Download Analysis of flower pigmentation mutants generated by random

The Plant Journal (1998) 13(1), 39–50
Analysis of flower pigmentation mutants generated by
random transposon mutagenesis in Petunia hybrida
Adèle van Houwelingen, Erik Souer, Kees Spelt,
Daisy Kloos, Joseph Mol and Ronald Koes*
Department of Genetics, Institute for Molecular Biological
Sciences, Vrije Universiteit, BioCentrum Amsterdam, de
Boelelaan 1087, 1081 HV Amsterdam, The Netherlands
Summary
Fifty new flower pigmentation mutants in Petunia hybrida
using endogenous transposable elements (TEs) as a
mutagen were generated. Forty-six mutants displayed
somatic and sporogenic instability indicating that they
were caused by a TE. Phenotypic analysis showed that
the mutation altered either anthocyanin biosynthesis (40
alleles for seven loci), the intracellular pH of petals (six
alleles for three loci) or the shape of petal cells (two
alleles for two loci). To identify the TEs reponsible for
the mutations, the authors subjected 16 alleles of
the anthocyanin-3 (an3) locus, encoding flavanone 3βhydroxylase, to molecular analysis. This showed that 11
out of 12 unstable an3 alleles harboured TE insertions
of a single family, dTph1, while one allele harboured a
new 177 bp TE designated dTph2. In addition, the
authors found one an3 allele (an3-W138A) in which a
dTph1 element had inserted 30 bp upstream the translation start, without inactivating the gene. This ‘cryptic’
element was responsible for the creation of a stable
recessive (untagged) an3 allele, where a large rearrangement inactivated the gene. These findings indicate that
mutants for novel loci are most likely tagged by dTph1
elements opening the way for their isolation.
Introduction
Transposable elements (TEs) are thought to be present
in virtually all species, though they have been studied
in only a few. The insertion of a TE in a gene often
results in the inactivation of that gene leading to a
mutant phenotype. This makes TEs valuable genetic tools
to generate mutants, to analyse cell-lineages (e.g. Scheres
et al., 1994; Vincent et al., 1995), and to study intercellular
trafficking of gene products and signals (Carpenter and
Coen, 1995; Hantke et al., 1995; Perbal et al., 1996). TEs
have proven to be particularly useful as a tag to isolate
Received 19 May 1997; revised 4 September 1997; accepted 8 September
1997.
*For correspondence (fax 131 20 4447155; e-mail [email protected]).
© 1998 Blackwell Science Ltd
new genes for which only a mutant phenotype is known
(for reviews see Kunze et al., 1997; Walbot, 1992).
Because this obviously requires that the TE is isolated,
transposon tagging in plants was initially limited to
maize and snapdragon. More recently, the maize Ac and
En/Spm elements have been introduced in other plant
species and successfully exploited to tag genes in the
novel host (reviewed by Kunze et al., 1997; Osborne and
Baker, 1995). We undertook the original approach and
characterized the endogenous transposons of Petunia
hybrida to exploit them for gene-tagging.
In P. hybrida eight unstable alleles, typical for transposon insertions, had been described for six flower pigmentation genes (for review see Gerats et al., 1989). The
P. hybrida line W138 contains an unstable allele at the
anthocyanin-1 (an1) locus and among W138 progeny
new unstable mutations are found at high incidence
(Doodeman et al., 1984). Of these unstable loci only the
rt locus, encoding an anthocyanin rhamnosyl-transferase,
had been isolated. Molecular analysis showed that the
unstable rt-Vu15 allele contained a 283 bp TE (Kroon
et al., 1994) highly homologous to the dTph1 element
originally identified in a polymorphic DNA fragment
containing the dfrC gene (Gerats et al., 1990).
To isolate mutants in which various steps in plant development were altered we conducted a self-pollination program of W138 plants. This yielded a collection of mutants
with alterations in growth characteristics, developmental
pattern formation, leaf pigmentation (unpublished data)
and flower pigmentation (this paper). The large majority
of these mutants appeared to be induced by TEs, as they
can somatically and sporogenically revert to wild-type.
Before novel loci can be isolated it was first necessary to
identify the TEs responsible for the mutations and study
their basic characteristics. Genes encoding enzymes from
the flavonoid pathway are particularly suited as a transposon trap as many of them are cloned (Holton and Cornish,
1995) and mutants for such genes are readily identified by
alterations in the flower colour.
In this paper we present an analysis of 50 new flower
pigmentation mutants. Phenotypic and genetic analyses
show that petal colour is influenced by (i) flavonoid metabolism, (ii) intracellular pH of petal cells, and (iii) the shape
of petal cells. Molecular analysis of 13 unstable an3 alleles,
encoding the flavonoid biosynthetic enzyme flavanone 3βhydroxylase (F3H), shows that most mutations are due to
insertions of dTph1 elements, while 1 unstable an3 allele
contained a novel transposon designated dTph2.
39
40 Adèle van Houwelingen et al.
Results
Isolation of flower pigmentation mutants by random
transposon-mutagenesis
The P. hybrida line W138 contains an unstable allele of the
an1 gene (Doodeman et al., 1984), encoding a regulatory
protein of the anthocyanin pathway (Beld et al., 1989; Huits
et al., 1994; Quattrocchio et al., 1993). It bears white flowers
with red or pink spots and sectors that result from somatic
excisions of a TE at the an1 locus (Figure 1b). The an1
gene appears to control anthocyanin synthesis cell-autonomously, because red cells neighbour white cells at the
borders of An11 revertant spots, giving the spots a ‘sharp’
appearance (Figure 1c). Sporogenic excisions result in full
or partial revertants with red or pink flowers respectively, or
to stable recessive mutants with white flowers (Doodeman
et al., 1984).
