JXB Advance Access originally published online on November 1, 2004
Journal of Experimental Botany 2005 56(409):65-72; doi:10.1093/jxb/eri007
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RESEARCH PAPER |
Effect of sucrose on ascorbate level and expression of genes involved in the ascorbate biosynthesis and recycling pathway in harvested broccoli florets
1The United Graduate School of Agricultural Science, Gifu University (Shizuoka University), Yanagido, Gifu, 501-1193 Japan
2Department of Biological Sciences, Faculty of Agriculture, Shizuoka University, 836 Ohya, Shizuoka, 422-8529 Japan
3Department of Citrus Research, National Institute of Fruit Tree Science, Shimizu-okitsunakacho, Shizuoka, 424-0292 Japan
* To whom correspondence should be addressed at the Department of Citrus Research: Fax: +81 543 69 2115. E-mail: fumien{at}affrc.go.jp
Received 13 May 2004; Accepted 12 August 2004
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Abstract |
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The relationship between sucrose (Suc) and ascorbate (AA) metabolism was investigated in harvested broccoli (Brassica oleracea L. var. italica) florets. Decreases in both Suc and AA content were observed in broccoli florets 48 h after all the leaves were excised, but none were observed when the plants were kept intact or with leaves attached in a room at 20 °C. In harvested broccoli plants without leaves and roots, continuous absorption of a 10% (w/v) Suc solution from the cut surface of the stem suppressed the degreening of sepals and the loss of AA content in florets. The expression of the genes related to AA metabolism in chloroplasts and its biosynthesis were up-regulated by Suc feeding in broccoli florets. These data suggest that a decline in Suc leads to considerable damage not only to AA biosynthesis but also to the hydrogen peroxide-scavenging system in chloroplasts. In addition, the cessation of the Suc supply from leaves can be the main factor of AA degradation in harvested broccoli florets.
Key words: Ascorbate, Brassica oleracea, gene expression, harvest, sucrose
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Introduction |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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Ascorbate (AA) plays an important role as an antioxidant and cofactor of many dioxygenases that relate to the key steps in cell metabolism (Noctor and Foyer, 1998
; Smirnoff et al., 2001). Furthermore, the AA level influences plant growth and development by modulating the expression of many defence genes and the abscisic acid signalling pathway (Pastori et al., 2003). The enzymes involved in the AA-recycling pathway have been reported (Noctor and Foyer, 1998), although relatively little information is available on other enzymes related to AA biosynthesis and dehydroascorbate (DHA) decomposition. The last step of AA synthesis, in which AA is synthesized from L-galactono-1,4-lactone (GL) catalysed by GL dehydrogenase (GLDH), has been reported to be in the mitochondria (Wheeler et al., 1998; Smirnoff et al., 2001; Millar et al., 2003). The major enzyme consuming AA is AA peroxidase (APX), which catalyses the reduction of hydrogen peroxide to water with simultaneous oxidation of AA with high specificity. To date, there have been many investigations of APX and Shigeoka et al. (2002) have reviewed much of the current 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. In the apoplast, AA oxidase (AO) catalyses AA oxidation in the presence of oxygen and it has been proposed that AO has a role in the control of growth (Pignocchi et al., 2003). AA oxidation always leads to monodehydroascorbate (MDA) which is normally converted to AA by MDA reductase (MDAR). MDA, if not rapidly reduced by MDAR, disproportionates non-enzymatically into AA and DHA. DHA is reduced to AA by the action of DHA reductase (DHAR), using glutathione (GSH) as the reducing substrate. Oxidized GSH is reduced by oxidized GSH reductase (GR). Thus, APX in combination with the effective AA–GSH cycle functions to prevent the accumulation of hydrogen peroxide.
Enzymes related to a hydrogen peroxide-scavenging system, such as APX, MDAR, DHAR, and GR, have isoenzymes localized in the cytosol or chloroplasts, which are encoded by discrete genes. It has been reported that the expression of these genes is influenced by environmental changes or hormone supply. Regulation by light was observed in the expression of cytosolic APX, GLDH, and AO (Karpinski et al., 1997; Yoshimura et al., 2000; Tabata et al., 2002; Pignocchi et al., 2003). Previous work by Nishikawa et al. showed that the transcripts of AA-related genes encoding enzymes located in the chloroplasts and mitochondria decreased upon harvesting or by treatment with methyl jasmonate or abscisic acid and increased by treatment with cytokinin in close association with the alteration of the AA level in harvested broccoli (Brassica oleracea L. var. italica) florets (Nishikawa et al., 2003b).
