Molecular characterization and expression studies during melon fruit development and ripening of...

RESEARCH PAPER

Molecular characterization and expression studiesduring melon fruit development and ripening ofL-galactono-1,4-lactone dehydrogenase

Irene Pateraki1,2, Maite Sanmartin1,2,3,*, Mary S. Kalamaki1, Dimitrios Gerasopoulos1,y

and Angelos K. Kanellis1,2,z

1 Group of Biotechnology of Pharmaceutical Plants, Laboratory of Pharmacognocy,Department of Pharmaceutical Sciences, Aristotle University of Thessaloniki,541 24 Thessaloniki, Greece2 Institute of Viticulture and Vegetable Crops, National Agriculture Research Foundation,PO Box 2229, 713 09 Heraklion, Crete, Greece3 Institute of Molecular Biology and Biotechnology, FORTH, PO Box 1527, 711 10 Heraklion, Crete, Greece

Received 10 February 2004; Accepted 27 April 2004

Abstract

The last step of ascorbic acid (AA) biosynthesis is

catalysed by the enzyme L-galactono-1,4-lactone dehy-

drogenase (GalLDH, EC 1.3.2.3), located on the inner

mitochondrial membrane. The enzyme converts L-gal-

actono-1,4-lactone to ascorbic acid (AA). In this work,

the cloning and characterization of a GalLDH full-length

cDNA from melon (Cucumis melo L.) are described.

Melon genomic DNA Southern analysis indicated that

CmGalLDH was encoded by a single gene. CmGalLDH

mRNA accumulation was detected in all tissues stud-

ied, but differentially expressed during fruit develop-

ment and seed germination. It is hypothesized that

induction of CmGalLDH gene expression in ripening

melon fruit contributes to parallel increases in the AA

content and so playing a role in the oxidative ripening

process. Higher CmGalLDH message abundance in

light-grown seedlings compared with those raised in

the dark suggests that CmGalLDH expression is re-

gulated by light. Finally, various stresses and growth

regulators resulted in no significant change in steady

state levels of CmGalLDH mRNA in 20-d-old melon

seedlings. To the authors’ knowledge, this is the first

report of GalLDH transcript induction in seed germina-

tion and differential gene expression during fruit

ripening.

Key words: Ascorbic acid, biosynthesis, Cucumis melo L., fruit

development, L-galactono-1,4-lactone dehydrogenase, melon,

ripening.

Introduction

L-Ascorbic acid (AA) plays a multifunctional role in bothplants and animals, and comprises one of the most abundantmetabolites in green tissues. Humans, plus other primatesand some birds and bats, are unable to synthesize AA.Consequently, they depend on dietary intake to cover theirrequirements (Chatterjee, 1973). Fruit and vegetables com-prise the major dietary sources of AA in humans and recentstudies suggest that increased AA intake (from 60 to 200mg d�1) may carry health benefits (Carr and Frei, 1999;Levine et al., 1999).

Much attention has focused on the antioxidant roleplayed by AA in both plants and animals. However, thecompound acts as a cofactor for a suite of enzymes,including 1-aminocyclopropane-1-carboxylic acid (ACC)oxidase, a key enzyme in the biosynthetic pathway ofethylene, the ‘ripening hormone’ (Ververidis and John,1991) and plays a vital role in many physiological pro-cesses (Smirnoff, 1996; Noctor and Foyer, 1998; Smirnoffand Wheeler, 2000). AA seems to affect the transcription ofa number of genes including PR (Pathogenesis Related)

* Present address: Departamento de Genetica Molecular de Plantas, Centro Nacional de Biotecnologıa CSIC, 28049 Madrid, Spain.y Present address: Department of Agriculture, Aristotle University of Thessaloniki, 541 24 Thessaloniki, Greece.z To whom correspondence should be addressed. Fax: +30 2310 997662, 997645. E-mail: [email protected]

Journal of Experimental Botany, Vol. 55, No. 403, pp. 1623–1633, August 2004

DOI: 10.1093/jxb/erh186 Advance Access publication 2 July, 2004

Journal of Experimental Botany, Vol. 55, No. 403, ª Society for Experimental Biology 2004; all rights reserved

by guest on May 7, 2014

http://jxb.oxfordjournals.org/D

ownloaded from

http://jxb.oxfordjournals.org/

proteins and those involved in the biosynthesis of abscisicacid (Pastori et al., 2003). AA directly scavenges reac-tive oxygen species (ROS) and regenerates lipophilic a-tocopherol to its reduced state (Noctor and Foyer, 1998;Asada, 1999). As a consequence, the compound plays a veryimportant role in protecting plant tissues against oxidativedamage provoked by a range of environmental stressesincluding excess light, soil water deficit, water logging,UV-B radiation, and gaseous pollutants (Conklin et al.,1996; Noctor and Foyer, 1998; Smirnoff and Wheeler,2000; Sanmartin et al., 2003). AA also maintains theprosthetic metal groups of various enzymes in the reducedstate ensuring their activity (Padh, 1990; Davey et al.,2000). Recent studies indicate that ascorbate is involvedin the process and regulation of both cell division andexpansion (Arrigoni, 1994; Kerk and Feldman, 1995;Smirnoff, 1996; Davey et al., 2000; Tabata et al., 2001).Work on transgenic potato plants expressing GDP-mannosepyrophosphorylase in the antisense orientation and thevtc-1 mutant of Arabidopsis thaliana reveal that a declinein AA content correlates with reduced cell growth andabnormalities in cell shape (Keller et al., 1999; Veljovic-Jovanovic et al., 2001). Ascorbate may modulate celldivision by controlling the transition from G1 to S duringthe cell cycle (Kerk and Feldman, 1995). Moreover, AAand its oxidation products in conjunction with the enzymeascorbate oxidase (AO) may comprise a regulatory systemcontrolling cell expansion (Smirnoff, 1996; Kato andEsaka, 1999, 2000), though the mechanism is poorlyunderstood (Pignocchi et al., 2003). Further, the AA redoxstate, controlled by AO activity levels, is mainly respon-sible for the apoplast capability of transmitting signalsrelated with environmental changes or defence processes(Pignocchi and Foyer, 2003; Sanmartin et al., 2003).In plants, AA is synthesized via the conversion of

mannose-6-P via GDP-mannose to GDP-L-galactose, a stepcatalysed by the enzyme GDP-mannose-39,59-epimerase,an enzyme purified and cloned from Arabidopsis thaliana(Wolucka et al., 2001). L-Galactose is produced from GDP-L-galactose (by uncharacterized enzymes), which is oxi-dized to L-galactono-1,4-lactone (GalL) by L-galactosedehydrogenase (GalDH) (Wheeler et al., 1998). An alter-native pathway, recently established in plants, involves theconversion of D-galacturonic acid (a component of cellwall pectins) to L-galactonic acid by D-galacturonic acidreductase (GalUR) (Agius et al., 2003). L-Galactonic acidis then converted to L-galactono-1,4-lactone. Expression ofGalUR correlates with increased ascorbate levels in ripen-ing strawberry fruit, and over-expression of GalUR intransgenic A. thaliana results in a 2–3-fold increase in AAcontent in selected transgenic lines (Agius et al., 2003).The final step in the AA biosynthetic pathway requires

the oxidation of GalL to AA, a step facilitated by theenzyme L-galactono-1,4-lactone dehydrogenase (GalLDH).This enzyme has been known for more than 40 years