Visual screening of about 2300 M2 families of W138
plants identified 32 (independent) families that segregated
for novel flower pigmentation patterns, indicating that a
new mutation had occurred. Some typical examples are
shown in Figure 1(d)–(h). In unstable an1-W138 plants
mutations in a second gene could be detected in different
ways. Unstable secondary mutations that resulted in loss
of pigmentation, typically limited the revertant red spots
(originating from reversions of one gene) to certain sectors
of the flower (originating from early reversions of the
second anthocyanin gene) (e.g. Figure 1d). Alternatively,
secondary mutations could change the colour of the
revertant spots, for instance, from predominantly red to
predominantly pink (Figure 1g). In An11 revertant plants
new unstable mutations were recognisable from a novel
spotting phenotype, for instance revertant sectors on a
pale background (an1-W138 flowers have a white background) or spots with diffuse boundaries (Figure 1e and f).
In addition, we screened about 200 000 plants (resulting
from about 4000 selfings) that were generated in the
breeding programs of a company (Novartis Seeds, Enkhuizen). This yielded another 18 pigmentation mutants which
were all in different genetic backgrounds.
All isolated mutants were subjected to a more detailed
phenotypic analysis, to better define the function of the
mutated locus.
Mutations altering flavonoid biosynthesis
Anthocyanins constitute the main flower pigments in P.
hybrida and are synthesized by one of the branches of
the flavonoid biosynthetic pathway (Figure 2). Flavonols,
colourless flavonoids that are synthesized by a different
sidebranch, serve as co-pigments that alter the absorption
spectrum of the anthocyanins.
For all flower pigmentation mutants we analysed the
Figure 1. Phenotypes of typical unstable flower pigmentation mutants of
P. hybrida.
(a) Flower of the line R27, the progenitor of line W138.
(b) Flower of line W138 harbouring the unstable an1-W138 allele.
(c) Phenotype of An11 revertant spots on a petal limb harbouring the
unstable an1-W138 allele.
(d) Flower of a double unstable mutant harbouring an1-W138 and an3-S205.
(e) Flower of a mutant harbouring the unstable an3-W2018 allele in a An11
revertant background.
(f) Phenotype of An31 revertant spots on a petal limb harbouring the
unstable an3-W2018 allele.
(g) Flower of a mutant harbouring the unstable alleles an1-W138 and ht1W2169. The coloured spots (An11 reversions) in the top right part of the
flower are red indicating that these cells are also Ht1 revertant.
(h) Flower of a double mutant harbouring the unstable allele an1-W138
and the stable recessive allele an3-S206.
(i) Flower of a mutant displaying instability at the rt locus.
(j) Flower displaying the unstable shp phenotype.
(k) Detail of revertant spots on an unstable shp flower.
(l) Flower harbouring the unstable mybPh1-X2368 allele in an unrelated
background.
(m) Flower of a mutant harbouring the unstable ph4-V2166 allele.
(n) Flower of a ph7-W2395 mutant just after opening of the bud.
(o) Similar flower as in (n) but about 3 days after opening of the bud.
amount and types of anthocyanin and flavonol endproducts
by thin layer chromatography (TLC) analysis (Table 1). This
showed that all the mutants with white or nearly white
flowers contained strongly reduced anthocyanin levels. In
© Blackwell Science Ltd, The Plant Journal, (1998), 13, 39–50
Transposon insertions in flower pigmentation genes 41
Table 1. Analysis of anthocyanin mutants
Flavonoid endproducts
Locus
Number
of alleles
Revertant
spots
Anthocyanin
Flavonol
Flavonoid
intermediates
Geneproduct*
an1
an3
an6
an9
an10
ht1
rt
12
15
3
2
1
6
1
Sharp
Fuzzy
Fuzzy
Fuzzy
Fuzzy
Fuzzy
Fuzzy
Reduced
Reduced
Reduced
Reduced
Reduced
Reduced
Altered
No effect
Reduced
No effect
No effect
Reduced
Altered
No effect
Dihydroquercetin
Eriodictyol
Dihydroquercetin
Unknown
Coumaric acid
Dihydrokaempferol
Delphinidin
transcription factor
F3H
DFR
GST-homolog
4CL?
F39H
ART
*For details and abbreviations see Figure 2 and text.
one subclass of mutants, the loss of anthocyanin correlated
with a loss of flavonols, indicating that the mutation blocked
an early biosynthetic step that is shared between the
anthocyanin and the flavonol pathway. In other anthocyanin mutants the flavonol levels were not affected, indicating that a late step was blocked. In a third class of mutants,
in which anthocyanin synthesis was diminished but not
completely blocked (pink flowers instead of red; Figure 1g),
the flavonol hydroxylation pattern was altered (accumulation of kaempferol instead of quercetin). To define more
precisely the step in the anthocyanin/flavonol pathway that
was blocked, we analysed the accumulation of (colourless)
pathway intermediates by TLC. The results of the flavonoid
analyses combined with the phenotype of revertant spots
indicated that the mutants represented at least six different
loci (Table 1).
