Plant Physiol. (1998) 117: 771-786
Overexpression of a Homeobox Gene, LeT6,
Reveals Indeterminate Features in the Tomato Compound
Leaf1
Bart-Jan Janssen,
Lance Lund, and
Neelima Sinha*
Section of Plant Biology, University of California, Davis,
California 95616
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ABSTRACT |
The cultivated tomato
(Lycopersicon esculentum) has a unipinnate compound
leaf. In the developing leaf primordium, major leaflet initiation is
basipetal, and lobe formation and early vascular differentiation are
acropetal. We show that engineered alterations in the expression of a
tomato homeobox gene, LeT6, can cause dramatic changes
in leaf morphology. The morphological states are variable and unstable
and the phenotypes produced indicate that the tomato leaf has an
inherent level of indeterminacy. This is manifested by the production
of multiple orders of compounding in the leaf, by numerous shoot,
inflorescence, and floral meristems on leaves, and by the conversion of
rachis-petiolule junctions into "axillary" positions where floral
buds can arise. Overexpression of a heterologous homeobox transgene,
kn1, does not produce such phenotypic variability. This
indicates that LeT6 may differ from the heterologous
kn1 gene in the effects manifested on overexpression,
and that 35S-LeT6 plants may be subject to alterations
in expression of both the introduced and endogenous LeT6
genes. The expression patterns of LeT6 argue in favor of
a fundamental role for LeT6 in morphogenesis of leaves
in tomato and also suggest that variability in homeobox gene expression
may account for some of the diversity in leaf form seen in nature.
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INTRODUCTION |
During vegetative growth most higher plants are indeterminate. The
SAM produces a radially symmetrical, acropetally differentiating shoot
axis (stem) and determinate, bilaterally symmetrical lateral organs
(leaves). Leaves can be one of two types, simple or compound, and the
nature of these has been a matter of debate. The ontogenetic relationship of the dicot compound leaf to the simple leaf is unclear
(Merrill, 1986
), with various researchers concluding that the basic
leaf form is simple (Eames, 1961) or compound pinnate (Hagemann, 1984),
or that compound leaves have some shoot-like features and may represent
a continuum between shoots and leaves (Sattler and Rutishauser, 1992;
Lacroix and Sattler, 1994).
To determine morphogenetic patterns and the level of indeterminacy in
compound leaves, we have used homeobox genes that were cloned from
tomato (Lycopersicon esculentum) (Chen et al., 1997; Janssen
et al., 1998). Homeobox genes encode transcription factors that control
the regulation of cell fate (McGinnis et al., 1984; Scott et al.,
1989). The first plant homeobox gene cloned was the maize
knotted1 (kn1) gene (Vollbrecht et al., 1991).
kn1-like homeobox (knox) genes have been placed
into two classes (Kerstetter et al., 1994). Although no specific
function has as yet been ascertained for class II knox genes
(Serikawa et al., 1997), class I knox genes play a role in
meristem maintenance and leaf and flower determination at the shoot
apex. Class I knox genes are not expressed in initiating
organ primordia, mature leaves, or floral organs in simple-leaved
species (Smith et al., 1992; Jackson et al., 1994; Long et al., 1996).
We have cloned a class I knox gene, LeT6
(L.
esculentum
T6), and shown that it is expressed in floral
and vegetative shoot apices and also in leaf and floral organ primordia
(Chen et al., 1997; Janssen et al., 1998). This gene has also been
referred to as Tomato knotted2 (Tkn2) by Parnis and coworkers (1997). The Tkn1 gene, also a class I
knox gene, similarly shows expression in leaf and floral
organ primordia of tomato (Hareven et al., 1996). Thus, in maize
(Zea mays) and Arabidopsis thaliana, which have
simple leaves, the class I knox genes are not expressed in
initiating leaf primordia, whereas in tomato, which has a compound
leaf, they are (Sinha, 1997).
The tomato leaf primordium produces major leaflet primordia in a
basipetal sequence, and these give rise to lobed leaflets (Dengler,
1984; Coleman and Grayson, 1976). Minor leaflets are produced
nonbasipetally, and early vascular differentiation follows an acropetal
sequence (Coleman and Grayson, 1976). We see LeT6 expression
in the preprimordium stage and in the initiating leaf primordia at the
shoot apex (Chen et al., 1997). If class I knox genes are
involved in morphogenesis of the compound leaf through their expression
in developing leaf primordia, what might be the consequences of
expressing a class I knox gene in mature leaves? Overexpression of the maize kn1 gene leads to lobed leaves
in tobacco (Nicotiana tabacum) (Sinha et al., 1993) and
Arabidopsis (Lincoln et al., 1994), and causes excessive leaflet
proliferation in tomato (Hareven et al., 1996). When the Arabidopsis
class I knox gene Knat1 is overexpressed in
Arabidopsis, stipules are produced in the sinuses of highly lobed
leaves, and ectopic shoots or floral primordia are seen (Chuck et al.,
1996).
Our phylogenetic analyses indicate that LeT6 is a possible
ortholog of the stm1 gene, whereas neither Tkn1
nor stm1 are orthologous to kn1 (G. Bharathan,
B.-J. Janssen, E.A. Kellogg, and N. Sinha, unpublished data). It was
unclear if there would be phenotypic differences between
35S-LeT6 and 35S-kn1
overexpression in tomato, since one is a class I knox gene
from a compound-leaved plant (LeT6), whereas the other
originates from a simple-leaved plant (kn1). The fact that
these are not orthologous genes and that expression of a homologous
transgene (LeT6) could result in cosuppression phenotypes
suggested that we might find novel morphological consequences from
overexpression of LeT6. Therefore, we generated transgenic tomato plants that overexpressed LeT6. The phenotypes in
35S-LeT6 tomato plants showed great variability.
Novel phenotypes not described for 35S-kn1 tomato
plants (Hareven et al., 1996) were seen. These phenotypes not only
indicated a role for LeT6 in leaf morphogenesis, but also
revealed the possible morphogenetic potential of the tomato compound
leaf.
