The Horticulture Journal 86 (4): 543–551. 2017. e Japanese Society for doi: 10.2503/hortj.MI-139 JSHS Horticultural Science http://www.jshs.jp/

Metabolite Composition of Grapefruit (Citrus paradisi) Grown in Japan Depends on the Growing Environment and Harvest Period

Yuta Kimura, Mari Naeshiro, Yuri Tominaga, Toyoaki Anai and Fuminori Komai*

Faculty of Agriculture, Saga University, Saga 847-0021, Japan

‘Sagan-Ruby’ is the first grapefruit (Citrus paradisi) cultivar to be developed in Japan and is used for food, cosmetics, and other purposes owing to its favorable characteristics, such as the absence of harmful pesticides and its long shelf life. The desired qualities of grapefruit depend on the specific use, and these qualities are influenced by the metabolite composition of the fruits. However, little is known about the influence of the growing environment or harvest period on the metabolite composition of the ‘Sagan-Ruby’ grapefruit. Therefore, we harvested fruits that were grown either in a plastic house without artificial heating or outdoors with rain cover from December, 2014 to April, 2015, on a monthly basis, and we investigated the composition of the primary metabolites such as sugars, organic acids, and amino acids, in the juice and peel of the fruit using gas chromatography mass spectrometry (GC/MS). We detected a total of 53 and 68 compounds in the juice and peel, respectively, and the first and second components of the principal component analyses of the detected metabolites of both juice and peel were associated with the growing environment and harvest period, respectively. Since we observed that glucose, fructose, sucrose, and citric acid were more concentrated in the juice of outdoor-grown fruits than in that of the house-grown fruits, especially in March and April, it is likely that the sweetness and acidity of the fruits are dependent on the growing environment. Similarly, the primary metabolite contents, including succinic acid and other organic acids, were higher in peels from outdoor-grown fruits. In addition, we also observed that the contents of proline, phenylalanine, and other amino acids in the juice increased continuously from December to April, and many sugars, including glucose and fructose, gradually decreased in peels from December to February and were lower from February to April. These results indicated that quality of the ‘Sagan-Ruby’ grapefruit varies with the harvest period.

Key Words: comprehensive analysis, GC/MS, primary metabolite, processing suitability, ‘Sagan-Ruby’.

chemicals are sprayed on the surface of citrus fruits, Introduction and some of these agrochemicals have been suggested Grapefruit (Citrus paradisi) is one of the most popu- to have detrimental health effects (Hiraga and Fujii, lar citrus fruits in Japan, but since it is typically culti- 1984) and have been shown to remain in citrus fruits vated in subtropical regions and is difficult to grow in even after storage and processing (Tsumura-Hasegawa Japan, most of the grapefruit consumed in Japan is im- et al., 1992). Since the safety of imported foods has re- ported. In fact, 100960 t of grapefruit were imported cently become a growing concern in Japan, the demand into Japan during 2015 (Overview of Foreign Trade of for domestically-grown grapefruit that are not treated Agricultural, Forestry, and Fishery Products of Japan with post-harvest agrochemicals is rising. (2015), http://www.maff.go.jp/j/tokei/kouhyou/kokusai/ The new grapefruit cultivar ‘Sashika-ichigou’ houkoku_gaikyou.html), and the fruit was third among (‘Sagan-Ruby’), which was developed by Saga Univer- the most imported fruits, after bananas and pineapples, sity (Saga, Japan), is the first cultivar to be bred and and first among imported citrus fruits. In addition, 95% variety-registered in Japan (registration no. 22466). The of the grapefruit in Japan was imported from North cultivar has excellent cold-resistance and is capable of America and South Africa. To suppress the deteriora- withstanding the climate of Japan. It is expected that tion in fruit quality during long-term transport, agro- this grapefruit cultivar will be used safely for various purposes without worrying about residual agrochemi- cals. In addition, this cultivar has a long shelf life and Received; December 29, 2015. Accepted; November 30, 2016. First Published Online in J-STAGE on January 25, 2017. can be shipped domestically from April to June, when * Corresponding author (E-mail: [email protected]). the supply of citrus fruits in Japan is usually low. We

