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Shoot Branching and Leaf Dissection in Tomato Are Regulated
by Homologous Gene Modules
W
Bernhard L. Busch,a,1 Gregor Schmitz,a,1 Susanne Rossmann,a Florence Piron,b Jia Ding,a
Abdelhafid Bendahmane,b and Klaus Theresa,2
a Max
Planck Institute for Plant Breeding Research, 50829 Cologne, Germany
de Recherche en Génomique Végétale, Unité Mixte de Recherche, Institut National de la Recherche Agronomique–
Centre National de la Recherche Scientifique, 91057 Evry cedex, France
b Unité
Aerial plant architecture is predominantly determined by shoot branching and leaf morphology, which are governed by
apparently unrelated developmental processes, axillary meristem formation, and leaf dissection. Here, we show that in
tomato (Solanum lycopersicum), these processes share essential functions in boundary establishment. Potato leaf (C), a key
regulator of leaf dissection, was identified to be the closest paralog of the shoot branching regulator Blind (Bl). Comparative
genomics revealed that these two R2R3 MYB genes are orthologs of the Arabidopsis thaliana branching regulator
REGULATOR OF AXILLARY MERISTEMS1 (RAX1). Expression studies and complementation analyses indicate that these
genes have undergone sub- or neofunctionalization due to promoter differentiation. C acts in a pathway independent of
other identified leaf dissection regulators. Furthermore, the known leaf complexity regulator Goblet (Gob) is crucial for
axillary meristem initiation and acts in parallel to C and Bl. Finally, RNA in situ hybridization revealed that the branching
regulator Lateral suppressor (Ls) is also expressed in leaves. All four boundary genes, C, Bl, Gob, and Ls, may act by
suppressing growth, as indicated by gain-of-function plants. Thus, leaf architecture and shoot architecture rely on a
conserved mechanism of boundary formation preceding the initiation of leaflets and axillary meristems.
INTRODUCTION
Shoot architecture of seed plants is characterized by multiple
repetitions of a basic module called the phytomer, which consists of an internode, a leaf, and an axillary meristem. Studies on
leaf evolution suggest that seed plant leaves evolved independent of other megaphylls from branched undifferentiated axes
(Stewart and Rothwell, 1993; Rothwell, 1999; Tomescu, 2009). At
about the same time in evolution, axillary branches appeared
(Galtier and Holmes, 1982; Galtier, 1999; Meyer-Berthaud et al.,
2000), providing the basis for the enormous plasticity observed in
seed plant shoot architecture (Galtier, 1999).
Leaves are initiated in a regular pattern from cells at the flank of
the shoot apical meristem (SAM). Recent studies have shown
that seed plants use similar mechanisms to regulate leaf primordium initiation at the SAM and to elaborate leaf form, especially in
plants with complex leaves (Blein et al., 2008; Rosin and Kramer,
2009). Leaf initiation at the SAM depends on regulated auxin flux
creating auxin maxima at the sites of primordium formation
(Reinhardt et al., 2003). Whereas loss of polar auxin transport
results in an arrest of primordium formation (Vernoux et al., 2000),
ectopic auxin application causes ectopic leaf development
1 These
authors contributed equally to this work.
correspondence to [email protected].
The author responsible for distribution of materials integral to the
findings presented in this article in accordance with the policy described
in the Instructions for Authors (www.plantcell.org) is: Klaus Theres
([email protected]).
W
Online version contains Web-only data.
www.plantcell.org/cgi/doi/10.1105/tpc.111.087981
2 Address
(Reinhardt et al., 2000). Similarly, the outgrowth of leaflets
requires the formation of auxin maxima along the rachis and
subsequent activation of auxin signaling (Barkoulas et al., 2008;
Koenig et al., 2009). In tomato (Solanum lycopersicum) compound leaf development, auxin maxima are found at positions
where leaflets initiate (Koenig et al., 2009), whereas auxin signaling needs to be repressed at positions where the rachis, a part
of the leaf axis without leaf blade, will form.
Class I KNOX gene activity is needed to maintain the SAM as a
population of pluripotent cells (Hay and Tsiantis, 2010). Furthermore, KNOX expression during leaf development is causally linked
to the elaboration of different leaf forms: In simple leaves, KNOX
genes are kept in a repressed state, but in many species with
complex leaf development, KNOX gene expression is reactivated
in a patterned fashion (Hay and Tsiantis, 2010). Loss of AUXIN/
INDOLE-3-ACETIC ACID gene activity, as in the tomato entire (e)
mutant (Zhang et al., 2007; Berger et al., 2009), leads to ectopic
activation of auxin responses throughout the tomato leaf margins.
As a consequence, ectopic leaf blade develops along the rachis
and leaflets are fused. Ectopic lamina growth on the rachis can also
be induced by auxin application to young leaf primordia (Koenig
et al., 2009). Expression of KNOX genes in ectopic positions, as in
the mutant Mouse ears (Me; Parnis et al., 1997) or when using
transgenic approaches (Hareven et al., 1996), results in highly
complex leaves with hundreds of leaflets. However, KNOX gene
expression can trigger leaflet formation only when it is induced at
the right developmental time (Shani et al., 2009).
NAC transcription factors of the CUC/NAM subfamily play an
important role in SAM function and organ separation. During leaf
development, CUC genes are expressed at positions where leaf
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blade indentations and leaflets will form (Blein et al., 2008; Berger
et al., 2009; Hasson et al., 2011). Expression of CUC genes
is detectable before indentations or leaflets are initiated at the
leaf margin, where CUC activity is needed to repress growth.
A mutation in the tomato Goblet (Gob) gene, the homolog of
Arabidopsis thaliana CUP-SHAPED COTYLEDON1 (CUC1)/
CUC2, results in leaves with a reduced number of leaflets
showing smooth margins and downregulation of KNOX gene
transcription (Blein et al., 2008; Berger et al., 2009).
After germination, new meristems initiate in the axils of leaves;
these develop into buds and finally into side-shoots, establishing
new axes of growth. In tomato, as well as in other species,
axillary meristem (AM) formation requires Lateral suppressor (Ls)
(Schumacher et al., 1999; Greb et al., 2003; Li et al., 2003), which
is expressed in the boundary region between the leaf primordium
and the stem. Tomato blind mutants are compromised in AM
formation during vegetative and reproductive development
(Schmitz et al., 2002). Blind and the homologous Arabidopsis
RAX genes (Keller et al., 2006; Müller et al., 2006) encode R2R3
MYB transcription factors that regulate AM formation in overlapping zones along the shoot axis. In Arabidopsis, recent
experiments have demonstrated that CUC1, CUC2, and CUC3
also play a role in AM establishment (Hibara et al., 2006; Raman
et al., 2008). CUC expression in the boundary regions of both leaf
and leaflet primordia indicates a functional similarity between the
two zones.
The genomes of most seed plants show signs of ancient
genome duplications. Most or all eudicots are derived from a
hexaploid ancestor (Van de Peer et al., 2009). During subsequent
evolution, many groups of dicot plants underwent whole-genome
duplications (WGD); for example, the Arabidopsis genome shows
signs of two rounds of WGD and tomato shows evidence for one
round of WGD. WGDs were followed by massive loss of duplicated
genes and by chromosome rearrangements, which are at least
partially connected to a reduction in chromosome number. Despite
these changes in chromosome organization, conservation of gene
arrangement can be detected over large evolutionary distances
and helps to identify evolutionary conserved functions.