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MATERIALS AND METHODS |
Transgenic Methods
LeT6 cDNA was cloned between the "double" CaMV 35S
5
region and the polyadenylation region from the Tml gene
in the vector pCGN2187 (Comai et al., 1990). This chimeric gene was
then cloned into the binary vector pCGN1549. pCGN1549 differs from
pCGN1547 only in the direction of transcription of the NptII
gene and in the order of sites in the polylinker. The empty pCGN1547
vector has been used in control tomato (Lycopersicon
esculentum cv NC8276) transformations (McBride and Summerfelt,
1990). The LeT6 construct in pCGN1549 was transformed into
Agrobacterium tumefaciens strain LBA4404 (Hoekema et al.,
1983) using the freeze/thaw method (An et al., 1988). Maize Kn1 cDNA
was cloned between the 430-bp CaMV 35S promoter and nopaline
synthase termination sequences, as described previously (Sinha et al.,
1993), and used in tomato transformations. Tomato cotyledons were
transformed and transgenic plants were regenerated as described by
Fillatti et al. (1987). 35S-kn1 tomato transformants utilizing this construct have also been described previously by Hareven and coworkers (1996). Some of the phenotypes that
we observed in the 35S-LeT6 transgenics were also
described by Parnis and coworkers (1997). Note, however, that the
LeT6 gene was called the Tkn2 gene in that
previous study. Tobacco (Nicotiana tabacum) leaf segments
were transformed according to the method of Sinha et al. (1993).
Histology and SEM
Fixed leaf tissue for thin-section examinations (8-10 µm) was
processed according to the method of Chen and coworkers (1997). For SEM
fresh tissue was fixed overnight in 3% glutaraldehyde in 0.02 M sodium phosphate buffer, pH 7.2 to 7.4, postfixed in 1%
osmium tetroxide, dehydrated in a graded ascending series of ethanol,
and critical point dried with CO2. Samples were
mounted on SEM stubs with epoxy and sputter coated with a 25-nm layer of gold. Samples were viewed with a scanning electron microscope (model
pSEM 501, Philips, Eindhoven, The Netherlands) at an accelerating voltage of 15 kV. Micrographs were taken on Polaroid 55 film directly from the microscope.
RNA and DNA Gel Blots
Fresh tomato leaf tissue was collected and frozen in liquid
nitrogen. Total RNA was extracted as described by Chomczynski and
Sacchi (1987). The RNA was further purified using standard ethanol
precipitation. RNA samples were resolved on a 1% phosphate glyoxal
gel, transferred to Hybond-N+ membranes
(Amersham), and hybridized as described previously (Sambrook et al.,
1989). Loading of RNA was estimated by hybridization with a labeled
Arabidopsis 18S rDNA probe (Pruitt and
Meyerowitz, 1986) or a cDNA clone of the tomato plastocyanin gene
(kindly provided by Neil Hoffman, Carnegie Institute of Washington,
Stanford, CA).
DNA was extracted using the method of Dellaporta and coworkers (1983),
with certain modifications (Chen et al., 1997). When necessary, DNA was
further purified by phenol/chloroform extraction and reprecipitation.
For DNA gel blots, genomic DNA (20 µg) was digested with restriction
enzymes in a large volume (400 µL) and then ethanol precipitated
before separation on a 0.8% agarose gel.
The LeT6 probe was a PCR fragment containing the entire LeT6 cDNA and
was amplified using T3 and T7 primers. Probes were
32P-labeled using a labeling system
(Prime-a-Gene, Promega) according to the manufacturer's instructions.
Hybridization, washes, and autoradiography for DNA and RNA gel blots
were as described previously (Chen et al., 1997). The RNA gel blots
were hybridized at approximately 15°C below the Tm, whereas the
washes were done at 5°C to 7°C above the calculated Tm (Sambrook et
al., 1989).
RNA in Situ Localizations
Slides for RNA in situ hybridization were labeled using
35S-riboprobes (Meyerowitz, 1987) and
digoxigenin-labeled riboprobes (Coen et al., 1990) according to
previous methods with some modifications. After anti-digoxigenin
antibody labeling and washes, slides were left from overnight to 2 d in wash buffer A (Coen et al., 1990) at 4°C before proceeding to
the detection steps. Sections were dehydrated through a graded-ethanol
series, cleared in Histoclear (National Diagnostics, Atlanta, GA), and
mounted in Permount (Fisher Scientific). Hybridization temperatures
were at or 8°C below the Tm, whereas washes were at 12°C above the
calculated Tm (Sambrook et al., 1989).
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RESULTS |
Development of the Wild-Type Leaf in Tomato
Tomato has a unipinnate compound leaf, and the presence of an
axillary bud demarcates the leaf base from the stem that bears it
(Figs. 1A and 2A). A compound leaf has
been considered a lateral determinate organ rather than a branched,
stem-like organ because axillary buds are only seen in the junction
between the petiole and the stem (the axil). The junction between the
rachis and the petiolule, for which we suggest the name
"pseudoaxil," does not normally bear axillary buds (Fig. 1A).
Certain aspects of development of the wild-type tomato leaf have been
described previously by Coleman and Grayson (1976), Dengler (1984), and
Chandra Shekhar and Sawhney (1990).
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| Figure 1.
Wild-type leaf development in tomato. A, Diagram
showing the principal features of simple and compound leaves. B,
Wild-type SAM with developing leaves. Leaf primordia are marked 1 through 4, from youngest to oldest. Leaflet primordia arise in
basipetal succession on leaves 3 and 4. Leaf 4 shows the formation of
two pairs of lateral leaflets (LL1 and LL2) in basipetal succession. The terminal leaflet (TL) on leaves 3 and 4 is shown producing lobes
(white arrowheads), which occurred in acropetal succession on the
terminal leaflet of leaf 4. C, Older wild-type leaf showing two lateral
leaflet pairs with lobes (arrows) initiating in a primarily acropetal
order. Leaflets are numbered LL2 (oldest) or LL3 (youngest). Leaflet
pair 1 and the terminal leaflet are not shown. A smaller minor leaflet
(sl) is seen to be arising at the base of the leaf, and another one can
be seen between leaflets 2 and 3. Size bars = 100 µm.