(Mapson and Breslow, 1958) and GalLDH activity hasbeen described in pea, cabbage, cauliflower, and sweetpotato (Mapson and Breslow, 1958; Oba et al., 1994;Mutsuda et al., 1995; Østergaard et al., 1997; Imai et al.,1998). The enzyme has been purified from cauliflower(Østergaard et al., 1997) and sweet potato (Imai et al.,1998) and cDNAs encoding GalLDH have been character-ized from cauliflower, sweet potato, strawberry, tomato,tobacco, and Arabidopsis (Østergaard et al., 1997; Imaiet al., 1998; Yabuta et al., 2000; Tamaoki et al., 2003).Recent studies demonstrate that GalLDH is an intrinsicmembrane protein linked to the inner mitochondrial mem-brane (Siendones et al., 1999; Bartoli et al., 2000) andemploys cytochrome c as a second substrate (Oba et al.,1995; Østergaard et al., 1997; Imai et al., 1998). DuringGalLDH activity, electrons are probably transferred to themitochondrial electron transport chain between complexesIII and IV (Bartoli et al., 2000).

A number of observations indicate that AA contentincreases during fruit ripening (Moser and Kanellis, 1994;Davey et al., 2000; Jimenez et al., 2002; Agius et al.,2003), implying cellular antioxidant status may play afundamental role in this process. However, very little isknown about the expression of AA biosynthetic enzymesduring this physiological process. In the present study, thecloning and characterization of a melon cDNA designatedCmGalLDH, is reported and its expression in fruit tissues isdescribed for the first time. It is demonstrated thatCmGalLDH is expressed in all tissues studied, but differ-entially regulated during fruit development and seed ger-mination. CmGalLDHmRNA expression is induced duringmelon fruit ripening concomitant with increased AA levels.In addition, the effect of various hormones and stresstreatments on CmGalLDH expression is examined anddiscussed.

Materials and methods

Plant culture and sampling

Melon (Cucumis melo L., variety reticulatus, F1 alpha hybrid) seedswere kindly provided by the Tezier Breeding Institute, Valence(France). Melon plants were grown in a naturally illuminatedglasshouse at 25 8C day/night temperature. Tissue from root, shoot,young leaves, apices, petals, stamens, and ovaries was collected,immediately frozen in liquid nitrogen, and stored at �80 8C. Fruitwere picked at various developmental stages (5, 10, 15, 20, 25, 30,32, 34, 36, 38, and 40 d after pollination [DAP]), the inner and outerareas of the fruit mesocarp were separated using a scalpel, frozen inliquid nitrogen, and then stored at �80 8C.For germination studies, seeds were germinated on damp cotton at

25 8C day/night temperature (in the dark). After 4 d, seedlings weretransferred to pots containing perlite and watered every 2 d witha commercial fertilizer. Seedlings were raised in growth chambersfor 16 h photoperiod (or continuous dark for etiolation treatment) at25 8C day/night.For feed and stress treatments, 20-d-old seedlings were grown as

described above. The growing medium was carefully removed bywashing the roots in tap water prior to transfer to 50 mM phosphate

1624 Pateraki et al.



ownloaded from


buffer, pH 7.0 (control) or solutions of 500 lM indole-3-acetic acid(IAA), 100 lM abscisic acid (6) cis-trans isomer (ABA), 50 lM(6)-jasmonic acid (JA), 50 lM salicylic acid (SA) or 50 lM ascorbicacid (AA). Plants were incubated in the light in the respectivesolutions for 12 h in the growth chambers described above. Allchemicals were purchased from Sigma-Aldrich Co (St Louis, USA).For the wounding treatment, leaves and cotyledons of 20-d-old

seedlings (5–6 plants per treatment) were crushed using sterileforceps and samples taken after 0, 6, 12, and 24 h. For heat-shockexperiments, seedlings were transferred to a growth chamber main-tained at 42 8C. Samples (5–6 plants per treatment) were collected 0,6, 12, or 24 h after transfer. Plant material was immediately frozen inliquid nitrogen and stored at �80 8C. Experiments were repeatedthree times and at least three replicates were used in each experiment.

Cloning of melon CmGalLDH

Total RNA isolated from melon ovaries was DNase I-treated(Promega GmbH, Mannheim, Germany) and used to synthesizesingle-stranded cDNA using the M-MuLV (New England Biolabs,Beverly, MA, USA) reverse transcriptase and oligodT primer [59-ACTAGTCTCGAG(T)19-39] according to the manufacturer’s in-structions. This cDNA was used as template in PCR for melonCmGalLDH cDNA amplification with degenerate primers [G-SEN2:59-A(CT)AT(ACT)CC(AGCT)TA(CT)AC(AGCT)GA(CT)(AG)C-39 and G-AS1: 59-CCA(AGCT)CC(AGCT)AC(AGCT)C(GT)(AG)-TA(AGCT)CC(CT)TC-39], designed based on the conserved regionsamong known GalLDH amino acid sequences (Østergaard et al.,1997; Imai et al., 1998). PCR amplification was performed for 40cycles (94 8C for 60 s, 55 8C for 90 s, and 72 8C for 90 s) followedby a final extension step of 10 min at 72 8C. PCR products were gelpurified and cloned into pGEM-T Easy (Promega GmbH, Man-nheim, Germany) plasmid vector. A LI-COR Long Readir 4200automated sequencer and a ‘Sequitherm EXCELII’ kit (Epicentre,Madison, WI, USA) were used to determine the nucleotide sequenceof the resulted clones. Based on the nucleotide sequence obtained,CmGalLDH-specific primers [G-SEN3: 59-TATCTCAAAATGGA-GAGGCCCTCCG-39 and G-AS3: 59-TCCTCCAGAACTCAGCCT-CTGCTTG-39] were designed for cloning the 59 and 39 remainingcDNA sequences. 59 and 39 RACE were performed using the ‘Mara-thon cDNA Amplification System’ (Clontech, Palo Alto, CA,USA). For full-length cDNA amplification a primer was designedfrom the 59 end containing the ATG translation initiation codon[G-ATG: 59-ATGCTCAACTTTCTCTCTCTTCGGCG-39]. PCRwas performed using the G-ATG and oligodT primers for 40 cyclesusing a touch-down strategy (10 cycles at 94 8C for 60 s, 55 8C! 45 8Cfor 90 s, and 72 8C for 180 s; 30 cycles at 94 8C for 60 s, 45 8C for90 s, and 72 8C for 180 s) followed by an extension step of 10 minat 72 8C. Double-stranded cDNA synthesized from melon ovaries’total RNA according to the ‘Marathon cDNA Amplification System’manual was employed as the template.

