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Changes in organic acids and acid metabolism enzymes in melon fruit during development
Scientia Horticulturae 123 (2010) 360–365
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Changes in organic acids and acid metabolism enzymes in melon fruit during development Mi Tang a, Zhi-long Bie a,*, Ming-zhu Wu b, Hong-ping Yi b, Jong-xin Feng b a b
College of Horticulture and Forestry, Huazhong Agricultural University/Key Laboratory of Horticultural Plant Biology (Ministry of Education), Wuhan 430070, PR China Research Center of Hami Melon, Xinjiang Academy of Agricultural Science, Wulumuqi 830000, PR China
A R T I C L E I N F O
A B S T R A C T
Article history: Received 25 March 2009 Received in revised form 7 July 2009 Accepted 2 November 2009
A novel melon cultivar (‘Flavor No. 3’) containing high acid concentration as well as high sugar content in the mature fruit was successfully bred out. The aim of this study is to determine the main organic acids present in this novel melon and to clarify the mechanisms of acid accumulation. Fruit development and acid accumulation patterns in ‘Flavor No. 3’ were compared with those of a conventional low-acid melon, ‘Xuelihong’. The titratable acidity (TA) and organic acids in fruits were measured at different developmental stages. There is a positive correlation between TA and citric acid content in ‘Flavor No. 3’, indicating that citric acid is the predominant organic acid present throughout the fruit development. The activities of the enzymes involved in citric acid metabolism, including phosphoenolpyruvate carboxylase (PEPC), citrate synthase (CS), aconitase (ACO), isocitrate dehydrogenase (IDH), malate dehydrogenase (MDH), and malic enzyme (ME), were determined. Although no single enzyme was responsible for citric acid accumulation throughout the fruit development, the activities of ACO and ME were positively correlated with citric acid concentration in the two cultivars from 5 to 25 days after pollination (DAP). Moreover, ACO and ME activities in ‘Flavor No. 3’ increased over time and were higher than those in ‘Xuelihong’, resulting in citric acid accumulation in the fruits of ‘Flavor No. 3’. Although a sharp decrease in ACO activity took place after 25 DAP in the fruits of ‘Flavor No. 3’, the earlier accumulation of citric acid was sufficient enough to maintain the high acid concentration until the melon fruits became mature. Crown Copyright ß 2009 Published by Elsevier B.V. All rights reserved.
Keywords: Citric acid Aconitase Malic enzyme Phosphoenolpyruvate carboxylase Citrate synthase Isocitrate dehydrogenase
1. Introduction Melons (Cucumis melo L.) are a very important commercial crop in China and in Mediterranean countries, with the harvested area and yield of melons around the world estimated to be at 1.3 million ha and 26 million ton, respectively (Faostat, 2007). Melon is favored by consumers mainly due to its refreshing, sweet taste and pleasant aroma. The sugar and acid contents in ripe fruits are crucial factors influencing taste and consumer demand. Many studies have been undertaken to ascertain compositional changes in melon fruit, and most of these studies have focused on the accumulation of sugars (Villanueva et al., 2004; Zhang and Li, 2005; Lester, 2008) and volatile compounds (Lamikanra et al., 2003; Bett-Garber et al., 2005). High acid content often reduces fruit quality, but a moderate concentration of acid can make the fruit more palatable. Organic
Abbreviations: ACO, aconitase; CS, citrate synthase; DAP, days after pollination; FW, fresh weight; GC, gas chromatography; IDH, isocitrate dehydrogenase; MDH, malate dehydrogenase; ME, malic enzyme; OAA, oxaloacetate; PEPC, phosphoenolpyruvate carboxylase; TA, titratable acidity; TCA, tricarboxylic acid; TSS, total soluble solids. * Corresponding author. Tel.: +86 27 87286908; fax: +86 27 87282010. E-mail address: [email protected] (Z.-l. Bie).
acids also play a crucial role in food nutrition and processing (Silva et al., 2004; Zampini et al., 2008). There are different organic acids in different kinds of fruits. For example, citric acid is the major organic acid found in citrus (Yamaki, 1989) and melon (Flores et al., 2001), while malic acid is the major organic acid found in apple (Yamaki, 1984) and loquat (Chen et al., 2009). The acid content of a fruit is determined by the balance of acid synthesis and degradation. Organic acids are intermediates in the tricarboxylic acid (TCA) cycle, one of the respiratory pathways that are ubiquitous throughout nature. Although the series of enzymes that participate in the pathway has long been known, their regulation and control are less understood. These enzymes include phosphoenolpyruvate carboxylase (PEPC, EC 4.1.1.31), citrate synthase (CS, EC 4.1.3.7), aconitase (ACO, EC 4.2.1.3), isocitrate dehydrogenase (IDH, EC 4.1.1.41), malate dehydrogenase (MDH, EC 1.1.1.37), and malic enzyme (ME, EC 1.1.1.40). However, little effort has been made to study acid metabolism in melons. This is mainly due to the limitations of current melon resources, most of which have high sugar content. Some studies have been performed on a novel melon cultivar containing both sugar and acid (Burger et al., 2003, 2006). Recently, a new melon cultivar, ‘Flavor No. 3’, the cross of two varieties belonging to C. melo L. var. reticulatus, was bred out at the Hami Melon Research Center in Xinjiang, China. This melon tastes both sour and sweet, in
0304-4238/$ – see front matter . Crown Copyright ß 2009 Published by Elsevier B.V. All rights reserved. doi:10.1016/j.scienta.2009.11.001
M. Tang et al. / Scientia Horticulturae 123 (2010) 360–365
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contrast to conventional cultivars that taste sweet only. This melon cultivar has been planted in some regions in China and is popular with consumers. Thus, this melon is of good commercial potential and worthy of further research. Previous works have shown that the ripe fruit of ‘Flavor No. 3’ contains high total soluble solids (TSS) (Tang et al., 2008). In the present study, ‘Flavor No. 3’ and a conventional melon ‘Xuelihong’ (C. melo L. var. reticulatus) were used to determine the differences in acid accumulation of the fruit. Likewise, the acid concentrations and the activities of enzymes involved in acid biosynthesis (PEPC, CS, ACO, IDH, MDH, and ME) were analyzed throughout fruit development.
liquid nitrogen, and stored at 74 8C. Reported data are means of the three replicates.
