
In contrast, nearly all axillary meristems of sfc-1 rosette leaves
were active, and activation resulted in production of leaves in
the configuration of a rosette. Axillary meristem derived leaves
also produced new axillary leaf primordia. This growth pattern
resulted in a plant that contained more than 100 leaves, and
that resembled a dense sphere (Fig. 8C).
sfc-1 and wild-type plants also differed in their transition to
flowering. Under our growth conditions, the inflorescence stem
was apparent in all wild-type plants by the end of the third
week. In contrast, less than 10% of sfc-1 plants had made the
transition to flowering after ten weeks, and the sfc mutants that
did bolt initiated this process at least two weeks later than wild
type. The sfc-1 flowering stem was short and produced few
flowers, yet the flowers that were produced contained normal
numbers and arrangements of floral organs (data not shown).
These flowers, however, senesced prior to anthesis, rendering
the mutant plants infertile. Taken together, these observations
Multiple genes function to specify continuity of
cotyledon secondary veins
In addition to SFC, mutations at four other loci (AXR6, CVP1,
CVP2 and MP) also define genes with functions in cotyledon
vein patterning (Hobbie et al., 2000; Carland et al., 1999;
Berleth and Jürgens, 1993). All these mutants show similar
cotyledon vein pattern defects; cotyledon primary veins are
largely intact, and cotyledon secondary veins are disrupted.
That primary veins are intact and secondary veins are
disrupted by mutations at five loci provides strong evidence
for separable pathways specifying patterning of these two
vein types.
Because auxin has been implicated in vein patterning, a
variety of experiments have been carried out to determine
whether auxin responses in these mutants are intact. Plants
mutant for mp show reduced auxin transport, pin-like flowers,
and the loss of structures (root, hypocotyl) associated with
auxin signaling (Przemeck et al., 1996). In contrast, cvp1 and
cvp2 mutants have normal auxin transport, normal flower
structures, and no morphological defects (Carland et al., 1999).
Auxin transport has not been analyzed in plants homozygous
mutant at the axr6 or sfc loci, however their vascular
phenotypes do not match the pattern observed for wild-type
plants treated with auxin polar transport inhibitors (Sieburth,
1999; Mattsson et al., 1999) or the pattern of plants mutant for
genes proposed to encode components of the polar auxin
transport machinery (Okada et al., 1991; Mattsson et al., 1999).
Auxin perception has been measured for axr6 and sfc mutants
using root elongation assays; whereas axr6 mutants show
reduced auxin response (Hobbie et al., 2000), sfc mutants show
enhanced auxin response. Thus, a clear picture of the
relationship between auxin, the products of these five genes,
and secondary vein patterning has yet to emerge.
The sfc mp double mutant phenotype suggests
overlapping roles
One approach to sorting out whether the genes identified by
this collection of mutants with discontinuous cotyledon veins
function in related processes is to analyze double mutants. We
have begun this analysis by characterizing sfc mp double
mutants. One element of the unique double mutant phenotype
was the sharp reduction in cotyledon primary vein provascular
tissue. In the mp single mutant, we observed primary vein
provascular tissue that extended into the distal quarter of
cotyledons, but when mp was combined with sfc, this
provascular tissue was essentially eliminated. These
observations suggest that SFC and MP have partially redundant
roles in cotyledon primary vein patterning. However, that some
3212 M. K. Deyholos and others
primary vein still remains in the double mutant indicates that
other genes also contribute to this process.
Another element of the unique sfc mp double mutant
phenotype was the small VIs that were apparently randomly
distributed. In sfc single mutants, secondary veins are replaced
by large VIs that mostly appear in positions similar to those of
secondary veins. The reduced size of VIs in the double mutant
suggests that MP contributes to VI formation in sfc single
mutants. That VI position does not completely match
secondary vein positions in the scf single mutant indicates a
defect in secondary vein patterning. Because this patterning
defect is so dramatically enhanced in the scf mp double mutant
suggests that SFC and MP also have partially redundant roles
in cotyledon secondary vein patterning.
SFC might function as a negative regulator of auxin
responses
Root elongation experiments indicated that sfc mutants showed
enhanced sensitivity to exogenous auxin. Because we isolated
six recessive mutant alleles with the same mutant phenotype,
it is reasonable to expect that these represent loss-of-function
alleles. One way to reconcile increased auxin sensitivity in a
loss-of-function allele is if SFC functions as a negative
regulator. Loss of a putative negative regulator would be
expected to allow increased activation of the affected pathway,
which in the case of auxin signaling might be detectable as
increased sensitivity.