Previous genetic and biochemical work had identified
several anthocyanin loci, some of which were shown to
control specific steps in the pathway (for review see Holton
and Cornish, 1995). Genetic complementation tests showed
that all eriodictyol and dihydrokaempferol accumulating
mutants represented new alleles of the an3 and ht1 locus
(Table 1). These loci are thought to encode or control the
enzymes flavanone 3β-hydroxylase (F3H) and flavanone
39-hydroxylase (F39H) respectively (Holton and Cornish,
1995). Two mutants represented unstable alleles of an9. In
particle bombardment assays an9 complements a Z. mays
bz2– mutant (M. Alfenito, E. Souer, D. Goodman, E. Buell,
J. Mol, R. Koes and V. Walbot; in preparation), and bz2
encodes a gluthathione S-transferase (GST) (Marrs et al.,
1995). The dihydroquercetin accumulating mutants represented either alleles of an1, encoding a regulatory protein,
or an6, encoding the enzyme dihydroflavonol 4-reductase
(Huits et al., 1994; Quattrocchio et al., 1993). The coumaric
acid accumulating mutant represented an allele of an10, a
poorly characterized locus. The observation that an10 –
flowers accumulate coumaric acid (Table 1), while pigmentation can be restored by external feeding of naringenin (data not shown), suggests that an10 encodes or
controls an enzyme in the general phenylpropanoid path© Blackwell Science Ltd, The Plant Journal, (1998), 13, 39–50
way, presumably the structural gene encoding 4 coumaroyl-CoA ligase (4CL).
One of the families in the Novartis Seeds breeding fields
segregated 3:1 for plants having purple flower corollas
and plants having ‘dull grey’ flower corollas with purple
revertant spots (Figure 1i). TLC analysis showed that the
purple flowers accumulated mainly malvidin, while the
main pigments in the spotted flowers were delphinidin,
indicating that the unstable mutation blocked one of the
steps between delphinidin and malvidin. Crosses of this
unstable mutant with an rt– line yielded progeny with
spotted flowers. Thus, the spotted phenotype was due to
instability of the rt locus. This locus encodes the enzyme
anthocyanin rhamnosyl-trasferase (ART; Kroon et al., 1994).
PCR and sequence analysis revealed that this unstable rt
allele harboured a dTph3 element (442bp) at the same
position and in the same orientation as was found previously in the stable recessive rt-R27 allele (Kroon et al.,
1994). In fact, most of the rt– P. hybrida cultivars contain
this rt-R27 allele, without showing any sign of genetic
instability. We therefore assumed that this dTph3 copy is
inactivated. However, the occurrence of rt-mutable plants
shows that this assumption is incorrect and that this
dTph3 copy is capable of transposition. Possibly, the stable
recessive character of this allele in most genetic backgrounds is due to the absence of trans-acting factors
required for transposition (e.g. a transposase source).
For the remaining set of flower pigmentation mutants
the accumulation pattern of flavonoids appeared to be
unaltered, suggesting that the mutation interfered with
another process. These mutants are discussed below.
Mutations altering cell shape
During the genetic complementation assays we crossed
W138 plants (with or without a new unstable mutation)
with the standard an3– tester line W62. Several of these
crosses yielded progeny with a novel unstable flower
pigmentation phenotype, called shrivelled-up (shp), that
42 Adèle van Houwelingen et al.
Figure 2. Simplified diagram of anthocyanin and flavonol biosynthesis.
The anthocyanin endproducts are in white lettering and the colour given
to the flower is indicated in brackets. The flavonol endproducts are boxed.
The enzymes catalysing specific steps in the pathway are indicated in
capitals and the genetic loci are indicated in italics. Abbreviations: 4CL, 4coumaroyl CoA ligase; CHS, chalcone synthase; CHI, chalcone flavanone
isomerase; F3H, flavanone 3β-hydroxylase; F39H, flavanone 39-hydroxylase;
F3959H, flavanone 3959-hydroxylase; DFR, dihydroflavonol 4-reductase; ART,
anthocyanin rhamnosyl transferase
was completely different from the an3 mutable phenotype
in the same genetic background.
Unstable shp flowers have a dull purplish background
with bright purple revertant spots and sectors (Figure 1j).
By TLC analysis we could not detect significant changes in
the anthocyanin and flavonol accumulation pattern. Closer
examination with a dissection microscope showed that all
cells in a Shp1 revertant sector were of the same bright
purple colour. Surprisingly, the dull purplish shp– mutant
background of the flower consisted of isolated (groups
of) bright purple cells on an apparently unpigmented
background (Figure 1k). To examine shp flowers at higher
resolution, we subjected them to Scanning Electron Microscopy (SEM). Figure 3(a) shows that epidermal cells in
wild-type flowers are all very similar and have a usually
pentagonal base with a conical tip. In the mutant background of shpmut flowers most of the cells have a ‘flat tyre’
appearance and are mingled with normal looking cells
(Figure 3b and c). In revertant sectors of a shpmut flower,
all cells have again a wild-type shape (Figure 3B). Thus,
the colour change in shp mutant flowers is caused by the
altered ‘shrivelled-up’ shape of a fraction of the epidermal cells.
Different W138 X W62 crosses yielded progeny that
segregated close to 1:1, 1:0 or 0:1 for wild-type and unstable
shp mutants, depending on the particular W138 plant that
was used. However, we could not detect clear differences
in cell shape between these different W138 plants. Even
some W138 plants that were separated since 10 generations
yielded unstable shp progeny after crossing to W62. Taken
Figure 3. Scanning Electron Microscopy of petal cells.
(a) Petal of a wild-type flower resulting from a cross between a W138 and
W62 plant.
(b) Petal of a flower displaying the unstable shp phenotype. Note the three
sectors of shp1 revertant cells (marked with R) amidst the shp– background.
(c) Detail of shp– cells.
(d) Petal of mybPh1-X2368 flower. Note the sector of five revertant cells
with conical tips amidst the background of flattened mutant cells.
The bar in panels (a), (c) and (d) equals 10 µm and in panel (b) 100 µm.
together it appears that a TE inserted in the shp locus of
W138 shortly after this line was established. However,
because the shp– phenotype is not expressed in the W138
background, the insertion went unnoticed until crosses to
W62 were made. This indicates that a yet unknown factor
in W62 is required for expression of the shp phenotype.