Sequence analysis

DNA sequence data were analysed using the National Centre ofBiotechnology (NCBI) web site (http://www.ncbi.nlm.nih.gov). TheBLAST program, developed by Altschul et al. (1990), was used tosearch for sequence homology to the EMBL plant DNA sequencedatabase and Swissprot protein database. The alignment of thededuced protein sequences and the phylogenetic tree were computedusing the ClustalW program (Thompson et al., 1994) employingstandard parameters.

Melon genomic DNA extraction and Southern blot analysis

Total genomic DNA was isolated from melon ovaries using themethod of Diallinas et al. (1997). Genomic DNA (10 lg) wasdigested with HindIII, EcoRI, and PstI, fractionated in 0.8% (w/v)

agarose gel, transferred on to nylon membrane (Nytran� 0.45,Schleicher & Schuell) and hybridized with a CmGalLDH fragment.Probe labelling was carried out with RadPrime DNA LabellingSystem (Invitrogen, Life Technologies, Madison, WI, USA) accord-ing to the manufacturer’s instructions. Blot hybridization andmembrane washing were performed as described by Church andGilbert (1984).

RNA blot analysis

Total RNA was isolated from vegetative tissues and ovaries of melonas described byWadsworth et al. (1988). Total RNA frommelon fruittissues was isolated using the method described by Smith et al.(1986). Total RNA (15 lg) was fractionated in formaldehydedenaturing agarose gels, transferred to nylon membranes (Nytran�

0.45, Schleicher and Schuell, GmbH, Dassel, Germany), stained with0.04% methylene blue (to observe equality of loading) and hybrid-ized with radio-labelled probes for CmGalLDH and melon ACOcDNAs. Alternatively, to ensure equal loading, RNA blots werehybridized with the 18S melon rRNA probe. RNA blot hybridizationand membrane washing were performed as described above.

Ascorbic acid analysis

One gram of frozen tissue from whole, inner and outer mesocarptissues were powdered in liquid nitrogen and extracted as describedby Foyer et al. (1983). Ascorbic acid was calculated as the differencein absorbance at 265 nm before and after the addition of 1 unit ml�1

of ascorbate oxidase (Sigma Chemical Co., St Louis, USA) in 100mM sodium phosphate buffer pH 5.6. Absorbance values wereevaluated against a standard curve constructed using L-ascorbic acid(Merck KGaA, Darmstadt, Germany) in the range of 0–100 nmol andreported as lmol g�1 FW.

Results

Cloning and characterization of CmGalLDH cDNA

Degenerate primers designed from conserved amino acidregions of cauliflower and sweet potato GalLDH (Øster-gaard et al., 1997; Imai et al., 1998) were used to amplifya 281 bp fragment by RT-PCR from melon ovary totalRNA. Sequence analysis revealed this fragment to encodea partial melon GalLDH. Based on this cDNA sequence,specific primers were designed to amplify the 59 and 39flanking regions using RACE techniques. Using G-ATGand oligodT primers, a 2097 bp cDNA clone (EMBLaccession number AF252339) was obtained. Nucleotide se-quence analysis of this cDNA revealed that it correspondedto a GalLDH full-length cDNA, termed Cucumis meloGalLDH (CmGalLDH). It contains a 58 bp 59 untranslatedregion followed by a 1770 bp open reading frame, codingfor a 589 amino acids polypeptide, and a 269 bp 39untranslated region including a 42 bp polyA tail. Nocanonical polyadenylation-processing signals (AATAAAor AATGAA) were found, which is common in most plantgenes (Graber et al., 1999).

In silico analysis of the CmGalLDH amino acid se-quence predicted a molecular weight of 67 059 Da. How-ever, this polypeptide contained a putative mitochondrial-targeting domain in its first 89 amino acids and acleavage site (FR/YA) similar to other known GalLDHs.

Characterization and expression of CmGalLDH 1625



ownloaded from

http://www.ncbi.nlm.nih.gov


Therefore, the mature CmGalLDH protein consists of 500amino acid residues having a calculated molecular weightof 56 731 Da and a pI of 6.71. Three possible trans-membrane regions were predicted in accordance with sweetpotato GalLDH (Bartoli et al., 2000). Amino acid sequencecomparison of CmGalLDH showed 79.2% identity withArabidopsis, 79.1% with strawberry, 78.9% with tobacco,78.2% with cauliflower, 78% with sweet potato, and 74%with tomato (Fig. 1A). Analysis of CmGalLDH amino acidsequence identified a putative FAD binding domain be-tween residues 111 and 162 (Fig. 1A), wherein is locateda region (139VGSGLSP145) common to all GalLDHscharacterized to date (Østergaard et al., 1997; Imai et al.,1998; Yabuta et al., 2000; Tamaoki et al., 2003).To investigate the evolutionary relationships between

GalLDH sequences and homologues, a phylogenetic anal-ysis was performed using the Clustal W program (Fig. 1B).All plant sequences analysed were clustered in three groups,according to their species relationship, while those of yeastD-arabino-1,4-lactone oxidase and rat L-gulono-c-lactoneoxidase formed a distal group, suggesting that these genesevolved independently, following species divergence.Melon total genomic DNA was digested with different

restriction enzymes (PstI, EcoRI and HindIII) and blottedon to nylon membrane. Only one positive band wasdetected in each lane suggesting that CmGalLDH is codedby a single gene (Fig. 2).

