JXB Advance Access originally published online on July 2, 2004
Journal of Experimental Botany 2004 55(403):1623-1633; doi:10.1093/jxb/erh186
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
RESEARCH PAPER |
Molecular characterization and expression studies during melon fruit development and ripening of L-galactono-1,4-lactone dehydrogenase
1Group of Biotechnology of Pharmaceutical Plants, Laboratory of Pharmacognocy, Department of Pharmaceutical Sciences, Aristotle University of Thessaloniki, 541 24 Thessaloniki, Greece
2Institute of Viticulture and Vegetable Crops, National Agriculture Research Foundation, PO Box 2229, 713 09 Heraklion, Crete, Greece
3Institute of Molecular Biology and Biotechnology, FORTH, PO Box 1527, 711 10 Heraklion, Crete, Greece
To whom correspondence should be addressed. Fax: +30 2310 997662, 997645. E-mail: kanellis{at}pharm.auth.gr
Received 10 February 2004; Accepted 27 April 2004
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and methodsResultsDiscussionReferences |
|---|
The last step of ascorbic acid (AA) biosynthesis is catalysed by the enzyme L-galactono-1,4-lactone dehydrogenase (GalLDH, EC 1.3.2.3 [EC] ), located on the inner mitochondrial membrane. The enzyme converts L-galactono-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 studied, but differentially expressed during fruit development 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 regulated 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 germination 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 |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
L-Ascorbic acid (AA) plays a multifunctional role in both plants and animals, and comprises one of the most abundant metabolites in green tissues. Humans, plus other primates and some birds and bats, are unable to synthesize AA. Consequently, they depend on dietary intake to cover their requirements (Chatterjee, 1973
). Fruit and vegetables comprise the major dietary sources of AA in humans and recent studies suggest that increased AA intake (from 60 to 200 mg d–1) may carry health benefits (Carr and Frei, 1999; Levine et al., 1999).
Much attention has focused on the antioxidant role played by AA in both plants and animals. However, the compound acts as a cofactor for a suite of enzymes, including 1-aminocyclopropane-1-carboxylic acid (ACC) oxidase, a key enzyme in the biosynthetic pathway of ethylene, the ‘ripening hormone’ (Ververidis and John, 1991) and plays a vital role in many physiological processes (Smirnoff, 1996; Noctor and Foyer, 1998; Smirnoff and Wheeler, 2000). AA seems to affect the transcription of a number of genes including PR (Pathogenesis Related) proteins and those involved in the biosynthesis of abscisic acid (Pastori et al., 2003). AA directly scavenges reactive oxygen species (ROS) and regenerates lipophilic 
-tocopherol to its reduced state (Noctor and Foyer, 1998; Asada, 1999). As a consequence, the compound plays a very important role in protecting plant tissues against oxidative damage provoked by a range of environmental stresses including 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 the prosthetic metal groups of various enzymes in the reduced state ensuring their activity (Padh, 1990; Davey et al., 2000). Recent studies indicate that ascorbate is involved in the process and regulation of both cell division and expansion (Arrigoni, 1994; Kerk and Feldman, 1995; Smirnoff, 1996; Davey et al., 2000; Tabata et al., 2001). Work on transgenic potato plants expressing GDP-mannose pyrophosphorylase in the antisense orientation and the vtc-1 mutant of Arabidopsis thaliana reveal that a decline in AA content correlates with reduced cell growth and abnormalities in cell shape (Keller et al., 1999; Veljovic-Jovanovic et al., 2001). Ascorbate may modulate cell division by controlling the transition from G1 to S during the cell cycle (Kerk and Feldman, 1995). Moreover, AA and its oxidation products in conjunction with the enzyme ascorbate oxidase (AO) may comprise a regulatory system controlling cell expansion (Smirnoff, 1996; Kato and Esaka, 1999, 2000), though the mechanism is poorly understood (Pignocchi et al., 2003). Further, the AA redox state, controlled by AO activity levels, is mainly responsible for the apoplast capability of transmitting signals related 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 step catalysed by the enzyme GDP-mannose-3',5'-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 oxidized to L-galactono-1,4-lactone (GalL) by L-galactose dehydrogenase (GalDH) (Wheeler et al., 1998). An alternative pathway, recently established in plants, involves the conversion of D-galacturonic acid (a component of cell wall pectins) to L-galactonic acid by D-galacturonic acid reductase (GalUR) (Agius et al., 2003). L-Galactonic acid is then converted to L-galactono-1,4-lactone. Expression of GalUR correlates with increased ascorbate levels in ripening strawberry fruit, and over-expression of GalUR in transgenic A. thaliana results in a 2–3-fold increase in AA content 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 the enzyme L-galactono-1,4-lactone dehydrogenase (GalLDH). This enzyme has been known for more than 40 years (Mapson and Breslow, 1958) and GalLDH activity has been described in pea, cabbage, cauliflower, and sweet potato (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 characterized from cauliflower, sweet potato, strawberry, tomato, tobacco, and Arabidopsis (Østergaard et al., 1997; Imai et al., 1998; Yabuta et al., 2000; Tamaoki et al., 2003). Recent studies demonstrate that GalLDH is an intrinsic membrane protein linked to the inner mitochondrial membrane (Siendones et al., 1999; Bartoli et al., 2000) and employs cytochrome c as a second substrate (Oba et al., 1995; Østergaard et al., 1997; Imai et al., 1998). During GalLDH activity, electrons are probably transferred to the mitochondrial electron transport chain between complexes III and IV (Bartoli et al., 2000).
