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First published online 28 January 2009
doi: 10.1242/dev.031625
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1 The Robert H. Smith Institute of Plant Sciences and Genetics in Agriculture
and The Otto Warburg Minerva Center for Agricultural Biotechnology, Faculty of
Agriculture, Hebrew University of Jerusalem, P.O. Box 12, Rehovot 76100,
Israel.
2 Department of Plant Sciences, Weizmann Institute of Science, Rehovot 76100,
Israel.
Author for correspondence (e-mail:
ori{at}agri.huji.ac.il)
Accepted 1 January 2009
 |
SUMMARY |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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Key words: CUC2, NAM, Boundary, Compound leaves, miR164, Tomato
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INTRODUCTION |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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;
Heisler et al., 2005;
Sablowski, 2007).
Plant leaves are initiated from the flanks of the shoot apical meristem
(SAM), and go through common developmental stages. However, very different
final leaf shapes and sizes result from tuning the timing, duration and
further patterning events within these stages
(Barkoulas et al., 2007;
Dengler and Tsukaya, 2001;
Efroni et al., 2008;
Kaplan, 2001;
Ori et al., 2007). A major
form of variation is illustrated by simple and compound leaves. In their
mature form, simple leaves consist of a proximal petiole and a distal
continuous blade. In compound leaves, such as those of tomato, subunits termed
leaflets are attached to a central rachis through petiolules (see
Fig. 1A). This pattern can be
reiterated to produce further orders of the same basic pattern
(Hareven et al., 1996). In
addition, leaf or leaflet margins can be smooth, serrated or lobed (see
Fig. 1A)
(Goliber et al., 1999).
However, similar final leaf shapes may result from very distinct early events.
For example, a mature simple leaf can result from early arrest of leaflet
initiation or from post-initiation leaflet fusion
(Bharathan et al., 2002). It
is therefore necessary to examine leaf ontology to define the underling
developmental program.
Traditionally, leaf development has been divided into three stages: (1)
initiation of the leaf at the flanks of the SAM; (2) primary morphogenesis,
during which secondary structures such as serrations or leaflets are produced;
and (3) histogenesis or secondary morphogenesis, in which cell expansion and
final differentiation occur (Dengler and
Tsukaya, 2001; Poethig,
1997). These stages are not synchronized throughout the leaf, such
that different leaf regions can be at different developmental stages at the
same time (Dengler, 1984;
Hagemann and Gleissberg, 1996;
Ori et al., 2007). Of
particular importance for leaf patterning is a region at the leaf margins, the
marginal blastozone, which maintains morphogenetic activity and is responsible
for the initiation of secondary structures such as leaflets
(Dengler and Tsukaya, 2001;
Hagemann and Gleissberg, 1996;
Reinhardt et al., 2007).
Sufficient temporal and spatial primary morphogenesis activity at this region
is required for the formation of elaborated structures such as leaflets, and
thus for the formation of a compound leaf. In tomato, the LANCEOLATE
(LA)-like gene family, which encodes TCP transcription factors,
promotes the transition from primary morphogenesis to the histogenesis stage,
defining the morphogenetic window within which leaflets can be formed
(Caruso, 1968;
Mathan and Jenkins, 1962;
Ori et al., 2007).
Several mechanisms have been shown to act within this developmental window
to promote leaf elaboration, many of which also play a role in SAM function.
Class I KNOTTED1-LIKE HOMEOBOX (KNOXI) transcription factors are essential for
SAM maintenance (Hake et al.,
2004), and also play a central role in the modulation of compound
leaves (Bharathan et al.,
2002; Hareven et al.,
1996; Hay and Tsiantis,
2006; Parnis et al.,
1997). In some legume species, such as pea and Medicago,
the orthologous genes UNIFOLIATA and SINGLE LEAFLET,
respectively, are also involved in leaf elaboration
(Hofer et al., 1997;
Wang et al., 2008). Likewise,
several plant hormones, such as auxin and gibberellic acid (GA), have also
been implicated in leaf elaboration, either via regulation of maturation or
through mediation of localized growth
(Barkoulas et al., 2008;
Bassel et al., 2008;
Hay et al., 2002;
Jasinski et al., 2008).
Leaf initiation at the flanks of the SAM is accompanied by the formation of
a boundary region between the initiating leaf and the SAM. This boundary
region is characterized by growth retardation and by the expression of
specific boundary genes, including a group of genes encoding NAC-domain
transcription factors, represented by the petunia NO APICAL MERISTEM
(NAM), the Antirrhinum CUP and the Arabidopsis CUC
genes (Aida et al., 1997;
Souer et al., 1996;
Weir et al., 2004). Single or
double mutants in these genes, depending on the species, result in the failure
to maintain proper structure and function of the embryonic SAM and to specify
boundary regions during organ initiation and development. Several NAC-domain
genes, including Arabidopsis CUC1 and CUC2 but not the
closely related CUC3, are subject to negative control by the microRNA
miR164. Analysis of the consequences of miR164 mutations and
overexpression, as well as of miR164-insensitive CUC forms,
has further emphasized the importance of these genes for boundary
specification, organ separation and proper plant development
(Baker et al., 2005;
Laufs et al., 2004;
Mallory et al., 2004;
Nikovics et al., 2006;
Peaucelle et al., 2007;
Raman et al., 2008;
Sieber et al., 2007).
Recently, the Arabidopsis CUC2 gene has also been implicated in
controlling the degree of elaboration of leaf serrations in its simple leaves
(Nikovics et al., 2006).