2. Materials and methods
TSS values were determined using refractometry beginning at 10 DAP because the fruits were too small and lacking juice to enable TSS measurement at 5 DAP. The pulp was ground with a mortar and pestle to extract the juice, which was titrated with 0.1N NaOH to pH 8.1. The results were expressed as percentage of citric acid (g of citric acid per 100 g FW) to show the value of titratable acidity (TA).
2.1. Plant materials and growth conditions The experiment was conducted from March to June 2006 in a greenhouse at the National Center for Vegetable Improvement at Huazhong Agricultural University, central China (latitude 308270 N, longitude 1148200 E, altitude 22 m). During the experiment, greenhouse conditions were maintained according to standard practices. Melon seeds were sown in plug trays containing peat and vermiculite substrate (2:1, v/v) on 15 March. On 6 April, at the two-true-leaf stage, the seedlings were transplanted into plastic containers (0.75 m 0.25 m 0.25 m) containing peat, vermiculite, and perlite (1:1:1, v/v/v). Three plants were grown in each container, which were arranged in 6 rows of 33 containers each. Three containers at either end comprised of guard rows to eliminate the surrounding effect. Double rows of plants were spaced 0.80 m apart with 0.35 m spacing within the rows to allow for plant density of approximately 6 plants per square meter. Three replicates were used in a randomized complete block design. There were 18 plots per replicate, and each plot consisted of 9 plants of the same cultivar. All plants were vertically trained to a single main vine, and female flowers were hand-pollinated and tagged. Finally, one fruit per plant was allowed to set on a similar node to ensure fruit uniformity. Water and fertilizer were supplied through drip irrigation, and the Shizuoka University nutrient solution formula for melon was used. ‘Flavor No. 3’ and ‘Xuelihong’ fruits ripened 35 and 40 days after pollination (DAP), respectively (Fig. 1). Developing fruits were harvested at 5-day intervals after pollination. The pulp was sliced into sections, quickly frozen in
2.2. Fruit growth measurements Fruits were gently harvested by hand and transported to the laboratory as quickly as possible for fresh weight (FW) determination. Each fruit was cut in half along the polar plane, with the polar and equatorial diameters measured immediately. 2.3. TSS and TA determinations
2.4. Organic acids analysis Organic acids were separated and quantified individually as described by Bartolozzi et al. (1997). Fruit pulp (2 g FW) was homogenized with 10 mL of pre-chilled extracting solution (80% methanol, v/v, 0.1 M imidazole, pH 7) and then heated at 80 8C for 15 min. The samples were centrifuged at 4000 g for 10 min, the supernatant collected, and the pellet re-centrifuged twice after the addition of more extracting solution. All supernatants were collected. A 1 mL internal standard (xylose) was added to each supernatant and then diluted to a volume of 50 mL with extracting solution. Of this stock, 0.3 mL was dried using a vacuum drier and then re-dissolved in a solution containing 2 g hydroxylamine hydrochloride in 100 mL pyridine. Hexamethyldisilazane and trimethylchlorosilane were added to convert the extracts to their derivatives for gas chromatography (GC) analysis. The extract derivatives were separated and detected by GC (Agilent 6890N, USA) with flame ionization detection. A capillary column (HP-5, 5% phenyl methylpolysiloxane, 30 m 0.32 mm i.d. 0.25 mm) was used. The temperatures of the injector and detector were 270 8C and 300 8C, respectively, and the split ratio was 30:1. Nitrogen was used as carrier gas at a flow rate of 1 mL min1. The temperature program was as follows: from an initial temperature of 130 8C, the temperature was increased to 152 8C at 8 8C min1, to 170 8C at 12 8C min1, to 198 8C at 16 8C min1, to 238 8C at 20 8C min1, and to 280 8C at 24 8C min1 and then held for 4 min. The average recovery rate was 98.2%, and the relative standard deviation of repeatability was less than 1.8% of this GC method (data not shown). Organic acid standards and internal standard were GC grade and obtained from Sigma (St. Louis, MO, USA). The organic acids were identified through a comparison of retention times using standard compounds. To obtain a linear curve, five concentrations of organic acid standards and the internal standard were prepared in the extracting solution and tested. The linear curves were used to quantitatively measure the organic acid contents of samples using the Agilent Chemstation software. Three replicates were analyzed for each sample. 2.5. Enzyme extraction and assay
Fig. 1. The novel melon cultivar Flavor No. 3 (A) and the conventional melon cultivar ‘Xuelihong’ (B) when ripe at 35 DAP and 40 DAP, respectively.
Crude enzymes were extracted from the fruit flesh through a modification of the method of Hirai and Ueno (1977). All procedures were conducted at 4 8C. Fruit pulp was extracted with 5 mL of grinding buffer [200 mM Tris–HCl (pH 8.2), 600 mM sucrose, 10 mM isoascorbic acid] and ground with a mortar and pestle. The mixture was centrifuged at 4000 g. The supernatant
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Fig. 2. Changes in fresh weight (A), polar diameter, and equatorial diameter (B) throughout fruit development in the two melon cultivars. (A) *—Xuelihong, *—Flavor No. 3; (B) &—equatorial diameter of Flavor No. 3, &—equatorial diameter of Xuelihong, *—polar diameter of Xuelihong and *—polar diameter of Flavor No. 3. Data shown in this and the following figures are means standard errors of three replications.
