Download ACC Synthase Genes are Polymorphic in Watermelon (Citrullus spp

Plant Cell Physiol. 49(5): 740–750 (2008)
doi:10.1093/pcp/pcn045, available online at www.pcp.oxfordjournals.org
ß The Author 2008. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists.
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ACC Synthase Genes are Polymorphic in Watermelon (Citrullus spp.)
and Differentially Expressed in Flowers and in Response to Auxin
and Gibberellin
Ayelet Salman-Minkov 1, Amnon Levi 2, Shmuel Wolf
3
and Tova Trebitsh
1,
*
1
Department of Life Sciences, Ben-Gurion University of the Negev, POB 653, Beer-Sheva 84105, Israel
2
USDA, ARS, US Vegetable Laboratory, 2700 Savannah Highway, Charleston, SC 29414, USA
3
The Institute of Plant Sciences and Genetics in Agriculture, The Hebrew University of Jerusalem, Faculty of Agricultural,
Food and Environmental Quality Sciences, PO Box 12, Rehovot 76100, Israel
Sequence data from this article can be found in the GenBank/
EMBL data libraries under accession numbers EF154455,
EF154456, EF154457 and EF154458.
The flowering pattern of watermelon species (Citrullus
spp.) is either monoecious or andromonoecious. Ethylene is
known to play a critical role in floral sex determination of
cucurbit species. In contrast to its feminizing effect in
cucumber and melon, in watermelon ethylene promotes male
flower development. In cucumber, the rate-limiting enzyme
of ethylene biosynthesis, 1-aminocyclopropane-1-carboxylate
(ACC) synthase (ACS), regulates unisexual flower development. To investigate the role of ethylene in flower development, we isolated four genomic sequences of ACS from
watermelon (CitACS1-4). Both CitACS1 and CitACS3 are
expressed in floral tissue. CitACS1 is also expressed in
vegetative tissue and it may be involved in cell growth
processes. Expression of CitACS1 is up-regulated by exogenous treatment with auxin, gibberellin or ACC, the immediate
precursor of ethylene. No discernible differential floral sexdependent expression pattern was observed for this gene. The
CitACS3 gene is expressed in open flowers and in young
staminate floral buds (male or hermaphrodite), but not in
female flowers. CitACS3 is also up-regulated by ACC, and is
likely to be involved in ethylene-regulated anther development.
The expression of CitACS2 was not detected in vegetative or
reproductive organs but was up-regulated by auxin. CitACS4
transcript was not detected under our experimental conditions.
Restriction fragment length polymorphism (RFLP) and
sequence tagged site (STS) marker analyses of the CitACS
genes showed polymorphism among and within the different
Citrullus groups, including watermelon cultivars, Citrullus
lanatus var. lanatus, the central subspecies Citrullus lanatus
var. citroides, and the desert species Citrullus colocynthis (L).
Introduction
Unisexual flower development is a process by which
separate male or female flowers are developed either on the
same plant (monoecious) or on separate plants (dioecious)
(Lebel-Hardenack and Grant 1997). Plant hormones are
implicated in various aspects of reproductive organ development both in hermaphrodite plants and in monoecious
and dioecious plants (Lebel-Hardenack and Grant 1997).
Generally, in dicots, higher levels of auxins, cytokinins and
ethylene are correlated with female sex expression, whereas
gibberellin favors differentiation of male sex organs
(Khryanin 2002).
In the Cucurbitaceae, a wide range of sex phenotypes
has evolved and, as in other unisexual plant species, floral
dimorphism is genetically controlled by sex-related loci and
modified by environmental and hormonal factors (LebelHardenack and Grant 1997, Roy and Saran 1990). In most
of the cucurbitaceous species, the gaseous plant hormone
ethylene is the principal hormone that regulates femaleness.
This occurs by increasing the ratio between pistillate flowers
(female or hermaphrodite) and male flowers or by reducing
the number of nodes with male flowers prior to the
appearance of the first pistillate flower (Roy and Saran
1990, Rudich 1990).
Watermelon (Citrullus lanatus var. lanatus) is a major
cucurbit crop and an important vegetable crop (FAO 1995).
The flowering pattern of watermelon Citrullus spp. is either
monoecious (male and female flowers) or andromonoecious
(male and hermaphrodite flowers) (Rudich and Zamski
1985). Typically, in watermelon cultivars, the number of
pistillate flowers (either female or hermaphrodite) is low,
with a phenological interval of 4–15 male flowers followed
by a pistillate flower, depending on the genotype. Genetic,
physiological and molecular data on watermelon reproduction are scarce, and no gynoecious, androecious or
Keywords: ACC synthase — Ethylene — Flower development — Restriction fragment length polymorphism —
Sequence tagged site markers.
Abbreviations: ACC, 1-aminocyclopropane-1-carboxylate;
ACS, ACC synthase; AVG, 1-aminoethoxyvinylglycine; RFLP,
restriction fragment length polymorphism; STS, sequence
tagged site.
*Corresponding author: E-mail, [email protected]; Fax, þ972-8-646-1710.
740
Characterization of the watermelon ACS gene family
hermaphrodite Citrullus plants have been reported (Rudich
and Zamski 1985). Production of seedless watermelon has
increased significantly in recent years, and 460% of the
watermelon produced in the USA in 2005 is of the seedless
type (NWPB 2006). Seed companies have great interest in
developing tetraploid breeding lines that produce female
flowers entirely, and could be used effectively in production of
triploid seeds through pollination with diploid parental lines.
Several physiological studies have been conducted to
decipher the hormonal regulation of sex expression in
watermelon. However, exogenous treatment with most of
the plant hormones resulted rather in a general growthinhibiting effect, with inconsistent reports concerning their
effect on sex expression (Rudich and Zamski 1985). On the
other hand, treatment with ethylene-releasing agents
resulted mostly in the suppression of ovary development
(Rudich and Zamski 1985). This observation is supported
by the finding that reducing the endogenous level of
ethylene, both by inhibitors of its biosynthesis and
perception, hastened the appearance of the first pistillate
flower and increased the pistillate to male flower ratio,
i.e. promoted femaleness (Rudich and Zamski 1985,
Sugiyama et al. 1998).
