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Stage-Specific Regulation of Solanum lycopersicumLeaf Maturation by Class 1 KNOTTED1-LIKEHOMEOBOX Proteins C W
Eilon Shani,a Yogev Burko,a Lilach Ben-Yaakov,a Yael Berger,a Ziva Amsellem,b Alexander Goldshmidt,b
Eran Sharon,c and Naomi Oria,1
a Robert H. Smith Institute of Plant Sciences and Genetics in Agriculture, Otto Warburg Minerva Center for Agricultural
Biotechnology, Hebrew University, Rehovot 76100, Israelb Department of Plant Sciences, Weizmann Institute of Science, Rehovot, 76100, Israelc Racah Institute of Physics, Hebrew University, Jerusalem, 91904, Israel
Class 1 KNOTTED1-LIKE HOMEOBOX (KNOXI) genes encode transcription factors that are expressed in the shoot apical
meristem (SAM) and are essential for SAM maintenance. In some species with compound leaves, including tomato
(Solanum lycopersicum), KNOXI genes are also expressed during leaf development and affect leaf morphology. To dissect
the role of KNOXI proteins in leaf patterning, we expressed in tomato leaves a fusion of the tomato KNOXI gene Tkn2 with a
sequence encoding a repressor domain, expected to repress common targets of tomato KNOXI proteins. This resulted in
the formation of small, narrow, and simple leaves due to accelerated differentiation. Overexpression of the wild-type form of
Tkn1 or Tkn2 in young leaves also resulted in narrow and simple leaves, but in this case, leaf development was blocked at
the initiation stage. Expression of Tkn1 or Tkn2 during a series of spatial and temporal windows in leaf development
identified leaf initiation and primary morphogenesis as specific developmental contexts at which the tomato leaf is
responsive to KNOXI activity. Arabidopsis thaliana leaves responded to overexpression of Arabidopsis or tomato KNOXI
genes during the morphogenetic stage but were largely insensitive to their overexpression during leaf initiation. These
results imply that KNOXI proteins act at specific stages within the compound-leaf development program to delay
maturation and enable leaflet formation, rather than set the compound leaf route.
INTRODUCTION
Leaves are lateral organs that are produced from the flanks of the
shoot apical meristem (SAM). Leaf development can be divided
into three continuous and overlapping phases: initiation, primary
morphogenesis (PM), and secondary morphogenesis (SM) or
histogenesis (Poethig, 1997; Dengler and Tsukaya, 2001; Holtan
andHake, 2003; Barkoulas et al., 2007; Efroni et al., 2008). During
leaf initiation, the leaf emerges from the flanks of the SAM. At PM,
the leaf expands laterally and elaborates by the initiation of
secondary structures from specific meristematic regions at the
leaf margin, termed marginal blastozones (Hagemann and
Gleissberg, 1996). SM is characterized by extensive cell expan-
sion and histogenesis. Theprogress of leafmaturation is followed
byplastochrons (P), such that P1 is the youngest leaf primordium;
it becomes P2 as the next primordium emerges, and so on. Plant
leaves can be either simple, with a single continuous lamina, or
compound, where lamina units termed leaflets are connected via
petiolules to a central rachis. Leaflets are initiated from the
marginal blastozone at the PM stage and go through similar
developmental stages as leaves (Ori et al., 2007; Barkoulas et al.,
2008;Berger et al., 2009). Thus, different regions in the leaf canbe
at different developmental stages at the same time.
Class I Knotted1-like homeobox (KNOXI) genes encode a
family of transcription factors that are expressed in the SAM and
have conserved functions in maintaining its activity (Hake et al.,
2004; Hay and Tsiantis, 2009). In plants with simple leaves, such
as Arabidopsis thaliana and maize (Zea mays), expression of
KNOXI genes is excluded from leaves throughout their develop-
ment (Lincoln et al., 1994; Long et al., 1996; Reiser et al., 2000;
Semiarti et al., 2001; Hake et al., 2004). In tomato (Solanum
lycopersicum), a plant with compound leaves, the KNOXI genes
Tomato KNOTTED1 (Tkn1) and Tkn2 (also called LeT6) are
expressed in young leaf primordia, slightly after their initiation,
in addition to their expression in the SAM. A dramatic increase in
leaf complexity results from KNOXI overexpression in leaves of
the dominant tomato mutantsMouse Ears and Curl, as well as in
a subset of transgenic plants overexpressing KNOXI genes
(Hareven et al., 1996; Chen et al., 1997; Parnis et al., 1997; Janssen
et al., 1998). In agreement, in Cardamine hirsuta, an Arabidopsis
relative with dissected leaves, KNOXI genes are expressed in
developing leaves, and their downregulation leads to the formation
of simple leaves, suggesting thatKNOXI genes are required for leaf
elaboration in this species (Hay and Tsiantis, 2006). Similarly,
1 Address correspondence to [email protected] author responsible for distribution of materials integral to thefindings presented in this article in accordance with the policy describedin the Instructions for Authors (www.plantcell.org) is: Naomi Ori ([email protected]).CSome figures in this article are displayed in color online but in blackand white in the print edition.WOnline version contains Web-only data.www.plantcell.org/cgi/doi/10.1105/tpc.109.068148
The Plant Cell, Vol. 21: 3078–3092, October 2009, www.plantcell.org ã 2009 American Society of Plant Biologists
additional species with compound leaves also express KNOXI
genes in leaf primordia (Bharathan et al., 2002). These observa-
tions suggest that KNOXI proteins are involved in compound leaf
patterning in some species but not others (Champagne et al.,
2007). In Arabidopsis, ubiquitous overexpression of KNOXI
genes led to leaf lobbing (Matsuoka et al., 1993; Sinha et al.,
1993; Lincoln et al., 1994; Chuck et al., 1996; Reiser et al., 2000;
Pautot et al., 2001; Gallois et al., 2002; Dean et al., 2004). Tomato
has been extensively used to study compound leaf development
due to the sensitivity of its leaf shape to genetic changes and the
large collection of mutants with altered leaf shape. This research
has led to important insights on the developmental differences
between simple and compound leaves (Hareven et al., 1996;
Chen et al., 1997; Parnis et al., 1997; Janssen et al., 1998; Brand
et al., 2007; Ori et al., 2007; Kimura et al., 2008; Berger et al.,
2009). However, the lack of KNOXI loss-of-function mutants in
tomato, as well as the inconsistent and sometimes opposite
phenotypes caused by KNOXI overexpression, hindered the
understanding of how KNOXI proteins affect leaf shape in this
and other species.