Is boundary specification by NAM-related genes utilized in the process of
leaflet formation during compound-leaf patterning? Here we address this
question by analyzing loss- and gain-of-function tomato goblet
(gob) alleles. SAMs of gob mutants terminate after the
production of two fused cotyledons, but occasionally recover to produce plants
with simpler leaves than the wild type
(Brand et al., 2007). We show
that GOB encodes a NAM homolog that is essential for the proper
specification of lateral organ boundaries at the apical meristem and of
leaflet boundaries in the developing compound leaf. We uncover new roles for
GOB in the timing of leaf maturation, spatial and temporal positioning of
leaflets, secondary-leaflet initiation and separation, and leaf margin
elaboration. These processes are coordinated by a quantitative balance between
GOB and miR164 which act locally to pattern leaf development at a
short distance.
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MATERIALS AND METHODS |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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)
(http://www.sgn.cornell.edu):
Gob-4d-e0042 and e2-n0741, e3-e0880,
e4-e3335. gob-1, 2 and 3 have been
described previously (Brand et al.,
2007). Tomato seeds were sown in a commercial nursery and grown
either during April to August in the field, or during October to March in a
greenhouse under natural daylight and regulated temperature (15-30°C).
Double-mutant combinations were confirmed by progeny tests in the F3
generation. Cardamine hirsuta plants [`Oxford strain', a kind gift of
Angela Hay and Miltos Tsiantis (Hay and
Tsiantis, 2006)] were grown under 18-hour cool white fluorescent
light at 18-22°C. Analysis of early leaf development in tomato was
performed by dissecting older leaves, exposing the appropriate developmental
stage (P2-6), and observing and photographing using a dissecting scope. For
each stage, at least eight seedlings were analyzed. Different plants were used
for each stage.
Identification of the molecular lesions in gob and entire alleles
The GOB and SlIAA9 (AJ937282) genes (including introns
that lie within the coding sequence) were amplified and sequenced from genomic
DNA of the respective mutants (Fig.
1B; Fig. 7A) using
the primers listed in Table
1.
|
), using
the GOBstart and GOBstop primers in conjunction with GOBmR' and
GOBmF' primers, respectively. The 35S::miR164b construct was
generated by transcriptional fusion of a genomic fragment encompassing the
Atpre-miR164b (Alvarez et al.,
2006) in front of the 35S promoter of the ART7 vector. Constructs
were subcloned into the pMLBART binary plasmid.
Tomato cotyledon transformation was performed as described
(McCormick, 1991). At least
three independent lines were assessed for each construct by crossing them to
the same promoter line and examining the transactivated F1s. Detailed
phenotypic analyses were performed with selected OP:gene responder lines that
were crossed to Promoter:LhG4 driver lines as described
(Lifschitz et al., 2006).
Transformation into C. hirsuta was performed by floral dipping using
the Agrobacterium tumefaciens GV3101 strain. C. hirsuta
transformants were selected on soil on the basis of resistance to the
herbicide BASTA.
In situ hybridization
Tissue preparation, sectioning and transcript detection were performed as
described (Pekker et al.,
2005), except that the fixation was performed in FAA (50% ethanol,
5% acetic acid, 3.7% formaldehyde). The whole-mount in situ procedure was
adapted from Hejátko et al.
(Hejatko et al., 2006), with
fixation of plant material without heptane. The GOB probe was
synthesized with UTP-DIG and expression of miR164 was detected using
a miR164-LNA probe (Exiqon, Denmark).
RNA isolation and analysis
Total RNA was isolated using the RNeasy Micro Kit (Qiagen, Hiden, Germany),
and cDNA was prepared from 1 µg total RNA with a poly(A) primer, using the
Verso Kit (ABgene, Epsom, UK). Relative gene expression was assayed by
hybridization and quantification of RT-PCR products, as described previously
(Brand et al., 2007), using a
GOB or a TUBULIN probe. Intact (uncleaved) GOB was
amplified using primers that span the miR164 recognition site. Primer
sequences, designed to exon-intron junctions, are shown in
Table 1.
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). Cloned cleavage products were sequenced.
Microscopy
In situ sections were photographed with an Olympus 1X81 microscope using
CellR software and whole-meristem images were captured using an
Olympus SZX7 binocular microscope. Scanning electron microscopy was performed
as described (Brand et al.,
2007).
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RESULTS |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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)
(Fig. 1M,N; see Fig. S4F in the
supplementary material). To study the link between the SAM and the marginal
blastozone activities, we identified the affected gene in gob
mutants. gob seedlings are reminiscent of the petunia nam
mutant and the Arabidopsis cuc1 cuc2 double mutant
(Aida et al., 1997;
Souer et al., 1996). A tomato
NAM and CUC homolog, termed GOB, to which the most
similar gene product in Arabidopsis is CUC2 (see Fig. S1 in
the supplementary material), was isolated by RT-PCR using primers
complementary to conserved regions in the NAM gene. GOB
co-segregated with the gob mutation, its mRNA levels were
dramatically reduced in gob-3 seedlings (see Fig. S2A in the
supplementary material) and sequence analysis revealed that all three
gob alleles, which display similar phenotypes, contain lesions in
this gene (Fig. 1B). These
findings indicated that impaired GOB function underlies the
gob phenotype. Similar to its homologs from other species, the
GOB mRNA contains a recognition site for the microRNA
miR164. RNA ligase-mediated (RLM)-RACE analysis confirmed the
presence of miR164-directed GOB cleavage products in tomato
seedlings (see Fig. S3 in the supplementary material). On this basis, we
screened available semi-dominant mutants for phenotypes that might represent
gain-of-function GOB alleles, such as the production of extra organs,
ectopic meristems and altered leaf shape. This led to the identification of
Gob-4d, which contains a point mutation in the miR164
recognition site (Fig. 1B,C)
and features elevated GOB mRNA levels (see Fig. S2B in the
supplementary material).