was then collected and re-centrifuged. Both supernatant and pellet were used to assay enzyme activity. The supernatant was diluted to 5 mL with an extracting buffer [200 mM Tris–HCl (pH 8.2), 10 mM isoascorbic acid, 0.1% Triton X-100]. Next, 2 mL of the diluted supernatant was centrifuged at 15,000 g for 15 min at 4 8C. The resulting pellet was diluted to 2 mL with an extracting buffer for ACO and IDH assays. The remaining 3 mL of the diluted supernatant was further diluted to 6 mL with the extracting buffer, after which 2 mL of this was used for MDH and ME assays. The remaining 4 mL underwent dialysis with the extracting buffer for 10 h and then used for PEPC and CS assays. ACO activity was determined in a 0.5 mL mixture composed of 40 mM Tris–HCl (pH 7.5), 100 mM NaCl, and 200 mM cis-aconitate. IDH activity was determined in a 0.5 mL mixture composed of 40 mM Hepes (pH 8.2), 800 mM NAD, 200 mM MnSO4, and 2 mM isocitrate. MDH activity was determined in a 0.5 mL mixture composed of 40 mM Tris–HCl (pH 8.2), 2 mM MgCl2, 10 mM KHCO3, 500 mM GSH, 150 mM NADH, and 2 mM oxaloacetate (OAA). ME activity was determined in a 0.5-mL mixture composed of 80 mM Tris–HCl (pH 7.4), 170 mM NADP, 200 mM MnSO4, and 2 mM malate. PEPC activity was determined in a 0.5 mL mixture composed of 40 mM Tris–HCl (pH 8.5), 2 mM MgCl2, 10 mM
KHCO3, 500 mM GSH, 150 mM NADH, and 2 mM PEP. CS activity was assayed in a 0.5 mL mixture composed of 40 mM Tris–HCl (pH 9.0), 40 mM DTNB, 40 mM acetyl-CoA, and 4 mM OAA. All the reactions were started by the addition of the respective enzyme extracts. The changes per minute in absorption were recorded by a spectrophotometer (Shimadzu UV-2401, Japan). Protein levels were determined using a dye-binding assay. Reagents used in enzyme assays were obtained from Sigma. 2.6. Statistical analysis Comparisons between cultivars were performed using the general linear means procedure, and means and standard errors calculated. Statistical analyses were performed using the SAS 8.1 software. 3. Results 3.1. Developmental changes in fruit size Fresh weights increased in both cultivars during development, and ‘Xuelihong’ was much heavier than ‘Flavor No. 3’ (Fig. 2A).
Fig. 3. Changes in total soluble solids (TSS) (A), titratable acidity (TA) (B), citric acid (C), and malic acid (D) contents throughout fruit development in the two melon cultivars. *—Xuelihong and *—Flavor No. 3.
M. Tang et al. / Scientia Horticulturae 123 (2010) 360–365 Table 1 Correlation analysis of citric acid and malic acid concentrations with titratable acidity (TA) during fruit development in two melon cultivars.
Table 2 Correlation analysis of citric acid concentration with activities of enzymes involved in the citric acid cycle in two melon cultivars.
ra
Citric acid–TA Malic acid–TA
363
ra
Xuelihong
Flavor No. 3
0.504 0.744*
0.956** 0.953**
a
Correlation coefficient. * Significance at the 0.05 level. ** Significance at the 0.01 level.
Citric Citric Citric Citric Citric Citric a
‘Xuelihong’ was nearly 2500 g while ‘Flavor No. 3’ was no more than 1500 g when they were ripe. The fruits expanded rapidly in the initial stage of development (from 5 to 15 DAP), as polar and equatorial diameters underwent a substantial increase, and then continued to grow at a slower rate until they ripened (Fig. 2B). The polar and equatorial diameters of ‘Flavor No. 3’ were nearly the same, around 13 cm, but in ‘Xuelihong’ these were 18 cm and 13 cm, respectively. Therefore, the fruit shapes of the two cultivars were round and oval, respectively (Fig. 1). 3.2. Developmental changes in TSS, TA and organic acid concentrations The two cultivars showed a similar pattern of change in TSS, but ‘Flavor No. 3’ increased earlier and reached a higher level than ‘Xuelihong’ (Fig. 3A). Moreover, the TSS in ‘Flavor No. 3’ was close to 18% while in ‘Xuelihong’ it was 14%. However, the patterns of change in TA differed greatly between the two cultivars (Fig. 3B). TA in ‘Flavor No. 3’ increased quickly from 15 to 25 DAP, reaching
b * **
acid-PEPC acid-CS acid-ACO acid-IDH acid-MDH acid-ME
Xuelihong (fruit development)
Flavor No. 3 (fruit development)
0.986* (5–20 DAPb) 0.865** (5–40 DAP) 0.879** (5–40 DAP) 0.956* (5–20 DAP) 0.980* (5–20 DAP) 0.834** (5–40 DAP)
0.346 (5–20 DAP) 0.489 (5–35 DAP) 0.946* (5–25 DAP) 0.235 (5–25 DAP) 0.976* (20–35 DAP) 0.888* (5–25 DAP)
Correlation coefficient. DAP, days after pollination. Significance at the 0.05 level. Significance at the 0.01 level.
its peak at 4.2 mg 100 g1 FW, and then leveled off until maturity, while TA in ‘Xuelihong’ held steady over time at a value of less than 1.0 mg 100 g1 FW. Further measurement of organic acid concentrations in the developing fruits revealed that citric and malic acids were the principal organic acids, and that citric acid was predominant after 20 DAP (Fig. 3C and D). A similar change occurred in both cultivars, with the citric acid concentration increasing and the malic acid concentration decreasing with the development of the melon fruits. Interestingly, the increasing rate of citric acid concentration in ‘Flavor No. 3’ was more sharp, from nearly 0 mg g1 FW at 15 DAP to its peak value of 9.9 mg g1 FW at 30 DAP, whereas in ‘Xuelihong’, the citric acid concentration increased to a peak value 8.3 mg g1 FW at 40 DAP.
Fig. 4. Changes in phosphoenolpyruvate carboxylase (PEPC) (A), citrate synthase (CS) (B), aconitase (ACO) (C), isocitrate dehydrogenase (IDH) (D), malate dehydrogenase (MDH) (E), and malic enzyme (ME) (F) throughout fruit development in the two melon cultivars. *—Xuelihong and *—Flavor No. 3.