© 2017 The Japanese Society for Horticultural Science (JSHS), All rights reserved. 544 Y. Kimura, M. Naeshiro, Y. Tominaga, T. Anai and F. Komai are now developing products using ‘Sagan-Ruby’ including sugars, organic acids, amino acids, and other grapefruit as a raw material by taking advantage of its primary metabolites, which together are directly re- increased food safety and long shelf life. The weight of sponsible for sweetness, acidity, and other characteris- fruits is over 300 g, which is large enough to eat fresh, tics and also indirectly influence bitterness, aroma, as and juices, liqueurs, and sweets that contain ‘Sagan- well as the functionality of secondary metabolites. The Ruby’ juice are already being consumed. Several cos- objective of the study was to evaluate the suitability of metics and fragrances that use the essential oil extracted ‘Sagan-Ruby’ grown in different environments and har- from the peel have also been developed. vested at different stages of ripening as foods and pro- The required quality and specific characteristics of cessing ingredients. grapefruit vary widely depending on the intended end- Materials and Methods use. For example, sweetness, acidity, and bitterness are important when consuming the fruit either fresh or in Plant materials drinks, whereas aroma is more important when using The ‘Sashika-ichigou’ (‘Sagan-Ruby’) grapefruit the fruit for cosmetics and fragrances. In addition, since trees were grafted to Citrus unshiu in 2002 (14 years grapefruit juice is reported to contain bioactive com- ago) and grown either in a plastic house without artifi- pounds that have high antioxidant potential and have a cial heating in northern Saga, Japan, or in an outdoor positive influence on plasma lipid metabolism field with rain cover in southern Saga, Japan. Fruits (Gorinstein et al., 2004), the functional aspects of were picked at approximately month-long intervals grapefruit are also attracting attention. A wide variety from December, 2014 to April, 2015 (December 8, of metabolites, such as sugars, organic acids, and flavo- January 6, February 5, March 5, and April 9). Each noids, influence the quality of grapefruit. Therefore, an month, we collected fruits from three individual trees as analysis of the metabolite composition of ‘Sagan-Ruby’ replicates and collected three fruits per replicate. After would be valuable for product development. During the collection, the fruits were weighed and then separated cultivation, fruits begin ripening on the trees in early into flesh and peel. The flesh was squeezed and filtered winter, but can be kept on the trees until spring in order to obtain juice, and the juice of the three fruits from to extend the shelf life and improve the fruit quality each tree were pooled, while the peels were separated (Suzuki et al., 1997). The changes with time in the into albedo and flavedo, and similar sized peel frag- chemical constituents of citrus fruit during the course of ments of the three fruits from each tree were pooled maturation and ripening have been studied (Bermejo into combined albedo and flavedo samples, which were and Cano, 2012; Iwagaki et al., 1981; Takebayashi then freeze-dried and powdered. et al., 1993), but there is no information about changes with time in the metabolite composition of grapefruit Extraction and derivatization grown in Japan. Furthermore, in Japan, citrus fruits are The metabolite analyses were conducted as described grown both outdoors and in plastic houses, with the aim by Roessner et al. (2000). For the juice, 200 μL filtered of stabilizing and improving fruit quality, and although juice was mixed with 600 μL methanol, 200 μL chloro- the influence of growing environment on fruit quality form, and 10 μL ribitol solution (10 mg·mL−1; used as and metabolite composition has been studied in other the internal standard) by vortexing for 5 min. Subse- citrus species (Izumi, 1999; Kamota, 1987; Morinaga quently, 400 μL distilled water was added, the samples and Sykes, 2001; Sakamoto and Okuchi, 1968; were centrifuged at 10000 × g for 5 min to separate the Sawamura et al., 1983; Takagi et al., 1994; Takebayashi polar and nonpolar phases, and 200 μL of the upper et al., 1992), no studies have compared the metabolite polar phase was transferred from each sample to a new composition of grapefruit grown outdoors to that of plastic tube. For the peels, 20 mg of either albedo or fla- grapefruit grown in plastic houses in Japan until the vedo powder was suspended in a mixture of 480 μL present study. methanol, 160 μL chloroform, 160 μL distilled water, Recently, metabolite profiling has been widely used and 8 μL ribitol solution (10 mg·mL−1), and the slurry to analyze the characteristics and qualities of agricultur- was mixed for 10 min. Subsequently, ~320 μL distilled al products, and gas chromatography-mass spectrome- water was added, the samples were centrifuged, and try (GC/MS) is suitable for the unbiased profiling of 