Plant Cell Physiol.

44(1): 8592 (2003) JSPP 2003

CvADH1, a Member of Short-Chain Alcohol Dehydrogenase Family, is Inducible by Gibberellin and Sucrose in Developing Watermelon Seeds
Joonyul Kim, Hong-Gyu Kang, Sung-Hoon Jun, Jinwon Lee, Jieun Yim and Gynheung An 1
National Research Laboratory of Plant Functional Genomics, Division of Molecular and Life Sciences, Pohang University of Science and Technology (POSTECH), Pohang, 790-784 Korea ;

To understand the molecular mechanisms that control seed formation, we selected a seed-preferential gene (CvADH1) from the ESTs of developing watermelon seeds. RNA blot analysis and in situ localization showed that CvADH1 was preferentially expressed in the nucellar tissue. The CvADH1 protein shared about 50% homology with short-chain alcohol dehydrogenase including ABA2 in Arabidopsis thaliana, stem secoisolariciresinol dehydrogenase in Forsythia intermedia, and 3b-hydroxysterol dehydrogenase in Digitalis lanata. We investigated gene-expression levels in seeds from both normally pollinated fruits and those made parthenocarpic via N-(2-chloro-4-pyridyl)-Nphenylurea treatment, the latter of which lack zygotic tissues. Whereas the transcripts of CvADH1 rapidly started to accumulate from about the pre-heart stage in normal seeds, they were not detectable in the parthenocarpic seeds. Treating the parthenogenic fruit with GA3 strongly induced gene expression, up to the level accumulated in pollinated seeds. These results suggest that the CvADH1 gene is induced in maternal tissues by signals made in the zygotic tissues, and that gibberellin might be one of those signals. We also observed that CvADH1 expression was induced by sucrose in the parthenocarpic seeds. Therefore, we propose that the CvADH1 gene is inducible by gibberellin, and that sucrose plays an important role in the maternal tissues of watermelon during early seed development. Keywords: Developing seed Gibberellin Parthenocarpic fruit Seed preferential Sucrose-watermelon.
Abbreviations: CPPU, 1-(2-chloro-4-pyrodyl)-3-phenylurea; DAP, days after pollination; DAT, days after CPPU treatment; DAI, days after imbibition; PCD, programmed cell death. Accession number: CvADH1, AB018559; Cvrp15a, AB018560.

Introduction
Because developing seeds contain more abundant gibberellin than other plant organs, they are frequently used in research to elucidate the biosynthetic pathways of gibberellin (Graebe 1987, Lange et al. 1997, MacMillan et al. 1997, Kang et al. 1999, Kang et al. 2002). Among the many derivatives,
1

biologically active gibberellins are crucial components in both early and late seed development. In maize (Zea mays), for example, an ABA/gibberellin ratio can become established that sufficiently suppresses germination and induces maturation (White et al. 2000, White and Rivin 2000). In the embryos and endosperms of the pea (Pisum sativum) lh-2 mutant, Swain et al. (1995) showed that endogenous gibberellin levels dropped a few days after anthesis, resulting in reduced seed weights and survival rates at harvest. In addition, Groot et al. (1987) reported that exogenous gibberellin increased seed weights and delayed their dehydration in the gibberellin-deficient ga-1 mutant of tomato (Lycopersicon esculentum). This suggests that gibberellins play a role even in the later stages of tomato fruit and seed development. Histochemical research of active gibberellins and the key enzymes involved in the biosynthetic pathway provide a clue in determining how gibberellins are partitioned in developing seeds (Kang et al. 1999, Nakayama et al. 2002). In particular, those studies have suggested that gibberellins occur in the integument, where they participate in the induction of hydrolytic enzymes required for the degradation of starch grains. Afterward, sugars can be released for either translocation or further seed development. Kim et al. (2002) have shown that GA3 also strongly induces CvSUS1, a putative sucrose synthase, which is preferentially expressed in the maternal tissues of developing watermelon seeds. However, little is known about other possible roles for gibberellins in the integument. Programmed cell death (PCD) in developing seeds occurs during developmental events, including the degeneration of the endosperm, nucellus, and inner integuments. Proteinases and nucleases participate in PCD by degrading proteins and nucleic acids, respectively, within the affected cells (Chen and Foolad 1997, Xu and Chye 1999, Young and Gallie 2000, Dominguez et al. 2001, Wan et al. 2002). In wheat grains, nucellar projection cells, with the characteristic morphology of transfer cells, form part of the pathway for nutrients to the endosperm cavity, where they are taken up through the modified aleurone cells to the developing endosperm (Wang et al. 1994, Wang et al. 1995a, Wang et al. 1995b, Wang and Fisher 1994, Wang and Fisher 1995, Weschke et al. 2000). Therefore, this maternal tissue plays an important role in nourishing the endosperm during grain development. However, other biochemical pathways and the signaling of PCD in maternal tissues are virtually unknown.