Harvesting can result in considerable damage due to the sudden disruption in water, energy, nutrient, and hormone supplies and can cause wounding stress in plants. In various fruits and vegetables, harvesting causes AA decomposition, which seems to induce serious damage to the plant cell (Lee and Kader, 2000). Broccoli is harvested at an immature stage before growth has ceased; therefore, its shelf life is short. Broccoli is known as an AA-rich vegetable, although rapid degradation of AA has been seen to occur in florets at ambient temperatures after harvest (Nishikawa et al., 2001, 2003b). In order to understand the regulation of the AA level, it is important to determine the alteration of AA-related enzyme activities or gene expressions under various conditions. It has been reported that mRNA abundance of AA-related enzymes in the cytosol and chloroplasts tended to act in a co-regulated, but distinct, manner after harvest and by treatment with several plant hormones in broccoli florets (Nishikawa et al., 2003b). In the present paper, changes in AA metabolism and its level in harvested broccoli florets are described as they relate to sucrose (Suc) concentration. The existence of more than 10% (w/v) Suc in plant phloem has been reported (Hayashi and Chino, 1990), and it is recognized that Suc is transported to sink tissue, such as florets, from source leaves under normal conditions. A rapid decline of sugars after harvest has been reported in various plants (King and Morris, 1994; Davies et al., 1996; Ichimura et al., 1999). Chlorophyll contents are kept at a higher level by Suc feeding in harvested broccoli florets (Coupe et al., 2003). It has been reported that Suc feeding in detached leaves increases the AA level, although the mechanism is still unclear (Smirnoff and Pallanca, 1996). Recently, sugar has been recognized as a signal to alter gene expression, a similar concept to that developed for hormones (Smeekens, 2000). The data from this study show that the Suc level can signal the alteration in gene expression of enzymes related to the chloroplastic AA metabolism and AA biosynthesis. In addition, the findings indicate that the AA degradation in harvested broccoli florets originates from a rapid decline of Suc.
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Materials and methods |
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Plant materials
Whole, intact broccoli (Brassica oleracea L. var. italica) plants with leaves and roots were harvested early in the morning from a campus farm and transported to the laboratory. Roots and/or all leaves were excised when needed, and the broccoli heads were incubated at 20 °C under 80% relative humidity and light conditions of 4.9 µmol m–2 s–1. When both roots and leaves were excised, the length of the stem was adjusted to the commercial length. At 0 h and 48 h after harvest, florets were excised from broccoli plants and immediately frozen in liquid nitrogen and stored at –80 °C until ready for use.
Suc application
Broccoli obtained from a local market was used for the treatments with Suc. Suc was absorbed continuously from the cut surface of the stem tissue. The stem ends were immersed in a 10% (w/v) Suc solution including 0.05% (v/v) Na-hypochlorite. In the control samples, 0.05% (v/v) Na-hypochlorite was treated in the same manner. The broccoli heads were incubated at 25 °C under 70% relative humidity and light conditions of 7.4 µmol m–2 s–1. The solution was replaced with new materials every 12 h. At 0, 12, 24, 36, 48, 60, and 72 h after absorption, the florets were excised, frozen in liquid N2, and stored at –80 °C until ready for use.
Assessment of yellowing broccoli
Floret colour, from green to yellow, was visually determined using the following scoring system: 5, all green: 4, 20% yellow: 3, 40% yellow: 2, 60% yellow: 1, 80% yellow: 0, completely yellow. The decrease in the scores (from 5 to 1) almost parallelled the decline in chlorophyll, which was extracted with ethanol and determined spectrophotometrically according to Hyodo et al. (1995).
