JXB Advance Access originally published online on September 25, 2003
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Journal of Experimental Botany, Vol. 54, No. 392, pp. 2439-2448,
November 1, 2003
© 2003 Oxford University Press
Ascorbate metabolism in harvested broccoli
Received 7 July 2003; Accepted 21 July 2003
1 The United Graduate School of Agricultural Science, Gifu University (Shizuoka University), Yanagido, Gifu, 501-1193 Japan
2 Department of Biological Sciences, Faculty of Agriculture, Shizuoka University, 836 Ohya, Shizuoka, 422-8529 Japan
3 Department of Citriculture, National Institute of Fruit Tree Science, Okitsu, Shimizu, 424-0292 Japan
* To whom correspondence should be addressed. Fax: +81 54 2373028. E-mail: w1611012{at}ipc.shizuoka.ac.jp
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Abstract |
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The ascorbate content declined rapidly in broccoli (Brassica oleracea L. var. italica) florets, but not in the stem tissue, during post-harvest senescence. Ascorbate peroxidase (APX), ascorbate oxidase (AO), L-galactono-1,4-lactone dehydrogenase (GLDH), monodehydroascorbate reductase (MDAR), dehydroascorbate reductase (DHAR), and glutathione reductase (GR) were investigated in gene expression after harvest in both florets and the stem tissue of broccoli. Cytosolic gene expressions (BO-APX 1, BO-APX 2, BO-AO, BO-MDAR 2, and BO-GR) were stimulated actively in broccoli florets after harvest. By contrast, it was observed that mRNA levels of chloroplastic APX, BO-sAPX and BO-tbAPX, had decreased by 12 h after harvest in broccoli florets, suggesting that the active oxygen species (AOS) scavenging system in chloroplasts was largely abolished in florets during the early hours of the post-harvest period. In addition, gene expressions in GLDH and other chloroplastic enzymes such as BO-MDAR 1 and BO-DHAR decreased rapidly within 24 h after harvest. Ethylene treatment had no effect on the ascorbate level and the expression of all genes investigated. The expressions of BO-GLDH and chloroplastic genes (BO-sAPX, BO-tbAPX, BO-MDAR 1, and BO-DHAR) mRNA were suppressed by treatment with methyl jasmonate (MJ) and abscisic acid (ABA) and were accompanied by the acceleration of ascorbate degradation. These data suggest that ascorbate metabolism tends to be inactivated in chloroplasts by transcriptional regulation, but not in the cytosol, when ascorbate decreases under stress conditions.
Key words: Active oxygen species, ascorbate, Brassica oleracea, gene expression, harvest.
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Introduction |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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Ascorbate is one of the most important components in both plants and animals. There have been several investigations on ascorbate metabolism and its function in plants which provide the major source of dietary vitamin C for humans (Noctor and Foyer, 1998; Smirnoff et al., 2001). Ascorbate is oxidized by enzymatic or non-enzymatic reactions. A well-recognized enzyme consuming ascorbate is ascorbate peroxidase (APX), which catalyses the reduction of hydrogen peroxide to water with the simultaneous oxidation of ascorbate with a high specificity (Fig. 1). Ascorbate oxidase (AO) also catalyses ascorbate oxidation in the presence of oxygen, although its physiological significance has been poorly understood. Ascorbate oxidation always leads to monodehydroascorbate (MDA) which normally has a short life span that, if not rapidly reduced by MDA reductase (MDAR), disproportionates into ascorbate and dehydroascorbate (DHA). DHA is reduced to ascorbate by the action of DHA reductase (DHAR), using glutathione as the reducing substrate. A biosynthetic pathway has been proposed in which ascorbate is synthesized from L-galactono-1,4-lactone (GL) dehydrogenase catalysed by GL dehydrogenase (GLDH) (Wheeler et al., 1998). Ascorbate and its related enzymes described above play an essential role in scavenging active oxygen species (AOS) produced under normal and stress conditions. AOS-scavenging enzymes such as APX, MDAR, DHAR, and glutathione reductase (GR) exist as isoenzymes distributed in distinct cellular compartments. To date, many investigations into APX isoenzymes have been presented to clarify the stress-tolerance responses and Shigeoka et al. (2002) has reviewed much of this information. At least four isoenzymes of APX have been demonstrated: thylakoid-bound APX (tbAPX) and stromal APX (sAPX) in chloroplasts, cytosolic APX, and microbody APX. The cDNAs encoding the enzymes of MDAR, DHAR, GR, AO, and GLDH were isolated from various plant species and have been characterized by many research groups (Mertz, 1961; Yamauchi et al., 1984; Ôba et al., 1994, 1995; Grantz et al., 1995; Diallinas et al., 1997; Østergaard et al., 1997; Stevens et al., 1997; Shimaoka et al., 2000; Urano et al., 2000; Smirnoff et al., 2001). Considering the changes in ascorbate pool size and the importance of scavenging AOS, it is necessary to understand the regulation of these isoenzymes in addition to APXs. However, very little work has been done on the simultaneous analysis of these enzymes in distinct cellular compartments.