The masculinizing effect of ethylene in watermelon is
in stark contrast to the feminizing effect of ethylene in
other cucurbits. Extensive physiological and molecular
studies in cucumber and melons have established the
role of ethylene as the female hormone (Rudich 1990,
Perl-Treves 1999). 1-Aminocyclopropane-1-carboxylate
(ACC) synthase (ACS, EC 4.4.1.14), which catalyzes the
conversion of S-adenosylmethionine to ACC, is considered
to be the rate-limiting enzyme in the ethylene biosynthetic
pathway and is encoded by a multigene family in most plant
species (Wang et al. 2002).
In common with members of gene families in general,
the ACS genes isolated from several cucurbits exhibit
differential regulation during plant and flower development
and in response to plant hormones and external stimuli
(Huang et al. 1991, Kamachi et al. 1997, Trebitsh et al.
1997, Shiomi et al. 1998, Rimon Knopf and Trebitsh 2006).
In cucumber, two of the four members of the ACS gene
family, CsACS1/G and CsACS2, are differentially expressed
in male and female flowers, with a higher expression in
female floral tissue (Kamachi et al. 1997, Yamasaki et al.
2003, Rimon Knopf and Trebitsh 2006). The importance of
ethylene and ACS genes in various developmental processes
combined with physiological and molecular studies contributed towards elucidating the molecular mechanisms of
sex determination in cucumber and to the development of
ethylene-linked molecular markers useful for the identification of female genotypes in cucumber or for the selection of
melon cultivars with long shelf life (Akashi et al. 2001,
Kahana et al. 1999, Zheng et al. 2002, Perl-Treves 1999,
741
Silberstein et al. 2003, Yamasaki et al. 2003, Mibus and
Tatlioglu 2004, Rimon Knopf and Trebitsh 2006).
We report the isolation and characterization of four
genomic sequences of ACS paralogs (CitACS1, CitACS2,
CitACS3 and CitACS4) and three cDNA sequences
(CitACS1, CitACS2 and CitACS3) that were isolated
from an F4 plant derived from a cross between
C. colocynthis (L.) Schrad [a desert-dwelling, crosscompatible relative of watermelon (C. lanatus)] and the
watermelon cultivar ‘Sugar Baby’ (C. lanatus var. lanatus).
The CitACS genes isolated in this study were differentially
expressed during male, female and hermaphrodite flower
development. The CitACS genes are also differentially
regulated in response to IAA, gibberellin and ACC
treatments. Analysis with restriction enzymes and primer
pairs specific to the CitACS gene sequences revealed several
restriction fragment length polymorphism (RFLP) and
sequence tagged site (STS) markers showing significant
differences between genotypes representing C. lanatus var.
lanatus (cultivated watermelon), C. lanatus var. citroides
(that has higher genetic diversity than the cultivated type)
and C. colocynthis.
Results
Isolation and characterization of watermelon ACS genes
Four ACS genes were amplified by PCR using
degenerate oligonucleotides as primers and genomic DNA
isolated from CL-F4 (colocynthis–lanatus F4) which exhibited a higher proportion of female to male flowers along the
main axis compared with either ‘Sugar Baby’ or the ‘Malali’
cultivars (0.45 vs. 0.15 pistillate/male flowers). The genomic
sequences of the ACS genes were designated CitACS1,
CitACS2, CitACS3 and CitACS4 (1,336, 891, 1,577 and
914 bp, respectively, Fig. 1). Based on the conserved
sequence pattern of the exon–intron junction and on the
genomic structure of known ACS genes, the CitACS
sequences amplified by the degenerate primers extend
between the second and fourth exon and could contain
two introns (Huang et al. 1991, Rottmann et al. 1991, Liang
et al. 1992, Reddy 2007). Indeed we found that the coding
sequences of CitACS1, CitACS2 and CitACS3 are interrupted by two introns at conserved positions (92 and
588 bp, 110 and 119 bp, and 94 and 830 bp, respectively,
Fig. 1). In contrast, the coding sequence of CitACS4 is
interrupted by a single intron (260 bp) and lacks the intron
that separates the second and third exon of ACS genes
(Fig. 1). To verify the deduced location of the exon–intron
junctions, we isolated the cDNA region corresponding to
the partial genomic sequences of CitACS1–3 as well as the
genomic sequence and the full-length cDNA of CitACS1
and CitACS3 (Supplementary Figs S1, S2). The conceptual
translations of the cDNA sequences confirmed the location
742
Characterization of the watermelon ACS gene family
Mval
2247
HindIII
26
Mval
495
Mval Mval
741 777
XbaI
1181
Mval MspI
1650 1752
Mval
2220
CitACS1
XbaI
MspI
517
707
Mval MspI MspI Mval
304 453
687
816
CitACS2
HindIII
426
MspI
Mval
223
451
AvaI Mval
658 729
MspI
690
MspI
1642
HindIII
909
AvaI
1625
Mval Mval
1979 2056
HindIII MspI
1881 2030
Mval
2632
HindIII AvaI
2616 2659
CitACS3
Mval Mval
158 194
Mspl
838
HindIII Mspl AvaI
544
718 837
CitACS4
100 bp
Fig. 1 Schematic representation of the four ACS genomic sequences isolated from watermelon. The exons are gray blocks, the introns are
thin lines between exons, and the 50 and 30 untranslated regions are thick lines. Restriction enzyme recognition sites and their location are
indicated for each gene.
of the exon–intron junctions. The genomic structure of
CitACS1 and CitACS3 is comprised of five exons
separated by four introns at conserved positions (CitACS1
111, 92, 588 and 120 bp; and CitACS3 108, 94, 830 and
305 bp, Fig. 1). As depicted in Fig. 2, the proteins encoded
by CitACS1 and CitACS3 (486 and 495 amino acids,
respectively) contain the seven highly conserved regions of
ACSs as well as the 12 amino acids of the active site of ACS
enzymes and the 11 invariant amino acids conserved among
ACSs (Huang et al. 1991, Rottmann et al. 1991, Liang et al.
