Download The REVOLUTA gene is necessary for apical meristem development

Development 121, 2723-2735 (1995)
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2723
The REVOLUTA gene is necessary for apical meristem development and for
limiting cell divisions in the leaves and stems of Arabidopsis thaliana
Paul B. Talbert, Haskell T. Adler, David W. Parks* and Luca Comai
University of Washington, Department of Botany, Box 355325, Seattle, WA 98195-5325, USA
*Present address: Camellia Forest Nursery, 125 Carolina Forest Rd, Chapel Hill, NC, 27516
SUMMARY
The form of seed plants is determined by the growth of a
number of meristems including apical meristems, leaf
meristems and cambium layers. We investigated five
recessive mutant alleles of a gene REVOLUTA that is
required to promote the growth of apical meristems and to
limit cell division in leaves and stems of Arabidopsis
thaliana. REVOLUTA maps to the bottom of the fifth chromosome. Apical meristems of both paraclades (axillary
shoots) and flowers of revoluta mutants frequently fail to
complete normal development and form incomplete or
abortive structures. The primary shoot apical meristem
sometimes also arrests development early. Leaves, stems
and floral organs, in contrast, grow abnormally large. We
show that in the leaf epidermis this extra growth is due to
extra cell divisions in the leaf basal meristem. The extent
of leaf growth is negatively correlated with the development of a paraclade in the leaf axil. The thickened stems
contain extra cell layers, arranged in rings, indicating that
they may result from a cambium-like meristem. These
results suggest that the REVOLUTA gene has a role in regulating the relative growth of apical and non-apical
meristems in Arabidopsis.
INTRODUCTION
cases axillary meristems appear to arise from the leaf primordium rather than from separate meristematic cells
(Majumdar, 1942; Irish and Sussex, 1992). Pioneering surgical
studies by Snow and Snow (1942) showed that the formation
of an axillary shoot was dependent on the subtending leaf primordium and inhibited by the apical meristem. These results
suggest that axillary meristems may form in response to positional information set up by the action of opposing morphogenetic gradients. Genetic or molecular data supporting this
model are currently lacking.
The flowering plant Arabidopsis thaliana has proved useful for
the molecular genetic analysis of developmental problems. Arabidopsis has a basal rosette of vegetative leaves and an erect
branched inflorescence. The branching pattern of the inflorescence is most easily described in terms of the phytomer concept.
A phytomer (Galinat, 1959) or metamer (White, 1984) is a reiterated module typically composed of an internode and a node
with its leaf and axillary branch. Many variations of this basic
pattern occur, such as the suppression of leaves or branches, or
their modification into structures specialized for assorted reproductive or vegetative functions. Three types of phytomers have
been described in the primary shoot of Arabidopsis (Schultz and
Haughn, 1991; Fig. 1A). All three are arranged in a spiral phyllotactic pattern. Type 1 comprises the basal rosette. Each type 1
phytomer has an extremely short internode, a rosette leaf, and an
axillary meristem that may form a branch. Type 2 comprises the
basal portion of the flowering stalk. These phytomers have long
internodes, cauline leaves and axillary branches. The first axillary
Elaboration of the plant body pattern depends primarily on the
proper regulation of cell division versus cell differentiation at
the growth sites, called meristems. In seed plants apical growth
is carried out by the apical meristems. Although structurally
identical, shoot apical meristems differ ontogenetically. A
primary shoot apical meristem originates during embryogenesis and becomes the apex of the primary shoot. Secondary
shoot apical meristems develop later on the sides of the
primary shoot and form lateral shoots. In many seed plants,
radial growth of the shoot is conferred by the cambium, a cylindrical meristematic layer in the shoot body. Growth of lateral
‘leafy’ organs (i.e. leaves, petals, etc.) occurs from transient
meristems formed on the flank of the apical meristem. Root
growth occurs from analogous apical and cambial meristems.
At present we understand very little of the regulation and interaction of these different types of meristems.
Morphological and developmental studies suggest that
different meristems interact. An example is provided by the
development of axillary meristems. These meristems form at
repeated positions along the shoot, known as nodes, that are
separated by sections of stem called internodes. A node
typically bears one or more leaves, each of which contains a
meristem in its axil that can form a branch shoot. Axillary
meristems are thought usually to originate directly from the
shoot apical meristem as detached meristematic cells in the
axils of the leaf primordia (Garrison, 1955). However, in some
Key words: meristem, branching, leaf growth, Arabidopsis,
REVOLUTA
2724 P. B. Talbert and others
comparisons of wild-type and mutant phenotypes were made on
branch originates in the uppermost leaf axil and others develop
cohorts of plants grown side-by-side under identical conditions.
basipetally through the type 2 and 1 phytomers (Alvarez et al.,
Cohort 1 was grown in May-July of 1994, cohort 2 in July-August
1992; Hempel and Feldman, 1994). In both of these phytomer
1994, cohort 3 in October-November 1994, cohort 4 in March-May
types, a second branch (accessory branch) may later develop
1994, and cohort 5 in October-December 1993. To achieve 100% gerbetween the axillary branch and its subtending leaf (Fig. 1A).
mination and uniform growth, cohort 4 was germinated on growth
Type 3 phytomers constitute the fertile terminal portion of the
medium containing 0.5× Murashige-Skoog salts (Gibco) under constalk, known as the main florescence (Weberling, 1989). Each
tinuous illumination with Philips 40 W Cool White lights (75 µE/m2
type 3 phytomer has an intermediate-length internode, no leaf and
sec) and transplanted to soil on day 14 after sowing.
a lateral flower. As observed by Goethe (1790), flowers are speMutagenesis and allelism tests
cialized shoots with floral organs in the place of leaves. Thus the
All five rev mutations were recovered from the M2 progeny of M1
flowers of Arabidopsis can be viewed as floral branches which
seeds mutagenized with ethyl methanesulfonate (EMS). Alleles revare serially homologous with the axillary branches. While the
1, rev-2 and rev-4 were each recovered from an independent batch of
floral branches have determinate growth and are composed of
M1 seeds of ecotype Nossen-0 (No-0) mutagenized in 10 mM EMS
special phytomer types (i.e. the whorls of four sepals, four petals,
for 17 hours. This dosage gave siliques with segregating M2 embryosix stamens and two carpels), the indeterminately growing
lethals on approximately half of the M1 plants. The rev-3 and rev-5
axillary and accessory branches repeat in part the pattern of
(=spitzen-1; Alvarez, 1994) alleles were induced in the Columbia
phytomers found on the primary shoot and are therefore termed
ecotype and generously supplied by Laura Conway and David Smyth,
paraclades (Weberling, 1989). The type 2 phytomers of a firstrespectively. Allelism of rev-1 and rev-2 was determined by their nonorder paraclade give rise to second-order paraclades, and these
complementation in multiple reciprocal crosses. Further complementation tests were carried out by pollinating a marked er ttg rev-2 stock
give rise to higher-order paraclades (Fig. 1A). The type 3
with a rev-3/REV, rev-4 /rev-4, or rev-5/rev-5 parent. Individuals that
phytomers of a paraclade make up a coflorescence (Weberling,
were Rev but not Er or Ttg made up half (15/32), all (7/7), and all
1989). The primary shoot distal to the rosette and the paraclades
(23/23), respectively, of the progeny in these three crosses. The revtogether make up the inflorescence.