Here, we addressed the role of KNOXI proteins in leaf pat-
terning by comparing the effects of overexpressing TKN proteins
and their fusion with a repressor domain. These experiments
revealed that KNOXI proteins affect leaf patterning in tomato by
delaying the progression of leaf maturation. KNOXI overexpres-
sion in specific domains in developing leaves implied that the
developmental window during which the leaf is responsive to
KNOXI activity is partially parallel between Arabidopsis and
tomato, suggesting that KNOXI proteins act within these frame-
works to affect leaf shape. Comparison of the effects of different
KNOXI genes revealed that their redundancy is limited to specific
developmental contexts.
RESULTS
Manipulation of KNOXI Activity in Developing Tomato
Leaves Distorts Leaf Shape and Size
To assess whether KNOXI function is required for compound leaf
development in tomato and address the mechanism of this
activity, we compared the effects of impaired KNOXI function
and its overexpression on leaf development. Attempts to identify
loss-of-function mutations through forward-genetic screens or
to downregulate KNOXI expression by synthetic microRNAs
(Alvarez et al., 2006; Schwab et al., 2006) were unsuccessful.
This could have resulted from redundant activities of homolo-
gous or nonhomologous proteins. We thus addressed the ques-
tion of KNOXI function in tomato leaf development using chimeric
repressor silencing technology (CRES-T) (Hiratsu et al., 2003). A
fusion of Tkn2 with the 12–amino acid long EAR repressor motif
of the Arabidopsis SUPERMAN gene (Ohta et al., 2001; Hiratsu
et al., 2002), termed SRDX, was expressed in tomato leaves. We
reasoned that this fusion protein would specifically repress the
transcription of common targets of tomato KNOXI proteins, in-
cluding TKN1-4, aswell as targets that are commonly regulatedby
potential nonhomologous redundant factors. In developmental
contexts in which KNOXI proteins act primarily as activators, this
will lead to the repression of downstream events (Markel et al.,
2002). To distinguish between the functions of KNOXI proteins in
SAMmaintenance and compound leaf development, expression
of Tkn2-SRDX was transactivated by the FIL promoter, which is
expressed throughout developing tomato leaves but not in the
SAM (Figures 1B and 1G) (Lifschitz et al., 2006). FILpro>>Tkn2-
SRDX tomato plants showed a pronounced alteration in leaf
structure (cf. Figures 1C and 1H to Figures 1A and 1F). The first
two leaves produced by these plants were simple and remark-
ably small. Later emerging leaves were small, narrow, and simple.
To further understand KNOXI function in compound leaf de-
velopment, we transactivated each of the tomato KNOXI genes
Tkn1 and Tkn2 specifically in developing leaves by the FIL
promoter. In contrast with the super-compound leaves of tomato
mutants with increased Tkn2 expression and a subset of trans-
genic plants ubiquitously overexpressing KNOXI genes (Hareven
et al., 1996; Chen et al., 1997; Parnis et al., 1997; Janssen et al.,
1998), FILpro>>Tkn1 and FILpro>>Tkn2 plants produced narrow
and simple leaves (Figures 1D, 1E, 1I, and 1J).
KNOXI Proteins Regulate Leaf Complexity by Timing
Leaf Maturation
Surprisingly, leaves of both FILpro>>Tkn1/2 and FILpro>>Tkn2-
SRDX were small, narrow, and simple (Figures 1D, 1E, 1I, and
1J). This prompted us to examine early leaf development in these
genotypes to understand the developmental basis for the pro-
duction of these final leaf shapes. FILpro>>Tkn2 leaves retained
morphological characteristics of very young leaf primordia at the
initiation stage for a prolonged period during their early devel-
opment (Figures 2A to 2D). Thus, at the P5 stage, these leaves
resembled elongated P2 wild-type primordia, with no lamina
expansion, and reduced and delayed trichome development. In
FILpro>>Tkn2 leaves of the plastochron equivalent of late-SM-
phase wild-type leaves, numerous initiation events of ectopic
meristems and leaflet primordia were observed at the leaf margin
(Figures 2H and 2I).
In striking contrast, FILpro>>Tkn2-SRDX leaf primordia showed
precocious leaf maturation, as revealed by the early expansion of
the leaf lamina, its precocious straightening, and the development
of trichomes on the entire surface of the leaf primordia from the P2
stage on (Figures 2E and 2F). Thus, the failure to develop leaflets
resulted from apparently opposite mechanisms in FILpro>>Tkn2
and FILpro>>Tkn2-SRDX: FILpro>>Tkn2 leaves failed to exit the
initiation stage, a transition that normally precedes leaflet initiation,
while in FILpro>>Tkn2-SRDX plants, leaf differentiation was ac-
celerated such that the PM stage was shortened, and leaves
progressed directly to the SM stage without leaflet initiation. In
tobacco (Nicotiana tabacum) and Arabidopsis, KNOXI proteins
have been shown to act as repressors of some of their targets,
such as the gibberellic acid biosynthesis geneGA20ox (Sakamoto
et al., 2001; Hay et al., 2002). Thus, some of the FILpro>>Tkn2-
SRDX phenotypes could represent overexpression phenotypes.
However, the complementary phenotypes of FILpro>>Tkn2-
SRDXandFILpro>>Tkn2 suggest that themajority of the observed
phenotypes resulted from downregulation of genes that are
normally activated by TKN2 (Matsui et al., 2008; Ikeda and
Ohme-Takagi, 2009). In agreement, while transactivation of
Regulation of Leaf Maturation by KNOX 3079
Tkn2-SRDX later in leaf development by theBLS promoter did not
affect leaf shape (see below), it almost entirely suppressed the
phenotypes caused by expression of either Tkn2 or Tkn1 through
the same promoter (see Supplemental Figure 1 online). This result
confirms that TKN2-SRDX represses the activity of both TKN1and
TKN2 and likely also that of additional redundant factors. This
emphasizes redundancy in the tomatoKNOXI family, demonstrat-
ing CRES-T as a powerful tool to address redundancy in tran-
scription factor activity. The opposing activity of TKN2-SRDX and
TKN2 was further confirmed by analysis of gene expression.