1992, Kende 1993) (Fig. 2). The deduced amino acid
sequences encoded by the partial sequences of CitACS2 and
CitACS4 (220 and 217 amino acids, respectively) contain
the expected five conserved regions that are between the
second and fourth exon of ACS genes (Fig. 2).
The polypeptides encoded by the watermelon ACSs
showed between 56 and 67% identity to one another:
CitACS1 and CitACS3 were most closely related to each
other (67% identity), whereas CitACS2 and CitACS3
showed the least degree of identity (56%). The CitACS
genes are more closely related to ACS polypeptides from
other cucurbit species than to each other (Fig. 3). Multiple
alignment analysis of ACS polypeptides (second to fourth
exon, Fig. 3) indicates that the CitACS1 polypeptide is most
closely related to the cucumber CsACS4, whereas CitACS2
is most closely related to CsACS1/G (in both cases 96%
identity between the watermelon and the respective
cucumber gene) (Trebitsh et al. 1997, Shiomi et al. 1998).
CitACS3 is 91% identical to the melon CMeACS1
(accession No. BAA93712) whereas CitACS4 is 95%
identical to the cucumber CsACS2 (Kamachi et al. 1997).
DNA blot hybridization was performed on genomic
DNA of two watermelon cultivars ‘Sugar Baby’ and
‘Malali’ (C. lanatus var. lanatus) and the wild relative of
watermelon, C. colocynthis. DNA was digested with
HindIII, blotted, and probed with the isolated fragment of
each gene (Fig. 4). No polymorphism was observed between
the two watermelon cultivars; however, all the genes except
CitACS2 showed HindIII RFLP between the cultivated
watermelon and the C. colocynthis. Each gene appeared as a
single copy gene, and no common restriction pattern was
observed among the different CitACS genes (Fig. 4). These
results indicate that the probes of CitACS1–4 do not crossreact with one another and could therefore be used as genespecific probes in the study of gene expression.
Expression of the ACS genes in vegetative and floral tissue
Expression of CitACS genes was examined by RNA blot
hybridization analysis. Of the four CitACS genes tested,
CitACS1 and CitACS3 showed detectable transcript levels in
floral tissues (Fig. 5), and CitACS1 was the only gene showing
detectable transcript levels in vegetative tissue (Fig. 6).
We compared the transcript abundance of the genes in
male, female and hermaphrodite flowers at four developmental stages, starting from a 3–4 mm floral bud to an open
flower (Fig. 5). Male and female floral tissue was collected
from the monoecious ‘Sugar Baby’ (Fig. 5a), while male
and hermaphrodite floral tissue was collected from the
Characterization of the watermelon ACS gene family
Cit-ACS2
Cit-ACS4
Cit-ACS3
Cit-ACS1
1
1
1
1
Cit-ACS2
Cit-ACS4
Cit-ACS3
Cit-ACS1
1
1
61
60
Cit-ACS2
Cit-ACS4
Cit-ACS3
Cit-ACS1
30
30
121
120
Cit-ACS2
Cit-ACS4
Cit-ACS3
Cit-ACS1
90
90
181
180
Cit-ACS2
Cit-ACS4
Cit-ACS3
Cit-ACS1
150
150
241
240
Cit-ACS2
Cit-ACS4
Cit-ACS3
Cit-ACS1
210
207
298
298
Cit-ACS2
Cit-ACS4
Cit-ACS3
Cit-ACS1
358
358
Cit-ACS2
Cit-ACS4
Cit-ACS3
Cit-ACS1
418
418
Cit-ACS2
Cit-ACS4
Cit-ACS3
Cit-ACS1
478
470
andromonoecious ‘Malali’ (Fig. 5b). The transcript level of
CitACS1 increased in both male and female floral buds up
to the stage of 8–10 mm then decreased in the open flower
(Fig. 5a). A similar expression pattern was observed in male
and hermaphrodite floral tissue, albeit with a lower
transcript level of CitACS1 (Fig. 5b). Unlike CitACS1, a
high CitACS3 transcript level was detected in young floral
buds and in the open flower, both in male and hermaphrodite floral tissue, but expression of CitACS3 was not
detectable in female floral tissue at the developmental stages
examined (Fig. 5). Altogether a steady increase of CitACS
transcript level is observed in male flowers compared with
female flowers, from a ratio of 1.4 in a 3–4 mm floral bud to
2.8 in an open flower (the ratio was calculated as the sum of
the CitACS1 plus CitACS3 transcript level in male flowers
to the CitACS1 transcript level in the female flower).
The CitACS genes were differentially expressed in
shoot apices, young leaves, tendrils, stems, cotyledons and
roots excised from watermelon plants. The highest CitACS1
transcript level was detected in tendrils (Fig. 6). Transcripts
of CitACS1 were also detected in young leaves and at barely
743
Fig. 2 Alignment of the deduced amino
acid sequences encoded by the isolated
CitACS1, CitACS2, CitACS3 and CitACS4.
Black and gray boxes indicate identities
and similarities among the different proteins, respectively. Protein characteristics
that are conserved in ACS proteins of plant
species are marked as follows: Roman
numbers above sequences mark conserved
regions, an asterisk below the sequences
marks the conserved amino acids among
ACSs, and an arrow marks the conserved
lysine residue in the active site.
detectable level in shoot apices, cotyledons and roots,
but not in stems (Fig. 6). The abundance of CitACS2–4
mRNA in these tissues was either barely detectable or not
detectable by RNA blot hybridization analysis.
Hormonal regulation of CitACS genes
The regulation of CitACS genes by auxin (IAA),
gibberellin (GA4þ7) and ACC, the immediate precursor
of ethylene, was examined in leaf discs of ‘Sugar Baby’.