Assay of AA content
AA content in both the reduced and oxidized forms was assayed using HPLC. Each frozen sample (0.5 g) was homogenized with a mortar and pestle in 5 ml of 2% (w/v) metaphosphoric acid. The homogenate was centrifuged at 3000 rpm for 15 min, and the supernatant was filtered through Miracloth (Calbiochem). The pH of the filtrate was adjusted by adding an equal volume of a 0.2 M K-phosphate buffer (pH 7.5). The total AA was assayed by adding 1 ml of 1 mM dithiothreitol to an aliquot of filtrate and incubating the mixture for 15 min (Masuda et al., 1988). After the sample was filtered through a 0.2 µm cellulose acetate filter (Advantec), a 20 µl aliquot was injected onto a TSK-GEL (Amide-80) column (TOSOH) attached to an LC-10AD pump (Shimadzu). The column kept at 20 °C was eluted with 80% (v/v) acetonitrile:0.04% (v/v) phosphoric acid at a flow rate of 1.0 ml min–1. AA 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 a dilution of stock AA to construct a standard curve. The AA content was determined in a similar manner without the addition of dithiothreitol. The DHA content was calculated by subtracting the AA value from that of the total AA.
Assay of sugar content
The contents of reducing sugars and total sugars were measured according to the Somogyi–Nelson method. The Suc content was calculated by subtracting the reducing sugar value from that of total sugars.
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 an 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 used for PCR were designed as described previously by Nishikawa et al. (2003b). 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 a GeneAmp PCR System 9600 (Perkin Elmer). The amplified cDNA was cloned with a TA Cloning Kit (Invitrogen). The sequences were determined using a Taq Dye Primer Cycle Sequencing Kit and a Taq Dye Terminator Cycle Sequencing Kit (Perkin Elmer) with a 373S DNA Sequencing System (Perkin Elmer).
Northern blot analysis
Northern blot analysis was performed as described by Nishikawa et al. (2003b). Total RNA (10 µg per lane) was separated in formaldehyde-agarose gels, and RNA was visualized with ethidium bromide under UV light to ensure equal loading of RNA in each lane. Probes were prepared using a DIG RNA labelling Kit (Roche), and the detection after blotting was performed by chemiluminescence with CDP-Star (Roche) and blots were exposed to Hyper film ECL (Amersham Pharmacia Biotech).
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Results |
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Changes in AA and sugar contents after excising roots or leaves from intact plants
To investigate the effect of excising leaves or roots from intact plants on plant senescence after harvest, broccoli plants were left intact, without roots, without leaves, or without either roots or leaves for 48 h at 20 °C, and then the AA and sugar contents were determined in the florets. After 48 h, the total AA contents in intact and root-removed florets were kept at almost the same level as those of 0 h. By contrast, the AA contents in the leaf-free samples declined substantially at 48 h (Fig. 1). The DHA content was less than 10% of the total AA in florets of all samples (data not shown). Both reducing sugars and Suc increased after 48 h in intact and root-separated plants (Fig. 2). In the leaf-free samples, the reducing sugar contents stayed at a level similar to those at 0 h and the Suc contents decreased 48 h after the removal of leaves.
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Changes in AA-related gene expression after harvest
The genes related to AA metabolism were previously cloned from broccoli; two cytosolic APX, BO-APX 1 (accession no. AB078599) and BO-APX 2 (accession no. AB078600), APX bound to the thylakoid membrane in chloroplasts, BO-tbAPX (accession no. AB125634), APX in the stroma of chloroplasts, BO-sAPX (accession no. AB125635), chloroplastic MDAR, BO-MDAR 1 (accession no. AB125636), cytosolic MDAR, BO-MDAR 2 (accession no. AB125637), chloroplastic DHAR, BO-DHAR (accession no. AB125638), cytosolic GR, BO-GR (accession no. AB125639), and mitochondrial GLDH, BO-GLDH (accession no. Z97060) (Nishikawa et al., 2003a
, b). Changes in the transcripts for these genes were detected using probes reported in a previous paper (Nishikawa et al., 2003a, b). In florets, the expressions of several of the genes investigated were influenced significantly by excising leaves (Fig. 3). An increase in the BO-GR mRNA level and decreases in BO-sAPX, BO-MDAR 1, BO-DHAR, and BO-GLDH transcripts were observed in leaf-excised plants, as compared with those in leaf-attached plants. The gene expressions of BO-APX 1 and BO-tbAPX were also suppressed slightly by the separation of leaves. The expression of several genes was altered at 48 h even in intact plants: BO-tbAPX, BO-sAPX, BO-MDAR 1, and BO-GLDH gene expression decreased markedly and BO-DHAR gene expression increased in florets when intact plants were incubated for 48 h at 20 °C.