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In the present study, tomato leaf primordia exhibited a basipetal order
of maturation. Leaflets arose as bumps on the adaxial marginal face of
the leaf primordium in a basipetal sequence (Fig. 1B). After the upper
pair of major lateral leaflet primordia was initiated, the middle pair
arose below it. A pair of bumps arose on the terminal leaflet,
representing the basal lobes. Smaller minor lateral leaflets (Fig. 1C)
are produced between the three large leaflet pairs in a nonbasipetal
sequence (Coleman and Grayson, 1976; Dengler, 1984). Our results
indicate that individual leaflets show a largely acropetal gradient of
morphogenesis, and that, although younger lobes can often be larger
than older lobes, lobes arise in acropetal succession on both the
terminal and the lateral leaflets (Fig. 1C). An early marked basipetal
gradient delimited the terminal leaflet and produced major lateral
leaflet primordia. A later acropetal gradient, perhaps coincident with
the gradient of early vascular differentiation in the leaf (Coleman and
Grayson, 1976), led to the production of marginal lobes on the
leaflets. Therefore, the wild-type tomato leaf exhibits several
developmental gradients that are not unidirectional.
Transgenic Plant Production and Phenotypic Analysis
A total of 27 independent tomato transformants for the
35S-kn1 construct (Fig.
2B) and 23 for the
35S-LeT6 construct were analyzed (Fig. 2, C-H;
Table I). The
35S-LeT6 transformed tobacco plants generated
resembled those transformed with 35S-kn1 (Sinha et al., 1993), and our 35S-kn1-transformed tomato
plants resembled those described by Hareven and coworkers (1996; Fig.
2B). However, the 35S-LeT6 transgenic tomato
plants exhibited many phenotypic differences from the
35S-kn1 tomato plants. Often, multiple plants were generated from each callus, and these were given an alphabetical designation within the number (Table I); therefore, "1a" and "1b" represent two individual plants regenerated from callus no. 1. We monitored the phenotype of plants that arose from the same callus
("clonal plants") and, presumably, from the same transformation event. This presumption was confirmed for most of the plants by DNA
gel-blot analysis using a probe specific to the NptII gene to determine the number of Kan loci in the plant (Table I). We saw one
instance of two independent transformation events from a "single"
callus (Table I; compare 9c with 9a and 9b).
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| Figure 2.
Comparison of
35S-kn1 and
35S-LeT6 phenotypes in tomato. A,
Wild-type tomato leaf showing a terminal leaflet and two pairs of major
lateral leaflets. These leaflets are all lobed. In addition, smaller
leaflets are seen between the major leaflets. B, A typical 35S-kn1 tomato leaf showing excessive
orders of pinnation. C to H, Phenotypes produced by
35S-LeT6 plants. C, Type I plant showing leaf-like structures with no expanded blade. D, Type II plant showing
excessive branching and proliferation of floral meristems. E, Type III
leaf showing the staghorn-fern-like shape. F, Type IV leaf showing no
expanded leaf blades. G, Type V leaf showing expanded blades on leaf
segments. H, Type VI leaf showing multiple phenotypes on a single leaf.
Size bars = 1 cm.
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Table I.
Categories of 35S-LeT6 tomato transformants
Copy number was determined by DNA gel-blot analysis of
HindIII-digested DNA probed with the NptII probe.
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Our 35S-LeT6 transgenic tomato plants fell into
six phenotypic categories (Table I; Fig. 2, C-H) that differed from
wild type (Fig. 2A): Type I, plants that produced simple leaves without any blade expansion whatsoever (Fig. 2C); Type II, plants that produced
excessively branched axes that terminated in floral structures (Fig.
2D); Type III, staghorn-fern-like leaves with little demarcation between blade and rachis (Fig. 2E); Type IV, compound leaves with no
blade expansion (Fig. 2F); Type V, bladed compound leaves (Fig. 2G);
and Type VI, variable leaf phenotypes on an individual plant (Fig. 2H).
A total of 33 plants represented 23 independent transformation events
(Table I). This was based on each primary callus being considered one
event (with the exception of callus no. 9). We noticed that clonal
plants exhibited enough differences in phenotype to be placed in
different categories and often showed different phenotypes during
development.
Based on the early and late phenotypic scores (as listed in Table I)
the bladed compound leaf category (Type V) was the most stable and had
the highest number of independent transformation events. The mature
leaves of these plants showed a high degree of compounding, rounded
leaflet bases, approximately palmate venation, and multiple leaflet
primordia on the rachis. In these aspects the leaves resembled most
closely leaves seen in a dominant leaf mutation in tomato called
Mouse ears (Me). Me plants have leaves with 2 to 3 orders of compounding, rounded leaflet bases, and palmate
venation. We have shown that this mutation is caused by the ectopic
overexpression of a fusion between LeT6 and the
pyrophosphate-dependent phosphofructokinase gene (Chen et al., 1997).
Similar phenotypes to the Type V described here have been reported in
another study (Parnis et al., 1997). The first two phenotypic
categories (Types I and II) did not survive transplantation. However,
we were able to analyze RNA, DNA, and tissue samples from a few
representative individuals.
The complete range of variability between the individual transgenic
plants (Types I-V) could be seen within some of the multiple-phenotype plants in Type VI. Segments from one leaf on such a multiple-phenotype plant (26a) are shown in Figure 3.