In this study, we elucidate the functions of members of the
Blind/RAX gene families in tomato. We show that the branching
regulator Blind and the regulator of complex leaf development
Potato leaf (C) are coorthologs of Arabidopsis RAX1 and that
they differ in their expression patterns. We connect side-shoot
development to complex leaf development by demonstrating
that the regulation by GOB and two homologous MYB proteins
is essential in both processes. These results are discussed in
the light of the current view of leaf and shoot evolution in seed
plants.
RESULTS
Blind-like2 and Blind Are Coorthologs of RAX1
To find additional factors regulating tomato shoot architecture, we
used database searches to identify eight tomato homologs of the
R2R3 MYB transcription factor Blind (Bl). We named these homologs Blind-like1 (Bli1) to Bli8. In addition to the nine genes in
tomato, we found six homologs in Arabidopsis (Müller et al., 2006)
and seven in grape (Vitis vinifera). As shown by phylogenetic
analysis, this group is clearly separate from other R2R3 repeat
MYB genes (Stracke et al., 2001; Wilkins et al., 2009). Alignment of
the amino acid sequences of these proteins showed that the
N-terminal part containing the two MYB repeats is highly conserved (see Supplemental Figure 1 online), but sequence similarity
in the C-terminal domains can be detected only between specific
pairs of sequences (Bl to Bli2 and Bli6, RAX3 to MYB68, and
MYB36 to Bli8) and is not significant between other pairs of genes.
Bli2 and Bli6 have the highest degree of sequence similarity to
each other and are separated by only 6373 bp in the genome,
indicating that they originated by tandem duplication. However,
the presence of at least five substitutions of highly conserved
amino acid residues in the MYB domain indicates that Bli6 is most
probably a pseudogene (see Supplemental Figure 1 online). This
conclusion is corroborated by the phenotype of a mutant carrying
a deletion of both Bli2 and Bli6 (c-2; see below), which is indistinguishable from that of other Bli2 loss-of-function mutants.
Phylogenetic analysis of either the full-length amino acid sequences or the MYB domains alone did not show any reliable
topology beyond the above described pairs, and bootstrap values
for most of the nodes were low. A one-to-one correlation between
tomato and Arabidopsis was detected for only one pair of genes
(MYB36 and Bli8). Another method to elucidate the relationships
among genes employs our knowledge of the evolutionary history
of both plant species. Most eudicots originated from an ancient
hexaploid progenitor (Van de Peer et al., 2009). During subsequent
evolution, the Brassicaceae underwent two rounds of WGD
(Simillion et al., 2002), tomato had one WGD (Fawcett et al.,
2009) and some species (e. g., grape) had no additional WGD, so
their genomes still represent the ancient hexaploid state (Figure
1C). Following each WGD, chromosomal rearrangements, loss
of gene copies, local gene amplification, and other events led
to differentiation of the duplicated chromosomes. To elucidate
orthology relationships, we determined collinearity of the genomic
regions spanning the nine tomato and the six Arabidopsis Bli
genes to the grape genome. This was done by running BLASTP
(Altschul et al., 1990) comparisons of the Bli gene–containing
tomato and Arabidopsis chromosome segments to grape proteins
and selecting the top five hits for each protein (see Supplemental
Data Set 1 online). Hits between all chromosome pairs were
plotted, and collinear regions were visually detected as diagonals
(Figure 1A; see Supplemental Figure 2 online). In each case, a
single grape genomic region clearly had the highest density of
conserved gene hits, and in most cases, two additional regions
with lower conservation could be identified (Figure 1B; see Supplemental Figure 2 online). In most collinear regions, frequent
smaller scale rearrangements that can be explained by inversions
and local translocations could be detected (see Supplemental
Figure 2 online). Similar recent rearrangements in closely related
species have been reported, for example, in the genus Solanum
(Livingstone et al., 1999; Doganlar et al., 2002).
These comparisons suggest that the progenitor of the core
dicots before hexaploidization possessed four Bli genes. Amplification during the subsequent rounds of genome duplication
was coupled to frequent gene losses. Four collinear groups with
diverse gene copy numbers could be identified in each species
Side-Shoot and Leaflet Formation
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Figure 1. Collinearity Reveals RAX1 as the Arabidopsis Ortholog of Bl and C (Bli2).
(A) Dot blot analysis of protein BLAST hits. Protein sequences of the tomato scaffolds containing Bl (scaffold 6004) and C (Bli2)/Bli6 (scaffold 6019) as
well as sequences from the region around the Arabidopsis RAX1 gene (At5g21000 to At5g25000) were compared with grape chromosomes. Grape
chromosome 6 showed the highest density of hits to all three regions. The positions of Bl, C/Bli2, and RAX1 are indicated on top, and the position of their
grape ortholog Vv6g16270 on the right of the plots (red arrows). Red circles mark the position of the different Bli genes. Numbers are gene numbers.
(B) List of grape chromosomes containing regions collinear to Bli genes from Arabidopsis (RAX1, RAX2, RAX3, MYB36, MYB68, and MYB87) and
tomato [Bl, C (Bli2), Bli1, Bli3, Bli4, Bli5, Bli7, and Bli8]. Collinearity groups are color coded. For the MYB36 region, collinearity to secondary hits was not
detectable.
(C) History of Bli genes in Arabidopsis, grape, and tomato derived from collinearity analysis. Color codes are as in (B). A seventh Bli gene in grape has
not been assigned to a linkage group.
(Figures 1B and 1C). Within the first group, RAX1 is the single
Arabidopsis gene derived from the same progenitor as Bl, Bli2
(C), and Bli6; therefore, RAX1 is the ortholog of these three
paralogous tomato genes (Figures 1A and 1C). Furthermore,
RAX2 and Bli1 are orthologs and originated from a gene that was
a paralog of Bl/Bli2/Bli6/RAX1 in the hexaploid ancestor. The fact
that the genomic segments harboring Bl and Bli2/Bli6 share a
higher density of conserved genes with each other than with
genomic regions of Arabidopsis or grape, respectively, indicates
that they originated during the genome duplication predicted for
the Solanaceae (Figure 1C). In a similar way, orthology relations
of the other genes could be resolved.
Analyses of microsynteny suggest that Bli2 is the strongest
candidate to have a redundant function to Bl. Additionally Bli1, as
the ortholog of RAX2, along with Bli3 and Bli7 as orthologs to
RAX3, are candidates for shoot branching regulators.
The Classical Tomato Trait C Is Caused by a Mutation in Bli2
To unravel the function of Bl’s closest paralog, Bli2, two independent mutants (bli2-1 and bli2-2) were isolated by TILLING
(McCallum et al., 2000). Both mutant alleles affected the codon for
the conserved Trp-58 in the MYB domain, which was changed
to either TTG (coding for Leu) or TAG (stop codon), most likely
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causing knock out mutations (Figure 2A). In populations segregating for either mutation, plants with simpler leaves cosegregated
with the homozygous mutant allele. This leaf morphology was
reminiscent of the phenotype of the classical mutant C (for cut leaf;
Price and Drinkard, 1908; Figures 2B to 2D).
To test the hypothesis that mutations in Bli2 cause the C
phenotype, we sequenced the Bli2 gene in different C alleles.
Indeed, all tested alleles carry lesions in Bli2 (Figure 2A; see
Supplemental Table 1 online). In the classical allele c-1, a Rider/
Kielia retrotransposon insertion disrupts the 39 region of the Bli2
open reading frame. The complete 4867-bp sequence of the
transposon is identical to the elements indentified in the Sun1642
fruit shape, fe inefficient, and yellow flesh mutants (Cheng et al.,
2009). The c-2 allele contains a 40,580-bp deletion that removes
Figure 2. Bli2 Is C, a Key Regulator of Leaf Architecture.