Expression of CmGalLDH and ascorbic acid levels invegetative tissues and shifts in fruit associated withdevelopment and ripening

GalLDH message accumulation was detected in seeds,roots, stems, leaves, shoot apices, petals, and stamens, al-though different levels of transcript abundance were ob-served (Fig. 3). CmGalLDH transcript levels were highestin stems and lowest in seeds and roots. In floral tissues,CmGalLDHmRNAwas abundant with stronger expressionin petals than in stamens (Fig. 3).CmGalLDH expression studies undertaken on total RNA

isolated from ovaries and 5, 10, 15, 20, 25, 30, 32, 34, 36,38, and 40 DAP fruit revealed a large accumulation of theCmGalLDH message in ovaries and lower levels in de-veloping green fruit (5, 10, 15, 20, and 25 DAP; Fig. 4A).However, after the onset of melon ripening, indicated byACO mRNA levels (30 DAP; Fig. 4C), there was a markedincrease in GalLDH transcript levels. The peak of GalLDHexpression paralleled the ACO message climacteric peakat 36 DAP. Moreover, CmGalLDH transcript levels werenotably higher in the outer mesocarp of maturing fruit (after30 DAP) compared with the inner mesocarp (Fig. 4A),whereas CmGalLDH mRNA levels were barely detectablein the inner mesocarp of ripening fruit (38 and 40 DAP).AA levels were determined in the same fruit tissues

probed for the expression of CmGalLDH. AA levels werehigh in ovaries, but declined progressively in developing

green fruit (5–25 DAP) (Fig. 4B). As fruit began to mature,AA levels increased in both the inner and outer mesocarpwith the highest levels observed at 34 DAP in the innermesocarp. In 40 DAP ripe fruit, AA levels were equal inboth the inner and outer mesocarp (Fig. 4B). It is interestingto note that AA concentration is higher in the inner than inthe outer mesocarp of mature and ripe fruit which isconverse to the CmGalLDH mRNA steady-state levels.

In parallel, ACO transcript abundance was also moni-tored in the tissues mentioned above. The ACO mRNAmessage was not detected in fruit younger than 30 DAP, buttranscripts accumulated in both the inner and outer meso-carp up to 38 DAP. Thereafter, transcript levels declinedand were barely detectable in ripe fruit (40 DAP) (Fig. 4C).In contrast to the differential expression of CmGalLDH inthe outer versus the inner mesocarp, ACO transcript levelsresponded similarly in the inner and outer mesocarp.

Effect of light on CmGalLDH expression during seedgermination and seedling development

To investigate whether CmGalLDH expression was dif-ferentially regulated during germination, blot analysis wasperformed of total RNA extracted from dry and germi-nating seeds, 1–4 d after they were imbibed. CmGalLDHtranscript levels were barely detectable in dry seeds, butaccumulated during germination, reaching the highest levelof expression in 3-d-old seedlings (Fig. 5). In seedlingsgrown in the light, CmGalLDH transcript levels increased,attaining a maximum level 10 d after seeds were imbibed.Conversely, seedlings raised in the dark exhibited a pro-nounced decline in the CmGalLDH message, to such anextent that transcript levels were barely detectable in 10-d-old seedlings (Fig. 5).

CmGalLDH gene expression in response to planthormones, signalling agents and stress conditions

CmGalLDH expression showed no significant change inmelon seedlings fed with IAA, AA, JA, SA, or ABA(Fig. 6). Moreover, there was no induction of CmGalLDHtranscript levels in melon leaves and cotyledons followingwounding or heat-shock (Fig. 7). It should be mentionedthat the above treatments (IAA, AA, JA, SA, or ABA) didnot cause any phenotypic effect in the treated seedlingswhile seedlings subjected to wounding or heat stress werevisibly wilted.

Discussion

The final step in AA biosynthesis in planta requires theoxidation of GalL by L-galactono-1,4-lactone dehydroge-nase (GalLDH). In this work, the cloning and character-ization of a cDNA encoding CmGalLDH are reported. Thededuced amino acid sequence presented 74–79% homologywith other known GalLDHs and lower levels of homology




ownloaded from


Fig. 1. Comparison of deduced amino acid sequences from melon CmGalLDHwith known sequences. Peptide sequence alignment (A) was created withthe ClustalW programme. The amino acid residues that are identical among the sequences from at least five species aligned are indicated in a black boxwhile similar residues are shown in grey boxes. The putative signal peptide cleavage site is shown by a hatched arrow. Gaps in the sequence created bythe programme to maximize similarities are shown as dashes. Suggested FAD-binding domain is indicated in asterisks. Amino acid sequence ofCmGalLDH (accession number AAF64319), cauliflower (CAB09796), sweet potato (BAA34995), tobacco (BAA87934), Arabidopsis (BAA95212),tomato (BAC11758), and strawberry (AAM48582), of rat L-gulono-c-lactone oxidase (AAA41164) and yeast (Saccharomyces cerevisiae) D-arabinono-1,4-lactone oxidase (AAC98538) are shown. The deduced protein sequences were subjected to phylogenetic analysis (B) using the same program.




ownloaded from


with (i) L-gulono-c-lactone oxidase (27%), the terminalenzyme mediating AA biosynthesis in animals, and (ii)yeast D-arabinono-1,4-lactone oxidase (26%), an enzymethat participates in the biosynthesis of the five-carbon AAanalogue D-erythroascorbic acid common in fungi (Loewus,1999). Homology was mainly restricted to the aminoterminal region of GalLDH. As suggested by Østergaardet al. (1997), this could illustrate the existence of a functionaldomain.Recent studies have demonstrated that GalLDH is an

intrinsic membrane protein linked to the innermitochondrial

membrane (Siendones et al., 1999; Bartoli et al., 2000). Theputative CmGalLDH polypeptide sequence presented amitochondrial targeting signal in the amino terminal end,rich in Ala, Leu, Arg, and Ser residues (7, 9, 8, and 12,respectively) and with relatively few Asp, Glu, Ile, and Valresidues (1, 2, 4, and 2, respectively). This composition issimilar to that reported for other known GalLDHs andmatched the characteristics of mitochondrial target pep-tides (von Heije, 1986). In addition, in silico analysis of theCmGalLDH sequence predicted three possible transmem-brane regions, located in similar positions to those identified

Fig. 1. (continued)




ownloaded from


in sweet potato GalLDH (Bartoli et al., 2000). Thesefindings lend further support to the contention that GalLDHspans the inner mitochondrial membrane with the active sitefacing the intermembrane space in a manner circumnavigat-ing the requirement for GalL and AA transport through theinner membrane (Bartoli et al., 2000). The existence ofa FAD binding domain suggests that the flavin group mightbe involved in the reaction catalysed byGalLDH (Oba et al.,1995).