1 allele was backcrossed to the wild type three times to eliminate any
The study of Snow and Snow (1942) suggests that genes
effects of additional mutations on the phenotype. The other alleles
involved in axillary meristem development might also have roles
have been backcrossed once.
in the primary apical meristem or in the development of leaves.
Mapping
We therefore speculated that mutations in Arabidopsis genes
affecting apical meristems might be identifiable by changes in leaf
Mapping of rev-1 used the polymerase chain reaction mapping
markers DFR (Konieczny and Ausubel, 1993) and nga129 (Bell and
morphology, as is true of the tomato gene lanceolata (Caruso,
1968), and screened for mutants accordingly. We describe here mutants
primary
defective in a gene, REVOLUTA (REV),
shoot
that is necessary to promote the normal
rev
REV
growth of apical meristems, including
primary
paraclade meristems, floral meristems
shoot
and the primary shoot apical meristem.
Simultaneously, REV has an opposing
3
cauline
effect on the meristems of leaves, floral
paraclade
organs and stems, being necessary to
limit their growth. Thus rev mutations
cauline
reveal a novel and fundamental regulaparaclade
tory feature of plant body pattern elaboration.
rosette
paraclade
2p
accessory
paraclade
MATERIALS AND METHODS
Plant growth conditions
Plants were grown in 5.0-cm pots in the
greenhouse in a buffered soil mix of 67%
peat moss and 33% pumice. Natural
sunlight was supplemented by 16 hours of
illumination with 1000 W high pressure
sodium lights. Plants were subirrigated
with alternating solutions of Peters PeatLite Special (20-10-20) or Peters Dark
Weather Food (15-0-15), both diluted to
100 ppm. Temperature control set points
were at 18-21˚C during the day and 9-13˚C
at night. To assist uniform germination,
wetted seeds were kept at 4˚C for 2 days
prior to planting. Because of the inherent
variability of greenhouse conditions, all
2p
2p
3p
*
2
3p
*
*
*
cauline
leaf
cauline
leaf
**
1
A
rosette leaf
rosette leaf
B
Fig. 1. Schematic representation of wild-type REV and mutant rev morphologies. (A) Wild type,
showing phytomer types (1, 2 and 3) and branching pattern. (B) rev, showing reduced growth of
primary shoot, paraclades and flowers, and increased growth of leaves and stems. s, flowers;
2p, second-order paraclade; 3p, third-order paraclade; *, axil lacking a paraclade.
Meristem development in Arabidopsis 2725
Ecker, 1994) on chromosome 5. DFR maps to 61.5 mu (AAtDB
version 3-4) and nga129 maps to approximately 86.4 mu (6.6 mu
below m435; Bell and Ecker, 1994). A rev-1 mutant (No-0 ecotype)
was crossed to wild-type Landsberg erecta and phenotypically Rev
F2 individuals were scored. We also mapped REV with respect to the
morphological marker lfy (Weigel et al., 1992). LFY maps to 89.5 mu
on the distal tip of chromosome 5 (AAtDB version 3-4). Rev F2 individuals from a rev-1 × lfy-6/+ cross were progeny tested for segregation of lfy rev F3 double mutants (Adler et al., unpublished data).
Map distances were corrected for double cross-overs using the
Kosambi mapping function (Koornneef and Stam, 1992), and the 95%
confidence interval was computed using the two tails of the binomial
distribution (Woolson, 1987).
Measurements and cell counts
Measurements of plant organ lengths were made with either a Measy
dial caliper (Max Mägerle, Sen., Switzerland), a calibrated Zeiss
reticle or a standard metric ruler. Leaf areas in cohort 4 were determined by harvesting leaves and tracing their outlines. The outlined
areas were measured using a Kurta digitizing tablet and SigmaScan
software (Neff and Van Volkenburgh, 1994). Epidermal peels of the
adaxial leaf surfaces were made using Revlon nail strengthener and
transparent tape. The peels were affixed to microscope slides and
three patches of cells from different parts of each leaf were traced in
a camera lucida, avoiding the midrib. Cells in each patch were counted
and areas of the patches were determined (Neff and Van Volkenburgh,
1994). The mean cell sizes of the three patches of each leaf were
averaged to get the mean cell size for that leaf. Similar results were
obtained when the three patches were grouped as a single patch.
Unpaired two-tailed Student’s t-tests on measurement sets were
conducted using the Statview Student statistical software package.
The P value for these tests is the probability that the true means of
the two genotypic populations are identical.
Determination of the region of leaf growth was made by marking
growing leaves from cohort 5 with dots of Sumi black calligraphy ink
(Yasutomo & Co.) placed 1-2 mm apart along the midrib and
measuring subsequent growth relative to the fixed dots.
Anatomical sections
Plant material for sectioning was fixed in formalin-aceto-alcohol,
dehydrated through an increasing tertiary butyl alcohol series and
embedded in Paraplast X-Tra (Oxford Labware) paraffin (Johansen,
1940). Serial 10 µm sections were cut on a Spencer microtome and
mounted on microscope slides with Haupt’s adhesive (Johansen,
1940). Paraffin was dissolved in xylene and the slides were hydrated
through a decreasing ethanol series before staining 1-2 minutes in
0.25% toluidine blue, or in safranin O and fast green FCF essentially
as described by Johansen (1940) except for the following: picric acid
and ammonia were omitted from the washes; 0.25 g of fast green was
dissolved in a mixture of 100 ml each of methyl cellosolve, absolute
ethanol and methyl salicylate; and clearing was in a mixture of 50%
methyl salicylate, 25% absolute ethanol, and 25% xylene. Sections
were photographed with a Nikon Microphot FX using Fujicolor 100
film.