(A) Mutations (indicated in red) in Bli2 in different c alleles. Coding sequences are represented by boxes, and introns are represented by lines. The MYB
domain is shown in blue.
(B) Number of leaflets initiated in leaves along the primary shoot of the c-2 mutant (green) and the wild type (Moneymaker; purple), demonstrating the
heteroblasty of both genotypes and a strong reduction of complexity in c leaves. Position 1 corresponds to the first leaf formed after the cotyledons
(error bars: confidence interval = 0.05; n = 22).
(C) Penultimate leaf of the primary shoot of the wild type (wt) (Moneymaker) and c-2, showing reduced leaflet formation, fusion of the distal three leaflets,
and lack of lobing and serration in the c-2 leaf.
(D) Scanning electron micrograph of a leaf primordium of the c-2 mutant showing the fusion (arrow) of the lateral leaflet primordium with the leaf blade of
the terminal leaflet.
(E) and (F) Close-up of the leaf tip of a c-2 mutant, showing a fusion of a terminal leaflet and lateral leaflets from the top (E) and the bottom (F).
(G) and (H) Scanning electron micrographs showing that formation of lateral leaflets in leaf primordia (P4/P5) of c-2 mutants (H) is severely delayed
compared with leaf primordia of equal length of wild-type (Moneymaker; [G]) plants. Lateral leaflet primordia (1 and 2) and leaf lobes (LB) are indicated.
Bars = 2 cm in (C) and (E), 1 mm in (D), and 100 mm in (G) and (H).
Side-Shoot and Leaflet Formation
the complete C gene and two neighboring genes. In the c-3
allele, the splice acceptor site of the second exon is mutated, and
the start codon in c-4 is changed to ATA. The c-5 and c-integerrima
alleles carry changes of conserved amino acids in the MYB
domain. Furthermore, the simple leaf mutant coalita (clt) (Stubbe,
1972) also carries an amino acid substitution in the MYB domain
of Bli2. Finally, genetic analyses confirmed that bli2-1 (c-z1) and
bli2-2 (c-z2) as well as clt are allelic to c. F1 plants obtained by
crossing these three mutants to c1 or c2 displayed the potato leaf
phenotype.
C was a well-known trait in tomato breeding used in the 19th
and 20th centuries. Today, potato-leafed cultivars, like the popular beefsteak cultivar Brandywine and the old elite cultivar
Matina, are still used by home growers and sold at farmers’ markets. Analyzing the c gene in 10 of these cultivars (see Methods)
revealed that they carry the classical allele c-1, confirming that C
had its origin in breeding. Additionally, we identified a new allele
of C in the cultivar Yellow Submarine (c-ys), a 286-bp deletion in
the 39 region of the gene (Figure 2A; see Supplemental Table
1 online). In total, 10 C alleles were characterized molecularly,
identifying seven single nucleotide polymorphisms, two deletions, and one transposon insertion. Thus, C, Bli2, clt, and
potato-leafed heirloom cultivars are allelic mutants.
C Regulates Leaf Complexity
The complexity of tomato leaves (Kessler and Sinha, 2004) is a
plastic trait dependent on growth conditions and cultivar background. The number of leaflets per leaf can vary drastically
between isogenic plants grown in different experiments (cf.
Figure 2B and Supplemental Figure 3A online). Additionally,
cultivars such as the greenhouse tomato Moneymaker produce
much simpler leaves than the field tomato M82. However, most
importantly, the complexity depends on the position of a leaf
along the shoot axis, where complexity increases acropetally
from the first leaf to the penultimate leaf of the primary shoot
(Figure 2B; Shani et al., 2010). c mutants show reduced leaf
complexity compared with the wild type, with ;70 to 80% fewer
leaflets, and the leaf blade margins are smooth, lacking lobes
and serrations. As in the wild type, the basal leaves of c mutants
are simpler than the more apical leaves (Figures 2B and 2C; see
Supplemental Figures 3A and 3B online). For example, in a c
mutant plant, the first two leaves after the cotyledons are usually
entire, with no indentations at all; however, the adult leaves do
make some leaflets and indentations, although many fewer than
the wild type. Thus, heteroblasty is still present in c mutant
plants, but the dissection of leaves is severely reduced at all
stages. All mutant alleles caused a similar reduction in leaf
complexity, except c-int, whose defects were milder (see Supplemental Figure 3B online). Differences were also observed
depending on the cultivar background and the growth conditions
but were always similar relative to the control (see Supplemental
Figure 3A online). Besides the defects in leaf development, c
plants develop normally, as would be expected for a trait used in
breeding.
Early development of c leaf primordia was analyzed using a
stereomicroscope and scanning electron microscopy. Leaflets
of c mutant leaf primordia emerge and develop with a delay
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compared with equally sized wild-type leaf primordia (Figures 2G
and 2H; see Supplemental Figure 3C online). Alternatively, c leaf
primordia elongate faster than those of the wild type. A similar
defect was found in the simple leaf mutant solanifolia (sf; Sinha,
1999). Furthermore, c mutants exhibit fusions of the terminal
leaflet with the two distal lateral leaflets, which are already visible
at early stages of development (Figures 2D to 2F). Taken together, the c mutant phenotypes of fusions, reduced leaflet
number, and smooth margins are very similar to the defects seen
in gob mutant leaves (Figures 6A to 6C; Berger et al., 2009).
Bl, Bli1, and Bli3 Regulate Shoot Branching
To learn more about the functions of the Bl/C gene family, we
used RNA interference (RNAi) to reduce the expression of two
genes, Bli1, the closest paralog of Bl and C, and Bli3, a close
relative of the Arabidopsis branching regulator RAX3 (Müller
et al., 2006). The resulting Bli1 and Bli3 RNAi plants frequently
lacked side-shoots, the branches that form in the leaf axils; this
phenotype was most pronounced between the second and sixth
leaf of the primary shoot (Figure 3; see Supplemental Figure 4A
online). Leaf axils of wild-type plants almost always harbored
axillary shoots (99.4%, n = 350 leaf axils). For the RNAi lines,
phenotype penetrance varied between experiments, a characteristic that was also described for the branching phenotype of bl
(Schmitz et al., 2002). In three independent experiments, transgenic populations lacked 2 to 10% of the side-shoots within the
first seven leaves for Bli1 RNAi and 2 to 5% for Bli3 RNAi (n = 12
to 80 plants/population). Double Bli1/Bli3 RNAi plants (three
populations, n = 15, 21, and 24) exhibited an enhanced branching defect with 5 to 40% of the first seven leaf axils lacking sideshoots, indicating that Bli1 and Bli3 act redundantly. In the same
experiments, bl and Bl RNAi (Schmitz et al., 2002) populations
lacked side-shoots in 40 to 70% of the first seven leaf axils (n = 12
to 18). Except for the most basal node, the relative distribution of
barren leaf axils in bl and Bl RNAi populations was complementary to that observed in Bli1 and Bli3 RNAi plants (Figure 3; see
Supplemental Figure 4B online).
Besides leaf axils completely lacking axillary buds, some bl
mutants as well as Bli1 and Bli3 RNAi plants developed determinate organs: pin-like structures, reduced leaves, or normal
leaves (Figure 3; see Supplemental Figure 4C online). Such
terminating axillary structures were not observed in the wild type.