Apices of 13-d-old wild-type, FILpro>>Tkn2, and FILpro>>Tkn2-
SRDX seedlings, which contained the SAM and the five youngest
leaf primordia, differed dramatically in their transcriptome (see
Supplemental Figure 2 online), providing molecular support to the
developmental phenotype. Specifically, in contrast with Tkn2,
whose expressionwas upregulated relative to thewild type in both
FILpro>>Tkn2 and FILpro>>Tkn2-SRDX apices (Figure 2G), S.
lycopersicumGA2ox4wasupregulated ninefold relative to thewild
type in FILpro>>Tkn2 but not in FILpro>>Tkn2-SRDX apices (see
Supplemental Figure 2C online). In agreement, GA2ox4 expres-
sion was shown to be elevated 23-fold in FILpro>>Tkn2 apices
relative to the wild type by quantitative real-time PCR verification
(see Supplemental Figure 2D online). A GA2ox gene was recently
shown tobeadirect target of KNOXI proteins inmaize (Bolduc and
Hake, 2009). While the wild-type apices used for the expression
profiling contained leaf primordia of various developmental
stages, primordia of FILpro>>Tkn2 apices were all at the initiation
stage, and FILpro>>Tkn2-SRDX primordia have differentiated
(Figures 2A, 2C, and 2E). Therefore, some of the differences in
gene expression among these genotypes are likely secondary to
these developmental differences.
Cumulatively, these observations imply that KNOXI proteins
promote compound leaf development by prolonging specific
stages of leaf development. Proper timing and balancing of
KNOXI activity during leaf development is therefore critical for
proper elaboration of the compound tomato leaves.
Differential Spatial and Temporal Competence of the
Developing Tomato Leaf to React to KNOXI Activity
The leaf phenotypes caused by Tkn2 overexpression or impaired
KNOXI activity throughout early leaf development suggested
that KNOXI activity is interpreted in a spatial- and temporal-
dependent manner during leaf development. To test this and
learn which stages in leaf development and which spatial do-
mains of the leaf primordium are sensitive to KNOXI activity, we
expressed Tkn2 and Tkn1 during a series of specific spatial and/
or temporal windows within the developing tomato leaf using
Arabidopsis promoters (Efroni et al., 2008; Shalit et al., 2009).
Tkn1 and Tkn2 affected leaf development similarly during the
tested developmental circumstances. The GH3.3 promoter di-
rected expression at the distal part of young leaf primordia
starting from the early P1 stage. In slightly older primordia, it
showed additional, lower-level expression in more proximal
domains, mainly in the vasculature (Figures 3B, 3F, 3H, and
4G; see Supplemental Figures 3A and 3B online). Expressing
Tkn1 or Tkn2 under the control of the GH3.3 promoter did not
lead to a visible alteration in final leaf shape (Figures 4A and 4D) in
contrast with the profound inhibition caused by their expression
throughout the young leaf primordia using the FIL promoter
(Figures 1, 2, 3A, 3E, and 3H). The distal part of the leaf, in which
the GH3.3 promoter is expressed, is the first to emerge and the
Figure 1. Alteration of KNOXI Activity in Developing Tomato Leaves Impairs Leaf Development.
(A) and (C) to (E) Four-week-old tomato plants.
(F) and (H) to (J) Mature fifth leaves.
(B) Expression pattern of the monomeric red fluorescent protein (mRFP) reporter (in red) driven by the FIL promoter.
(G) Schematic illustration of the expression pattern directed by the FIL promoter (in red) in the SAM and three youngest leaf primordia.
The genotypes are indicated above each column. FILpro>>Tkn2-SRDX, FILpro>>Tkn1, and FILpro>>Tkn2 overexpress Tkn1, Tkn2, and Tkn2-SRDX,
respectively, under the control of the FIL promoter. P1-P3, initiating leaf primordia, such that P1 is the youngest primordium. Bars = 1 cm (A), (C) to (F),
and (H) to (J) and 0.5 mm in (B).
3080 The Plant Cell
first to differentiate. Similarly, expressing Tkn1 or Tkn2 under the
control of the 650 promoter, which directs expression starting
from the P4 stage in a gradient that increases toward the distal
end of the leaf, and in the leaf vasculature (Figures 3D, 3H, and 4I;
see Supplemental Figure 3D online) (Shalit et al., 2009) had no
obvious effect on leaf structure (Figures 4C and 4F). By contrast,
expressing these genes using the BLS promoter caused a
substantial increase in the degree of leaflet reiteration and an
elaboration of the marginal lobes (Figures 4B and 4E). The BLS
promoter drives expression starting at the P4 stage, in both
abaxial and adaxial domains. It is expressed in leaflets shortly
after their initiation and is nearly absent from both proximal and
distal domains; it thus roughly represents domains that are in the
PM stage (Figures 3C, 3H, and 4H; see Supplemental Figure 3C
online) (Shalit et al., 2009). Primary leaflets were formed relatively
normally in BLSpro>>Tkn1/2 plants, as expected from the BLS
expression domain, but increased number of higher-order leaf-
lets were produced (see Supplemental Figure 4 online). The
differential response of leaf primordia to KNOXI overexpression
by the different promoters could result from either the distinct
expression domains or from differences in expression levels. To
distinguish between these possibilities, we compared the ex-
pression levels driven by these promoters in six locations
representing different spatial and temporal developmental do-
mains (Figures 3G and 3H). Expression from theGH3.3 promoter
was weaker than that from FIL in all locations (Figure 3H; see
Figure 2. TKN2 Inhibits Leaf Maturation.
(A), (C), and (E) Scanning electron micrographs of shoot apices and initiating leaf primordia.
(B), (D), and (F) Successive stages of early leaf development, at stages P2 to P5, as indicated at the top of each column. The genotypes of each row are
indicated at the left. P1 to P5 are as in Figure 1G.
(G) Comparison of the Tkn2 mRNA levels in 13-d-old apices by quantitative RT-PCR. Standard errors (n = 3) are indicated for each genotype.
(H) Scanning electron micrographs of a FILpro>>Tkn2 leaf at a later developmental stage, showing curved growth and an ectopic meristem (arrow,
enlarged in the inset) that was formed at the distal end of the leaf and produced leaf and flower primordia.
(I) A scanning electron micrograph showing multiple events of lateral initiation at the leaf margin.
Bars = 200 mm in (A), (C), (E), (H), the inset, and (I) and 1 mm in (B), (D), and (F).
[See online article for color version of this figure.]
Regulation of Leaf Maturation by KNOX 3081
Supplemental Figure 5A online). The differential effect of Tkn2
overexpression driven by these promoters could thus stem from
either the expression domain or from the expression level.