One mutant isolated from the Novartis breeding fields
displayed purple flowers with a shining metallic hue that
were dotted with somewhat darker purple revertant spots,
without the metallic hue (Figure 1l). SEM analysis showed
that the revertant cells had the typical wild-type shape,
while the mutant cells were flattened and lacked the conical
tip (Figure 3d). This phenotype strongly resembles that of
mixta mutants of Antirrhinum majus (Noda et al., 1994).
Genetic analysis indicated that this mutation is an allele of
the mybPh1 locus, which is thought to be the P. hybrida
ortholog of mixta (Avila et al., 1993; Mur, 1995).
Mutations altering intracellular pH
A set of six mutants was obtained where the colour shifted
from red to more bluish (Figure 1m–o). By TLC analysis
no significant alterations in the flavonoid accumulation
pattern could be detected. De Vlaming et al. (1983) previously described five loci that co-segregated with a blue
flower colour and a high pH in flower limb homogenates.
It was assumed that in such mutants the pH of the vacuole
© Blackwell Science Ltd, The Plant Journal, (1998), 13, 39–50
Transposon insertions in flower pigmentation genes 43
was increased, resulting in a change of the anthocyanin
absorption spectrum and a more blue flower colour and
therefore these loci were named ph1 to ph5 (de Vlaming
et al., 1983). More recently a sixth ph locus was identified
(ph6; de Vlaming unpublished data; see also Chuck et al.,
1993).
Corolla limb homogenates of the unstable mutants have
a pH of 6.2 6 0.1, while those of the wild-type progenitor
have a pH of 5.2 6 0.1. Genetic complementation tests
showed that the six mutants represented at least three
different loci. Four mutants represented alleles of the ph4
locus, while one mutant represented a new allele of the
ph3 locus. The sixth mutant (W2395) differed from all other
ph mutants, because its phenotype is expressed late during
corolla development. Corolla limbs that have just opened
still display the wild-type red colour (Figure 1n). Upon
aging of the flower the colour of the corolla changes to a
more purplish colour and a pattern of red revertant spots
and sectors develops (Figure 1o). This suggests that this
mutant represents a novel ph locus. Allelism tests ruled
out that the mutant represents an allele of ph1 to ph6
indicating that it defines a new locus (ph7).
Molecular analysis of unstable an3 alleles
In order to identify the TEs that were responsible for the
generated mutations, we focussed on those mutants where
we suspected that a gene had been hit for which a probe
was available. The TLC analyses indicated that the an3
locus contains the structural gene for F3H. We, therefore,
isolated the P. hybrida gene encoding F3H using a cDNA
clone that was isolated by Britsch et al. (1991). Sequence
analysis showed that this gene contains two introns
(Figure 4a), in identical positions as in the f3h gene from
Dianthus caryophyllus (Dedio et al., 1995) and Arabidopsis
(Pelletier and Shirley, 1996). The transcription start was
not mapped, but a putative TATA box was found 162 bp
upstream of the translation start. As shown below, the
isolated gene indeed derived from the an3 locus and is
therefore referred to as an3.
Amplification of the wild-type an3 alleles (an3-R27, an3W138A, an3-W138B) with primers 1 and 12 yielded a
product of 1.2 kb, while the unstable alleles an3-R134, an3S205, an3-W2018, an3-W2140, an3-V2221, an3-W29, an3W2298, an3-W2303 and an3-X2092 all yielded a 1.5 kb
product and variable amounts of the 1.2 kb product
(Figure 4b). Sequence analysis showed that these alleles
all harboured a 284 bp dTph1 element at different positions
in the an3 gene, that was flanked by an 8 bp target site
duplication (TSD) in all cases (not shown). For an3-W2258
we could not detect an insertion in the coding sequence.
However, a 0.6 kb fragment was amplified with primers 4
and 14 (Figure 4c, lane 25), while a 0.3 kb fragment (lane
19) was found in wild-type. Sequence analysis showed
© Blackwell Science Ltd, The Plant Journal, (1998), 13, 39–50
that a dTph1 element had inserted ~100 bp upstream of
the translation start, which may explain the leaky character
of this an3-W2258 allele.
To verify whether the detected dTph1 elements were
indeed responsible for the mutant phenotype we analysed
revertant (An31) progeny by PCR. This showed that reversion of the mutant phenotype for all alleles correlated with
excision of dTph1 (data not shown).
DNA gel blot analysis of an3-S857 homozygotes and
derived revertants indicated that this allele contained two
independent insertions of about 300 bp in the 59 end of
the gene (not shown). However, the amplification products
obtained with primers 4 and 14 were inconsistent with
the DNA gel blot data, apparently because this region
contained a structure that could not be amplified (not
shown). Further analysis showed that this region of an3S857 contained two 300 bp TEs that could be amplified
separately with the primer pairs 2114 and 413 (Figure 4b,
lane 18 and 4c, lane 36). Sequencing showed these were
two dTph1 copies in an inverted orientation that were
separated by only 40 bp (Figure 4a). When we examined
the other an3 alleles we found that an3-R134, an3-S205
and an3-X2092 also harboured a second 300 bp TE at the
59 end of the gene. Analysis of some parental An31 W138
plants that were used at the time of isolation of these an3
alleles showed that they also contained this insertion
(Figure 4c, lane 20; an3-W138A). Therefore, it appears that
in some W138 plants a dTph1 element inserted 30 bp
upstream the translation start codon, giving rise to an3W138A, without being noticed because it does not cause
a mutant phenotype. Unstable an3 mutants derived from
an3-W138A, thus, have a second TE elsewhere in the gene
that is responsible for the mutant phenotype. Revertants
of these alleles invariably lost the downstream element,
while the upstream element remained present (data not
shown). Plants that lost the dTph1 element upstream of
the ATG and retained the downstream copy kept a mutant
phenotype.