200 μL of the upper polar phase was transferred from primary metabolites in plants (Roessner et al., 2000). In each sample to a new tube. All of the extracts were fact, GC/MS-based metabolite profiling has already dried completely using a centrifugal evaporator, and for been applied to tomatoes (Solanum lycoperstcum), oxime-derivatization, the dried samples were resolved strawberries (Fragaria × ananassa), and peaches with 20 μL methoxyamine hydrochloride (pyridine so- (Prunus persica), in order to analyze the dynamics of lution, 40 mg·mL−1) and incubated at 30°C for 120 min. metabolite composition during fruit development (Fait Finally, 10 μL of each sample was transferred to an in- et al., 2008; Lombardo et al., 2011; Mintz-Oron et al., dividual glass vial containing an insert (0.2 mL, within 2008; Zhang et al., 2011). The present study focused on a 1.5-mL glass vial), mixed with 40 μL N-methyl- the low molecular weight metabolites of grapefruit, trifluoroacetamide (MSTFA, reagent for producing a Hort. J. 86 (4): 543–551. 2017. 545 silylated derivative), and incubated at 37°C for 30 min. constant, with only a few exceptions, indicating that the growth and development of the fruit were already com- GC/MS experiments and data analyses plete by early winter and that later harvesting only in- The derivatized sample (1 μL) was injected into an creases the duration of the fruit’s ripening period. In Agilent GC/MSD 5977A, using the 1:5 split mode. The contrast, the peel color of the fruit depended on the har- type of column, oven temperature, and other GC/MSD vested period, with a lemon yellow in December that parameters were set for metabolite identification using gradually changed to reddish yellow in February, re- the Fiehn GC/MS metabolomics RTL library (Agilent turning to pale yellow in April (Fig. 1). Technologies, Inc., California, USA). The identification and quantitative estimation of each of the metabolites Effects of environment and harvest period on metabolite were conducted using MassHunter software (Agilent composition Technologies, Inc.). Before statistical analysis, the data A total of 53 compounds (15 amino acids, 14 organic were normalized using the peak area of the internal acids, 15 sugars, and 9 unidentified compounds) were standard, ribitol. The peak areas of all compounds de- detected from the grapefruit juices, and a total of 68 tected in the juices and peels were subjected to the prin- compounds (15 amino acids, 17 organic acids, 18 sug- cipal component analysis (PCA) and other multivariate ars, 4 others, and 14 unidentified compounds) were de- analysis. Significantly differences (P-value calculated tected from the grapefruit peels (albedo and flavedo). by ANOVA was lower than 0.05) were detected among ANOVA between outdoor-grown fruits and house- growing environments (outdoor field or plastic house, grown fruits in all harvest months showed that the con- each group consisted of 15 samples) or harvest months tent of 24, 23, and 25 compounds were significantly (December, January, February, March, or April, each influenced by growing environments in the juice, albe- group consisted of 6 samples). These analyses were do, and flavedo, respectively (Table 2). ANOVA among conducted using R ver 3.1 (https://www.r-project.org/) fruits harvested in different month, regardless of grow- and MetaboAnalyst 3.0 (http://www. metaboanalyst.ca/ ing environment, showed that the contents of 25, 25, faces/home.xhtml). The contents of glucose, fructose, and 27 compounds were significantly influenced by the sucrose, citric acid, and malic acid in juice were calcu- harvest period in the juice, albedo, and flavedo, respec- lated by comparing their peak areas with those of com- tively. pounds with known concentrations. In PCA of the juice metabolites, the first principal Results Fruit characteristics December February April The ‘Sagan-Ruby’ trees grown in the outdoor field yielded heavier fruits than those grown in the plastic Outdoor house, except for December (Table 1), and during the harvesting period (December to April) no marked in- field creases in fruit weight were observed in either of the growing environments, except for that of the outdoor- grown fruits from December to January. Meanwhile, the ratio of peel weight to whole fruit weight remained Plastic constant from December to March in the outdoor- house grown fruits and from December to February in the house-grown fruits and subsequently increased until Fig. 1. Changes in the color of ‘Sagan-Ruby’ grapefruit peels dur- April in the fruits from both growing environments. ing the ripening period. Photographs of peels from fruits grown Therefore, the fruit weight and peel ratio were mainly in an outdoor field (upper) or in a plastic house (lower) and har- Table 1. Effect of the growing environment on the temporal changes in ‘Sagan-Ruby’vested grapefruit in December whole fruit(left), weight February and (center),separated and peel April weight. (right).