However, the expression from GH3.3 in P3 primordia and in
distal domains of P5 primordia was stronger than that of BLS
(Figures 3B, 3C, 3F, and 3H), and expression of BLS in initiating
leaflets and of 650 in distal and abaxial domains were compa-
rable to that of FIL (Figures 3H). The differential responses of
leaves to KNOXI expression driven by these promoters thus
likely stem from the distinct location and timing of expression
rather than from the expression level. In agreement, the differ-
ences in phenotypic severity between BLSpro>>Tkn2 and
650pro>>Tkn2 leaves correlated with the expression domains
directed by these promoters (Figures 3C, 3D, 4E, 4F, 4H, and 4I)
rather than with the relative expression levels of the Tkn2mRNA
(see Supplemental Figure 5B online). These results indicate that
a spatial and temporal gradient of morphogenetic potential
exists within the developing tomato leaf and that only very
specific regions and developmental stages are competent to
react to KNOXI activity. Thus, KNOXI overexpression during the
initiation stage inhibits the progress to the PM stage, while in
regions at the PM stage, KNOXI proteins appear to maintain the
morphogenetic potential. By contrast, regions at the SM stage
are insensitive to KNOXI activity. An alternative interpretation is
Figure 3. Characterization of the Expression Directed by a Series of Leaf-Specific Promoters.
(A) to (D) Confocal microscope images of P5 primordia of the indicated genotypes.
(E) to (F) Confocal microscope images of the SAM and three youngest leaf primordia of the indicated genotypes.
(G) Location of six sites at which the fluorescence signal was quantified and compared among the four leaf-specific promoters, in P3 or P5 primordia.
(H)Relative fluorescence intensity from RFP, expressed by the indicated promoters at six spatial domains of P3 or P5 primordia, as shown in (G). LMFIL,
local mean fluorescence intensity level (see Methods). Standard error (n = 3) is indicated for each combination of genotype and location.
The mRFP fluorescence is shown in green or yellow. Bars = 200 mm.
3082 The Plant Cell
that proximal and adaxial leaf domains show increased KNOXI
responsiveness. These results explain the variability of the KNOXI
constitutive overexpression phenotypes by demonstrating that
they act differentially during distinct developmental windows.
To examine when KNOXI function is required during normal
leaf development, we expressed Tkn2-SRDX using a subset of
the stage-specific promoters. No obvious alteration of leaf shape
relative to the wild type was observed in plants that expressed
Tkn2-SRDX via either the GH3.3, BLS, or 650 promoters. This
implies that KNOXI proteins are normally active only in very
young regions of the developing tomato leaf.
A Similar Developmental Window of KNOXI Sensitivity in
Simple and Compound Leaves
Strong constitutive overexpression of KNOXI in Arabidopsis
simple leaves leads to leaf lobing and rumpling (Figure 5A,
compare with 5G) (Sinha et al., 1993; Lincoln et al., 1994; Chuck
et al., 1996; Hake et al., 2004). Is the spatial and temporal window
of KNOXI responsiveness similar between simple and com-
pound leaves? To address this question, we overexpressed in
Arabidopsis each of the four Arabidopsis KNOXI genes, BREVI-
PEDICELLUS (BP), KNOTTED-like from Arabidopsis thaliana 2
(KNAT2), KNAT6, and SHOOT MERISTEMLESS (STM), using
each of five representative promoters, which drive expression
transiently at successive stages of leaf development (Efroni
et al., 2008) (Figures 5B to 5F). The effect of transient expression
of the Arabidopsis KNOXI genes at early stages of leaf develop-
ment resulted in mild or no change in leaf shape, depending on
the specific combination of the gene being expressed and the
expression window (Figures 5B and 5C). By contrast, expressing
these genes by theBLS promoter, which drives expression at the
P5-P7 stages of leaf development, approximating the PM stage
(Efroni et al., 2008), altered leaf development (cf. Figures 5D and
5H). Overexpression of STM and BP using the BLS promoter
caused mainly rumpling, whereas KNAT2 and KNAT6 over-
expression with this promoter resulted in the formation of three
lobes (Figure 5D; see Supplemental Figure 6 online). No altera-
tion in leaf shape resulted from KNOXI expression at late stages
of leaf development (Figures 5E and 5F). Roughly, the sensitivity
Figure 4. Developmental Context–Specific Response to Tkn1 and Tkn2 Overexpression.
Tkn1 or Tkn2 overexpression was directed by a series of leaf-specific promoters. Promoters are indicated at the top of each column and the expressed
genes at the left of each row. P1 to P5 are as in Figure 1. Asterisk indicates the SAM. Bars = 5 cm.
(A) to (F) Fifth mature leaves.
(G) to (I) Schematic illustration of the expression domains of the different Arabidopsis promoters.
[See online article for color version of this figure.]
Regulation of Leaf Maturation by KNOX 3083
to KNOXI overexpression shows a peak at the PM stage, and the
sensitivity decreases with the temporal distance from this peak.
These results suggest that all Arabidopsis KNOXI genes can only
affect the simple leaf structure during a short developmental
window in leaf patterning. However, the specific effect on leaf
development and the exact time window of KNOXI responsive-
ness differed among the four Arabidopsis KNOXI genes. To
confirm that these differences stemmed from differential activity
rather than from the genomic context of the insertion site in the
specific responder line, we crossed the BLS driver line to six to
eight independent responder lines for each of the KNOXI genes.
While the different responder lines of each KNOXI gene pro-
duced a range of phenotypic severities, the phenotypes were
similar among independent lines of each KNOXI, and the
Figure 5. Stage-Specific KNOXI Responsiveness of Developing Arabidopsis Leaves.
Arabidopsis plants expressing the four different Arabidopsis KNOXI genes at different stages of leaf development. The promoters and their respective
expression domains, as deduced from the literature (Efroni et al., 2008), drawn on schemes of transverse sections, are indicated at the top of each
column. The expressed genes are indicated at the left of each row. Bars = 1 cm.
(A) Whole plants.
(B), (C), (E), and (F) Fifth leaves.
(D) Second to seventh rosette leaves of the indicated genotypes. White arrows point to lobes in BLSpro>>KNAT2 and BLSpro>>KNAT6 leaves.
(G) Whole plant of wild-type Landsberg erecta.
(H) A leaf series of wild-type Landsberg erecta.
(I) An illustration indicating the location of the SAM (M) and the different leaf primordia (P1 to P7) on a scheme of a transverse section as those shown in
(A) to (F).
[See online article for color version of this figure.]