Furthermore, bl also affects organ separation, similar to c. For
example, bl mutants developed concaulescent fusions of sideshoots, mainly affecting the fast developing sympodial shoot
(see Supplemental Figures 5A to 5D online; Schmitz et al., 2002).
Thus, Bl, Bli1, and Bli3 regulate the initiation of AMs in a zonal and
partially complementary fashion, similar to RAX1, 2, and 3 in
Arabidopsis (Müller et al., 2006).
Bl and C Are Expressed in Prospective and
Actual Boundaries
The Bl and C genes originated from a recent duplication event
but have distinct mutant phenotypes; this contrast raises the
question of their expression patterns. Quantitative RT-PCR
(qRT-PCR) analysis revealed that both genes are expressed in
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Figure 3. Bl, Bli1, and Bli3 Regulate Shoot Branching.
Analyses of branch formation in bl mutant, Bli1 Bli3 double transgenic RNAi, and wild-type plants. Each column represents a single plant. Each square
represents an individual leaf axil starting from the cotyledons (Cot) at the lowermost rows up to the last leaf formed by the primary shoot. Green denotes
the presence of an axillary bud (i.e., a side-shoot), orange the presence of a terminating structure like a leaf, and yellow a barren leaf axil. bl plants
formed side-shoots most frequently between the first and the fourth leaf, in contrast with Bli1 Bli3 RNAi plants, which lacked side-shoots most
frequently in the leaf axils two to six.
the shoot apex but not, or at very low levels, in mature leaves (see
Supplemental Table 2 online). RNA in situ hybridizations on
tomato shoot tips detected C transcripts in young leaf primordia
(Figures 4A to 4F; see Supplemental Figure 6 online), with
expression strongest in P2 to P4 and no hybridization signal
observed outside of leaf primordia. C was expressed in three
well-defined domains: (1) at incipient lateral leaflet primordia
preceding organ emergence (Figures 4A to 4F), (2) at the proximal and distal boundaries of young leaflet primordia (Figures 4A
to 4E; see Supplemental Figure 6 online), and (3) in the sinuses of
developing leaf lobes (Figure 4A; see Supplemental Figure 6
online). The different expression domains were ball- to bandshaped with one to six cells of strong expression in the largest
expanse and were just below the outermost cell layer. Thus, C
expression marks prospective and actual boundaries during
compound leaf development. Therefore, both the phenotype of c
and the pattern of C expression in leaf primordia coincide with
that of Gob (Blein et al., 2008; Berger et al., 2009).
Bli1 and Bli3 transcripts could not be detected by RNA in situ
hybridization, probably due to the detection limits of the technique. Bl expression was detected in prospective and actual
boundaries in the shoot apex, just like C expression in leaf
primordia. During the vegetative phase, Bl mRNA accumulated
at the position of incipient leaf primordia (P0), preceding organ
emergence (Figures 4G and 4H; see Supplemental Figure 7
online). This expression domain usually comprised two to six
cells in all three dimensions. Furthermore, Bl was strongly
expressed adaxially of the leaf primordia. In transverse sections,
this oval- to band-shaped domain covered at least half of the
boundary between the SAM and the leaf primordium (Figure 4H;
see Supplemental Figure 7 online). The hybridization signal
usually extended from the L1 through all cell layers of the SAM,
fading out in the region where cells start to become vacuolated
(Figure 4G; see Supplemental Figure 7 online). Expression of Bl
was detected from P0 to the oldest leaf axil analyzed (P6) (see
Supplemental Figure 7 online). Bl expression was also detected
on the adaxial side of young AMs, separating them from the
parental shoot, which correlates with the function of Bl to prevent
concaulescent fusions (see Supplemental Figure 5E online). In
summary, C and Bl are expressed in different organs according
to their functions but show similar domains of activity predicting
boundaries and organ formation in leaf primordia, shoot apices,
and leaf axils.
The fact that Gob and the MYB genes Bl and C are expressed
in the boundary zones essential for the formation of AMs and
leaflets prompted us to test whether the branching regulator Ls is
also expressed in leaves. Although mutations in the Ls gene
manifest only in a lack of AMs, Ls mRNA accumulates in distinct
domains of the leaf axil and at the distal boundary of leaflets
(Figures 4I to 4L). This finding supports the view that the two
domains have a similar identity.
Bl and C Show Functional Conservation
The expression studies suggest that Bl and C have undergone
sub- or neofunctionalization due to promoter differentiation. The
almost identical amino acid sequence of the Bl and C DNA
binding domains (see Supplemental Figure 1 online) indicates
that both proteins target similar downstream genes. To examine
Bl and C function, we made transgenic tomato plants overexpressing Bl or C and found that plants with enhanced expression
of Bl or C displayed similar developmental defects characterized by retarded growth and delayed development (Figures
5A and 5B; see Supplemental Figure 8 online). All organs remained
smaller throughout development. Scanning electron microscopic
analysis of the dwarf 35S:Bl plants showed that epidermal cells
were either reduced in length (rachis cells) or in number (internode
cells; Figures 5C to 5F). Leaf lobes and serrations were more
elaborated with sharper lobe and serration tips in both C- and
Bl-overexpressing lines, suggesting that Bl can also regulate leaf
dissection (Figures 5A and 5B). A lack of functional promoter
constructs for both Bl and C hindered successful promoter swap
Side-Shoot and Leaflet Formation
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experiments to test directly for the supposed conserved function
of the proteins. Instead, we introduced the 35S:Bl construct into
the c mutant background. The ectopic expression of Bl reestablished serration of leaf margins in c mutants (Figures 5G to 5J).
When crossed to sf (Sinha, 1999), another simple leaf tomato
mutant (Figure 5K), the resulting 35S:Bl sf plants developed
as dwarfs but retained the smooth leaf margins of the sf parent
(Figure 5L). Hence, 35S:Bl expression specifically complemented
the loss of leaf serration caused by a mutation in the C gene,
supporting the conclusion that C and Bl are functionally conserved.
C and Bl Act in Parallel to the Gob/KNOX Pathway
Figure 4. C, Bl, and Ls Expression Domains Define Boundaries.
Tomato has a single ortholog of the Arabidopsis CUC1 and CUC2
genes, named Gob (Blein et al., 2008; Berger et al., 2009), and
lacks the partially redundant CUC3 function. gob mutants show
very similar leaf development phenotypes as c mutants: Both
mutants exhibit loss of most second-order and intercalary leaflets
and of leaf lobes and serrations (Figures 6B and 6C). Furthermore,
the expression pattern of Gob (Blein et al., 2008; Berger et al.,
2009) resembles the combined patterns of C and Bl (Figure 4). In
addition, Gob also functions in organ separation. Identical to c, the
three distal most leaflets of gob leaves are usually fused (Berger
et al., 2009; Figure 6C). Because of these similarities, two questions arose: Do gob mutants have the same developmental
defects as bl mutants, namely, the lack of AMs, and does Gob
act in one pathway with the MYB genes C and Bl? In a total of 80
regenerated gob-3 shoots analyzed, all completely lacked axillary
shoots with the exception of infrequently formed sympodial
shoots. Leaf axils remained barren throughout the life span of
these plants and did not produce any axillary structures (Figure
6D). This result demonstrates that Gob combines the regulation of
two different processes: leaflet formation and AM initiation.