RESULTS
rev alleles and mapping
We recovered three independent mutants with a syndrome consisting of revolute (downwardly curled) leaves, reduced
branching and many infertile flowers from the M2 self-progeny
of the No-0 ecotype plants mutagenized with EMS. The first
mutant was backcrossed to the wild type and its mutant
phenotype segregated as a simple recessive mutation in the F2
generation (160 wild type: 52 mutant; χ2 = 0.02; P = 0.9), indicating that the mutation is likely to be a loss-of-function. Complementation tests showed that the mutations defined a single
locus that we call REVOLUTA represented by three alleles
(rev-1, rev-2 and rev-4). Two EMS-induced alleles (rev-3 and
rev-5) were available in the Columbia ecotype.
The rev-1, rev-2 and rev-4 alleles all had indistinguishable
phenotypes. The rev-3 and rev-5 mutants were more and less
severely affected than these alleles, respectively, but it is not
yet clear whether these differences are due to the properties of
the latter alleles or to modifying factors from the mutageneses
or ecotype. The phenotype of rev-1 was stably transmitted
through three backcrosses with only a slight reduction in
severity and this allele was therefore selected for careful
description.
REV showed linkage to the fifth chromosome markers DFR
(11 recombinants/32) and nga129 (13 recombinants/64). Six of
the 11 recombinants for DFR were also recombinant for
nga129, suggesting that REV is distal to nga129. This placed
REV approximately 21 mu from nga129 at approximately 108
mu (99 mu ≤ actual location ≤124 mu) on the bottom tip of
chromosome 5 (Koornneef and Stam, 1992). This map location
was verified by REV showing tight linkage to LFY (2 recombinants/33). This second experiment placed REV approximately 6.1 mu from the LFY locus. Because the nga129 data
suggest that REV is distal to LFY, this experiment gives a map
position for REV of approximately 95.6 mu (90 mu ≤ actual
location ≤110 mu) on the bottom tip of chromosome 5.
Overview of the Rev vegetative phenotype
The rev mutations were pleiotropic and strongly affected both
vegetative and reproductive shoot development (Fig. 1B). In
contrast, no differences from wild-type REV controls were
discerned in the roots of rev-1 and rev-2 mutants up to 2-3
weeks of age. Older roots have not been examined. We will
first describe the effects of the rev-1 mutation on the vegetative phytomers (types 1 and 2) before describing its effects on
the reproductive phytomers (type 3).
The rev-1 mutation caused overgrowth of both rosette and
cauline leaves. The rosette leaves of rev-1 plants were not
readily distinguishable from wild-type No-0 leaves prior to
bolting. As bolting began, however, the youngest rosette leaves
became abnormally large and distorted or uneven in shape as
they matured. This overgrowth was even more dramatic in the
cauline leaves. The latter were longer and narrower than wildtype cauline leaves, had rolled-under margins, and curved
downward along their longitudinal axes (Fig. 2A,B). Both the
leaves and the primary shoots of rev-1 mutants were often
darker green than those of wild type. Paraclades frequently
failed to develop in both rosette and cauline leaf axils (Fig. 2B).
The axils instead appeared empty, contained thin filamentous
structures usually bearing branched trichomes, or bore leaves
with or without a visible supporting stem (Fig. 2C,E-G).
Paraclade formation is reduced in rev-1 mutants
Table 1 shows the mean numbers of vegetative nodes, leaves
and paraclades on the primary shoots and first-order paraclades
of REV and rev-1 plants from cohort 1. The rev-1 plants had
an average of about one more rosette leaf and one less cauline
leaf on the primary shoot than did the REV plants, but only the
2726 P. B. Talbert and others
Fig. 2. Morphology of wild-type and rev-1 plants. (A) REV plant. (B) rev-1 plant. (C) REV cauline leaf axil with paraclade. (D) REV
florescence apex. (E-G) rev-1 cauline leaf axils: (E) empty axil; (F) axillary filament; (G) axillary leaf. (H) rev-1 florescence apex with flowers
and tapered filaments. (I) rev-1 florescence apex with clustered tapered filaments. (J) rev-1 tapered filaments subtended by knobs and a filament
(arrow). (K) rev-3 arrested florescence apex. f, fertile flower; k, knobs; s; sterile flower: t; tapered filament.
Meristem development in Arabidopsis 2727
Fig. 3. Anatomy of cauline leaves and axils. (A-G) Longitudinal axillary sections. (A) Immature REV axil. (B) Near-mature REV axil.
(C) Empty REV axil. (D-G) rev-1 axils with: (D) a leaf and terminal knob on a short stem; (E) a filament; (F) club-like projections;
(G) trichomes. (H-K) Midlength leaf cross-sections. (H) REV leaf midvein. (I) rev-1 leaf midvein. (J) REV leaf margin. (K) rev-1 leaf margin.
Bars, (A-D,F,H,I) 20 µm; (E,G,J,K) 100 µm. l, leaf; p, paraclade; s, stem.
2728 P. B. Talbert and others
Table 1. Mean numbers of leaves and paraclades in wildtype REV and mutant rev-1 plants
Structures
REV
rev-1
P value of t-test
Primary shoots*
Vegetative nodes
Rosette leaves
Cauline leaves
Rosette paraclades
Cauline paraclades
Accessory rosette paraclades
Accessory cauline paraclades
8.2
6.1
2.1
4.4
2.1
0.2
1.8
8.5
7.1
1.4
1.3
0.85
0
0
0.50
0.06
0.002
0.0001
0.0001
0.33
0.0001
First-order paraclades†
Vegetative nodes
Cauline leaves
Cauline paraclades
Accessory cauline paraclades
2.7
2.6
2.7
1.1
2.1
2.1
0.14
0
0.01
0.06
0.0001
0.0001
*Data are from the primary shoots of 13 REV and 13 rev-1 48-day-old
plants (cohort 1).
†Data are from 55 REV and 30 rev-1 secondary shoots of cohort 1,
comprised of approximately equal numbers of rosette and cauline paraclades.