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Effect of Suc application on post-harvest senescence
The contents of reducing sugars and Suc increased remarkably in florets when a 10% (w/v) Suc solution was fed from the cut surface of the stem tissue in harvested broccoli (Fig. 4). In the control samples, reducing sugars gradually decreased and Suc contents stayed at a low level during storage.
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The yellowing of sepals was observed from 24 h in the control samples, while Suc-treated samples remained almost entirely green during storage (Fig. 5). The total AA contents were kept at a relatively higher level in Suc-treated samples even at 72 h storage, compared with the control samples in which a linear decrease in AA was observed (Fig. 5).
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Effect of Suc on AA-related gene expression
Suc feeding altered the expression of the several genes investigated (Fig. 6). Distinct increases in the transcripts of BO-sAPX, BO-MDAR 1, BO-DHAR, and BO-GLDH genes were observed, while BO-GR gene expression was suppressed in the Suc-treated broccoli florets (Fig. 6). The gene expression of BO-APX 1 showed a slight increase by Suc feeding. The results of the expression of other genes investigated yielded no distinct differences between the control and the Suc-treated samples.
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Discussion |
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Several investigations have been carried out on the regulation of the AA pool size in plants. It is well known that AA biosynthesis is strongly controlled by light (Smirnoff, 2000
). Using isotope-labelled AA, it has been demonstrated that AA synthesis is subject to feedback inhibition by its pool size and that its turnover rate is faster and directly proportional to its pool size in pea seedlings (Pallanca and Smirnoff, 2000). Tabata et al. (2002) have reported that AA loading decreased the expression of the genes related to AA biosynthesis. Furthermore, it has been observed that the increase in DHAR gene expression increased the foliar and kernel AA levels and its redox state in tobacco and maize (Chen et al., 2003). A group of cDNA clones encoding AA-related enzymes were previously isolated from broccoli and the changes in the AA level and the expression of the AA-related genes were studied under various conditions (Nishikawa et al., 2003a, b). Previous data on this subject suggest that it is important to understand the changes in each isoenzyme localized in different cellular compartments. However, separate assays of each isoenzyme activity have not been established, except for those of APX isoenzymes (Amako et al., 1994). The studies have focused on gene expression because it might be helpful to anticipate the changes in the activity of each isoenzyme, which were assumed to show opposite patterns in the cytosol and chloroplast under stress conditions. In this paper, it was examined whether changes in the level of sugars, which can be supplied from the plant leaves to growing sites, such as florets, can control the expression of AA-related genes and AA levels in harvested broccoli florets. The results showed that Suc feeding from the cut stem strongly enhanced the expression of GLDH and some genes related to AA metabolism in chloroplasts, but not the accumulation of cytosolic APX, MDAR, and GR transcripts. This is the first report to demonstrate that Suc can regulate the expression of AA-related genes.
Effect of removing leaves on plant senescence
In broccoli florets, the decreases in either Suc or AA contents were caused by removing leaves from intact plants, indicating that these components may be transported from leaves to florets under normal conditions. It has been demonstrated that more than 10% (w/v) Suc exists in the phloem sap (Hayashi and Chino, 1990). AA has also been found in the phloem sap of Arabidopsis although it is unknown how much AA is present in the phloem sap (Franceschi and Tarlyn, 2002). In florets of intact and root-removed plants, increases in sugars were observed at 48 h, suggesting that florets can accumulate a high concentration of sugars that can be utilized to satisfy their energy requirements.
The mRNA level of the genes related to AA metabolism in chloroplasts and the mitochondria changed markedly at 48 h after transfer to a room at 20 °C, even in florets from broccoli plants that had been kept intact. These changes observed in the florets of intact plants are assumed to be the result of environmental factors, such as temperature, water, and light conditions. In intact or root-removed plants, the mRNA abundance of BO-DHAR increased markedly, while decreases in the BO-tbAPX, BO-sAPX, BO-MDAR 1, and BO-GLDH mRNA levels were seen at 48 h in florets, as compared with those at 0 h (Fig. 3). The increase in DHAR could have compensated for the suppression of other enzymes and helped to keep the balance of AA synthesis and breakdown, which may be the reason that the AA contents remained at high levels in these samples. However, removing leaves resulted in a dramatic suppression of all chloroplastic and mitochondrial genes investigated, indicating that there is no way to compensate for the AA levels. As a consequence, the hydrogen peroxide-scavenging system in chloroplasts could largely have been abolished and oxidized AA could be further metabolized without reduction to AA. The suppression of BO-GLDH indicates a decrease in the rate of AA biosynthesis. These results agree with previous observations of harvested broccoli florets (Nishikawa et al., 2003b). The collapse in the balance of expression of those genes caused by removing leaves at harvest seems to have led to AA degradation.