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TA was positively correlated with citric acid in ‘Flavor No. 3’ and the correlation coefficient was 0.956 (Table 1), suggesting that the higher citric acid concentration in ‘Flavor No. 3’ was the key contributor to its higher TA. 3.3. Developmental changes in key enzymes involved in citric acid metabolism The two cultivars exhibited a similar pattern of changes in PEPC activity, first increasing and later decreasing (Fig. 4A), but PEPC activity in ‘Flavor No. 3’ was higher than that in ‘Xuelihong’ during most periods, except toward ripening. The activity of CS in ‘Flavor No. 3’ increased and was higher than that in ‘Xuelihong’ from 5 to 25 DAP, then decreased (Fig. 4B). Meanwhile, CS activity in ‘Xuelihong’ increased steadily and peaked at maturity. ACO activity in ‘Xuelihong’ changed little, remaining at a low level throughout development (Fig. 4C). In ‘Flavor No. 3’, ACO activity increased from 5 to 25 DAP, then rapidly decreased. IDH and MDH showed similar patterns (Fig. 4D and E); their activities in ‘Flavor No. 3’ changed little, while in ‘Xuelihong’ they were notably higher at 20 DAP. ME activity in ‘Xuelihong’ increased gradually. Before 25 DAP, ME activity in ‘Flavor No. 3’ was higher than that in ‘Xuelihong’, reaching its peak value at 25 DAP (Fig. 4F) and subsequently decreasing to a lower level than that in ‘Xuelihong’. The activities of PEPC, CS, IDH, and MDH were correlated with the changes in citric acid concentration in ‘Xuelihong’ but not in ‘Flavor No. 3’ (Table 2). The activities of ACO and ME were positively correlated with citric acid concentration in both cultivars. 4. Discussion This work showed that citric acid is abundant in melon fruit pulp, especially at the later stages (Fig. 3C). This reflected the predominance of citric acid as the fruit develops. In ‘Flavor No. 3’, TA and citric acid increased throughout development (Fig. 3B and C) and TA was strongly correlated with citric acid concentration (Table 1). PEPC activity in the two cultivars increased during the early stages and decreased later (Fig. 4A). This was in agreement with results obtained in loquat (Chen et al., 2009). The data revealed a correlation between PEPC activity and citric acid concentration in ‘Xuelihong’ from 5 to 20 DAP, but the correlation was not significant in ‘Flavor No. 3’ (Table 2). Thus, PEPC may participate in controlling citric acid accumulation during fruit development in ‘Xuelihong’. In ‘Flavor No. 3’, PEPC activity increased with fruit development from 5 to 15 DAP, but the citric acid concentration changed little during the same period. This is consistent with the finding of Moing et al. (2000) that PEPC activity does not correlate with levels of acids in peach. The first step in the TCA cycle is that CS directly synthesizes citric acid by catalyzing the condensation of OAA and acetyl Co-A. Citric acid concentration in ‘Xuelihong’ increased linearly with increasing CS activity (Table 2), implying that CS may play a significant role in citric acid biosynthesis. However, citric acid concentration in ‘Flavor No. 3’ did not correlate with CS activity (Table 2). Therefore, the difference in citric acid concentration between the two cultivars may not be attributable to the difference in CS activity. Similar results have been obtained in high- and lowacid citrus (Sadka et al., 2001; Canel et al., 1996) and pineapple (Saradhuldhat and Paull, 2007). Table 2 shows that the citric acid concentration in both cultivars increased linearly with increasing ACO activity. Therefore, the difference in citric acid concentration may result from a difference in ACO activity between the two cultivars. From 5 to 25 DAP, ACO activity increases with fruit development and is much higher in
‘Flavor No. 3’ than in ‘Xuelihong’ (Fig. 4C). As a result, citric acid concentration was higher in the former cultivar (Fig. 3C). Interestingly, citric acid is degraded by ACO in the TCA cycle; thus, citric acid concentration is expected to decrease as ACO activity increases. Our results were in conflict with this expectation. Even after 25 DAP, when ACO activity in ‘Flavor No. 3’ declined sharply, the citric acid concentration did not change but remained as high as that at 25 DAP until it ripened. During the same period, ACO activity in ‘Xulihong’ slightly increased. This is in conflict with earlier findings that metabolic reductions in ACO activity played a role in citric acid accumulation (Bogin and Wallace, 1966; Sadka et al., 2000). Citric acid concentration increased linearly with increasing IDH and MDH activities from 5 to 20 DAP (Table 2) in ‘Xuelihong’, but not in ‘Flavor No. 3’. This indicated that the difference in citric acid concentration between the two cultivars could not be explained by the differences in IDH and MDH activities. Similarly, a study on peach has shown that expression patterns of genes involved in organic acid metabolism (including CS, IDH, and MDH) are not necessarily correlated with changes in organic acid content (Etienne et al., 2002). There was also a statistically significant positive correlation between ME activity and citric acid concentration. Pulp that contained a higher level of ME had a greater citric acid concentration in both cultivars (Table 2). During the early stages of fruit development, the ME activity increase was larger in ‘Flavor No. 3’ than in ‘Xuelihong’ (Fig. 4F). Therefore, it can be concluded that the higher citric acid concentration in the ripe pulp of ‘Flavor No. 3’ may also be related to the fact that it had higher ME activity during the early stages. Chen et al. (2009) found out that ME may play a significant role in decreasing malic acid concentrations during the ripening of loquats, but it has no correlation with citric acid in pineapples (Saradhuldhat and Paull, 2007). In conclusion, no single enzyme was correlated with citric acid accumulation throughout fruit development in ‘Flavor No. 3’. However, the fact that activity levels of ACO and ME were positively correlated with citric acid concentration in the two cultivars from 5 to 25 DAP could partially explain the different acid-accumulation patterns. Interestingly, it is proposed that the evolution of melon fruit taste is a sequential selection, as a low-acid mutant preceded the high sucrose accumulation (Burger et al., 2003); however, TSS in ‘Flavor No. 3’ was also higher (Fig. 3A). Thus, this novel melon cultivar ‘Flavor No. 3’, a combination of two tastes (sour and sweet), may broaden the gene pool of breeding and contribute to fruit quality improvement because the balance between acid and sugar is an important aspect of fruit quality and taste. Acknowledgements This work was supported by the National Key Technology R&D Program of China (2006BAD01A7–6) and the earmarked fund for Modern Agro-industry Technology Research System. References Bartolozzi, F., Bertazza, G., Bassi, D., Cristoferi, G., 1997. Simultaneous determination of soluble sugars and organic acids as their trimethylsilyl derivatives in apricot fruits by gas–liquid chromatography. J. Chromatogr. A 758, 99–107. Bett-Garber, K.L., Lamikanra, O., Lester, G.E., Ingram, D.A., Watson, M.A., 2005. Influence of soil type and storage conditions on sensory qualities of freshcut cantaloupe (Cucumis melo). J. Sci. Food Agric. 85, 825–830. Bogin, E., Wallace, A., 1966. Organic acid synthesis and accumulation in sweet and sour lemon fruit. J. Am. Soc. Hortic. Sci. 89, 182–194. Burger, Y., Sa’ar, U., Distelfeld, A., Katzir, N., Yeselson, Y., Shen, S., Schaffer, A.A., 2003. Development of sweet melon (Cucumis melo) genotypes combining high sucrose and organic acid content. J. Am. Soc. Hortic. Sci. 128, 537–540.
