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l -ascorbic acid metabolism in spinach (Spinacia oleracea L.) during postharvest storage in light and dark
Postharvest Biology and Technology 28 (2003) 47 /57 www.elsevier.com/locate/postharvbio
L-ascorbic
acid metabolism in spinach (Spinacia oleracea L.) during postharvest storage in light and dark
Merry Evelyn A. Toledo, Yoshinori Ueda *, Yoshihiro Imahori, Mitsuko Ayaki 1 Laboratory of Postharvest Physiology and Quality Control, Graduate School of Agriculture and Biological Sciences, Osaka Prefecture University, 1-1 Gakuen-cho, Sakai, Osaka 599-8531, Japan Received 2 April 2002; accepted 4 July 2002
Abstract L-ascorbic acid metabolism in spinach (Spinacia oleracea L.) leaves during postharvest storage in low intensity white light (20 /25 mmol m 2 s 1) and the dark was investigated. The endogenous pool of ascorbic acid (AsA) in both young and mature leaves showed higher values in the light than in the dark. The higher content of AsA in the light could not be explained by the activities of the different redox enzymes involved in AsA metabolism. The enzyme activities did not show marked differences between those stored under light and those in the dark. On the other hand, AsA catabolism as indicated by oxalic acid contents was not indicative of the effects of light and darkness since oxalic acid contents did not quantitatively relate well with AsA contents. Feeding of spinach leaf disks with L-galactono-1,4-lactone showed similar activities of L-galactono-1,4-lactone dehydrogenase during both light and dark incubation; however, production of AsA was higher in the light-incubated than in the dark-incubated leaf disks. Thus it is possible that light could enhance AsA synthesis in spinach leaves. The total soluble carbohydrates and glucose contents of leaves in the light were higher than those in leaves in the dark. These results suggest that continuous illumination of white light effectively supports the photosynthetic capacity of spinach leaves during postharvest storage thereby increasing the availability of soluble carbohydrates, especially glucose, enabling them to contribute to the control of the AsA pool size. # 2002 Elsevier Science B.V. All rights reserved.
Keywords: Spinach; Spinacia oleracea L.; Postharvest storage; Ascorbic acid; Soluble carbohydrates; Ascorbic acid metabolism; Light; Dark
1. Introduction L-ascorbic acid (Vitamin C) is a molecule of dietary importance to humans. It plays an im-
* Corresponding author: Tel./fax: /81-72-254-9418 E-mail address: [email protected] (Y. Ueda). 1 Present address: Marudai Shokuhin Co. Ltd, Japan.
portant role in photosynthesis (Foyer, 1993; Smirnoff, 1996), provides significant biochemical functions as an antioxidant, enzyme co-factor, electron donor and acceptor in electron transport, and a precursor for oxalate and tartrate synthesis, acts as a defense against oxidative stress and plays a possible role in cell wall metabolism and expansion (Smirnoff, 1996).
0925-5214/02/$ - see front matter # 2002 Elsevier Science B.V. All rights reserved. PII: S 0 9 2 5 - 5 2 1 4 ( 0 2 ) 0 0 1 2 1 - 7
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Ascorbic acid (AsA) is synthesized via a sequence of hexose precursors that primarily involve D-glucose (Loewus et al., 1956; Loewus, 1963). However, the metabolic pathway leading to AsA biosynthesis in higher plants has not been conclusively established. Several possible pathways have been proposed. One pathway which is backed by strong biochemical and molecular genetic evidence proceeds through the intermediates GDP-D-mannose and L-galactose, culminating in the final conversion of L-galactono-1,4-lactone (L-Gal) to AsA (Smirnoff et al., 2001) by a mitochondrial enzyme, L-galactono-1,4-lactone dehydrogenase (GLDH) (Oba et al., 1994). Another pathway involves the conversion of glucose to D-glucosone, then to L-sorbosone and finally to AsA (Saito et al., 1990). Moreover, other alternative pathways of AsA biosynthesis via uronic acids have also been proposed (Smirnoff et al., 2001). AsA then enters the ascorbate-glutathione cycle (Smirnoff and Pallanca, 1996) or the Asada-Halliwell pathway (Shimaoka et al., 2000) for AsA regeneration. Finally, AsA catabolism occurs with tartrate and oxalate as major products (Smirnoff and Pallanca, 1996). AsA occurs abundantly in many horticultural crops. A wide range of factors such as genotype, and preharvest and postharvest conditions influence AsA content (Lee and Kader, 2000). Some basic research (Goldthwaite and Laetsch, 1967; Foyer et al., 1983; Smirnoff and Pallanca, 1996) and field experiments (Shinohara and Suzuki, 1981; Gillham and Dodge, 1987) have demonstrated the effect of light on AsA content. During postharvest storage, low intensity light at around 1000 /3000 lux (22 /44 mmol m 2 s 1) has also been reported to positively affect AsA content of komastuna (Brassica campestris L. var. komastuna ) (Hosoda et al., 1981a,b). Hosoda et al. (2000) on the other hand, claimed that a high energy reaction like photosynthesis is responsible for AsA maintenance. However, this regulatory mechanism and perhaps other possible regulatory mechanisms of light during AsA biosynthesis, metabolism and catabolism have not been well demonstrated. Furthermore, the effect of light, especially that of low intensity, on the AsA
contents during postharvest storage of other crops, has not yet been established. Spinach (Spinacia oleracea L.) is a cool season annual herb that belongs to the Goosefoot family along with Swiss chard and beets. It is a popular vegetable that is eaten raw, boiled or baked into various dishes. Spinach is low in calories and is a good source of vitamin C, vitamin A and minerals, especially iron. Spinach has been used extensively in studies involving AsA metabolism. Yang and Loewus (1975) have observed the metabolic conversion of AsA to oxalic acid in spinach seedlings while Hossain and Asada (1984), Hossain et al. (1984) and Miyake and Asada (1992) reported the presence of the different AsA metabolizing enzymes in spinach chloroplasts. The presence of GLDH in the mitochondrial membrane in spinach leaves was observed by Mastuda et al. (1995) suggesting the occurrence of the biosynthetic pathway of AsA via L-Gal. This paper aims to determine the effects of low intensity light (20 /25 mmol m 2 s 1) and dark storage on (a) AsA metabolism in spinach leaves during postharvest storage, (b) AsA biosynthesis via L-Gal and (c) AsA catabolism as indicated by oxalic acid contents.
2. Materials and methods 2.1. Plant material and storage methods Spinach (Spinacia oleracea L. cv. Atlas) plants were either grown in open culture at the University of Osaka Prefecture Farm, Sakai, Osaka, Japan during late autumn or bought from local stores close to the university during spring. Plants planted at the university farm were harvested after 50 days (January) and these were called winterharvested spinach whereas those bought from stores during spring were called spring-harvested spinach. Winter-harvested spinach was used for all experimental assays except for the feeding trials of spinach leaf disks with L-Gal which used springharvested spinach. Harvested spinach plants were washed in running water and the roots were immediately covered
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with water-soaked cotton. The plants were then arranged individually in slanting position in trays without binding stocks and stored under continuous white fluorescent light (20 /25 mmol m 2 s 1; measured at the top of the plant) or in the dark at 8 8C. Trays were covered with polypropylene plastic to minimize desiccation and atomized water was administered every day for light-stored spinach and every other day for darkstored spinach for a period of 24 days. For light storage, trays were wrapped with reflective aluminum foil so as to facilitate exposure of lower stem parts to light. Spinach plants stored in light and dark conditions were removed at scheduled sampling dates (at harvest and at 4, 8, 16 and 24 days storage). During sampling, composite leaf tissues of young fully emerged (but not yet fully expanded) inner leaves and of mature leaves (3 /4 young or mature leaves per plant) were used for all assays. Analytical samples were cut into small pieces ( :/0.5 /1 cm2), pre-weighed, placed inside aluminum foil sachets, frozen in liquid nitrogen and stored at /75 8C prior to analysis. Data were gathered from 3 sets of plants, each containing 2 plants per set. All measurements were repeated at least twice. For the feeding trials of young and mature spinach leaves with L-Gal, disks about 20 mm in diameter were punched from areas between the midrib of the leaf using a metal cylindrical borer. AsA assay was obtained from 30 leaf disks floated on Petri dishes containing about 25 ml of 25 mM L-Gal. GLDH activity was measured from another set of about 100 /150 leaf disks (total of 5 g) floated on transparent plastic trays containing about 500 ml of 25 mM L-Gal. Each plate/plastic tray contained representative disks from every leaf chosen for the activity. Plates were incubated at the same storage conditions as described above for whole spinach plants. AsA contents in leaf disks which had been frozen in liquid nitrogen and stored at /75 8C were measured from samples taken just before incubation (0 h) and every 2 h thereafter. GLDH activity was measured after 0 and at 8 h incubation using fresh leaf disks. Each activity assay was triplicated and all measurements were done at least twice.