GOB sets boundaries throughout plant development
To understand how GOB is utilized for boundary specification at the SAM and
during compound-leaf patterning, we compared the effects of GOB loss-
and gain-of-function mutations. In contrast to the fused cotyledons and
terminated SAM observed in gob-3 mutants, homozygous Gob-4d
seedlings initiated more cotyledons (Fig.
1D-G). These effects were already apparent during early embryo
development (Fig. 1H,I).
Heterozygous Gob-4d seedlings showed an intermediate phenotype, with
two to three cotyledons (Fig.
1F), suggesting that GOB affects cotyledon initiation, or
partitioning of the embryo apical domain, in a quantitative manner. Likewise,
Gob-4d flowers had more floral organs per whorl, whereas
gob-3 flowers produced extended and fused organs (see Fig. S4E-I in
the supplementary material). In contrast to the reduced SAM activity in
gob-3 plants, Gob-4d plants showed increased indeterminacy
throughout development. The SAM often split into two or more parallel SAMs
(Fig. 1J,K) and, under certain
environmental conditions, ectopic meristems and leaves were initiated from the
rachis of older leaves (see Fig. S4C in the supplementary material). In
Gob-4d fruits, ectopic carpels were often produced inside the
gynoecium, whereas gob-3 mutants produced fruits with fewer locules
than the wild type (Fig. 1S-U).
Plant architecture was severely altered in Gob-4d plants owing to
variable stem elongation and the tendency of the SAM to split (see Fig. S4A,B
in the supplementary material). However, at the level of leaf initiation,
spiral phyllotaxis was maintained, similar to Arabidopsis transgenic
plants overexpressing a miR164-resistant CUC2 or that are
mutant in all three miR164-coding loci
(Peaucelle et al., 2007;
Sieber et al., 2007).
GOB sets boundaries during compound-leaf patterning
Alterations in GOB activity dramatically affected leaf shape. Leaves of
gob-3 seedlings that recovered following cotyledon removal produced
only primary leaflets with smooth margins, compared with the compound
wild-type tomato leaves that have primary, secondary and intercalary leaflets
and lobed leaflet margins. Moreover, primary gob-3 leaflets were
often fused (Fig. 1A,L-N,Q,R).
Later leaves showed severe fusion of leaflets and petiolules, resulting in a
malformed leaf (see Fig. S4D in the supplementary material). By contrast,
Gob-4d leaflet margins were deeply lobed, and the lobe sinuses were
wider than those of the wild type (Fig.
1O-R). Compared with the wild-type, leaf petioles were shorter in
gob-3 and longer in Gob-4d. Strikingly, the level of leaflet
reiteration was reduced in both loss- and gain-of-function gob
alleles: in both cases there was a reduction in the number of distinct
secondary leaflets (Fig. 1L-R),
but this resulted from different underlying causes and was manifested in very
different mature leaf shapes. Whereas in gob-3 no secondary leaflets
were observed, in Gob-4d initiation events from the primary leaflets
developed into lobes rather than secondary leaflets, which is likely to be due
to their fusion (Fig. 1O-R). As
a result, the gob-3 primary leaflets were flat and simple, whereas
those of Gob-4d were buckled and deeply lobed. Intercalary leaflets
were absent and leaflet petiolules were shorter in both the loss- and
gain-of-function alleles.
GOB expression marks leaflet boundaries in the leaf margin
gob phenotypes suggest that the precise timing and location of GOB
activity might be required for proper formation and separation of lateral
organs and leaflets. We therefore analyzed the spatial distribution of
GOB mRNA by in situ hybridization. Similar to its petunia and
Arabidopsis orthologs, GOB mRNA was expressed in stripes at
the boundaries between the SAM and initiating organs
(Fig. 2A,D). Stripes of
GOB mRNA expression additionally marked the flanks of initiating
primary and secondary leaflets (Fig.
2D,G,J,K). Initially, GOB expression appeared in a single
band at the margin of an early P3 (P, plastochron number) leaf primordium,
prior to visible leaflet initiation (Fig.
2D,J). Following leaflet initiation, two bands could be detected
on each side of the initiating leaflet
(Fig. 2J). The presence of
miR164-directed GOB cleavage products in wild-type plants
and the phenotypes of Gob-4d indicated that miR164 is a
negative regulator of GOB. We examined the spatial distribution of
miR164 in wild-type tomato plants. miR164 expression was
observed at the flanks of the SAM and in young leaf primordia, but was
downregulated at the boundary between the initiating leaves and the SAM
(Fig. 2C,F). Strong
miR164 expression appeared in leaflets shortly after their
initiation, and its expression was downregulated between initiating leaflets
(Fig. 2F,I,L). Thus, the
miR164 and GOB expression domains are largely
complementary.
To further understand GOB expression and function, we examined how
the Gob-4d mutation affects GOB spatial expression. In
Gob-4d seedlings, GOB expression marked the SAM leaf and the
leaflet boundaries as in the wild type, but was also expanded to include part
of the SAM peripheral zone and the leaf base
(Fig. 2B,E). Within the
developing leaf, the edges of the GOB expression domain were more
diffuse than in the wild type (Fig.
2E,H). In addition, GOB levels appeared higher in
Gob-4d, and patches of ectopic expression were occasionally observed.