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Scientia Horticulturae journal homepage: www.elsevier.com/locate/scihorti
Changes in organic acids and acid metabolism enzymes in melon fruit during development Mi Tang a, Zhi-long Bie a,*, Ming-zhu Wu b, Hong-ping Yi b, Jong-xin Feng b a b
College of Horticulture and Forestry, Huazhong Agricultural University/Key Laboratory of Horticultural Plant Biology (Ministry of Education), Wuhan 430070, PR China Research Center of Hami Melon, Xinjiang Academy of Agricultural Science, Wulumuqi 830000, PR China
A R T I C L E I N F O
A B S T R A C T
Article history: Received 25 March 2009 Received in revised form 7 July 2009 Accepted 2 November 2009
A novel melon cultivar (‘Flavor No. 3’) containing high acid concentration as well as high sugar content in the mature fruit was successfully bred out. The aim of this study is to determine the main organic acids present in this novel melon and to clarify the mechanisms of acid accumulation. Fruit development and acid accumulation patterns in ‘Flavor No. 3’ were compared with those of a conventional low-acid melon, ‘Xuelihong’. The titratable acidity (TA) and organic acids in fruits were measured at different developmental stages. There is a positive correlation between TA and citric acid content in ‘Flavor No. 3’, indicating that citric acid is the predominant organic acid present throughout the fruit development. The activities of the enzymes involved in citric acid metabolism, including phosphoenolpyruvate carboxylase (PEPC), citrate synthase (CS), aconitase (ACO), isocitrate dehydrogenase (IDH), malate dehydrogenase (MDH), and malic enzyme (ME), were determined. Although no single enzyme was responsible for citric acid accumulation throughout the fruit development, the activities of ACO and ME were positively correlated with citric acid concentration in the two cultivars from 5 to 25 days after pollination (DAP). Moreover, ACO and ME activities in ‘Flavor No. 3’ increased over time and were higher than those in ‘Xuelihong’, resulting in citric acid accumulation in the fruits of ‘Flavor No. 3’. Although a sharp decrease in ACO activity took place after 25 DAP in the fruits of ‘Flavor No. 3’, the earlier accumulation of citric acid was sufficient enough to maintain the high acid concentration until the melon fruits became mature. Crown Copyright ß 2009 Published by Elsevier B.V. All rights reserved.
Keywords: Citric acid Aconitase Malic enzyme Phosphoenolpyruvate carboxylase Citrate synthase Isocitrate dehydrogenase
1. Introduction Melons (Cucumis melo L.) are a very important commercial crop in China and in Mediterranean countries, with the harvested area and yield of melons around the world estimated to be at 1.3 million ha and 26 million ton, respectively (Faostat, 2007). Melon is favored by consumers mainly due to its refreshing, sweet taste and pleasant aroma. The sugar and acid contents in ripe fruits are crucial factors influencing taste and consumer demand. Many studies have been undertaken to ascertain compositional changes in melon fruit, and most of these studies have focused on the accumulation of sugars (Villanueva et al., 2004; Zhang and Li, 2005; Lester, 2008) and volatile compounds (Lamikanra et al., 2003; Bett-Garber et al., 2005). High acid content often reduces fruit quality, but a moderate concentration of acid can make the fruit more palatable. Organic
Abbreviations: ACO, aconitase; CS, citrate synthase; DAP, days after pollination; FW, fresh weight; GC, gas chromatography; IDH, isocitrate dehydrogenase; MDH, malate dehydrogenase; ME, malic enzyme; OAA, oxaloacetate; PEPC, phosphoenolpyruvate carboxylase; TA, titratable acidity; TCA, tricarboxylic acid; TSS, total soluble solids. * Corresponding author. Tel.: +86 27 87286908; fax: +86 27 87282010. E-mail address: [email protected] (Z.-l. Bie).
acids also play a crucial role in food nutrition and processing (Silva et al., 2004; Zampini et al., 2008). There are different organic acids in different kinds of fruits. For example, citric acid is the major organic acid found in citrus (Yamaki, 1989) and melon (Flores et al., 2001), while malic acid is the major organic acid found in apple (Yamaki, 1984) and loquat (Chen et al., 2009). The acid content of a fruit is determined by the balance of acid synthesis and degradation. Organic acids are intermediates in the tricarboxylic acid (TCA) cycle, one of the respiratory pathways that are ubiquitous throughout nature. Although the series of enzymes that participate in the pathway has long been known, their regulation and control are less understood. These enzymes include phosphoenolpyruvate carboxylase (PEPC, EC 4.1.1.31), citrate synthase (CS, EC 4.1.3.7), aconitase (ACO, EC 4.2.1.3), isocitrate dehydrogenase (IDH, EC 4.1.1.41), malate dehydrogenase (MDH, EC 1.1.1.37), and malic enzyme (ME, EC 1.1.1.40). However, little effort has been made to study acid metabolism in melons. This is mainly due to the limitations of current melon resources, most of which have high sugar content. Some studies have been performed on a novel melon cultivar containing both sugar and acid (Burger et al., 2003, 2006). Recently, a new melon cultivar, ‘Flavor No. 3’, the cross of two varieties belonging to C. melo L. var. reticulatus, was bred out at the Hami Melon Research Center in Xinjiang, China. This melon tastes both sour and sweet, in
0304-4238/$ – see front matter . Crown Copyright ß 2009 Published by Elsevier B.V. All rights reserved. doi:10.1016/j.scienta.2009.11.001
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contrast to conventional cultivars that taste sweet only. This melon cultivar has been planted in some regions in China and is popular with consumers. Thus, this melon is of good commercial potential and worthy of further research. Previous works have shown that the ripe fruit of ‘Flavor No. 3’ contains high total soluble solids (TSS) (Tang et al., 2008). In the present study, ‘Flavor No. 3’ and a conventional melon ‘Xuelihong’ (C. melo L. var. reticulatus) were used to determine the differences in acid accumulation of the fruit. Likewise, the acid concentrations and the activities of enzymes involved in acid biosynthesis (PEPC, CS, ACO, IDH, MDH, and ME) were analyzed throughout fruit development.
liquid nitrogen, and stored at 74 8C. Reported data are means of the three replicates.
