Department of Biological Sciences, University of Lethbridge, Lethbridge, AB, TIK 3M4, Canada
* Author for correspondence (e-mail: schultz{at}uleth.ca)
Accepted 3 June 2003
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SUMMARY |
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Key words: Arabidopsis thaliana, forked1, forked2, mp, axr6, pin1, Vascular patterning, Leaf venation, Auxin
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Introduction |
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Both classes of angiosperms, the monocots and dicots, have a complex, branched and closed leaf venation pattern. In monocots the pattern is called striate with a series of longitudinal veins running approximately parallel to each other, meeting at the apex of the leaf and interconnecting through a multitude of smaller transverse veins. In dicots, the pattern is reticulate with a basic pattern of secondary veins branching from the midvein and connecting to one another or to higher order (tertiary and quaternary) veins at their distal ends near the leaf margin.
The primary model to explain vascular patterning, the auxin canalization
model, is supported by evidence indicating a central role for auxin in
vascular differentiation within the stem
(Sachs, 1981;
Sachs, 1989
;
Sachs, 1991
), in isolated
mesophyll cells (Church, 1993
;
Berleth and Sachs, 2001
) and in
leaves (Mattson et al., 1999; Sieburth,
1999
; Aloni, 2001
).
According to the auxin canalization model, an initially homogenous field
emanates from auxin sources, but random fluctuations expose some cells to
increased auxin leading to vascular cell differentiation and increased
efficiency in auxin transport. The increased transport results in two classes
of cells: (1) those to which auxin is transported form vascular tissue and (2)
those from which auxin is drained form nonvascular tissue.
Auxin is thought to be transported in a primarily basipetal manner
throughout the plant body from primary sources of synthesis
(Wallroth-Marmonr and Harte,
1988; Lomax et al.,
1995
), although recent studies indicate that the shoot apical
meristem and very young leaf primordia are auxin sinks to which auxin is
acropetally transported (Reinhardt et al.,
2000
; Avsian-Kretchmer et al.,
2002
). Basipetal transport from young developing leaves is
proposed to direct stem vasculature since removal of leaves eliminates
vascular differentiation in the stem below and can be compensated by exogenous
auxin (Sachs, 1981
). Leaf
venation is proposed to be directed by basipetal transport of auxin from the
leaf margin (Mattson et al., 1999;
Sieburth, 1999
;
Aloni, 2001
;
Avsian-Kretchmer et al., 2002
),
a mechanism consistent with auxin response patterns in regions that will
become procambium (Mattsson et al.,
2003
). Leaves in which polar auxin transport is inhibited either
chemically (Goto et al., 1991
;
Okada et al., 1991
;
Gälweiler et al., 1998
;
Sieburth, 1999
), or
genetically, through eliminating PIN-FORMED 1 (PIN1), a component of the auxin
efflux carrier (Gälweiler et al.,
1998
; Müller et al.,
1998
; Mattsson et al.,
1999
; Steinmann et al.,
1999
), develop increased vascularization adjacent to the leaf
margin.
Consistent with a role for auxin in vascular patterning, a number of
Arabidopsis mutants known to affect aspects of auxin transport or
response have defects in vascular development. Mutations in MONOPTEROS
(MP), BODENLOS (BDL) or AUXIN RESISTANT 6 (AXR6) show early
defects in embryonic apical basal patterning followed by a reduction in
cotyledon venation (Berleth and Jurgens,
1993; Hamann et al.,
1999
; Hobbie et al.,
2000
). MP encodes ARF5, an auxin response factor that
activates auxin response targets, while BDL encodes IAA12, a protein
that has been shown to bind to ARF5 and prevent its activity
(Hamann et al., 2002
).
AXR6 encodes the protein cullin, a subunit of the ubiquitin ligase
complex SCF (Hobbie et al.,
2002
). Mutations in SCARFACE (SFC) result in
plants that are more sensitive to auxin and show reduction and discontinuity
of venation within foliar organs (Deyholos
et al., 2000
). Plants mutant for PINOID, which encodes a
serine-threonine kinase believed to affect either auxin signaling or auxin
transport, show altered venation within floral organs, while mutants in
LOPPED (LOP) are defective in basipetal auxin transport and
alter leaf venation (Carland and McHale,
1996
; Christensen et al.,
2000
; Benjamins et al.,
2001
). Alleles of GNOM (EMB30), such as
van7 (vascular network 7)
(Koizumi et al., 2000
), result
in discontinuous venation in cotyledons and increased marginal venation in
leaves, a leaf phenotype similar to pin1 and consistent with the
proposed role of GNOM in PIN1 localization
(Steinmann et al., 1999
).