A number of observations indicate that AA content increases 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 a fundamental role in this process. However, very little is known about the expression of AA biosynthetic enzymes during this physiological process. In the present study, the cloning and characterization of a melon cDNA designated CmGalLDH, is reported and its expression in fruit tissues is described for the first time. It is demonstrated that CmGalLDH is expressed in all tissues studied, but differentially regulated during fruit development and seed germination. CmGalLDH mRNA expression is induced during melon fruit ripening concomitant with increased AA levels. In addition, the effect of various hormones and stress treatments on CmGalLDH expression is examined and discussed.
|
Materials and methods |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Plant culture and sampling
Melon (Cucumis melo L., variety reticulatus, F1 alpha hybrid) seeds were kindly provided by the Tezier Breeding Institute, Valence (France). Melon plants were grown in a naturally illuminated glasshouse at 25 °C 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 °C. Fruit were picked at various developmental stages (5, 10, 15, 20, 25, 30, 32, 34, 36, 38, and 40 d after pollination [DAP]), the inner and outer areas of the fruit mesocarp were separated using a scalpel, frozen in liquid nitrogen, and then stored at –80 °C.
For germination studies, seeds were germinated on damp cotton at 25 °C day/night temperature (in the dark). After 4 d, seedlings were transferred to pots containing perlite and watered every 2 d with a commercial fertilizer. Seedlings were raised in growth chambers for 16 h photoperiod (or continuous dark for etiolation treatment) at 25 °C day/night.
For feed and stress treatments, 20-d-old seedlings were grown as described above. The growing medium was carefully removed by washing the roots in tap water prior to transfer to 50 mM phosphate buffer, pH 7.0 (control) or solutions of 500 µM indole-3-acetic acid (IAA), 100 µM abscisic acid (±) cis-trans isomer (ABA), 50 µM (±)-jasmonic acid (JA), 50 µM salicylic acid (SA) or 50 µM ascorbic acid (AA). Plants were incubated in the light in the respective solutions for 12 h in the growth chambers described above. All chemicals 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 sterile forceps and samples taken after 0, 6, 12, and 24 h. For heat-shock experiments, seedlings were transferred to a growth chamber maintained at 42 °C. Samples (5–6 plants per treatment) were collected 0, 6, 12, or 24 h after transfer. Plant material was immediately frozen in liquid nitrogen and stored at –80 °C. Experiments were repeated three 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 synthesize single-stranded cDNA using the M-MuLV (New England Biolabs, Beverly, MA, USA) reverse transcriptase and oligodT primer [5'-ACTAGTCTCGAG(T)19-3'] according to the manufacturer's instructions. This cDNA was used as template in PCR for melon CmGalLDH cDNA amplification with degenerate primers [G-SEN2: 5'-A(CT)AT(ACT)CC(AGCT)TA(CT)AC(AGCT)GA(CT)(AG)C-3' and G-AS1: 5'-CCA(AGCT)CC(AGCT)AC(AGCT)C(GT)(AG)TA(AGCT)CC(CT)TC-3'], designed based on the conserved regions among known GalLDH amino acid sequences (Østergaard et al., 1997; Imai et al., 1998). PCR amplification was performed for 40 cycles (94 °C for 60 s, 55 °C for 90 s, and 72 °C for 90 s) followed by a final extension step of 10 min at 72 °C. PCR products were gel purified and cloned into pGEM-T Easy (Promega GmbH, Mannheim, Germany) plasmid vector. A LI-COR Long Readir 4200 automated sequencer and a ‘Sequitherm EXCELII’ kit (Epicentre, Madison, WI, USA) were used to determine the nucleotide sequence of the resulted clones. Based on the nucleotide sequence obtained, CmGalLDH-specific primers [G-SEN3: 5'-TATCTCAAAATGGAGAGGCCCTCCG-3' and G-AS3: 5'-TCCTCCAGAACTCAGCCTCTGCTTG-3'] were designed for cloning the 5' and 3' remaining cDNA sequences. 5' and 3' RACE were performed using the ‘Marathon cDNA Amplification System’ (Clontech, Palo Alto, CA, USA). For full-length cDNA amplification a primer was designed from the 5' end containing the ATG translation initiation codon [G-ATG: 5'-ATGCTCAACTTTCTCTCTCTTCGGCG-3']. PCR was performed using the G-ATG and oligodT primers for 40 cycles using a touch-down strategy (10 cycles at 94 °C for 60 s, 55 °C 
45 °C for 90 s, and 72 °C for 180 s; 30 cycles at 94 °C for 60 s, 45 °C for 90 s, and 72 °C for 180 s) followed by an extension step of 10 min at 72 °C. 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 of Biotechnology (NCBI) web site (http://www.ncbi.nlm.nih.gov). The BLAST program, developed by Altschul et al. (1990), was used to search for sequence homology to the EMBL plant DNA sequence database and Swissprot protein database. The alignment of the deduced protein sequences and the phylogenetic tree were computed using the ClustalW program (Thompson et al., 1994) employing standard parameters.