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Ascorbate has been proposed to have roles in the regulation of photosynthesis (Noctor and Foyer, 1998), cell expansion (Smirnoff, 1996) and trans-membrane electron transport (Horemans et al., 1994), besides its well-established antioxidant role. In many vegetables and fruit, harvesting causes ascorbate degradation, which seems to induce serious damage to the plant cell after harvest (Lee and Kader, 2000). In a previous paper, a rapid degradation of ascorbate was seen to occur in broccoli (Brassica oleracea L. var. italica) florets after harvest, but not in the stem tissue, in which the changes in both activities and gene expressions of APX after harvest were studied (Nishikawa et al., 2001, 2003). In the present paper, the changes are described in gene expression of the enzymes involved in ascorbate synthesis and breakdown in several portions of harvested broccoli. The results showed that the mRNA abundance of ascorbate-related enzymes in chloroplasts and the cytosol acted in a co-regulated, but distinct, manner after harvest and treatment with several plant hormones (methyl jasmonate (MJ), 6-benzylaminopurine (BA), and abscisic acid (ABA)) in broccoli florets. APX, MDAR and DHAR transcript levels in chloroplasts were strongly suppressed by 24 h after harvest. On the other hand, the expressions of the genes related to ascorbate metabolism in the cytosol were actively stimulated in harvested broccoli florets. Similar patterns were obtained in the treatment with MJ or ABA. In association with the decline of GLDH mRNA level within 12 h after harvest, these results may correlate with ascorbate degradation in harvested broccoli florets.
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Materials and methods |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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Plant materials
Whole, intact broccoli plants, with roots, were harvested on a farm and transported to the laboratory where the stem ends were cut. Harvested broccoli heads were incubated at 20 °C under humidified condition (RH >100). At 0, 2, 4, 6, 12, 24, 48, and 72 h after harvest, samples of stem tissue (2 mm thick) for the first and second layers, and basal portion (c. 2 mm thick) of curds, and florets were excised from broccoli (Fig. 2). The outer portion (green part) of the stem tissue was excised and used for RNA extraction (Nishikawa et al., 2001). The excised stem tissue and florets were immediately frozen in liquid nitrogen and stored at –80 °C until ready for use.
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Treatments with plant hormones
Broccoli obtained from a local market was used for treatment with chemical compounds. For 2,5-norbornadiene (NBD) and ethylene treatments, broccoli heads were exposed to 4000 µl l–1 NBD or 50 µl l–1 ethylene for 12 h and then florets were removed and frozen in liquid N2. For BA and MJ treatment, samples were dissected into curds (segments averaging 17.1 g) which were immersed with gentle stirring for 1 h in a solution of 0.22 mM BA or 1 mM MJ containing 1% ethanol and 0.1% Tween 20. Control broccoli curds were treated in the same way with 1% ethanol and 0.1% Tween 20. These curds were removed from the solution and held at 20 °C under high humidity. After 23 h, florets were separated from the curds and used for the subsequent analyses. For treatment with ABA, florets excised from a harvested broccoli head were floated in 1 mM ABA or water with gentle agitation for 24 h. After the treatment, the florets were frozen in liquid N2 and stored at –80 °C until ready for use.
Assay of ascorbate content and ethylene production
Ascorbate content in the reduced and oxidized form was assayed using HPLC. Each frozen sample (0.5 g) was homogenized with a mortar and pestle in 5 ml of 2% metaphosphoric acid. The homogenate was centrifuged at 3000 rpm for 15 min, and then the supernatant was filtered through Mirachloth (Calbiochem). The pH of the filtrate was adjusted by adding an equal volume of 0.2 M K-phosphate buffer (pH 7.5). Total ascorbate was assayed by adding 1 ml of 1 mM dithiothreitol (DTT) to an aliquot of filtrate and incubating the mixture for 15 min (Masuda et al., 1988). After the sample was filtered through 0.2 µm cellulose acetate filter (Advantec), a 20 µl aliquot was injected onto a TSK-GEL (Amide-80) column (TOSOH) attached to a LC-10AD pump (Shimadzu). The column kept at 20 °C was eluted with 80% acetonitrile:0.04% phosphoric acid at a flow rate of 1.0 ml min–1. Ascorbate was monitored at 245 nm (retention time 5.3 min) with an SPD-10A spectrophotometric detector (Shimadzu) attached to a chart recorder (C-R6A, Shimadzu). Peaks were converted to concentrations by using the dilution of stock ascorbate to construct a standard curve. Ascorbate content was determined in a similar manner without the addition of DTT. Dehydroascorbate content was calculated by subtracting the ascorbate value from the total ascorbate.