In agreement with the results presented in Fig. 6 (nonwounded leaves), CitACS2 and CitACS3 are not expressed
in the control leaf discs and the level of CitACS1 in the
control leaf discs is comparable with its level of expression in
the non-wounded leaf shown in Fig. 6 (Fig. 7). This indicates
that CitACS1–3 do not respond to wounding within 4 h
(Figs 6, 7). The CitACS1 transcript level increased markedly
following a 4 h treatment with either 50 or 100 mM IAA or
with 100 mM ACC (Fig. 7a, b). Relative to the untreated
control, the CitACS1 transcript level was lower after
treatment with 1-aminoethoxyvinylglycine (AVG), an inhibitor of ACS activity, indicating that CitACS1 may be
Characterization of the watermelon ACS gene family
LeACS1B
LeACS4
LeACS2
AtACS7
CsACS2
CitACS4
LeACS5
CmACS2
CitACS2
CMeACS3
CsACS1/G
CmACS4
AtACS5
AtACS9
LeACS3
CitACS4
M
SB
Cc
CitACS3
Cc
CitACS2
M
SB
Plant
Cc
CitACS1
Probe
M
SB
AtACS11
AtACS6
CpACS1B
CpACS1A
CmACS1
CitACS3
CMeACS1
CsACS3
AtACS2
AtACS1
CmACS3
CitACS1
CMeACS2
CsACS4
LeACS6
LeACS1A
M
SB
Cc
744
kb
kb
kb
kb
21.2
21.2
21.2
21.2
5.0
10.8
8.7
6.3
5.0
10.8
8.7
6.3
5.0
2.0
2.0
1.4
1.0
1.4
1.0
5.0
4.2
3.5
2.0
2.0
1.4
1.0
0.6
0.6
0.4
0.4
Fig. 4 DNA blot hybridization analysis of C. lanatus var. lanatus
cv. ‘Malali’ (M) and cv. ‘Sugar Baby’ (SB), and C. colocynthis (L.)
Schrad. (Cc). Genomic DNA (20 mg per lane) was digested with
HindIII, separated on a 0.8% agarose gel and probed with the
entire CitACS 32P-labeled inserts as indicated (Probe).
Fig. 3 Comparison of CitACS deduced amino acids with ACS
polypeptides from Arabidopsis thaliana, tomato (Lycopersicon
esculentum) and several cucurbits. A cladogram of a multiple
alignment of the ACS polypeptides (second to fourth exon) was
generated using the ClustalW program (Thompson et al. 1994) (EBI
services). An arrowhead marks the CitACS amino acid sequences.
GenBank accession numbers of the genes encoding the polypeptides are given in parentheses: CpACS1A and CpACS1B, Cucumis
pepo (M61195); CmACS1, CmACS2, CmACS3 and CmACS4,
Cucurbita maxima (D01032, D01033, AB038559 and AB038558,
respectively); CMeACS1, CMeACS2 and CMeACS3, Cucumis melo
(AB032935, AB032936 and D86241, respectively); CsACS1/G,
CsACS2, CsACS3 and CsACS4, Cucumis sativus (DQ839406,
D89732, AB006803 and AB006804, respectively); CitACS1,
CitACS2, CitACS3 and CitACS4, Citrullus (EF154455-EF154458,
respectively); AtACS1, AtACS2, AtACS4, AtACS5, AtACS6,
AtACS7, AtACS8, AtACS9 and AtACS11, Arabidopsis thaliana
(U26543, AF334719, U23481, L29261, AF361097, NM118753,
AF334712, AF332391 and AF332405, respectively); LeACS1A,
LeACS1B, LeACS2, LeACS3, LeACS4, LeACS5, LeACS6, LeACS7
and LeACS8, Lycopersicon esculentum (U18056, U18057,
AY326958, U17972, AJ842054, AF179246, AF179249, AF43122
and AF179247, respectively).
ethylene. Similarly to CitACS1, expression of CitACS2
increased markedly in response to IAA treatment (Fig. 7a).
No increase in CitACS2 transcript level was observed in
response to ACC, but a marked increase in transcript level
was induced in response to the combined treatment of AVG
and IAA, indicating that transcription of CitACS2 is
positively regulated by auxin but possibly not by ethylene
(Fig. 7b). A slight increase in CitACS3 transcript level was
observed in response to treatment with 100 mM IAA or
ACC, relative to the control, and no CitACS3 transcript was
detected by combined treatment with IAA and AVG
(Fig. 7a, b). The finding that treatment with IAA in
combination with AVG did not result in a higher transcript
level is an indication that transcription of CitACS3 might be
positively regulated by ethylene but not by IAA (Fig. 7b).
Thus, the slightly higher transcript level that was observed
in the IAA treatment probably originated from auxininduced ethylene and was not a direct effect of IAA on the
transcription of CitACS3. Of the four genes, only CitACS1
responded to treatment with gibberellin, and its expression
increased markedly following a 4 h treatment with 250 or
500 mM GA4þ7 (Fig. 7c). The CitACS4 transcript level was
not detectable in the control nor in any of the examined
treatments.