Melon genomic DNA extraction and Southern blot analysis
Total genomic DNA was isolated from melon ovaries using the method of Diallinas et al. (1997). Genomic DNA (10 µg) was digested 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 Labelling System (Invitrogen, Life Technologies, Madison, WI, USA) according to the manufacturer's instructions. Blot hybridization and membrane washing were performed as described by Church and Gilbert (1984).
RNA blot analysis
Total RNA was isolated from vegetative tissues and ovaries of melon as described by Wadsworth et al. (1988). Total RNA from melon fruit tissues was isolated using the method described by Smith et al. (1986). Total RNA (15 µg) was fractionated in formaldehyde denaturing agarose gels, transferred to nylon membranes (Nytran® 0.45, Schleicher and Schuell, GmbH, Dassel, Germany), stained with 0.04% methylene blue (to observe equality of loading) and hybridized with radio-labelled probes for CmGalLDH and melon ACO cDNAs. Alternatively, to ensure equal loading, RNA blots were hybridized with the 18S melon rRNA probe. RNA blot hybridization and membrane washing were performed as described above.
Ascorbic acid analysis
One gram of frozen tissue from whole, inner and outer mesocarp tissues were powdered in liquid nitrogen and extracted as described by Foyer et al. (1983). Ascorbic acid was calculated as the difference in absorbance at 265 nm before and after the addition of 1 unit ml–1 of ascorbate oxidase (Sigma Chemical Co., St Louis, USA) in 100 mM sodium phosphate buffer pH 5.6. Absorbance values were evaluated against a standard curve constructed using L-ascorbic acid (Merck KGaA, Darmstadt, Germany) in the range of 0–100 nmol and reported as µmol g–1 FW.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Cloning and characterization of CmGalLDH cDNA
Degenerate primers designed from conserved amino acid regions of cauliflower and sweet potato GalLDH (Østergaard et al., 1997
; Imai et al., 1998) were used to amplify a 281 bp fragment by RT-PCR from melon ovary total RNA. Sequence analysis revealed this fragment to encode a partial melon GalLDH. Based on this cDNA sequence, specific primers were designed to amplify the 5' and 3' flanking regions using RACE techniques. Using G-ATG and oligodT primers, a 2097 bp cDNA clone (EMBL accession number AF252339) was obtained. Nucleotide sequence analysis of this cDNA revealed that it corresponded to a GalLDH full-length cDNA, termed Cucumis melo GalLDH (CmGalLDH). It contains a 58 bp 5' untranslated region followed by a 1770 bp open reading frame, coding for a 589 amino acids polypeptide, and a 269 bp 3' untranslated region including a 42 bp polyA tail. No canonical polyadenylation-processing signals (AATAAA or AATGAA) were found, which is common in most plant genes (Graber et al., 1999).
In silico analysis of the CmGalLDH amino acid sequence predicted a molecular weight of 67 059 Da. However, this polypeptide contained a putative mitochondrial-targeting domain in its first 89 amino acids and a cleavage site (FR/YA) similar to other known GalLDHs. Therefore, the mature CmGalLDH protein consists of 500 amino acid residues having a calculated molecular weight of 56 731 Da and a pI of 6.71. Three possible transmembrane regions were predicted in accordance with sweet potato GalLDH (Bartoli et al., 2000). Amino acid sequence comparison of CmGalLDH showed 79.2% identity with Arabidopsis, 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 acid sequence identified a putative FAD binding domain between residues 111 and 162 (Fig. 1A), wherein is located a region (139VGSGLSP145) common to all GalLDHs characterized 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 analysis 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 yeast D-arabino-1,4-lactone oxidase and rat L-gulono-
-lactone oxidase formed a distal group, suggesting that these genes evolved independently, following species divergence. Melon total genomic DNA was digested with different restriction enzymes (PstI, EcoRI and HindIII) and blotted on to nylon membrane. Only one positive band was detected in each lane suggesting that CmGalLDH is coded by a single gene (Fig. 2).