To test whether C and Gob act redundantly or in a hierarchical
order, we performed double mutant analysis. Whereas c and gob
single mutants display acropetally increasing leaf complexity
(Figure 6I), c gob double mutants produced simple leaves
throughout development, drastically altering the appearance
of these plants (Figures 6G to 6I). The lack of lateral leaflets
demonstrates that the two boundary genes are partially redundant, and, although expressed only in a very limited number of
cells, their functions are essential for compound tomato leaf
development. In agreement with the double mutant results, qRTPCR analysis of C expression in leaf primordia of Gob-4d, a
microRNA-resistant gain-of-function mutant (Berger et al., 2009),
as well as of Gob in c tissues, did not reveal any regulatory
interaction between the two genes (see Supplemental Table 3
RNA in situ hybridizations with C ([A] to [F]), Bl ([G] and [H]), and Ls ([I] to
[L]) antisense probes. Longitudinal ([A], [C], [D], [G], [I], and [K]) and
transverse ([B], [E], [F], [H], [J], and [L]) sections through tomato shoot
apices. Arrows mark the earliest expression domains detected for C and
Bl, preceeding the formation of lateral leaflets and AMs, respectively.
Arrowheads point to expression domains in the distal (black) and
proximal (red) boundaries of developing leaflets. The blue arrowhead in
(A) points to the weak expression of C at an initiating leaf lobe groove.
Asterisks indicate the SAM. Bars = 100 mm in (A) to (D), (G), and (H) to (K)
and 50 mm in (E), (F), and (L).
(C) and (D) Two consecutive partial sections through an old leaf primor-
dium showing a large lateral leaflet primordium.
(E) and (F) Two cross sections through a stage 3 primordium (P3)
separated by 56 mm.
(G) and (H) Bl mRNA accumulates in the axils of leaf primordia.
(I) to (L) Ls is expressed in the axils of leaf primordia ([I] and [J]) and at
the distal boundary of leaflets ([I] to [L]).
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The Plant Cell
mutants displayed simple c-like basal leaves (Figure 6J); however,
from leaf four onwards leaf complexity increased, similar to Me/+
(Figure 6K; see Supplemental Figures 9A to 9D online). The double
mutants initiated many higher order leaflets, but the leaflets
showed characteristics of c mutant leaves, like fusions of leaflets
and smooth margins (Figure 6K; see Supplemental Figure 9B
online). In summary, the Me/+ c double mutants showed phenotypic characteristics of both single mutants, suggesting that C and
Me/TKn2 act independently to influence leaf complexity. This is
supported by qRT-PCR analyses showing that expression of Me/
TKn2 was not significantly altered in c leaves (see Supplemental
Table 3 online). Finally, we observed that Me/+ enhanced branching. In contrast with the wild type, Me/+ plants frequently produced
accessory side-shoots, indicating that together with Gob, Me also
plays a role in both leaf development and AM initiation (see
Supplemental Figure 9E online).
C Acts in an Independent Pathway Regulating
Leaf Dissection
Figure 5. Protein Function Is Conserved between Bl and C.
(A) and (B) Leaves of transgenic plants ectopically expressing Bl (Bl-ox)
or C (C-ox) compared with wild-type (wt) leaves at the same developmental stage. Note the pronounced pointedness of lobe and serration
tips of the transgenic leaves.
(C) and (D) Scanning electron micrographs of wild-type and 35S:Bl
abaxial epidermis cells of leaf rachises (leaves as in [A], central part of
rachis). The 35S:Bl plants have little cell elongation.
(E) and (F) Scanning electron micrographs of internode epidermises of a
wild-type and an ;50% smaller 35S:Bl internode of equal age and
developmental stage showing equal cell sizes.
(G) to (J) Leaf and close-up of a leaflet from a c-z2 mutant ([G] and [H])
and from a c-z2 mutant expressing the 35S:Bl construct resulting in a
reestablishment of marginal serration ([I] and [J]).
(K) and (L) Leaf of a sf mutant (K). Top-down view on a wild-type plant
(left) and an sf mutant (right) both expressing the 35S:Bl construct (L).
Note the smooth leaf margins of the sf mutant.
Bars = 2 cm in (A) and (K), 2 cm in B, 100 mm in (C) and (D), 100 mm in (E)
and (F), 5 cm in (G) and (J), 2 cm in (H) and (I), and 12 cm in (L).
online). Hence, despite the similarity in expression and function,
Gob does not act in a pathway with C or Bl.
An important regulator of leaf complexity known to act in
concert with Gob is the KNOXI gene Me/Tomato Knotted2
(TKn2) (Blein et al., 2008). Heterozygous Me mutants (Me/+)
generate a large number of higher order leaflets resulting in
enormously ramified leaves (Parnis et al., 1997). Me/+ c double
To check for genetic interactions of c with other known leaf
complexity regulators, double mutants with e, trifoliate (tf), and
Lanceolate (La), were analyzed. e is characterized by a reduced
number of distinct leaflets due to an excess of lamina outgrowth
(Zhang et al., 2007; Berger et al., 2009; Figures 7A and 7B). The
different defects in e and c mutants are already obvious in basal
leaves (Figure 7A). In double mutants, basal leaves showed no
lobing and no serration; also, leaflets were further reduced
compared with both single mutants (Figure 7A). Similarly, adult
c e double mutant leaves were simpler than leaves of both
parents, lacking most leaflets, lobes, and serrations, which
resulted in an enhanced simple leaf defect (Figure 7B).
tf leaves usually develop a barren rachis with two lateral
leaflets at the distal end (Figure 7C). c tf plants mostly produced
completely simple leaves, lacking leaflets, lobes, and serrations
(Figure 7C). Fifth and sixth leaves of c-z2 tf and c-2 tf double
mutants (n = 12 and 20) produced no lateral leaflets, whereas tf
leaves (n = 19) initiated 2.0 6 0.4 lateral leaflets. Few adult leaves
of the double mutants developed rudimentary lateral leaflets.
Heterozygous and homozygous La mutants develop leaves
with only a few small leaflets and lobes (Ori et al., 2007). In La/+ c
(Figure 7D) and La/La c (see Supplemental Figure 9F online)
double mutants, these small leaflets and lobes were missing,
resulting in an even simpler leaf phenotype. In summary, c
enhanced the simple leaf phenotypes of gob, e, tf, and La and
modified the defects of Me/+. The lack of epistasis demonstrates
that C acts in a previously unknown pathway regulating leaf
dissection, independent of other described leaf complexity regulators.
DISCUSSION
Uncovering the Molecular Cause of a Classical Trait
“Potato leafed” is an old and well known trait in tomato breeding,
discussed in the scientific literature by White (1901) and described as a recessive Mendelian gene by Price and Drinkard
Side-Shoot and Leaflet Formation
9 of 15
Figure 6. Gob and Me Act Redundantly to C and Bl.
(A) to (C) Adult leaves of wild-type (wt) (M82), c-z1, and gob plants.
(D) gob mutant plant lacking axillary shoot formation (arrows).
(E) to (H) Plants of indicated genotypes. Leaflets are formed in c-2 and gob mutants but are completely lacking in the c-2 gob double mutant ([E], [F],
and [H], top-down views; [G], side view).
(I) Leaflets initiated in gob (magenta), c-2 (blue), and c-2 gob (green) mutants. The “1” reflects the first leaf formed after regeneration from hypocotyl (n =
16, 15, and 34, for gob, c, and double mutants, respectively; the length of the lines corresponds to the average leaf number before flowering for each
genotype, error bars, confidence interval = 0.05).
(J) Second leaf after cotyledons of wt, c-2, Me/+, and c-2 Me/+ plants. c is epistatic over Me/+ at this stage.
(K) Sixth leaf of Me/+, c-2 Me/+, and c-2 mutant plants displaying an epistasis of Me/+ in the double mutant.