positively regulated by ethylene (Fig. 7b). The combined
treatment with AVG and IAA resulted in an increased
transcript level, indicating that the transcriptional regulation
of CitACS1 by IAA is independent of its regulation by
Polymorphism in CitACS gene sequences
RFLP analysis was performed to determine whether
the ACS genomic sequences that were isolated from CL-F4
originated from the colocynthis genome or the cultivated
LeACS7
LeACS8
AtACS4
AtACS8
Characterization of the watermelon ACS gene family
(b)
Bud 3-4 mm
Bud 5-6 mm
Bud 8-10 mm
Hermaphrodite
Bud 3-4 mm
Bud 5-6 mm
Bud 8-10 mm
Male
Bud 3-4 mm
Bud 5-6 mm
Flower
Bud 8-10 mm
Female
Bud 3-4 mm
Bud 5-6 mm
Flower
Bud 8-10 mm
Male
Andromonoecious
Flower
Monoecious
Flower
(a)
745
CitACS1
CitACS3
rRNA
s
Roo
ts
don
yle
Cot
Ste
ms
s
dril
Ten
ung
Yo
Ap
ices
leav
es
Fig. 5 Expression of CitACS1 and CitACS3 in male, female and hermaphrodite flowers of watermelon. RNA was isolated from male and
female flowers of the monoecious C. lanatus var. lanatus cv. ‘Sugar Baby’ (a) or male and hermaphrodite flowers of the andromonoecious
C. lanatus var. lanatus cv. ‘Malali’ (b) that were excised at four developmental stages as indicated in the figure. Each lane contains 20 mg of
total RNA. RNA blots were hybridized to 32P-labeled CitACS1 or CitACS3 as indicated. rRNA stained by ethidium bromide indicates the
amount of RNA loaded in each lane.
CitACS1
rRNA
Fig. 6 Expression of CitACS1 in shoot apices, young leaves,
tendrils, stems, cotyledons and roots of watermelon. Total RNA was
extracted from tissue collected from C. lanatus var. lanatus cv.
‘Malali’. The RNA blot (20 mg of total RNA per lane) was hybridized
to 32P-labeled CitACS1. rRNA stained by ethidium bromide
indicates the amount of RNA loaded in each lane.
watermelon genome. This analysis was conducted by
comparing CL-F4 with the parental lines, ‘C.c.10’
(C. colocynthis) and ‘Sugar Baby’ (C. lanatus var. lanatus)
(Fig. 8). A single XbaI restriction site is present in the
isolated CitACS1 sequence (Fig. 1). Accordingly, two DNA
fragments were detected in the XbaI digest of the cultivated
watermelon genomic DNA and the CL-F4, whereas a single
DNA fragment was detected in the C. colocynthis genome
(Figs 1, 8). These results indicate that the CL-F4 contains
the CitACS1 gene of the cultivated watermelon. Following
the same guidelines, we conclude that the CitACS2 and
CitACS3 genes also originated from the cultivated watermelon (Figs 1, 8). However, based on the HindIII RFLP,
the CL-F4 contained the CitACS4 fragment unique to
C. colocynthis (Figs 1, 8). Additional RFLP markers
between the watermelon cultivars ‘Sugar Baby’ and
‘Malali’ to C. colocynthis were detected for each of the
four genes (MspI RFLP for CitACS1, XbaI RFLP for
CitACS2, and MspI, MvaI or XhoI RFLP for CitACS3,
data not shown). No polymorphism was observed between
the two watermelon cultivars using RFLP analysis.
Polymorphism between watermelon genotypes was
also examined by STS marker analysis. Primer pairs (PPs)
were designed for each of the CitACS gene sequences and
were used in PCR amplification with nine watermelon
genotypes (Table 1, Supplementary Fig. S3). All five
PPs produced PCR products (designated here as STS
markers) that were polymorphic between C. colocynthis
and C. lanatus var. citroides) (Table 1). STS polymorphism
was also detected between: the cultivar Malali to the other
cultivars (CitACS4); between lanatus species (var. lanatus or
citroides) and C. colocynthis (CitACS1-PP2, CitACS2 and
CitACS3); between the watermelon cultivars (var. lanatus)
and Griffin 14113 or PI296341 (var. citroides) (CitACS1PP1, CitACS2 and CitACS3); between the two citroides
subspecies (Griffin 14113 and PI296341) (CitACS2-4); and
between the two C. colocynthis (CitACS3-4).
Discussion
In most of the cucurbitaceous species the gaseous plant
hormone ethylene is the major feminizing hormone (Roy
and Saran 1990, Rudich 1990). In cucumber and melon, a
high level of endogenous ethylene, increasing ethylene
production by genetic engineering or foliar application of
ethylene-releasing agents were correlated with increased
femaleness, while inhibitors of ethylene biosynthesis or
perception reduced pistillate flower production (Roy and
Saran 1990, Rudich 1990, Perl-Treves 1999, Yamasaki et al.
2003, Papadopoulou et al. 2005). Molecular studies aimed
at elucidating the mechanisms governing female flower
production revealed the pivotal role of ACS genes in the
process (Kamachi et al. 1997, Yamasaki et al. 2003, Mibus
Characterization of the watermelon ACS gene family
AVG+IAA
500
250
100
GA4+7 (µM)
0
ACC
Control
IAA 50 µM
Control
(c)
AVG
(b)
IAA 100 µM
(a)
50
746
Cit-ACS1
Cit-ACS2
Cit-ACS3
rRNA
kb
kb
6.8
5.0
6.8
5.0
3.5
1-c
Cc
SB
kb
21.1
21.1
6.8
5.0
3.5
6.8
5.0
3.5
2.0
1.6
2.0
1.2
1.2
3.5
2.0
1.6
2.0
1.6
1.2
1.2
1.6
0.6
0.5
0.5
0.5
R.E
CitACS4
1-c
Cc
SB
kb
21.1
21.1
CitACS3
1-c
SB
Cc
Plant
CitACS2
1-c
CitACS1
Cc
Probe
SB
Fig. 7 Expression of CitACS1, CitACS2 and CitACS3 in response to chemical treatments. Leaf discs (10 mm) were excised from fully
expanded leaves of C. lanatus var. lanatus cv. ‘Sugar Baby’. Total RNA was extracted from leaf discs treated for 4 h with or without IAA,
1-aminocyclopropane-1-carboxylate (ACC), gibberellin (GA4þ7) or 1-aminoethoxyvinyl glycine (AVG), an inhibitor of ACS activity. The
response to IAA was examined at the concentrations indicated in (a); responses to 100 mM ACC, 10 mM AVG or 100 mM IAA þ 10 mM AVG
are shown in (b); response to GA4þ7 was examined at the concentrations indicated in (c). RNA blots (20 mg of total RNA per lane) were
hybridized to 32P-labeled CitACS1, CitACS2 or CitACS3 as indicated. rRNA stained by ethidium bromide indicates the amount of RNA
loaded in each lane.