Ethylene production was measured according to the procedures described by Nishikawa et al. (2001).
RNA extraction, RT-PCR, cloning, and sequencing
Total RNA was isolated according to the method of Kato et al. (2000). The first strand cDNA was synthesized from 5 µg of the total RNA by reverse transcriptase with Oligo-(dT) primer according to the instructions of the Ready-To-Go T-Primed First Strand Kit (Amersham Pharmacia Biotech). RT-PCR was performed in a total volume of 100 µl containing 1 µl of the first strand cDNA reaction products, 10 µl of 10x PCR buffer II, 2.5 mM MgCl2, 200 µM deoxynucleotides, 100 pmol of primers, and 2.5 units of AmpliTaq GoldTM DNA polymerase (Perkin Elmer). The primers were designed by the common sequences in the genes of various plants. For BO-sAPX and BO-tbAPX the primers were designed by the common sequences based on Arabidopsis thaliana sAPX (accession no. X98925
[GenBank]
) and tbAPX (accession no. X98926
[GenBank]
), Spinacia oleracea chloroplastic APX (accession no. AB002467
[GenBank]
), Nicotiana tabacum sAPX (accession no. AB022274
[GenBank]
) and Cucurbita cv. Kurokawa Amakuri sAPX (accession no. D88420
[GenBank]
). BO-GLDH was isolated with the primers designed on the basis of the common sequences in sweet potato (accession no. AB017357
[GenBank]
) and cauliflower (accession no. Z97060
[GenBank]
) GLDH (Table 1). The primers for BO-MDAR 1 were designed by the common sequences in A. thaliana (accession no. D84417
[GenBank]
), Brassica juncea (accession no. AF109695
[GenBank]
) and S. oleracea (accession no. AB063289
[GenBank]
) MDAR, and for BO-MDAR 2 were designed on the basis of A. thaliana (accession no. AF360197
[GenBank]
) and Oryza sativa (accession no. D85764
[GenBank]
) and tomato (accession no. L41345
[GenBank]
) MDAR nucleotide sequences. DHAR cDNA was isolated using the primers designed by the common sequences in A. thaliana (accession no. AF301597
[GenBank]
) and S. oleracea (accession no. AF195783
[GenBank]
) and O. sativa (accession no. AB037970
[GenBank]
) DHAR. BO-AO was isolated with the primers designed by the common sequences in A. thaliana (accession no. AB004798
[GenBank]
), B. juncea (accession no. AF206721
[GenBank]
) and Cucumis melo (accession no. AF233593
[GenBank]
) AO. The primers for GR were designed on the basis of the common sequences in A. thaliana (accession no. AY040029
[GenBank]
), Brassica rapa (accession no. AF008441
[GenBank]
), O. sativa (accession no. D85751
[GenBank]
), Pisum sativum (accession no. X98274
[GenBank]
) and S. oleracea (accession no. D37870
[GenBank]
) GR and used for RT-PCR. The PCR procedure started with 10 min at 95 °C and was carried out for 35 cycles of 30 s at 95 °C, 30 s at 52 °C and 30 s at 72 °C, and ended with 10 min at 72 °C with GeneAmp PCR System 9600 (Perkin Elmer). The amplified cDNA was cloned with TA Cloning Kit (Invitrogen). The sequences were determined using the Taq Dye Primer Cycle Sequencing kit and the Taq Dye Terminator Cycle Sequencing kit (Perkin Elmer) with 373S DNA Sequencing System (Perkin Elmer).
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Northern blot analysis
Northern blot analysis was performed as described by Nishikawa et al. (2003). Total RNA (10 µg per lane) was separated in formaldehyde–agarose gels and RNA was visualized with ethidium bromide under UV light to ensure the equal loading of RNA in each lane. Probes were prepared using DIG RNA labelling Kit (Roche) and the detection after blotting was performed by chemiluminescence with CDP-Star (Roche) exposed to Hyper film ECL (Amersham Pharmacia Biotech).
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Results |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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Changes in ascorbate content
Ascorbate content was measured in four parts of broccoli: the first layer of the cut stem (0–2 mm, cut surface layer), the second layer (2–4 mm, adjacent to the first layer), the basal portion of curds and florets (Fig. 2).