CmGalLDH expression was detectable in all tissuesstudied (Fig. 3). This is not an unexpected finding, sinceAA is present in practically all plant tissues and recentstudies have shown a strong correlation between GalLDHexpression patterns and AA levels (Tabata et al., 2001,2002). In the present study, organ-specific and novel fruit-ripening-associated accumulation of CmGalLDH transcriptlevels was shown. In general, ripening-associated increasesin CmGalLDH mRNA levels were accompanied by in-creases in AA content in melon. However, there appearedto be spatial segregation in the fruit mesocarp between theincreased expression of CmGalLDH and increases in AAcontent between the outer and inner pericarp, respectively.The ripening of melon fruit, as with other fruit (Moser andKanellis, 1994; Jimenez et al., 2002; Agius et al., 2003;present study), is commonly accompanied by an increasein AA content, although the mechanistic significance ofthis observation is poorly understood. Fruit ripening is a

developmental process where oxidative processes take placeand a variety of ROS accumulates (Brennen and Frenkel,1977; Jimenez et al., 2002). The observed increase in AAcontent during this developmental stage may be part of aglobal antioxidant response that accompanies the ripeningprocess (Jimenez et al., 2002). Moreover, ACO, the finalenzyme in the synthesis of ethylene, the ‘ripening hor-mone’ (Abeles et al., 1992), requires AA as a cofactor. Thisrequirement may explain the pattern of expression ob-served in CmGalLDH and why there is a need of increasedAA levels in mature fruit cells. In contrast to the prefer-ential expression of CmGalLDH in the outer mesocarp,ACO transcripts accumulated equally in both inner andouter fruit mesocarp (Fig. 4A, C). This implies that theinduction of CmGalLDH gene expression during melonfruit development and ripening is independent of ethylenebiosynthesis and, consequently, ethylene action.

Alternatively, the activation of the CmGalLDH geneduring melon fruit ripening may constitute part of a broaderstrategy to conserve carbon losses following cell wallbreakdown (Davey et al., 2000). Fruit ripening is charac-terized by a progressive loss of mesocarp firmness pro-voked by fruit cell wall disassembly (Brummell andHarpster, 2001). Changes in cell wall structure includesolubilization and depolymerization of pectins, usuallyabundant in fruit cell walls (Redgwell et al., 1997; Roseet al., 1998). In the latter stages of melon fruit ripening,polygalacturonase-dependent pectin degradation can occur(Rose et al., 1998) resulting in the concomitant liberation ofD-galacturonic acid, a compound proposed many years ago(Isherwood et al., 1954), and shown recently (Agius et al.,2003), to be an alternative precursor of AA. It is possible,as suggested by the discoverers of this alternative AAbiosynthetic pathway, that this newly-identified means ofproduction may be activated in a developmental manner,influencing AA content.

The apparently reciprocal accumulation of AA andCmGalLDH mRNA levels in the inner and outer fruitmesocarp, respectively, may be explained by differences inthe spatial expression of ascorbate oxidase (CMAO1).Indeed, Sanmartin (2002) showed that throughout fruitripening levels of CMAO1 were markedly higher in theouter versus the inner mesocarp. Consequently, AA formed

Fig. 2. Southern blot analysis of CmGalLDH gene. Genomic DNA(;10 lg) isolated from melon ovaries was digested with HindIII, PstI,and EcoRI, fractionated in 0.8% agarose-TAE gel, transferred to HybondN-membrane and hybridized with specific 32P-radiolabelled probes forCmGalLDH.

Fig. 3. ExpressionofCmGalLDH in vegetative tissues. TotalRNA(;20lg)from various melon tissues was fractionated in denaturing agarosegels, transferred to Hybond N-membranes and hybridized with 32P-radiolabelled probes for CmGalLDH. Equal loading, integrity, andtransfer were checked by hybridizing the membranes with melon 18Sribosomal probe. SD, seeds; RT, roots; LV, leaves; ST, stems; AP, shootapices; PE, petals; SM, stamens.




ownloaded from


via the action of CmGalLDH may be rapidly oxidized byAO in the apoplast, thus preventing the accumulation ofAA in the outer mesocarp. In the inner mesocarp, becauseAO expression is low, AA could be accumulated, eventhough CmGalLDH message accumulation is low. Afterthe onset of ripening, AA redox (AA/total AA) steadystatus remains unchanged through the concerted action ofCmGalLDH and AO. The higher AA content in the innerfruit mesocarp compared with the outer part could alsocontribute to cell wall loosening, since cell wall polysac-charides could suffer non-enzymatic scission in the pres-ence of AA under certain conditions (Fry, 1998).There is accumulating evidence that AA levels strongly

influence cell division and expansion (Arrigoni, 1994; Katoand Esaka, 1999, 2000; Tabata et al., 2001). AA concen-tration was low in seeds, but exhibited a rapid rise duringgermination, especially during radicle emergence (Pallancaand Smirnoff, 1999). It was found here that CmGalLDHmessage levels correlated well with the initial stages ofgermination and the highest degree of CmGalLDH

mRNA expression was observed during radicle emergence(3 d after imbibition), a developmental stage characterizedmainly by cell enlargement (Bewley and Black, 1983). It ispossible therefore, that activation of CmGalLDH transcriptaccumulation is required to ensure sufficient AA availabil-ity during the cell expansion process. In this context, itis known that AA, and its radical MDHA, stimulate theelongation of onion roots, although the mechanism is farfrom completely understood (Gonzalez-Reyes et al., 1995;Smirnoff, 2000).