Bars = 2 cm in (A) to (D) and (J) and 5 cm in (E) to (H) and (K).
(1908). In this work, we show that C is an R2R3 MYB transcription
factor and the closest paralog of the branching regulator Blind.
We identified a Rider retrotransposon disrupting the C gene in
the classical allele c-1 and lesions in nine other alleles of C. Rider
elements originated from a horizontal gene transfer into tomato
(Cheng et al., 2009). Outside of the Lycopersicon section, the
closest homologs are found in Beta vulgaris and Arabidopsis, but
not in other Solanaceae or in any other Asterids, as determined
by searching the National Center for Biotechnology Information
(NCBI) nr, htgs, gss, and wgs databases (http://blast.ncbi.nlm.
nih.gov). Interestingly, the element found in c-1 is identical in
sequence (4867 bp) to the Rider elements responsible for mutations in the genes Sun, Fer, and R (Cheng et al., 2009). This
active Rider element (Jiang et al., 2009) may also be responsible
for some of the many spontaneous mutants collected at the
Tomato Genetics Resource Center (TGRC).
Doganlar et al. (2002) proposed C as a quantitative trait locus
candidate gene influencing the difference in leaf morphology
between the simple leafed eggplant (Solanum melongena) and
its wild relative Solanum linnaeanum, which is strongly lobed and
serrated. Furthermore, it was speculated that C might cause the
leaf phenotype of potato (Solanum tuberosum) plants, as the
gene name indicates. However, we found intact copies of C in
eggplant and in potato, contrary to this hypothesis (see Supplemental Figure 1 online). Furthermore, C is not likely to be the
gene responsible for these leaf form differences, considering
the fusion phenotypes seen in c mutants but not in potato or
eggplant leaves. The tomato gene Sf, with yet unknown molecular identity, might be a much better candidate, as sf mutant
leaves are indeed highly similar to those of potato plants.
Initiation of Leaflets and Shoot Branches Depends on
Homologous Gene Modules
This work revealed that identical and homologous boundary
genes are essential for the initiation of AMs and the initiation of
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fail to initiate vegetative AMs. Gob orthologs in the NAM/CUC
family play a conserved role in compound leaf development
in Aquilegia caerulea, Cardamine hirsuta, Pisum sativum, and
tomato (Blein et al., 2008). In addition, this gene family also
regulates the formation of axillary shoots in several species
besides tomato. NAM together with its paralogs in Petunia
hybrida (Souer et al., 1998), CUP in Antirrhinum majus (Weir
et al., 2004), and CUC1, CUC2, and CUC3 in Arabidopsis (Hibara
et al., 2006; Raman et al., 2008) are all crucial for the formation of
AMs. Gob is the first example where a NAM/CUC orthologous
function simultaneously regulates AM formation and compound
leaf development within the same species.
The Bl/C gene pair and the Gob gene provide evidence for the
presence of homologous functions regulating AM and leaflet
initiation. Additional observations support this link between the
two processes and will therefore be the subject of future
research, as they suggest that a larger set of genes is involved
in this common mechanism. For example, Ls, a major regulator
of axillary branching, is expressed in leaflet axils, similar to Gob
and C. Furthermore, we observed several correlations between
leaflet and axillary branch formation, although we do not know
the molecular basis for this phenomenon. For example, the
tomato cultivar M82 produces both more accessory side-shoots
Figure 7. C Action Is Independent of e, tf, and La.
(A) and (B) Additive phenotypes of c and e in second (A) and adult leaves
(B).
(C) and (D) Leaves of tf single and tf c double mutants ([C], fifth leaves)
and of heterozygous La (La/+) single and La/+ c double mutants ([D],
adult leaves). Both double mutants showed strongly enhanced simple
leaf phenotypes, almost completely lacking lateral leaflet initiation.
Bars = 2 cm.
lateral leaflets (Figure 8), which was unexpected considering the
obviously distinct nature of leaflets and axillary shoots. Collinearity analyses revealed that a recent genome duplication in
tomato evolution led to the establishment of the two paralogous
genes, Bl and C, regulating branching and leaflet initiation,
respectively. Functional differentiation between Bl and C appears to be based on different promoter activities, as our data
demonstrate that Bl and C have distinct mRNA expression
patterns but conserved protein functions. We can only speculate
about whether the ancestral gene had both functions (i.e.,
whether the duplicated genes have undergone subfunctionalization or whether one underwent neofunctionalization).
Indeed, another boundary gene unites both promoter activities
and gene functions, indicating that Bl and C may have become
subfunctionalized. This other gene, Gob, is a leaf complexity
regulator (Blein et al., 2008; Berger et al., 2009), and we showed
that Gob is also essential for axillary shoot formation. gob
mutants have severe defects in leaflet initiation and completely
Figure 8. Boundary Genes Regulate Both AM and Leaflet Initiation.
The transcriptional regulators Bl, C, Gob, and Ls are expressed in regions
where AMs (Bl, Gob, and Ls) and leaflets (C, Gob, and Ls) will be initiated.
Loss of Bl, Gob, or Ls function compromises AM formation. Lack of C or
Gob expression leads to a reduction in leaf complexity; c gob double
mutants have almost completely simple leaves.
Side-Shoot and Leaflet Formation
and more leaflets compared with the cultivar Moneymaker.
Additionally, we were able to identify a number of tomato
mutants, such as tf, that display reduced leaf complexity and
reduced AM formation simultaneously. Molecular analyses of
these observations will further extend the understanding of the
commonalities of AM and leaflet initiation.
All three types of boundary genes, Gob, Bl/C, and Ls, may have
at least one function in common: growth repression. Gob-4D
mutants, expressing a microRNA-resistant transcript, exhibit retarded growth (Berger et al., 2009) similar to plants misexpressing
Bl and C (see Supplemental Figure 8 online) and similar to 35S:Ls
plants (G. Schmitz and K. Theres, unpublished). Repression of
growth is an obvious requirement at boundary regions, which
separate two areas of high growth rates (Hussey, 1971). Interestingly, the expression of the boundary genes Bl/C, Gob, and Ls
precedes the physical formation of boundaries and probably
defines these areas of inhibited growth, enabling organ separation. All the respective mutants display organ fusions. Furthermore, this repression seems to keep cells in an undifferentiated
state with the potential for new morphogenesis, as bl, c, gob, and
ls lose organogenic potential, lacking leaflet and/or AM initiation.
A New Branch in the Leaf-Shoot Comparison
Our observations add a new branch to the recently discussed
idea of leaf and shoot homologies and to the leaf shoot continuum hypothesis (Sinha, 1999; Rosin and Kramer, 2009; Blein
et al., 2010; Canales et al., 2010; Floyd and Bowman, 2010).
Recent publications emphasize the homology between leaf
primordium initiation at the shoot apex and leaflet primordium
initiation at the marginal blastozone of young leaves: Polar PIN
localization leads to auxin response maxima, inducing outgrowth
of leaves and leaflets and adjacent CUC expression (reviewed in
Rosin and Kramer, 2009). Our data demonstrate parallels
between shoot formation and leaflet formation. The initiation
of lateral meristems in leaf axils and the initiation of leaflets at
the border of existing leaflets share common gene modules
(Figure 8).
Even though the boundary genes Gob and Bl are expressed at
prospective axillary positions before leaf primordia are detectable, no obvious defect in early leaf development were observed
in the respective single mutants and in gob bl double mutants.