While the interactions amongst these genetic factors are not yet well
understood, the frequent association of defective auxin signaling or transport
with defective vascular patterning clearly points to a primary role for auxin
in establishing vascular pattern within shoots.
We report the isolation and characterization of mutants in two novel Arabidopsis thaliana genes, FORKED1 (FKD1) and FORKED2 (FKD2), crucial to the formation of the closed leaf vascular pattern characteristic of dicot leaves. Recessive mutations in either FKD1 or FKD2 result in a failure of distal portions of the vascular bundles to form connections with the remaining leaf vascular network, resulting in an open leaf venation pattern reminiscent of primitive vascular plants. Our analysis suggests that FKD1 responds to a particular auxin threshold and allows vascular development, and that this action is redundant to that of FKD2 except at the lowest auxin levels. The function of these genes is of particular interest as the closed leaf vascular pattern is ubiquitous within the angiosperms and appears to have been important in their evolution.
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Materials and methods |
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Growth conditions and analysis
Seed were sown on Metromix 200 (W. R. Grace Co., Marysville, OK) in 100
cm2 pots or on A. thaliana (AT) growth medium
(Wilson et al., 1990) with or
without 30 µM naphthylphthalamic acid (NPA; Sigma Chemical Company) in 78.5
cm2 Petri plates. Following 4-5 days stratification, seeds were
transferred (considered as the day of germination: 0 Day After
Germination; DAG) to growth chambers (Percival Scientific, Perry, IA)
set for 21°C, 60% relative humidity, and continuous light (130 mol
second-1 m-2) provided by a combination of Sylvania Cool
White, Gro Lux, and incandescent bulbs (Osram Sylvania Inc., Danvers, MA). To
assess root growth, 5-day old seedlings vertically grown on plates were
transferred to medium containing either 1.0x10-6,
1.0x10-7 or 1.0x10-8 M
2,4-dichlorophenoxyacetic acid (2,4-D). Root growth was measured from the
position of the root tip at transfer to the position 4 days later.
For isotopic analysis, wild-type and fkd1 plants were greenhouse
grown (16-hour days) on soil (as described above) and leaves were harvested
when the shoot was 2 cm long. A 1-2 mg subsample of ground leaf material was
sealed in a tin capsule and loaded into the elemental analyzer (NC2500, CE
Instruments, ThermoQuest Italia, Milan, Italy) and the diatomic nitrogen and
carbon dioxide gases generated therein were separated in a gas chromatographic
column and passed directly, using a helium stream, to the inlet of the mass
spectrometer (Delta Plus, Finnigan Mat, San Jose, CA, USA) for quantification
and measurement of stable isotope ratios. Carbon stable isotope ratios
(13C/12C) were expressed in delta notation (
values presented in parts per thousand) where the international standard is
CO2 from Pee Dee Belemnite (PDB) limestone
(Farquhar et al., 1989
).
Mutant screening
Approximately 6000 EMS-mutagenized M2 seed were sown at a
density of 50 seed per pot. A cotyledon and first leaf was taken 14 DAG,
mounted in low viscosity Cytoseal (Stephen's Scientific, Kalamazoo, MI) and
screened for abnormalities in vascular patterning using a dissecting
microscope (Stemi 2000, Carl Zeiss Inc., Thornwood, NY). M3 seed of
potential mutants was collected and re-screened. Lines with heritable
phenotypes were backcrossed, using wild type as the female, at least twice
before characterization.
Morphological and anatomical characterization
Wild-type and fkd1 seeds (20 per plate) were sown on AT medium
with or without NPA and plants taken every 24 hours from 1 DAG. Histochemical
localization of GUS activity and subsequent clearing was performed as
described previously (Kang and Dengler,
2002). For photography, mature cotyledons (14 DAG) and first
leaves (21 DAG) of all genotypes, and fully open fkd1, fkd2 and
fkd2 fkd1 flowers were removed and cleared in a solution of 3:1
ethanol:acetic acid for 2-4 hours, 70% ethanol for 1 hour, 95% ethanol
overnight and 5% NaOH for 1 hour at 60°C. Dissections were performed in a
50% aqueous glycerol solution and samples viewed with a compound light
microscope (Eclipse E600, Nikon, Mississauga, ON).
Cotyledons (14 DAG) and first leaves (21 DAG) of all genotypes were mounted in Cytoseal, images captured with a CCD camera (RS-170, Cohu Inc., Electronics Division, San Diego, CA) attached to a dissecting microscope (Stemi 2000, Carl Zeiss Inc., Thornwood, NY), and assessed using NIH Image (http://rsb.info.nih.gov/nih-image/). Whole cotyledons were scored for numbers of secondaries (veins attached at least at one point to the midvein) and cotyledons and half leaves were scored for number of branch points (two or more veins meeting), vein endings and areoles (any area of the leaf blade completely bounded by veins). Cleared cotyledons and leaves were scored for the percentage showing vascular islands (VIs). Wild-type, fkd1 and fkd2 plants (4 per pot) were scored for germination, total number of leaves, number of secondary stems and time to flowering (days). Statistical differences were determined using Student's t-test.