The AA content of plant tissues seems to be influencedby light (Gatzek et al., 2002; Tamaoki et al., 2003).CmGalLDH mRNA abundance was shown to decrease inseedlings kept in the dark. This finding is consistent withrecent reports that GalLDH and GDP-D-mannose pyrophos-phorylase (GMPase) transcript levels are reduced in dark-ened tobacco leaves (Tabata et al., 2002). In Arabidosisthaliana, GalLDH expression and protein activity plus AAlevels have been shown to exhibit light-mediated diurnalchanges in mature plants, but not in young rosette leaves

Fig. 4. Shifts in CmGalLDH expression and AA content during melon fruit development and ripening. Total RNA (;20 lg) from melon ovaries (OV)and fruit at different developmental stages (5, 10, 20, 25, 30, 34, 36, 38, and 40 d after pollination) was fractionated in denaturing agarose gels,transferred to Hybond N-membranes and hybridized with 32P-radiolabelled probe for melon GalLDH (A) and melon ACO (C). Equal loading, integrity,and transfer were observed by methylene blue staining. IN indicates the inner part while OUT indicates the outer part of fruit mesocarp. Reduced AAlevels (B) were monitored during fruit development and ripening and are reported as concentration (lmol g�1 FW) from whole fruit (hatched bars), inner(solid bars), and outer (clear bars) mesocarp tissues. Error bars represent one standard deviation above the mean based on three replicates.




ownloaded from


(Tamaoki et al., 2003). Similar light-mediated shifts in leafAA content have been observed in Arabidopsis, tobacco,and barley (Smirnoff and Pallanca, 1996; Grace and Logan,1996; Conklin et al., 1997; Gatzek et al., 2002). Bycontrast, Pignocchi et al. (2003) did not observe any dif-ferences in GalLDH transcript accumulation in tobaccoplants grown in light or dark conditions. Taken together,these results suggest that light may regulate AA bio-synthesis. However, further studies are needed to unravelpossible species-to-species variation or specific growthconditions effects in the regulation of AA biosynthesis bylight.

Apoplastic AA is suggested to act as a first line ofdefence against hostile environments and to behave as apossible link between environmental stresses and adaptiveresponses in plants (Noctor et al., 2000; Sanmartin et al.,2003). Oxidative stress, (rapid production of ROS) is

experienced by plants exposed to different stresses. AArequirements to cope with stress conditions are clearlyunderstood in Arabidopsis thaliana mutants. Mutant vtc1with only 30% of wild-type AA content is ozone-, UV-B-and sulphur dioxide-sensitive (Conklin et al., 1996). More-over, this mutant showed a higher GalLDH capacity thanthe wild-type plants (Conklin et al., 1997). However, nomodification of CmGalLDH message accumulation wasdetected in melon seedlings exposed to high temperature,wounding or wound-signalling compounds (JA, SA, andABA) at least under these experimental conditions. It washypothesized that the regulation of AA levels under thesestress conditions does not require de novo synthesis ofCmGalLDH transcript.

In conclusion, the molecular characterization ofCmGalLDH has been reported and the expression of thisgene has been investigated in vegetative and reproductiveplant tissues during development and under stressed con-ditions. To current knowledge, the CmGalLDH differentialgene expression during melon fruit development andripening is reported for the first time. However, theseresults combined with future in vivo labelling studiesmay explain the mechanism of ascorbate biosynthesis inripening fruit.

Acknowledgements

We are grateful to J Barnes (University of Newcastle) for editing andfor his constructive comments and N Smirnoff (University of Exeter)for comments on the manuscript. We thank C Balague for providing

Fig. 7. Expression of CmGalLDH gene in response to stress. Total RNA(;20 lg) from 20-d-old melon seedlings wounded or exposed to heat-shock for 0, 6, 12, or 24 h was fractionated in denaturing agarose gels,transferred to Hybond N-membranes and hybridized with specific 32P-radiolabelled probes for CmGalLDH. Equal loading and transfer wereverified by hybridizing the membranes with melon 18S ribosomal probe.

Fig. 6. Expression of CmGalLDH gene in response to different growthregulators and signalling agents. 20-d-old melon seedlings were in-cubated in 50 mM phosphate buffer (CON, control), 500 lM indole-3-acetic acid (IAA), 100 lM abscisic acid (6) cis-trans isomer (ABA), 50lM [6]-jasmonic acid (JA), 50 lM salicylic acid (SAL) or 50 lMascorbic acid (AA) for 12 h. Total RNA (;20 lg) was isolated fromwhole seedlings and fractionated in denaturing agarose gels, transferredto Hybond N-membranes and hybridized with specific 32P-radiolabelledprobes for CmGalLDH gene. Staining with methylene blue was per-formed to verify the evenness of RNA loading on the gel.

Fig. 5. Effect of light on CmGalLDH expression during seed germina-tion and seedling development. (A) Seed germination and melonseedlings at different developmental stages grown in light (16/8 h light/dark photoperiod) or dark conditions. (B) Total RNA (;20 lg) wasisolated from seeds (S, 0 d) and dark-grown seedlings at differentdevelopmental stages (1, 2, 3, 4, 6, 8, and 10 d) after seeds were allowedto imbibe, as well as light-grown seedlings (6, 8, 10, 15, and 20 d) afterallowing seeds to imbibe, fractionated in denaturing agarose gels,transferred to Hybond N-membranes and hybridized with specific 32P-radiolabelled probes for CmGalLDH. Equal loading, integrity, andtransfer were monitored by hybridizing the blots with melon 18Sribosomal probe.




ownloaded from


the melon ACO cDNA (pMEL-1 plasmid). The work was supportedin part by grants awarded to AKK (EU-FAIR-CT-97-5021, GR-NAGREF-DIMITRA, GR-GSRT). MS was a TMR fellow partiallysupported by EU-FAIR-CT-97-5021.

References

Abeles FB, Morgan PW, Saltveit KE. 1992. In: Ethylene in plantbiology, 2nd edn. San Diego: Academic Press Inc.

Agius F, Gonzalez-Lamothe R, Caballero JL, Munoz-Blanco J,Botella MA, Valpuesta V. 2003. Engineering increased vitaminC levels in plants by overexpressing of a D-galacturonic acidreductase. Nature Biotechnology 21, 177–181.

Altschul SF, Gish W, Miller W, Meyers EW, Lipman DJ. 1990.Basic local alignment search tool. Journal of Molecular Biology215, 403–410.

Arrigoni O. 1994. Ascorbate system in plant development. Journalof Bioenergetics and Biomembranes 26, 407–419.

Asada K. 1999. The water-water cycle in chloroplast: scavenging ofactive oxygens and dissipation of excess photons. Annual Reviewof Plant Physiology and Plant Molecular Biology 50, 601–639.

Bartoli CG, Pastori GM, Foyer CH. 2000. Ascorbate biosynthesisin mitochondria is linked to the electron transport chain betweencomplexes III and IV. Plant Physiology 123, 335–343.

Bewley JD, Black M. 1983. Development, germination and growth.In: Bewley JD, Black M, eds. Physiology and biochemistry ofseeds in relation to germination, Vol. 1. Berlin: Springer-Verlag,120–123.