Portions of leaves on plant 26a developed relatively normal, unlobed
leaflets with pinnate venation (Fig. 3A). Some of the leaflets
developed a fasciated rachis and produced leaf-like primordia from the
edges of the leaflets and rachis (Fig. 3, B and C; this phenotype was
also described by Parnis and coworkers [1997]). Portions of the leaf showed a marked difference in complexity between the pairs of leaflets
arising on opposite sides of a rachis (Fig. 3D). Staghorn-fern-like leaves with no distinction between petiolule and leaflet were often
seen as well (Fig. 3E). Segments bearing leaflets often showed a
fiddlehead-fern-like structure, with immature regions of the leaf
segment at the tip (Figs. 3F and 6C). Gel-blot analysis of DNA from
four phenotypically distinct portions of plant 26a confirmed that all
regions of this plant contained the same T-DNA (data not shown).
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| Figure 3.
Details of leaf phenotypes seen on a single
multiple-phenotype leaf. A, Bladed compound part showing leaflets with
very reduced marginal lobes (arrow). B, The margin of a leaflet
producing leaflet primordia (arrow). C, Close-up of leaflet in B
showing foliar primordia arising at the edge of the blade (arrows). D,
Terminal segment showing leaflets (L) developing on one side of the
rachilla, whereas on the other side segments are undergoing further
orders of leaflet production. E, Staghorn-fern-like segment on the leaf with no distinction between blade and rachis. F, Pinnate segments arising in a coiled manner from the edge of the rachis (arrows).
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| Figure 6.
SEM of 35S-LeT6 phenotypes in
tomato. A, Bladed compound leaf with a leaflet primordium (lp; arrow)
developing from the mature region of the rachis. B, Compounded leaf
structure arising from a pseudoaxil. The first-order branch on the left
shows acropetal differentiation; the tip is still immature and has
smaller cells. The compounded structure arising from the pseudoaxil
also has an acropetal differentiation of segments. C,
Fiddlehead-fern-like structure with delayed differentiation at the tip
(arrows). D, Stomata on raised columns of cells (arrows) on the adaxial
leaf surface. E, Wild-type SAM (meri) producing leaf primordia in
succession. A differentiating trichome is seen at the tip of the
plastochron 2 leaf (arrowhead). F and G, Meristems on
35S-LeT6 plants producing leaf primordia
in succession. The leaf primordium in F does not show basipetal
differentiation, whereas the leaf primordium in G does. Differentiating
trichomes are marked (arrowheads). H, Leaf blade of wild type showing
elongated cells in the leaf margin (m) and over the vasculature (v).
Arrows point to stomata on the blade. I, Leaf blade of a
35S-Kn plant showing small epidermal cells with angular margins. Arrows point to stomata on the blade. J,
Leaf blade of a 35S-LeT6 plant showing
elongated cells in the margin and blade regions. Arrows point to the
stomata interspersed between these cells. Scale bars = 100 µm
(except in G, which is 15 µm).
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In addition to kanamycin resistance, DNA gel-blot analysis was used to
confirm the presence and copy number of the transgene in the plants we
analyzed (Fig. 4). Digestion of genomic
DNA with HindIII was used to estimate copy number by
hybridization with the NptII probe, and the results were confirmed by
stripping and rehybridizing the blots with an LeT6 probe
(Fig. 4B). Using this probe the intensity of the T-DNA-specific 0.3-kb
band (Fig. 4C) could be compared with the intensity of the endogenous
0.8-kb LeT6 band (Fig. 4D) to give an estimate of copy
number. One to five copies of the T-DNA were present in the 20 plants
examined, and there was no consistent correlation between phenotypic
stability or severity and locus copy number (Table I; Fig. 4, A and B). We reasoned that the unstable phenotypes could be the result of cosuppression phenomena or of regeneration of chimeric plants.
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| Figure 4.
DNA gel-blot analysis of the
35S-LeT6 plants. A, NptII gene used as a probe.
NptII-hybridizing bands correspond to the number of
integrated copies of T-DNA. B, Same blot as in A stripped and rehybridized with an LeT6 probe. DNA size markers (in
kilobases) are on the right. LeT6 probe hybridizes to
both the endogenous LeT6 (bands at 800 bp, 5.5 kb, 9 kb,
and 11 kb) and the introduced gene (bands at 300 bp and 2.2 kb). The
DNA was digested with HindIII and DNA from an
untransformed (UT) plant shows only the endogenous bands. A comparison
of band intensity differences between the 0.8-kb endogenous and 0.3-kb
transgene HindIII fragments correlates with transgene
copy number in the transformants. C and D, Map of the transgene locus
(C) and the LeT6 locus (D) in tomato. Restriction sites
for HindIII are shown.
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The analysis of DNA from individual parts of the plants confirmed that
plants were not chimeric and that clonal plants derived from the same
callus showed identical restriction patterns for the transgene (data
not shown). For these plants the phenotypic instability could be the
result of variation in tissue- or age-specific transgene expression, or
it could be caused by an interaction between the endogenous and
introduced LeT6 gene at either the posttranscriptional
(Flavell, 1994; Jorgensen, 1995; Que et al., 1997) or the DNA level
(Jorgensen, 1995; Matzke and Matzke, 1995; Park et al., 1996).
Levels of LeT6 Expression in Leaves of Transformed
Tomato Plants
To determine if transgenic phenotypes correlated with levels of
transgene expression, we analyzed the expression of LeT6 RNA in mature leaves of our transformants using
RNA gel blots. LeT6 transcript is not detected in mature
wild-type leaves (Chen et al., 1997; Janssen et al., 1998), but we
found LeT6 transcript in leaves from 27 transformed plants
(Fig. 5A). Our RNA gel blots showed
multiple LeT6-hybridizing bands that did not result from
general RNA degradation (plastocyanin [Fig. 5B], 18S rDNA,
and LeT12 [not shown] hybridized to single bands of RNA).
We used a tomato plastocyanin cDNA as a control probe to determine the
levels of RNA loaded on the gel (Fig. 5B). In general, we did not
notice any strict correlation between transcript levels and phenotypes
or between transcript levels and T-DNA copy number. However, the simple
midrib (Type I) and the branched floral (Type II; Fig. 5A, lane
42b) phenotypes showed very high levels of LeT6 RNA. We
concluded from our RNA gel-blot analysis that variability in phenotypes
between individuals could not simply be attributed to overall levels of
LeT6 RNA.