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2.2. Ascorbic acid assay Frozen leaf tissues (2.5 g) were ground in an icecold mortar and pestle containing 10 ml of 5% metaphosphoric acid. After the tissues had been well homogenized, 12.5 ml of distilled water was added. The homogenate was then filtered and the clear supernatant was collected for AsA analysis. AsA (also known as total AsA) and dehydroascorbic acid was analyzed following the 2,4-dinitrophenylhydrazine method of Roe et al. (1948) using L-AsA (Wako Pure Chemicals, Ind.) as a standard. Reduced AsA was calculated from the difference between AsA and dehydroascorbic acid. 2.3. Extraction and assay of ascorbic acid metabolizing enzymes Crude enzymes from spinach leaves were extracted following the method of Imahori et al. (2000). Frozen leaf tissues (2.5 g) were homogenized in a cooled mortar and pestle with 12.5 ml of ice-cold 50 mM potassium phosphate buffer (pH 7.0), containing 1 mM ascorbate, 1 mM EDTA and 5% polyvinylpyrrolidone. The homogenate was filtered through two layers of miracloth and the filtrate was centrifuged at 10 000/g for 10 min at 4 8C. Aliquots of the supernatant were assayed for activities of AsA metabolizing enzymes. Ascorbate peroxidase (APX) and ascorbate oxidase (AOX) activities were assayed according to Nakano and Asada (1981) and Nishikawa et al. (2001), respectively. APX activity was determined at 25 8C following the H2O2 /dependent decomposition of ascorbate at 290 nm using a spectrophotometer (Shimadzu Co. Ltd). AOX activity was assayed in the same manner except that H2O2 was not added into the assay mixture. An absorption coefficient of 2.8 mM 1 cm 1 of ascorbate was used for calculating both APX and AOX activities. Dehydroascorbate reductase (DHAR) activity was also assayed at 25 8C by following the increase in absorbance of ascorbate at 265 nm due to glutathione-dependent production of ascorbate (Hossain and Asada, 1984). DHAR activity was calculated using an absorption coefficient of 14 mM 1 cm 1. Finally, monodehydroascorbate re-
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ductase (MDHAR) activity was assayed following the method of Hossain et al. (1984) by monitoring the decrease in absorbance at 340 nm at 30 8C due to reduction of NAD(P)H to NADP during ascorbate regeneration. MDHAR activity was calculated using an absorption coefficient of 6.2 mM 1 cm 1. Data for the activities of the different AsA metabolizing enzymes were obtained from fresh unstored leaves (0 day storage) and from leaves stored for 16 days when the mature leaves started yellowing.
2.4. Protein determination Protein in crude enzyme extracts was determined by the method of Bradford (1976) using bovine serum albumin (Sigma Chemical Co.) as a standard.
2.5. Soluble carbohydrate contents Frozen tissues (5 g) of spinach leaves were placed into a reflux tube containing a boiling solution of 20 ml 99% ethanol, boiled for 15 min and then cooled in running water. The resulting extract was filtered and the residue was homogenized in a Vortex homogenizer, filtered and washed with 80% ethanol until the total volume reached 50 ml. Thirty milliliters of the alcohol extract was then evaporated under vacuum at 50 8C until the ethanol was completely removed. The residue was then suspended in 1 ml distilled water, centrifuged for 25 min at 13 000 /g and the resulting supernatant passed through a Sep Pak C18 cartridge (Waters, Milford, MA) prior to analysis. A 20-ml eluate was injected into an HPLC with a Shodex Asahipak NH2P-50 4E column, a refractive index detector (Shimadzu Co. Ltd) with a flow rate of 0.5 ml min 1 using 75% acetronitrile as eluent and a column temperature of 35 8C. Individual sugars were quantified by comparison with peak areas of individual sugar standards. Total soluble carbohydrate was computed as the sum of sucrose, glucose and fructose.
2.6. Soluble oxalic acid content Frozen leaf tissues (2.5 g) were homogenized in 10 ml of distilled water using a mortar and pestle. The homogenate was left undisturbed for 30 min, filtered through 2 layers of gauze and then centrifuged at 3700/g for 10 min. About 2.5 ml of the supernatant was passed through a 0.45 mm Dismic microfilter, then through a Toyopak IC-SP cartridge (Tosoh Corp., Tokyo). Soluble oxalic acid was analyzed using a Conductivity HPLC (Hitachi Corp.). A 20-ml sample was injected into an HI-30 (Ion-Exchange) column. Flow rate was 2.0 ml min 1 using a combined carbonate-bicarbonate eluent composed of 1.8 mM Na2CO3 and 1.7 mM NaHCO3. Column temperature was 40 8C. Quantification was made using the peak area of an oxalic acid standard (Wako Pure Chemicals Ind.). 2.7. Extraction and assay of GLDH activity in LGal fed leaf disks Enzyme extraction was by the method of Oba et al. (1995) with slight modifications. All procedures were carried out at 0 /4 8C. Five grams of spinach leaf disks were grated gently with 0.1 M potassium phosphate buffer (pH 7.4) containing 0.4 M sucrose and 30 mM mercaptoethanol. The extract was passed through 2 layers of miracloth and then centrifuged at 500/g for 10 min. The resulting supernatant was centrifuged at 12 000 /g for 20 min. The pellet was suspended in 2.5 ml of the above buffer and centrifuged at 12 000 /g for 20 min. Finally, the pellet was resuspended gently using a horsehair brush in 150 ml of 0.1 M potassium phosphate buffer containing 20% (w/v) glycerol and 5 mM glutathione. The assay for GLDH activity was by the method of Siendones et al. (1999) with some modifications. The assay mixture, in a final volume of 1.0 ml, was composed of enzyme solution (40 ml), Cyt c (0.068 mM with 0.1 mM KCN) and L-Gal (4.48 mM) in 10 mM potassium phosphate buffer with 0.03% Triton X-100. The increase in absorbance at 550 nm at 30 8C was followed immediately after the addition of L-Gal. One unit of activity is defined as the amount of
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extract required to oxidize 1 mmol of L-Gal (equivalent to the formation of 2 mmol of reduced Cyt c ) per minute. A molar coefficient of 17.3 mM 1 cm 1 for the difference between oxidized and reduced form of Cyt c was used.
3. Results 3.1. Ascorbic acid During postharvest storage of whole spinach plants, leaves stored in the light showed a slower decline in reduced AsA compared to those stored in the dark (Fig. 1A). Reduced AsA content in young leaves in the light decreased by 32% from the pre-storage level (61.98 mg 100 g1 fresh weight) after 16 days but overcame this decline by 19% after another 8 days of storage. In contrast, reduced AsA content in the dark-held counterpart decreased by 56% from the prestorage level after 24 days and did not show any variation in this pattern of change. For the reduced AsA contents in mature leaves, lightstored leaves had a slower decrease by 44% compared to dark-stored leaves which exhibited
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a rapid decline of 90% from their pre-storage level (63.44 mg 100 g1 fresh weight) after 24 days. In terms of dehydroascorbic acid contents, values only showed clear differences on the 16th day when the onset of yellowing was observed (Fig. 1B). At this stage, dehydroascorbic acid contents in light-stored mature leaves were higher than their dark-stored counterparts. However, in young leaves, dehydroascorbic acid levels did not vary markedly through to the end of the storage period. 3.2. Soluble carbohydrates Soluble carbohydrates in spinach were composed of 45 /60% sucrose, 25 /35% glucose and 15/20% fructose. During storage, the concentration of total soluble carbohydrates decreased more rapidly in dark-stored leaves than in their lightstored counterpart (Fig. 2A). The soluble carbohydrates in young leaves stored in the light decreased to about 37% of the pre-storage level (1.61 g 100 g1 fresh weight) after 16 days but overcame this decline by 35% after another 8 days in storage. The dark-stored leaves however, showed a decline of about 54% from the pre-
Fig. 1. Changes in reduced AsA (A) and dehydroascorbic acid (B) contents in spinach leaves during storage at 8 8C for 24 days. Symbols: young leaves in light ( /I /), young leaves in dark ( /j /), mature leaves in light ( /k /), mature leaves in dark ( /m /). Mean9/S.E. from three replications and are not shown when the values are smaller than the symbol. Arrow denotes onset of yellowing at the upper leafy part of outermost leaves which has been observed in 25% of all stored plants. FW, fresh weight.