Table 1. Effect of the growing environment on the temporal changes in ‘Sagan-Ruby’ grapefruit whole fruit weight and separated peel weight.

Outdoor field Plastic house Harvested month Fruit (g) Peel (g) Peel/Fruitz Fruit (g) Peel (g) Peel/Fruitz December 330 ± 23 99 ± 6 30.1 ± 0.5 320 ± 13 105 ± 7 32.7 ± 1.2 January 380 ± 22 119 ± 2 31.4 ± 1.2 301 ± 17 95 ± 6 31.3 ± 0.6 February 375 ± 1 121 ± 2 32.4 ± 0.5 288 ± 18 94 ± 8 32.7 ± 2.1 March 359 ± 12 112 ± 4 31.4 ± 0.2 289 ± 24 100 ± 10 34.6 ± 2.5 April 382 ± 12 138 ± 3 36.2 ± 1.7 330 ± 22 119 ± 8 36.2 ± 1.2 Values are means ± SE (n = 3). z Ratio of peel weight to whole fruit weight. Table546 2. Principal component analysis (PCA)Y. Kimura,factor loading M. Naeshiro, of metabolites Y. Tominaga, with PC1 T. Anaiand PC2 and ofF. ‘Sagan-Ruby’Komai grapefruit juice, albedo, and flavedo samples. Table 2. Principal component analysis (PCA) factor loading of metabolites with PC1 and PC2 of ‘Sagan-Ruby’ grapefruit juice, albedo, and flavedo samples. Juice Albedo Flavedo Metabolites PC1 PC2 Env. Mon. PC1 PC2 Env. Mon. PC1 PC2 Env. Mon. Amino acid Alanine 0.43 −0.56 * 0.79 0.23 0.84 0.11 * Asparagine −0.07 0.19 0.85 0.22 * 0.71 0.11 Aspartic acid 0.30 0.60 * 0.66 0.04 0.76 −0.03 * beta-Alanine 0.62 −0.68 * — — — — gamma-Aminobutyric acid 0.58 −0.69 * 0.75 0.05 0.77 0.00 * Glutamic acid 0.58 −0.39 0.25 −0.26 0.28 −0.39 Glutamine — — 0.80 0.17 0.72 0.08 * Glycine — — 0.74 0.04 0.74 0.39 Isoleucine 0.45 −0.81 * — — — — Ornithine — — — — −0.36 −0.03 * * Phenylalanine 0.70 −0.41 * * 0.33 −0.68 * * 0.46 −0.54 * Proline 0.34 −0.85 * * 0.15 −0.49 * 0.17 −0.45 * Putrescine 0.26 −0.52 −0.12 0.23 * * −0.35 0.34 * Serine 0.44 −0.19 * 0.62 0.39 * 0.69 0.53 * Threonine 0.55 −0.73 * 0.64 0.05 0.72 0.20 Tryptophan 0.36 0.12 0.12 −0.65 * * 0.27 −0.15 Tyrosine 0.65 0.06 0.00 0.00 — — Valine 0.89 −0.21 * * 0.71 0.03 0.72 0.30 Organic acid alpha-Ketoglutaric acid −0.28 0.37 * — — — — Benzoic acid 0.69 0.25 * — — — — cis-Aconitic acid 0.11 0.76 * * 0.32 −0.16 0.51 0.31 Citric acid 0.51 0.68 * * 0.38 −0.14 0.19 −0.26 Dehydroascorbic acid — — −0.27 −0.80 * 0.01 −0.93 * * Fumaric acid — — 0.77 −0.03 * 0.88 −0.06 * Galactonic acid 0.79 0.33 * −0.11 −0.24 * 0.31 −0.67 * * Gluconic acid 0.85 −0.26 * * 0.36 −0.42 * 0.60 −0.44 * Glyceric acid 0.51 −0.50 * 0.21 −0.24 * 0.67 0.30 * * keto-Gluconic acid — — 0.33 −0.78 * * 0.71 −0.43 * Maleic acid — — 0.66 −0.34 * 0.77 −0.22 * Malic acid −0.21 −0.17 * 0.77 −0.10 * 0.78 −0.01 * Mucic acid 0.76 −0.54 * * 0.79 −0.14 * 0.88 −0.07 * Phosphoric acid 0.84 0.04 * * 0.48 −0.17 0.48 0.09 Quinic acid 0.63 0.50 * 0.75 −0.15 * 0.84 −0.09 * * Ribonic acid — — 0.70 −0.17 * * 0.76 −0.39 * Saccharic acid 0.72 0.41 * 0.83 0.17 * 0.88 0.06 * Succinic acid 0.18 −0.49 * 0.37 −0.49 0.57 −0.35 * * Threonic acid 0.63 −0.45 * 0.41 0.01 * 0.33 −0.07 * * Sugar allo-Inositol 0.67 0.44 * 0.69 0.19 * 0.78 −0.41 * Arabinose 0.12 0.67 * −0.47 0.47 −0.05 −0.28 * Cellobiose — — 0.25 −0.66 0.60 −0.42 * Fructose 0.93 0.20 * −0.23 −0.88 * * −0.16 −0.90 * Galactinol — — −0.20 −0.08 * 0.25 0.27 * Glucose 0.92 0.22 * −0.34 −0.79 * * −0.17 −0.87 * Glucose-6P 0.51 −0.66 * 0.19 −0.26 0.39 −0.01 Isomaltose — — 0.52 −0.60 * * 0.05 −0.72 * * Lactitol 0.81 0.49 * −0.41 −0.67 * −0.26 −0.77 * Lactose 0.77 0.50 * −0.45 −0.81 * * −0.19 −0.90 * Leucrose 0.83 0.40 * −0.46 −0.65 * * −0.30 −0.67 * Maltitol 0.69 0.34 * −0.16 −0.75 * −0.20 −0.80 * Mannitol −0.29 −0.32 * * — — — — Melibiose 0.65 −0.54 * * −0.49 −0.75 * * −0.16 −0.87 * myo-Inositol 0.83 0.38 * 0.03 0.47 0.22 −0.48 * * Raffinose 0.52 −0.05 * * 0.25 −0.09 * * 0.17 −0.12 * Sucrose 0.73 −0.01 * 0.21 0.66 * 0.18 0.66 * Trehalose — — −0.40 −0.56 * * −0.19 −0.75 * Xylose 0.25 0.08 −0.67 0.14 * −0.18 −0.34 * Others Ethanolamine — — −0.03 0.47 0.26 0.21 * Glycerol — — 0.45 −0.21 0.41 −0.32 Glycerol-1P — — 0.18 −0.70 * 0.13 −0.28 * Urea — — 0.02 −0.72 * 0.07 −0.56 * Asterisks indicate components that were significantly affected (P < 0.05) by growing environments (Env.) or harvest month (Mon.). The P-values were calculated by ANOVA. Hort. J. 86 (4): 543–551. 