2. Materials and methods
TSS values were determined using refractometry beginning at 10 DAP because the fruits were too small and lacking juice to enable TSS measurement at 5 DAP. The pulp was ground with a mortar and pestle to extract the juice, which was titrated with 0.1N NaOH to pH 8.1. The results were expressed as percentage of citric acid (g of citric acid per 100 g FW) to show the value of titratable acidity (TA).
2.1. Plant materials and growth conditions The experiment was conducted from March to June 2006 in a greenhouse at the National Center for Vegetable Improvement at Huazhong Agricultural University, central China (latitude 308270 N, longitude 1148200 E, altitude 22 m). During the experiment, greenhouse conditions were maintained according to standard practices. Melon seeds were sown in plug trays containing peat and vermiculite substrate (2:1, v/v) on 15 March. On 6 April, at the two-true-leaf stage, the seedlings were transplanted into plastic containers (0.75 m 0.25 m 0.25 m) containing peat, vermiculite, and perlite (1:1:1, v/v/v). Three plants were grown in each container, which were arranged in 6 rows of 33 containers each. Three containers at either end comprised of guard rows to eliminate the surrounding effect. Double rows of plants were spaced 0.80 m apart with 0.35 m spacing within the rows to allow for plant density of approximately 6 plants per square meter. Three replicates were used in a randomized complete block design. There were 18 plots per replicate, and each plot consisted of 9 plants of the same cultivar. All plants were vertically trained to a single main vine, and female flowers were hand-pollinated and tagged. Finally, one fruit per plant was allowed to set on a similar node to ensure fruit uniformity. Water and fertilizer were supplied through drip irrigation, and the Shizuoka University nutrient solution formula for melon was used. ‘Flavor No. 3’ and ‘Xuelihong’ fruits ripened 35 and 40 days after pollination (DAP), respectively (Fig. 1). Developing fruits were harvested at 5-day intervals after pollination. The pulp was sliced into sections, quickly frozen in
2.2. Fruit growth measurements Fruits were gently harvested by hand and transported to the laboratory as quickly as possible for fresh weight (FW) determination. Each fruit was cut in half along the polar plane, with the polar and equatorial diameters measured immediately. 2.3. TSS and TA determinations
2.4. Organic acids analysis Organic acids were separated and quantified individually as described by Bartolozzi et al. (1997). Fruit pulp (2 g FW) was homogenized with 10 mL of pre-chilled extracting solution (80% methanol, v/v, 0.1 M imidazole, pH 7) and then heated at 80 8C for 15 min. The samples were centrifuged at 4000 g for 10 min, the supernatant collected, and the pellet re-centrifuged twice after the addition of more extracting solution. All supernatants were collected. A 1 mL internal standard (xylose) was added to each supernatant and then diluted to a volume of 50 mL with extracting solution. Of this stock, 0.3 mL was dried using a vacuum drier and then re-dissolved in a solution containing 2 g hydroxylamine hydrochloride in 100 mL pyridine. Hexamethyldisilazane and trimethylchlorosilane were added to convert the extracts to their derivatives for gas chromatography (GC) analysis. The extract derivatives were separated and detected by GC (Agilent 6890N, USA) with flame ionization detection. A capillary column (HP-5, 5% phenyl methylpolysiloxane, 30 m 0.32 mm i.d. 0.25 mm) was used. The temperatures of the injector and detector were 270 8C and 300 8C, respectively, and the split ratio was 30:1. Nitrogen was used as carrier gas at a flow rate of 1 mL min1. The temperature program was as follows: from an initial temperature of 130 8C, the temperature was increased to 152 8C at 8 8C min1, to 170 8C at 12 8C min1, to 198 8C at 16 8C min1, to 238 8C at 20 8C min1, and to 280 8C at 24 8C min1 and then held for 4 min. The average recovery rate was 98.2%, and the relative standard deviation of repeatability was less than 1.8% of this GC method (data not shown). Organic acid standards and internal standard were GC grade and obtained from Sigma (St. Louis, MO, USA). The organic acids were identified through a comparison of retention times using standard compounds. To obtain a linear curve, five concentrations of organic acid standards and the internal standard were prepared in the extracting solution and tested. The linear curves were used to quantitatively measure the organic acid contents of samples using the Agilent Chemstation software. Three replicates were analyzed for each sample. 2.5. Enzyme extraction and assay
Fig. 1. The novel melon cultivar Flavor No. 3 (A) and the conventional melon cultivar ‘Xuelihong’ (B) when ripe at 35 DAP and 40 DAP, respectively.
Crude enzymes were extracted from the fruit flesh through a modification of the method of Hirai and Ueno (1977). All procedures were conducted at 4 8C. Fruit pulp was extracted with 5 mL of grinding buffer [200 mM Tris–HCl (pH 8.2), 600 mM sucrose, 10 mM isoascorbic acid] and ground with a mortar and pestle. The mixture was centrifuged at 4000 g. The supernatant
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Fig. 2. Changes in fresh weight (A), polar diameter, and equatorial diameter (B) throughout fruit development in the two melon cultivars. (A) *—Xuelihong, *—Flavor No. 3; (B) &—equatorial diameter of Flavor No. 3, &—equatorial diameter of Xuelihong, *—polar diameter of Xuelihong and *—polar diameter of Flavor No. 3. Data shown in this and the following figures are means standard errors of three replications.