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Fig. 2. Changes in total soluble carbohydrates (A) and glucose (B) contents in spinach leaves during storage at 8 8C for 24 days. Symbols: young leaves in light ( /I /), young leaves in dark ( /j /), mature leaves in light ( /k /), mature leaves in dark ( /m /). Mean9/S.E. from three replications and are not shown when the values are smaller than the symbol. Arrow denotes onset of yellowing at the upper leafy part of outermost leaves which has been observed in 25% of all stored plants. FW, fresh weight.
storage level after 24 days of storage. In lightstored mature leaves, the pre-storage level of soluble carbohydrates (1.39 g 100 g1 fresh weight) decreased by 54% while those stored in the dark decreased by 75% after 24 days. Glucose levels during storage followed the same trend as that of total soluble carbohydrates for all leaf samples (Fig. 2B). Glucose levels of young leaves stored in the light gradually decreased by 32% from the pre-storage level (0.55 g 100 g1 fresh weight) after 16 days of storage and then increased by 64% at 24 days of storage. On the other hand, glucose levels in young dark-stored leaves exhibited a continuous decrease to 56% of the pre-storage level at the end of the storage period. In light-stored mature leaves, the decrease in the pre-storage level of glucose (0.35 g 100 g1 fresh weight) was much lower (29%) than in darkstored leaves (75%) after 24 days. 3.3. Soluble oxalic acid Generally, mature leaves contained higher oxalic acid levels than young leaves. Soluble oxalic acid in young leaves showed increasing levels with
storage time (Fig. 3). At the end of the storage period, the soluble oxalic acid content in light-
Fig. 3. Changes in soluble oxalic acid in spinach leaves during storage at 8 8C for 24 days. Symbols: young leaves in light ( / I /), young leaves in dark ( /j /), mature leaves in light ( / k /), mature leaves in dark ( /m /). Mean9/S.E. from three replications and are not shown when the values are smaller than the symbol. Arrow denotes onset of yellowing at the upper leafy part of outermost leaves which has been observed in 25% of all stored plants. FW, fresh weight.
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stored leaves increased by 57% compared with the 40% increase in the dark-stored leaves relative to the pre-storage level (0.68 g 100 g1 fresh weight). In mature leaves, the initial oxalic acid level (1.25 g 100 g1 fresh weight) slightly decreased by 20% on the 8th day of storage. Thereafter, the oxalic acid levels of light-stored leaves sharply increased to about 66% while dark-stored leaves had an increase of 23% only after 24 days. 3.4. Activities of ascorbic acid-metabolizing enzymes The AsA metabolizing enzymes (AOX, DHAR and MDHAR) except for APX, showed decreased activities upon yellowing of leaves (Table 1). However, after 16 days of storage in the light and the dark, the activities of these enzymes did not show any marked differences, both in young and in mature leaves. APX activities in young and mature leaves were either maintained or slightly increased after storage for 16 days. However, the APX activities in spinach leaves stored under light or dark conditions did not show substantial differences after 16 days. 3.5. Effect of feeding spinach leaf disks with L-Gal on ascorbic acid pool size and GLDH activity Feeding of spinach leaf disks with L-Gal resulted in an increased AsA pool size after 8 h of
Fig. 4. Changes in AsA contents in L-Gal fed spinach leaf disks during incubation at 8 8C for 8 h. Symbols: young leaves in light ( /I /), young leaves in dark ( /j /), mature leaves in light ( /k /), mature leaves in dark ( /m /). Mean9/S.E. from three replications. FW, fresh weight.
incubation for all leaf disk samples (Fig. 4). AsA in light-stored leaves increased to about 67 and 49% after incubation in young and in mature leaves, respectively. In contrast, the pool size in dark-stored leaves only increased by 29 and 17%, respectively after the incubation period. In a previous experiment, AsA content in spinach leaf disks which had been floated in distilled water
Table 1 Enzyme activities involving AsA metabolism in spinach leaves Enzymea
Fresh b
Ascorbate oxidased Ascorbate oxidase peroxidased Dehydroascorbate reductased Monodehydroascorbate reductasee a b c d e
c
In light, 16th day
In dark, 16th day
Y
M
Y
M
Y
M
0.209/0.01 0.749/0.08 4.629/0.46 2.869/0.14
0.179/0.02 0.369/0.04 2.629/0.04 2.109/0.14
0.069/0.01 0.849/0.01 2.679/0.43 0.569/0.07
0.03/0.004 0.559/0.02 2.279/0.38 0.649/0.05
0.05/0.005 0.749/0.05 2.179/0.23 0.569/0.06
0.0239/0.004 0.489/0.06 1.729/0.17 0.689/0.02
Data presented as mean9/S.E. from 3 replications. Young leaves. Mature leaves. mmolAsA min 1 mg1protein. mmolNADH min 1mg1protein.
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remained unchanged after 8 h of incubation in light and darkness (data not shown). The GLDH activities of spinach leaf disks in light and dark storage were comparable after 8 h of incubation (Table 2). Moreover, comparable GLDH activities were also observed between young and mature leaf disks.
4. Discussion Continuous illumination of spinach with low intensity white light (20 /25 mmol m 2 s1) has been shown to effectively minimize the decrease of AsA content of attached leaves during storage. By contrast, storage in the dark did not favor AsA maintenance in spinach leaves. White light (66 mmol m 2 s 1) has also been reported to minimize the decrease in AsA levels of attached komastuna leaves after storage for 3 days at various temperatures (Hosoda et al., 1981b). In contrast, storage of boiled broccoli florets in light (30 mmol m 2 s 1) has been reported to exhibit decreased AsA contents (Kotani et al., 1999). Moreover, in an earlier study, Foyer et al. (1983) reported that ascorbate content in the protoplasts and chloroplasts of spinach leaves was maintained at approximately the same concentrations during storage in both light (red light at 1320 mmol m 2 s 1) and dark. The effect of light on AsA was more pronounced in mature leaves than in young leaves before the onset of yellowing. However, after this stage, AsA contents in both light-stored leaves
Table 2 Activity of
GLDH
in L-Gal fed spinach leaf disks Activitya
Fresh In light, 8th h In dark, 8th h a
Young leaves
Mature leaves
16.29/2.3 12.39/2.7 12.69/4.2
13.99/4.9 15.69/4.4 17.29/2.6
Data presented as mean9/S.E. from 3 replication; nmol AsA min 1 (g fresh weight)1.