Possible function of SFC as a negative regulator might
explain the presence of VIs in sfc mp double mutants. mp
mutants generally do not have VIs (Przemeck et al., 1996),
however VIs were a regular feature of sfc mp double mutants.
Molecular characterization of MP indicates that it encodes a
transcription factor that is likely to regulate gene expression in
response to auxin (Hardtke and Berleth, 1998). The numerous
morphological defects observed in mp mutants, including vein
axial defects, are thus explained by the loss of auxin-derived
positive signals. Residual positive signals for secondary veins
or VIs might be amplified in sfc mp double mutants if
sfc mutations result in the loss of negative regulation. This
possibility leads to the question of the origin of possible
residual positive signals. Because mpT370 is believed to be a
null allele, VI formation in the mpT370 sfc-1 double mutant
suggests the existence of other positive signals.
Reconciling a role for SFC as a negative regulator with its
redundancy with MP is more problematic. MP is proposed to
control gene expression in the context of providing positive
auxin signaling (Hardtke and Berleth, 1998). The simplest
extrapolation from our observation of redundancy is that SFC
also provides positive signaling functions. Recently, the
characterization of shy2 mutants also revealed phenotypic traits
with apparently opposing auxin related phenomena (Tian and
Reed, 1999).
Apical dominance and transport of leaf-generated
signals
Apical dominance has been linked to auxin signaling, although
its precise role in this process continues to be elusive (Cline,
1994; Stirnberg et al., 1999). The loss of apical dominance in
sfc mutants could be the result of defects in dominancepromoting signals reaching the axillary meristems, or defects
within the axillary meristems themselves. Little is known about
how axillary meristems become activated, although a role for
auxin as a positive signal has been proposed based on increased
auxin levels in axillary buds following dominance release
(Hillman et al., 1977). If auxin does function positively in
axillary meristem activation, then the increased auxin
sensitivity of sfc mutants might allow activation regardless of
the presence of dominance-promoting signals. However, auxin
is also considered a likely candidate for at least part of the
signalling pathway that promotes apical dominance, and the
loss of apical dominance in mutants is often accompanied by
reduced auxin or reduced auxin responses (e.g. see Ruegger et
al., 1997). In stems, auxin polar transport occurs in specialized
cells that are associated with vascular tissue. The interrupted
veins in sfc mutants might also interrupt delivery of auxin or
other apical dominance-promoting signals. Vascular tissue has
also been proposed to transport leaf-generated signals for floral
induction (King and Zeevaart, 1973; Lang et al., 1977). The
strong reduction in flowering of sfc mutants might be explained
if sfc vascular discontinuities also decreased the delivery of
putative leaf-generated floral-induction signals to the shoot
apical meristem.
Models for vein patterning
Two models have been proposed for patterning of veins
(reviewed by Nelson and Dengler, 1997). One model, the
canalization of auxin flow hypothesis, suggests that linear paths
for veins are first specified by the direction of auxin polar
transport (Sachs, 1991). An alternative model, the diffusionreaction prepattern hypothesis, proposes that initially random
signals are refined by the combined action of an autocatalytic
activator and a long-distance suppressor (Meinhardt, 1982;
Koch and Meinhardt, 1994). The presence of VIs, and the
malleability of their positions within cotyledon lamina in
different genetic backgrounds, is difficult to explain in terms
of the canalization model. Molecular and more extensive
genetic characterizations of SFC, CVP1, CVP2 and AXR6
should provide the data for distinguishing between these two
models, or might suggest new models to explain the
developmental events that underlie vein patterning.
We thank Gary Drews for critical reading of this manuscript, and
Candace Waddell and members of the Waddell lab for discussion of
this work while it was in progress. We are also grateful for having
received seed stocks from Thomas Berleth (mp), Candace Waddell
and Aaron Windsor (rty-5), and the Arabidopsis Biological Resource
Center (aux and axr seed stocks). We are grateful to both NSF and
NSERC for support of this work (NSF 99-82876 to L. E. S. and an
NSERC PGSB fellowship awarded to M. K. D.).
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