These results imply that GOB expression is controlled both
transcriptionally and post-transcriptionally, and that miR164
spatially and quantitatively sharpens and tunes the GOB expression
domain. This is compatible with the emerging picture of the combined
quantitative effects of transcriptional control and miR164 action on
the spatial and temporal activity of the Arabidopsis CUC1 and
CUC2 genes (Baker et al.,
2005; Nikovics et al.,
2006; Sieber et al.,
2007). The importance of accurate expression of GOB is
revealed by the dramatic phenotypic changes that are caused by the expansion
and blurring of the GOB expression domain in Gob-4d.
The combined observed effects of the Gob-4d mutation on GOB expression and leaf development imply that proper leaflet separation requires a sharp boundary between high GOB expression and no GOB expression in adjacent leaf marginal regions. gob-3 and Gob-4d both impair the sharpness of this boundary, and this might underlie their common effects in simplifying the leaf.
miR164 overexpression eliminates secondary leaflets and lobes
The complementary expression of GOB and miR164, and the
effect of Gob-4d on GOB expression and leaf structure,
suggested that GOB is negatively regulated by miR164. In
agreement with this, strong constitutive overexpression of an Arabidopsis
miR164 precursor in tomato resulted in a gob-like phenotype
(Alvarez et al., 2006). The
simpler leaf phenotype of gob-3 could be an indirect consequence of
the dramatic SAM function defect or might represent a separate role for GOB in
compound-leaf patterning. To distinguish between these possibilities, and to
test the combined role of miR164 targets in leaf development, we
expressed an Arabidopsis miR164 precursor via the FIL
promoter, which directs expression specifically in lateral organs and is not
expressed in the SAM (Lifschitz et al.,
2006; Ori et al.,
2007). As expected, FIL>>miR164 leaves showed expanded
miR164 expression and a dramatic reduction of GOB expression
in developing leaves, but not in the SAM
(Fig. 3A-D; see Fig. S2B in the
supplementary material). FIL>>miR164 transgenic plants had normal
SAM function (not shown) but simpler leaves
(Fig. 3E,F) that lacked
secondary leaflets and had smooth margins, similar to gob-3 leaves.
Thus, GOB expression, either before or during leaflet initiation, is necessary
for the development of secondary leaflets.
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). These
results imply that balancing indeterminate and determinate fates requires
restriction of GOB function.
GOB affects the rate of leaf maturation, leaflet elaboration and secondary-leaflet initiation
A final leaf shape can result from different early events
(Champagne and Sinha, 2004).
We followed early leaf development in backgrounds with altered GOB activity to
further understand the role of GOB in the ontogeny of the tomato compound leaf
(Fig. 5). In the wild-type
tomato leaf, primary leaflets were initiated at the P3 stage from the marginal
blastozone, which was characterized by distinct meristem-like cell morphology
and the lack of trichomes. In this manner, leaflet initiation from the leaf
margin resembled leaf initiation from the flanks of the SAM
(Fig. 5A,D,G). At the P5 stage,
secondary leaflets were initiated from the marginal blastozone of the primary
leaflet, and, slightly later, intercalary leaflets were initiated along the
rachis (Fig. 5J,K,Q). The
initiation of primary leaflets appeared normal in FIL>>miR164
leaves; however, no initiation of secondary leaflets and intercalary leaflets
was observed (Fig.
5C,F,I,N,O,R). Moreover, basal primary leaflets arose at an
earlier developmental stage than in the wild type. Early leaf development in
recovered gob-3 mutants was similar to that of
FIL>>miR164, although an accurate assignment of a developmental
stage to these mutants was impossible owing to the abnormal leaf initiation
(see Fig. S7F,I in the supplementary material). The marginal blastozone of
primary FIL>>miR164 and gob-3 leaflets was visible, but
narrower than that of the wild type. Developing
FIL>>GOBm leaf primordia failed to properly expand at
their distal end. Overall, these leaves showed prolonged blastozone activity
and appeared younger than wild-type leaves of the same developmental stage, as
judged by delayed trichome formation and chlorophyll accumulation
(Fig. 5B,E,H,L,M). In addition,
FIL>>GOBm failed to retain proper spatial and temporal
spacing between initiation events. At the P3 stage, these primordia were
shorter than those of the wild type, and had already initiated three or more
primary leaflets, in comparison to a single one in the wild type
(Fig. 5D,E). Soon after their
initiation, primary FIL>>GOBm leaflets initiated
numerous secondary outgrowths, which in turn precociously initiated
additional, tertiary outgrowths (Fig.
5H,L,M). All these structures remained connected and developed
into lobes rather than distinct leaflets. Thus, the final
FIL>>GOBm leaf shape is a combination of inhibition of
leaf differentiation, ectopic or precocious lateral initiation events and
improper spatial and temporal spacing between them. Young Gob-4d
leaves initiated fewer primary leaflets than the wild type, in agreement with
their final leaf shape (see Fig. S7A-E in the supplementary material).
Initiating primary leaflets were shorter and wider than in the wild type, and
precocious initiation events from the margins of their terminal leaflet were
observed, possibly reflecting the more diffuse GOB expression. Secondary
outgrowths were clearly initiated from the primary leaflet margin (see Fig.
S7G,H in the supplementary material).
These results demonstrate that precise regulation of spatial and temporal GOB activity affects the progression of leaf maturation, the location and timing of leaflet initiation sites and leaflet separation. The elaboration of higher order initiation events into distinct leaflets appears to require a sharp boundary between adjacent regions that feature high versus no GOB expression.