XbaI
MvaI
AvaI
HindIII
Fig. 8 RFLP analysis of DNA isolated from CL-F4 and its paternal lines. Citrullus lanatus var. lanatus cv. ‘Sugar Baby’ (SB), C. colocynthis
(L.) Schrad. (Cc) and the CL-F4 (l-c) that was used to isolate the CitACS genomic sequences. DNA (20 mg per lane) was digested with the
restriction enzymes indicated below the figure (R.E.), separated on a 0.8% agarose gel and probed with the entire CitACS 32P-labeled
inserts as indicated (Probe).
and Tatlioglu 2004, Papadopoulou et al. 2005, Rimon
Knopf and Trebitsh 2006).
Isolation and characterization of the CitACS gene family
Bearing in mind the role of ethylene as a sex hormone
and the focal role of ACS genes in cucumber with
respect to unisexual flower development, we isolated four
watermelon CitACS paralogs from a colocynthis–lanatus
F4 plant with enhanced femaleness characteristics. The
structure of three genes shows the expected exon–intron
pattern characteristic of ACS genes of other species,
except for CitACS4, which lacks one intron. Variations in
the typical structure of ACS genes have also been observed
in other ACS genomic sequences such as the zucchini
CpACS1 that has an additional intron located in the last
Characterization of the watermelon ACS gene family
Table 1
genes
747
Summary of the sequence tagged site (STS) markers produced for several watermelon genotypes using the CitACS
Genotype
CitACS1
CitACS2
CitACS3
CitACS4
PI 296341 (C)
Griffin 14113 (C)
Charleston Gray (L)
N. H. Midget (L)
Malali (L)
Sugar Baby (L)
PI 386015 (O)
Cc (O)
CL-F4 (L, O)
PP1, PP2
266, 344
266, 344
264, 344
264, 344
264, 344
264, 344
264, 345
264, 345
264, 344
348
347
346
346
346
346
337
337
346
361
360
344
344
344
344
353
351
344, 351
348
349
341
341
348
341
348
336
336
The watermelon genotypes and the species or subspecies they belong to: Citrullus lanatus var. lanatus (designated as L), Citrullus lanatus
var. citroides (designated as C) and Citrullus colocynthis (designated as O). For reaction details see Materials and Methods and
Supplementary Fig. S4.
PP, primer pair.
exon of most ACS genes, or the cucumber CsACS1/G that
similarly to the CitACS4 gene lacks the second intron found
in other ACS genes (Huang et al. 1991, Rimon Knopf and
Trebitsh 2006). The deduced amino acid sequences of the
four CitACSs have higher homology to ACSs from other
cucurbit species than to each other. The fact that ACS
polypeptides from the same species are more closely related
to ACSs from other species than to those from the same
species has also been observed in Arabidopsis and other
plant species (Liang et al. 1992, Wang et al. 2002). This is
consistent with the studies that demonstrate differential
regulation of the different ACS isoforms (Wang et al. 2002).
Differential expression and hormonal regulation of the
CitACS genes
Typical of multigene families, the ACS genes in
numerous plant species have both unique and overlapping
expression patterns during plant development and in
response to internal and external stimuli (Wang et al.
2002, Tsuchisaka and Theologis 2004).
We found that the CitACS1 gene is expressed in
vegetative tissue and we were able to detect it in cotyledons,
young leaves and roots. A distinctly high transcript level of
CitACS1 was detected in tendrils or in leaf discs treated
with IAA, ACC or GA4þ7. Auxin and, to a lesser extent,
ethylene have been implicated in tendril growth and coiling
(Reinhold 1967, Jaffe 1970). Since auxin induces ethylene
production, it was proposed, ‘when a tendril is mechanically
stimulated, auxin is ventrally translocated in a basipetal
direction, causing ethylene to be produced ventrally as it
moves’ (Jaffe 1970). Reducing the endogenous level of
ethylene by inhibitors of its biosynthesis or in mutants did
not prevent coiling, therefore it appears that ethylene is not
a direct signal for coiling but rather is involved in the radial
expansion of tendrils in the coiling response (Braam 2005).
Interestingly, in cucumber, GA4þ7 induced tendril formation both in vitro and in vivo (Rudich and Halevy 1974,
Ameha et al. 1998). The high transcript level of CitACS1
found in tendrils together with the observation that ACC,
IAA and GA4þ7 positively regulate the expression of this
gene may imply a possible role for CitACS1 during tendril
growth and coiling.
Two of the four genes, CitACS1 and CitACS3, are
differentially expressed in floral tissues. CitACS1 is
expressed in young floral buds but not in the open flower,
and no discernible differences were observed between male,
female and hermaphrodite floral tissue. Interestingly, in
Cucumis and grapes, both tendrils and flowers are proposed
to develop from the same uncommitted primordia, one at
the expense of the other (Whitaker and Davis 1962, Ameha
et al. 1998, Calonje et al. 2004). In the tendrilless watermelon, mutant flowers develop instead of tendrils and
vegetative buds (Guner and Wehner 2004). Since no sexdependent differential expression pattern was observed, we
suggest that CitACS1 may be involved in the cell growth
processes of floral organs, consistent with its high expression in tendrils and its possible role in tendril growth.
In contrast to CitACS1, CitACS3 is expressed in both male
and hermaphrodite young floral buds and then again in the
open flower, but no expression was detected in female
floral tissue. CitACS3 is positively regulated by ACC, the
immediate precursor of ethylene that is considered to be a
masculinizing hormone in watermelon. In tobacco, anther
dehiscence was shown to be ethylene regulated and in
petunia a pollen-specific PhACS2 was detected in mature
pollen grains (Lindstrom et al. 1999, Rieu et al. 2003). Thus,
CitACS3 that is up-regulated by ACC and preferentially
expressed in staminate flowers but not in female flowers is
implicated in anther development, and further studies are
748
Characterization of the watermelon ACS gene family
being conducted to elucidate its role in flower development
in watermelon.
