cvadh1, a member of short-chain alcohol dehydrogenase family, is

8/3/2019 CvADH1, A Member of Short-Chain Alcohol Dehydrogenase Family, Is

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Plant Cell Physiol. 44(1): 85–92 (2003)

JSPP © 2003

85

CvADH1, a Member of Short-Chain Alcohol Dehydrogenase Family, isInducible 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 Ara-

bidopsis thaliana, stem secoisolariciresinol dehydrogenase

in Forsythia intermedia, and 3b-hydroxysterol dehydroge-

nase in Digitalis lanata. We investigated gene-expressionlevels in seeds from both normally pollinated fruits and

those made parthenocarpic via N-(2-chloro-4-pyridyl)- N ¢-

phenylurea treatment, the latter of which lack zygotic tis-

sues. 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. Treat-

ing 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 water-

melon 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 gib-

berellin 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,

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 anddelayed 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 hydro-

lytic enzymes required for the degradation of starch grains.

Afterward, sugars can be released for either translocation orfurther seed development. Kim et al. (2002) have shown that

GA3

also strongly induces CvSUS1, a putative sucrose syn-

thase, 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 projec-

tion 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 tis-

sue 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.

1 Corresponding author: e-mail, [email protected]; Fax, +82-54-279-2199.

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A GA- and sucrose-inducible gene from watermelon86

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 water-

melon seeds

We isolated a seed-preferential EST clone from a develop-

ing-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. Tran-

script 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-

cult to isolate RNA from seeds after 20 DAP. During imbibi-

tion, 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 ribos-

omal 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 short-

chain alcohol dehydrogenase/reductase (SDR) family. Espe-

cially, 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 dehydro-

genase 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 thedatabases, using the ClustalX and MEGA2 programs. Signifi-

cant sequence similarity was revealed between the predicted

CvADH1 protein and a family of short-chain alcohol dehydro-

genases (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 hybrid-

ized 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 par-

thenocarpic 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-(2-

chloro-4-pyridyl)- N ¢-phenylurea (CPPU), a synthetic cytoki-

nin. Although zygotic tissues do not form due to parthenocarpy,

seed coats do develop normally in the early stages (Kang et al.

Fig. 1 RNA gel-blot analyses of temporal and spatial expression of

CvADH1 (accession No. AB018559). Total RNA (25 mg) was gel-

fractionated, blotted, and proved with the cDNA fragment of CvADH1.

The cDNA of Cvrp15a (accession No. AB018560) was used as a con-

trol. (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 from0.5 to 3 d after imbibition.

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A GA- and sucrose-inducible gene from watermelon 87

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 bind-ing 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 pro-

tein 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 dehydroge-

nase, or 3b HSD (AJ345026), Pisum sativum sadA (AF053638), Lactuca sativa alcohol dehydrogenase (JC4320), and Solanum tuberosum CB12

(AF349916).

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2002). In seeds that were fertilized normally, the maternal tis-

sues 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 partheno-

carpic 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. Alterna-

tively, 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 pat-

tern of the gene, we examined transcript levels in the micropy-

Fig. 4 Gel-blot analysis of genomic DNA from CvADH1, as digestedwith 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. 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.

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, micro-

pylar; M, middle; Ch, chalazal portions.

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lar, middle, and chalazal portions of the seeds. Transcript

amounts were almost equal in all three sections, which pre-

cludes the possibility that the clone is expressed only in the

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 gib-

berellins in seeds makes it difficult to study the effects of exog-

enous 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).

Discussion

We have previously reported that differential expression

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 liter –1 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 condi-

tions are as described for (A).

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 pre-

pared 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

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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 gibberel-

lins 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 pref-

erentially 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 con-

tents 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 parthe-

nocarpic seeds, it is likely that its expression in normally devel-

oping 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 exam-

ple, the gad3 gene from tomato is down-regulated by gibberel-

lin 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 etal. 1993, Calderon-Urrea and Dellaporta 1999). Recently, Ara-

bidopsis ABA2, which is closely related to CvADH1 in a phyl-

ogenetic 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 Arabi-

dopsis ABA2 gene expression has not been observed, it is pos-

sible 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 endog-

enous ABA level.

CvADH1 from watermelon appears to be a member of the

short-chain alcohol dehydrogenases family that reduces ring-

alcohol 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 dehydroge-

nase (which has the highest homology with CvADH1) pro-

duces matairesinol, a central precursor of numerous lignans,

including that of the important antiviral and anticancer agent,

podophyllotoxin (Xia et al. 2001). This compound is responsi-

ble 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 elimi-nation or biosynthesis of an unknown signaling molecule possi-

bly related to steroids. It also had been speculated that StGAN

is involved in a biochemical route for the breakdown of gib-

berellin (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 biolog-ically 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 bio-

logically active gibberellins in the integuments of developing

morning glory seeds coincides with expression of PnAmy1.

The results from both of these studies suggests that biologi-

cally 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 eluci-

date its biochemical activity, and provide new clues about its

biological role during seed development.

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

30–35°C during the day and 20–25°C at night. Fertile fruits were

induced through hand-pollination, while parthenocarpic fruit develop-

ment was instigated by applying 100 mg liter –1 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 por-

tions 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-

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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

–70°C.

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 4°C. 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 10–20 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 etha-

nol, infiltrated with xylene, and covered permanently. We used a

Nikon Labophoto-2 (Nikon Inc.) for light microscopy.

In situ hybridizationDeveloping 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 45°C. The riboprobes were

prepared with the DIG nucleic acid labeling kit (Roche Molecular Bio-

chemicals, Mannheim, Germany), according to the manufacturer’s pro-

tocol. Antisense and sense probes of the cDNA were synthesized using

T7 and T3 RNA polymerases, and hydrolyzed in bicarbonate at 60°C

for 30 min. The sections were treated at 37°C for 30 min with a solu-

tion containing 20 mg ml –1 proteinase K. Samples were acetylated and

hybridized at 50°C for 18 h in a solution comprising 50% (v/v) forma-

mide, 0.3 M NaCl, 20 mM Tris-HCl (pH 7.5), 5 mM EDTA, 5 mM

Na 2HPO

4, 10% (w/v) dextran sulfate, 1´ Denhardt’s solution, 0.5 mg

ml –1 yeast tRNA, 80 mg ml –1 salmon-sperm DNA, and 300 ng ribo-

probe ml –1. After this incubation, the slides were treated with RNase

(20 mg ml –1 at 37°C for 30 min) to remove the free RNA probes, and

were then washed at 55°C 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 Bio-

chemicals).

DNA sequencing and computer analyses of sequences

We performed dye-terminator sequencing of the cDNAs, using

an automated sequencing system (373A, Perkin-Elmer/Applied Bio-

systems) according to the manufacturer’s recommendations. Multiple

alignments of the conceptual amino acid sequences were generated

with the ClustalX program (Thompson et al. 1997). We then con-

structed the neighbor-joining (NJ) tree using the computer programMEGA2 (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¢-TTTCGTCTTGGATATCGGCG-

3¢) 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 95°C, 50 s at 57°C, and 1 min 30 s at 72°C, 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 – .

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 forabout 1 h at 65°C in a solution containing 0.5 M Na

2HPO

4(pH 7.2),

1 mM EDTA, 1% BSA, and 7% SDS. Hybridization was performed

for 20 h at 65°C 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 65°C.

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 60°C.

Acknowledgments

We thank Seonghoe Jang for comments on the manuscript andPriscilla 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 Genet-

ics 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).

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

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cvadh1, a member of short-chain alcohol dehydrogenase family, is

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