Table 2. Mean dimensions of leaves and internodes in
REV and rev-1 plants
Organ
Dimension
Cotyledon blade*
length (cm)
1st rosette leaf*
length (cm)
3rd rosette leaf*
length (cm)
Longest rosette leaf†
length (cm)
1st cauline leaf‡
length (cm)
2nd cauline leaf
length (cm)
1st cauline leaf
width (cm)
2nd cauline leaf
width (cm)
1st cauline leaf
area (cm2)
2nd cauline leaf
area (cm2)
1st leaf epidermal cell
area (µm2)
2nd leaf epidermal cell area (µm2)
1st cauline internode
length (cm)
1st cauline internode§ diameter (mm)
REV
rev-1
P value
of t-test
0.27
1.52
2.62
4.9
3.7
2.9
1.3
0.9
3.5
2.1
2100
2000
7.4
1.05
0.34
1.91
2.93
6.8
6.9
6.3
1.1
0.7
5.1
3.4
2200
1600
3.8
1.24
0.0001
0.0001
0.0238
0.0001
0.0001
0.0001
0.10
0.0008
0.0059
0.0016
0.70
0.031
0.0012
0.0083
*Data from cohort 3, consisting of 17 REV and 20 rev-1 28-day-old plants.
†Data from cohort 2, consisting of 21 REV and 29 rev-1 37-day-old plants.
Nearly identical mean lengths were made on a rev-3 cohort (data not shown).
‡All cauline leaf measurements and internode length were measured on
cohort 4, consisting of 12 REV plants and 14 rev-1 plants, on material
harvested on day 49. See Materials and Methods and the text for further
details.
§Internode diameter was measured just above the rosette on 53-day-old
plants from cohort 1.
leaf on the primary shoot than did the REV plants, but only the
latter difference was significant at the 95% confidence level.
On the primary shoots of REV plants, paraclades developed
from the axils of 100% of the cauline leaves and 72% of the
rosette leaves. In sharp contrast, the primary shoots of rev-1
mutants formed paraclades in 61% of cauline leaf axils and in
18% of rosette leaf axils (Table 1). Accessory paraclades were
visible in the axils of about 85% of the REV cauline leaves, but
in only about 4% of rosette leaf axils. No accessory paraclades
were formed in rev-1 mutants. Rosette paraclades had more
vegetative nodes than cauline paraclades in both genotypes
(REV: 3.5 vs. 1.9, P = 0.0001; rev-1: 2.5 vs. 1.6, P = 0.002).
Second-order paraclades normally developed in the cauline
leaf axils of the REV first-order paraclades, and accessory par-
Table 3. Growth in the basal portion of leaves
Measurement
REV
rev-1
Length of first cauline leaf, day 21 (mm)
Length of basal region*, day 21 (mm)
Percentage of total leaf length in basal region, day 21
Increase in total leaf length, day 21-56 (mm)
Increase in length of basal region, day 21-56 (mm)
Percentage of total increase occurring in basal region
12.3
2.3
19
7.3
6.5
89
12.3
2.7
22
20.4
18.1
89
*The basal region is the arbitrary region of the leaf that is proximal to all
ink dots placed on the expanding 21-day-old leaves (cohort 5) to serve as
position markers.
aclades developed in 41% of these axils. However, some of the
vegetative nodes of REV first-order paraclades (10/151)
developed second-order paraclades even though they failed to
form a macroscopic leaf. Such second-order paraclades were
subtended by putative vestigial leaf tissue, similar to that
described by Hempel and Feldman (1994) on the primary
shoots of Columbia and Landsberg erecta plants. Rarely (2/151
nodes), a second-order paraclade failed to develop in the axil
of a cauline leaf on a first-order paraclade. Thus about 93% of
the vegetative nodes on REV first-order paraclades bore cauline
leaves and about 99% bore second-order paraclades.
On the rev-1 first-order paraclades, all vegetative nodes had
cauline leaves, but only 4/62 (6%) also bore second-order paraclades. A leaf, with or without a visible supporting stem, grew
from 3 of the 80 rev-1 cauline leaf axils on the primary and
secondary shoots.
Data from other cohorts of rev-1, rev-2 and rev-3 plants
generally confirmed the results shown in Table 1, except that
the number of accessory branches in wild-type plants was
generally less and the rev mutants in these cohorts had even
lower rates of paraclade formation, with the fraction of primary
cauline leaf axils with paraclades ranging from 0% to 36%
(data not shown).
Decapitation of rev-1 plants
We tested whether the failure of paraclade formation in rev-1
plants could be relieved by decapitation of the primary shoot,
as might be expected if it resulted from extreme apical
dominance. A strain of rev-1 (from a Landsberg erecta
outcross) was used which typically forms no paraclades.
Fourteen flowering rev-1 plants were decapitated just above the
rosette on day 31, and 19 sibs were left undecapitated. 20 days
later no rosette paraclades had formed on the decapitated
plants, and only one rosette paraclade was present on the undecapitated plants. An analogous experiment with a normal rev1 strain also failed to detect a stimulation of paraclade
formation by decapitation (data not shown).
Anatomy of rev-1 axils
To understand better the nature of the defect in rev-1 paraclade
growth, we made serial longitudinal sections through wild-type
and rev-1 cauline leaf axils. In a cauline leaf axil from a 17day-old wild-type plant from cohort 2 (just prior to visible
bolting), the developing paraclade appeared to arise from the
base of the leaf (Fig. 3A). Procambium was differentiating in
the leaf and primary shoot, and procambial strands were
evident by their darker staining in the developing paraclade. In
the leaf, vacuolation was more advanced in the abaxial cells
Meristem development in Arabidopsis 2729
than in the adaxial cells, as has been noted in other angiosperm
species (Steeves and Sussex, 1989). On the edges of the leaves
were heavily stained club-like stipules (data not shown). In a
more mature (bolting) axil (Fig. 3B), vascular tissue was well
developed in the leaf, stem and paraclade. Adaxial leaf cells
were vacuolated; however, differentiation of the palisade layer
and spongy mesophyll had not yet taken place.
As mentioned above, some axils on wild-type first-order paraclades were found that lacked second-order paraclades. We
sectioned four such mature axils and found all four lacked any
apparent axillary bud or mound of meristematic cells (Fig. 3C).
A cryptic meristem of a few cells lacking obvious organization
could still be present.