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Corresponding author: e-mail, genean@postech.ac.kr; Fax, +82-54-279-2199. 85

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cult to isolate RNA from seeds after 20 DAP. During imbibition, the transcript was weakly present in the first half day, but then decreased to an undetectable level after 1 d (Fig. 1C). As a control, transcript levels of Cvrp15a (Citrullus vulgaris ribosomal protein subunit 15a) also were examined. The seed-preferential clone is highly homologous to alcohol dehydrogenase Because the clone was partial, a full-length ORF clone was isolated from the developing-seed cDNA library. That clone encodes a protein sharing high homology with shortchain alcohol dehydrogenase/reductase (SDR) family. Especially, it shared 48% homology with Arabidopsis ABA2, which catalyzed the conversion of xanthoxin to abscisic aldehyde in ABA biosynthesis (Gonzalez-Guzman et al. 2002), 51% homology with Forsythia intermedia stem secoisolariciresinol dehydrogenase, an enzyme that catalyzes the enantiospecific conversion of ()-secoisolariciresinol into ()-matairesinol (Xia et al. 2001), 50% homology with secoisolariciresinol dehydrogenase of Podophyllum peltatum (Xia et al. 2001), and 49% with 3b-hydroxysteroid dehydrogenase of Digitalis lanata (Finsterbusch et al. 1999), respectively (Fig. 2). The primary structure of the clone showed three sequence motifs that are common to members of the cD1d subfamily in the classical SDR family (Kallberg et al. 2002). The protein contained the NAD(H)-binding motif (I), the presumed active site motif (II) (Jornvall et al. 1995, Gonzalez-Guzman et al. 2002), the putative substrate binding motif (III) (Jornvall et al. 1995, Gonzalez-Guzman et al. 2002), and an uncharacterized motif (Fig. 2). We designated this clone as CvADH1 (Citrullus vulgaris alcohol dehydrogenase 1), and compared it with the databases, using the ClustalX and MEGA2 programs. Significant sequence similarity was revealed between the predicted CvADH1 protein and a family of short-chain alcohol dehydrogenases (Fig. 3). Genomic DNA blot analyses DNA gel-blot analysis of the watermelon genomic DNA was conducted to identify the copy number of the CvADH1 gene. This analysis was performed with the full-length cDNA probe at low- and high-stringent conditions, and similar hybridized patterns were obtained from both. The CvADH1 probe hybridized to one or two major bands, indicating that one or, at most, two copies of the CvADH1 genes are present in the watermelon genome (Fig. 4). Expression patterns of the CvADH1 gene in pollinated and parthenocarpic seeds To investigate the expression patterns of CvADH1 in the maternal organs, we used parthenocarpic seeds that were induced by treating unfertilized female ovaries with N-(2chloro-4-pyridyl)-N-phenylurea (CPPU), a synthetic cytokinin. Although zygotic tissues do not form due to parthenocarpy, seed coats do develop normally in the early stages (Kang et al.

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Fig. 1 RNA gel-blot analyses of temporal and spatial expression of CvADH1 (accession No. AB018559). Total RNA (25 mg) was gelfractionated, blotted, and proved with the cDNA fragment of CvADH1. The cDNA of Cvrp15a (accession No. AB018560) was used as a control. (A) Spatial expression pattern of CvADH1. Transcript levels were examined from inner seed tissues at 10 DAP (S), outer part of the seed (I), female flowers (W), fruit at 2 DAP (F), leaves (L), and roots (R) of 14-day-old plants, and seedlings grown 7 d after imbibition (Y). (B) Developing seeds at 3, 6, 13, and 15 DAP. (C) Germinating seeds from 0.5 to 3 d after imbibition.

Here, we report the identification of a putative short-chain alcohol dehydrogenase cDNA, CvADH1, that is preferentially expressed in developing seeds and inducible by GA3 and sucrose.

Results
Isolation of a seed-preferential clone from developing watermelon seeds We isolated a seed-preferential EST clone from a developing-seed cDNA library. RNA gel-blot analysis showed that the clone was strongly expressed 10 d after pollination (DAP) in seeds from which the integuments had been removed. Transcript of the clone was not detectable in the outer portion of the seeds, the female flowers, young fruit and leaves, roots, or seedlings (Fig. 1A). The temporal expression pattern of this clone was studied during seed development. Transcript was not present until 6 DAP, after which a high level was then observed until 15 DAP. Further expression patterns could not be studied during the late stage of fruit development because it was diffi-

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Fig. 2 Alignment of CvADH1 and related protein sequences. Underlined sequences are the typical motifs which are shown in the cD1d subfamily of the classical SDR family. Arrow above the alignment shows the residue forming hydrogen bonds to the 2- and 3-hydroxyl groups of the adenine ribose moiety. The additional conserved region is boxed. I, NAD(H)-binding region; II, catalytic site; III, presumed substrate binding site.