was then collected and re-centrifuged. Both supernatant and pellet were used to assay enzyme activity. The supernatant was diluted to 5 mL with an extracting buffer [200 mM Tris–HCl (pH 8.2), 10 mM isoascorbic acid, 0.1% Triton X-100]. Next, 2 mL of the diluted supernatant was centrifuged at 15,000 g for 15 min at 4 8C. The resulting pellet was diluted to 2 mL with an extracting buffer for ACO and IDH assays. The remaining 3 mL of the diluted supernatant was further diluted to 6 mL with the extracting buffer, after which 2 mL of this was used for MDH and ME assays. The remaining 4 mL underwent dialysis with the extracting buffer for 10 h and then used for PEPC and CS assays. ACO activity was determined in a 0.5 mL mixture composed of 40 mM Tris–HCl (pH 7.5), 100 mM NaCl, and 200 mM cis-aconitate. IDH activity was determined in a 0.5 mL mixture composed of 40 mM Hepes (pH 8.2), 800 mM NAD, 200 mM MnSO4, and 2 mM isocitrate. MDH activity was determined in a 0.5 mL mixture composed of 40 mM Tris–HCl (pH 8.2), 2 mM MgCl2, 10 mM KHCO3, 500 mM GSH, 150 mM NADH, and 2 mM oxaloacetate (OAA). ME activity was determined in a 0.5-mL mixture composed of 80 mM Tris–HCl (pH 7.4), 170 mM NADP, 200 mM MnSO4, and 2 mM malate. PEPC activity was determined in a 0.5 mL mixture composed of 40 mM Tris–HCl (pH 8.5), 2 mM MgCl2, 10 mM
KHCO3, 500 mM GSH, 150 mM NADH, and 2 mM PEP. CS activity was assayed in a 0.5 mL mixture composed of 40 mM Tris–HCl (pH 9.0), 40 mM DTNB, 40 mM acetyl-CoA, and 4 mM OAA. All the reactions were started by the addition of the respective enzyme extracts. The changes per minute in absorption were recorded by a spectrophotometer (Shimadzu UV-2401, Japan). Protein levels were determined using a dye-binding assay. Reagents used in enzyme assays were obtained from Sigma. 2.6. Statistical analysis Comparisons between cultivars were performed using the general linear means procedure, and means and standard errors calculated. Statistical analyses were performed using the SAS 8.1 software. 3. Results 3.1. Developmental changes in fruit size Fresh weights increased in both cultivars during development, and ‘Xuelihong’ was much heavier than ‘Flavor No. 3’ (Fig. 2A).
Fig. 3. Changes in total soluble solids (TSS) (A), titratable acidity (TA) (B), citric acid (C), and malic acid (D) contents throughout fruit development in the two melon cultivars. *—Xuelihong and *—Flavor No. 3.
M. Tang et al. / Scientia Horticulturae 123 (2010) 360–365 Table 1 Correlation analysis of citric acid and malic acid concentrations with titratable acidity (TA) during fruit development in two melon cultivars.
Table 2 Correlation analysis of citric acid concentration with activities of enzymes involved in the citric acid cycle in two melon cultivars.
ra
Citric acid–TA Malic acid–TA
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ra
Xuelihong
Flavor No. 3
0.504 0.744*
0.956** 0.953**
a
Correlation coefficient. * Significance at the 0.05 level. ** Significance at the 0.01 level.
Citric Citric Citric Citric Citric Citric a
‘Xuelihong’ was nearly 2500 g while ‘Flavor No. 3’ was no more than 1500 g when they were ripe. The fruits expanded rapidly in the initial stage of development (from 5 to 15 DAP), as polar and equatorial diameters underwent a substantial increase, and then continued to grow at a slower rate until they ripened (Fig. 2B). The polar and equatorial diameters of ‘Flavor No. 3’ were nearly the same, around 13 cm, but in ‘Xuelihong’ these were 18 cm and 13 cm, respectively. Therefore, the fruit shapes of the two cultivars were round and oval, respectively (Fig. 1). 3.2. Developmental changes in TSS, TA and organic acid concentrations The two cultivars showed a similar pattern of change in TSS, but ‘Flavor No. 3’ increased earlier and reached a higher level than ‘Xuelihong’ (Fig. 3A). Moreover, the TSS in ‘Flavor No. 3’ was close to 18% while in ‘Xuelihong’ it was 14%. However, the patterns of change in TA differed greatly between the two cultivars (Fig. 3B). TA in ‘Flavor No. 3’ increased quickly from 15 to 25 DAP, reaching
b * **
acid-PEPC acid-CS acid-ACO acid-IDH acid-MDH acid-ME
Xuelihong (fruit development)
Flavor No. 3 (fruit development)
0.986* (5–20 DAPb) 0.865** (5–40 DAP) 0.879** (5–40 DAP) 0.956* (5–20 DAP) 0.980* (5–20 DAP) 0.834** (5–40 DAP)
0.346 (5–20 DAP) 0.489 (5–35 DAP) 0.946* (5–25 DAP) 0.235 (5–25 DAP) 0.976* (20–35 DAP) 0.888* (5–25 DAP)
Correlation coefficient. DAP, days after pollination. Significance at the 0.05 level. Significance at the 0.01 level.
its peak at 4.2 mg 100 g1 FW, and then leveled off until maturity, while TA in ‘Xuelihong’ held steady over time at a value of less than 1.0 mg 100 g1 FW. Further measurement of organic acid concentrations in the developing fruits revealed that citric and malic acids were the principal organic acids, and that citric acid was predominant after 20 DAP (Fig. 3C and D). A similar change occurred in both cultivars, with the citric acid concentration increasing and the malic acid concentration decreasing with the development of the melon fruits. Interestingly, the increasing rate of citric acid concentration in ‘Flavor No. 3’ was more sharp, from nearly 0 mg g1 FW at 15 DAP to its peak value of 9.9 mg g1 FW at 30 DAP, whereas in ‘Xuelihong’, the citric acid concentration increased to a peak value 8.3 mg g1 FW at 40 DAP.
Fig. 4. Changes in phosphoenolpyruvate carboxylase (PEPC) (A), citrate synthase (CS) (B), aconitase (ACO) (C), isocitrate dehydrogenase (IDH) (D), malate dehydrogenase (MDH) (E), and malic enzyme (ME) (F) throughout fruit development in the two melon cultivars. *—Xuelihong and *—Flavor No. 3.