In contrast to the all-or-none development of paraclades in
wild-type axils, rev axils frequently contained unusual intermediate structures. In 29 rev-1 cauline leaf axils, 3% of the
axils bore a leaf (Fig. 2G), 24% bore thin filaments (Fig. 2F),
and 62% of the axils appeared empty except for small bumps
of tissue present in about half of them. In 44 rev-3 axils, 23%
bore leaves, 23% bore filaments and 43% were empty or had
small bumps. Seven rev-1 axils from bolting plants were
chosen for sectioning. One axil contained a paraclade indistinguishable from a wild-type paraclade (data not shown), while
another contained an apparent abortive paraclade consisting of
a leaf and terminal knob separated from their subtending leaf
by a short stem-like region (Fig. 3D). One axil contained a
short filament about five cell layers in diameter (Fig. 3E). Four
others contained one or more deeply staining club-shaped projections, often on a bulge of tissue in the position normally
occupied by a paraclade (Fig. 3F). These were reminiscent of
stipules, but were generally larger, more irregularly shaped,
and were not located at the leaf edges, where normal stipules
were present. These projections appear to correspond to the
small axillary bumps visible macroscopically. Two of these
axils also supported unbranched trichomes (Fig. 3G), which
were not observed in wild-type axils. These unusual structures
may indicate a premature differentiation of the presumptive
rev-1 axillary meristem.
The REV gene is required to limit cell division in
leaves
In contrast to the abortive development of paraclades, the
leaves of rev-1 mutants grew abnormally large. The difference
in leaf size between wild-type and rev-1 plants was not obvious
in the earlier rosette leaves, but we measured significant size
differences in the cotyledons and first and third leaves from
cohort 3 (Table 2). Later leaves differed more dramatically: the
mean length of the longest rosette leaf (ordinarily the youngest
leaf) of rev-1 plants was about 39% longer than wild-type
controls, and rev-1 cauline leaves became up to twice as long
as their wild-type counterparts.
To determine the timing of this excessive leaf growth, we
measured the lengths of the first and second cauline leaves of
wild-type and rev-1 plants in cohort 4, daily from first visible
bolting on day 33 until the growth of the cauline leaves of all
wild-type and most rev-1 plants had terminated on day 45.
(Bolting in this cohort was delayed more than a week relative
to other cohorts because seeds were germinated on plates and
transplanted to soil on day 14.) Over the first week of measurement the average growth rate of the first cauline leaves was
about 80% greater for rev-1 plants than for wild-type plants
(5.8 mm/day vs. 3.2 mm/day), and rev-1 second cauline leaves
grew more than twice as fast as their wild-type counterparts
(Fig. 4A). Most wild-type leaves quit growing by day 43, but
most rev-1 leaves continued to grow at least 2 days longer.
To determine the respective contributions of cell expansion
and increased numbers of cells to the excessive growth of rev1 cauline leaves, we harvested the first and second cauline
leaves from cohort 4 on day 49 and compared the adaxial
surface areas of REV and rev-1 leaves, as well as the average
surface areas of the epidermal cells within these leaves (see
Materials and methods). As shown in Table 2, both the first
and second rev-1 cauline leaves had significantly larger surface
areas (about 45% and 63% larger, respectively) than the wildtype controls. Despite these size differences, the average
surface areas of individual epidermal cells from the rev-1 first
cauline leaves and from wild-type first and second cauline
leaves were not significantly different. The rev-1 second
cauline leaf cells were smaller, perhaps indicating that not all
of them had completed expansion by day 49. Both the first and
second rev-1 cauline leaves must therefore contain more
epidermal cells than their wild-type counterparts. We estimate
the average number of epidermal cells on the adaxial surfaces
of the first and second wild-type cauline leaves to be 1.7×105
and 1.1×105, respectively. The corresponding estimates for
rev-1 leaves are 2.3×105 and 2.2×105 epidermal cells. This
implies that one function of the wild-type REV gene must be
to limit cell division in the leaves.
Cell division in rev-1 leaves is predominantly in the
basal region of the leaf
In an expanding wild-type leaf, cell division is confined to the
basal region (Pyke et al., 1991). To determine if the extra
growth in rev-1 leaves was also confined to the basal regions
or spread over other parts of the leaves, expanding 21-day-old
first cauline leaves of plants in cohort 5 were marked along
their midvein with dots of ink. The positions of dots were
determined on day 21 and again on day 56. Table 3 compares
a representative REV leaf with a rev-1 leaf with a similar initial
size and placement of dots. Both leaves had about 90% of their
growth in the basal fifth of the leaf. Similar results with other
leaves (data not shown) confirm that cell divisions in both REV
and rev-1 leaves are confined to the basal regions of the leaves.
rev-1 leaf size is diminished when paraclades form
To see whether the overgrowth of leaves in rev-1 mutants is
related to the failure to form paraclades, we compared the
lengths of mature cauline leaves which subtended paraclades
and those that lacked paraclades on 25 rev-1 plants from cohort
2. Although rev-1 cauline leaves were enlarged whether or not
they subtended paraclades, the mean length of 11 first cauline
leaves that lacked paraclades was 24% greater than the mean
length of 14 first cauline leaves that subtended paraclades (6.8
cm vs. 5.5 cm, respectively; P = 0.014). Similarly, 15 second
cauline leaves that lacked paraclades were 37% longer on
average than 6 that had them (5.6 cm vs. 4.1 cm; P = 0.037).
This suggests that the growth of a leaf is antagonistically
related to the growth of the meristem in its axil.
rev-1 leaf structure
We cross-sectioned six REV and six rev-1 mature cauline
leaves near their midlength. The wild-type leaves were 5 to 9
2730 P. B. Talbert and others
rev-1 shoots have extra cell layers that arise late in
development
To investigate the cause of thicker stems in rev-1 mutants,
cross-sections of wild-type and rev-1 stems from cohort 2 were
made just above the rosettes of 17-day-old plants (just prior to
visible bolting) and 40-day-old plants (that had completed
cauline leaf expansion). Individuals with six to eight procambial strands or vascular bundles were observed in both
genotypes. In four REV and four rev-1 17-day-old plants protoxylem elements were visible but other tissues were poorly
differentiated (Fig. 5A-D). A protodermal layer and about five
cortical cell layers were found outside the procambial strands.
The most obvious difference between the genotypes at this
stage was that paraclades were present in the axils of wild-type
rosette leaves but were frequently missing or reduced to
filaments in rev-1 axils.