Conserved role for miR164 in leaf elaboration
To investigate the effect of overexpressing the GOB regulator
miR164 on leaflet formation in a different species with complex
leaves, we ectopically overexpressed miR164 in Cardamine
hirsuta plants. C. hirsuta leaves become gradually divided with
age, owing to significant heteroblasty in primary leaflet number and shape
(Fig. 6A). 35S::AtmiR164b
C. hirsuta transformants exhibited extensive fusion phenotypes, which are
characteristic of equivalent transformants in Arabidopsis
(Laufs et al., 2004),
including cotyledon fusion and a loss of the embryonic apical meristem in
strong lines, and fusion between leaves, leaflets and same-whorl floral organs
in less severe lines (see Fig. S8 in the supplementary material). As in
tomato, AtmiR164 overexpression resulted in extensive leaflet fusion
and the smoothing of serrated margins, a phenotype that was particularly
prominent in cauline leaves (Fig.
6B,C). Unlike in tomato leaves, a primary effect of
miR164 overexpression on C. hirsuta leaves was a reduction
in the number of primary leaflets (Fig.
6).
|
). In
agreement, mutations in the tomato putative auxin-response repressor
ENTIRE (E) (also known as SlIAA9) were shown to
cause a severe reduction in leaflet separation
(Wang et al., 2005;
Zhang et al., 2007). Several
new e alleles have been identified in a screen of a mutant population
generated in the same genetic background as gob-1 to -4
(Menda et al., 2004)
(http://zamir.sgn.cornell.edu/mutants).
Of these, we identified the mutation in two alleles of the E gene
(Fig. 7A), and confirmed
allelism with E for two additional alleles. Leaves of e and
recovered gob mutants show a striking resemblance (compare
Fig. 1M with
Fig. 7B). Moreover, in
Arabidopsis, auxin has been proposed to negatively regulate
CUC2, possibly via positive regulation of miR164
(Furutani et al., 2004;
Guo et al., 2005;
Heisler et al., 2005;
Vernoux et al., 2000). To test
whether this is the basis for the phenotypic similarity between gob-3
and e, we examined the genetic and molecular interaction between
e and gob. If the e leaf phenotype results from
upregulation of miR164, the Gob-4d phenotype is expected to
be epistatic to the e phenotype. However, leaves of e Gob-4d
double mutants appeared very similar to those of e, although some
features of the Gob-4d phenotype were evident
(Fig. 7B-D). In agreement, in
situ hybridization revealed no obvious change in GOB expression in
e leaves (Fig. 7G-I),
arguing against E as a positive regulator of GOB expression. Finally,
FIL>>miR164 or a reduction in GOB copy number (in e
gob-3/+) enhanced the e phenotype
(Fig. 7E,F). These results
suggest that E and GOB might act through partially independent pathways,
However, as no mutation was identified in the E coding region in
e-2 mutants, the possibility remains that it represents a weak allele
that is enhanced by gob-3/+, in which case both genes might act in
the same pathway. The relationship of both regulators with auxin-mediated leaf
elaboration warrants more detailed examination. |
DISCUSSION |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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). The
absence of any secondary-leaflet initiation in gob-3 and
FIL>>miR164 leaves in spite of the maintenance of an
undifferentiated marginal blastozone (Fig.
5R; see Fig. S7I in the supplementary material) might indicate
that these are distinct effects.
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|
Context-dependent effect of GOB on leaf shape
The terminal, distal-most leaflet of the compound tomato leaf is initiated
directly from the flanks of the SAM. As such, it is largely equivalent to a
simple leaf. Initiation of the terminal leaflet is largely insensitive to GOB
expression, but GOB is required for elaboration of its marginal serrations and
to prevent its fusion with the two lateral primary leaflets formed
subsequently (Fig. 1M,N).
Similarly, many simple lateral organs in the tomato, such as the cotyledons or
sepals, require GOB to inhibit their congenital fusion with primordia in the
same whorl. The requirement for GOB in leaflet separation is reduced in the
context of the other primary tomato leaflets
(Fig. 8B). The response of
leaflets of different order to GOB further emphasizes the distinct nature of
the different leaf units: whereas the effect of impaired GOB-like function on
the initiation of primary leaflets is relatively mild, it completely abolishes
the initiation of secondary and higher order leaflets. Finally, GOB is
essential in all leaflets for the elaboration of lamina margins. GOB activity
thus helps to define the unique properties of the different leaf units, and
illustrates how individual leaflets are independently regulated within a
compound leaf.
The context-dependent role of GOB in leaf patterning is further exemplified
by comparing the effects of altered NAM-like activity in species with
different leaf shapes. Such a comparison might hint at the relationship
between these different leaf forms. The role of CUC2 in the formation of leaf
serrations in Arabidopsis
(Nikovics et al., 2006)
resembles the effect of GOB on leaflet margins, suggesting that leaflets and
simple leaves are at least partially equivalent structures. C.
hirsuta leaves lack higher order leaflets, and the formation of its
primary leaflets was more susceptible to miR164 overexpression than
in tomato, suggesting that primary leaflets in these two species represent
partially distinct structures. Together, these observations suggest that GOB
is utilized in leaf patterning in a context-dependent manner. The degree of
leaf elaboration appears to be determined by both GOB-dependent and
GOB-independent mechanisms. The potentially redundant involvement of other
NAM/CUC transcription factors, such as the orthologs of Arabidopsis
CUC3 (Aida et al., 1997;
Hibara et al., 2006;
Vroemen et al., 2003), in
compound-leaf patterning remains to be determined.
|
; Nikovics et al.,
2006), the punctuate GOB expression pattern and its role in
boundary specification (Fig.
8), it is somewhat surprising that relatively uniform GOB
expression in developing leaves did not inhibit leaflet initiation altogether.