They have normal phyllotaxis, and young leaf primordia largely
resemble wild-type primordia. Defects are observed only later,
affecting the initiation of new shoots from the boundary region,
just like the defects of c and gob mutants affect leaflet initiation
(Figure 8), suggesting that Gob and Bl are not involved in leaf
primordium initiation.
Deep Homology or True Homology?
These data raise the question why two different structures like
leaflets and AMs employ homologous modules in their morphogenesis. At least two possibilities are conceivable. First, a
boundary gene set could have evolved for one of the processes
and later during evolution been recruited for the other. This would
be called deep homology, the co-option of a preexisting regulatory program or circuit (Gaunt, 1997; Shubin et al., 2009).
11 of 15
Homology is limited to the involved genes, but the resulting
organs or structures do not share common ancestry. This seems
very likely in the case of leaflets and AMs.
However, although highly speculative, another scenario is
supported by the fossil record. True axillary branching is unique
for seed plants (Galtier, 1999; Tomescu, 2006). Equally, seed
plants evolved leaves independently from other plant lineages
(Sanders et al., 2009; Tomescu, 2009; Floyd and Bowman, 2010).
The development of both structural novelties may even have
been intimately linked, as both probably appeared simultaneously, 375 to 345 million years ago, in the late Devonian
and lowermost Carboniferous (cf. Stewart and Rothwell, 1993;
Galtier, 1999; Meyer-Berthaud et al., 2000; Beerling and Fleming,
2007; Sanders et al., 2009). The earliest seed plants had leafless
three dimensionally branched shoot systems generated by
overtopped dichotomous branching from which leaves with
leaflets have evolved. Although the fossil record is limited and
difficult to interpret, axillary shoots probably coevolved with
leaves and may also be derived from overtopped dichotomous
branches. The idea that, like leaves and leaflets, axillary shoots
were derived from unequal and overtopped dichotomous
branches is supported by the fact that Gob, Bl, and Ls, like their
orthologs in Arabidopsis, are all expressed already within the
SAM. However, no matter what the exact origin of AMs might be,
the likely common origin of leaves with leaflets and axillary
shoots in ancestral seed plants indicates a homology of these
structures at an ancient, very basal level. This would be true
homology due to descent from a common ancestral structure,
the branches of the leafless ancestor of seed plants.
METHODS
Plant Materials and Growth Conditions
Seeds of c-1 (LA3168), c-2 (3-345), c-3 (3-604), c-4 (3-609), c-5 (3-626),
c-int (LA0611 and LA3728A), c-clt (LA2026), bl-2 (LA0980), La (LA335),
Me (LA 324), and sf (2-311) were obtained from the TGRC (Davis, CA);
c-z1 (e2978), c-z2 (e2986), gob-3 (n5126-m1), Gob-4d (e0042), e-2
(n0741), tf-z (e0761), and M82 cultivar from http://zamir.sgn.cornell.
edu/mutants (Menda et al., 2004); and MM from Kiepenkerl. gob-3,
Gob-4d, e-2, e-2 c-z1, and e-2 c-z2 mutants were kindly provided by
Naomi Ori (Rehovot, Israel). Seeds of the c-1, heirloom tomatoes
Brandywine “Sudduth Strain,” Oaxacan Jewel, Stupice, and Malinowy
Ozarowski were obtained from Tomatenundanderes (Eltendorf, Austria);
Bloody Butcher, Black Sea Man, Black Pear from Semillas La Palma (El
Paso, Spain); Matina, Olena Ukrainian, Brandywine Sherry, and seeds of
the c-ys Yellow Submarine were obtained from Reinsaat KG.
Plants were grown under standard glasshouse conditions with additional artificial light (16-h photoperiod) when needed. Shoots of gob-3,
c-2 gob-3, and bl gob-3 mutants were regenerated from wound-induced
callus (Berger et al., 2009).
Phenotyping
Parts of the leaf, consisting of a continuous leaf blade and separated from
other parts of the leaf by a piece of rachis, were counted as leaflets.
Leaflet sizes ranged from a few millimeters to 20 cm. Whenever “initiated”
or “lateral” leaflets are mentioned, this by definition excludes the terminal
leaflet. “Adult leaves” indicates leaves that were generated three plastochrons prior to flowering or later. For phenotypic comparisons, sister
12 of 15
The Plant Cell
plants from segregating populations were used when possible. In addition, different alleles and/or backgrounds were used. In all experiments,
at least one biological replicate was performed. bl-2 and wild-type plants
in Figure 3 are from segregating F2 populations (cv LUxMM), and Bli1 Bli3
double transgenic plants are from an F1 population. gob-3 mutants in
Figures 6E to 6H had a mixed background (MM:M82, 1:1), and gob c-2
double mutants were from an F3 population of c-2 (MM) crossed with
gob-3 (M82). Cultivar abbreviations are used as in TGRC databases.
Scanning electron microscopy was performed on a DSM 940 (Zeiss)
using fresh tissue.
TILLING
To perform the screening by TILLING (McCallum et al., 2000), we
exploited the tomato ethyl methanesulfonate–mutagenized population
of M82 (Menda et al., 2004). Screening for mutations in the coding
sequence of Bli2 was performed as described by Piron et al. (2010).
Characterization of C Alleles
The Rider element insertion in c-1 was identified by inverse PCR (iPCR)
using C-specific primers (see Supplemental Table 4 online). Sequence
analysis revealed that the Rider element in c-1 is identical to the SUNRider element (accession number EU195798). A tandem transposon
insertion cannot be excluded, as a single PCR spanning the complete
insertion was not possible. Transposon sequences were amplified using
Advantage GC 2 polymerase (Clontech Laboratories).
The genomic flanking sequence of c-2 was obtained by iPCR and
fosmid and BAC sequencing. Publically available genome sequence data
enabled the final identification of the 40,580-bp deletion.
The provisional alleles c-prov2, -3, -4, and -5 were confirmed as c
alleles. 3-631 (c-prov6) was found not to be allelic to c-4.
DNA Sequencing and Analysis
Plant DNA was prepared as described (Brandstädter et al., 1994) or using
the DNeasy 96 plant kit and BioSprint 96 automated DNA extraction
apparatus (Qiagen). Standard techniques were performed according to
Sambrook and Russel (2001), unless otherwise stated.
PCR and iPCR products were treated with ExoSAP-IT (USB Corporation) prior to sequencing. DNA sequences were determined by the Max
Planck Genome Centre Cologne on Applied Biosystems ABI Prism 377,
3100, and 3730 sequencers using BigDye-terminator v3.1 chemistry.
Premixed reagents were from Applied Biosystems. Oligonucleotides
were purchased from Eurofins MWG Operon.
Primers were designed using Primer3 (Rozen and Skaletsky, 2000). The
primers used for genotyping of plants are listed in Supplemental Table 4
online.
BLAST analyses were performed on Sol Genomics Network (Mueller
et al., 2005) and GenBank databases (NCBI).
RNA in Situ Hybridization
Sample preparations and in situ hybridizations were performed as described by Müller et al. (2006). The C and Bl antisense probes contained
the nucleotides +909 to +338 and +948 to 22, respectively, relative to the
ATG. The C probe was transcribed using T7 RNA polymerase (Ambion)
from AflII-linearized pCR-Blunt-II-TOPO vector (Invitrogen) and Bl probe
from NdeI-linearized pGEM-T Easy (Promega). A PCR product containing
the nucleotides +112 to +1224 relative to the ATG was used as template
for synthesis of an Ls antisense probe (primers Lsfo and Lsrext; see
Supplemental Table 4 online). Slides were imaged using bright-field
microscopy or differential interference contrast microscopy.
