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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. All rights reserved. For permissions, please email: [email protected] 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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Genet. 105: 397–403. (Received December 11, 2007; Accepted March 20, 2008)