Transcripts of two genes, CitACS2 and CitACS4, were
not detected in vegetative or floral tissue. However,
CitACS2 is transcriptionally active in response to IAA
while no CitACS4 transcript was detected under our
experimental conditions. Treatment of watermelon plants
with various auxins did not have a consistent effect on floral
sex, but rather had a general growth-inhibiting effect
(Rudich and Zamski 1985). Based on the present data, no
putative role can be suggested for CitACS2. The expression
of CitACS4 might be below the threshold level of detection
by RNA blot hybridization. Alternatively, CitACS4 may
either have a particular spatio-temporal expression pattern
not tested here or it may be a non-functional ACS paralog.
Polymorphism in CitACS gene sequences
The low polymorphism in watermelon cultivars makes
it difficult to build a genetic map important for studying
inheritance and linkage of various traits important in crop
management (Zamir et al. 1984, Katzir et al. 1996, Levi
et al. 2002, Levi et al. 2004, Levi et al. 2006). DNA markers
linked to genes controlling horticultural qualities can be
used effectively in genomic studies and in breeding
programs of plants.
In a previous study we mapped the CitACS3 gene to
linkage group XVII in a test cross-mapping population
(Levi et al. 2006). Here we assessed the polymorphism of the
CitACS genes by RFLP and STS analysis. We show that the
CitACS genes are polymorphic among Citrullus species
(lanatus vs. var. colocynthis) and varieties (var. lanatus vs.
var. citroides), as well as between genotypes belonging to the
same varieties (var. lanatus or var. citroides).
The flowering pattern of watermelons is either monoecious or andromonoecious, with no hermaphrodite,
androecious or gynoecious lines (Rudich and Zamski
1985). The lack of diversity in the sex phenotype of
cultivated watermelon has so far hampered the development
of female-enhanced watermelon plants and the production
of female tetraploid plant lines that could greatly facilitate
triploid seed production. In the majority of cucumber
cultivars, the femaleness trait was introduced from the
Japanese cultivar Shogoin female PI 220860 (Galun 1961,
Shifriss 1961). Gynoecy in cucumber appears to have
evolved through the gene duplication of CsACS1 that
gave rise to a female-specific twin gene CsACS1G that
was mapped to the Female (F) locus of cucumber (Trebitsh
et al. 1997, Mibus and Tatlioglu 2004, Rimon Knopf
and Trebitsh 2006). The studies in cucumber led to
the development of CsACS-linked molecular markers
useful for the identification of female (F–) genotypes
(Mibus and Tatlioglu 2004, Rimon Knopf and Trebitsh
2006, Xiang et al. 2006). At least two genetic sources could
be used to broaden the genetic base of cultivated watermelon and to introduce improved traits: C. lanatus var.
citroides, the wild relative of cultivated watermelon that
has higher genetic diversity, and C. colocynthis, a desert
species with wide genetic and geographical diversity (Zamir
et al. 1984, Katzir et al. 1996, Levi et al. 2001a, Levi et al.
2001b, Simmons and Levi 2002). The CL-F4 used for
the isolation of the CitACS genes exhibited a higher
proportion of female to male flowers along the main axis
compared with either ‘Sugar Baby’ or the ‘Malali’ cultivars.
Unlike in cucumber, each of the four CitACS genes appears
as single-copy genes in the watermelon cultivars, the
C. colocynthis and the CL-F4. Thus, we propose that the
molecular mechanism leading to the enhanced femaleness
observed in CL-F4 might be other than the ACS gene
duplication event observed in cucumber. The markers
described here could be tested for linkage to ethylenerelated traits and exploited for phylogenetic analyses,
measurement of genetic diversity in populations and in
DNA fingerprinting of breeding lines and cultivars of
watermelon.
Materials and Methods
Plant material
An F4 plant (colocynthis–lanatus) of a cross between ‘C.c.10’
[C. colocynthis (L.) Schrad (accession No. 10 collected in the Negev
Desert, Israel)], a desert-dwelling, cross-compatible relative of
watermelon (C. lanatus), and the watermelon cultivar C. lanatus
var. lanatus cv. ‘Sugar Baby’ was produced in our laboratory
(referred to as CL-F4). Seeds of two commercial cultivated
watermelon lines (C. lanatus var. lanatus cv. ‘Malali’ and cv.
‘Sugar Baby’) were obtained from Hazera Genetics, Ltd (Berurim
M.P. Shikmim, Israel).
For STS marker analysis, the following genotypes were used:
U.S. PI 386015 (C. colocynthis), Griffin 14113 (C. lanatus var.
citroides) provided by R. Jarret (USDA Plant Genetic Resources
Conservation Unit, Griffin, GA), U.S. PI 296341 (C. lanatus var.
citroides), and the watermelon cultivars New Hampshire Midget
and Charleston Gray (C. lanatus var. lanatus).
DNA and RNA blot hybridization analysis
Upon harvesting, or the termination of chemical treatments,
all plant tissues were frozen in liquid nitrogen and stored at –808C
for genomic DNA and RNA extraction. Genomic DNA was
extracted from young leaves by cetyltrimethylammonium bromide
(CTAB) extraction according to Levi and Thomas (1999). Genomic
DNA (20 mg) was digested with selected restriction enzymes (AvaI,
HindIII, MspI, MvaI, XbaI and XhoI) (Fermentas, Vilnius,
Lithuania) and separated by electrophoresis on a 0.8% agarose
gel. DNA blot hybridization analysis was performed as previously
described (Trebitsh et al. 1997).
