Impaginato 13 Adv. Hort. Sci., 2022 36(1): 13­26 DOI: 10.36253/ahsc­11437 Profiling of primary metabolites of Averrhoa carambola, Spondias dulcis and Syzygium malaccense fruits revealed underpinning markers during “on­tree” maturation and ripening stages T.­A. Smith 1, J. Vásquez­Martínez 2 †, E. Mellado­Mojica 2, K. Vaidya 1, M. G. Lopez 2, N. Benkeblia 1, 3 (*) 1 Department of Life Sciences, University of the West Indies, Mona Campus, Kingston 7, Jamaica. 2 Biotechnology and Biochemistry Department, CINVESTAV‐Unidad Irapuato, Km 9.6 Libramiento Norte Carr., Irapuato‐León 36821, Irapuato Gto., Mexico. 3 Laboratory of Tree Fruit and Aromatic Crops, The Biotechnology Centre, Faculty of Science and Technology, University of the West Indies, Mona Campus, Kingston 7, Jamaica. Key words: Acids, Carambola, June plum, maturation, otaheite, ripening, sugars. Abstract: The study aimed to profile and quantify sugars and organic acids metabolites in carambola, June plum and otaheite fruits during three different “on tree” stages: immature, green­mature and ripe stages. Metabolites were profiled and quantified by gas chromatography­mass spectrometry (GC­MS). Results showed that glucose, fructose, galactose, arabinose, and the sugar alco­ hol myo­inositol were detected in all fruits, while sucrose was detected in carambola and June plum only. Organic acids identified in all fruits were malic acid, citric acid, propanoic acid, and acetic acid. Comparatively, June plum showed the highest content of total sugars and carambola the lowest, while the highest total in organic acids content was found in otaheite and the lowest in carambola. On the other hand, most sugars increased during ripening of the three fruits, while organic acids decreased. Total sugars increased by 37%, 8% and 46% in ripe carambola, June plum and otaheite, respectively. Total organic acids decreased by 20% and 49% in ripe carambola and otaheite, while they slightly increased by 3% in ripe June plum. Furthermore, sugars/organic acids ratio in all fruits increased during maturation and ripening stages. Principal component analysis (PCA) showed two main groups of highly scoring metabo­ lites, while the hierarchical cluster analysis (HCA) showed that the metabolites were grouped into three main clusters. Conclusively, results showed that glu­ cose, fructose, malic acid and tartaric acids were the key marker metabolites of the maturation and ripening stages of the three fruits. 1. Introduction Carambola (Averrhoa carambola) belongs to the family Oxalidaceae (*) Corresponding author: noureddine.benkeblia@uwimona.edu.jm † Present address: Biochemistry Department, Superior Institute of Technology of Irapuato (ITESI), TecNM., Irapuato Gto., Mexico. Citation: SMITH T.­A., VÁSQUEZ­MARTÍNEZ J., MELLADO­ MOJICA E., VAIDYA K., LOPEZ M.G., BENKEBLIA N., 2022 ­ Profiling of primary metabolites of Averrhoa carambola, Spondias dulcis and Syzygium malaccense fruits revealed underpin‐ ning markers during “on‐tree” maturation and ripening stages. ­ Adv. Hort. Sci., 36(1): 13­26. Copyright: © 2022 Smith T.­A., Vásquez­Martínez J., Mellado­Mojica E., Vaidya K., Lopez M.G., Benkeblia N. This is an open access, peer reviewed article published by Firenze University Press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Competing Interests: The authors declare no competing interests. Received for publication 30 June 2021 Accepted for publication 20 September 2021 AHS Advances in Horticultural Science https://doi.org/10.36253/ahsc-11437 http://www.fupress.net/index.php/ahs/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ Adv. Hort. Sci., 2022 36(1): 13­26 14 originated in Asia but has since developed a toler­ ance for tropical climates (Shui and Leong, 2006). Presently, carambola is cultivated extensively in India and China (Narain et al., 2001), but Malaysia is the largest exporter (Abdullah et al., 2007). Work has been done to improve carambola cultivars in the United States in the 1930s, and since then, the fruit became more popular, while some sub species of star fruit exist in the Caribbean, Central America and tropical West Africa (Neto et al., 2009). Unripe carambola is moderately sour, but when ripe it is very sweet and used for juice, fruit salads, chutney, stewed fruits, garnish drinks and dishes, or adding it to fruit smoothies. Physiologically, carambola does not exhibit climacteric ripening behaviour even though it continues to synthesise carotenoids and develop its yellow­orange colour (Warren, 2009). June plum (Spondias dulcis Forst. Syn. Spondias cytherea Sonn.), a drupe belonging to the family Anacardiacea, is native to the Society Islands of the South Pacific ranging from Melanesia to Polynesia and was first introduced to Jamaica in the year 1782, and later in 1792 by Captain Bligh (Graham et al., 2004 a). Although the fruit is well known, there are no named cultivars, but both forms exist, one is with a thick mesocarp and a more pleasant taste, and another has long spines, a woody endocarp with a pungent and resinous taste (Daulmerie, 1994). Ripe June plum is eaten mostly raw but is also used in making refreshing drinks, jam, chutney, sauce or served with meat and seafood. When harvested green and mature, June plum ripens, and studies showed that respiratory pattern is typical of a climac­ teric fruit (Daulmerie, 1994; Graham et al., 2004 b). Otaheite (Syzygium malaccense) is a berry and belongs to the family Myrtaceae. It is thought to be native to the Indo­Malay or Southeast Asian region (Whistler and Elevitch, 2006). Otaheite is, however distributed in many tropical countries