Brennen T, Frenkel C. 1977. Involvement of hydrogen peroxidein the regulation of senescence in pear. Plant Physiology 59,411–416.

Brummell DA, Harpster MH. 2001. Cell wall metabolism in fruitsoftening and quality and its manipulation in transgenic plants.Plant Molecular Biology 47, 311–340.

Carr AC, Frei B. 1999. Toward a new recommended dietaryallowance for vitamin C based on antioxidant and health effects inhumans. American Journal of Clinical Nutrition 69, 1086–1107.

Chatterjee IB. 1973. Evolution and the biosynthesis of ascorbicacid. Science 182, 1271–1272.

Church GM, Gilbert W. 1984. Genomic sequencing. Proceedingsof the National Academy of Sciences, USA 81, 1991–1995.

Conklin PL, Pallanca JE, Last RL, Smirnoff N. 1997. L-ascorbicacid metabolism in the ascorbate-deficient Arabidopsis mutantvtc1. Plant Physiology 115, 1277–1285.

Conklin PL, Williams EH, Last RL. 1996. Environmental stresssensitivity of an ascorbic acid-deficient Arabidopsis mutant.Proceedings of the National Academy of Sciences, USA 93,9970–9974.

Davey MD, Van Montagu M, Inze D, Sanmartin M, Kanellis AK,Smirnoff N, Benzie IJJ, Strain JJ, Favell D, Fletcher J. 2000.Plant L-ascorbic acid: chemistry, function, metabolism, bioavail-ability, and effects of processing. Journal of the Science of Foodand Agriculture 80, 825–860.

Diallinas G, Pateraki I, Sanmartin M, Scossa A, Stilianou E,Panopoulos NJ, Kanellis AK. 1997. Melon ascorbate oxidase:cloning of a multigene family, induction during fruit develop-ment and repression by wounding. Plant Molecular Biology 34,759–70.

Foyer C, Rowell J, Walker D. 1983. Measurement of the ascorbatecontent of spinach leaf protoplasts and chloroplasts during illumi-nation. Planta 157, 239–244.

Fry SC. 1998. Oxidative scission of plant cell wall polysaccharidesby ascorbate-induced hydroxyl radicals. The Biochemical Journal332, 507–515.

Gatzek S, Wheeler GL, Smirnoff N. 2002. Antisense suppressionof L-galactose dehydrogenase in Arabidopsis thaliana providesevidence for its role in ascorbate synthesis and reveals lightmodulated L-galactose synthesis. The Plant Journal 30, 541–553.

Gonzalez-Reyes JA, Alcain FJ, Caler JA, Serrano A, Cordoba F,Navas P. 1995. Stimulation of onion root elongation by ascorbateand ascorbate free radical in Allium cepa L. Protoplasma 184,31–35.

Graber JH, Cantor CR, Mohr SC, Smith TF. 1999. In silicodetection of control signals: mRNA 39-end-processing sequencesin diverse species. Proceedings of the National Academy ofSciences, USA 96, 14055–14060.

Grace SC, Logan BA. 1996. Acclimation of foliar antioxidantsystems to growth irradiance in 3 broad-eaved evergreen species.Plant Physiology 112, 1631–1640.

Imai T, Karita S, Shiratori G, Hattori M, Nunome T, Oba K,Hirai M. 1998. L-galactono-gamma-lactone dehydrogenase fromsweet potato: purification and cDNA sequence analysis. Plant CellPhysiology 39, 1350–1358.

Isherwood FA, Chen YT, Mapson LW. 1954. Synthesis of L-ascorbic acid in plants and animals. Biochemical Journal 56, 1–21.

Jimenez A, Creissen G, Kular B, Firmin J, Robinson S,Verhoeyen M, Mullineaux P. 2002. Changes in oxidative pro-cesses and components of the antioxidant system during tomatofruit ripening. Planta 214, 751–758.

Kato N, Esaka M. 1999. Changes in ascorbate oxidase geneexpression and ascorbate levels in cell division and cell elongationin tobacco cells. Physiologia Plantarum 105, 321–329.

Kato N, Esaka M. 2000. Expansion of transgenic tobacco proto-plasts expressing pumpkin ascorbate oxidase is more rapid thanthat of wild-type protoplasts. Planta 210, 1018–1022.

Keller R, Renz FS, Kossmann J. 1999. Antisense inhibition of theGDP-mannose pyrophosphorylase reduces the ascorbate content intransgenic plants leading to developmental changes during senes-cence. The Plant Journal 19, 131–141.

Kerk NM, Feldman LJ. 1995. A biochemical model for theinitiation and maintenance of the quiescent centre: implicationsfor organization of root meristems. Development 121, 2825–2833.

Levine M, Rumsey SC, Daruwala R, Park JB, Wang YH. 1999.Criteria and recommendations for vitamin C intake. Journal of theAmerican Medical Association 281, 1415–1423.

Loewus FA. 1999. Biosynthesis and metabolism of ascorbic acid inplants and of analogs of ascorbic acid in fungi. Phytochemistry 52,193–210.

Mapson LW, Breslow E. 1958. Biological synthesis of ascorbicacid: conversion of derivatives of D-galacturonic acid into L-ascorbic acid by plant extracts. Biochemical Journal 64, 151–157.

Moser O, Kanellis AK. 1994. Ascorbate oxidase of Cucumis meloL. var. reticulatus: purification, characterization and antibodyproduction. Journal of Experimental Botany 45, 717–724.

Mutsuda M, Ishikawa T, Takeda T, Shigeoka S. 1995. Subcellularlocalization and properties of L-galactono-c-lactone dehydroge-nase.Bioscience, Biotechnology, andBiochemistry 59, 1983–1984.

Noctor G, Foyer CH. 1998. Ascorbate and glutathione: keepingactive oxygen under control. Annual Review of Plant Physiologyand Plant Molecular Biology 49, 249–279.

Noctor G, Veljovic-Jovanovic S, Foyer CH. 2000. Peroxideprocessing in photosynthesis: antioxidant coupling and redoxsignaling. Philosophical Transactions: Biological Sciences 355,1465–1475.