Fig. 3 Phylogenetic tree of various proteins related to CvADH1, as generated with the MEGA2 program. Proteins are as follows (accession numbers in parentheses): CvADH1 from Citrullus vulgaris (AB018559), SADH from Forsythia intermedia secoisolariciresinol dehydrogenase (AF352735), ADH from Nicotiana tabacum short-chain alcohol dehydrogenase (AJ223177), SADH1 from Ipomoea trifida short-chain alcohol dehydrogenase (AF072447), rhizome SADH from Podophyllum peltatum rhizome secoisolariciresinol dehydrogenase (AF352734), CPRD12 protein from Vigna unguiculata (D88121), Arabidopsis thaliana ABA2 (AY082345), Zea mays TASSELSEED2 (L20621), Arabidopsis thaliana ATA1 (U76501), Silene latifolia STA1-2 (U53827), Lycopersicon esculentum GAD3 (U21801), Digitalis lanata 3b-hydroxysteroid dehydrogenase, or 3b HSD (AJ345026), Pisum sativum sadA (AF053638), Lactuca sativa alcohol dehydrogenase (JC4320), and Solanum tuberosum CB12 (AF349916).

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Fig. 4 Gel-blot analysis of genomic DNA from CvADH1, as digested with EcoRI (E) or HindIII (H), and resolved on a 0.8% agarose gel. The cDNA cloned by EST analysis was used as a probe.

Fig. 6 (A) RNA gel-blot analysis using RNA samples prepared from whole seeds of fertilized fruit and CPPU-treated fruit. 4, 4 DAP; 8, 8 DAP; 10, 10 DAP; 4T, 4 DAT; 8T, 8 DAT; 10T, 10 DAT. (B) RNA was extracted from three sections of equally dissected seeds at 10 DAP. The probe was the same as that described above. Mp, micropylar; M, middle; Ch, chalazal portions.

2002). In seeds that were fertilized normally, the maternal tissues between the endosperm and inner integuments rapidly deteriorated (Fig. 5). However, we observed no such tissue degradation in the unfertilized, parthenocarpic seeds. RNA gel-blot analyses of both fertilized and parthenocarpic seeds did not detect CvADH1 transcript in the latter (Fig. 6A). Therefore, we suggest that the CvADH1 gene is expressed

in zygotic tissues such as the embryo and endosperm. Alternatively, the gene is being expressed in maternal tissues such as the nucellus, but signals generated from the zygotic tissues are required for that to occur. To deduce the spatial expression pattern of the gene, we examined transcript levels in the micropy-

Fig. 5 Longitudinal sections of watermelon seeds. (A) is a pollinated seed at 12.5 DAP and (B) that from a CPPU-treated parthenocarpic fruit at 15 DAT. Sections were made perpendicular to the plane of the seed. n, nucellus; ii, inner layer of integument; oi, outer layers of integument; e, endosperm. Bars = 200 mm.

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Fig. 7 Localization of the CvADH1 mRNA in watermelon seeds by in situ hybridization. Samples were obtained from seeds at 10 DAP (heart embryo stage) and sectioned longitudinally in parallel with seed plane. Sections were hybridized to the antisense probe. RNA probe of the entire cDNA was labeled with digoxigenin. (A) Almost entire seed showing embryo (e), endosperm (en), and nucellus (nu). (B) Middle portion of seed. (C) Enlargement of rectangle in B. Hybridization with the sense CvADH1 probe did not show any significant hybridization to the section prepared at the same stage of seed development (data not shown). nu, nucellus; ii, inner layer of integument; oi, outer layers of integument; en, endosperm; e, embryo. Bars = 200 mm

embryo or endosperm (Fig. 6B). We conducted in situ hybridization experiments to localize the tissue types that express the gene in the developing seeds. There, the antisense probe hybridized preferentially to the degenerating nucellus tissue and transfer cells (Fig. 7). Because these tissues are maternal, we speculate that signals from the zygotic tissues would be needed to induce expression in those tissues. Induction of CvADH1 by GA3 and sucrose Because gibberellins play an important role during seed development, we examined their effects on seed morphology and expression of the CvADH1 gene. The high level of gibberellins in seeds makes it difficult to study the effects of exogenous applications. Therefore, we used parthenocarpic seeds, in which the amount of gibberellin is inherently quite low (Kang et al. 1999). Treatment with GA3 significantly increased expression of CvADH1 (Fig. 8A). We also evaluated the effects of sucrose in parthenocarpic seeds (Fig. 8A), and found that CvADH1 expression was also induced at higher concentrations. We studied the kinetics of CvADH1 transcript levels as affected by GA3 and sucrose treatments. In response to the former, the amount of CvADH1 mRNA reached a maximum in 30 min, then decreased rapidly after 2 h (Fig. 8B). In contrast, the CvADH1 transcript level was slowly increased by sucrose, reaching a maximum after 6 h of treatment and maintaining a high level for several hours (Fig. 8B).