Figure 5(a) shows that the different trapped dTph1 elements were all very similar. They were all 284 bp in size
and sequence polymorphisms were found at only 19 sites
scattered throughout the element. The highest divergence
(13 mismatches) is found between dTph1 elements of
groups I and V. The an3 target sites surrounding the dTph1
insertions did not show any apparent sequence homology.
Renckens et al.(1996) suggested that dTph1 elements may
preferentially integrate in regions with 4–6 bp inverted
repeats. We found similar repeats in 10 out of the 13 dTph1
target sequences in an3, but also in 4 out of 5 randomly
chosen an3 sequences, indicating that this finding is of
little significance. Therefore, if dTph1 has a target site
preference, it is not immediately recognisable from the
DNA sequence.
44 Adèle van Houwelingen et al.
© Blackwell Science Ltd, The Plant Journal, (1998), 13, 39–50
Transposon insertions in flower pigmentation genes 45
site duplication. Although the 14 bp imperfect terminal
inverted repeats are partially homologous to those of
dTph1 and dTph3, the internal sequences show no further
homology. Apparently this insertion represents a new
family of TEs and was named dTph2. DNA gel blot analyses
of six different P. hybrida lines, including W138, showed
that each contained about 20 dTph2 copies (data not
shown)
Analysis of stable recessive an3 alleles
Figure 5. Sequence of dTph1 elements and dTph2.
(a) The full sequence of the dTph1 element isolated from dfrC (Gerats
et al., 1990) is shown at the top. The nucleotides marked with a dot are
polymorphic in other dTph1 copies. The lower part shows the sequences
at these polymorphic sites for dTph1 elements of six distinct groups. Group
I: an3-W138A, an3-S205, and an3-T3631 (see Koes et al., 1995 for the latter
allele); group II: an3-W2258, an3-S857, an3-W2018, an3-X2092, an3-R134,
an3-V2221, and an3-W2140; group III: an3-W2303, and dfrC-W138; group
IV: an3-W29; group V: an3-W2298; group VI: rt-Vu15 (see Kroon et al., 1994).
(b) Sequence of the dTph2 element isolated from an3-GSm.
Terminal inverted repeats are boxed. The ∆ indicates a missing nucleotide.
The an3-GSm allele contains a novel TE
PCR amplification of the an3-GSm allele with primers 1
and 12 yielded a 1.4 kb PCR product in addition to a small
amount of 1.2 kb product (Figure 4b, lane 14), indicating
the insertion of a TE of about 200 bp. Sequencing showed
that this TE was 177 bp in size with 14 bp imperfect terminal
inverted repeats (Figure 5b) and flanked by an 8 bp target
The an3-W62 reference allele originated from a spontaneous event in a genetic background unrelated to W138.
When genomic DNA of an3-W62 homozygotes was used
to amplify the f3h coding sequence or the 59 flanking region
by PCR, no products could be obtained (Figure 4b, lane 15
and Figure 4c, lane 33). Genomic DNA gel blot analysis
showed that this was due to the complete absence of
sequences hybridising to the f3h cDNA and the f3h 59
flanking region (Figure 6a). Thus, the an3-W62 allele
appears to be a complete deletion of the an3 gene
(Figure 6c), explaining the complete absence of f3h transcripts in this mutant (data not shown).
In the W138 populations we identified one mutant, S206,
with nearly white flowers in which the an1 spotting pattern
was still faintly visible (Figure 1h). Further analysis showed
that the mutation had occurred in the an3 locus and
resulted in the absence of f3h transcripts (data not shown).
However, the an3-S206 allele never displayed somatic or
sporogenic instability and thus appears to be a stable
recessive allele. DNA from an3-S206 homozygotes could
be used as a template to amplify the f3h coding sequence
with primers 1 and 12 (Figure 4b, lane 5), but not the 59
flanking region with primers 4 and 14 (Figure 4c lane 23).
DNA gel blot analysis indicated that this was due to a
rearrangement at the 59 end of the an3 gene. Hybridization
of the f3h cDNA to the wild-type progenitor allele an3-R27
detected a 2.3 kb XbaI fragment carrying the 39 part and a
1.3 kb fragment carrying the 59 part of the f3h gene
(Figure 6b, lane 8). In an3-S206 homozygotes the 2.3 kb
fragment could still be detected, but the 1.3 kb fragment
was absent (lane 7). Hybridization of the f3h 59 flanking
Figure 4. Molecular analysis of unstable an3 alleles.
(a) Diagram showing the structure of the wild-type and unstable an3 alleles. Boxes represent exons and the filled regions translated sequences. The greyfilled triangles depict the dTph1 insertions responsible for the phenotype of the indicated mutants. The orientation of dTph1 is indicated by arrowheads; to
the right if the orientation is as in Figure 5(a) or to the left if the orientation is opposite.
The dTph1 insertions reponsible for the mutant phenotype of an3-S857, an3-R134, an3-X2092 and an3-S205 are shown on long bar, to indicate that these
alleles derived from an3-W138A which harbours a dTph1 insertion (open triangle) that does not cause a mutant phenotype. The black filled triangle represents
the dTph2 insertion in an3-GSm.
The scheme below the an3 gene shows the primers used for PCR analysis (arrowheads) and the DNA fragments that were amplified (connecting lines).
(b) Ethidium bromide stained PCR products amplified from DNA extracted from leaves of an3 mutants with the primer pairs 1112, or 2114.