showed substantial differences from their darkstored counterparts. In fact, after the onset of yellowing in mature leaves, young leaves (which were then still green) in the light showed increased AsA contents while those in the dark continued to show decreased AsA contents. These trends were likewise exhibited in the soluble carbohydrate contents of light- and dark-stored spinach leaves. This may be due to the translocation of metabolites from the roots and stems of the plant to the younger leafy parts which might have possessed a high sink strength. A study on the storage of cut chrysanthemum revealed that even when yellowing and wilting of leaves had occurred, the capitula still maintained its quality for 3 weeks (Adachi et al., 1999). Adachi et al. (1999) suggested that the sugars in the stems of the cut chrysanthemum could have provided carbohydrates for the maintenance of the capitula. The activity of redox enzymes (APX, AOX, DHAR and MDHAR) involved in AsA metabolism did not show marked differences between leaves stored in light and dark even when yellowing had begun i.e. on the 16th day. Thus, the idea that higher AsA levels during light is caused by either decreased activity of an enzyme that causes its oxidation or by an increased ability to reduce monodehydroascorbate or dehydroascorbate back to AsA (Conklin et al., 1997) cannot be supported. Moreover, minimal contents of dehydroascorbic acid compared to reduced AsA contents in both light and dark-stored leaves during storage imply that these redox enzymes have enough activity to maintain AsA in its reduced form. This may then suggest that the AsA pool could either be influenced by a decrease in catabolism or an increase in biosynthesis of AsA in the light. AsA catabolism has been suggested to be regulated in response to AsA pool size (Conklin et al., 1997). However, a close positive relationship between AsA pool size and oxalic acid content was not clearly demonstrated in this study. In terms of AsA, young leaves had higher contents than mature leaves. But mature leaves had higher oxalic acid contents than young leaves. The latter trend was also observed by Suyama et al. (1996) when they investigated the differences in oxalic acid contents of mature and young spinach leaves using
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the leaf disk method. Results further show that the levels of AsA do not seem to quantitatively relate well to the levels of oxalic acid. Even from the beginning of the storage period, oxalic acid contents were already at least 20-fold greater than AsA contents. While the metabolic conversion of AsA to oxalic acid has been established through radiolabeling studies (Yang and Loewus, 1975; Nuss and Loewus, 1978), the biogenesis of oxalic acid also occurs via other pathways (Chang and Beevers, 1968) which would include glycolic and glyoxylic acids as immediate precursors (Millerd et al., 1963). Thus, although the results of this study showed relatively higher contents of oxalic acid in the light-stored leaves than in their dark-stored counterparts, this may not be a reliable indication of the degree of AsA catabolism in either light or dark conditions, since light may also have enhanced oxalic acid biosynthesis via other pathways. Spinach leaf disks fed with 25 mM sodium ascorbate during incubation in the light and darkness for 24 h at 8 8C did not show any variation in oxalic acid contents as compared to their controls (floated in distilled water) (data not shown). Loewus (1988) has stated that only about 20 /25% of the oxalic acid pool comes from AsA, which might then suggest the presence of a possible regulatory mechanism controlling oxalic acid biosynthesis via AsA, hence the results of the feeding trials. To date however, the pathway of AsA catabolism and the enzymes involved have yet to be established and identified (Smirnoff and Pallanca, 1996). Feeding of spinach leaf disks with L-Gal resulted in similar activity of GLDH during both light and dark incubation. It is interesting to note however, that even though GLDH activity did not show apparent differences, higher production of AsA was observed in the light-incubated than in the dark-incubated leaf disks. This suggests a probable role of light in enhancing AsA biosynthesis in spinach leaves. L-Gal oxidation to AsA by intact leaves has been reported by Smirnoff (2000) to be faster in high-light acclimated leaves and also enhanced by high light. Thus, it was suggested that light contributes to the control of AsA pool size in the terminal step of AsA biosynthesis.
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Total soluble carbohydrates exhibited higher levels in the light-stored than in the dark-stored leaves. A concurrent trend in glucose levels was likewise observed. These results suggest that continuous illumination of light at 20 /25 mmol m 2 s 1 supports the photosynthetic capacity of spinach leaves during postharvest storage. Since AsA is synthesized from a hexose precursor (Smirnoff and Pallanca, 1996; Wheeler et al., 1998), the increased availability of these sugars, especially that of glucose, could then contribute to the control of AsA pool size. This relationship has been observed in the AsA and soluble carbohydrate contents of barley leaf segments of different ages which have been grown at low light (80 mmol m 2 s 1) and high light (360 mmol m 2 s 1) intensities (Smirnoff and Pallanca, 1996). The present study demonstrated the ability of low intensity light (20 /25 mmol m 2 s 1) in effectively minimizing the decline in AsA content during postharvest storage of spinach. A possible role of light in enhancing AsA biosynthesis is postulated while a close positive relationship between the soluble carbohydrate and AsA contents in spinach leaves during light and dark storage has been established.
Acknowledgements The authors are grateful to Dr Naoki Yamauchi, Professor of Yamaguchi University, Japan for constructive discussions and to Dr Nelly S. Aggangan, University Researcher of the University of the Philippines */Los Ban˜os, Philippines for critical reading of the manuscript.
References Adachi, M., Kawabata, S., Sakiyama, R., 1999. Changes in carbohydrate content in cut chrysanthemum (Dendranthema /grandiflorum (Ramat.) Kitamura) ‘Shuho-no-chikara’ stems kept at different temperatures during anthesis and senescence. J. Japan Soc. Hort. Sci. 68, 505 /511. Bradford, M.M., 1976. A rapid and sensitive method for the quantification of microgram quantities of protein utilizing
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the principle of protein dye-binding. Anal. Biochem. 72, 248 /254. Chang, C., Beevers, H., 1968. Biogenesis of oxalate in plant tissues. Plant Physiol. 43, 1821 /1828. Conklin, P.L., Pallanca, J.E., Last, R.L., Smirnoff, N., 1997. LAscorbic acid metabolism in the ascorbate-deficient Arabidopsis mutant vtc1 . Plant Physiol. 115, 1277 /1285. Foyer, C.H., 1993. Ascorbic acid. In: Alscher, R.G., Hess, J.L. (Eds.), Antioxidants in Higher Plants. CRC Press, Boca Raton, pp. 31 /58. Foyer, C., Rowell, J., Walker, D., 1983. Measurement of the ascorbate content of spinach leaf protoplasts and chloroplasts during illumination. Planta 157, 239 /244. Gillham, D.J., Dodge, A.D., 1987. Chloroplast superoxide and hydrogen peroxide scavenging systems from pea leaves: seasonal variations. Plant Sci. 50, 105 /109. Goldthwaite, J.J., Laetsch, W.M., 1967. Regulation of senescence in bean leaf discs by light and chemical growth regulators. Plant Physiol. 42, 1757 /1762. Hosoda, H., Nawa, Y., Kurogi, M., 1981. Effect of light on postharvest quality in vegetables. Part 1. Changes in chemical components in detached komastuna leaves during storage. Rept. Natl. Food Res. Inst. 38, 33 /39 (In Japanese with an English abstract). Hosoda, H., Nawa, Y., Kurogi, M., 1981. Effect of light on postharvest quality in vegetables. Part 2. Changes in chemical components in attached komastuna leaves during storage. Rept. Natl. Food Res. Inst. 38, 40 /45 (In Japanese with an English abstract). Hosoda, H., Nawa, Y., Kurogi, M., 2000. Effect of light quality on postharvest changes of chemical components in komastuna (Brassica campestris L. var. komastuna ) leaves. Food Preservation Sci. 26, 15 /18 (In Japanese with an English abstract). Hossain, M.A., Asada, K., 1984. Purification of dehydroascorbate reductase from spinach and its characterization as a thiol enzyme. Plant Cell Physiol. 25, 85 /92. Hossain, M.A., Nakano, Y., Asada, K., 1984. Monodehydroascorbate reductase in spinach chloroplasts and its participation in regeneration of ascorbate for scavenging hydrogen peroxide. Plant Cell Physiol. 25, 385 / 395. Imahori, Y., Kanetsune, Y., Ueda, Y., Chachin, K., 2000. Changes in hydrogen peroxide content and antioxidative enzyme activities during the maturation of sweet pepper (Capsicum annuum L.) fruit. J. Japan Soc. Hort. Sci. 69, 690 /695. Kotani, M., Yamauchi, N., Ueda, Y., Imahori, Y., Chachin, K., 1999. Chlorophyll degradation in boiled broccoli florets during storage in the light. Food Sci. Technol. Res. 5, 35 / 38. Lee, S.K., Kader, A.A., 2000. Preharvest and postharvest factors influencing vitamin C content of horticultural crops. Postharvest Biol. Technol. 20, 207 /220. Loewus, F.A., 1963. Tracer studies on ascorbic acid formation in plants. Phytochemistry 2, 109 /128.