The six stems of 40-day-old wild-type plants (Fig. 5E,F,I)
differed from the younger stems primarily in cell enlargement
and in the presence of well-differentiated tissues. The
epidermal layer surrounded a cortex of about five layers of
chlorenchyma. The vascular bundles contained metaphloem
and heavily lignified metaxylem elements. Between the xylem
and phloem were a few layers of small cells that may represent
a fascicular cambium (Fig. 5I). Between and connecting the
vascular bundles were a few cell layers of lignified (redstaining) interfascicular cells forming a continuous ring of
sclerified tissue adjacent to the chlorenchyma.
The stems of 40-day-old rev-1 plants (Fig. 5G,H,J) had a
starkly contrasting anatomy. Inside the epidermis were several
additional cortical layers not present in wild-type or younger
Growth of cauline leaves
8
r
7
rev-1 1st
rev-1 2nd
d
r
r
5
r
r
4
r
3
r
1
r
e
d
s
e
r
e
e
d
e
r
2
d
d
s
s
34
35
e
e
d
s
s
s
s
d
d
d
d
d
d
d
d
r
r
r
r
6
Length (cm)
rev-1 cauline internodes are shortened and
thickened
The cauline leaves of rev-1 mutants were often lower on the
primary shoot than wild-type cauline leaves. We measured the
length of the first cauline internode (between the rosette and
the first cauline leaf) on the bolting plants of cohort 4. Both
wild-type and rev-1 internodes completed growth in about the
same time interval, but rev-1 internodes became only slightly
more than half as long as wild-type internodes on average (Fig.
4B). This suggests that the failure of cauline internode
elongation may account for the average of one more rosette
leaf and one less cauline leaf on rev-1 shoots compared to wildtype shoots (Table 1). The first cauline internodes of rev-1
plants were also 18% larger in diameter (Table 2). Despite this
increased diameter, which was also seen in subsequent internodes (data not shown), the shoots of rev-1 mutants were weaker
and tended to fall over earlier and more frequently than wildtype shoots.
A
e
e
s
s
e
e
e
e
s
s
s
s
e
REV
REV
s
1st
2nd
0
33
36
37
38
39
40
41
42
43
44
45
Days
B
Growth of first cauline internode
9
8
e
7
REV
6
Length (cm)
cell layers thick between vascular bundles while rev-1 leaves
ranged from 6 to 12 cell layers. The rev-1 leaves also had more
vascular elements in the midvein, with about 30 tracheary
elements in the xylem versus about 15 in REV leaves (Fig.
3H,I). The leaf margins tapered in wild-type leaves (Fig. 3J)
and some rev-1 leaves, but were blunt and thickened in other
rev-1 leaves that had more cell layers and large intercellular
spaces (Fig. 3K). The continued division of cells on the adaxial
side of the leaf after vacuolation begins on the abaxial side may
account for the thickening of leaf margins and the downward
curling of rev-1 leaves.
e
e
r
r
r
r
rev-1
e
e
5
e
4
r
3
r
e
r
2
1
e
e
r
r
e
r
0
33
34
35
36
37
38
39
40
41
Days
Fig. 4. Growth of REV and rev-1 cauline leaves and internodes.
(A) First and second cauline leaves. For simplicity, standard error
bars are shown in only one direction. (B) First cauline internode.
rev-1 stems, which included some very large parenchymatous
cells, especially capping the phloem. The additional layers of
chloroenchyma probably account for the darker color of rev-1
stems. Inside these extra layers the vascular bundles were
broader and appeared to have more xylem and phloem cells.
Meristem development in Arabidopsis 2731
Fig. 5. Stem cross-sections immediately above the rosette. (A-D) Stems of 17-day-old plants: (A,B) REV, (C,D) rev-1. Some leaves were
damaged by dissection. (E-J) Stems of 40-day-old plants: (E,F,I) REV, (G,H,J) rev-1. F and H are backlit. Bars, (A,C,E-H) 100 µm; (B,D,I,J)
20 µm. Arrowheads, protoxylem elements; arrows, cambium-like zone; c, cortex; e, epidermis; if, interfascicular region; l, leaf; p, paraclade;
pc, procambial strand; pd: protoderm; ph: phloem; v, vascular bundle; x, xylem; *, axillary filaments.
The interfascicular cells did not appear to be as heavily
lignified as those in the wild type. This may account for the
weaker stems of rev-1 mutants. Between the interfascicular
cells and the cortical layers and between the xylem and phloem
was a ring of many small cells in files resembling a cambial
zone. We suppose that periclinal divisions in this zone gave
rise to the extra layers of cells. Since the extra cell layers were
not present in the 17-day-old rev-1 plants, in which the apical
2732 P. B. Talbert and others
meristems had already initiated 10-15 flowers, these layers
must have arisen from cells that retained a capacity to divide
at a considerable distance behind the apical meristem.
Floral branch types in rev-1 florescences
In addition to the striking effects rev-1 had on vegetative
growth, it also dramatically affected the reproductive structures. In describing these rev-1 mutants, we will use the term
‘floral branches’ to refer both to the flowers and to those
abnormal structures lacking floral organs that occurred in the
position of flowers. The phyllotaxy of the floral branches along
rev-1 florescence axes was commonly irregular, and internode
length was often reduced. This reduction, together with the
shorter vegetative internodes, gave plants an overall shorter
stature: the mean length of the primary shoot of 30 rev-1 plants
in cohort 3 was 24.1 cm, whereas 22 REV plants had a mean
length of 46.0 cm (P = 0.0001). The rev-1 floral branches
exhibited a range of abnormalities which could be grouped into
three branch types (Fig. 2D,H): fertile flowers that were larger
than normal, sterile and usually misshapen flowers that lacked
pistils, and tapered filamentous structures.
Frequency of floral branch types
The relative frequency of these three types of floral branches
in an inflorescence varied widely even among siblings in the
same cohort. Most rev-1 florescences bore some fertile flowers,
especially on the first few floral nodes. In extreme cases,
however, rev-1 florescences could consist almost entirely of
closely spaced tapered filaments and have a brush-like appearance (Fig. 2I). Table 4 gives the relative frequencies of the
different floral branch types for cohort 1. The number of fertile
flowers per rev-1 main florescence ranged from 0 to 34 in this
cohort. The overall fraction of fertile rev-1 flowers among the
floral branches was about 32% for the main florescences and
about 24% for the coflorescences, compared to over 90% for
all florescences in the wild type. Although sterile flowers were
observed in wild-type florescences, these were always wellformed, in contrast to the sterile flowers of rev-1 plants which
lacked pistils. None of the tapered filaments seen in rev-1 florescences were observed in wild-type plants.