This implies that GOB acts in the context of additional factors that function
in the laminar GOB-less region to specify proper leaflet initiation and
growth. Such factors might include growth and differentiation factors such as
LANCEOLATE or the tomato orthologs of genes such as FILAMENTOUS FLOWER, as
well as auxin. Auxin has been implicated in the development of serrations,
lobes and leaflets in different species
(Avasarala et al., 1996;
Barkoulas et al., 2008;
Hay et al., 2006;
Wang et al., 2005;
Zhang et al., 2007). The
enhancement of FIL>>miR164 by e might imply that GOB and
auxin interact in leaflet positioning.
Similarities and differences between leaf and leaflet initiation
Morphological and genetic evidence point to striking similarities between
leaf initiation from the flanks of the SAM and leaflet initiation from the
leaf margin (Barkoulas et al.,
2008; Brand et al.,
2007; Hagemann and Gleissberg,
1996; Mathan and Jenkins,
1962; Ori et al.,
2007; Sachs,
1969). Here we show that NAM-like genes, shown to be
involved in boundary specification during lateral organ formation from the
SAM, are also utilized for boundary specification at the site of leaflet
initiation. However, leaflet initiation is also very different from leaf
initiation, both morphologically and in molecular terms. Whereas the SAM
continues to form many more leaves, the marginal blastozone forms a finite
number of leaflets. In addition, whereas in the SAM GOB expression
marks the boundary between the adaxial side of the leaf and the SAM, during
leaflet initiation it first marks the distal lateral domain of the leaflet
(Fig. 2). This implies a
conserved but flexible role for GOB in boundary specification.
Our results demonstrate how plants adapt a common boundary-specification program to modulate different developmental processes in a context-specific manner. It remains to be seen how the boundary specification by GOB interacts with additional factors in the context of compound-leaf patterning.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/136/5/823/DC1
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Footnotes |
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* These authors contributed equally to this work 
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REFERENCES |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
|---|
Aida, M. and Tasaka, M. (2006). Genetic control
of shoot organ boundaries. Curr. Opin. Plant Biol.
9, 72-77.[CrossRef][Medline]
Aida, M., Ishida, T., Fukaki, H., Fujisawa, H. and Tasaka,
M. (1997). Genes involved in organ separation in Arabidopsis:
an analysis of the cup-shaped cotyledon mutant. Plant
Cell 9,841
-857.
Alvarez, J. P., Pekker, I., Goldshmidt, A., Blum, E., Amsellem,
Z. and Eshed, Y. (2006). Endogenous and synthetic microRNAs
stimulate simultaneous, efficient, and localized regulation of multiple
targets in diverse species. Plant Cell
18,1134
-1151.
Arazi, T., Talmor-Neiman, M., Stav, R., Riese, M., Huijser, P.
and Baulcombe, D. C. (2005). Cloning and characterization of
micro-RNAs from moss. Plant J.
43,837
-848.[CrossRef][Medline]
Avasarala, S., Yang, J. and Caruso, J. L.
(1996). Production of phenocopies of the lanceolate mutant in
tomato using polar auxin transport inhibitors. J. Exp.
Bot. 47,709
-712.
Baker, C. C., Sieber, P., Wellmer, F. and Meyerowitz, E. M.
(2005). The early extra petals1 mutant uncovers a role for
MicroRNA miR164c in regulating petal number in arabidopsis. Curr.
Biol. 15,303
-315.[CrossRef][Medline]
Barkoulas, M., Hay, A., Kougioumoutzi, E. and Tsiantis, M.
(2008). A developmental framework for dissected leaf formation in
the Arabidopsis relative Cardamine hirsuta. Nat.
Genet. 40,1136
-1141.[CrossRef][Medline]
Barkoulas, M., Galinha, C., Grigg, S. P. and Tsiantis, M.
(2007). From genes to shape: regulatory interactions in leaf
development. Curr. Opin. Plant Biol.
10,660
-666.[CrossRef][Medline]
Bassel, G. W., Mullen, R. T. and Bewley, J. D.
(2008). Procera is a putative DELLA mutant in tomato (Solanum
lycopersicum): effects on the seed and vegetative plant. J. Exp.
Bot. 59,585
-593.
Bharathan, G., Goliber, T. E., Moore, C., Kessler, S., Pham, T.
and Sinha, N. R. (2002). Homologies in leaf form inferred
from KNOXI gene expression during development. Science
296,1858
-1860.
Brand, A., Shirding, N., Shleizer, S. and Ori, N.
(2007). Meristem maintenance and compound-leaf patterning utilize
common genetic mechanisms in tomato. Planta
226,941
-951.[CrossRef][Medline]
Caruso, J. L. (1968). Morphogenetic aspects of
a leafless mutant in tomato I. General patterns in development. Am.
J. Bot. 55,1169
-1176.[CrossRef]
Champagne, C. and Sinha, N. (2004). Compound
leaves: equal to the sum of their parts? Development
131,4401
-4412.
Dengler, N. G. (1984). Comparison of leaf
development in Normal (+/+), Entire (E/E), and Lanceolate (La/+) plants of
tomato, Lycopersicon esculentum Ailsa Craig. Bot.
Gaz. 145,66
-77.
Dengler, N. G. and Tsukaya, H. (2001). Leaf
morphogenesis in dicotyledons: current issues. Int. J. Plant
Sci. 162,459
-464.[CrossRef]
Efroni, I., Blum, E., Goldshmidt, A. and Eshed, Y.
(2008). A protracted and dynamic maturation schedule underlies
arabidopsis leaf development. Plant Cell
20,2293
-2306.