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| Figure 5.
RNA gel-blot analysis of mature leaf tissue from
various 35S-LeT6 transgenic plants. The
blots shown in A and B were hybridized first with a PCR-labeled
fragment specific to the LeT6 gene (A), then stripped
and rehybridized with a tomato plastocyanin probe (B) to determine the
amount of RNA loaded. Leaf RNA from an untransformed, wild-type plant
(UT WT) does not show any LeT6-hybridizing transcript. The type classification for the individuals used in RNA extraction is
indicated at the top of the lanes. The sizes are in kilobases.
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Leaf Maturation Is Altered in
35S-LeT6 Leaves
Four of the six phenotypic categories in our transgenic plants
produced leaves with many orders of compounding. In one instance we saw
6 orders of pinnation in a leaf. Mature leaflets had very reduced or no
marginal lobes (Fig. 3A) and often showed aberrant vasculature,
including the absence of a prominent midvein (Fig. 3B). Mature leaves
continued to produce leaflet primordia, which often arose either on the
leaflet margin or on the rachis (Fig. 3, B-F). SEM analysis revealed
that these primordia were regions of immature,
undifferentiated cells in an otherwise differentiated region of the
leaf (Fig. 6, A-C). This would indicate
that even mature 35S-LeT6 leaves retain a
potential for organogenesis. The leaflets produced often showed marked
coiling like that seen in fern fiddleheads (Figs. 3F and 6, B and C),
with a markedly acropetal mode of differentiation and a persistent
apical meristematic zone.
Initiating wild-type tomato leaves showed a basipetal maturation
gradient (Figs. 1B and 6E). Ectopic expression of LeT6 (and kn1 [Hareven et al., 1996]) in tomato sometimes enhanced
the late, acropetal maturation pattern, with the basal region maturing
first while the apical (terminal) region remained immature and devoid of trichomes for long periods of time (Figs. 3F, 6B, 6C, and 6F). Such
a reversal in leaf maturation is also seen in a naturally occurring
gene fusion that causes LeT6 overexpression (Chen et al.,
1997). However, meristems bearing leaf primordia with early basipetal
differentiation were also seen on 35S-LeT6 plants
(Fig. 6G). Meristems in all transgenic plants appeared flattened
compared with wild type, regardless of the kind of leaf primordia they had (compare Fig. 6E with 6F and 6G).
The leaves on the 35S-LeT6 plants lacked marginal
lobes. When observed under a scanning electron microscope, lobe-like
structures were seen to initiate, but these structures matured into
leaflets rather than lobes (Figs. 3D and 6B). This suggests that there was no fundamental difference between lobe and leaflet initiation. Delayed differentiation was also seen in cells surrounding the stomata.
The last cell divisions in the leaf occur when guard cells are formed
in the stomatal complexes (Coleman and Grayson, 1976). In the
35S-LeT6 plants, stomata on the adaxial surface were raised on mounds of cells several layers high (Fig. 6D), presumably as a result of LeT6 expression delaying
differentiation and prolonging cell division in the adaxial
subepidermal and epidermal layers. Furthermore, cell shapes and
arrangements were perturbed in these plants. Wild-type leaves had
elongated epidermal cells in the margins and above veins, whereas the
region of the blade had large cells shaped like pieces of a puzzle and
with interspersed stomata (Fig. 6H). In contrast,
35S-Kn plants had very small cells with angular
margins (Fig. 6I). In the 35S-LeT6 transgenics,
leaf epidermal cells either resembled the 35S-Kn
type (but with stomata raised on mounds; Fig. 6D) or consisted mostly
of narrow, elongated cells (Fig. 6J), indicating the presence of a more
extended, margin-like domain in these leaves.
Ectopic Meristems Are Initiated on
35S-LeT6 Leaves
Leaves on 35S-LeT6 plants often produced
ectopic meristems. In Type I and Type II plants these meristems were
made early in development and often formed floral primordia (Fig.
7, A and D). In Type I plants, the SAM
terminated early, and axillary meristems were therefore also
precociously activated (Fig. 7, B and C). Leaves on these plants were
bladeless and simple, with their normal phyllotactic patterns appearing
disrupted prior to SAM termination (Fig. 7C), and ectopic sympodial
meristems bearing floral meristems being produced on some leaves (Fig.
7A). Leaves on Type IV and Type VI plants could also produce ectopic
meristems on the blade (Fig. 7E) in a manner similar to that described
for the 35S-Kn "shooty" phenotype in tobacco (Sinha et
al., 1993). Very rarely, leaflet primordia would also bear meristems
along their margins. These appeared flattened, as described earlier
(Fig. 6, F and G), and produced leaf primordia in succession (Fig. 7F).
In addition, a subset of plants produced meristems in the axils of
leaflets (pseudoaxils), which sometimes bore leaf primordia (Fig. 7G), but more often produced inflorescence or floral structures (Fig. 8, A and B).
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| Figure 7.
A through C, Type I plant showing leaves with no
blade expansion. A, Shoot apex region showing three radial leaf
primordia (a, b, and c) and a floral meristem (d) on the left.
Sympodial meristems (m) arise on the adaxial surface of older leaf
primordia. B and C, Other views of the same shoot apex as in A showing
absence of the SAM at the predicted position (arrowhead). Axillary
meristems (am) develop precociously. The corresponding structures have
the same identifying letters in A, B, and C. The floral meristem (d) is
part of an inflorescence meristem produced from the sympodium (m) on
the adaxial face of the leaf primordium. D, Type II phenotype showing
clusters of meristems developing into mostly solitary flowers (f). E,
Vegetative meristem (arrow) arising from the surface of a leaflet (lf).