|
10 µg) isolated from melon ovaries was digested with HindIII, PstI, and EcoRI, fractionated in 0.8% agarose-TAE gel, transferred to Hybond N-membrane and hybridized with specific 32P-radiolabelled probes for CmGalLDH.Expression of CmGalLDH and ascorbic acid levels in vegetative tissues and shifts in fruit associated with development and ripening
GalLDH message accumulation was detected in seeds, roots, stems, leaves, shoot apices, petals, and stamens, although different levels of transcript abundance were observed (Fig. 3). CmGalLDH transcript levels were highest in stems and lowest in seeds and roots. In floral tissues, CmGalLDH mRNA was abundant with stronger expression in 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 the CmGalLDH message in ovaries and lower levels in developing green fruit (5, 10, 15, 20, and 25 DAP; Fig. 4A). However, after the onset of melon ripening, indicated by ACO mRNA levels (30 DAP; Fig. 4C), there was a marked increase in GalLDH transcript levels. The peak of GalLDH expression paralleled the ACO message climacteric peak at 36 DAP. Moreover, CmGalLDH transcript levels were notably higher in the outer mesocarp of maturing fruit (after 30 DAP) compared with the inner mesocarp (Fig. 4A), whereas CmGalLDH mRNA levels were barely detectable in 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 were high 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 mesocarp with the highest levels observed at 34 DAP in the inner mesocarp. In 40 DAP ripe fruit, AA levels were equal in both the inner and outer mesocarp (Fig. 4B). It is interesting to note that AA concentration is higher in the inner than in the outer mesocarp of mature and ripe fruit which is converse to the CmGalLDH mRNA steady-state levels.
In parallel, ACO transcript abundance was also monitored in the tissues mentioned above. The ACO mRNA message was not detected in fruit younger than 30 DAP, but transcripts accumulated in both the inner and outer mesocarp up to 38 DAP. Thereafter, transcript levels declined and were barely detectable in ripe fruit (40 DAP) (Fig. 4C). In contrast to the differential expression of CmGalLDH in the outer versus the inner mesocarp, ACO transcript levels responded similarly in the inner and outer mesocarp.
Effect of light on CmGalLDH expression during seed germination and seedling development
To investigate whether CmGalLDH expression was differentially regulated during germination, blot analysis was performed of total RNA extracted from dry and germinating seeds, 1–4 d after they were imbibed. CmGalLDH transcript levels were barely detectable in dry seeds, but accumulated during germination, reaching the highest level of expression in 3-d-old seedlings (Fig. 5). In seedlings grown 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 pronounced decline in the CmGalLDH message, to such an extent that transcript levels were barely detectable in 10-d-old seedlings (Fig. 5).
|
CmGalLDH gene expression in response to plant hormones, signalling agents and stress conditions
CmGalLDH expression showed no significant change in melon seedlings fed with IAA, AA, JA, SA, or ABA (Fig. 6). Moreover, there was no induction of CmGalLDH transcript levels in melon leaves and cotyledons following wounding or heat-shock (Fig. 7). It should be mentioned that the above treatments (IAA, AA, JA, SA, or ABA) did not cause any phenotypic effect in the treated seedlings while seedlings subjected to wounding or heat stress were visibly wilted.
|
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
The final step in AA biosynthesis in planta requires the oxidation of GalL by L-galactono-1,4-lactone dehydrogenase (GalLDH). In this work, the cloning and characterization of a cDNA encoding CmGalLDH are reported. The deduced amino acid sequence presented 74–79% homology with other known GalLDHs and lower levels of homology with (i) L-gulono-
-lactone oxidase (27%), the terminal enzyme mediating AA biosynthesis in animals, and (ii) yeast D-arabinono-1,4-lactone oxidase (26%), an enzyme that participates in the biosynthesis of the five-carbon AA analogue D-erythroascorbic acid common in fungi (Loewus, 1999). Homology was mainly restricted to the amino terminal region of GalLDH. As suggested by Østergaard et al. (1997), this could illustrate the existence of a functional domain.
Recent studies have demonstrated that GalLDH is an intrinsic membrane protein linked to the inner mitochondrial membrane (Siendones et al., 1999; Bartoli et al., 2000). The putative CmGalLDH polypeptide sequence presented a mitochondrial 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 Val residues (1, 2, 4, and 2, respectively). This composition is similar to that reported for other known GalLDHs and matched the characteristics of mitochondrial target peptides (von Heije, 1986). In addition, in silico analysis of the CmGalLDH sequence predicted three possible transmembrane regions, located in similar positions to those identified in sweet potato GalLDH (Bartoli et al., 2000). These findings lend further support to the contention that GalLDH spans the inner mitochondrial membrane with the active site facing the intermembrane space in a manner circumnavigating the requirement for GalL and AA transport through the inner membrane (Bartoli et al., 2000). The existence of a FAD binding domain suggests that the flavin group might be involved in the reaction catalysed by GalLDH (Oba et al., 1995).