3084 The Plant Cell
phenotype shown in Figure 5D is representative of the relatively
severe phenotype of each responder.
Comparing the results of transient KNOXI overexpression
between tomato and Arabidopsis suggests that leaves of both
species react to KNOXI activity during a comparable develop-
mental window during the PM stage. However, tomato leaves
show an additional window of KNOXI responsiveness during leaf
initiation. Whereas KNOXI overexpression apparently causes an
extension of the initiation stage in tomato plants, initiating
Arabidopsis leaves are comparatively insensitive to KNOXI mis-
expression.
Context-Specific Similarities and Differences in
KNOXI Function
To testwhether tomato andArabidopsis KNOXIgenes differ in their
effect on leaf development in addition to having differences in
expression, we expressed the tomato Tkn1 and Tkn2 genes in
Arabidopsis leavesusing theBLSpromoter.StrongBLSpro>>Tkn2
lines were highly rumpled and lobed (Figures 6E and 6F compared
with Figures 6A and 6B), and higher-order lobeswere initiated from
the margins of the leaf lobes, which partially resembled initiating
leaflets (Figures 6G to 6I). Such a phenomenon of ectopic initiation
eventswasnot observedwithanyof theArabidopsisKNOXIgenes,
implying functional differences between tomato and Arabidopsis
KNOXI proteins. Interestingly, BLSpro>>Tkn1 leaves had a very
mild phenotype with rounded shape but minor lobing (Figures 6C
and 6D), suggesting that while tomato leaves respond similarly to
Tkn1 and Tkn2 overexpression, Arabidopsis leaves display differ-
ential sensitivity to these tomato genes. Comparison of six to eight
independent responder lines each for Tkn1, Tkn2, and the four
Arabidopsis KNOXI genes confirmed that the distinct responses
were due to differential activity rather than an effect of the specific
responder line (data not shown). Tkn1 and Tkn2 expression only at
Figure 6. Overexpression of the Tomato KNOXI Gene Tkn2 but Not Tkn1 Severely Distorts Leaf Development in Arabidopsis.
Genotypes are indicated on the bottom left corner. Bars = 1 cm in (A) to (F) and 500 mm in (G) to (I).
(A), (C), and (E) Whole plants.
(B), (D), and (F) First to eighth rosette leaves of the indicated genotypes.
(G) to (I) Scanning electron micrographs of the initiation events (white arrows) from the leaf lobes of BLS>>Tkn2 leaves. The black rectangle in (G)
indicates the region magnified in (H).
(H) Magnification of part of the leaf shown in (G).
[See online article for color version of this figure.]
Regulation of Leaf Maturation by KNOX 3085
late stages of Arabidopsis leaf development using the 7470
promoter (Figure 5E) did not show any aberrant leaf shape
phenotypes (see Supplemental Figure 7 online). Cumulatively,
misexpression of the different Arabidopsis and tomato KNOXI
genes through stage-specific promoters in both species argues
against differences in KNOXI activity as the basis for the
distinction between simple and compound leaves. Rather,
KNOXI proteins are used within the compound leaf develop-
ment program to delay differentiation and enable leaflet forma-
tion, rather than set the compound leaf route.
A comparison of the stage-specific effects of tomato and
Arabidopsis KNOXI genes on leaf shape suggests that these
proteins have evolved overlapping but partially distinct activities
at different developmental circumstances. To expand this com-
parison to additional developmental contexts, we tested the
ability of each Arabidopsis KNOXI gene to substitute for BP by
expressing them using the BP promoter in the bp-1 mutant
background. The responder lines that were used for these
experiments all showed a relatively strong phenotype when
crossed to the BLS driver line. bp-1 mutants have impaired
inflorescence stem elongation, short internodes, and downward-
pointing siliques (Douglas et al., 2002; Venglat et al., 2002)
(cf. Figure 7F to 7G). BPpro>>BP, BPpro>>STM, and BPpro>>
KNAT6 partially rescued the bp-1mutant phenotype (Figures 7B
to 7D). This was manifested by taller plant stature, longer
internodes, and partially recovered pedicel-stem angles rela-
tive to bp-1 plants. Interestingly, BPpro>>KNAT2 bp-1 plants
were indistinguishable from bp-1 mutants (Figure 7E). Quanti-
fication of the degree of rescue by measuring the angle be-
tween the first four pedicels and the main inflorescence
stem confirmed these results (Figure 7A). The genotype of
BPpro>>KNAT2 bp-1 plants was confirmed by PCR and by
crossing BPpro>>KNAT2 to the BLSpro driver line, which
resulted in the expected phenotype of lobed leaves (Figure
5D). Similarly, the different KNOXI responder lines that were used
in this evaluation produced the expected phenotype when
crossed to the BLSpro driver line. Thus, while the KNAT2 and
KNAT6 proteins display very similar effects on leaf shape, they
have acquired different functions in the context of inflorescence
development.
DISCUSSION
Developmental Stage–Specific KNOXI Responsiveness
Previously, ubiquitous KNOXI overexpression in tomato was
found to result in variable and even opposite leaf phenotypes
(Hareven et al., 1996; Chen et al., 1997; Parnis et al., 1997;
Janssen et al., 1998). Here, Tknmisexpression in a series of leaf-
specific developmental windows enabled us to dissect these
phenotypes into several context-specific effects. Tkn overex-
pression affected leaf shape only when expressed during the
early initiation or PM stages of leaf development. However, the
effect of Tknoverexpressionwas different between those stages.
Overexpression during leaf initiation prevented further progress
of leaf development, apparently by extending the initiation phase,
while during PM it caused an expansion of the morphogenetic
Figure 7. STM and KNAT6 but Not KNAT2 Can Substitute for BP Function in Arabidopsis.
The four different Arabidopsis KNOXI genes were expressed by the BP promoter in the bp-1 mutant background.
(A) A quantitative representation of the complementation level, presented as the angle between the pedicels and the main stem. Relatively high angles
similar to the wild type represent relatively strong complementation, while low angles similar to those in bp represent little or no complementation.
Standard deviation is shown for each genotype (n = 20 for bp-1, the wild type, BPpro>>BP, BPpro>>STM, and BPpro>>KNAT2; n = 8 for
BPpro>>KNAT6).
(B) to (G) Inflorescences of the indicated genotypes. Wild type, Landsberg erecta. Bars = 1 cm.
[See online article for color version of this figure.]