Oba K, Fukui M, Imai Y, Iriyama S. Nogami K. 1994.L-Galactono-c-lactone dehydrogenase: partial characterization, in-duction of activity and role in the synthesis of ascorbic acid inwounded white potato tuber tissue. Plant Cell Physiology 35, 473–478.




ownloaded from


Oba K, Ishikawa S, Nishikawa M, Mizuno H, Yamamoto T. 1995.Purification and properties of L-galactono-gamma-lactone dehy-drogenase, a key enzyme for ascorbic acid biosynthesis, fromsweet potato roots. Journal of Biochemistry 117, 120–124.

Østergaard J, Persiau G, Davey MW, Bauw G, Van Montagu M.1997. Isolation of a cDNA coding for L-galactono-c-lactonedehydrogenase, an enzyme involved in the biosynthesis of ascorbicacid in plants. Purification, characterization, cDNA cloning, andexpression in yeast. Journal of Biological Chemistry 272, 30009–30016.

Padh H. 1990. Cellular functions of ascorbic acid. Biochemistry andCell Biology 68, 1166–1173.

Pallanca JE, Smirnoff N. 1999. Ascorbic acid metabolism in peaseedlings. A comparison of D-glucosone, L-sorbosone, and L-galactone-1,4-lactone as ascorbate precursors. Plant Physiology120, 453–461.

Pastori GM, Kiddle G, Antoniw J, Bernard S, Veljovic-Jovanovic S, Verrier PJ, Noctor G, Foyer CH. 2003. Leafvitamin C contents modulate plant defense transcripts and regulategenes controlling development through hormone signalling. ThePlant Cell 15, 1212–1226.

Pignocchi C, Fletcher JM, Wilkinson JE, Barnes J, Foyer CH.2003. The function of ascorbate oxidase in tobacco (Nicotianatabacum L.). Plant Physiology 132, 1631–1641.

Pignocchi C, Foyer CH. 2003. Apoplastic ascorbate metabolism andits role in the regulation of cell signalling. Current Opinion inPlant Biology 6, 379–389.

Redgwell RJ, MacRae E, Hallett J, Fischer M, Perry J, HarkerR. 1997. In vivo and in vitro swelling of cell walls during fruitripening. Planta 203, 162–173.

Rose JKC, Hadfield KA, Labavitch JM, Bennett A. 1998.Temporal sequence of cell wall disassembly in rapidly ripeningmelon fruit. Plant Physiology 117, 345–361.

Sanmartin M. 2002. Regulation of melon ascorbate oxidase geneexpression and effect of its modification in transgenic tobacco andmelon plants. PhD Thesis, University of Valencia, Spain.

Sanmartin M, Drogoudi PD, Lyons T, Pateraki I, Barnes J,Kanellis AK. 2003. Over-expression of ascorbate oxidase in theapoplast of transgenic tobacco results in altered ascorbate andglutathione redox states and increased sensitivity to ozone. Planta216, 918–928.

Siendones E, Gonzalez-Reyes JA, Santos-Ocana C, Navas P,Cordoba F. 1999. Biosynthesis of ascorbic acid in kidney bean. L-Galactono-c-lactone dehydrogenase is an intrinsic protein locatedat the mitochondrial inner membrane. Plant Physiology 120, 907–912.

Smirnoff N. 1996. The function and metabolism of ascorbic acid inplants. Annals of Botany 78, 661–669.

Smirnoff N. 2000. Ascorbic acid: metabolism and functions ofa multi-facetted molecule. Current Opinion in Plant Biology 3,229–235.

Smirnoff N, Pallanca JE. 1996. Ascorbate metabolism in relation tooxidative stress. Biochemical Society Transactions 24, 472–478.

Smirnoff N, Wheeler GL. 2000. Ascorbic acid in plants: bio-synthesis and function. Critical Review in Biochemistry andMolecular Biology 35, 291–314.

Smith CJS, Slater A, Grierson D. 1986. Rapid appearance of anmRNA correlated with ethylene synthesis encoding a protein ofmolecular weight 35000. Planta 168, 94–100.

Tabata K, Oba K, Suzyki K, Esaka M. 2001. Generation andproperties of ascorbic acid-deficient transgenic tobacco cellsexpressing antisense RNA for L-galactono-1,4-lactone dehydroge-nase. The Plant Journal 27, 139–148.

Tabata K, Takaoka T, Esaka M. 2002. Gene expression of ascorbicacid-related enzymes in tobacco. Phytochemistry 61, 631–635.

Tamaoki M, Mukai F, Asai N, Nakajima N, Kubo A, Aono M,Saji H. 2003. Light-controlled expression of a gene encoding L-galactono-c-lactone dehydrogenase which affects ascorbate poolsize in Arabidopsis thaliana. Plant Science 164, 1111–1117.

Thompson JD, Higgins DG, Gibson TJ. 1994. ClustalW: improv-ing the sensitivity of progressive multiple sequence alignmentsthrough sequence weighting, position-specific gap penalties andweight matrix choice. Nucleic Acids Research 22, 4673–4680.

Veljovic-Jovanovic SD, Pignocchi C, Noctor G, Foyer CH. 2001.Low ascorbic acid in the vtc-1 mutant of Arabidopsis is associatedwith decreased growth and intracellular redistribution of theantioxidant system Plant Physiology 127, 426–435.

Ververidis P, John P. 1991. Complete recovery in vitro of ethyleneforming enzyme activity. Phytochemistry 30, 725–727.

von Heije G. 1986. Mitochondrial targeting sequences may formamphiphilic helices. EMBO Journal 5, 1335–1342.

Wadsworth GJ, Redinbaugh MG, Scandalios JG. 1988. A pro-cedure for the small-scale isolation of plant RNA suitable for RNAblot analysis. Analytical Biochemistry 172, 279–283.

Wheeler GL, Jones MA, Smirnoff N. 1998. The biosyntheticpathway of Vitamin C in higher plants. Nature 393, 365–369.

Wolucka BA, Persiau G, Van Doorsselaere J, Davey MW, DemolH, Vandekerckhove J, Van Montagu M, Zabeau M, BoerjanW. 2001. Partial purification and identification of GDP-mannose39,59-epimerase of Arabidopsis thaliana, a key enzyme of the plantvitamin C pathway. Proceedings of the National Academy ofSciences, USA 98, 14843–14848.

Yabuta Y, Yoshimura K, Takeda T, Shigeoka S. 2000. Molecularcharacterization of tobacco mitochondrial L-galactono-c-lactonedehydrogenase and its expression in Escherichia coli. Plant CellPhysiology 41, 666–675.




ownloaded from


Molecular characterization and expression studies during melon fruit development and ripening of...

Documents

Transcript of Molecular characterization and expression studies during melon fruit development and ripening of...