CmGalLDH expression was detectable in all tissues studied (Fig. 3). This is not an unexpected finding, since AA is present in practically all plant tissues and recent studies have shown a strong correlation between GalLDH expression patterns and AA levels (Tabata et al., 2001, 2002). In the present study, organ-specific and novel fruit-ripening-associated accumulation of CmGalLDH transcript levels was shown. In general, ripening-associated increases in CmGalLDH mRNA levels were accompanied by increases in AA content in melon. However, there appeared to be spatial segregation in the fruit mesocarp between the increased expression of CmGalLDH and increases in AA content between the outer and inner pericarp, respectively. The ripening of melon fruit, as with other fruit (Moser and Kanellis, 1994; Jimenez et al., 2002; Agius et al., 2003; present study), is commonly accompanied by an increase in AA content, although the mechanistic significance of this observation is poorly understood. Fruit ripening is a developmental process where oxidative processes take place and a variety of ROS accumulates (Brennen and Frenkel, 1977; Jimenez et al., 2002). The observed increase in AA content during this developmental stage may be part of a global antioxidant response that accompanies the ripening process (Jimenez et al., 2002). Moreover, ACO, the final enzyme in the synthesis of ethylene, the ‘ripening hormone’ (Abeles et al., 1992), requires AA as a cofactor. This requirement may explain the pattern of expression observed in CmGalLDH and why there is a need of increased AA levels in mature fruit cells. In contrast to the preferential expression of CmGalLDH in the outer mesocarp, ACO transcripts accumulated equally in both inner and outer fruit mesocarp (Fig. 4A, C). This implies that the induction of CmGalLDH gene expression during melon fruit development and ripening is independent of ethylene biosynthesis and, consequently, ethylene action.
Alternatively, the activation of the CmGalLDH gene during melon fruit ripening may constitute part of a broader strategy to conserve carbon losses following cell wall breakdown (Davey et al., 2000). Fruit ripening is characterized by a progressive loss of mesocarp firmness provoked by fruit cell wall disassembly (Brummell and Harpster, 2001). Changes in cell wall structure include solubilization and depolymerization of pectins, usually abundant in fruit cell walls (Redgwell et al., 1997; Rose et 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 of D-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 AA biosynthetic pathway, that this newly-identified means of production may be activated in a developmental manner, influencing AA content.
The apparently reciprocal accumulation of AA and CmGalLDH mRNA levels in the inner and outer fruit mesocarp, respectively, may be explained by differences in the spatial expression of ascorbate oxidase (CMAO1). Indeed, Sanmartin (2002) showed that throughout fruit ripening levels of CMAO1 were markedly higher in the outer versus the inner mesocarp. Consequently, AA formed via the action of CmGalLDH may be rapidly oxidized by AO in the apoplast, thus preventing the accumulation of AA in the outer mesocarp. In the inner mesocarp, because AO expression is low, AA could be accumulated, even though CmGalLDH message accumulation is low. After the onset of ripening, AA redox (AA/total AA) steady status remains unchanged through the concerted action of CmGalLDH and AO. The higher AA content in the inner fruit mesocarp compared with the outer part could also contribute to cell wall loosening, since cell wall polysaccharides could suffer non-enzymatic scission in the presence of AA under certain conditions (Fry, 1998).
There is accumulating evidence that AA levels strongly influence cell division and expansion (Arrigoni, 1994; Kato and Esaka, 1999, 2000; Tabata et al., 2001). AA concentration was low in seeds, but exhibited a rapid rise during germination, especially during radicle emergence (Pallanca and Smirnoff, 1999). It was found here that CmGalLDH message levels correlated well with the initial stages of germination and the highest degree of CmGalLDH mRNA expression was observed during radicle emergence (3 d after imbibition), a developmental stage characterized mainly by cell enlargement (Bewley and Black, 1983). It is possible therefore, that activation of CmGalLDH transcript accumulation is required to ensure sufficient AA availability during the cell expansion process. In this context, it is known that AA, and its radical MDHA, stimulate the elongation of onion roots, although the mechanism is far from completely understood (Gonzalez-Reyes et al., 1995; Smirnoff, 2000).
The AA content of plant tissues seems to be influenced by light (Gatzek et al., 2002; Tamaoki et al., 2003). CmGalLDH mRNA abundance was shown to decrease in seedlings kept in the dark. This finding is consistent with recent reports that GalLDH and GDP-D-mannose pyrophosphorylase (GMPase) transcript levels are reduced in darkened tobacco leaves (Tabata et al., 2002). In Arabidosis thaliana, GalLDH expression and protein activity plus AA levels have been shown to exhibit light-mediated diurnal changes in mature plants, but not in young rosette leaves (Tamaoki et al., 2003). Similar light-mediated shifts in leaf AA 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). By contrast, Pignocchi et al. (2003) did not observe any differences in GalLDH transcript accumulation in tobacco plants grown in light or dark conditions. Taken together, these results suggest that light may regulate AA biosynthesis. However, further studies are needed to unravel possible species-to-species variation or specific growth conditions effects in the regulation of AA biosynthesis by light.
Apoplastic AA is suggested to act as a first line of defence against hostile environments and to behave as a possible link between environmental stresses and adaptive responses in plants (Noctor et al., 2000; Sanmartin et al., 2003). Oxidative stress, (rapid production of ROS) is experienced by plants exposed to different stresses. AA requirements to cope with stress conditions are clearly understood in Arabidopsis thaliana mutants. Mutant vtc1 with only 30% of wild-type AA content is ozone-, UV-B- and sulphur dioxide-sensitive (Conklin et al., 1996). Moreover, this mutant showed a higher GalLDH capacity than the wild-type plants (Conklin et al., 1997). However, no modification of CmGalLDH message accumulation was detected in melon seedlings exposed to high temperature, wounding or wound-signalling compounds (JA, SA, and ABA) at least under these experimental conditions. It was hypothesized that the regulation of AA levels under these stress conditions does not require de novo synthesis of CmGalLDH transcript.