2017. 547 component (PC1) accounted for 38% of the total var- Since PC1 and PC2 sufficiently reflected the differ- iance and mainly discriminated between the samples ences in the metabolites of ‘Sagan-Ruby’ grapefruit grown in the outdoor field and those grown in the plas- from different growing environments and harvest peri- tic house (Fig. 2A). Meanwhile, the second principal ods, we used factor loading scores that were calculated component (PC2) accounted for 20% of the total var- along with each of the PCs to determine which metabo- iance, and the December and April samples had higher lites were influenced by environmental and seasonal and lower PC2 scores, respectively (Fig. 2A), whereas changes (Table 2). According to the PC1 loading scores the January, February, and March samples had inter- of the juices, many of the identified metabolites were mediate PC2 scores, suggesting that the changes in me- more prevalent in juices from outdoor-grown fruits, and tabolite contents were proportional to the duration of the PC2 loading scores of the juices indicated that the the ripening period of fruits (from December to April) content of many amino acids and some organic acids and were reflected in PC2. increased with the duration of the ripening period, In contrast, in the albedo and flavedo, PC1 only ac- whereas the content of other organic acids decreased. counted for 22% and 25% of the total variance, respec- At each of the harvest periods, glucose, sucrose, sac- tively, although it still mainly discriminated between charic acid, quinic acid, and other metabolites, which samples from different growing environments (Fig. 2B, had positive PC1 scores and were significantly affected C), and PC2 accounted for 18% and 16% of the total by the growing environment, were more concentrated in variance in the metabolite contents of the albedo and juices from outdoor-grown fruits (Fig. 3). From January flavedo samples, respectively. Furthermore, we also to February, the contents of these metabolites were ele- found that the albedo samples from December, January vated in the juice of outdoor-grown fruits and from and April, and February and March had relatively high, February to March were much lower in the juice from intermediate, and low PC2 scores, respectively, and that house-grown fruits. The contents of phenylalanine, pro- flavedo samples from December, April, and the three line, threonine, mucic acid, and other metabolites, months in between had high, moderately high, and which had negative PC2 scores and were significantly moderately low PC2 scores, respectively (Fig. 2B, C). affected by the harvest period, increased with the dura- These results indicated that PC2 reflected the change in tion of fruit ripening, and from February to April, those metabolites and that the individual metabolites either that had positive PC1 scores increased more in the juice increased from December to February and decreased from the outdoor-grown fruits than that from the house- from February to April (ridge-shaped pattern) or de- grown fruits. creased from December to February and increased from Meanwhile, the PC1 and PC2 loading scores of the February to April (valley-shaped pattern), with a greater peels indicated that the differences in metabolite con- change from December to February than from February tents caused by the growing environment and harvest to April. Meanwhile, the third principal component period were similar in the albedo and flavedo tissues (PC3) accounted for 13% and 12% of the total variance (Table 2). Some amino acids and many organic acids of the metabolite contents of albedo and flavedo and were more concentrated in the peels of outdoor-grown mainly discriminated the April samples from the other fruits, whereas the contents of many sugars in peels samples. were only affected by the harvest period. The contents