Total RNA was extracted from different plant tissues by an
EZ-RNA extraction kit according to the manufacturer’s instructions (Biological Industries, Beit Haemek, Israel). RNA (20 mg) was
fractionated by electrophoresis in formaldehyde–agarose gels, and
RNA blot hybridization analysis was performed as previously
described (Trebitsh et al. 1997). The ACS transcript level in male
Characterization of the watermelon ACS gene family
749
and female flowers was calculated using the image processing and
analysis software ImageJ (http://rsb.info.nih.gov/ij/).
DNA and RNA gel blot hybridization and RFLP analysis
were performed at 608C in a solution of 0.5 M sodium phosphate
pH 7.2, 7% SDS, 1 mM EDTA pH 7.0, 0.2 mg ml–1 salmon sperm
DNA (Applichem, Darmstadt, Germany) and CitACS1, CitACS2,
CitACS3 or CitACS4 inserts. Inserts were labeled with [32P]dCTP
by random priming (Random Primer DNA Labeling Mix,
Biological Industries, Beit Haemek, Israel).
pooling 0.3 ml of the D4 reaction, 0.3 ml of the D3 reaction, 0.5 ml
of the D2 reaction, 0.28 ml of the D1 size standard and 30 ml of
de-ionized formamide in a 96-well PCR plate. Samples were run on
the CEQ8800 (Beckman Coulter) capillary sequencer and analyzed
using the built-in fragment analysis software provided with the
CEQ8800 system. Fragments that ranged in size from 75 to 400 bp
could be scored with high confidence. PCR fragments that
were reproduced at the three annealing temperatures are presented
in Table 1.
Isolation of CitACS genes
Isolation of genomic sequences. The degenerate oligonucleotides ACS/2F and ACS/6R correspond to the highly conserved amino
acid sequences FQDYHGL (region 2) and KMSSFG (region 6) of
various ACS polypeptides (Rottmann et al. 1991, Kende 1993,
Trebitsh et al. 1997). Degenerate PCRs, using 125 ng of each primer
and 25 ng of genomic DNA of the CL-F4 plant, were performed
using Taq DNA polymerase (Fermentas, Vilnius, Lithuania). DNA
fragments obtained by PCR were cloned into the pGEM T-easy
vector (Promega, Madison, WI, USA) and sequenced.
Full-length genomic sequences of CitACS1 and CitACS3
were isolated by direct PCR (as above) using primers derived from
the cDNA end sequences.
Isolation of CitACS cDNA sequences. The full-length cDNA
of CitACS1 and CitACS3 were isolated by 50 and 30 rapid
amplification of cDNA ends (FirstChoice RLM-RACE Kit,
Ambion, Huntingdon, UK) using nested primers derived from
the partial genomic sequence that were isolated by PCR in
conjugation with the primers provided in the kit. Partial cDNA
of CitACS2 was obtained by direct PCR using primers based on
the partial genomic sequence. For primer sequences, see
Supplementary Fig. S4.
Chemical treatments
Discs (10 mm in diameter) were excised from fully expanded
leaves of C. lanatus var. lanatus cv. ‘Sugar Baby’ using a cork borer.
Twenty discs were incubated for 4 h in a 100 ml flask containing 5 ml
of a solution of 50 mM citrate-phosphate buffer (pH 4.5) and 50 mM
sucrose with or without 50 or 100 mM IAA, 100 mM ACC (SigmaAldrich Co., St Louis, MO, USA), 10 mM AVG (commercial name
‘ReTain’; Valent Biosciences Co., Libertyville, IL, USA) or
gibberellin GA4þ7 (Duchefa, Haarlem, The Netherlands) at 50,
100, 250 and 500 mM. At the end of incubation period, discs were
frozen in liquid nitrogen and were kept at –808C for RNA isolation.
Each treatment was repeated at least three times.
DNA sequence analysis
The PCR-amplified fragments were sequenced in both
directions to ensure sequence authenticity, and for each gene a
single contig was constructed using the Sequencher program V. 4.5
(Gene Codes Co., Ann Arbor, MI, USA). Sequence analysis was
carried out using BLAST searches (http://www.ncbi.nlm.nih.gov/
BLAST/) (Altschul et al. 1997). Nucleotide and amino acid
sequences were aligned using ClustalW at the EBI web server
(http://www.ebi.ac.uk/cgi-bin/clustalw/) (Thompson et al. 1994).
STS marker analysis
Primer pairs were designed using each of the CitACS genomic
sequences (for primer sequences, see Supplementary Fig. S4). The
primer pairs were tested in nine genotypes (Table 1). PCR was
performed in 10 ml reactions containing 1 PCR buffer (Promega),
2.5 mM MgCl2, 100 mM dNTPs (Sigma, St Louis, MO, USA),
0.4 U of Taq DNA polymerase (Promega), 30 ng of reverse primer
(IDT, Coraville, IA, USA) and 25 ng of DNA. For visualization
with a capillary DNA sequencer (CEQ8800; Beckman Coulter,
Fullerton, CA, USA), each of the forward primers was labeled with
one of three WellRED dyes (D2, D3 or D4) (Proligo, Boulder, CO,
USA). The forward primers were added in the following amounts:
15 ng for a D4 primer; 30 ng for a D3 primer; or 75 ng for a D2
primer. PCR amplification included 5 min of DNA denaturation
at 948C, five cycles of: 1 min of denaturing at 948C, 1 min of
annealing at 49, 53 or 548C, and 2 min of elongation at 728C,
followed by 30 cycles with the annealing temperature increased to
558C, and a final elongation step of 5 min at 728C. The PCR
products were analyzed using the CEQ8800 capillary sequencer, by
Supplementary material
Supplementary material mentioned in the article is available
to online subscribers at the journal website www.pcp.oxford
journals.org.
Acknowledgments
The authors thank Anat Preiss for the production and
selection of the CL-F4 line, and Ruth van-Oss and Laura Pence for
their assistance in the molecular analysis. Professors Dina Raveh
and Dudy Bar-Zvi are gratefully acknowledged for the critical
reviewing of the manuscript.
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(Received December 11, 2007; Accepted March 20, 2008)