Furutani, M., Vernoux, T., Traas, J., Kato, T., Tasaka, M. and
Aida, M. (2004). PIN-FORMED1 and PINOID regulate boundary
formation and cotyledon development in Arabidopsis embryogenesis.
Development 131,5021
-5030.
Goliber, T., Kessler, S., Chen, J. J., Bharathan, G. and Sinha,
N. (1999). Genetic, molecular, and morphological analysis of
compound leaf development. Curr. Top. Dev. Biol.
43,259
-290.[Medline]
Guo, H. S., Xie, Q., Fei, J. F. and Chua, N. H.
(2005). MicroRNA directs mRNA cleavage of the transcription
factor NAC1 to downregulate auxin signals for arabidopsis lateral root
development. Plant Cell
17,1376
-1386.
Hagemann, W. and Gleissberg, S. (1996).
Organogenetic capacity of leaves: the significance of marginal blastozones in
angiosperms. Plant Syst. Evol.
199,121
-152.[CrossRef]
Hake, S., Smith, H. M., Holtan, H., Magnani, E., Mele, G. and
Ramirez, J. (2004). The role of knox genes in plant
development. Annu. Rev. Cell Dev. Biol.
20,125
-151.[CrossRef][Medline]
Hareven, D., Gutfinger, T., Parnis, A., Eshed, Y. and Lifschitz,
E. (1996). The making of a compound leaf: genetic
manipulation of leaf architecture in tomato. Cell
84,735
-744.[CrossRef][Medline]
Hay, A. and Tsiantis, M. (2006). The genetic
basis for differences in leaf form between Arabidopsis thaliana and
its wild relative Cardamine hirsuta. Nat.
Genet. 38,942
-947.[CrossRef][Medline]
Hay, A., Kaur, H., Phillips, A., Hedden, P., Hake, S. and
Tsiantis, M. (2002). The gibberellin pathway mediates
KNOTTED1-type homeobox function in plants with different body plans.
Curr. Biol. 12,1557
-1565.[CrossRef][Medline]
Hay, A., Barkoulas, M. and Tsiantis, M. (2006).
ASYMMETRIC LEAVES1 and auxin activities converge to repress BREVIPEDICELLUS
expression and promote leaf development in Arabidopsis.
Development 133,3955
-3961.
Heisler, M. G., Ohno, C., Das, P., Sieber, P., Reddy, G. V.,
Long, J. A. and Meyerowitz, E. M. (2005). Patterns of auxin
transport and gene expression during primordium development revealed by live
imaging of the Arabidopsis inflorescence meristem. Curr.
Biol. 15,1899
-1911.[CrossRef][Medline]
Hejatko, J., Blilou, I., Brewer, P. B., Friml, J., Scheres, B.
and Benkova, E. (2006). In situ hybridization technique for
mRNA detection in whole mount Arabidopsis samples. Nat.
Protoc. 1,1939
-1946.[CrossRef][Medline]
Hibara, K., Karim, M. R., Takada, S., Taoka, K., Furutani, M.,
Aida, M. and Tasaka, M. (2006). Arabidopsis CUP-SHAPED
COTYLEDON3 regulates postembryonic shoot meristem and organ boundary
formation. Plant Cell
18,2946
-2957.
Hofer, J., Turner, L., Hellens, R., Ambrose, M., Matthews, P.,
Michael, A. and Ellis, N. (1997). UNIFOLIATA regulates leaf
and flower morphogenesis in pea. Curr. Biol.
7, 581-587.[CrossRef][Medline]
Jasinski, S., Tattersall, A., Piazza, P., Hay, A.,
Martinez-Garcia, J. F., Schmitz, G., Theres, K., McCormick, S. and Tsiantis,
M. (2008). PROCERA encodes a DELLA protein that mediates
control of dissected leaf form in tomato. Plant J.
56,603
-612.[CrossRef][Medline]
Kaplan, D. R. (2001). Fundamental concepts of
leaf morphology and morphogenesis: a contribution to the interpretation of
molecular genetic mutants. Int. J. Plant Sci.
162,465
-474.[CrossRef]
Laufs, P., Peaucelle, A., Morin, H. and Traas, J.
(2004). MicroRNA regulation of the CUC genes is required for
boundary size control in Arabidopsis meristems.
Development 131,4311
-4322.
Lifschitz, E., Eviatar, T., Rozman, A., Shalit, A., Goldshmidt,
A., Amsellem, Z., Alvarez, J. P. and Eshed, Y. (2006). The
tomato FT ortholog triggers systemic signals that regulate growth and
flowering and substitute for diverse environmental stimuli. Proc.
Natl. Acad. Sci. USA 103,6398
-6403.
Mallory, A. C., Dugas, D. V., Bartel, D. P. and Bartel, B.
(2004). MicroRNA regulation of NAC-domain targets is required for
proper formation and separation of adjacent embryonic, vegetative, and floral
organs. Curr. Biol. 14,1035
-1046.[CrossRef][Medline]
Mathan, D. S. and Jenkins, J. A. (1962). A
morphogenetic study of Lanceolate, a leaf shape mutant in the tomato.
Am. J. Bot. 49,504
-514.[CrossRef]
McCormick, S. (1991). Transformation of tomato
with Agrobacterium tumifaciens. In Plant Tissue Culture
Manual, vol. B6 (ed. K. Lindsey), pp.1
-9. Dordrecht, The Netherlands: Kluwer Academic
Publishers.
Menda, N., Semel, Y., Peled, D., Eshed, Y. and Zamir, D.