Effect of Suc treatment on plant senescence
A loss of sugars has generally been observed in vegetables and flowers after harvest (King and Morris, 1994; Davies et al., 1996; Ichimura et al., 1999). Supplying Suc exogenously has been found to increase the longevity of broccoli and cut flowers (Ichimura et al., 1999; Coupe et al., 2003). In the present experiments, feeding of 10% (w/v) Suc delayed the yellowing of sepals (Fig. 5), suggesting that Suc concentration is one of the factors controlling chlorophyll breakdown. The broccoli used in this experiment was purchased at a local market and it seems that more than 24 h had passed since harvest. Therefore it is assumed that the rapid decrease in Suc after harvest was nearly complete at 0 h in the control sample, which showed the steady low level of Suc contents throughout the experimental period (Fig. 4).
It is possible that Suc feeding can have an osmotic effect in broccoli. It was thought that suppression of yellowing and AA loss by Suc treatment was due to the alteration of osmotic pressure, since the feeding solution contained a Suc concentration of about 0.3 M. However, feeding 0.3 M glucose or sorbitol in the same manner as Suc failed to suppress the yellowing of sepals and AA degradation in harvested broccoli florets (F Nishikawa et al., unpublished data). Therefore, the effect of Suc feeding on post-harvest senescence is attributable to factors other than osmotic pressure.
The mRNA transcripts of BO-sAPX, BO-MDAR 1, BO-DHAR, and BO-GLDH were up-regulated by Suc application although it is quite hard to distinguish between the reduced rate of degradation of the transcripts in the presence of Suc and the increased rate of transcription. These results indicate that the rate of AA regeneration in chloroplasts and its biosynthesis in the mitochondria were improved. As a result, the decrease in AA may have been suppressed in the Suc-treated samples. It is widely accepted that a major pathway for AA biosynthesis in plants is via mannose-1-P and L-galactose from glucose (Wheeler et al., 1998; Smirnoff et al., 2001). Suc fed into harvested broccoli can be hydrolysed to fructose and glucose, which may serve as a substrate of AA biosynthesis. Millar et al. (2003) have demonstrated the control of AA biosynthesis in plants by respiratory activity in the mitochondria. The rate of AA biosynthesis may be increased because of the increased rate of respiration by Suc feeding. Sugars can replenish NAD(P)H, which is needed by MDAR and GR, suggesting that decreases in sugar contents could result in the inactivation of an AA-regenerating system. On the other hand, sugars have important signalling functions throughout all the stages of a plant's life cycle and they control gene expression (Smeekens, 2000). Harvest-related genes such as asparagine synthetase and ß-galactosidase have been shown to be regulated by the Suc level (Davies et al., 1996). Suc-induced signal transduction, whose mechanism is not fully understood, may lead to the up-regulation observed in AA-related gene expression. Furthermore, it was found that the pattern of the changes in gene expression observed in Suc-treated samples was the opposite of that observed in leaf-excised samples while identical to that observed in leaf-attached samples. These findings support the idea that the changes in AA metabolism after harvest are due to the cessation of Suc supply from leaves.
In leaves, it has been observed that the AA pool size is light-dependent (Smirnoff, 2000). In normal conditions, plants may control the AA level and chloroplastic AA metabolism in response to various light intensities through changes in the Suc concentration, which relies on photosynthetic activity. However, the AA levels still declined somewhat and did not reach the 0 h levels even with the addition of Suc, suggesting that AA levels are influenced by additional factors, such as direct light control of the biosynthetic and/or recycling pathway besides the Suc concentration.
The results summarized above suggest that Suc can be a limiting factor of the gene expression of the enzymes involved in the chloroplastic AA metabolism and mitochondrial AA biosynthesis, which could affect the AA level significantly in photosynthetic organs. It seems that rapid growth of broccoli florets is largely supported by the Suc supply from leaves, which is likely to keep the homeostasis of chloroplast metabolism in the cell. Therefore, the cessation of the Suc supply from leaves could be the main factor of AA degradation in harvested broccoli florets.
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