Loewus, F.A., 1988. Ascorbic acid and its metabolic products. In: Priess, J. (Ed.), The Biochemistry of Plants, vol. 14. Academic Press, New York, pp. 85 /107. Loewus, F.A., Jang, R., Seegmiller, C.G., 1956. The conversion of C14-labeled sugars to L-ascorbic acid in ripening strawberries. J. Biol. Chem. 222, 649 /664. Mastuda, M., Ishikawa, T., Takeda, T., Shigeoka, S., 1995. Subcellular localization and properties of L-galactono-g lactone dehydrogenase in spinach leaves. Biosci. Biotech. Biochem. 59, 1983 /1984. Millerd, A., Morton, R.K., Wells, J.R.E., 1963. Oxalic acid synthesis in shoots of Oxalis pes-caprae (L.). Biochem. J. 86, 57 /62. Miyake, C., Asada, K., 1992. Thylakoid bound ascorbate peroxidase in spinach chloroplasts and photoreduction of its primary oxidation product monodehydroascorbate radicals in thylakoids. Plant Cell Physiol. 33, 541 /553. Nakano, Y., Asada, K., 1981. Hydrogen peroxide is scavenged by ascorbate-specific peroxidase in spinach chloroplasts. Plant Cell Physiol. 22, 867 /880. Nishikawa, F., Kato, M., Kamo, T., Wang, R., Hyodo, H., Ikoma, Y., et al., 2001. Enzymic catabolism of ascorbate in florets of harvested broccoli during senescence. J. Japan Soc. Hort. Sci. 70, 709 /715. Nuss, R.F., Loewus, F.A., 1978. Further studies on oxalic acid biosynthesis in oxalate-accumulating plants. Plant Physiol. 61, 590 /592. Oba, K., Fukui, M., Imai, Y., Iriyama, S., Nogami, K., 1994. Lgalactono-1,4-lactone dehydrogenase: partial characterization, induction of activity and role in the synthesis of ascorbic acid in wounded white potato tuber tissue. Plant Cell Physiol. 35, 473 /478. Oba, K., Ishikawa, S., Nishikawa, M., Mizuno, H., Yamamoto, T., 1995. Purification and properties of L-galactono-g -lactone dehydrogenase, a key enzyme for ascorbic acid biosynthesis from sweet potato roots. J. Biochem. 117, 120 /124. Roe, J.H., Mills, M.B., Oesterling, M.J., Damron, C.M., 1948. The determination of diketo-L-gulonic acid, dehydro-Lascorbic acid, and L-ascorbic acid in the same tissue extract by the 2,4-dinitrophyenylhydrazine method. J. Biol. Chem. 174, 201 /208. Saito, K., Nick, J.A., Loewus, F.A., 1990. D-glucosone and Lsorbosone, putative intermediates of L-ascorbic acid biosynthesis in detached bean and spinach leaves. Plant Physiol. 94, 1496 /1500. Shimaoka, T., Yokota, A., Miyake, C., 2000. Purification and characterization of chloroplast dehydroascorbate reductase from spinach leaves. Plant Cell Physiol. 41, 1110 /1118. Shinohara, Y., Suzuki, Y., 1981. Effects of light and nutritional conditions on the ascorbic acid content of lettuce. J. Japan Soc. Hort. 50, 239 /246. Siendones, E., Gonzalez-Reyes, J.A., Santos-Ocan˜a, C., Navas, P., Cordoba, F., 1999. Biosynthesis of ascorbic acid in kidney bean. L-galactono-g -lactone dehydrogenase is an intrinsic protein located at the mitochondrial inner membrane. Plant Physiol. 120, 907 /912.
M.E.A. Toledo et al. / Postharvest Biology and Technology 28 (2003) 47 /57 Smirnoff, N., 1996. The function and metabolism of ascorbic acid in plants. Ann. Bot. 78, 661 /669. Smirnoff, N., 2000. Ascorbate biosynthesis and function in photoprotection. Phil. Trans. R. Soc. Biol. Sci. 355, 1455 / 1464. Smirnoff, N., Pallanca, J.E., 1996. Ascorbate metabolism in relation to oxidative stress. Biochem. Soc. Trans. 24, 472 / 478. Smirnoff, N., Conklin, P.L., Loewus, F.A., 2001. Biosynthesis of ascorbic acid in plants: a renaissance. Ann. Rev. Plant Physiol. Plant Mol. Biol. 52, 441 /467.
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Suyama, N., Tone, S., Sakai, T., 1996. Differences of oxalic acid content among cultivars in spinach leaf blades (Spinacia oleracea L.) by the leaf disk method. Bull. Yamaguchi Agric. Expt. Stn. 47, 41 /46 (In Japanese with an English abstract). Yang, J.C., Loewus, F.A., 1975. Metabolic conversion of Lascorbic acid to oxalic acid in oxalate-accumulating plants. Plant Physiol. 56, 283 /285. Wheeler, G.L., Jones, M.A., Smirnoff, N., 1998. The biosynthetic pathway of vitamin C in higher plants. Nature 393, 365 /369.
L-ascorbic
acid metabolism in spinach (Spinacia oleracea L.) during postharvest storage in light and dark
Merry Evelyn A. Toledo, Yoshinori Ueda *, Yoshihiro Imahori, Mitsuko Ayaki 1 Laboratory of Postharvest Physiology and Quality Control, Graduate School of Agriculture and Biological Sciences, Osaka Prefecture University, 1-1 Gakuen-cho, Sakai, Osaka 599-8531, Japan Received 2 April 2002; accepted 4 July 2002
Abstract L-ascorbic acid metabolism in spinach (Spinacia oleracea L.) leaves during postharvest storage in low intensity white light (20 /25 mmol m 2 s 1) and the dark was investigated. The endogenous pool of ascorbic acid (AsA) in both young and mature leaves showed higher values in the light than in the dark. The higher content of AsA in the light could not be explained by the activities of the different redox enzymes involved in AsA metabolism. The enzyme activities did not show marked differences between those stored under light and those in the dark. On the other hand, AsA catabolism as indicated by oxalic acid contents was not indicative of the effects of light and darkness since oxalic acid contents did not quantitatively relate well with AsA contents. Feeding of spinach leaf disks with L-galactono-1,4-lactone showed similar activities of L-galactono-1,4-lactone dehydrogenase during both light and dark incubation; however, production of AsA was higher in the light-incubated than in the dark-incubated leaf disks. Thus it is possible that light could enhance AsA synthesis in spinach leaves. The total soluble carbohydrates and glucose contents of leaves in the light were higher than those in leaves in the dark. These results suggest that continuous illumination of white light effectively supports the photosynthetic capacity of spinach leaves during postharvest storage thereby increasing the availability of soluble carbohydrates, especially glucose, enabling them to contribute to the control of the AsA pool size. # 2002 Elsevier Science B.V. All rights reserved.
Keywords: Spinach; Spinacia oleracea L.; Postharvest storage; Ascorbic acid; Soluble carbohydrates; Ascorbic acid metabolism; Light; Dark
1. Introduction L-ascorbic acid (Vitamin C) is a molecule of dietary importance to humans. It plays an im-
* Corresponding author: Tel./fax: /81-72-254-9418 E-mail address: [email protected] (Y. Ueda). 1 Present address: Marudai Shokuhin Co. Ltd, Japan.
portant role in photosynthesis (Foyer, 1993; Smirnoff, 1996), provides significant biochemical functions as an antioxidant, enzyme co-factor, electron donor and acceptor in electron transport, and a precursor for oxalate and tartrate synthesis, acts as a defense against oxidative stress and plays a possible role in cell wall metabolism and expansion (Smirnoff, 1996).