Fig. 8 (A) Induction of CvADH1 by exogenous treatment with GA3 and sucrose in CPPU-treated parthenocarpic fruit. Experiments were performed using in vitro-cultured seeds that were collected from the fruit at 7 DAT, and incubated at room temperature under dim light in a medium (2% sorbitol and 100 mg liter1 myo-inositol) containing 0 or 50 mM GA3 and 0, 250, or 300 mM sucrose. (B) Response kinetics of CvADH1 to 50 mM GA3 and 300 mM sucrose. Experimental conditions are as described for (A).

lar, middle, and chalazal portions of the seeds. Transcript amounts were almost equal in all three sections, which precludes the possibility that the clone is expressed only in the

Discussion
We have previously reported that differential expression

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of gibberellin 20-oxidase and gibberellin 3b-hydroxylase in developing watermelon seeds influences the partitioning of biologically active gibberellins, and that these active gibberellins are generated primarily in zygotic tissues (Kang et al. 1999, Kang et al. 2002). In the present study, we identified a putative short-chain alcohol dehydrogenase gene, CvADH1, that is specifically expressed in the maternal tissues, especially the nucellus and transfer cells. The CvADH1 gene is turned on at a developmental stage similar to that when expression of the gibberellin 3b-hydroxylase gene is first detected (Kang et al. 2002). This gibberellin 3b-hydroxylase gene is expressed preferentially in the zygotic tissues, providing the possibility that active gibberellins are synthesized primarily in those cells (Kang et al. 2002). Our comparison of active gibberellin contents between parthenocarpic and normal seeds supports the hypothesis that active gibberellins are synthesized primarily in zygotic tissues (Kang et al. 2002). Because the CvADH1 gene is inducible by exogenous gibberellin application in the parthenocarpic seeds, it is likely that its expression in normally developing seeds is dependent on active gibberellins synthesized in the zygotic tissues. A number of plant genes that show significant homology to CvADH1 also are affected by active gibberellins. For example, the gad3 gene from tomato is down-regulated by gibberellin application (Jacobsen and Olszewski 1996). Likewise, the ADH-type gene from lettuce (Lactuca sativa) is induced in seeds by gibberellin (Toyamasu et al. 1995). Finally, antisense expression of Stgan (CB12) affects the levels of biologically active gibberellins and their respective precursors to become elevated (Bachem et al. 2001), while the tasselseed2 gene apparently has an effect on gibberellin metabolism (De Long et al. 1993, Calderon-Urrea and Dellaporta 1999). Recently, Arabidopsis ABA2, which is closely related to CvADH1 in a phylogenetic tree (Fig. 3), has been characterized as a critical enzyme in ABA biosynthetic pathway (Gonzalez-Guzman et al. 2002, Cheng et al. 2002). Although the regulation of Arabidopsis ABA2 gene expression has not been observed, it is possible that the gene is up regulated by exogenous gibberellin application to keep the balance between gibberellin and ABA. Ikeda et al. (2002) supported this postulation with the evidence that constitutive activation of the gibberellin signal transduction pathway by the slr1-1 mutation in rice promotes the endogenous ABA level. CvADH1 from watermelon appears to be a member of the short-chain alcohol dehydrogenases family that reduces ringalcohol substrates, including a variety of steroids, at a number of positions (Persson et al. 1991). Developing seeds contain multiple short-chain alcohols, such as gibberellins and ABAs, that could be substrates for CvADH1. Experimental evidence supports the theory that this family is involved in cell division or differentiation. For example, secoisolariciresinol dehydrogenase (which has the highest homology with CvADH1) produces matairesinol, a central precursor of numerous lignans, including that of the important antiviral and anticancer agent,