(c) Ethidium bromide stained PCR products amplified from DNA extracted from leaves of an3 mutants with the primer pairs 4114 or 413.
Note that PCR products containing a TE insertion form a heteroduplex with the wild-type sized excision products, which can be seen as an intermediate
sized band (Huits et al., 1994).
© Blackwell Science Ltd, The Plant Journal, (1998), 13, 39–50
46 Adèle van Houwelingen et al.
Figure 6. DNA gel blot analysis of the stable recessive an3-W62 and an3S206 allele.
(a) DNA gel blot analysis of EcoRI digested DNA of the lines W62 (an3W62) and W137 (An31-R27). Left panel: f3h cDNA probe; middle panel: f3h
59 probe; right panel: chsA cDNA probe (control).
(b) DNA gel blot analysis of XbaI digested DNA of an3-S206 and An31-R27
homozygotes. Left panel: f3h cDNA probe; right panel: f3h 59 probe.
(c) Genomic map of the An31-R27 wild-type allele, an3-W62, and an3-S206.
Exons are indicated as boxes; primers used for PCR analysis as arrows.
The bars above the map show the probes used for DNA gel blot analysis.
X: XbaI site; zigzag line: rearrangement at the 59 end of the gene; -( )-:
absence of an3 sequences.
PCR analyses which showed that the region in between
primers 15 and 18 (see Figure 6c) could still be amplified
(data not shown). Taken together these data suggest that
the an3-S206 allele resulted from a chromosomal
rearrangement, presumably an inversion or translocation,
that separated the 59 flanking region of an3 from the protein
coding region.
To establish the exact breakpoint of this rearrangement
we isolated part of the an3-S206 allele from a genomic
library and sequenced from the ATG translation start into
the region upstream. This showed that in an3-S206 the
region upstream nt.-32 has been replaced by a new,
unknown, sequence. Because this is precisely the site
where in the wild-type progenitor allele a dTph1 element
was present (Figure 4a, an3-W138A), we believe that this
element caused the observed rearrangement.
One plant from a cross between an3-R134 and an3-S206
homozygotes failed to yield an3 amplification products in
PCR reactions with the primer pair 4 and 14, indicating a
rearrangement at the 59 end of the original an3-R134
allele. This new allele, an3-W1006, was subsequently made
homozygous and further analysed by PCR. These homozygotes (an3-W1006/an3-W1006) failed to yield PCR products
with the primer pairs 4114, 15118 and 1112 (data not
shown; see Figures 4a and 6c for the position of the
primers), while the regions downstream of the dTph1
insertions in an3-R134 (nt. 429–1421) could still be amplified. This indicates that the sequences upstream the dTph1
insertion in the first exon of an3-R134 are missing in
an3-W1006.
Discussion
region to an3-R27 DNA also detects this 1.3 kb fragment
and in addition a 3.6 kb promoter fragment (lane 10). In
an3-S206 homozygous plants, however, the 1.3 kb fragment
was absent and two new fragments of 2.3 and 1.0 kb were
found (lane 9). The 3.6 kb fragment appeared unaltered,
suggesting that the an3 promoter region was still present
in the genome of an3-S206 plants. This was confirmed by
Mutagenesis is a powerful strategy to identify genes that
control specific processes. We exploited P. hybrida lines
with highly active TEs to generate a set of mutants with
alterations in flower development. As described in this
paper we first focused on flower pigmentation mutants to
isolate the TEs from cloned target genes, to enable the
subsequent isolation of novel loci using these TEs.
© Blackwell Science Ltd, The Plant Journal, (1998), 13, 39–50
Transposon insertions in flower pigmentation genes 47
Flower pigmentation genes as a transposon trap
The isolation of TEs from Z. mays and A. majus was
strongly facilitated because of the large collection of
unstable alleles of various loci that had been collected over
decades of genetic work. In species where such a mutant
collection was not available other strategies had to be
designed to trap TEs in specific target genes. Attempts to
trap TEs in transgenes have been largely unsuccessful; in
most of the mutants the transgene was inactivated by
methylation and not by mutation (Meyer et al., 1992;
Renckens et al., 1992). Mutations that alter the nitrate
reduction can be directly selected because they cause
resistance to chlorate. This enabled the trapping of TEs in
the nitrate reductase gene of Nicotiana tabacum
(Grandbastien et al., 1989), N. plumbaginifolia (Meyer et al.,
1994) and P. hybrida (Renckens et al., 1996) and the
chlorate1 gene of Arabidopsis (Tsay et al., 1993).
One of the advantages of using flower pigmentation
genes as a TE trap is that it involves multiple loci scattered
throughout the genome. This increases the efficiency and
the chances of trapping independent TEs. On the other
hand it requires some further analysis of the mutants to
identify the mutated gene. We found mutations that affected three seemingly unrelated factors: (i) the biosynthesis
of flavonoid pigments, (ii) the intracellular pH, and (iii) the
shape of petal cells. Because virtually all structural genes
required for anthocyanin synthesis have been isolated, the
first category of mutants is particularly suitable for the
isolation of novel TEs. For most mutants it takes only a
visual inspection of the mutant flower (to determine the
colour of mutant cells and the phenotype of revertant
spots) and some simple TLC analyses of the accumulated
flavonoids to determine the gene that is most likely mutated. Thus, the molecular analyses can usually be limited
to one or at most two loci.
Plant breeding companies produce for many species
large populations of greenhouse or field grown plants, that
can be exploited for mutant screens. Although we were
only interested in P. hybrida mutants, it took little effort to
identify unstable flower pigmentation mutants in breeding
programs for other species like for instance Pelargonium.
Therefore, flower pigmentation genes are likely to be
convenient TE traps in a wide variety of species.