0925-5214/02/$ - see front matter # 2002 Elsevier Science B.V. All rights reserved. PII: S 0 9 2 5 - 5 2 1 4 ( 0 2 ) 0 0 1 2 1 - 7
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Ascorbic acid (AsA) is synthesized via a sequence of hexose precursors that primarily involve D-glucose (Loewus et al., 1956; Loewus, 1963). However, the metabolic pathway leading to AsA biosynthesis in higher plants has not been conclusively established. Several possible pathways have been proposed. One pathway which is backed by strong biochemical and molecular genetic evidence proceeds through the intermediates GDP-D-mannose and L-galactose, culminating in the final conversion of L-galactono-1,4-lactone (L-Gal) to AsA (Smirnoff et al., 2001) by a mitochondrial enzyme, L-galactono-1,4-lactone dehydrogenase (GLDH) (Oba et al., 1994). Another pathway involves the conversion of glucose to D-glucosone, then to L-sorbosone and finally to AsA (Saito et al., 1990). Moreover, other alternative pathways of AsA biosynthesis via uronic acids have also been proposed (Smirnoff et al., 2001). AsA then enters the ascorbate-glutathione cycle (Smirnoff and Pallanca, 1996) or the Asada-Halliwell pathway (Shimaoka et al., 2000) for AsA regeneration. Finally, AsA catabolism occurs with tartrate and oxalate as major products (Smirnoff and Pallanca, 1996). AsA occurs abundantly in many horticultural crops. A wide range of factors such as genotype, and preharvest and postharvest conditions influence AsA content (Lee and Kader, 2000). Some basic research (Goldthwaite and Laetsch, 1967; Foyer et al., 1983; Smirnoff and Pallanca, 1996) and field experiments (Shinohara and Suzuki, 1981; Gillham and Dodge, 1987) have demonstrated the effect of light on AsA content. During postharvest storage, low intensity light at around 1000 /3000 lux (22 /44 mmol m 2 s 1) has also been reported to positively affect AsA content of komastuna (Brassica campestris L. var. komastuna ) (Hosoda et al., 1981a,b). Hosoda et al. (2000) on the other hand, claimed that a high energy reaction like photosynthesis is responsible for AsA maintenance. However, this regulatory mechanism and perhaps other possible regulatory mechanisms of light during AsA biosynthesis, metabolism and catabolism have not been well demonstrated. Furthermore, the effect of light, especially that of low intensity, on the AsA
contents during postharvest storage of other crops, has not yet been established. Spinach (Spinacia oleracea L.) is a cool season annual herb that belongs to the Goosefoot family along with Swiss chard and beets. It is a popular vegetable that is eaten raw, boiled or baked into various dishes. Spinach is low in calories and is a good source of vitamin C, vitamin A and minerals, especially iron. Spinach has been used extensively in studies involving AsA metabolism. Yang and Loewus (1975) have observed the metabolic conversion of AsA to oxalic acid in spinach seedlings while Hossain and Asada (1984), Hossain et al. (1984) and Miyake and Asada (1992) reported the presence of the different AsA metabolizing enzymes in spinach chloroplasts. The presence of GLDH in the mitochondrial membrane in spinach leaves was observed by Mastuda et al. (1995) suggesting the occurrence of the biosynthetic pathway of AsA via L-Gal. This paper aims to determine the effects of low intensity light (20 /25 mmol m 2 s 1) and dark storage on (a) AsA metabolism in spinach leaves during postharvest storage, (b) AsA biosynthesis via L-Gal and (c) AsA catabolism as indicated by oxalic acid contents.
2. Materials and methods 2.1. Plant material and storage methods Spinach (Spinacia oleracea L. cv. Atlas) plants were either grown in open culture at the University of Osaka Prefecture Farm, Sakai, Osaka, Japan during late autumn or bought from local stores close to the university during spring. Plants planted at the university farm were harvested after 50 days (January) and these were called winterharvested spinach whereas those bought from stores during spring were called spring-harvested spinach. Winter-harvested spinach was used for all experimental assays except for the feeding trials of spinach leaf disks with L-Gal which used springharvested spinach. Harvested spinach plants were washed in running water and the roots were immediately covered
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with water-soaked cotton. The plants were then arranged individually in slanting position in trays without binding stocks and stored under continuous white fluorescent light (20 /25 mmol m 2 s 1; measured at the top of the plant) or in the dark at 8 8C. Trays were covered with polypropylene plastic to minimize desiccation and atomized water was administered every day for light-stored spinach and every other day for darkstored spinach for a period of 24 days. For light storage, trays were wrapped with reflective aluminum foil so as to facilitate exposure of lower stem parts to light. Spinach plants stored in light and dark conditions were removed at scheduled sampling dates (at harvest and at 4, 8, 16 and 24 days storage). During sampling, composite leaf tissues of young fully emerged (but not yet fully expanded) inner leaves and of mature leaves (3 /4 young or mature leaves per plant) were used for all assays. Analytical samples were cut into small pieces ( :/0.5 /1 cm2), pre-weighed, placed inside aluminum foil sachets, frozen in liquid nitrogen and stored at /75 8C prior to analysis. Data were gathered from 3 sets of plants, each containing 2 plants per set. All measurements were repeated at least twice. For the feeding trials of young and mature spinach leaves with L-Gal, disks about 20 mm in diameter were punched from areas between the midrib of the leaf using a metal cylindrical borer. AsA assay was obtained from 30 leaf disks floated on Petri dishes containing about 25 ml of 25 mM L-Gal. GLDH activity was measured from another set of about 100 /150 leaf disks (total of 5 g) floated on transparent plastic trays containing about 500 ml of 25 mM L-Gal. Each plate/plastic tray contained representative disks from every leaf chosen for the activity. Plates were incubated at the same storage conditions as described above for whole spinach plants. AsA contents in leaf disks which had been frozen in liquid nitrogen and stored at /75 8C were measured from samples taken just before incubation (0 h) and every 2 h thereafter. GLDH activity was measured after 0 and at 8 h incubation using fresh leaf disks. Each activity assay was triplicated and all measurements were done at least twice.
49
2.2. Ascorbic acid assay Frozen leaf tissues (2.5 g) were ground in an icecold mortar and pestle containing 10 ml of 5% metaphosphoric acid. After the tissues had been well homogenized, 12.5 ml of distilled water was added. The homogenate was then filtered and the clear supernatant was collected for AsA analysis. AsA (also known as total AsA) and dehydroascorbic acid was analyzed following the 2,4-dinitrophenylhydrazine method of Roe et al. (1948) using L-AsA (Wako Pure Chemicals, Ind.) as a standard. Reduced AsA was calculated from the difference between AsA and dehydroascorbic acid. 2.3. Extraction and assay of ascorbic acid metabolizing enzymes Crude enzymes from spinach leaves were extracted following the method of Imahori et al. (2000). Frozen leaf tissues (2.5 g) were homogenized in a cooled mortar and pestle with 12.5 ml of ice-cold 50 mM potassium phosphate buffer (pH 7.0), containing 1 mM ascorbate, 1 mM EDTA and 5% polyvinylpyrrolidone. The homogenate was filtered through two layers of miracloth and the filtrate was centrifuged at 10 000/g for 10 min at 4 8C. Aliquots of the supernatant were assayed for activities of AsA metabolizing enzymes. Ascorbate peroxidase (APX) and ascorbate oxidase (AOX) activities were assayed according to Nakano and Asada (1981) and Nishikawa et al. (2001), respectively. APX activity was determined at 25 8C following the H2O2 /dependent decomposition of ascorbate at 290 nm using a spectrophotometer (Shimadzu Co. Ltd). AOX activity was assayed in the same manner except that H2O2 was not added into the assay mixture. An absorption coefficient of 2.8 mM 1 cm 1 of ascorbate was used for calculating both APX and AOX activities. Dehydroascorbate reductase (DHAR) activity was also assayed at 25 8C by following the increase in absorbance of ascorbate at 265 nm due to glutathione-dependent production of ascorbate (Hossain and Asada, 1984). DHAR activity was calculated using an absorption coefficient of 14 mM 1 cm 1. Finally, monodehydroascorbate re-
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ductase (MDHAR) activity was assayed following the method of Hossain et al. (1984) by monitoring the decrease in absorbance at 340 nm at 30 8C due to reduction of NAD(P)H to NADP during ascorbate regeneration. MDHAR activity was calculated using an absorption coefficient of 6.2 mM 1 cm 1. Data for the activities of the different AsA metabolizing enzymes were obtained from fresh unstored leaves (0 day storage) and from leaves stored for 16 days when the mature leaves started yellowing.
2.4. Protein determination Protein in crude enzyme extracts was determined by the method of Bradford (1976) using bovine serum albumin (Sigma Chemical Co.) as a standard.
2.5. Soluble carbohydrate contents Frozen tissues (5 g) of spinach leaves were placed into a reflux tube containing a boiling solution of 20 ml 99% ethanol, boiled for 15 min and then cooled in running water. The resulting extract was filtered and the residue was homogenized in a Vortex homogenizer, filtered and washed with 80% ethanol until the total volume reached 50 ml. Thirty milliliters of the alcohol extract was then evaporated under vacuum at 50 8C until the ethanol was completely removed. The residue was then suspended in 1 ml distilled water, centrifuged for 25 min at 13 000 /g and the resulting supernatant passed through a Sep Pak C18 cartridge (Waters, Milford, MA) prior to analysis. A 20-ml eluate was injected into an HPLC with a Shodex Asahipak NH2P-50 4E column, a refractive index detector (Shimadzu Co. Ltd) with a flow rate of 0.5 ml min 1 using 75% acetronitrile as eluent and a column temperature of 35 8C. Individual sugars were quantified by comparison with peak areas of individual sugar standards. Total soluble carbohydrate was computed as the sum of sucrose, glucose and fructose.