F, Leaflet (lf) bearing meristems (m) on its margin that are producing
leaf primordia (lp). G, Leaflet (lf) bearing a pseudoaxillary meristem
(px) at the junction with the rachis (r). The meristem is shown
producing leaf primordia. Size bars = 100 µm.
|
|
View larger version (139K):
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| Figure 8.
Reproductive structures in
35S-LeT6 tomato transgenics. A, Leaf with
several floral inflorescences (arrowheads) developing in the
pseudoaxils. B, An inflorescence with two floral buds (arrow) arising
from the pseudoaxil of a leaf. C, Wild-type tomato flower with an outer
whorl of green sepals, a whorl of yellow petals, and a column of
anthers (arrowhead). D, Fasciated flower with normal sepals, a large
number of petals (p), very few stamens (arrowhead), and a cluster of
sepaloid organs in the center (arrow). E, Bifurcated fasciated fruit
developing from an abnormal flower. Arrow points to a notch in the
fruit. F and G, SEM image of a leaflet (L) showing clusters of
developing flowers. F, These flowers show the normal order of organ
production, with outer sepals (s) and an inner ring of petals (p), but
the sizes and numbers are abnormal. A central bifurcation into two
units (arrow) is visible in each flower. G, Leaflet showing
proliferation of ectopic inflorescence meristems (im) on its surface.
These meristems are shown generating floral buds (f).
|
|
In Type II plants these leaf-borne meristems were often determined to
be inflorescence meristems (Figs. 7D and 8G). Meristems in the later
plastochrons of Type IV and Type VI plants were also shown to be
inflorescence meristems (Fig. 8, A and B). The inflorescence meristems formed flower primordia that produced floral organs in the
normal sequence. However, the flowers were not normal (Fig. 8D), often
having larger meristems that produced more organ primordia than normal
and that rarely attained full maturity or fertility. Flowers produced
in the normal location on these plants also showed similar defects.
Alterations in meristem size, fractionation, and organ differentiation,
rather than ectopic location, were the likely causes of abnormal flower
structures in 35S-LeT6 plants.
On the floral meristems five to six sepal-like structures enclosed a
large number of petaloid and green sepaloid organs (Fig. 8D),
reminiscent of mutations in C function genes in Arabidopsis (Bowman et
al., 1991). Often, the central whorls were bifurcated into two units
(Fig. 8, D and F). Upon fertilization, these produced fasciated double
or multiple fruit (Fig. 8E) reminiscent of the clavata
phenotypes in Arabidopsis (Clark et al., 1993). Thus, overexpression of
LeT6 appeared to enhance the phase of SAM proliferation in
relation to lateral organ inception so that when organs finally formed,
the meristem was able to produce more organs than normal.
Extra organs were produced on the majority of
35S-LeT6 leaves. When these organs were present
on the leaflet margins or on the rachis they usually differentiated
into leaflets. However, when they were produced at the junction between
the petiolule and the rachis (the pseudoaxil), they often resulted in
meristems that produced organs (Fig. 8, A and B). There must be an
inherent difference between the organogenic potential in the leaflet
margin and the rachis compared with the pseudoaxil. The former usually produced determinate, lateral structures (although meristems were seen
in rare instances, Fig. 7F), whereas the latter gave rise to
indeterminate meristems.
Patterns of LeT6 Expression in Wild-Type and
Transgenic Leaves
In situ hybridization was used to determine whether
LeT6 expression was localized to specific regions of the
developing leaves and leaflets in wild-type and
35S-LeT6 plants. LeT6 is expressed in
the SAM and initiating leaf primordia (Chen et al., 1997). In older
wild-type leaf primordia we saw high LeT6 expression in
initiating leaflet primordia (Fig. 9A),
indicating that the leaf and leaflet primordium could be equivalent
structures. Vascular tissue in leaves and the subtending axis also
showed strong expression of LeT6 (Fig. 9A). Expression in
the axis vascular tissue has also been seen for kn1 (Smith
et al., 1992; Jackson et al., 1994) and Knat1 (Lincoln et
al., 1994; Chuck et al., 1996). In addition, the developing leaflet
margin showed high levels of LeT6 expression (Fig. 9B), a
feature not seen in simple-leaved plants. Analysis of the transgenic
leaves revealed high levels of expression in vascular tissue in the
marginal blastozone regions (Hagemann and Gleissberg, 1996) (Fig. 9C),
and in the junctions between the rachis and petiolule, the pseudoaxils
(Fig. 9D).
View larger version (71K):
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| Figure 9.
Analysis of expression patterns of homeobox genes
in wild-type and 35S-LeT6 tomato. A and
B, Expression of LeT6 in developing leaves. A,
Longitudinal section through a developing leaf primordium (Lf)
on the right shows a basipetal order of leaflet production. Each
leaflet primordium (Lp) has high levels of LeT6
expression (arrows). Expression is also high in the vascular trace (V)
in the leaf. B, Transverse section through an older wild-type leaf shows the basal rachis and three pairs of lateral leaflet primordia. Expression (arrows) is seen in the vascular traces and developing leaflet blade margins (m). The region of high expression on the rachis
(arrow) probably represents a site of minor leaflet initiation. C and
D, Expression of LeT6 in
35S-LeT6 developing leaves. C, Transverse
section through rachis of a multiple-phenotype leaf showing a
developing leaflet on the left and a developing compound-leaf-like structure with leaflet primordia in the center. Expression (arrows) is
seen in the vascular tissue and in the marginal blastozones (m) from
which blades will develop. D, Transverse section through the rachis and
leaflet primordia of a multiple-phenotype leaf showing regions of
high expression in the pseudoaxils (Px), vascular traces, and
edges of the leaf blade (arrow). E, Expression of LeT12
in a tissue section adjacent to that shown in D. Transverse section
through the rachis and leaflet primordia of a multiple-phenotype leaf
showing regions of high expression in the pseudoaxils, vascular traces,
and edges of the leaf blade (arrow).