In conclusion, the molecular characterization of CmGalLDH has been reported and the expression of this gene has been investigated in vegetative and reproductive plant tissues during development and under stressed conditions. To current knowledge, the CmGalLDH differential gene expression during melon fruit development and ripening is reported for the first time. However, these results combined with future in vivo labelling studies may explain the mechanism of ascorbate biosynthesis in ripening fruit.
|
Acknowledgements |
|---|
We are grateful to J Barnes (University of Newcastle) for editing and for his constructive comments and N Smirnoff (University of Exeter) for comments on the manuscript. We thank C Balague for providing the melon ACO cDNA (pMEL-1 plasmid). The work was supported in part by grants awarded to AKK (EU-FAIR-CT-97-5021, GR-NAGREF-DIMITRA, GR-GSRT). MS was a TMR fellow partially supported by EU-FAIR-CT-97-5021.
|
Footnotes |
|---|
* Present address: Departamento de Genética Molecular de Plantas, Centro Nacional de Biotecnología CSIC, 28049 Madrid, Spain.
Present address: Department of Agriculture, Aristotle University of Thessaloniki, 541 24 Thessaloniki, Greece.
|
References |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Abeles FB, Morgan PW, Saltveit KE. 1992. In: Ethylene in plant biology, 2nd edn. San Diego: Academic Press Inc.
Agius F, Gonzalez-Lamothe R, Caballero JL, Muñoz-Blanco J, Botella MA, Valpuesta V. 2003. Engineering increased vitamin C levels in plants by overexpressing of a D-galacturonic acid reductase. Nature Biotechnology 21, 177–181.[CrossRef][ISI][Medline]
Altschul SF, Gish W, Miller W, Meyers EW, Lipman DJ. 1990. Basic local alignment search tool. Journal of Molecular Biology 215, 403–410.[CrossRef][ISI][Medline]
Arrigoni O. 1994. Ascorbate system in plant development. Journal of Bioenergetics and Biomembranes 26, 407–419.[CrossRef][ISI][Medline]
Asada K. 1999. The water-water cycle in chloroplast: scavenging of active oxygens and dissipation of excess photons. Annual Review of Plant Physiology and Plant Molecular Biology 50, 601–639.[CrossRef][ISI][Medline]
Bartoli CG, Pastori GM, Foyer CH. 2000. Ascorbate biosynthesis in mitochondria is linked to the electron transport chain between complexes 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 of seeds in relation to germination, Vol. 1. Berlin: Springer-Verlag, 120–123.
Brennen T, Frenkel C. 1977. Involvement of hydrogen peroxide in the regulation of senescence in pear. Plant Physiology 59, 411–416.
Brummell DA, Harpster MH. 2001. Cell wall metabolism in fruit softening and quality and its manipulation in transgenic plants. Plant Molecular Biology 47, 311–340.[CrossRef][ISI][Medline]
Carr AC, Frei B. 1999. Toward a new recommended dietary allowance for vitamin C based on antioxidant and health effects in humans. American Journal of Clinical Nutrition 69, 1086–1107.
Chatterjee IB. 1973. Evolution and the biosynthesis of ascorbic acid. Science 182, 1271–1272.
Church GM, Gilbert W. 1984. Genomic sequencing. Proceedings of the National Academy of Sciences, USA 81, 1991–1995.
Conklin PL, Pallanca JE, Last RL, Smirnoff N. 1997. L-ascorbic acid metabolism in the ascorbate-deficient Arabidopsis mutant vtc1. Plant Physiology 115, 1277–1285.[Abstract]
Conklin PL, Williams EH, Last RL. 1996. Environmental stress sensitivity of an ascorbic acid-deficient Arabidopsis mutant. Proceedings of the National Academy of Sciences, USA 93, 9970–9974.
Davey MD, Van Montagu M, Inzé D, Sanmartin M, Kanellis AK, Smirnoff N, Benzie IJJ, Strain JJ, Favell D, Fletcher J. 2000. Plant L-ascorbic acid: chemistry, function, metabolism, bioavailability, and effects of processing. Journal of the Science of Food and Agriculture 80, 825–860.[CrossRef]
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 development and repression by wounding. Plant Molecular Biology 34, 759–70.[CrossRef][ISI][Medline]
Foyer C, Rowell J, Walker D. 1983. Measurement of the ascorbate content of spinach leaf protoplasts and chloroplasts during illumination. Planta 157, 239–244.[CrossRef][ISI]
Fry SC. 1998. Oxidative scission of plant cell wall polysaccharides by ascorbate-induced hydroxyl radicals. The Biochemical Journal 332, 507–515.[ISI][Medline]
Gatzek S, Wheeler GL, Smirnoff N. 2002. Antisense suppression of L-galactose dehydrogenase in Arabidopsis thaliana provides evidence for its role in ascorbate synthesis and reveals light modulated L-galactose synthesis. The Plant Journal 30, 541–553.[CrossRef][ISI][Medline]
Gonzalez-Reyes JA, Alcain FJ, Caler JA, Serrano A, Cordoba F, Navas P. 1995. Stimulation of onion root elongation by ascorbate and ascorbate free radical in Allium cepa L. Protoplasma 184, 31–35.[CrossRef]
Graber JH, Cantor CR, Mohr SC, Smith TF. 1999. In silico detection of control signals: mRNA 3'-end-processing sequences in diverse species. Proceedings of the National Academy of Sciences, USA 96, 14055–14060.