Juice Albedo Flavedo A 6 B C 5 3 3

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-6 -6 -5 0 5 -5 0 5 10 -5 0 5 10 PC1 (39%) PC1 (22%) PC1 (25%)

Grown in outdoor field December January February March April Grown in plastic house December January February March April

Fig. 2. Sample scores of the first (PC1) and second (PC2) principal components from a principal component analysis (PCA) of metabolites detected in ‘Sagan-Ruby’ grapefruit juice (A), albedo (B), and flavedo (C). 548 Y. Kimura, M. Naeshiro, Y. Tominaga, T. Anai and F. Komai of quinic acid, malic acid, and other metabolites, which In the juices, we found that glucose, fructose, and had positive PC1 scores, were higher in the peels of sucrose yielded relatively high GC/MS peaks compared outdoor-grown fruits, and the contents of glucose, fruc- to the other sugars, which suggested that the sweetness tose, and other sugars, which had negative PC2 scores, of ‘Sagan-Ruby’ grapefruit juice is greatly affected by gradually increased from December to February and these sugars, while the content of sucrose was higher then gradually decreased from February to April than that of glucose or fructose (Fig. 4). The environ- (Fig. 3). Exceptionally, sucrose had positive PC2 scores mental and seasonal changes in glucose were the same and exhibited a valley-shaped change with time. The as those of fructose and were similar to those of su- concentrations of sugars, which had negative PC1 crose, and the growing environment only had a small scores, were higher in the albedo of house-grown fruits, effect on the content of these sugars from December to and the content of xylose, arabinose, and other sugars, February. However, from February to March, we found which had negative PC2 and PC3 scores, continuously that levels of these three sugars decreased in the house- increased in the flavedo from December to April. grown fruits, which resulted in the differences in the

Juice Albedo Flavedo Outdoor field Plastic house Outdoor field Plastic house Outdoor field Plastic house Threonicacid Serine Raffinose Valine Sucrose Putrescine Phenylalanine Glycericacid Sucrose Gluconicacid Putrescine Serine Mucicacid Xylose Glycericacid Melibiose Asparagine Alanine Threonine Fumaricacid Fumaricacid beta-Alanine Malicacid Malicacid GABA Quinicacid Threonicacid Glycericacid Saccharicacid Galactinol Glucose-6P allo-Inositol Asparticacid Alanine Maleicacid GABA Isoleucine Phenylalanine Cellobiose Proline keto-Gluconicacid Maleicacid Serine Raffinose Mucicacid Raffinose Proline keto-Gluconicacid Benzoicacid Galactonicacid Quinicacid myo-Inositol Galactinol Saccharicacid Sucrose Threonicacid Phenylalanine Phosphoricacid Ribonicacid Gluconicacid Fructose Mucicacid Ribonicacid Glucose Gluconicacid allo-Inositol Lactose Isomaltose Glutamine Lactitol Lactitol Succinicacid Leucrose Leucrose Ornithine allo-Inositol Dehydroascorbicacid Maltitol Quinicacid Glucose Trehalose Saccharicacid Fructose Arabinose Galactonicacid Lactose Xylose Maltitol Melibiose Isomaltose Asparticacid Trehalose myo-Inositol Arabinose Tryptophan Glycerol-1P cis-Aconiticacid Urea Urea Citricacid Glycerol-1P Lactitol Ketoglutaricacid Maltitol Leucrose Malicacid 12 1 2 3 4 12 1 2 3 4 Dehydroascorbicacid Mannitol Harvested month Melibiose Fumaricacid Lactose Succinicacid Fructose 12 1 2 3 4 12 1 2 3 4 Glucose Harvested month 2 1 0 -1 -2 Proline Galactonicacid relative content 12 1 2 3 4 12 1 2 3 4 Harvested month Fig. 3. Effect of the growing environment and harvesting period on the metabolite contents of ‘Sagan-Ruby’ grapefruit juice, albedo, and flave- do. Only the metabolites for which we observed significant effects of growing environment or harvest period (refer to Table 2) are shown. Colors indicate the relative metabolite contents of each sample point in relation to the mean content of all samples; dark blue indicates the lowest ratios, and dark red indicates the highest ratios. The metabolites with similar patterns are arranged close together.

Glucose Fructose Sucrose Citric acid Malic acid

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C 8 9 20 30 0 Dec. Jan. Feb. Mar. Apr. Dec. Jan. Feb. Mar. Apr. Dec. Jan. Feb. Mar. Apr. Dec. Jan. Feb. Mar. Apr. Dec. Jan. Feb. Mar. Apr.