Cotyledons (14 DAG) and first leaves (21 DAG) of wild type and fkd1 were prepared for sectioning by vacuum infiltration in FAA (18:1:1 70% ethanol:formalin:glacial acetic acid), stored at 4°C overnight, dehydrated through an ethanol series, and embedded in Spurr's resin. Five µm sections were cut using a glass knife and stained with Toluidine Blue before being viewed with a compound light microscope (Eclipse E600, Nikon, Mississauga, ON).
Mapping of FKD1 and FKD2
Plants mutant for FKD1 and FKD2 were crossed to Landsberg
erecta (Ler) ecotype and DNA was extracted from
F2 plants exhibiting the fkd1 or fkd2 phenotype
(Edwards et al., 1991).
Mapping using simple sequence length polymorphisms (SSLPs) between the Col and
Ler backgrounds was done using standard PCR conditions
(Bell and Ecker,1994
) and
primers (Research Genetics Inc., Huntsville, AL).
Generation of double mutants
Double mutants were generated as described in
Table 1. F3 progeny
from axr2 fkd1, aux1-7 fkd1 and fkd2 fkd1 double
mutants, F3 progeny segregating for pin1-1 fkd-1, mp
fkd1 or axr6-2 fkd1 double mutants and F4 progeny
from axr1-3 fkd1 were characterized. To generate the fkd1
DR5::GUS line, fkd1 plants were crossed to plants homozygous for
DR5::GUS. F2 progeny were screened on plates with 10
µg/ml kanamycin, surviving plants showing the fkd1 phenotype were
allowed to self, and three F3 families entirely resistant to
kanamycin were characterized.
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Results |
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For mapping, fkd1 and fkd2 plants were crossed into the
Ler background (Bell and Ecker,
1994). The FKD1 gene mapped to 89.48 cM on chromosome III
based on recombination with nga112 (1.6%, n=562 chromosomes) and nga6
(3.8%, n=210 chromosomes). FKD2 mapped to 30.2 cM on
chromosome V based on recombination with ciw8 (11.8%, n=152
chromosomes) and nga106 (14.0% n=50 chromosomes). This places
FKD2 near a previously described gene involved in vascular
patterning, SFC (Deyholos et al.,
2000
). Plants mutant for SFC have severely altered
morphology and leaves with highly disrupted vascular patterning, including
VIs. Unfortunately, sfc seed was not available so we were unable to
determine through complementation if fkd2 is a weak allele of
sfc or a mutation in a novel gene. For this reason, detailed
developmental and double mutant characterization was performed only on
fkd1 plants.
Cotyledon vascular pattern development
In order to assess the differences between wild-type, fkd1 and
fkd2 mature cotyledons, we first quantified wild-type numbers of vein
branch points, areoles, freely ending veins and secondary veins
(Table 2). Most commonly, the
vascular pattern of the mature wild-type cotyledon consists of a midvein and 4
secondary veins (two distal and two proximal) that meet with the midvein and
one another to generate four closed loops
(Fig. 1E).
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The lack of distal vein meeting is evident early in fkd1 cotyledon development. At 1 DAG, the provascular tissue of one or both of the distal secondary veins fails to join the midvein (Table 3, Fig. 1C, Fig. 3C,D). Furthermore, fkd1 proximal secondary veins initiate at a point distant from the existing distal secondary vein (Fig. 3G) and initiation is delayed relative to wild type (Table 3). Other aspects of fkd1 vein development are similar to wild type, except that all secondary veins connect proximally with the midvein.
First leaf vascular pattern development
The vascular pattern of wild-type first leaves consists of a midvein from
which regularly spaced secondary veins extend to the leaf margin where they
join one another (Fig. 2A).
Tertiary veins form connections between the secondary veins or occasionally
end freely in the lamina, while quaternary veins usually end freely in the
lamina. To compare the fkd1 and fkd2 leaf venation pattern
to that of wild type, we quantified the number of branch points, freely ending
veins and areoles of first leaves 21 DAG
(Table 2).
The midvein of the wild-type leaf differentiates acropetally until it reaches the distal tip of the developing leaf 5 DAG where the distal secondary veins are initiated (Table 3, Fig. 4A). These develop basipetally, meeting the midvein between 6 and 7 DAG (Table 3, Fig. 4B). Most remaining secondary and tertiary veins initiate from previously formed, more distal veins and develop basipetally to join the vascular network at their proximal end (Fig. 4C). At least one vein (excluding quaternary) in 53% (n=38) of leaves at 21 DAG fails to rejoin the vascular network proximally.