|
|
LeT12 Expression in Transgenic and Wild-Type Leaves
Since animal homeobox genes have been shown to be coordinately
regulated (Magli et al., 1991) and also to regulate other homeobox genes (Carroll and Vavra, 1989), we used a class II
knox gene, LeT12, as a hybridization probe
on RNA gel blots and in situ-hybridization experiments to determine if
similar events occur in plant tissues. The LeT12 transcript
is 200 bases larger than the LeT6 transcript (1600 bp) and present at
moderately high levels in all wild-type tissues we have examined
(Janssen et al., 1998). On RNA gel blots we saw a moderate
up-regulation of LeT12 levels in transgenic leaves that also
showed high levels of LeT6 (data not shown). We used in
situ-hybridization experiments to see if the two homeobox genes were
expressed in similar regions of the adjacent tissue sections in these
leaves. Regions of elevated LeT6 expression also showed
elevated LeT12 expression. These were vascular regions in
the rachis and blade, pseudoaxillary locations, and leaflet margins
(compare Fig. 9, D and E). Our results suggest that in the
35S-LeT6 plants, either LeT6
overexpression led to LeT12 overexpression, or these two
genes were coordinately regulated and LeT12 also had a role
in morphogenetic events in the tomato leaf.
| |
DISCUSSION |
Equivalence of Leaflets and Lobes
The tomato leaf is unipinnately compound, producing a terminal
leaflet and usually three pairs of lobed, lateral major leaflets. An
early basipetal gradient leads to the production of major leaflet primordia, and a later acropetal gradient leads to the production of
marginal lobes and early vascular differentiation. Engineered or
natural perturbations in growth and differentiation in this system can
interplay with the potential for phenotypic plasticity and lead to
complex morphologies.
Leaflet primordia arise from previously undifferentiated basal regions
of the leaf and retain the morphogenetic potential to produce lobes,
whereas lobe primordia form on regions with reduced morphogenetic
potential and quickly form a differentiated blade edge. We hypothesize
that leaflets and leaflet lobes are equivalent structures that take on
different morphogenetic trajectories by virtue of their position on the
leaf (i.e. lobes are prematurely differentiated leaflets). If this is
indeed the case, then delaying differentiation in the leaf as a whole
should allow initiating lobe primordia to take on a leaflet fate. This
seems to be one of the consequences of 35S-LeT6
(and 35S-kn1) overexpression in tomato. These
transgenic plants produce leaves with numerous orders of unlobed
leaflets. Leaflets beyond the first order (i.e. those produced over and
above the "normal" number) are produced in acropetal succession,
just as leaflet lobes are, and lobing is usually missing or reduced on
these leaves. Furthermore, the initial basipetal gradient indicates
that regions closer to the base of the tomato leaf may be the last to
differentiate and may retain morphogenetic potential. If this is indeed
the case, then one would expect more than just three major ranks of
secondary lateral branching in the leaf (the three pairs of
leaflets seen in the wild type). This is seen in a number of
transformants, indicating that leaflets (or secondary lateral branches)
continue to arise from the base of the leaf.
Indeterminate Features in the Tomato Leaf
The compound leaf has sometimes been equated to a structure
intermediate between a stem and a leaf (Sattler and Rutishauser, 1992;
Lacroix and Sattler, 1994). Although this proposal may have to be
evaluated on a case-by-case basis, we have analyzed the complex
morphologies produced in our 35S-LeT6 plants for
similarities to shoot systems. The junction between the rachis and the
petiolule on these transgenic leaves often takes on a measure of
indeterminacy and shows axillary characteristics. In the more extreme
phenotypes, these pseudoaxils produce floral and vegetative meristems,
a characteristic feature of leaf axils. In the less-extreme phenotypes,
unipinnate or multipinnate, determinate, leaf-like structures are
produced from these locations. Incidentally, mutations in tomato that
fail to produce axillary meristems often cause the production of
ectopic shoots in pseudoaxillary locations on an otherwise normal leaf (Rick and Butler, 1956).
A similar difference in organogenic potential was seen between the
sinus region and the rest of the leaf in Arabidopsis plants overexpressing knat1 (Chuck et al., 1996). We have two
alternate explanations for these phenotypes. Either these locations
have some atavistic axillary features that are enhanced by
35S-LeT6 overexpression or there is some special
characteristic in these locations that allows for elevated
LeT6 expression, leading to the production of meristems or
leaf-like organs. Since the CaMV 35S promoter causes high
levels of expression in vascular tissues (Benfey et al., 1989),
vascular junctions at these pseudoaxils might have levels of
LeT6 above the threshold required for development of shoots.
Phenotypic Variability and Cosuppression
When comparing the results of overexpression of the maize
kn1 gene in tomato with overexpression of the endogenous
LeT6 gene, the extreme variability of phenotypes observed in
the 35S-LeT6 plants stands out. This phenotypic
variability is probably the result of several factors acting
individually or in combination. Variability of expression from the CaMV
35S promoter and tissue or cell-type specificity of RNA
expression may have contributed to these phenotypes. The endogenous
gene LeT6 could have unique features that result in
phenotypic changes, such as the initiation of meristems at the leaf
margins, that are not seen with overexpression of the maize
kn1 gene. Phenotypic instability in these transgenic plants
may be the result of threshold-dependent cosuppression, a phenomenon
dependent on a high level of sequence identity between the transgene
and the endogenous gene (de Carvahlo Niebel et al., 1995). Because
maize kn1 has only 49% similarity to LeT6 at the nucleotide level, and the Arabidopsis ortholog stm1 is only
65% similar to LeT6, it is unlikely that the
35S-kn1 plants would be able to show such
cosuppression of LeT6, Tkn1, or some other knox class I gene. We suggest that endogenous gene effects
such as silencing and cosuppression, rather than differences in the constructs, may play a role in the phenotypic effects of
LeT6 overexpression in tomato. Distinct roles for the
various class I knox genes have also been postulated
elsewhere based on transgenic data (Parnis et al., 1997).