Grace SC, Logan BA. 1996. Acclimation of foliar antioxidant systems to growth irradiance in 3 broad-eaved evergreen species. Plant Physiology 112, 1631–1640.[Abstract]
Imai T, Karita S, Shiratori G, Hattori M, Nunome T, Oba K, Hirai M. 1998. L-galactono-gamma-lactone dehydrogenase from sweet potato: purification and cDNA sequence analysis. Plant Cell Physiology 39, 1350–1358.
Isherwood FA, Chen YT, Mapson LW. 1954. Synthesis of L-ascorbic acid in plants and animals. Biochemical Journal 56, 1–21.[ISI][Medline]
Jimenez A, Creissen G, Kular B, Firmin J, Robinson S, Verhoeyen M, Mullineaux P. 2002. Changes in oxidative processes and components of the antioxidant system during tomato fruit ripening. Planta 214, 751–758.[CrossRef][ISI][Medline]
Kato N, Esaka M. 1999. Changes in ascorbate oxidase gene expression and ascorbate levels in cell division and cell elongation in tobacco cells. Physiologia Plantarum 105, 321–329.
Kato N, Esaka M. 2000. Expansion of transgenic tobacco protoplasts expressing pumpkin ascorbate oxidase is more rapid than that of wild-type protoplasts. Planta 210, 1018–1022.[CrossRef][ISI][Medline]
Keller R, Renz FS, Kossmann J. 1999. Antisense inhibition of the GDP-mannose pyrophosphorylase reduces the ascorbate content in transgenic plants leading to developmental changes during senescence. The Plant Journal 19, 131–141.[CrossRef][ISI][Medline]
Kerk NM, Feldman LJ. 1995. A biochemical model for the initiation and maintenance of the quiescent centre: implications for organization of root meristems. Development 121, 2825–2833.[Abstract]
Levine M, Rumsey SC, Daruwala R, Park JB, Wang YH. 1999. Criteria and recommendations for vitamin C intake. Journal of the American Medical Association 281, 1415–1423.
Loewus FA. 1999. Biosynthesis and metabolism of ascorbic acid in plants and of analogs of ascorbic acid in fungi. Phytochemistry 52, 193–210.[CrossRef]
Mapson LW, Breslow E. 1958. Biological synthesis of ascorbic acid: 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 melo L. var. reticulatus: purification, characterization and antibody production. Journal of Experimental Botany 45, 717–724.
Mutsuda M, Ishikawa T, Takeda T, Shigeoka S. 1995. Subcellular localization and properties of L-galactono--lactone dehydrogenase. Bioscience, Biotechnology, and Biochemistry 59, 1983–1984.
Noctor G, Foyer CH. 1998. Ascorbate and glutathione: keeping active oxygen under control. Annual Review of Plant Physiology and Plant Molecular Biology 49, 249–279.[CrossRef][ISI][Medline]
Noctor G, Veljovic-Jovanovic S, Foyer CH. 2000. Peroxide processing in photosynthesis: antioxidant coupling and redox signaling. Philosophical Transactions: Biological Sciences 355, 1465–1475.[Medline]
Oba K, Fukui M, Imai Y, Iriyama S. Nogami K. 1994. L-Galactono--lactone dehydrogenase: partial characterization, induction of activity and role in the synthesis of ascorbic acid in wounded white potato tuber tissue. Plant Cell Physiology 35, 473–478.
Oba K, Ishikawa S, Nishikawa M, Mizuno H, Yamamoto T. 1995. Purification and properties of L-galactono-gamma-lactone dehydrogenase, a key enzyme for ascorbic acid biosynthesis, from sweet 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--lactone dehydrogenase, an enzyme involved in the biosynthesis of ascorbic acid in plants. Purification, characterization, cDNA cloning, and expression in yeast. Journal of Biological Chemistry 272, 30009–30016.
Padh H. 1990. Cellular functions of ascorbic acid. Biochemistry and Cell Biology 68, 1166–1173.[ISI][Medline]
Pallanca JE, Smirnoff N. 1999. Ascorbic acid metabolism in pea seedlings. A comparison of D-glucosone, L-sorbosone, and L-galactone-1,4-lactone as ascorbate precursors. Plant Physiology 120, 453–461.