Identification of TEs responsible for genetic instability in
P. hybrida; implications for gene-tagging
Our data show that the high incidence of mutations found
in progeny of the line W138 is largely due to the high
activity of dTph1 elements. Most of the recovered mutant
alleles are unstable and all molecularly analysed alleles
harbour insertions of dTph1. This includes nine an3 alleles
(Figures 4 and 5), two alleles of aberrant leaf and flower
© Blackwell Science Ltd, The Plant Journal, (1998), 13, 39–50
(alf; Souer, 1997) and single alleles for no apical meristem
(nam, Souer et al., 1996), and an11 (de Vetten et al., 1997).
The activity of dTph1 elements is so high, that on two
occasions insertions were found without any (phenotypic)
selection (an3-W138A, Figure 4 and dfrC, Gerats et al.,
1990; Huits et al., 1994). Such dTph1 insertions are easily
overlooked as they do not give a phenotype by themselves,
but can have a profound influence on the further genetic
behaviour of a plant. They can, for instance, cause subsequent rearrangements resulting in the recovery of
(untagged) stable recessive alleles (cf. an3-S206) or alter
the behaviour of a nearby dTph1 copy (van Houwelingen
et al., in preparation).
In genetic backgrounds other than W138 the majority of
mutations are again due to dTph1 elements (three out of
four an3 alleles), but also other elements like dTph2 (this
paper) or dTph3 (Kroon et al., 1994) were found. The same
trend is seen for the insertions in an1; all unstable alleles
isolated in W138 harboured dTph1 insertions, while novel
elements (dTph5 and dTph6) could only be trapped in
other backgrounds (Spelt et al., unpublished).
DNA gel blot analyses showed that the copy number of
dTph2 is 10–20 in all P. hybrida lines tested including
W138 (unpublished data). Furthermore, these experiments
showed that dTph2 elements are mobile in W138. The
copy number of dTph1 elements is 10–30 in most P. hybrida
lines, but is unusually high (.200) in R27 derivatives
including W138 (Gerats et al., 1990; Huits et al., 1994). This
specific amplification of dTph1 elements may explain why
unstable alleles isolated in the W138 background harbour
mainly dTph1 insertions, while in other backgrounds the
chances of trapping different TEs are higher.
Based on small sequence variations the 12 dTph1 elements that inserted in an3 can be divided in five groups
with sequences that differ in at most 13 positions. Seven
independent an3 alleles isolated in the W138 background
contain a dTph1 element with identical sequence (group II).
One possibility is that these alleles result from preferential
transposition of one element from a single donor site into
an3. Close linkage of donor and acceptor sites may, for
instance, contribute to preferential insertion of a specific
copy, as has been shown for Ac of maize (Dooner and
Belachew, 1989; Jones et al., 1990; Smith et al., 1996).
Given the specific amplification of dTph1 elements in
W138, it seems equally possible that seven different donor
dTph1 copies were involved. In fact, the dTph1 copy located
just 30 bp upstream the an3 translation start in many of
the progenitor W138 plants (an3-W138A) was apparently
not involved in creating new insertions elsewhere in the
gene (an3-R134; an3-S857 and an3-X2092) as it belongs to
a different group (I).
The finding that TE insertions in known loci involved
dTph1 elements, indicates that the unstable alleles for
other loci are very likely to contain dTph1 insertions as
48 Adèle van Houwelingen et al.
well. Starting from this premise we were indeed able to
isolate four new loci, the pattern formation gene no apical
meristem (Souer et al., 1996) and the regulatory anthocyanin loci an11 (de Vetten et al., 1997), an2 (Quattrocchio,
1994), and an1 (Spelt et al., unpublished data), using a
novel method to quickly sort out the .200 dTph1 elements
in W138 generated mutants (Souer et al., 1995).
Classification of TEs in families
The P. hybrida TEs isolated so far are remarkably small. In
fact, dTph2 is until now the smallest plant TE isolated that
is still capable of transposition. Tourist elements found in
monocot plants are even smaller (133 bp in average), but
do not appear to be mobile any longer (Bureau and Wessler,
1992). This indicates that the P. hybrida TEs are all nonautonomous and depend for their mobility on a transposase source elsewhere in the genome. Genetic mapping
showed that the mobility of dTph1 elements is controlled
by the Act1 locus which itself appears to be immobile
(Huits et al., 1994). We consider dTph3 to represent a novel
family because (i) the homology between dTph1 and dTph3
is limited to a few nucleotides in the terminal inverted
repeats and (ii) the dTph3 copy at the rt locus does not
transpose in W138, even though it is mobile in other
backgrounds. This indicates that dTph1 and dTph3 require
different trans-acting factors for mobility. The homology
of dTph2 to either dTph1 or dTph3 is equally limited.
Therefore, we assume dTph2 to represent a third family of
elements, although we do not have direct evidence that it
depends on a third set of transacting factors for mobility.
Based on sequence homology of the terminal inverted
repeats and the size of the target site duplication (8 bp)
found after integration, a group of related TEs, called the
Ac-family, has been classified that are thought to originate
from a common ancestor (Calvi et al., 1991). This family
includes Ac (Z. mays), Tam3 (A. majus), but also TEs of
animal origin such as the P and hobo elements of Drosophila melanogaster. According to these criteria, dTph1,
dTph2 and dTph3 can be included into this family. The
termini of these P. hybrida elements share respectively
7 bp, 6 bp, and 6 bp sequence identity with the 11 bp
terminal inverted repeat of Ac. Moreover, they all create
an 8 bp target site duplication upon integration.