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In fkd1 leaves, development of the midvein, initiation of distal secondary veins and their proximal joining with the midvein is slightly delayed relative to wild type (Table 3). Initiation of proximal secondary veins and subsequent secondary and tertiary veins occurs at a point distant from the previously formed distal secondary veins (Fig. 4G,H), but further development of all veins is normal (Fig. 4G,H).
Plant morphology
Given the crucial function of the vascular system, we expected that the
loss of the reticulate venation pattern might result in a decreased growth
rate or photosynthetic capacity. All characters analyzed were
indistinguishable between wild type and fkd1. In contrast,
fkd2 plants produced fewer rosette leaves, flowered later, and
produced less seed (Table 4).
The similar photosynthetic capacities and growth rate in fkd1 and
wild-type plants suggests either that the altered vascular pattern has no
effect or that the fkd1 plants are compensating for their non-meeting
venation by altering another component of the transpiration mechanism. Leaf
carbon isotope composition (expressed using notation with units of
parts per thousand [
13C,%thou]) provides information about
the ratio of photosynthetic capacity to stomatal conductance: higher
13C values (less negative) mean a lower stomatal conductance
in relation to photosynthetic capacity
(Farquhar et al., 1989
). The
significantly lower
13C in fkd1 than in wild type
suggests that the fkd1 vascular pattern provides less efficient water
delivery that is compensated by increased stomatal conductance.
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Effect of exogenous auxin on Fkd1 roots
One explanation for the fkd1 phenotype is that the mutation
affects a component of auxin canalization and compromises distal vein meeting.
If correct, one might expect that fkd1 plants would show altered
sensitivity to changes in auxin levels, whether introduced exogenously or
through double mutant combinations. Since altered sensitivity of root growth
to 2,4-D indicates altered auxin response or transport
(Wilson et al., 1990), we
compared root growth of fkd1 to that of wild type at four 2,4-D
concentrations. No significant difference was observed at any concentration
(data not shown), suggesting that fkd1 plants do not show a global
change in auxin sensitivity.
While the auxin root assay effectively detects defects in the general auxin pathway, it may not detect defects specific to leaf vascular pattern formation. We therefore grew plants on auxin transport inhibitors and assessed alterations in leaf vascular pattern. As well, we generated double mutants between fkd1 and various auxin mutants known to have alterations in leaf morphology and/or leaf vascular patterning.
Effect of an auxin transport Inhibitor on Fkd1 leaf vascular
patterning
The proliferation of veins adjacent to the margin in wild-type leaves
treated with auxin transport inhibitors suggests that the leaf margin is a
major source of auxin within the developing leaf
(Mattsson et al., 1999;
Sieburth, 1999
). To assess
whether, in the presence of an auxin transport inhibitor, fkd1 alters
the ability of marginal veins to meet, we grew fkd1 and wild-type
seedlings on 30 µM NPA. Mature (14 DAG) fkd1 cotyledons had the
same vascular pattern when grown with or without NPA (data not shown).
Extensive marginal venation at the distal leaf tip was evident in both
wild-type and fkd1 leaves by 5 DAG, and vascular strands began to
develop basipetally from the marginal region to the proximal leaf blade at 7
DAG. However, by 8 DAG, the interior of the wild-type leaf blade showed
extensive vein branching, whereas in fkd1 no branching was evident in
the leaf interior. The marginal regions of fkd1 and wild-type 21 DAG
leaves were indistinguishable, however, significantly fewer secondary veins
extended from the marginal region to the proximal leaf blade in fkd1
(14.74±0.88) than in wild type (20.5±0.78) and tertiary veins
were rare (Fig. 2G,H).
Furthermore, 20% (n=23) of fkd1 leaves had secondary veins
that did not connect proximally, whereas all veins in all wild type leaves
were connected (n=25). The ability of veins to meet distally in
marginal areas of fkd1 leaves where auxin is increased owing to
reduced transport suggests that FKD1 either acts in response to auxin
to direct vascular tissue or is necessary for the auxin response that directs
vascular tissue.