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TA was positively correlated with citric acid in ‘Flavor No. 3’ and the correlation coefficient was 0.956 (Table 1), suggesting that the higher citric acid concentration in ‘Flavor No. 3’ was the key contributor to its higher TA. 3.3. Developmental changes in key enzymes involved in citric acid metabolism The two cultivars exhibited a similar pattern of changes in PEPC activity, first increasing and later decreasing (Fig. 4A), but PEPC activity in ‘Flavor No. 3’ was higher than that in ‘Xuelihong’ during most periods, except toward ripening. The activity of CS in ‘Flavor No. 3’ increased and was higher than that in ‘Xuelihong’ from 5 to 25 DAP, then decreased (Fig. 4B). Meanwhile, CS activity in ‘Xuelihong’ increased steadily and peaked at maturity. ACO activity in ‘Xuelihong’ changed little, remaining at a low level throughout development (Fig. 4C). In ‘Flavor No. 3’, ACO activity increased from 5 to 25 DAP, then rapidly decreased. IDH and MDH showed similar patterns (Fig. 4D and E); their activities in ‘Flavor No. 3’ changed little, while in ‘Xuelihong’ they were notably higher at 20 DAP. ME activity in ‘Xuelihong’ increased gradually. Before 25 DAP, ME activity in ‘Flavor No. 3’ was higher than that in ‘Xuelihong’, reaching its peak value at 25 DAP (Fig. 4F) and subsequently decreasing to a lower level than that in ‘Xuelihong’. The activities of PEPC, CS, IDH, and MDH were correlated with the changes in citric acid concentration in ‘Xuelihong’ but not in ‘Flavor No. 3’ (Table 2). The activities of ACO and ME were positively correlated with citric acid concentration in both cultivars. 4. Discussion This work showed that citric acid is abundant in melon fruit pulp, especially at the later stages (Fig. 3C). This reflected the predominance of citric acid as the fruit develops. In ‘Flavor No. 3’, TA and citric acid increased throughout development (Fig. 3B and C) and TA was strongly correlated with citric acid concentration (Table 1). PEPC activity in the two cultivars increased during the early stages and decreased later (Fig. 4A). This was in agreement with results obtained in loquat (Chen et al., 2009). The data revealed a correlation between PEPC activity and citric acid concentration in ‘Xuelihong’ from 5 to 20 DAP, but the correlation was not significant in ‘Flavor No. 3’ (Table 2). Thus, PEPC may participate in controlling citric acid accumulation during fruit development in ‘Xuelihong’. In ‘Flavor No. 3’, PEPC activity increased with fruit development from 5 to 15 DAP, but the citric acid concentration changed little during the same period. This is consistent with the finding of Moing et al. (2000) that PEPC activity does not correlate with levels of acids in peach. The first step in the TCA cycle is that CS directly synthesizes citric acid by catalyzing the condensation of OAA and acetyl Co-A. Citric acid concentration in ‘Xuelihong’ increased linearly with increasing CS activity (Table 2), implying that CS may play a significant role in citric acid biosynthesis. However, citric acid concentration in ‘Flavor No. 3’ did not correlate with CS activity (Table 2). Therefore, the difference in citric acid concentration between the two cultivars may not be attributable to the difference in CS activity. Similar results have been obtained in high- and lowacid citrus (Sadka et al., 2001; Canel et al., 1996) and pineapple (Saradhuldhat and Paull, 2007). Table 2 shows that the citric acid concentration in both cultivars increased linearly with increasing ACO activity. Therefore, the difference in citric acid concentration may result from a difference in ACO activity between the two cultivars. From 5 to 25 DAP, ACO activity increases with fruit development and is much higher in
‘Flavor No. 3’ than in ‘Xuelihong’ (Fig. 4C). As a result, citric acid concentration was higher in the former cultivar (Fig. 3C). Interestingly, citric acid is degraded by ACO in the TCA cycle; thus, citric acid concentration is expected to decrease as ACO activity increases. Our results were in conflict with this expectation. Even after 25 DAP, when ACO activity in ‘Flavor No. 3’ declined sharply, the citric acid concentration did not change but remained as high as that at 25 DAP until it ripened. During the same period, ACO activity in ‘Xulihong’ slightly increased. This is in conflict with earlier findings that metabolic reductions in ACO activity played a role in citric acid accumulation (Bogin and Wallace, 1966; Sadka et al., 2000). Citric acid concentration increased linearly with increasing IDH and MDH activities from 5 to 20 DAP (Table 2) in ‘Xuelihong’, but not in ‘Flavor No. 3’. This indicated that the difference in citric acid concentration between the two cultivars could not be explained by the differences in IDH and MDH activities. Similarly, a study on peach has shown that expression patterns of genes involved in organic acid metabolism (including CS, IDH, and MDH) are not necessarily correlated with changes in organic acid content (Etienne et al., 2002). There was also a statistically significant positive correlation between ME activity and citric acid concentration. Pulp that contained a higher level of ME had a greater citric acid concentration in both cultivars (Table 2). During the early stages of fruit development, the ME activity increase was larger in ‘Flavor No. 3’ than in ‘Xuelihong’ (Fig. 4F). Therefore, it can be concluded that the higher citric acid concentration in the ripe pulp of ‘Flavor No. 3’ may also be related to the fact that it had higher ME activity during the early stages. Chen et al. (2009) found out that ME may play a significant role in decreasing malic acid concentrations during the ripening of loquats, but it has no correlation with citric acid in pineapples (Saradhuldhat and Paull, 2007). In conclusion, no single enzyme was correlated with citric acid accumulation throughout fruit development in ‘Flavor No. 3’. However, the fact that activity levels of ACO and ME were positively correlated with citric acid concentration in the two cultivars from 5 to 25 DAP could partially explain the different acid-accumulation patterns. Interestingly, it is proposed that the evolution of melon fruit taste is a sequential selection, as a low-acid mutant preceded the high sucrose accumulation (Burger et al., 2003); however, TSS in ‘Flavor No. 3’ was also higher (Fig. 3A). Thus, this novel melon cultivar ‘Flavor No. 3’, a combination of two tastes (sour and sweet), may broaden the gene pool of breeding and contribute to fruit quality improvement because the balance between acid and sugar is an important aspect of fruit quality and taste. Acknowledgements This work was supported by the National Key Technology R&D Program of China (2006BAD01A7–6) and the earmarked fund for Modern Agro-industry Technology Research System. References Bartolozzi, F., Bertazza, G., Bassi, D., Cristoferi, G., 1997. Simultaneous determination of soluble sugars and organic acids as their trimethylsilyl derivatives in apricot fruits by gas–liquid chromatography. J. Chromatogr. A 758, 99–107. Bett-Garber, K.L., Lamikanra, O., Lester, G.E., Ingram, D.A., Watson, M.A., 2005. Influence of soil type and storage conditions on sensory qualities of freshcut cantaloupe (Cucumis melo). J. Sci. Food Agric. 85, 825–830. Bogin, E., Wallace, A., 1966. Organic acid synthesis and accumulation in sweet and sour lemon fruit. J. Am. Soc. Hortic. Sci. 89, 182–194. Burger, Y., Sa’ar, U., Distelfeld, A., Katzir, N., Yeselson, Y., Shen, S., Schaffer, A.A., 2003. Development of sweet melon (Cucumis melo) genotypes combining high sucrose and organic acid content. J. Am. Soc. Hortic. Sci. 128, 537–540.
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