podophyllotoxin (Xia et al. 2001). This compound is responsible for reducing malignancies (Jenab and Thompson 1996). The tasselseed2 gene is involved in PCD, regulating the elimination of pistils during early development in the male inflorescence of maize. Calderon-Urrea and Dellaporta (1999) have suggested that this process may be the result of the elimination or biosynthesis of an unknown signaling molecule possibly related to steroids. It also had been speculated that StGAN is involved in a biochemical route for the breakdown of gibberellin (Bachem et al. 2001). Such a role would be consistent with data showing that CvADH1 is gibberellin-inducible. Although we did not determine the biochemical action of CvADH1, the protein may be involved in generating the inner structure of developing seeds, thereby providing space and nutrients to the newly forming endosperm and embryo. Note that in the parthenocarpic seeds, where the CvADH1 gene was not expressed, the nucellus tissue remained intact. Recently, two independent groups have shown that biologically active gibberellins regulate carbohydrate metabolism. First, Kim et al. (2002) reported that Cvsus1, a gene expressed primarily in the maternal tissues of young watermelon seeds, is induced by gibberellin during early seed development. Second, Nakayama et al. (2002) demonstrated that localization of biologically active gibberellins in the integuments of developing morning glory seeds coincides with expression of PnAmy1. The results from both of these studies suggests that biologically active gibberellins function as part of a process to release sugars for either translocation or further seed development. Interestingly, most of the ABA-deficient mutants screened based on sugar responsiveness were identified as aba2 alleles. (Zhou et al. 1998, Arenas-Huertero et al. 2000, Laby et al. 2000, Rook et al. 2001) and these mutants were rescued by ABA treatment (Cheng et al. 2002). It seems that gibberellin/ ABA ratio in developing seeds plays a role regulating sugar concentration in maternal tissues surrounding embryo. Our report here on the induction of CvADH1 by GA3 and sucrose is coincident with the studies described above. Further experiments with the isolated CvADH1 protein will help elucidate its biochemical activity, and provide new clues about its biological role during seed development.

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Materials and Methods

Plant materials and treatments F1 hybrid plants of watermelon (Citrullus vulgaris Thunb. var Country Home) were grown in a greenhouse that was maintained at 3035C during the day and 2025C at night. Fertile fruits were induced through hand-pollination, while parthenocarpic fruit development was instigated by applying 100 mg liter1 N-(2-chloro-4-pyridyl)N-phenylurea (CPPU) in a 0.1% Tween 20 solution to the ovaries at the flowering stage (Hayata et al. 1995). The coats and the inner portions of the seeds, including the embryo, endosperm, nucellus, and transfer cells, were separately collected after splitting the seeds with a razor blade. The roots were washed thoroughly in tap water and blotted on a paper towel. Leaf samples were harvested from 2-week-old seed-

A GA- and sucrose-inducible gene from watermelon lings. Seven days after the CPPU treatment (DAT), the fruit was dipped in either a 50 mM GA3 solution (in 0.1% Tween 20) or water for 2 h, then left for 21 h in the greenhouse (Kim et al. 2001). All plant materials were quickly frozen in liquid N2 post-harvest, and stored at 70C. Microscopic techniques Watermelon seeds were fixed in an FAA solution (37% formaldehyde : glacial acetic acid : 95% ethanol : water = 10 : 5 : 50 : 35) for 15 h at 4C. Afterward, they were dehydrated with ethanol, infiltrated with xylene, and embedded in paraffin (Paraplast x-tra, OXFORD Labware: Mckhann and Hirsch 1993). Ten-mm sections were then attached to gelatin-coated glass slides, deparaffinized in xylene, and rehydrated in a graded ethanol and water series. Sections were incubated for 1020 s in a 0.05% toluidin blue solution (0.05% toluidin blue and 0.1% sodium borate). After the excess stain was rinsed away with tap water, the samples were dehydrated with ethanol, infiltrated with xylene, and covered permanently. We used a Nikon Labophoto-2 (Nikon Inc.) for light microscopy. In situ hybridization Developing seeds were fixed in the FAA solution and embedded in paraffin as described above. Afterwards, 8- to 10-mm sections were transferred to Vectabond-coated (Vector Laboratories, Burlingame, CA, U.S.A.) slides and dried overnight at 45C. The riboprobes were prepared with the DIG nucleic acid labeling kit (Roche Molecular Biochemicals, Mannheim, Germany), according to the manufacturers protocol. Antisense and sense probes of the cDNA were synthesized using T7 and T3 RNA polymerases, and hydrolyzed in bicarbonate at 60C for 30 min. The sections were treated at 37C for 30 min with a solution containing 20 mg ml1 proteinase K. Samples were acetylated and hybridized at 50C for 18 h in a solution comprising 50% (v/v) formamide, 0.3 M NaCl, 20 mM Tris-HCl (pH 7.5), 5 mM EDTA, 5 mM Na2HPO4, 10% (w/v) dextran sulfate, 1 Denhardts solution, 0.5 mg ml1 yeast tRNA, 80 mg ml1 salmon-sperm DNA, and 300 ng riboprobe ml1. After this incubation, the slides were treated with RNase (20 mg ml1 at 37C for 30 min) to remove the free RNA probes, and were then washed at 55C for 1 h each in 1 SSC and 0.1 SSC. The hybridized probes were visualized by an anti-digoxigenin-alkaline phosphatase conjugate plus the color substrates nitroblue tetrazolium and 5-bromo-4-chloro-3-indolyl phosphate (Roche Molecular Biochemicals). DNA sequencing and computer analyses of sequences We performed dye-terminator sequencing of the cDNAs, using an automated sequencing system (373A, Perkin-Elmer/Applied Biosystems) according to the manufacturers recommendations. Multiple alignments of the conceptual amino acid sequences were generated with the ClustalX program (Thompson et al. 1997). We then constructed the neighbor-joining (NJ) tree using the computer program MEGA2 (Kumar et al. 2001) with p-distance, complete deletion of gaps, and 500 bootstrap re-samplings (Kumar et al. 2001). Isolation of full-length ORF cDNA clones We used the T3 primer (5-AATTAACCCTCACTAAAGGG-3) and the CvADH1-specific primer (5-TTTCGTCTTGGATATCGGCG3) to isolate, by PCR, the 5 region of the CvADH1 cDNA from a developing-seed cDNA library (Kim et al. 2001). The PCR profile included 1 min at 95C, 50 s at 57C, and 1 min 30 s at 72C, for a total of 35 cycles. Amplified fragments were double-digested with Pst I and Spe I (the site near the CvADH1-specific primer), and were cloned in pBluescript SK.