The amount of total ascorbate in florets was the highest of all the portions measured at 0 h (Fig. 3). It stayed at a relatively high level until about 12 h, followed by a rapid decline to a low level during senescence. In the stem tissue, the levels of ascorbate remained almost unchanged or increased slightly as time progressed. DHA content exhibited less than 10% of total ascorbate in all portions throughout the post-harvest period (data not shown). These results were consistent with those obtained previously (Nishikawa et al., 2001).
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Isolation and identification of the cDNAs involved in ascorbate metabolism
To determine the changes in the levels of transcripts corresponding to APX, AO, GLDH, MDAR, DHAR, and GR, each cDNA was isolated from broccoli by RT-PCR with the primers shown in Table 1 and used as a template for probes in northern blot analysis. The sequences of the cDNAs isolated showed close similarities with those of Arabidopsis genes. Two chloroplastic APX cDNAs were isolated by RT-PCR using the primers designed by the common sequences in chloroplastic APXs reported in various plants. The nucleotide sequences, BO-sAPX and BO-tbAPX, showed a high similarity to those of stromal and thylakoid bound APX from Arabidopsis (accession nos AY056319 [GenBank] , X98926 [GenBank] ), respectively. BO-sAPX and BO-tbAPX cDNAs shared a high nucleotide identity in the coding regions so that probes were made using 3'-untranslated regions in which they shared a lower nucleotide identity of less than 70%. The nucleotide sequence obtained of BO-GLDH was identical with that of cauliflower (accession no. Z97060 [GenBank] ). Two distinct clones having a high nucleotide identity to the sequences encoding MDAR were isolated from broccoli. The partial cDNA obtained for BO-MDAR 1 and BO-MDAR 2 were 328 and 411 bp long, respectively, and shared less than 60% nucleotide identity. The nucleotide sequence of BO-MDAR 1 showed more than 90% nucleotide identity with those of A. thaliana (accession no. D84417 [GenBank] ) and B. juncea (accession no. AF109695 [GenBank] ) MDAR. BO-MDAR 2 cDNA gave values of 92.7% and 77.1% identity with those of A. thaliana (accession no. AF360197 [GenBank] ) and O. sativa (accession no. D85764 [GenBank] ) MDAR, respectively. BO-DHAR cDNA obtained showed more than 70% identity with those of A. thaliana (accession no. AF301597 [GenBank] ) and B. juncea (accession no. AF536329 [GenBank] ) and S. oleracea (accession no. AF195783 [GenBank] ). BO-AO cDNA showed more than 80% nucleotide identity with A. thaliana (accession no. AB004798 [GenBank] ) and B. juncea (accession no. AF206721 [GenBank] ) AO. BO-GR obtained showed more than 90% nucleotide identity with A. thaliana (accession no. AY040029 [GenBank] ) and B. rapa (AF008441 [GenBank] ) GR. According to the similarity with the sequences from other plants, putative localization of their encoding protein was estimated. BO-sAPX, BO-tbAPX, BO-MDAR 1, and BO-DHAR were supposed to encode the proteins in the chloroplasts and BO-MDAR 2 and BO-GR showed a high similarity to the nucleotide sequences of the proteins in the cytosol.
Changes in mRNA levels
Changes in the transcript levels for BO-APX 1 (accession no. AB078599
[GenBank]
) and BO-APX 2 (accession no. AB078600
[GenBank]
) were detected using the probes previously reported by Nishikawa et al. (2003). Expressions of both genes were induced markedly just after harvest in all the portions examined (Fig. 4). The mRNA levels of both BO-sAPX and BO-tbAPX in florets remained high until 6 h after harvest and then diminished rapidly. Transcripts of both chloroplastic APXs increased gradually in the stem tissue except that BO-sAPX in the basal portion of curds showed a similar pattern to that in florets. Transcript abundance of BO-GLDH in florets and the basal portion of curds stayed at a high level until 6 h after harvest and then decreased rapidly. In the 1st and 2nd layers of the stem, mRNA levels of BO-GLDH were induced and maintained at a comparatively higher level over the experimental period. The mRNA levels of both BO-MDAR 1 and BO-MDAR 2 in the 1st and 2nd layers were induced gradually after harvest with a peak at 12 h and 24 h, respectively. In florets, BO-MDAR 1 was highly expressed until 6 h and then declined rapidly at 12 h, while BO-MDAR 2 transcripts were detected at a low level over the first 24 h and increased markedly at 48 h and 72 h. The BO-DHAR mRNA level in florets showed a rapid increase until 12 h after harvest, but it became almost undetectable at 24 h. Transcripts of BO-DHAR in the three portions of the stem tissue exhibited almost the same pattern, showing a gradual increase during the experimental period.