3086 The Plant Cell
activity, resulting in a dramatically increased number of leaflet
reiteration orders (Figure 4). Thus, TKN activity is interpreted in a
spatial- and temporal-specific manner. That repression of po-
tential TKN targets affects leaf development only during early
stages of leaf development suggests that TKN is normally active
only transiently during leaf development and further empha-
sizes the context-specific interpretation of KNOXI action. How-
ever, the lack of aberrant phenotype in BLSpro>>Tkn2-SRDX
is surprising, as this promoter acts in tissues approximately at
the PM stage (Figure 3C). This observation suggests that the
BLS promoter drives expression slightly later than the develop-
mental timing at which KNOXI genes act to extend PM, which is
in agreement with its expression slightly following leaflet initia-
tion (Figure 3C).
How Do KNOXI Proteins Affect Leaf Shape?
While KNOXI proteins were shown to play major roles in the
development of compound leaves (Bharathan et al., 2002; Hay
and Tsiantis, 2006; Champagne et al., 2007; Jasinski et al., 2007),
their exact role in compound leaf development is still unclear.
For example, KNOXI activity in leaves could underlie the devel-
opmental choice between simple and compound leaves, be
required for leaflet initiation, or maintain the morphogenetic
competence at the leaf margin by prolonging the indeterminate
developmental phase. Comparing the outcome of overexpress-
ing Tkn1/2 and repressing TKN2 activity at specific stages of
tomato leaf development suggests that TKNs inhibit the progress
of leaf maturation (Figure 8). Thus, when overexpressed at
Figure 8. A Proposed Model for KNOXI Function in Tomato Compound Leaf Development.
The panels present the proposed developmental interpretation of the different genotypes. Left: Schematics describing the progress of leaf
development. The color codes of the different stages are indicated in the wild type. I, initiation. Right: Scanning electron micrographs of shoot apices of
the different genotypes, color coded for the different stages of leaf development. TKN2 is proposed to inhibit the transition between stages during leaf
development. In the wild type, TKN2 acts to extend PM. Repression of TKN2-like function in developing leaves of FILpro>>Tkn2-SRDX plants leads to a
prompt progression to the SM phase. By contrast, overexpressing wild-type Tkn2 throughout leaf development in FILpro>>Tkn2 plants results in an
extended initiation stage and in the failure to progress to PM. Tkn2 expression specifically at the PM stage of developing leaflets in BLSpro>>Tkn2
extends leaflet PM, resulting in higher-order reiteration of leaflet formation. White arrows point to primary leaflets, which are situated in the position
shown in red on the diagram to the left of the micrographs. Numbers 1 to 4 indicate leaflet reiteration levels.
[See online article for color version of this figure.]
Regulation of Leaf Maturation by KNOX 3087
initiation, TKNs delay the onset of PM and thus prevent leaf
elaboration, while expression in leaflets at PMextends this stage,
resulting in additional levels of reiteration (Figure 8). The rapid
transition from the initiation to the SM phase caused by the
repression of TKN activity is compatible with this model. This,
together with the profound effect of KNOXI overexpression
during leaf initiation, and the narrow window of sensitivity to
TKN2-SRDX, suggests that during normal leaf development,
KNOXI proteins act specifically at the PM stage. The similar
developmental window at which tomato and Arabidopsis
leaves respond to KNOXI activity further supports the model
that KNOXI proteins affect leaf development by inhibiting dif-
ferentiation. The observation that overexpression of KNOXI
genes during leaf initiation in Arabidopsis has very minor
consequences for leaf development in contrast with the dra-
matic effect in tomato may imply that part of the difference
between simple and compound leaves stems from differences
at the initiation stage. This and the observation that in Arabi-
dopsis secondary initiation events caused by Tkn2 overexpres-
sion occur after the lamina has started to expand, leading to
reiteration of lobes rather than leaflets, further support the
conclusion that KNOXI genes are used for leaf elaboration
within the simple or compound developmental programs,
rather than for distinguishing between these developmental
programs.
LANCEOLATE (LA) activity was shown to promote the transi-
tion from the PM to the SM stage of leaf development in tomato.
Elevation of LA activity at early stages of leaf development in the
dominant Lamutant caused precocious leaf differentiation and a
failure to properly initiate leaflets from the leaf margin (Ori et al.,
2007). This phenotype is similar to that of FILpro>>Tkn2-SRDX.
Interestingly, the La mutant phenotype is epistatic to the super-
compound leaf phenotype caused by KNOXI overexpression
(Hareven et al., 1996; Kessler et al., 2001; Ori et al., 2007). This
genetic interaction seems to stem from a developmental rather
than molecular mechanism, as LA downregulation and KNOXI
upregulation display an additive genetic interaction (Ori et al.,
2007). Thus, KNOXI proteins and LA appear to act antagonisti-
cally to define the morphogenetic window in the developing
tomato leaf. This window requires both KNOXI activity and
downregulation of LA activity. The boundary NAC domain gene
GOBLET (GOB) has been recently implicated in the elaboration
of compound leaves (Blein et al., 2008; Berger et al., 2009).
However, overexpressing or downregulating GOB using the FIL
promoter affects mainly the interval between secondary leaflets
and leaflet margin elaboration and has a minor effect on the
initiation stage. This suggests that while balancing the activities
of KNOXI and LA proteins defines the length of the PM stage,
GOB affects the location and timing of leaflet initiation within
the PM.
HowdoKNOXI proteinsmaintain themorphogenetic activity of
the leaf margin? One way by which these proteins regulate the
morphogenetic activity of the SAM is by modulating hormone
levels (Shani et al., 2006). KNOXI genes have been shown to
negatively regulate gibberellic acid levels (Sakamoto et al., 2001;
Hay et al., 2002; Chen et al., 2004; Jasinski et al., 2005) and
positively regulate cytokinin biosynthesis (Jasinski et al., 2005;
Yanai et al., 2005).
Similarities and Differences in KNOXI Function
Our results imply that functional redundancy among KNOXI
proteins is not directly correlated to their sequence homology.
Furthermore, the functional relationships among the Arabi-
dopsisKNOXI proteins appear to be context specific, such that
each specific activity is shared by different subsets of family
members. The different functions might relate to differ-
ential interactors and/or targets (Krizek and Fletcher, 2005;
Sablowski, 2007). The very different effect of the tomato genes
Tkn1 and Tkn2 in Arabidopsis leaves despite their very similar
effects in tomato is intriguing in this respect. The complex
functional relationship among KNOXI proteins, as revealed by
this and previous studies exemplifies how plants use gene
families flexibly for both backup of essential functions and
acquirement of novel activities and processes, as has been
also shown for other families of developmentally related tran-
scription factors (Emery et al., 2003; Ditta et al., 2004; Hibara
et al., 2006).