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DNA and RNA gel-blot analyses We used the cetyltrimethylammonium bromide method to isolate total genomic DNA from leaves of 2-week-old watermelon plants. Eight mg of the genomic DNA was digested with EcoR1 or HindIII, separated on a 0.8% agarose gel, blotted onto a Hybond-N+ filter (Amersham), and hybridized with a 32P-labeled probe prepared by the random priming method (Amersham). The filter was prehybridized for about 1 h at 65C in a solution containing 0.5 M Na2HPO4 (pH 7.2), 1 mM EDTA, 1% BSA, and 7% SDS. Hybridization was performed for 20 h at 65C in a prehybridization solution supplemented with the labeled probe. The filter was washed three times for 5 min in 0.2 SSPE and 0.1% SDS at 65C. Total RNA was isolated from various organs using Tri Reagent (Molecular Research Center, Inc.). We used 25 mg of RNA from each sample for the gel-blot analysis. After RNA transfer onto a Hybond-N+ filter, the blot was prehybridized, hybridized, and washed as described for the DNA analysis, except that the temperature was 60C.

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Acknowledgments
We thank Seonghoe Jang for comments on the manuscript and Priscilla Licht for critical reading of the manuscript. Watermelon seeds were kindly provided by the Dongbu Hannong Seeds Co. This work was funded, in part, by a grant from SRC for Plant Molecular Genetics and Breeding Research, Korea Science and Engineering Program, and by a grant from the National Research Laboratory Program of the Korea Institute of Science and Technology Evaluation and Planning (KISTEP).

References
Arenas-Huertero, F., Arroyo, A., Zhou, L., Sheen, J. and Leon, P. (2000) Analysis of Arabidopsis glucose insensitive mutants, gin5 and gin6, reveals a central role of the plant hormone ABA in the regulation of plant vegetative development by sugar. Genes Dev. 14: 20852096. Bachem, C.W., Horvath, B., Trindade, L., Claassens, M., Davelaar, E., Jordi, W. and Visser, R.G. (2001) A potato tuber-expressed mRNA with homology to steroid dehydrogenases affects gibberellin levels and plant development. Plant J. 25: 595604. Calderon-Urrea, A. and Dellaporta, S.L. (1999) Cell death and cell protection genes determine the fate of pistils in maize. Development 126: 435441. Chen, F. and Foolad, M.R. (1997) Molecular organization of a gene in barley which encodes a protein similar to aspartic protease and its specific expression in nucellar cells during degeneration. Plant Mol. Biol. 35: 821831. Cheng, W.H., Endo, A., Zhou, L., Penney, J., Chen, H.C., Arroyo, A., Leon, P., Nambara, E., Asami, T., Seo, M., Koshiba, T. and Sheen, J. (2002) A unique short-chain dehydrogenase/reductase in Arabidopsis glucose signaling and abscisic acid biosynthesis and functions. Plant Cell 14: 27232743. De Long, A., Calderon-Urrea, A. and Dellaporta, S.L. (1993) Sex determination gene TASSELSEED2 of maize encodes a short-chain alcohol dehydrogenase required for stage-specific floral organ abortion. Cell 74: 757768. Dominguez, F., Moreno, J. and Cejudo, F.J. (2001) The nucellus degenerates by a process of programmed cell death during the early stages of wheat grain development. Planta 213: 352360. Finsterbusch, A., Lindemann, P., Grimm, R., Eckerskorn, C. and Luckner, M. (1999) Delta (5)-3-beta-hydroxysteroid dehydrogenase from Digitalis lanata Ehrh. A multifunctional enzyme in steroid metabolism? Planta 209: 478 486. Gonzalez-Guzman, M., Apostolova, N., Belles, J.M., Barrero, J.M., Piqueras, P., Ponce, M.R., Micol, J.L., Serrano, R. and Rodriguez, P.L. (2002) The shortchain alcohol dehydrogenase ABA2 catalyzes the conversion of xanthoxin to abscisic aldehyde. Plant Cell 14: 18331846. Graebe, J.E. (1987) Gibberellin biosynthesis and control. Annu. Rev. Plant Physiol. 38: 419465. Groot, S.P.C., Bruinsma, J. and Karssen, C.M. (1987) The role of endogenous gibberellin in seed and fruit development of tomato: Studies with a gibberel-