Fertile flowers and seeds are enlarged in rev-1
plants
We investigated in more detail the structures of the three floral
branch types. Fertile rev-1 flowers generally had a complete
set of floral organs and a normal shape, but the floral organs
were consistently larger than those of wild-type flowers (Table
5). Sepals, petals and stamens from rev-1 flowers actively
shedding pollen (stage 15 of Smyth et al., 1990) were found to
be 37%, 58% and 28% longer, respectively, than wild-type
controls. Since pedicels and pistils are actively expanding at
stage 15, we compared mature REV and rev-1 pedicels and
siliques just prior to dehiscence. Pedicels were about 28%
larger in rev-1 compared to wild type. No difference was seen
in REV and rev-1 silique length, but rev-1 siliques were 56%
larger in diameter. These measurements suggest that the rev-1
floral organs experience an overgrowth analogous to that seen
for leaves. We also measured the size of REV and rev-1 seeds
from cohort 3. The rev-1 seeds were 15% longer than REV
seeds (Table 5). Although we did not measure seed diameter,
this appeared to be increased proportionally in rev-1 seeds.
Table 4. Occurrence of floral branch types in REV and
rev-1 plants
Florescence
Floral branch type
REV
rev-1
P value
of t-test
Fertile flowers
Sterile flowers
Tapered filaments
26.5
2.9
0
14
22
8
0.0049
0.0001
0.0022
Fertile flowers
Sterile flowers
Tapered filaments
22.1
0.7
0
8.0
13.6
12.2
0.0001
0.0001
0.0001
Main florescences
Coflorescences
The mean numbers of fertile flowers, sterile flowers and tapered filaments
on 13 REV and 12 rev-1 main florescences and on 52 REV and 29 rev-1
coflorescences from 48-day-old plants in cohort 1 are shown.
Table 5. Mean dimensions of floral organs and seeds in
REV and rev-1 fertile flowers
Dimension (mm)
REV
rev-1
P value of t-test
Length of sepal*
Length of petal*
Length of long stamen*
Length of mature silique†
Width of mature silique†
Length of long pedicel†
Length of seed‡
1.9
2.6
2.5
14.8
0.9
13.0
0.46
2.6
4.1
3.2
14.7
1.4
16.7
0.53
0.0001
0.0001
0.0001
0.85
0.0001
0.0001
0.0001
*Lengths of one sepal, one petal and one of the four long stamens were
measured from a stage 15 flower from each of 20 REV and 22 rev-1 plants
from cohort 2 on day 38.
†Measurements on mature siliques and pedicels were made on three of the
largest siliques and pedicels (ordinarily from the most basal floral branches on
the main florescence except when these were sterile) from each of 12 REV
and 12 rev-1 plants from cohort 1 on day 48.
‡Five seeds were measured from basal siliques of each of 9 REV and 9 rev1 plants from cohort 3 on day 80.
Sterile rev-1 flowers are missing floral organs
The floral organs of the rev-1 sterile flowers ranged in size
from as large as the fertile flowers to extremely dwarfed forms
with incomplete development. The sepals in these sterile
flowers usually were of varying sizes, giving an asymmetrical
appearance to the flowers. The sepals sometimes bore branched
trichomes, which are normally found only on leaves in wildtype plants. In both sterile and fertile rev-1 flowers, rare individual organs of either the first or second whorl were found
with a hybrid identity that was part sepal and part petal. Dissection of 50 sterile flowers revealed that they were missing a
variable number of floral organs in an acropetally increasing
manner: the mean numbers of organs found were 2.1 sepals,
0.88 petals, 0.08 stamens and no carpels. Filamentous structures were found in 26% of the flowers. Two adjacent organs
in a whorl were fused proximally in 6/50 sepal whorls, in 2/27
petal whorls, and 1/2 stamen whorls. One of the two organs
involved in a fusion was usually filamentous or reduced in size.
A similar dissection of 17 rev-4 flowers gave means of 3.0
sepals, 1.9 petals, 0.24 stamens and no carpels, with filamentous structures in 29% of the flowers. The acropetally increasing loss or reduction of floral organs suggests that the rev-1
and rev-4 floral meristems become exhausted prematurely.
Tapered filaments and the rev-1 florescence apex
The tendency to form incomplete flowers and incomplete floral
Meristem development in Arabidopsis 2733
organs appeared to reach its extreme in the third type of floral
branch, the tapered filaments. These resembled pedicels that
terminated development before forming any normal floral
organs. Both tapered filaments and pedicels could be subtended
by knobs somewhat resembling leaf bases (Fig. 2H-J) that are
absent from wild-type pedicels. In addition to the knobs, the
tapered filaments could also be subtended or flanked by additional filaments (Fig. 2J). The positions of these additional
filaments are suggestive of homology to leaves and stipules.
Some of the tapered filaments bore branched trichomes. This
may indicate that the filaments had partial leaf identity;
however, we have infrequently observed branched trichomes
on wild-type pedicels. The phyllotaxy of the floral branches
tended to be more irregular for the tapered filaments than for
rev-1 pedicels (Fig. 2H), with the internode distance often
severely reduced, as in the brush-like florescence in Fig. 2I.
The excessive production of tapered filaments was followed by
the premature termination of growth of the rev-1 florescence
apex.
Premature termination also occurred in other contexts that
are not well defined. Sporadic rev-1 florescences terminated
abruptly in a filament. In a significant fraction of rev-3 plants,
apical growth terminated only a few nodes above the rosette in
a cluster of primordia (Fig. 2K). A similar phenotype was
observed in preliminary experiments on rev-1 plants grown at
29˚C (unpublished data). These observations indicate that the
REV gene has a role in the maintenance of the primary shoot
apical meristem.
To investigate the structures of the tapered filaments and the
rev-1 primary apical meristem, we made sections through the
apices of 17-day-old and 40-day-old REV and rev-1 plants
from cohort 2. No difference in size or organization of the
primary shoot apical meristem could be found between REV
and rev-1 plants at day 17 (Fig. 6A,B). The densely cytoplasmic cells of the apical meristem stained deeply in both
genotypes, as did the procambial strands. In contrast, the apices
of the 40-day-old REV and rev-1 plants (Fig. 6C,D) had
markedly different staining. The REV plant had a domed,
deeply staining meristem surrounded by new floral primordia
and developing flowers (Fig. 6C). The rev-1 plant had a more
flattened apex with severely reduced staining and dramatically
larger cells (Fig. 6D). The apex was surrounded by arrested
primordia. We suggest that the rev-1 apical meristem cells had
stopped dividing and were far along in cell enlargement
compared to the dividing cells in the wild-type apex. We also
sectioned some other arrested rev-1 apices. Arrested primordia
and tapered filaments with their subtending knobs exhibited
light staining (Fig. 6E), indicating a lack of meristematic and
vascular tissues and suggesting that the development of the
rev-1 floral primordia arrested quite early.