Outdoor field Plastic house

Fig. 4. Effect of growing environment and harvest period on the glucose, fructose, sucrose, citric acid, and malic acid contents of ‘Sagan-Ruby’ grapefruit juice. Hort. J. 86 (4): 543–551. 2017. 549 sugar contents in fruits from the two growing environ- ported that satsuma mandarin grown in plastic houses ments in March and April. Meanwhile, the level of have better yield, sugar content, and fruit quality than citric acid in the juices was extremely high when those grown outdoors (Suzuki et al., 1997). However, compared to the levels of malic acid and other organic Sawamura et al. (1983) reported that the amount of acids, and the content of citric acid was more than sev- sugars, organic acids, and other metabolites was sub- eral ten-fold that of malic acid (Fig. 4), which sug- stantially lower in juices from house-grown satsuma gested that citric acid was the main contributor to the mandarin than in juices from satsuma mandarin grown acidity of ‘Sagan-Ruby’ grapefruit juice. The influence outdoors, and with the exception of satsuma mandarin, of the growing environment and harvest period on citric the sugar content of citrus grown in plastic houses is not acid was similar to their influence on glucose. On the different from that of citrus grown outdoors (Kamota, other hand, the content of malic acid in juices exhibited 1987). In the present study, we demonstrated that the a valley-shaped change with time. content of sugars, organic acids, amino acids, and other metabolites were lower in the juices of house-grown Discussion ‘Sagan-Ruby’ grapefruits (Table 2). The concentrations Factors affecting metabolite composition of many metabolites, including glucose, fructose, and To investigate whether growing environment and citric acid, were higher in juices from outdoor-grown harvest period contributed to the variation in primary fruits, owing to the reduction in the metabolites in the metabolites of ‘Sagan-Ruby’ grapefruit, we conducted juice of house-grown fruits from February to March. metabolite profiling of the fruit and peel of ‘Sagan- Average air temperature near the cultivated region was Ruby’ grapefruit using GC/MS. A PCA of the detected constant from December of 2014 to February of 2015 metabolite contents revealed that the most influential and increased in March and April of 2015 (Japan factor for the metabolite composition of both juice and Meteorological Agency, http://www.jma.go.jp/). It is peel was the growing environment (Fig. 2). predicted that the higher temperature of plastic houses The differences between the PC1 scores of juices enhances the respiration activity of fruit flesh and the from outdoor-grown fruits collected in December and metabolism of sugars and organic acids as substrates, January and those of house-grown fruits were smaller and in addition, several reports have demonstrated that than differences between the PC1 scores of juices from high temperatures during fruit development reduce the outdoor-grown fruits collected in February, March, and acidity and total sugar content of juice from satsuma April and those from house-grown fruits (Fig. 2A). This mandarin (Sakamoto and Okuchi, 1968; Takagi et al., suggests that the influence of growing environment on 1994). the metabolite composition of the juice is more pro- The present study revealed that the content of phe- nounced during late winter and early spring. It is possi- nylalanine, proline, gamma-aminobutyric acid ble that the difference in weight of fruit from the two (GABA), and various amino acids in juices increased growing environments, which was small in December with the duration of the ripening period (Table 2; and large thereafter (Table 1), affects these different Fig. 3). Arginine, alanine, glutamine, proline, and metabolite contents of the juices. GABA are reported to increase with development and In contrast, the metabolite composition of peels from maturation of satsuma mandarin fruit (Iwagaki et al., fruit collected in early winter was sensitive to the grow- 1981). These results suggest that the change with time ing environment (Fig. 2B, C), and we found that ridge- of amino acids in ‘Sagan-Ruby’ is similar to that of and valley-shaped changes with time were more other citrus fruits. From late winter to early spring, the prevalent in the albedo and flavedo samples than in the ripening and aging of the fruit progressed, and the de- juice samples (Fig. 3). In addition, the difference be- creased production of new proteins and increased deg- tween the metabolite contents of the peel samples from radation of old proteins may have promoted the April and those from other months was represented by accumulation of amino acids. The present study demon- PC3, which indicated that the metabolic mechanism in strated that the content of many organic acids and sug- peels was greatly different between fruits harvested dur- ars remain relatively constant during fruit ripening, ing early and mid-winter and fruits harvested during especially in outdoor-grown fruit (Fig. 3), and similar late winter and early spring. From February to April, patterns have been reported in grapefruit cultivated in both the metabolite change in a direction opposite to the the Mediterranean region (Bermejo and Cano, 2012). change from December to February, and a metabolite Takebayashi et al. (1993) explained that citrus species change unique to this period, were observed in peels. and cultivars are divided on the basis of patterns in their accumulation of sugar during fruit development and Effect of growing environment and harvest period on maturation. In this scheme, grapefruit is categorized the metabolite composition of juice into a group that contains citrus fruits in which the in- In Japan, the influence of growing environment on crease with time and final accumulation of sugars are the quality of citrus fruits has been investigated using small. Our results correspond to this grouping. satsuma mandarin (Citrus unshiu), and it has been re- 550 Y. Kimura, M. Naeshiro, Y. Tominaga, T. Anai and F. Komai