The context-specific activity of KNOXI proteins is further
emphasized by the observation that ubiquitous STM expression
failed to rescue the bp-1 phenotype (Scofield et al., 2007), in
contrast with its expression driven by the BP promoter (this
study). Furthermore, while in the Landsberg erecta background
the bp-1 phenotype is suppressed by expression ofKNAT6 in the
BP expression domain (this study), in the Columbia background,
BP was reported to negatively regulate KNAT2 and KNAT6
(Ragni et al., 2008). Genetic background was shown to dramat-
ically affect the phenotypes of KNOXI loss-of-function mutants
and KNOXI overexpressing lines (Felix et al., 1996; Mele et al.,
2003; E. Shani and N. Ori, unpublished data). Further character-
ization of the effect of genetic background on KNOXI function
might contribute to the understanding of the context-dependent
function of KNOXI proteins.
In conclusion, these results imply that highly controlled but
flexible tuning of the spatial and temporal progress of organ
maturation is one of themechanisms used by plants to produce a
highly diverse array of organ shapes.
METHODS
Plant Material
Tomato (Solanum lycopersicum cv M82) plants were grown in green-
house conditions under temperatures ranging between 18 and 258C.
Tomato plants were in an sp background. All described transgenic
genotypes were produced by the LhG4 transactivation system (Moore
et al., 1998). This system comprises driver lines that express the
synthetic transcription factor LhG4 under the control of a specific
promoter (PRO:LhG4). In a responder line, a gene of interest is ex-
pressed under the control of several copies of the Escherichia coli
Operator (OP), which is recognized by LhG4 (OP:GENE). A cross
between a driver and a responder line results in the expression of the
gene of interest under the control of the specific promoter. The tomato
driver and responder lines FILpro:LhG4, 650pro:LhG4, and OP:mRFP
have been described (Efroni et al., 2008; Shalit et al., 2009).Arabidopsis
thaliana plants were grown under long-day fluorescent light (16 h light,
218C). The described Arabidopsis plants are all in the Landsberg erecta
background. The Arabidopsis driver lines KAN1pro:LhG4, GH3.3pro:
LhG4, BLSpro:LhG4, 7470pro:LhG4, 650pro:LhG4, and ANTpro:LhG4
3088 The Plant Cell
have been described (Schoof et al., 2000; Efroni et al., 2008). BPpro:
LhG4 was a gift from Yutaka Sato (Nagoya University). The Arabidopsis
lines OP:STM, OP:BP, OP:KNAT2, OP:KNAT6, OP:Tkn1, and OP:Tkn2
and the tomato lines OP:Tkn1, OP:Tkn2, OP:Tkn2-SRDX, GH3pro:
LhG4, and BLSpro:LhG4 were generated during this research as
described below.
Plasmids and cDNA Clones and Plant Transformation
To generate OP:Tkn2-SRDX, assembly PCR was used to introduce the
36-nucleotide-long SRDX motif: CTCGATCTGGATCTAGAACTCCGTTT-
GGGTTTCGCT + TAA stop codon (Hiratsu et al., 2003) in the C terminus
of the TKN2 protein. Tkn2-SRDX, Tkn1, Tkn2, STM, BP, KNAT2, and
KNAT6 were cloned downstream to an OP array (Moore et al., 1998) and
transferred into the binary pMLBART or pART27 vectors (Eshed et al.,
2001). The GH3.3 and BLS promoters were cloned upstream of LhG4
(Moore et al., 1998) and subsequently cloned into the pART27 binary
vector. Primers used to clone the different cDNAs and promoters are
described in Supplemental Table 1 online. Cotyledon transformation in
tomato was performed according to McCormick (1991). Arabidopsis
transgenic lines were generated by the floral dip method (Clough and
Bent, 1998), and BASTA- or kanamycin-resistant transformants were
selected in soil by spraying. Between 8 and 10 independent lines of each
construct were examined, and further analysis was performed with one
strong and one weak line.
Plant Genetics
Detailed phenotypic analyses were performed with selected OP:GENE
responder lines that were crossed to promoter:LhG4 driver lines. Six to
eight independent responder lines of each KNOXI gene were crossed to
the BLS driver line, and a representative line is presented in Figure 5. To
generate BPpro>>KNOXI bp-1, plants homozygous for BPpro:LhG4
were crossed to plants homozygous for the bp-1 mutation. Similarly,
relatively strong lines of OP:STM, OP:BP, OP:KNAT2, and OP:KNAT6,
selected by the phenotype of the corresponding BLSpro>>KNOXI line,
were each crossed to bp-1. F2 BPpro:LhG4 bp-1 plants, selected by
resistance to BASTA and the bp-1 phenotype, were crossed to each
of the four similarly selectedOP:KNOX1 bp-1plants. All F1 plants showed
the bp-1 phenotype except those possessing both the BPpro:LhG4 and
the OP:KNOXI transgenes, which showed a suppressed phenotype.
Genomic DNA was extracted and analyzed by PCR to verify BPpro>>
KNAT2 bp-1 plants.
Phenotype Quantification
The WinFOLIA image analysis software was used to measure leaf area
and leaf perimeter. The angles between the first four pedicels and the
main stem in Arabidopsis were measured in five individuals using the NIH
ImageJ software program.
RNA Analysis
Thirteen-day-old seedlings were harvested at comparable developmen-
tal stages. Total RNA was isolated using the Qiagen’s RNeasy Mini and
Micro kits, and cDNA was prepared from 1 mg total RNA with a poly(A)
primer using the ABgene Verso RT-PCR kit (AB-1453/B). Quantitative
real-time RT-PCR analysis was performed using the TaKaRa SYBR
Premix Ex Taq II (RR081Q) kit. Reactions were performed using a Corbett
Research Rotor-Gene 6000 cycler. For each gene tested, at least one of
the primers used spanned an exon-exon border to avoid amplification of
genomic DNA. A standard curve was obtained for each gene using
dilutions of a cDNA sample. Quantification of each gene was performed
using the Corbett Research Rotor-Gene software. At least four indepen-
dent technical repeats were performed for each cDNA sample, and at
least three biological repeats were used for each genotype. Relative
expression of each sample was calculated by dividing the expression
level by that of tubulin (in arbitrary units). Gene/tubulin ratios were then
averaged and presented as a ratio of a control treatment, the value of
which was set to 1. Primer sequences are detailed in Supplemental
Table 1 online.