DISCUSSION
REV performs similar roles in vegetative and
reproductive phytomers
We have recovered mutations in a gene, REVOLUTA, which
is necessary to regulate basic cell division patterns in Arabidopsis. The rev mutations are pleiotropic and affect all aerial
parts of the plant. Although the effects of rev-1 on vegetative
and reproductive structures appear to be quite different, they
share some notable similarities when they are examined in light
of the phytomer concept.
Both the vegetative and reproductive phytomers of rev-1
mutants undergo excessive leaf or leaf-homologous growth
and simultaneously have reduced branch structures. In the rev1 vegetative phytomers, leaves grow too large and formation
of many paraclades is drastically reduced to club-shaped
objects and filaments. In the type 3 phytomers rev-1 mutants
grow knobs, filaments and stipule-like objects beneath their
floral branches in the positions homologous to those of leaves
and stipules in vegetative phytomers. The floral branches are
often reduced to incomplete flowers or tapered filaments.
Within the flower itself, floral organs are regarded as homologous to leaves, and rev-1 floral organs are enlarged like rev-1
leaves. Thus the same growth-altering processes appear to act
on all phytomers. This contrasts with other known mutations
affecting axillary meristem development such as the tomato
mutants lateral suppressor (Malayer and Guard, 1964) and
torosa-2 (Mapelli and Kinet, 1992), which affect branch
formation in only a specific subset of vegetative phytomers and
have no reciprocal effect on leafy organ growth.
REV limits the growth of non-apical meristems
At least two disparate aspects of the Rev phenotype, the overgrowth of leaves and the thickening of the stems, are due to
the presence of extra cells. This is compelling evidence that
REV is involved in limiting cell division. Extra cell divisions
in rev-1 leaves are largely confined to the base of the leaf as
in the wild type. The extra cell layers in rev-1 stems have no
counterpart in greenhouse-grown wild-type plants, but are not
due to a generalized deregulation of cell division. The apparent
derivation of the extra cells from a cambium-like layer may
reveal a potential for secondary growth in Arabidopsis stems.
Secondary growth has been observed in the roots of wild-type
plants (Dolan et al., 1993). A structure resembling a fascicular
cambium is visible in wild-type stems. A cryptic interfascicular cambium may also be present in wild-type stems and both
cambia may be deregulated in rev-1 mutants. If this is true,
both leaf and stem growth in rev-1 plants may arise from the
enhancement of normal meristematic activities.
REV is required to maintain apical meristem growth
Although rev-1 mutants have enhanced cell division in leaves
and stems, they have a defect in apical (including floral)
meristem activity that results in the premature termination of
the rev-1 shoot apex and in the formation of abnormal or
incomplete structures in place of paraclades and flowers. These
incomplete structures imply the defect is in apical meristem
organization or maintenance, in contrast to the failure to
initiate meristematic growth that is seen in empty wild-type
axils. There is a hierarchy to the probability of apical meristem
failure: the primary apical meristem is least likely to fail, while
first-order paraclades and flowers fail more frequently and the
second-order paraclades fail even more frequently (Table 1).
This suggests that the partitioning of some resource may
influence the probability of meristem failure.
Models of REV function
What is the role of REV in maintaining apical meristems? It is
unlikely that REV is required for general cell viability or
2734 P. B. Talbert and others
Fig. 6. Longitudinal sections of primary florescence apices. (A,B) Apical meristems of 17-day-old plants: (A) REV, (B) rev-1. (C,D) Apices of
40-day-old plants: (C) REV, (D) rev-1. (E) Backlit rev-1 arrested apex. Bars, (A-D) 20 µm; (E) 100 µm. f, flower or floral primordium; kt,
knobs and tapered filaments; m, apical meristem.
division, since rev-1 leaves and stems grow excessively and
rev-1 plants are vigorous. Although the effects of rev-1
mutations on growth resemble the pleiotropic actions of phytohormones, the particular suite of defects in rev-1 mutants are
not easily interpretable in terms of the known actions of any
one hormone. REV appears to regulate cell proliferation with
opposing effects on apical versus non-apical meristems.
Explaining these opposing effects is crucial to understanding
REV function.
One possibility is that REV controls the partitioning of
nutrients or growth factors between apical and non-apical
meristems, either by direct regulation of nutrient allocation or
by indirect re-allocation to non-apical meristems caused by
lack of competition for nutrients from the failed apical
meristems. This model requires the unusual assumption that
the formation of de novo stipules and knobs below floral
branches is normally limited by nutritional partitioning.
Another possibility is that REV directs meristematic cells to be
incorporated into the axillary (or floral) meristem instead of
contributing to the formation of leaves (or stipules, knobs and
filaments). This possibility is particulary interesting because
axillary meristems are clonally related to their subtending
leaves in Arabidopsis (Furner and Pumfrey, 1992; Irish and
Sussex, 1992). However, it is difficult to explain the overgrowth of cotyledons and floral organs by this model. A third
possibility is that REV could promote or inhibit cell division
according to the presence of cofactors that differ between
apical and non-apical meristems. Finally, REV could control
the production of or response to a morphogen necessary to
maintain apical growth and to inhibit non-apical growth.
Regardless of which model is correct, REV clearly has
profound regulatory effects on the development of meristems.
Together with other mutations affecting meristem growth, the
rev mutations offer the exciting possibility of dissecting the
processes involved in the regulation and interactions of
meristems that result in plant morphogenesis.
Meristem development in Arabidopsis 2735
We thank Laura Conway, Elliot Meyerowitz, David Smyth and Ry
Meeks-Wagner for supplying seeds. We thank Kevin Lease, Anne
Paul, Arp Schnittger, Gene Tanimoto, Doug Ewing and the greenhouse staff for technical assistance. This work was supported by University of Washington Royalty Research Fund 629 to L. C. and
National Institute of Health Postdoctoral Fellowship GM14355-02 to
H. T. A.
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(Accepted 24 June 1995)