Effect of auxin mutants on the fkd1 phenotype
pin1-1 fkd1
Like chemical inhibition of auxin transport, the loss-of-function allele,
pin1-1, in the auxin efflux carrier results in a proliferation of
marginal venation (Mattsson et al.,
1999; Galweiler et al.,
1998
). pin1-1 cotyledons and leaves are similar to wild
type in size and shape, although the cotyledons are slightly rounder, the
leaves are slightly smaller, and are sometimes fused
(Fig. 2E and
Table 5). The venation pattern
of the cotyledons shows no difference from wild type while the leaf pattern is
quite distinct, being simpler (fewer areoles and branch points) and having
fewer freely ending veins (Table
2).
pin1-1 fkd1 cotyledons show no significant difference from pin1-1 cotyledons for any of the characters measured (Table 2). Relative to pin1-1, the first leaves of pin1-1 fkd1 have more freely ending veins and fewer areoles, consistent with the increased distal non-meeting of fkd1. Relative to fkd1, they show a decrease in freely ending veins, consistent with the increased vein meeting of pin1-1. Specifically, the increased meeting in double mutants occurs along the margin while the distal non-meeting occurs in the leaf blade (Fig. 2F). Therefore, like treatment of fkd1 leaves with NPA, loss of PIN1 compensates for lack of FKD1 at the leaf margin, but not in the internal regions of the leaf.
axr1-3 fkd1; axr2 fkd1; aux1-7 fkd1
Plants mutant for any of AXR1, AXR2 or AUX1 are auxin
resistant, with various defects in morphology. AXR1 acts in an
ubiquitin-like pathway that responds to the presence of auxin by targeting
proteins for degradation (del Pozo and
Estelle, 1999; Gray and
Estelle, 2000
), AXR2 belongs to the IAA family of genes
that are inducible by auxin (Nagpal et
al., 2000
) and AUX1 encodes a membrane protein that is
believed to be a component of the auxin influx carrier
(Bennett et al., 1996
;
Marchant et al., 1999
;
Swarup et al., 2000
). We found
that both axr1-3 and axr2 cotyledons and leaves are smaller
than wild type (Table 5) with a
simpler vascular pattern (Table
2, Fig. 2M) that in
axr1-3 is combined with frequent proximal non-meeting
(Table 2). In contrast,
aux1-7 cotyledons and leaves are significantly larger but maintain
the wild-type vascular pattern, and aux1-7 first leaves are more
elongate (Table 5).
In double mutant combinations between these auxin resistant mutants and fkd1, both leaf morphology and leaf vascular patterning show essentially additive phenotypes (Tables 2, 5). Relative to the respective single mutants, cotyledons and first leaves of axr1-3 fkd1, axr2 fkd1 and aux1-7 fkd1 have significantly fewer branch points and areoles, consistent with the double mutant phenotypes combining the distal non-meeting of fkd1 with the simplified vascular pattern of the auxin mutants (Table 2 and Fig. 2N).
mp fkd1 and axr6-2 fkd1
Plants with loss-of-function MP alleles or gain-of function
AXR6 alleles do not produce the basal embryonic structures, hypocotyl
and root, but produce normal apical embryonic structures, including shoot
apical meristem and cotyledons (Berleth and
Jurgens, 1993; Przemeck et
al., 1996
; Hobbie et al.,
2000
). AXR6 mutants rarely form a small number of rosette
leaves; mp seedlings do not normally form leaves, but seedlings
carrying weak MP alleles, such as mpG92, can be
induced by wounding to form roots, and the rooted seedlings go on to form
leaves and inflorescences (Przemeck et
al., 1996
). Within these structures, defects occur in the leaf
venation, including simplification of pattern and discontinuities in vascular
strands. We found that mpG92 and axr6-2
cotyledons have few distal secondaries arising from the midvein, and that
these do not connect proximally with the midvein
(Fig. 1I).
Many of the shoot defects seen in mpG92 and axr6-2 plants are suppressed in the mpG92 fkd1 or axr6-2 fkd1 double mutants, while the morphology and development of double mutant basal structures is indistinguishable from mpG92 or axr6-2 respectively. The vascular pattern of mpG92 fkd1 cotyledons is simpler than that of mpG92, while the vascular pattern of axr6-2 fkd1 cotyledons is slightly more extensive than axr6-2 (Fig. 1J,L, Table 2) with increased frequency of VIs.
The remainder of the double mutant shoot structure diverges markedly from that of mpG92 or axr6-2: while mpG92 or axr6-2 plants grown under the same conditions only rarely (mpG92 -16.2%, n=37; axr6-2 -10%, n=19) develop a very small first leaf with highly reduced venation (Fig. 2I,K), 64.8% (n=54) of mpG92 fkd1 double mutants and 39% (n=33) of axr6-2 fkd1 double mutants show one to four small but well developed leaves, with extensive venation (Fig. 2J,L). Because mpG92 fkd1 and axr6-2 fkd1 double mutants rarely survived to 21 DAG, at which point we consider first leaves to be fully expanded, we could not make a quantitative comparison of their vascular pattern to either wild type or fkd1. However, examination of 20 mpG92 fkd1 and 12 axr6-2 fkd1 first leaves indicate that they show a simplified vascular pattern with reduced numbers of tertiary and quaternary veins (Fig. 2J,L), and that, as in fkd1 leaves, the distal ends of veins often do not meet. Moreover, if mpG92 fkd1 plants survive on medium for 21 days, 70% (n=10) form inflorescences, usually consisting of a single terminal flower, often reduced to a pistil.