(2004). In silico screening of a saturated mutation library of
tomato. Plant J. 38,861
-872.[CrossRef][Medline]
Nikovics, K., Blein, T., Peaucelle, A., Ishida, T., Morin, H.,
Aida, M. and Laufs, P. (2006). The balance between the
MIR164A and CUC2 genes controls leaf margin serration in Arabidopsis.
Plant Cell 18,2929
-2945.
Ori, N., Cohen, A. R., Etzioni, A., Brand, A., Yanai, O.,
Shleizer, S., Menda, N., Amsellem, Z., Efroni, I., Pekker, I. et al.
(2007). Regulation of LANCEOLATE by miR319 is required for
compound-leaf development in tomato. Nat. Genet.
39,787
-791.[CrossRef][Medline]
Parnis, A., Cohen, O., Gutfinger, T., Hareven, D., Zamir, D. and
Lifschitz, E. (1997). The dominant developmental mutants of
tomato, Mouse-ear and Curl, are associated with distinct modes of abnormal
transcriptional regulation of a Knotted gene. Plant
Cell 9,2143
-2158.[Abstract]
Peaucelle, A., Morin, H., Traas, J. and Laufs, P.
(2007). Plants expressing a miR164-resistant CUC2 gene reveal the
importance of post-meristematic maintenance of phyllotaxy in Arabidopsis.
Development 134,1045
-1050.
Pekker, I., Alvarez, J. P. and Eshed, Y.
(2005). Auxin response factors mediate Arabidopsis organ
asymmetry via modulation of KANADI activity. Plant
Cell 17,2899
-2910.
Pilcher, R. L., Moxon, S., Pakseresht, N., Moulton, V., Manning,
K., Seymour, G. and Dalmay, T. (2007). Identification of
novel small RNAs in tomato (Solanum lycopersicum).
Planta 226,709
-717.[CrossRef][Medline]
Poethig, R. S. (1997). Leaf morphogenesis in
flowering plants. Plant Cell
9,1077
-1087.[CrossRef][Medline]
Raman, S., Greb, T., Peaucelle, A., Blein, T., Laufs, P. and
Theres, K. (2008). Interplay of miR164, CUP-SHAPED COTYLEDON
genes and LATERAL SUPPRESSOR controls axillary meristem formation in
Arabidopsis thaliana. Plant J.
55, 65-76.[CrossRef][Medline]
Reinhardt, B., Hanggi, E., Muller, S., Bauch, M., Wyrzykowska,
J., Kerstetter, R., Poethig, S. and Fleming, A. J. (2007).
Restoration of DWF4 expression to the leaf margin of a dwf4 mutant is
sufficient to restore leaf shape but not size: the role of the margin in leaf
development. Plant J.
52,1094
-1104.[Medline]
Sablowski, R. (2007). The dynamic plant stem
cell niches. Curr. Opin. Plant Biol.
10,639
-644.[CrossRef][Medline]
Sachs, T. (1969). Regeneration experiments on
the determination of the form of leaves. Israel J.
Botany 18,21
-30.
Sieber, P., Wellmer, F., Gheyselinck, J., Riechmann, J. L. and
Meyerowitz, E. M. (2007). Redundancy and specialization among
plant microRNAs: role of the MIR164 family in developmental robustness.
Development 134,1051
-1060.
Souer, E., van Houwelingen, A., Kloos, D., Mol, J. and Koes,
R. (1996). The no apical meristem gene of Petunia is required
for pattern formation in embryos and flowers and is expressed at meristem and
primordia boundaries. Cell
85,159
-170.[CrossRef][Medline]
Takada, S., Hibara, K., Ishida, T. and Tasaka, M.
(2001). The CUP-SHAPED COTYLEDON1 gene of Arabidopsis regulates
shoot apical meristem formation. Development
128,1127
-1135.[Abstract]
Vernoux, T., Kronenberger, J., Grandjean, O., Laufs, P. and
Traas, J. (2000). PIN-FORMED 1 regulates cell fate at the
periphery of the shoot apical meristem. Development
127,5157
-5165.[Abstract]
Vroemen, C. W., Mordhorst, A. P., Albrecht, C., Kwaaitaal, M. A.
and De Vries, S. C. (2003). The CUP-SHAPED COTYLEDON3 gene is
required for boundary and shoot meristem formation in Arabidopsis.
Plant Cell 15,1563
-1577.
Wang, H., Jones, B., Li, Z., Frasse, P., Delalande, C., Regad,
F., Chaabouni, S., Latche, A., Pech, J. C. and Bouzayen, M.
(2005). The tomato Aux/IAA transcription factor IAA9 is involved
in fruit development and leaf morphogenesis. Plant
Cell 17,2676
-2692.
Wang, H., Chen, J., Wen, J., Tadege, M., Li, G., Liu, Y.,
Mysore, K. S., Ratet, P. and Chen, R. (2008). Control of
compound leaf development by FLORICAULA/LEAFY ortholog SINGLE LEAFLET1 in
Medicago truncatula. Plant Physiol.
146,1759
-1772.
Weir, I., Lu, J. P., Cook, H., Causier, B., Schwarz-Sommer, Z.
and Davies, B. (2004). CUPULIFORMIS establishes lateral organ
boundaries in Antirrhinum. Development
131,915
-922.
Zhang, J., Chen, R., Xiao, J., Qian, C., Wang, T., Li, H.,
Ouyang, B. and Ye, Z. (2007). A single-base deletion mutation
in SlIAA9 gene causes tomato (Solanum lycopersicum) entire mutant.
J. Plant Res. 120,671
-678.[CrossRef][Medline]
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