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According to the putative localization of the encoding proteins, the genes examined were classified into three groups of cytosol, mitochondra and chloroplast. BO-APX 1 and BO-APX 2 were previously reported as cytosolic APX (Nishikawa et al., 2003). BO-AO was classified in the cytosolic group because of the AO localization proposed in the cytosol as well as the apoplast. When the mRNA expression patterns in florets after harvest were divided into two groups of cytosol and others (chloroplast and mitochondrion), their patterns were quite similar to each group (Fig. 5A, B). Gene expression of the cytosolic enzymes (BO-AO, BO-APX 1, BO-APX 2, BO-MDAR 2, and BO-GR) were actively stimulated within 4 h after harvest in florets. After a transient decrease almost to the initial level, they increased significantly after 12 h (Fig. 5A). Changes in the mRNA of chloroplastic genes (BO-sAPX, BO-tbAPX, BO-MDAR1, and BO-DHAR) and of the mitchondrial gene (BO-GLDH) in florets after harvest tended to exhibit a similar pattern of the mRNA level increasing within 6 h, and then decreasing rapidly at 6–12 h after harvest (Fig. 5B). In the later period of storage, this group of genes showed a lower expression in the transcript levels.
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Effect of treatments with NBD, ethylene, BA, MJ and ABA in mRNA level
To investigate the factors that may have played a role in altering the ascorbate pool size and the transcript levels of the genes related to ascorbate metabolism after harvest, broccoli florets were treated with NBD, ethylene, BA, MJ, and ABA. The NBD treatment enhanced the levels of most mRNAs detected with the exception for BO-GR, while the ethylene treatment had no effect on ascorbate content and gene expression involved in ascorbate metabolism (Fig. 6). Ascorbate degradation was suppressed slightly by the BA treatment and evidently accelerated by both MJ and ABA (Fig. 6). The mRNA levels of BO-GLDH, BO-sAPX, BO-tbAPX, and BO-MDAR 1 increased by the application of BA, whereas those of BO-GLDH, BO-sAPX, BO-tbAPX, BO-MDAR 1, and BO-DHAR decreased by the treatments with MJ and ABA in correlation with the changes in ascorbate concentration in florets.
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Discussion |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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In a previous paper it was reported that ascorbate degradation occurred in florets but not in the stem tissue of harvested broccoli (Nishikawa et al., 2001). To understand the effect of harvesting on ascorbate metabolism in broccoli plants, several isoenzymes involved in ascorbate metabolism (APX, MDAR, DHAR, GR, AO, and GLDH) were investigated. This study concentrated on gene expression because it was difficult to assay the activity of each isoenzyme separately, except for APX isoenzymes. These results help to understand the changes in the activity of each isoenzyme, which may have opposite patterns in the cytosolic patterns and chloroplastic ones under stress conditions. The finding that is led by the grouping of the genes indicates that the genes in the chloroplasts and the cytosol act in a co-regulated but distinct manner under stress conditions (Figs 5, 6). It seems that these changes could effect the AOS and ascorbate levels and ascorbate redox status in each compartment.
Changes in cytosolic gene expressions related to ascorbate metabolism under stress conditions
Increases after harvesting and treatment with MJ or ABA and decreases after BA treatment were observed in the expressions of the cytosolic genes related to ascorbate metabolism in broccoli florets. It seems likely that ascorbate metabolism in the cytosol is regulated in the same way at the transcriptional level and that the increases after harvesting and treatment with MJ or ABA may be a response to oxidative damage caused by the treated stresses. Generally, the AOS-scavenging system in the cytosol is considered to play a role in the protection of important cellular compartments from oxidative stress caused by environmental changes. It is well known that the formation of AOS is accelerated under stress conditions such as chilling, high temperature, intensive light and wounding, which are all associated with the process of senescence (Okuda et al., 1991; Foyer et al., 1994, 1997; Dat et al., 1998; Watanabe and Sakai, 1998). Harvesting could also cause an increase of AOS in the cell, relating to the activation of the AOS-scavenging system in the cytosol. Many have reported that cytosolic APX is induced by environmental changes (Mittler and Zilinskas, 1992; Morita et al., 1999; Yoshimura et al., 2000). Under stress conditions, the plant cell tends to scavenge AOS and protect cellular compartments by activating ascorbate-regenerating systems besides APX in the cytosol.