RNAi and Overexpression Constructs and Tomato Transformation
The T-DNA cassette of pJawohl17 (provided by Imre Somsich and Bekir
Ulker, Cologne, Germany) was cloned as a PmeI-RsrII fragment into the
binary plant transformation vector pPZP212 backbone (GenBank accession number U10462; Hajdukiewicz et al., 1994), resulting in pJaZP. cDNA
fragments of Bli1 (254 to +892) and Bli3 (+56 to +1025) were cloned in
reverse orientation into pDONR201 (Invitrogen) and subsequently into
pJaZP using the Gateway cloning system.
For overexpression of Bl (Bl-ox), the Bl open reading frame was cloned
as a NotI-AscI fragment into pRT-/Not/Asc (Überlacker and Werr, 1996)
between the cauliflower mosaic virus 35S promoter/TMV- leader and the
cauliflower mosaic virus 35S polyadenylation sequences. This cassette
was introduced into the AscI site of plant transformation vector pGPTVKan-Asc (Überlacker and Werr, 1996). For overexpression of C, a genomic fragment starting 2456 bp upstream of the ATG and ending 746 bp
downstream of the stop codon was introduced into the SalI/SacI site of
pGPTV-Kan (Becker et al., 1992). A double 35S-enhancer element (327
bp each; Benfey et al., 1989) was cloned in reverse orientation into the
SalI/SbfI site 59 of the C promoter.
Agrobacterium tumefaciens–mediated transformation of tomato (Solanum lycopersicum) leaf explants of cultivar MM was performed as
described (Knapp et al., 1994). At least four independent transgenic lines
exhibiting similar developmental defects were characterized further. For
double RNAi experiments, individual T-DNA insertion lines were crossed.
Colinearity Analyses
Genome-wide pairwise BLASTP alignments (Altschul et al., 1990) were
performed using protein sequences from Arabidopsis thaliana (ftp://ftp.
Arabidopsis.org/home/tair/Genes/TAIR9_genome_release/TAIR9_sequences/
TAIR9_pep_20090619), tomato (ftp://ftp.solgenomics.net/genomes/Solanum_
lycopersicum/wgs/assembly/build_1.00/S_lycopersicum_scaffolds.1.00.fa.
gz), and grape (Vitus vinifera; ftp://ftp.psb.ugent.be/pub/plaza/plaza_
public_01/Fasta/). The five best BLAST hits for each protein were selected, irrespective of the e-values. Chromosomal regions containing
members of the Bl subfamily in tomato (complete scaffolds) and in
Arabidopsis (starting around 200 genes upstream of the corresponding
MYB gene and ending ;200 genes downstream; e.g., for RAX1,
At5g23000, the region between At5g21000 and At5g25000) were used
as queries. For each tomato or Arabidopsis region, the three grape
chromosomes with the highest number of hits were selected (see Supplemental Data Set 1 online). Collinearity was visualized by plotting the
positions of query and target genes.
qRT-PCR
Primordia of fourth and fifth leaves after cotyledons (at a size of 1 to 2 mm
and 2 to 5 mm), 2-cm leaves (second leaf after cotyledons), and vegetative shoot apices were harvested. RNA was extracted with the RNeasy
plant mini kit (Qiagen), including on-column DNase treatment (DNA-free
kit; Applied Biosystems/Ambion) or a subsequent DNaseI treatment
(Roche Diagnostics). One microgram of RNA was used for cDNA synthesis with the RevertAid H Minus M-MuLV reverse transcriptase kit
(Fermentas). qPCR was performed using the PowerSYBR-Green PCR
Master Mix (Applied Biosystems). Primers are described in Supplemental
Table 4 online. Relative quantification was done using internal standard
curves and correcting by the use of reference genes (TIP41, GAPDH1,
and CAC; Expósito-Rodrı́guez et al., 2008).
Accession Numbers
Sequence data from this article can be found in the Sol Genomics
Network data library under the following accession numbers: Bl,
Side-Shoot and Leaflet Formation
Solyc11g069030; C, Solyc06g074910; Bli3, Solyc04g077260; Bli4,
Solyc12g008670; Bli5, Solyc08g065910; Bli6, Solyc06g074920; Bli7,
Solyc02g091980; Bli8, Solyc07g006750; and Ls, Solyc07g066250.
Supplemental Data
The following materials are available in the online version of this article.
Supplemental Figure 1. Alignment of Blind-Related MYB Transcription Factors.
Supplemental Figure 2. Analysis of Collinearity between Regions
Containing Bl, C/Bli6, or Bli1 and Grape Chromosomes.
Supplemental Figure 3. Leaflet Development in c Mutants.
Supplemental Figure 4. Zonal Branching Defects and Determinate
Axillary Structures in bl, bli1, and bli3 Loss-of-Function Plants.
Supplemental Figure 5. Concaulescent Fusions in bl.
Supplemental Figure 6. C Expression Analysis in Serial Sections
through a Young Leaf.
Supplemental Figure 7. Bl Transcript Accumulation.
Supplemental Figure 8. Ectopic Expression of Bl and C Leads to
Repression of Growth.
Supplemental Figure 9. Genetic Interaction of C with Me and La.
Supplemental Table 1. Alleles of C.
Supplemental Table 2. Relative Expression Levels of Bl, C, Bli1, and
Bli3 in Shoot Apices and Leaves.
Supplemental Table 3. qRT-PCR Analyses of C, Gob, and TKn2.
Supplemental Table 4. Primers.
Supplemental Data Set 1. BLAST Hits between Selected Chromosomal Regions.
ACKNOWLEDGMENTS
We thank the Tomato Genetics Resource Center (Davis, CA) for providing seed stocks. We thank Naomi Ori (Rehovot, Israel) for seeds of
gob-3, Gob-4d, and e as well as for providing the double mutants e c-z1
and e c-z2. We also thank Imre Somsich and Bekir Ülker (Max Planck
Institute for Plant Breeding Research, Cologne, Germany) for the vector
pJawohl17, Rolf-Dieter Hirtz (Max Planck Institute for Plant Breeding
Research, Cologne, Germany) for help with scanning electron microscopy, and Lukas Scharfenberger (University of Cologne, Germany) for
characterization of C genes in eggplant and potato. We thankAlexandra
Kalde (Max Planck Institute for Plant Breeding Research, Cologne,
Germany) and Ursula Pfordt (Max Planck Institute for Plant Breeding
Research, Cologne, Germany) for excellent technical assistance and
Bodo Raatz (Max Planck Institute for Plant Breeding Research, Cologne,
Germany) for critical reading of the manuscript. This research was supported in part by the Max-Planck-Society and in part by the European
Union SOL Integrated Project FOOD-CT-2006-016214.
AUTHOR CONTRIBUTIONS
B.L.B., G.S., and K.T. designed the research. B.L.B., G.S., S.R., and F.P.
performed the research. B.L.B., G.S., S.R., J.D., F.P., A.B., and K.T.
analyzed the data. B.L.B., G.S., and K.T. wrote the article.
Received June 6, 2011; revised September 19, 2011; accepted October
17, 2011; published October 28, 2011.
13 of 15
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Shoot Branching and Leaf Dissection in Tomato Are Regulated by Homologous Gene Modules
Bernhard L. Busch, Gregor Schmitz, Susanne Rossmann, Florence Piron, Jia Ding, Abdelhafid
Bendahmane and Klaus Theres
Plant Cell; originally published online October 28, 2011;
DOI 10.1105/tpc.111.087981
This information is current as of June 18, 2017
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