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starch biosynthetic gene expression by abscisic acid signalling. Plant J. 26: 421433. Swain, S.M., Ross, J.J., Reid, J.B. and Kamiya, Y. (1995) Gibberellins and pea seed development: Expression of the lhi, ls and le5839 mutations. Planta 195: 426433. Thompson, J.D., Gibson, T.J., Plewniak, F., Jeanmougin, F. and Higgins, D.G. (1997) The ClustalX windows interface: Flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucl. Acids Res. 24: 48764882. Toyamasu, T., Yamauchi, T., Yamane, H., Murofushi, N. and Inoue, Y. (1995) cDNA cloning and characterization of gibberellin-responsive genes in photoblastic lettuce seeds. Biosci. Biotechnol. Biochem. 59: 18461849. Wan, L., Xia, Q., Qiu, X. and Selvaraj, G. (2002) Early stages of seed development in Brassica napus: A seed coat-specific cysteine proteinase associated with programmed cell death of the inner integument. Plant J. 30: 110. Wang, H.L., Offler, C.E. and Patrick, J.W. (1995a) The cellular pathway of photosynthate transfer in the developing wheat grain. II. A structural analysis and histochemical studies of the pathway from the crease phloem to the endosperm cavity. Plant Cell Environ. 18: 373388. Wang, H.L., Offler, C.E., Patrick, J.W. and Ugalde, T.D. (1994) The cellular pathway of photosynthate transfer in the developing wheat grain. I. Delineation of a potential transfer pathway using fluorescent dyes. Plant Cell Environ. 17: 257266. Wang, H.L., Patrick, J.W. and Offler, C.E. (1995b) The cellular pathway of photosynthate transfer in the developing wheat grain. III. A structural analysis and physiological studies of the pathway from the endosperm cavity to the starchy endosperm. Plant Cell Environ. 18: 389407. Wang, N. and Fisher, D.B. (1994) The use of fluorescent tracers to characterize the post-phloem transport pathway in maternal tissues of developing wheat grains. Plant Physiol. 104: 1727. Wang, N. and Fisher, D.B. (1995) Sucrose release into the endosperm cavity of wheat grains apparently occurs by facilitated diffusion across the nucellar cell membranes. Plant Physiol. 109: 579585. Weschke, W., Panitz, R., Sauer, N., Wang, Q., Neubohn, B., Weber, H. and Wobus U. (2000) Sucrose transport into barley seeds: Molecular characterization of two transporters and implications for seed development and starch accumulation. Plant J. 21: 455467. White, C.N., Proebsting, W.M., Hedden, P. and Rivin, C.J. (2000) Gibberellins and seed development in maize. I. Evidence that gibberellin/abscisic acid balance governs germination versus maturation pathways. Plant Physiol. 122: 10811088. White, C.N. and Rivin, C.J. (2000) Gibberellins and seed development in maize. II. Gibberellin synthesis inhibition enhances abscisic acid signaling in cultured embryos. Plant Physiol. 122: 10891097. Xia, Z.Q., Costa, M.A., Pelissier, H.C., Davin, L.B. and Lewis, N.G. (2001) Secoisolariciresinol dehydrogenase purification, cloning, and functional expression. Implications for human health protection. J. Biol. Chem. 276: 1261412623. Xu, F.X. and Chye, M.L. (1999) Expression of cysteine proteinase during developmental events associated with programmed cell death in brinjal. Plant J. 17: 321327. Young, T.E. and Gallie, D.R. (2000) Programmed cell death during endosperm development. Plant Mol. Biol. 44: 283301. Zhou, L., Jang, J.C., Jones, T.L. and Sheen, J. (1998) Glucose and ethylene signal transduction crosstalk revealed by an Arabidopsis glucose-insensitive mutant. Proc. Natl. Acad. Sci. USA 95: 1029410299.