Changes in chloroplastic gene expressions related to ascorbate metabolism under stress conditions
The down-regulation of chloroplastic genes observed after harvest and MJ or ABA treatment could lead to a breakdown of the AOS-scavenging system in chloroplasts, resulting in the inhibition of CO2 fixation in the Calvin cycle (Kaiser, 1976). These results indicate that APX, MDAR, and DHAR in the chloroplasts can be regulated at the mRNA level and that not only APXs but also the ascorbate-regenerating system including MDAR and DHAR could be inactivated in chloroplasts under the stress conditions. As a consequence of this down-regulation, chloroplasts may start to disintegrate at an earlier stage before the occurrence of chlorophyll degradation during senescence because the degreening of sepals was observed visually at 48 h after harvest in this experiment. It has been reported that chloroplastic APX isoenzymes are inactivated by the excess generation of hydrogen peroxide and the depletion of ascorbate (Shikanai et al., 1998; Mano et al., 2001). APX may be inactivated in chloroplasts before the suppression of their transcription in harvested broccoli florets because of the excess AOS caused by harvesting or the ascorbate redistribution from chloroplasts to the cytosol which may have a high demand for ascorbate to protect the cells from oxidative damage during senescence. It has been reported that tbAPX activity decreased rapidly under photo-oxidative stress (Mano et al., 2001). Yabuta et al. (2002) have demonstrated that tbAPX is a limiting factor of antioxidative systems under photo-oxidative stress using transgenic tabacco plants that expressed tbAPX cDNA. Therefore, it is thought that the loss of tbAPX activity is a more important factor in post-harvest senescence. However, it seems that the disruption of the stromal AOS-scavenging system, including sAPX, MDAR and DHAR, is also an important factor in the alteration of AOS and ascorbate levels under stress conditions and that the down-regulation of MDAR and DHAR in chloroplasts leads directly to ascorbate degradation.
Changes in ascorbate biosynthesis under stress conditions
BO-GLDH exhibited a similar expression pattern to the chloroplastic genes in harvested broccoli florets, which showed a rapid decrease at the early stages of post-harvest, indicating a decrease in the rate of ascorbate biosynthesis. A decrease in ascorbate biosynthesis may be expected because of sugar deficiency caused by an inactivation of photosynthesis in florets and/or the cessation of its supply to florets from leaves after harvest. The rapid decline of sucrose has been observed in broccoli florets (F Nishikawa et al., unpublished data) and asparagus spears (Davies et al., 1996) just after harvest. The suppressed expression of BO-GLDH after harvest may be related to the loss of sugars.
Changes in gene expressions related to ascorbate metabolism in the stem tissue of harvested broccoli
In the basal portion of curds, which is part of the stem tissue far from the cut surface, the ascorbate level remained unchanged over the experimental period, despite the fact that the expression patterns of many genes showed a high similarity to those of florets. Compared with the stem parts with florets, a big difference may be in the number of chloroplasts, since florets are rich in chlorophyll in their sepals. It has been observed that post-harvest changes in broccoli florets show many similarities to the processes of developmental leaf senescence (Page et al., 2001). Assuming that a major source of ascorbate degradation in florets is primarily caused in chloroplasts, it could be supposed that the loss of ascorbate after harvest is influenced by the amount of chloroplasts and the regulation of chloroplastic gene expression involved in the AOS-scavenging system. The constitutive levels of ascorbate in the stem tissue, including the basal portion of curds, may be attributable to the lower number of chloroplasts or to the different response to florets in BO-tbAPX and BO-DHAR gene expression.
The effect of hormone supplies on ascorbate metabolism and ascorbate level
Harvesting disrupts water, energy, nutrient, and hormone supplies, and also causes wounding stress. Heads of broccoli are harvested at the immature stage and show rapid yellowing during storage. It has been observed that the rate of ethylene production in florets increased to a maximum and then declined in a pattern almost paralleling that of ACC oxidase activity (Kato et al., 2002). Ethylene production regulates the yellowing of sepals after harvest based on chlorophyll degradation. The activation of APX by ethylene has also been documented (Mehlhorn, 1990; Ievinsh et al., 1995). In the present study, the rate of ethylene production began to increase at 12 h after harvest and the degreening of sepals became obvious after 48 h (data not shown). To understand the relationship between ascorbate catabolism and ethylene production, harvested broccoli was treated with NBD, an inhibitor of ethylene action, and with ethylene. However, there were no substantial effects on ascorbate metabolism with ethylene treatment, indicating that ascorbate degradation in harvested broccoli florets was not regulated by ethylene. Increases in the expression of some genes involved in ascorbate metabolism by NBD treatment suggest that these genes could be suppressed to some extent by ethylene. It was reported that the MJ and ABA were induced by wounding and water stress, respectively (León et al., 2001; Zhu, 2002). The changes in transcript levels of mRNA related to ascorbate metabolism after harvest seemed related to those observed in the samples treated with MJ and ABA, suggesting that MJ and/or ABA may be related to the post-harvest response of florets.