Auxin response in fkd1 mutants
To test the hypothesis that the fkd1 phenotype is the result of
reduced ability to respond to auxin, we introduced the synthetic AuxRE
reporter gene construct (DR5::GUS) into fkd1 plants. The
DR5::GUS line contains a composite promoter with seven tandem repeats
of the AuxRE TGTCTC fused to a minimal cauliflower mosaic virus 35S
promoter-GUS reporter gene construct
(Ulmasov et al., 1997).
Initial reporter gene expression in the subapical cells of the 3 DAG
fkd1 leaves was indistinguishable from wild type (not shown).
However, reporter gene expression in fkd1 cells destined to be
secondary veins was reduced in both intensity and duration relative to wild
type (Fig. 6). Moreover,
whereas in wild type, reporter gene expression at the distal junctions of
secondary veins was somewhat reduced or delayed relative to the rest of the
vein (see arrows, Fig. 6A,C)
(Mattsson et al., 2003
), in
fkd1, such cells never expressed the reporter gene. In support of the
hypothesis that auxin transport inhibition alleviates the fkd1
phenotype by increasing auxin levels and allowing increased auxin response,
both wild-type and fkd1 leaves treated with NPA showed a broad band
of intense reporter gene expression (Fig.
6I,J) that preceded the formation of vascular tissue
(Fig. 6K,L).
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Discussion |
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FKD1 directs vascular differentiation in response to an
auxin threshold
Several models for the role of FKD1 in vascular pattern formation
are consistent with the fkd1 phenotype: (1) FKD1 is a
component of the auxin synthesis or transport pathway that controls
distribution of auxin; (2) FKD1 is necessary for differentiation of
vascular tissue in response to auxin; (3) FKD1 is necessary for the
auxin response that results in vascular differentiation; or (4) FKD1
acts independently of, but in conjunction with, auxin canalization to induce
vascular tissue in areas where auxin alone is insufficient. To distinguish
amongst the possibilities, we assessed auxin responsive reporter gene
expression (DR5::GUS) in fkd1, the response of fkd1
to synthetic auxin and auxin transport inhibitors and the response of
fkd1 to mutant backgrounds that affect auxin response or
transport.
Our data provide the most support for Model 3. The expression of DR5::GUS is reduced in duration and extent throughout fkd1 leaves, suggesting that FKD1 is required for the auxin response. The reduced auxin response is sufficient to direct vascular development throughout most of the leaf, although it results in a slight developmental delay. At distal junctions, the level of auxin is sufficiently low that no auxin response occurs in the absence of FKD1 and distal junctions do not form. When fkd1 seedlings are grown on medium containing an auxin transport inhibitor or when double mutants are made between fkd1 and the auxin transport mutant pin1-1, distal meeting of marginal veins in either cotyledons or leaves is more similar to wild type. Moreover, when auxin transport is inhibited, DR5::GUS expression in fkd1 leaves is indistinguishable to that in wild type. Thus, the increased auxin at the margin following auxin transport inhibition reestablishes auxin response even in the absence of FKD1 function. Our data suggest that FKD1 activity is restricted to the leaves and is not present globally; however, the additive phenotypes of double mutants between fkd1 and the auxin response mutants axr1-3, axr2 and aux1-7, as well as the root response of fkd1 plants grown in 2,4-D, may be the result of redundant activities in other tissues.
The hypothesis that FKD1 is necessary for the auxin response is
further supported by the effect of fkd1 on both
mpG92 and axr6-2 phenotypes. Plants homozygous
for mpG92 or axr6-2 show a similar,
seedling lethal phenotype, forming no root, an abnormal, stubby hypocotyl,
cotyledons with reduced venation and rarely a first leaf. Double mutants with
fkd1 show no change in the root or hypocotyl phenotype, but like
mp sfc double mutants, mpG92 fkd1 show
enhanced vascular defects in the cotyledons. More surprisingly, axr6-2
fkd1 or mpG92 fkd1 double mutants exhibit
suppression of the mpG92 or axr6-2 leaf
phenotypes, producing several well-expanded leaves and in the case of
mpG92 fkd1, an inflorescence. MP encodes
the auxin response protein ARF5 (Hardtke
and Berleth, 1998), while AXR6 encodes the protein
cullin, a subunit of the ubiquitin ligase complex SCF
(Hobbie et al., 2002
), The
similarities in mp, axr6 and bdl phenotypes suggest that
AXR6 may degrade IAA12 (product of BDL) and allow activity of ARF5.