Microarray Analysis
Microarray expression analysis was performed with total RNA from 13-d-
old apices that contained the SAM and the five youngest leaf primordia.
Labeled RNAwas hybridized to an Affymetrix GeneChip TomatoGenome
Array (900738) containing >10,000 probe sets. Two biological repeats
were analyzed for each genotype. Robust multichip average analysis
(Irizarry et al., 2003) was used to simultaneously normalize data from all
groups, and Partek software was used for statistical analysis. One-way
analysis of variance was applied to compare the three genotypes: wild-
type, FILpro>>Tkn2-SRDX, and FILpro>>Tkn2. Contrasts were calcu-
lated between each pair of genotypes. False discovery rate was used to
identify differentially expressed genes. Genes with step-up (P value <
0.05) that met at least one of the following criteria were used for cluster
analysis: filter 1, genes with fold change higher than two between the wild
type and FILpro>>Tkn2; filter 2, genes with fold change higher than two
between the wild type and FILpro>>Tkn2-SRDX; filter 3, genes with fold
change higher than two between FILpro>>Tkn2 and FILpro>>Tkn2-
SRDX. The filtered set of 1221 genes was clustered by the SPC algorithm
into eight unique clusters, and the samples were sorted by the SPIN
algorithm for clearer visualization of the clusters (Blatt et al., 1996; Kela
et al., 2009).
Imaging and Microscopy
Dissected apices and primordia were immediately placed into drops of
buffer (75 mM n-propylgallate, 60% glycerol) on glass microscope slides
and covered with cover slips. The pattern of mRFP expression was
detected by a confocal laser scanning microscope (CLSM model
LSM510; Zeiss) with the argon laser set at 488 nm for excitation, a
long-pass 560-nm filter for chlorophyll emission, and HeNe laser set at
543 nm for excitation, band-pass 560- to 615-nm filter for mRFP emis-
sion. Optical sections from successive focal planes of each primordia
tract region were collected and projected as a reconstructed three-
dimensional image using the LSM image browser (version 3.5.0.376).
Image collection and parameter settings were identical for each of the
different tract regions analyzed. Fluorescence was quantified using the
Image J software by the local mean fluorescence intensity level ap-
proach (Heifetz and Wolfner, 2004). Briefly, fluorescence intensity was
quantified in four different locations of identical areas within each leaf
region and averaged. The background fluorescence intensity was
subtracted. Three biological repeats were tested for each combination
of genotype and location. Stereoscope images were captured by an
Olympus SZX12 zoom stereo microscope using a dsRED florescence
filter set for mRFP. Tissue sectioning was performed as described
(Goldshmidt et al., 2008) and imaged with a Nikon Eclipse E800
microscope using a 4’,6-diamidino-2-phenylindole filter set for flores-
cent brighter and dsRED filter set for mRFP. Scanning electron micros-
copy was performed using a JEOL 5410 LV microscope as described
(Brand et al., 2007).
Accession Numbers
Sequence data from this article can be found in the Arabidopsis Genome
Initiative or GenBank/EMBL databases under the following accession
Regulation of Leaf Maturation by KNOX 3089
numbers: KAN1, At5g16560; GH3.3, At2g23170; BLS, At3g49950; 7470,
At2g17470; 650, At1g13650; ANT, AT4G37750; BP, AT4G08150; STM,
AT1G62360; KNAT2, AT1G70510; KNAT6, AT1G23380; Tkn1, U32247;
and Tkn2, U76407.
Supplemental Data
The following materials are available in the online version of this article.
Supplemental Figure 1. Tkn2-SRDX Coexpression Represses the
Phenotypes of Both BLSpro>>Tkn1 and BLSpro>>Tkn2.
Supplemental Figure 2. FILpro>>Tkn2 and FILpro>>Tkn2-SRDX
Seedlings Show Opposing Molecular Phenotypes.
Supplemental Figure 3. Characterization of the Expression Directed
by Three Leaf-Specific Promoters.
Supplemental Figure 4. Relative Expression Levels of Tkn2 mRNA in
Seedlings Overexpressing Tkn2 by the Indicated Promoters.
Supplemental Figure 5. Tkn2 Expression in Tomato Leaflets Delays
Differentiation and Enables the Formation of Several Higher-Order
Leaflets.
Supplemental Figure 6. Diverse Levels of Leaf Lobing Caused by
Expression of Different KNOXI Family Members.
Supplemental Figure 7. Tkn1 and Tkn2 Expression in Arabidopsis at
Late Stages of Development Does Not Alter Leaf Shape.
Supplemental Table 1. Primers Used in This Study.
Supplemental Table 2. Differentially Expressed Genes of Cluster 1.
ACKNOWLEDGMENTS
We thank Yuval Eshed and Yutaka Sato for providing genetic material,
Osnat Yanai, Roni Shalev, Ido Shwartz, and Hagay Kohay for help with
cloning and plant transformation, members of the Ori lab for technical
help and stimulating discussions, Itai Kela for help with the microarray
analysis, Einat Zelinger for help with the confocal microscopy analysis,
and David Weiss, John Alvarez, Yuval Eshed, Smadar Harpaz-Saad,
and Sharona Shleizer for critical reading of the manuscript. This work
was supported by grants from U.S.–Israel Binational Agricultural Re-
search and Development Fund (IS 3453-03 and IS 04140-08C), Israel
Science Foundation (689/05), and the Israeli Ministry of Agriculture (837-
0010-06) to N.O. and from the Israel Science Foundation Bikura Pro-
gram (1437/04) to E.S and N.O.
Received April 22, 2009; revised September 6, 2009; accepted Septem-
ber 17, 2009; published October 9, 2009.
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3092 The Plant Cell
DOI 10.1105/tpc.109.068148; originally published online October 9, 2009; 2009;21;3078-3092Plant Cell
Eran Sharon and Naomi OriEilon Shani, Yogev Burko, Lilach Ben-Yaakov, Yael Berger, Ziva Amsellem, Alexander Goldshmidt,
KNOTTED1-LIKE HOMEOBOX Proteins Leaf Maturation by Class 1Solanum lycopersicumStage-Specific Regulation of
This information is current as of November 24, 2020
Supplemental Data /content/suppl/2009/10/09/tpc.109.068148.DC1.html
References /content/21/10/3078.full.html#ref-list-1
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