lin-deficient mutant. Physiol. Plant. 71: 184190. Hayata, Y., Niimi, Y. and Iwasaki, N. (1995) Synthetic cytokinin-1-(2-chloro-4pyridyl)-3-phenylurea (CPPU) promotes fruit set and induces parthenocarpy in watermelon. J. Amer. Soc. Hort. Sci. 120: 9971000. Ikeda, A., Sonoda, Y., Vernieri, P., Perata, P., Hirochika, H. and Yamaguchi, J. (2002) The slender rice mutant, with constitutively activated gibberellin signal transduction, has enhanced capacity for abscisic acid level. Plant Cell Physiol. 43: 974979. Jacobsen, S.E. and Olszewski, N.E. (1996) Gibberellins regulate the abundance of RNAs with sequence similarity to proteinase inhibitors, dioxygenases and dehydrogenases. Planta 198: 7886. Jenab, M. and Thompson, L.U. (1996) The influence of flaxseed and lignans on colon carcinogenesis and beta-glucuronidase activity. Carcinogenesis 17: 13431348. Jornvall, H., Persson, B., Krook, M., Atrian, S., Gonzalez-Duarte, R., Jeffery, J. and Ghosh D. (1995) Short-chain dehydrogenases/reductases (SDR). Biochemistry 34: 60036013. Kallberg, Y., Oppermann, U., Jornvall, H. and Persson, B. (2002) Short-chain dehydrogenases/reductases (SDRs). Eur. J. Biochem. 269: 44094417. Kang, H.G., Jun, S.H., Kim, J., Kawaide, H., Kamiya, Y. and An, G. (1999) Cloning and molecular analyses of a gibberellin 20-oxidase gene expressed specifically in developing seeds of watermelon. Plant Physiol. 121: 373382. Kang, H.G., Jun, S.H., Kim, J., Kawaide, H., Kamiya, Y. and An, G. (2002) Cloning of gibberellin 3 beta-hydroxylase cDNA and analysis of endogenous gibberellins in the developing seeds in watermelon. Plant Cell Physiol. 43: 152158. Kim, J., Jun, S.H., Kang, H.G., Lee, J. and An, G. (2002) Molecular characterization of a GA-inducible gene, Cvsus1, in developing watermelon seeds. Mol. Cells 14: 255260. Kim, J., Jun, S.H., Lee, J., Kang, H.G. and An, G. (2001) Analyses of expressed sequence tags and mRNA expression levels of the tagged cDNAs from the watermelon anthers and developing seeds. J. Plant Biol. (formerly Kor. J. Bot.) 44: 172177. Kumar, S., Tamura, K., Jakobsen, I.B. and Nei, M. (2001) MEGA2: Molecular evolutionary genetics analysis software. Bioinformatics 17: 12441245. Laby, R.J., Kincaid, M.S., Kim, D. and Gibson, S.I. (2000) The Arabidopsis sugar-insensitive mutants sis4 and sis5 are defective in abscisic acid synthesis and response. Plant J. 23: 587596. Lange, T., Robatzek, S. and Frisse, A. (1997) Cloning and expression of a gibberellin 2b,3b-hydroxylase cDNA from pumpkin endosperm. Plant Cell 9: 14591467. MacMillan, J., Ward, D.A., Phillips, A.L., Sanchez-Beltran, M.J., Gaskin, P., Lange, T. and Hedden, P. (1997) Gibberellin biosynthesis from gibberellin A12-aldehyde in endosperm and embryos of Marah macrocarpus. Plant Physiol. 113: 13691377. Mckhann, H.I. and Hirsch, A.M. (1993) In situ localization of specific mRNAs in plant tissues. In Methods in Plant Molecular Biology and Biotechnology. Edited by Glick, B.R. and Thompson, J.E. pp. 179205. CRC Press, Boca Raton, FL. Nakayama, A., Park, S., Zheng-Jun, X., Nakajima, M. and Yamaguchi, I. (2002) Immunohistochemistry of active gibberellins and gibberellin-inducible alphaamylase in developing seeds of morning glory. Plant Physiol. 129: 1045 1053. Persson, B., Krook, M. and Jornvall, H. (1991) Characteristics of short-chain alcohol dehydrogenases and related enzymes. Eur. J. Biochem. 200: 537543. Rook, F., Corke, F., Card, R., Munz, G., Smith, C. and Bevan, M.W. (2001) Impaired sucrose-induction mutants reveal the modulation of sugar-induced

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(Received August 28, 2002; Accepted November 10, 2002)