2.6. Soluble oxalic acid content Frozen leaf tissues (2.5 g) were homogenized in 10 ml of distilled water using a mortar and pestle. The homogenate was left undisturbed for 30 min, filtered through 2 layers of gauze and then centrifuged at 3700/g for 10 min. About 2.5 ml of the supernatant was passed through a 0.45 mm Dismic microfilter, then through a Toyopak IC-SP cartridge (Tosoh Corp., Tokyo). Soluble oxalic acid was analyzed using a Conductivity HPLC (Hitachi Corp.). A 20-ml sample was injected into an HI-30 (Ion-Exchange) column. Flow rate was 2.0 ml min 1 using a combined carbonate-bicarbonate eluent composed of 1.8 mM Na2CO3 and 1.7 mM NaHCO3. Column temperature was 40 8C. Quantification was made using the peak area of an oxalic acid standard (Wako Pure Chemicals Ind.). 2.7. Extraction and assay of GLDH activity in LGal fed leaf disks Enzyme extraction was by the method of Oba et al. (1995) with slight modifications. All procedures were carried out at 0 /4 8C. Five grams of spinach leaf disks were grated gently with 0.1 M potassium phosphate buffer (pH 7.4) containing 0.4 M sucrose and 30 mM mercaptoethanol. The extract was passed through 2 layers of miracloth and then centrifuged at 500/g for 10 min. The resulting supernatant was centrifuged at 12 000 /g for 20 min. The pellet was suspended in 2.5 ml of the above buffer and centrifuged at 12 000 /g for 20 min. Finally, the pellet was resuspended gently using a horsehair brush in 150 ml of 0.1 M potassium phosphate buffer containing 20% (w/v) glycerol and 5 mM glutathione. The assay for GLDH activity was by the method of Siendones et al. (1999) with some modifications. The assay mixture, in a final volume of 1.0 ml, was composed of enzyme solution (40 ml), Cyt c (0.068 mM with 0.1 mM KCN) and L-Gal (4.48 mM) in 10 mM potassium phosphate buffer with 0.03% Triton X-100. The increase in absorbance at 550 nm at 30 8C was followed immediately after the addition of L-Gal. One unit of activity is defined as the amount of
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extract required to oxidize 1 mmol of L-Gal (equivalent to the formation of 2 mmol of reduced Cyt c ) per minute. A molar coefficient of 17.3 mM 1 cm 1 for the difference between oxidized and reduced form of Cyt c was used.
3. Results 3.1. Ascorbic acid During postharvest storage of whole spinach plants, leaves stored in the light showed a slower decline in reduced AsA compared to those stored in the dark (Fig. 1A). Reduced AsA content in young leaves in the light decreased by 32% from the pre-storage level (61.98 mg 100 g1 fresh weight) after 16 days but overcame this decline by 19% after another 8 days of storage. In contrast, reduced AsA content in the dark-held counterpart decreased by 56% from the prestorage level after 24 days and did not show any variation in this pattern of change. For the reduced AsA contents in mature leaves, lightstored leaves had a slower decrease by 44% compared to dark-stored leaves which exhibited
51
a rapid decline of 90% from their pre-storage level (63.44 mg 100 g1 fresh weight) after 24 days. In terms of dehydroascorbic acid contents, values only showed clear differences on the 16th day when the onset of yellowing was observed (Fig. 1B). At this stage, dehydroascorbic acid contents in light-stored mature leaves were higher than their dark-stored counterparts. However, in young leaves, dehydroascorbic acid levels did not vary markedly through to the end of the storage period. 3.2. Soluble carbohydrates Soluble carbohydrates in spinach were composed of 45 /60% sucrose, 25 /35% glucose and 15/20% fructose. During storage, the concentration of total soluble carbohydrates decreased more rapidly in dark-stored leaves than in their lightstored counterpart (Fig. 2A). The soluble carbohydrates in young leaves stored in the light decreased to about 37% of the pre-storage level (1.61 g 100 g1 fresh weight) after 16 days but overcame this decline by 35% after another 8 days in storage. The dark-stored leaves however, showed a decline of about 54% from the pre-
Fig. 1. Changes in reduced AsA (A) and dehydroascorbic acid (B) contents in spinach leaves during storage at 8 8C for 24 days. Symbols: young leaves in light ( /I /), young leaves in dark ( /j /), mature leaves in light ( /k /), mature leaves in dark ( /m /). Mean9/S.E. from three replications and are not shown when the values are smaller than the symbol. Arrow denotes onset of yellowing at the upper leafy part of outermost leaves which has been observed in 25% of all stored plants. FW, fresh weight.
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Fig. 2. Changes in total soluble carbohydrates (A) and glucose (B) contents in spinach leaves during storage at 8 8C for 24 days. Symbols: young leaves in light ( /I /), young leaves in dark ( /j /), mature leaves in light ( /k /), mature leaves in dark ( /m /). Mean9/S.E. from three replications and are not shown when the values are smaller than the symbol. Arrow denotes onset of yellowing at the upper leafy part of outermost leaves which has been observed in 25% of all stored plants. FW, fresh weight.
storage level after 24 days of storage. In lightstored mature leaves, the pre-storage level of soluble carbohydrates (1.39 g 100 g1 fresh weight) decreased by 54% while those stored in the dark decreased by 75% after 24 days. Glucose levels during storage followed the same trend as that of total soluble carbohydrates for all leaf samples (Fig. 2B). Glucose levels of young leaves stored in the light gradually decreased by 32% from the pre-storage level (0.55 g 100 g1 fresh weight) after 16 days of storage and then increased by 64% at 24 days of storage. On the other hand, glucose levels in young dark-stored leaves exhibited a continuous decrease to 56% of the pre-storage level at the end of the storage period. In light-stored mature leaves, the decrease in the pre-storage level of glucose (0.35 g 100 g1 fresh weight) was much lower (29%) than in darkstored leaves (75%) after 24 days. 3.3. Soluble oxalic acid Generally, mature leaves contained higher oxalic acid levels than young leaves. Soluble oxalic acid in young leaves showed increasing levels with
storage time (Fig. 3). At the end of the storage period, the soluble oxalic acid content in light-
Fig. 3. Changes in soluble oxalic acid in spinach leaves during storage at 8 8C for 24 days. Symbols: young leaves in light ( / I /), young leaves in dark ( /j /), mature leaves in light ( / k /), mature leaves in dark ( /m /). Mean9/S.E. from three replications and are not shown when the values are smaller than the symbol. Arrow denotes onset of yellowing at the upper leafy part of outermost leaves which has been observed in 25% of all stored plants. FW, fresh weight.
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53
stored leaves increased by 57% compared with the 40% increase in the dark-stored leaves relative to the pre-storage level (0.68 g 100 g1 fresh weight). In mature leaves, the initial oxalic acid level (1.25 g 100 g1 fresh weight) slightly decreased by 20% on the 8th day of storage. Thereafter, the oxalic acid levels of light-stored leaves sharply increased to about 66% while dark-stored leaves had an increase of 23% only after 24 days. 3.4. Activities of ascorbic acid-metabolizing enzymes The AsA metabolizing enzymes (AOX, DHAR and MDHAR) except for APX, showed decreased activities upon yellowing of leaves (Table 1). However, after 16 days of storage in the light and the dark, the activities of these enzymes did not show any marked differences, both in young and in mature leaves. APX activities in young and mature leaves were either maintained or slightly increased after storage for 16 days. However, the APX activities in spinach leaves stored under light or dark conditions did not show substantial differences after 16 days. 3.5. Effect of feeding spinach leaf disks with L-Gal on ascorbic acid pool size and GLDH activity Feeding of spinach leaf disks with L-Gal resulted in an increased AsA pool size after 8 h of
Fig. 4. Changes in AsA contents in L-Gal fed spinach leaf disks during incubation at 8 8C for 8 h. Symbols: young leaves in light ( /I /), young leaves in dark ( /j /), mature leaves in light ( /k /), mature leaves in dark ( /m /). Mean9/S.E. from three replications. FW, fresh weight.