The enhancement of the mpG92 cotyledon phenotype by
fkd1 suggests that in cotyledons, FKD1 acts redundantly with
MP. While it is difficult to find a simple explanation for the
suppression of mpG92 and axr6-2 leaf phenotypes
in a fkd1 background, the most likely interpretation is that, like
MP and AXR6, FKD1 is involved in the auxin response. While
it is tempting to speculate that FKD1, MP and AXR6 act as a complex required
for auxin response, the function of which is restored if pairs of genes are
mutated, the proposed activities of MP and AXR6 products and
the fact that mpG92 introduces a nonsense codon indicate
that this is unlikely. A second possibility is that the suppression is due to
indirect changes in auxin distribution caused by altered source/sink
relationships resulting from mpG92 and axr6-2
morphological defects. In wild-type seedlings, the initial source of auxin is
probably the cotyledons (Ljung et al.,
2001
; Bhalerao et al.,
2002
; Marchant et al.,
2002
), while the region below the quiescent centre (QC) acts as a
sink for auxin (Sabatini et al.,
1999
) that controls a gradient of auxin distribution
(Casimiro et al., 2001
;
Marchant et al., 2002
) and
auxin-dependent patterning (Friml et al.,
2002
). Disrupting either auxin source or sink alters auxin
distribution. In stm1 seedlings, disrupting the auxin source alters
auxin gradients within the root (Casimiro
et al., 2001
). One might predict a corresponding alteration in
seedlings lacking an auxin sink, such as the rootless
mpG92 and axr6-2 seedlings. The lack of a sink
could cause higher than normal auxin gradients within the shoot that would
disrupt formation of vascular tissue and leaf development. Such a disruption
would provide a possible explanation for the suppression of the
mpG92 and axr6-2 phenotypes in a fkd1
background: while auxin levels are too high to allow the function of wild-type
FKD1, they allow the product of fkd1 to function and enable
differentiation of vascular tissue in developing leaves.
Our analysis suggests that FKD1 is necessary to induce an auxin response at low auxin levels and enable differentiation of vascular tissue. The product of fkd1 cannot initiate a response to low auxin concentrations but can to higher levels as demonstrated by increased marginal vein meeting and DR5::GUS expression when auxin transport from the leaf margin is prevented. In contrast, one explanation consistent with the defective venation in mpG92 and axr6-2 plants and suppression of this defect in a fkd1 background is that the product of FKD1 cannot function if auxin concentrations are too high.
Vascular pattern is driven by sequential dynamic auxin sources
If FKD1 is necessary for responses to auxin concentrations below a
particular threshold and direct vascular cell differentiation, then the
fkd1 phenotype may represent a leaf in which only higher auxin
concentrations are driving vascular formation and provide insight into how
auxin is distributed within the developing leaf. Based on mutant phenotypes,
we suggest that three different sources of auxin are sequentially required for
vascular pattern development within the leaf: (1) acropetally directed auxin
directs midvein development; (2) a dynamic marginal source directs secondary
vein development; (3) internal auxin sources direct tertiary and quaternary
vein development.
Auxin from the first source is transported acropetally into the meristem
and young leaf primordia (Reinhardt et
al., 2000; Avsian-Kretchmer et
al., 2002
) where it directs differentiation of the midvein. The
control of midvein differentiation by a different mechanism to that of
subsequent veins is supported by its unique, acropetal developmental pattern
and by the observation that all vascular pattern mutants described thus far
affect subsequent veins but do not affect midvein development
(Carland et al., 1999
;
Deyholos et al., 2000
;
Koizumi et al., 2000
).
Moreover, exposure to auxin transport inhibitors eliminates acropetal midvein
development (Mattsson et al.,
1999
; Seiburth, 1999;
Avsian-Kretchmer et al.,
2002
).