Experimental procedures
Transposon mutagenesis program
To generate novel transposon induced mutations we conducted
a self-pollination program using the line P. hybrida W138. Typically,
M1 plants (up to a maximum of 20 plants per family) were grown
to flowering in small pots and they were self-pollinated again to
obtain M2 seeds. For each M2, 10–20 plants were grown to
flowering in small jiffy pots and visually screened for segregating
novel mutations. Because W138 contains mutant alleles at the
hf1, hf2 and rt loci, mutations that alter downstream steps in
anthocyanin synthesis could not be detected in this background.
In addition we visually screened about 200 000 P. hybrida plants
that were generated during the breeding programs of S&G Seeds
(Enkhuizen, The Netherlands) for novel unstable phenotypes.
Interesting mutants were self-pollinated to maintain the new
allele and crossed to tester lines to establish allelism with previously identified mutants.
Thin layer chromatography analysis of flavonoids
To determine the nature of flavonol or anthocyanin endproducts,
the limbs and occasionally also the tube of mutant flower corollas
were extracted in 2N HCl. After boiling for 20 min the flavonol
and anthocyanin aglycones were concentrated by extraction with
iso-amylalcohol and separated on cellulose TLC plates (Merck) in
water:acetic acid:HCl 10:30:3. Defined flavonol or anthocyanin
compounds, obtained by extraction of well characterized P. hybrida
lines served as markers.
Colourless intermediates of the flavonol and anthocyanin pathway were analysed as described by Koes et al. (1995). Mutantspecific spots were identified by running flavonoid extracts from
mutants and related wild-types in parallel. Their identity was
established by co-migrating purified flavonoids (obtained from
Apin Ltd. and Sigma)
Scanning electron microscopy
For Scanning Electron Microscopy pieces of petal tissue with a
diameter of about 5 mm were fixed, dehydrated, critical point
dried and sputtered with gold as described previously (Souer
et al., 1996). Specimens were examined in a JEOL JSM6400 SEM
using accelerating voltages of 10 kV.
Determination of the pH of petal limb extracts
For pH measurements two corolla limbs were ground with pestle
and mortar in 2 ml. of distilled water. The pH was measured
within 5 min after grinding with a pH electrode.
DNA and RNA methodology
P. hybrida DNA was extracted from leaves following a modified
version (Souer et al., 1995) of the protocol described by Dellaporta
et al. (1983). The f3h gene was amplified from P. hybrida W138
by PCR with primers complementary to the ends of the f3h cDNA
(Britsch et al., 1991), cloned in pBluescript-KS and sequenced. A
genomic library of the An31 line W137 was constructed in lambdaGEM 11 and screened with a f3h cDNA probe. One positive
genomic clone was selected to subclone and sequence a 2.1 kb
fragment containing 1500 bp promoter sequence and 600 bp
coding sequence.
Analysis of an3 mutants was performed by amplifying different
parts of the f3h gene from genomic DNA by PCR. The primers
used (see Figure 4A for their position in the f3h gene) have the
following sequences: an3-1: 59-dGTTGTGGATCATGGGGTTG-39;
an3-2: 59-dGGAATTCCGGAGGGTTGTAAATGGCAC-39; an3-3: 59dGCCATTTACAACCCTCC-39; an3-4: 59-dGGAATTCCACGCTTACATGCAAC-39; an3-12: 59-dATGACCGTGGTCACCAAGATTG-39;
an3-14: 59-dCGGGATCCCATTGCTGAATTGGTTGT-39. Sequence of
other primers used (see Figure 6C for their position) for PCR
© Blackwell Science Ltd, The Plant Journal, (1998), 13, 39–50
Transposon insertions in flower pigmentation genes 49
analysis of stable recessive an3 alleles: an3-15: 59-dAAGGCCTGAGCCTTCTTGTG-39; an3-18: 59-dCGGAATTCGGTACTGTCAGTGATTGT-39. PCR fragments were cut at restriction sites in the
primer ends and cloned in pBluescript-KS for sequence analysis.
To clone the an3-S206 allele, a genomic library of 15–20 kb
Sau3AI fragments was made in lambda-GEM 11 and screened
with the f3h cDNA probe. One positive clone was restriction
mapped and a fragment carrying the an3 protein coding region
plus the upstream sequence was subcloned and partially
sequenced.
Sequence analysis was performed using asymmetric PCR with
fluorescent M13 primers, employing an Applied Biosystems DNA
sequencer model 370 A.
For DNA gel-blot analysis 10 µg of genomic P. hybrida DNA was
cut with a restriction enzyme, size-separated by agarose gel
electrophoresis, and blotted on Hybond-N membranes (Amersham). Membranes were hybridized with 32P-labelled DNA fragments for 18–36 h at 60°C in a buffer containing: 0.5 M Na2HPO4,
pH 7.2, 1 mM EDTA, 7% SDS, 1% BSA and washed in 0.1xSSC/
0.1% SDS at 60°C prior to autoradiography.
Acknowledgements
We are grateful to Dr Johan Oud from Novartis Seeds, Enkhuizen,
The Netherlands for giving us the opportunity to screen the
company’s breeding fields for unstable mutants. We thank Saskia
Kars from the Faculty of Earth Sciences for her help with the SEM
analysis. Thanks are also due to Pieter Hoogeveen and Martina
Meesters for their care of the plants and Wim Bergenhenegouwen
and Fred Schuurhof for photography. A.H. and E.S were supported
by the Netherlands Technology Foundation (STW) with financial
aid from the Netherlands Organisation for the Advancement of
Research (NWO).
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EMBL Data Library accession numbers AFO22142 (anthocyanin -3 gene) and AFO22143 (dTph2).
© Blackwell Science Ltd, The Plant Journal, (1998), 13, 39–50