Effect of growing environment and harvest period on of juice and liquor, the characteristics of sweetness, the metabolite composition of peel acidity, and bitterness are probably the most important. In the albedo and flavedo, we found that the seasonal In grapefruit juice, sweetness and acidity depend on the changes in metabolite contents were often ridge- or content of sugars such as glucose, fructose, and sucrose, valley-shaped (Fig. 3). In the subtropics, citrus fruits and organic acids, especially citric acid. The contents of degreen during the cool winter season and, when left on glucose, fructose, and citric acid in juices squeezed the tree, some varieties regreen during the ensuing from outdoor-grown fruit were about 1.2 to 1.6 times spring (Hortensteiner, 2006). The peel color of ‘Sagan- those in juices squeezed from house-grown fruit during Ruby’ changed from yellow to reddish yellow and then March and April, and other sugars and organic acids returned to yellow as the ripening period progressed tended to be found at high content in juices of outdoor- (Fig. 1). Thus, it is considered that the change with time grown fruit (Figs. 3 and 4). Thus, it is likely that of metabolites in peels is associated with degreening outdoor-grown fruits have a ‘stronger taste’ than house- and regreening processes, which, in turn, are strongly grown fruits. In satsuma mandarin, house-grown fruits influenced by the sugar (mainly reducing sugars) con- were preferred over outdoor-grown fruits, even though centration in peels (Hortensteiner, 2006). In the present the content of sugars and organic acids was lower study, the content of several kinds of sugars in the albe- (Sawamura et al., 1983). This evaluation is thought to do and flavedo, including glucose and fructose, in- be attributed to the increased sugar-acid ratio that re- creased from December to February and decreased sults from the reduced acidity of the house-grown from February to April. The low and high concentra- fruits. Therefore, the house-grown fruit may also be tions of sugars promote the regreening and degreening considered ‘sweeter’ than the outdoor-grown fruit. In of peels, respectively (Ahmed, 2009; Iglesias et al., fruit cultivation, drought stress is known to reduce fruit 2001; Takagi et al., 1994). It is possible that the peel weight and to increase the sugar content of fruit (Suzuki color change of ‘Sagan-Ruby’ was induced by the et al., 1997). Plastic houses are suitable for managing ridge-shaped change with time of sugar contents. Al- drought stress and producing high-quality fruits. Al- though sucrose exhibited a valley-shaped change with though the sugar content of juice from house-grown time in both albedo and flavedo, sucrose has a relatively fruits was lower in the present study, proper irrigation small effect on citrus degreening compared to reducing would likely improve the quality of house-grown sugars (Takagi et al., 1994), and the content in ‘Sagan- ‘Sagan-Ruby’ grapefruits. Ruby’ peels is lower than either glucose or fructose Compared to sugars and organic acids, the number of (data not shown). These results suggest that sucrose has reports that have investigated the relationship between an insignificant effect on the change of peel color when amino acids and citrus taste is small. Takebayashi et al. compared to glucose and fructose. (1993) reported that good-tasting citrus fruits have One of the amino acids, ornithine, was not detected higher concentrations of proline. In the present study, in the peels from fruits collected during December and we found that the content of proline in juices increased January, and the ornithine content of the flavedo was about two times from December to April, and the con- significantly elevated from February to April (Fig. 3). tent of other amino acids was increased with the dura- In higher plants, ornithine is the precursor of arginine, tion of fruit ripening. Thus, it is predicted that the taste which serves as a main nitrogen storage compound, and of ‘Sagan-Ruby’ is influenced by the harvest period. In increases in ornithine content induce tolerance to salt addition, the content of GABA in juices more than dou- and drought stresses (Kalamaki et al., 2009). In addi- bled from December to April. GABA is reported to tion, ornithine is utilized in the biosynthesis of poly- possess anti-stress activity, and the GABA content of amine, alkaloids, and other plant secondary metabolites grapefruit is the second highest among citrus fruits, fol- (Shargool et al., 1988). In the present study, it is unclear lowing oranges (Citrus sinesis; Shimizu and Sawai, whether the elevation of ornithine content was the result 2008). When ‘Sagan-Ruby’ grapefruit with high GABA of preparation for the synthesis of secondary metabo- content is required, fruits harvested in the early spring lites or if the elevation of ornithine content results from would be ideal. the suspension of biosynthesis pathways that utilize or- The bitterness of citrus fruits is caused by a variety of nithine as a substrate. However, the reduced content of secondary metabolites, such as flavonoid and limonoid proline, an ornithine metabolite, in the peels of fruits (Hasegawa et al., 1995). Unfortunately, we were unable collected from February to April supports the second to analyze the composition of secondary metabolites. hypothesis, that is, ornithine content is elevated as a re- However, we found that the contents of phenylalanine, sult of suspended biosynthesis. benzoic acid, and quinic acid, which are associated with the metabolism of phosphoenolpyruvate to flavonoid, in Effect of the growing environment and harvest period juices squeezed from outdoor-grown fruit were about on quality of ‘Sagan-Ruby’ grapefruit 1.4, 1.2, and 1.8 times those in juices squeezed from Since the flesh of ‘Sagan-Ruby’ grapefruit is mainly house-grown fruit, respectively. Moreover, the content used for eating raw or as an ingredient in the production of phenylalanine in juices more than doubled from Hort. J. 86 (4): 543–551. 2017. 551

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