incubation for all leaf disk samples (Fig. 4). AsA in light-stored leaves increased to about 67 and 49% after incubation in young and in mature leaves, respectively. In contrast, the pool size in dark-stored leaves only increased by 29 and 17%, respectively after the incubation period. In a previous experiment, AsA content in spinach leaf disks which had been floated in distilled water
Table 1 Enzyme activities involving AsA metabolism in spinach leaves Enzymea
Fresh b
Ascorbate oxidased Ascorbate oxidase peroxidased Dehydroascorbate reductased Monodehydroascorbate reductasee a b c d e
c
In light, 16th day
In dark, 16th day
Y
M
Y
M
Y
M
0.209/0.01 0.749/0.08 4.629/0.46 2.869/0.14
0.179/0.02 0.369/0.04 2.629/0.04 2.109/0.14
0.069/0.01 0.849/0.01 2.679/0.43 0.569/0.07
0.03/0.004 0.559/0.02 2.279/0.38 0.649/0.05
0.05/0.005 0.749/0.05 2.179/0.23 0.569/0.06
0.0239/0.004 0.489/0.06 1.729/0.17 0.689/0.02
Data presented as mean9/S.E. from 3 replications. Young leaves. Mature leaves. mmolAsA min 1 mg1protein. mmolNADH min 1mg1protein.
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54
remained unchanged after 8 h of incubation in light and darkness (data not shown). The GLDH activities of spinach leaf disks in light and dark storage were comparable after 8 h of incubation (Table 2). Moreover, comparable GLDH activities were also observed between young and mature leaf disks.
4. Discussion Continuous illumination of spinach with low intensity white light (20 /25 mmol m 2 s1) has been shown to effectively minimize the decrease of AsA content of attached leaves during storage. By contrast, storage in the dark did not favor AsA maintenance in spinach leaves. White light (66 mmol m 2 s 1) has also been reported to minimize the decrease in AsA levels of attached komastuna leaves after storage for 3 days at various temperatures (Hosoda et al., 1981b). In contrast, storage of boiled broccoli florets in light (30 mmol m 2 s 1) has been reported to exhibit decreased AsA contents (Kotani et al., 1999). Moreover, in an earlier study, Foyer et al. (1983) reported that ascorbate content in the protoplasts and chloroplasts of spinach leaves was maintained at approximately the same concentrations during storage in both light (red light at 1320 mmol m 2 s 1) and dark. The effect of light on AsA was more pronounced in mature leaves than in young leaves before the onset of yellowing. However, after this stage, AsA contents in both light-stored leaves
Table 2 Activity of
GLDH
in L-Gal fed spinach leaf disks Activitya
Fresh In light, 8th h In dark, 8th h a
Young leaves
Mature leaves
16.29/2.3 12.39/2.7 12.69/4.2
13.99/4.9 15.69/4.4 17.29/2.6
Data presented as mean9/S.E. from 3 replication; nmol AsA min 1 (g fresh weight)1.
showed substantial differences from their darkstored counterparts. In fact, after the onset of yellowing in mature leaves, young leaves (which were then still green) in the light showed increased AsA contents while those in the dark continued to show decreased AsA contents. These trends were likewise exhibited in the soluble carbohydrate contents of light- and dark-stored spinach leaves. This may be due to the translocation of metabolites from the roots and stems of the plant to the younger leafy parts which might have possessed a high sink strength. A study on the storage of cut chrysanthemum revealed that even when yellowing and wilting of leaves had occurred, the capitula still maintained its quality for 3 weeks (Adachi et al., 1999). Adachi et al. (1999) suggested that the sugars in the stems of the cut chrysanthemum could have provided carbohydrates for the maintenance of the capitula. The activity of redox enzymes (APX, AOX, DHAR and MDHAR) involved in AsA metabolism did not show marked differences between leaves stored in light and dark even when yellowing had begun i.e. on the 16th day. Thus, the idea that higher AsA levels during light is caused by either decreased activity of an enzyme that causes its oxidation or by an increased ability to reduce monodehydroascorbate or dehydroascorbate back to AsA (Conklin et al., 1997) cannot be supported. Moreover, minimal contents of dehydroascorbic acid compared to reduced AsA contents in both light and dark-stored leaves during storage imply that these redox enzymes have enough activity to maintain AsA in its reduced form. This may then suggest that the AsA pool could either be influenced by a decrease in catabolism or an increase in biosynthesis of AsA in the light. AsA catabolism has been suggested to be regulated in response to AsA pool size (Conklin et al., 1997). However, a close positive relationship between AsA pool size and oxalic acid content was not clearly demonstrated in this study. In terms of AsA, young leaves had higher contents than mature leaves. But mature leaves had higher oxalic acid contents than young leaves. The latter trend was also observed by Suyama et al. (1996) when they investigated the differences in oxalic acid contents of mature and young spinach leaves using
M.E.A. Toledo et al. / Postharvest Biology and Technology 28 (2003) 47 /57
the leaf disk method. Results further show that the levels of AsA do not seem to quantitatively relate well to the levels of oxalic acid. Even from the beginning of the storage period, oxalic acid contents were already at least 20-fold greater than AsA contents. While the metabolic conversion of AsA to oxalic acid has been established through radiolabeling studies (Yang and Loewus, 1975; Nuss and Loewus, 1978), the biogenesis of oxalic acid also occurs via other pathways (Chang and Beevers, 1968) which would include glycolic and glyoxylic acids as immediate precursors (Millerd et al., 1963). Thus, although the results of this study showed relatively higher contents of oxalic acid in the light-stored leaves than in their dark-stored counterparts, this may not be a reliable indication of the degree of AsA catabolism in either light or dark conditions, since light may also have enhanced oxalic acid biosynthesis via other pathways. Spinach leaf disks fed with 25 mM sodium ascorbate during incubation in the light and darkness for 24 h at 8 8C did not show any variation in oxalic acid contents as compared to their controls (floated in distilled water) (data not shown). Loewus (1988) has stated that only about 20 /25% of the oxalic acid pool comes from AsA, which might then suggest the presence of a possible regulatory mechanism controlling oxalic acid biosynthesis via AsA, hence the results of the feeding trials. To date however, the pathway of AsA catabolism and the enzymes involved have yet to be established and identified (Smirnoff and Pallanca, 1996). Feeding of spinach leaf disks with L-Gal resulted in similar activity of GLDH during both light and dark incubation. It is interesting to note however, that even though GLDH activity did not show apparent differences, higher production of AsA was observed in the light-incubated than in the dark-incubated leaf disks. This suggests a probable role of light in enhancing AsA biosynthesis in spinach leaves. L-Gal oxidation to AsA by intact leaves has been reported by Smirnoff (2000) to be faster in high-light acclimated leaves and also enhanced by high light. Thus, it was suggested that light contributes to the control of AsA pool size in the terminal step of AsA biosynthesis.
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Total soluble carbohydrates exhibited higher levels in the light-stored than in the dark-stored leaves. A concurrent trend in glucose levels was likewise observed. These results suggest that continuous illumination of light at 20 /25 mmol m 2 s 1 supports the photosynthetic capacity of spinach leaves during postharvest storage. Since AsA is synthesized from a hexose precursor (Smirnoff and Pallanca, 1996; Wheeler et al., 1998), the increased availability of these sugars, especially that of glucose, could then contribute to the control of AsA pool size. This relationship has been observed in the AsA and soluble carbohydrate contents of barley leaf segments of different ages which have been grown at low light (80 mmol m 2 s 1) and high light (360 mmol m 2 s 1) intensities (Smirnoff and Pallanca, 1996). The present study demonstrated the ability of low intensity light (20 /25 mmol m 2 s 1) in effectively minimizing the decline in AsA content during postharvest storage of spinach. A possible role of light in enhancing AsA biosynthesis is postulated while a close positive relationship between the soluble carbohydrate and AsA contents in spinach leaves during light and dark storage has been established.
Acknowledgements The authors are grateful to Dr Naoki Yamauchi, Professor of Yamaguchi University, Japan for constructive discussions and to Dr Nelly S. Aggangan, University Researcher of the University of the Philippines */Los Ban˜os, Philippines for critical reading of the manuscript.
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