Pastori GM, Kiddle G, Antoniw J, Bernard S, Veljovic-Jovanovic S, Verrier PJ, Noctor G, Foyer CH. 2003. Leaf vitamin C contents modulate plant defense transcripts and regulate genes controlling development through hormone signalling. The Plant Cell 15, 1212–1226.
Pignocchi C, Fletcher JM, Wilkinson JE, Barnes J, Foyer CH. 2003. The function of ascorbate oxidase in tobacco (Nicotiana tabacum L.). Plant Physiology 132, 1631–1641.
Pignocchi C, Foyer CH. 2003. Apoplastic ascorbate metabolism and its role in the regulation of cell signalling. Current Opinion in Plant Biology 6, 379–389.[CrossRef][ISI][Medline]
Redgwell RJ, MacRae E, Hallett J, Fischer M, Perry J, Harker R. 1997. In vivo and in vitro swelling of cell walls during fruit ripening. Planta 203, 162–173.[CrossRef][ISI]
Rose JKC, Hadfield KA, Labavitch JM, Bennett A. 1998. Temporal sequence of cell wall disassembly in rapidly ripening melon fruit. Plant Physiology 117, 345–361.
Sanmartin M. 2002. Regulation of melon ascorbate oxidase gene expression and effect of its modification in transgenic tobacco and melon 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 the apoplast of transgenic tobacco results in altered ascorbate and glutathione redox states and increased sensitivity to ozone. Planta 216, 918–928.[ISI][Medline]
Siendones E, Gonzalez-Reyes JA, Santos-Ocana C, Navas P, Cordoba F. 1999. Biosynthesis of ascorbic acid in kidney bean. L-Galactono--lactone dehydrogenase is an intrinsic protein located at the mitochondrial inner membrane. Plant Physiology 120, 907–912.
Smirnoff N. 1996. The function and metabolism of ascorbic acid in plants. Annals of Botany 78, 661–669.
Smirnoff N. 2000. Ascorbic acid: metabolism and functions of a multi-facetted molecule. Current Opinion in Plant Biology 3, 229–235.[ISI][Medline]
Smirnoff N, Pallanca JE. 1996. Ascorbate metabolism in relation to oxidative stress. Biochemical Society Transactions 24, 472–478.[ISI][Medline]
Smirnoff N, Wheeler GL. 2000. Ascorbic acid in plants: biosynthesis and function. Critical Review in Biochemistry and Molecular Biology 35, 291–314.
Smith CJS, Slater A, Grierson D. 1986. Rapid appearance of an mRNA correlated with ethylene synthesis encoding a protein of molecular weight 35000. Planta 168, 94–100.[CrossRef]
Tabata K, Oba K, Suzyki K, Esaka M. 2001. Generation and properties of ascorbic acid-deficient transgenic tobacco cells expressing antisense RNA for L-galactono-1,4-lactone dehydrogenase. The Plant Journal 27, 139–148.[CrossRef][ISI][Medline]
Tabata K, Takaoka T, Esaka M. 2002. Gene expression of ascorbic acid-related enzymes in tobacco. Phytochemistry 61, 631–635.[CrossRef][ISI][Medline]
Tamaoki M, Mukai F, Asai N, Nakajima N, Kubo A, Aono M, Saji H. 2003. Light-controlled expression of a gene encoding L-galactono--lactone dehydrogenase which affects ascorbate pool size in Arabidopsis thaliana. Plant Science 164, 1111–1117.
Thompson JD, Higgins DG, Gibson TJ. 1994. ClustalW: improving the sensitivity of progressive multiple sequence alignments through sequence weighting, position-specific gap penalties and weight 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 associated with decreased growth and intracellular redistribution of the antioxidant system Plant Physiology 127, 426–435.
Ververidis P, John P. 1991. Complete recovery in vitro of ethylene forming enzyme activity. Phytochemistry 30, 725–727.[CrossRef]
von Heije G. 1986. Mitochondrial targeting sequences may form amphiphilic helices. EMBO Journal 5, 1335–1342.[ISI][Medline]
Wadsworth GJ, Redinbaugh MG, Scandalios JG. 1988. A procedure for the small-scale isolation of plant RNA suitable for RNA blot analysis. Analytical Biochemistry 172, 279–283.[CrossRef][ISI][Medline]
Wheeler GL, Jones MA, Smirnoff N. 1998. The biosynthetic pathway of Vitamin C in higher plants. Nature 393, 365–369.[CrossRef][Medline]
Wolucka BA, Persiau G, Van Doorsselaere J, Davey MW, Demol H, Vandekerckhove J, Van Montagu M, Zabeau M, Boerjan W. 2001. Partial purification and identification of GDP-mannose 3',5'-epimerase of Arabidopsis thaliana, a key enzyme of the plant vitamin C pathway. Proceedings of the National Academy of Sciences, USA 98, 14843–14848.
Yabuta Y, Yoshimura K, Takeda T, Shigeoka S. 2000. Molecular characterization of tobacco mitochondrial L-galactono--lactone dehydrogenase and its expression in Escherichia coli. Plant Cell Physiology 41, 666–675.[ISI][Medline]