Two phenotypic classes of 35S-LeT6 plants are
suggestive of cosuppression. The Type I plants show suppression of the
SAM and their leaf blades fail to expand. In these plants expression of the endogenous LeT6 gene in the SAM combined with expression
of the 35S-LeT6 transgene may result in
cosuppression, leading to failure of SAM maintenance. In this respect
these plants are strongly reminiscent of the stm mutation in
Arabidopsis (Long et al., 1996). The bladeless leaves produced in the
Type I plants may result from cosuppression in the edge domain, where
both endogenous and 35S-LeT6 gene expression
would be expected to be high. Petunia flowers show a similar variation
of cosuppression between the edge and internal domains (Que et al.,
1997). Type II plants show suppression of the vegetative stage of SAM
activity and an almost complete loss of leaf-like structures.
Because expression from the 35S promoter is known to be weak
in meristems (H. Klee, personal communication) and higher in differentiating tissues, cosuppression phenomena may be different in
these two regions. As the leaf primordium begins to form,
35S-LeT6 expression may increase to a level
sufficient to trigger cosuppression, halting cell division and
completing differentiation before the leaf primordium can completely
form. In these differentiated tissues the endogenous LeT6
expression level would decrease, perhaps below the threshold required
for cosuppression, causing induction of a new meristem by
overexpression of the 35S-LeT6 transgene.
Complete suppression of the vegetative meristem program may cause the
plant to shift to the production of floral meristems (Shannon and
Meeks-Wagner, 1993).
Coordinate Regulation of Homeobox Genes
Although class I knox genes are expressed at high
levels in meristems of Arabidopsis (Lincoln et al., 1994; Long et al.,
1996), the class II knox gene Knat 3 has been
shown to be expressed at low levels in the SAM and is postulated to
have diverse roles in the plant (Serikawa et al., 1997). In contrast,
the expression patterns of LeT6, a class I knox
gene, and LeT12, a class II knox gene, appear to
be very similar in wild-type reproductive structures in tomato (Janssen
et al., 1998). This could be due to coincidence, to coordinate
regulation of these two genes in the organs examined, or to regulation
of expression of one gene by the other. Expression in ectopic locations
by the CaMV 35S promoter has allowed us to monitor the
expression patterns of LeT12 relative to LeT6 in
adjacent tissue sections.
High levels of LeT6 expression are coincident with high
levels of LeT12 expression in 35S-LeT6
plants. Either the 35S-LeT6 plants produce
ectopic organs and both genes are expressed in these organs (as
they would be in wild-type organs of a similar nature) or
LeT6 overexpression leads to LeT12 overexpression
as part of a developmental cascade of events. This indicates that, as
has previously been observed in animal systems (Carroll and Vavra,
1989; Magli et al., 1991), plant homeobox genes may participate in
morphogenetic events through an interrelated regulatory network. This
may be a way to fine-tune expression patterns and morphogenetic fields.
Previous attempts at identifying such regulatory relationships have
utilized probes from the class I knox genes only (Jackson et
al., 1994). Our results hint that there may be regulation of a class II
gene by a class I gene. The nature of this regulation, i.e. direct or
indirect, and the consequence of coordinate expression of the two genes
in tomato remains to be explored.
Evolutionary Considerations
Compound leaves may have arisen multiple times in the dicots. Leaf
morphology seems to be evolutionarily plastic, with variations in
degree of pinnation and the order of initiation of leaflets. We see
that alterations in levels of a class I knox gene can lead not only to increased orders of pinnation, but can also alter the order
in which leaflets are generated. This suggests that some of the natural
variation in compound leaf morphology could be explained by alterations
in the expression patterns of the class I knox genes. The
Type II transgenic class bears resemblance to genera in the aquatic
Angiosperm family Podostemaceae, in which most members have
"thalloid" bodies that produce clusters of floral branches
(Rutishauser, 1995). Analysis of homeobox gene expression in these
genera and in organisms with leaves bearing ectopic meristems (e.g.
Kalanchoe) may be useful in elucidating the causes of these unique morphologies.
Overexpression of the class I knox gene LeT6 in
tomato has revealed an important role for this gene in morphogenesis of
the compound leaf. These transgenic leaves produce meristems in ectopic locations analogous to axils between the junction of the leaf and the
stem. In addition, gradients of maturation are altered in these leaves,
leading to conversion of lobes into leaflets. These features indicate
that the tomato leaf has some indeterminate stem-like features, and
that leaves, leaflets, and lobes all represent a continuum of
structures that are distinguishable only by virtue of timing and
maturation patterns of the organ as a whole. The phenotypes that we
observed, as well as the changes in determinacy in the transgenic
plants, suggest that alterations in homeobox gene expression may
provide one explanation for the natural range of variability in leaf
shape and plant form.
| |
FOOTNOTES |
1
This work was funded by the National Science
Foundation (grant no. IBN-96-32013).
*
Corresponding author; e-mail nrsinha{at}ucdavis.edu; fax
1-530-752-5410.
Received December 10, 1997;
accepted March 13, 1998.
| |
ABBREVIATIONS |
Abbreviations:
CaMV, cauliflower mosaic virus.
SAM, shoot
apical meristem.
SEM, scanning electron microscopy.
| |
ACKNOWLEDGMENTS |
We are grateful to John Harada for advice and encouragement, to
Rich Jorgensen for helpful discussions on cosuppression, and to Sharon
Kessler, Tom Goliber, and Kim Snowden for critical reading of the
manuscript. We thank Dr. Charlie Rick and the Tomato Genetics Resource
Center, University of California, Davis (UCD), for seed stocks, and we
are especially grateful to Virginia Ursin and Maureen Daley of Calgene,
Inc., for allowing us to perform the tomato transformations at their
facility with assistance from Danny Lee, Huy Nguyen, and Joy Philipson.
We also thank Andrina Williams for help with the SEM and in situ
analyses, and Vivek Pai (National Science Foundation Young Scholars
Program at UCD) and Bhaskar Sinha for help with the tobacco
transformations.
| |
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Abstract
Introduction