The second source arises at the margin, beginning at the distal tip and
proceeding proximally with marginal cell differentiation, and directs
differentiation of secondary veins. This idea is supported by this study and a
number of previous studies. If auxin transport from the marginal source is
inhibited, auxin accumulates at the distal leaf tip
(Avsian-Kretchmer et al., 2002)
and vascular development begins with a band of vascular tissue parallel to the
margin of the leaf near the distal tip which lengthens along the margin as the
leaf develops (Mattsson et al.,
1999
; Sieburth,
1999
). Moreover, high levels of auxin have been found to move
basipetally during leaf development
(Avsian-Kretchmer et al.,
2002
), coincident with highest rate of cell division and onset of
vascular differentiation (Ljung et al.,
2001
). The sequential development of distally unconnected
secondary vascular bundles originating at the margin of fkd1 leaves
also supports a dynamic auxin source. Moreover, the fkd1 phenotype
combined with reduced DR5:GUS expression at distal junctions in
wild-type leaves suggest that auxin depletion of neighbouring cells by
secondary veins results in low auxin concentrations at the point of distal
vein connection. Interestingly, despite changes to marginal venation in
pin1, fkd1 and fkd2 mutants, marginal vascular tissue always
forms at a point distant from the margin, suggesting (1) that a maximum is
reached at a point distant from the margin and/or (2) that cells at this point
have prior competence for the vascular fate.
The third source of auxin is within the leaf blade, and directs formation
of tertiary and quaternary veins (Aloni,
2001; Aloni et al.,
2003
). In fkd1 and fkd2 leaves the marginal
auxin source alone is sufficient to explain the generation of these veins,
since the open venation pattern would allow auxin from the margin to reach the
interior leaf blade. However, the wild-type leaf blade interior is separated
from the marginal auxin source by a continuous loop of vascular tissue,
prompting the proposal that an internal source of auxin directs formation of
tertiary and quaternary veins (Aloni,
2001
; Aloni et al.,
2003
). The reduction in these veins in both axr1-3 and
axr2 suggests that the internal source of auxin is lower than the
marginal source so that the partial loss-of-function gene products can respond
to the marginal source but not the internal source.
FKD1 acts redundantly with FKD2 to allow vascular
cell formation
We have identified a second gene, FKD2, whose mutant phenotype is
very similar to fkd1, being distinguished only by a higher frequency
of VIs in the distal portion of the leaf blade. FKD2 maps very close
to a previously characterized gene, SFC
(Deyholos et al., 2000).
Plants carrying mutations in SFC show severely disrupted vascular
pattern with a very high frequency of VIs, and correspondingly severe defects
in morphology, a phenotype consistent with fkd2 being a weak allele
of SFC. Furthermore, double mutants between fkd1 and
fkd2 exhibit a phenotype that is very similar to that reported for
SFC mutants. The double mutant phenotype suggests that both
FKD1 and FKD2 are essential for distal vein meeting,
presumably because auxin concentrations are extremely low in these areas.
Within other regions of developing veins where auxin levels are higher, the
two genes act redundantly such that partial loss of function of either gene is
insufficient to alter vein morphology, but partial loss of function of both
genes results in severe disruption to the vascular integrity. We suggest that
both FKD1 and FKD2 are required to respond to auxin and
allow vascular differentiation. In the partial absence of one gene product,
only the lowest thresholds are not recognized, disrupting vascular
differentiation at vein junctions. In the absence of both gene products,
response to a range of thresholds is faulty, and vascular discontinuities
occur throughout the leaf veins.
FKD1 and FKD2 may provide an evolutionary link
between open and closed venation patterns
If the fkd1 and fkd2 leaf pattern, which is similar to
that of lower vascular plants, represents a leaf in which only high levels of
auxin are able to initiate vascular differentiation, it is plausible that the
vascular pattern seen in lower plants results from auxin canalization but that
these plants have decreased sensitivity to auxin thresholds. Sztein et al.
(Sztein et al., 1995) have
shown that primitive vascular plants have only simple IAA conjugates and the
increasing conjugate complexity occurring in higher vascular plants is
associated with increasingly complex vascular tissue. It has been well
established that leaves of conifers, Gingko and ferns are sources of
auxin and that this auxin is at least partly responsible for the vascular
pattern found within the stem of these plants
(Gunckel and Wetmore, 1946
;
Steeves and Sussex, 1989
;
Wang et al., 1997
).
Additionally, lycopod leaves have been shown to have an effect upon the stem
vasculature but it has not been conclusively demonstrated that auxin is
responsible for this phenomenon (Freeberg
and Wetmore, 1967
). In conifers, it has also been shown that a
concentration gradient of auxin is required for proper differentiation and
patterning of xylem and phloem from the cambium
(Nix and Wodzicki, 1974
;
Uggla et al., 1996
). Given
that auxin in primitive plants has a similar, if not identical, role to that
in more advanced plants, it is likely that an auxin canalization mechanism is
responsible for leaf vascular patterning in these groups, but that higher
plants have evolved mechanisms to respond to a wider range of auxin
thresholds. We suggest that FKD1 and FKD2 enable distal
meeting of the vascular bundles in areas of low auxin concentration to form a
complete, closed vascular pattern.
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ACKNOWLEDGMENTS |
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