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Acknowledgements |
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We thank Professor Kazuko Ôba for helpful suggestions and for critical reading of this manuscript.
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References |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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Diallinas G, Pateraki I, Sanmartin M, Scossa A, Stilianou E, Panopoulos NJ, Kanellis AK. 1997. Melon ascorbate oxidase: cloning of multigene family, induction during fruit development and repression by wounding. Plant Molecular Biology 34, 759–770.[CrossRef][Web of Science][Medline]
Dat JF, Lopez-Delgado H, Foyer CH, Scott IM. 1998. Parallel changes in H2O2 and catalase during thermotolerance induced by salicylic acid or heat acclimation in mustard seedlings. Plant Physiology 116, 1351–1357.
Davies KM, Seelye JF, Irving DE, Borst WM, Hurst PL, King GA. 1996. Sugar regulation of harvest-related genes in asparagus. Plant Physiology 111, 877–883.[Abstract]
Foyer CH, Lelandais M, Kunert KJ. 1994. Photooxidative stress in plants. Physiologia Plantarum 92, 696–717.[CrossRef]
Foyer CH, Lopez-Delgado H, Dat JF, Scott IM. 1997. Hydrogen peroxide- and glutathione-associated mechanisms of acclimatory stress tolerance and signalling. Physiologia Plantarum 100, 241–254.[CrossRef]
Grantz AA, Brummell DA, Bennett AB. 1995. Ascorbate free radical reductase mRNA levels are induced by wounding. Plant Physiology 108, 411–418.[Abstract]
Horemans N, Asard H, Caubergs RJ. 1994. The role of ascorbate free radical as an electron acceptor to cytochrome b-mediated trans-plasma membrane transport in higher plants. Plant Physiology 104, 1455–1458.[Abstract]
Kaiser WM. 1976. The effect of hydrogen peroxide on CO2 fixation of isolated intact chloroplasts. Biochimica et Biophysica Acta 440, 476–482.[Medline]
Ievinsh G, Valcina A, Ozola D. 1995. Induction of ascorbate peroxidase activity in stressed pine (Pinus sylvestris L.) needles: a putative role for ethylene. Plant Science 112, 167–173.[CrossRef]
Kato M, Hayakawa Y, Hyodo H, Ikoma Y, Yano M. 2000. Wound-induced ethylene synthesis and expression and formation of 1-aminocyclopropane-1-carboxylate (ACC) synthase, ACC oxidase, phenylalanine ammonia-lyase, and peroxidase in wounded mesocarp tissue of Cucurbita maxima. Plant and Cell Physiology 41, 440–447.
Kato M, Kamo T, Wang R, Nishikawa F, Hyodo H, Ikoma Y, Sugiura M, Yano M. 2002. Wound-induced ethylene synthesis in stem tissue of harvested broccoli and its effect on senescence and ethylene synthesis in broccoli florets. Postharvest Biology and Technology 24, 69–78.
Lee SK, Kader AA. 2000. Preharvest and postharvest factors influencing vitamin C content of horticultural crops. Postharvest Biology and Technology 20, 207–220.[CrossRef]
León J, Rojo E, Sánchez-Serrano JJ. 2001. Wound signalling in plants. Journal of Experimental Botany 52, 1–9.
Mano J, Ohno C, Domae Y, Asada K. 2001. Chloroplastic ascorbate peroxidase is the primary target of methylviologen-induced photooxidative stress in spinach leaves: its relevance to monodehydroascorbate radical detected with in vivo ESR. Biochimica et Biophysica Acta 1504, 275–287.[Medline]
Masuda R, Hayakawa A, Kakiuchi N, Iwamoto M. 1988. HPLC determination of total ascorbic acid in fruits and vegetables. Report of National Food Research Institute 52, 30–35.
Mehlhorn H. 1990. Ethylene-promoted ascorbate peroxidase activity protects plants against hydrogen peroxide, ozone and paraquat. Plant, Cell and Environment 13, 971–976.[CrossRef]
Mertz D. 1961. Distribution and cellular localization of ascorbic acid oxidase in the maize root tip. American Journal of Botany 48, 405–413.[CrossRef]
Mittler R, Zilinskas BA. 1992. Molecular cloning and characterization of a gene encoding pea cytosolic ascorbate peroxidase. Journal of Biological Chemistry 267, 21802–21807.
Morita S, Kaminaka H, Masumura T, Tanaka K. 1999. Induction of rice cytosolic ascorbate peroxidase mRNA by oxidative stress; the involvement of hydrogen peroxide in oxidative stress signalling. Plant and Cell Physiology 40, 417–422.
Nishikawa F, Kato M, Kamo T, Wang R, Hyodo H, Ikoma Y, Sugiura M, Yano M. 2001. Enzymatic catabolism of ascorbate in florets of harvested broccoli during senescence. Journal of Japan Society of Horticultural Science 70, 709–715.
Nishikawa F, Kato M, Wang R, Hyodo H, Ikoma Y, Sugiura M, Yano M. 2003. Two ascorbate peroxidases from broccoli: identification, expression and characterization of their recombinant proteins. Postharvest Biology and Technology 27, 147–156.[CrossRef]
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][Web of Science]
Ôba 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 and Cell Physiology 35, 473–478.
Ôba K, Ishikawa S, Nishikawa M, Mizuno H, Yamamoto T. 1995. Purification and properties of L-galactono--lactone dehydrogenase, a key enzyme for ascorbic acid biosynthesis, from sweet potato roots. Journal of Biochemistry 117, 120–124.
Okuda T, Matsuda Y, Yamanaka A, Sagisaka S. 1991. Abrupt increase in the level of hydrogen peroxide in leaves of winter wheat is caused by cold treatment. Plant Physiology 97, 1265–1267.
Østergaard J, Persiau G, Davey MW, Bauw G, Montagu MV. 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.
Page T, Griffiths G, Buchanan-Wollaston VV. 2001. Molecular and biochemical characterization of post-harvest senescence in broccoli. Plant Physiology 125, 718–727.
Shigeoka S, Ishikawa T, Tamoi M, Miyagawa Y, Takeda T, Yabuta T, Yoshimura K. 2002. Regulation and function of ascorbate peroxidase isoenzymes. Journal of Experimental Botany 53, 1305–1319.
Shikanai T, Takeda T, Yamauchi H, Sano S, Tomizawa K, Yokota A, Shigeoka S. 1998. Inhibition of ascorbate peroxidase under oxidative stress in tobacco having bacterial catalase in chloroplasts. FEBS Letters 428, 47–51.[CrossRef][Web of Science][Medline]
Shimaoka T, Yokota A, Miyake C. 2000. Purification and characterization of chloroplast dehydroascorbate reductase from spinach leaves. Plant and Cell Physiology 41, 1110–1118.
Smirnoff N. 1996. The function and metabolism of ascorbic acid in plants. Annals of Botany 78, 661–669.
Smirnoff N, Conklin PL, Loewus FA. 2001. Biosynthesis of ascorbic acid in plants: a renaissance. Annual Review of Plant Physiology and Plant Molecular Biology 52, 437–467.[CrossRef][Web of Science][Medline]
Stevens RG, Creissen GP, Mullineaux PM. 1997. Cloning and characterization of a cytosolic glutathione reductase cDNA from pea (Pisum sativum L.) and its expression in response to stress. Plant Molecular Biology 35, 641–654.[CrossRef][Web of Science][Medline]
Urano J, Nakagawa Y, Maki Y, Masumura T, Tanaka K, Murata N, Ushimaru T. 2000. Molecular cloning and characterization of a rice dehydroascorbate reductase. FEBS Letters 466, 107–111.[CrossRef][Web of Science][Medline]
Watanabe T, Sakai S. 1998. Effects of active oxygen species and methyl jasmonate on expression of the gene for a wound-inducible 1-aminocyclopropane-1-carboxylate synthase in winter squash (Cucurbita maxima). Planta 206, 570–576.[CrossRef]
Wheeler GL, Jones MA, Smirnoff N. 1998. The biosynthetic pathway of vitamin C in higher plants. Nature 393, 365–369.[CrossRef][Medline]
Yabuta Y, Motoki T, Yoshimura K, Takeda T, Ishikawa T, Shigeoka S. 2002. Thylakoid membrane-bound ascorbate peroxidase is a limiting factor of antioxidative systems under photo-oxidative stress. The Plant Journal 32, 915–925.[CrossRef][Web of Science][Medline]
Yamauchi N, Yamawaki K, Ueda Y, Chachin K. 1984. Subcellular localization of redox enzymes involving ascorbic acid in cucumber fruits. Journal of the Japan Society of Horticultural Science 53, 347–353.
Yoshimura K, Yabuta Y, Ishikawa T, Shigeoka S. 2000. Expression of spinach ascorbate peroxidase isoenzymes in response to oxidative stresses. Plant Physiology 123, 223–233.
Zhu JK. 2002. Salt and drought stress signal transduction in plants. Annual Review of Plant Biology 53, 247–273.[CrossRef][Medline]
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