throughout the world, particularly in Africa and South America (Oliveira et al., 2011). Other English common names for this fruit include: Malay apple, mountain apple, pomerac, and rose apple (Batista et al., 2017). Two colour forms exist: one which produces red flowers and fruits and another, less common variety, which produces white flowers and fruit (Whistler and Elevitch, 2006). Ripe otaheite fruit is not very sweet, and often eaten raw. However, in some tropical countries, it is stewed with sugar to make jam or wine and refreshing drinks. No study is recorded on whether otaheite is a climacteric or non­climacteric fruit except the work of Basanta (1998) who reported that otaheite is a non­climacteric fruit. Ripening can be defined as the total changes of fruit tissue metabolism, leading to the production of an attractive fruit which can be consumed, aiding in the release and dispersal of the seed (Adams­Phillips et al., 2004). The ripening process is characterized by softening of fruit tissue and an increase in volatile compounds as well as pigments such as carotenoids and flavonoids which results in a more appealing fruit (Giovannoni, 2001). The concentration of sugars and organic acids in fruits varies depending on the fruit variety and the environmental conditions of the par­ ent plant (Haruenkit, 2004). Overall, there is a gener­ al decrease in organic acids and an increase in sugar content as fruit development progresses, due to decarboxylation of organic acids and breakdown of stored carbohydrates to produce sugars (Batista­Silva et al., 2018). According to Etienne et al. (2013), using advanced technologies, i.e. proteomics, transcrip­ tomics and metabolomics, studies have shown evi­ dence of a shift from the accumulation of organic acids to sugar synthesis in the final stage of fruit development in several species of fruit. Thus, the res­ piratory pathways commonly involved in the reduc­ tion of fruit sugars are glycolysis, oxidative pentose phosphate (OPP) pathway, and the tricarboxylic acid (TCA) pathway (Tucker, 2012). Because most fruits reach their best sensorial and commercial quality attributes when they ripen on the plants, the correct maturity for harvest of fruits impacts their postharvest shelf­life and quality attributes during storage (Thompson, 2003). Gene expression resulting from natural processes and trig­ gering fruit ripening induces many metabolic pro­ cesses leading to the formation of hundreds and even thousands of different metabolites (Pech et al., 2013). Although extensive literature is readily available on the metabolic changes during the maturation, ripening and senescence of fresh crops, few researches reported on changes in metabolite pro­ files during postharvest ripening and senescence (Benkeblia, 2016). However, a limited work was car­ ried out on the metabolites variation during the development and ripening of peach (Lombardo et al., 2011), strawberry (Zhang et al., 2011), pear (Oikawa et al., 2015), and pitaya (Wu et al., 2019), while scarce work was reported on some tropical fruit (Fabi Smith et al. ‐ Ripening metabolome of tropical fruits 15 et al., 2010). In the present work, in order to explore the varia­ tion of the metabolic profile of three tropical fruits commonly consumed in the tropics, we performed a profiling study of primary metabolites which are the main indicators of the maturation and the ripening of fruits. For this purpose, we selected three fruits namely carambola (sweet type), June plum and ota­ heite. One of the goals of this study was to assess how metabolically different the “on tree” maturation and ripening stages of these three fruits are and to find out if there is any particular metabolic profile which could be associated with these two stages of the fruits. On the other hand, by evaluating the metabolomic pattern at both maturation and ripen­ ing stages of the three fruits. Overall, this study is aiming to explore a part of the chemical potential of carambola (sweet type), June plum and Otaheite which will aid in the future to know the primary metabolites of these fruits that may correlate to dif­ ferent stages and determine which metabolites might be used as maturation and ripening markers. 2. Materials and Methods Fruits collection For the purpose of the present study, three physi­ ological stages of the fruits were investigated: green­ immature, mature, and ripe stages (Fig. 1). The colour and softness of the fruits were the two criteria used for discriminating the different maturation and ripening stages. A period varying from seven to ten days elapsed between each harvesting (sampling) stage. The commercial (optimal) harvesting stage of carambola is stage 3 (ripe), while for June plum and otaheite is stage 2 (mature). The fruits carambola, June plum, and otaheite of the three stages of each fruit were collected from three trees of same loca­ tion. The fruit June plum was collected from a farm in St. Elizabeth. Otaheite fruits were collected from a local farm in Mona, Kingston, and carambola samples were collected from Orange River Research Station in St. Mary. The three stages differentiated and sam­ pled based on their size and colour. For each stage, three samples were collected from three different trees, and each sample consisted of at least six fruits. Fruits collected were controlled for absent of any defect, wound or disease. Immediately after being collected, fruits were placed in plastic bags, and the bags were placed on ice in a cooler and transported to the laboratory within few hours. Then, fruits were washed with mild detergent and rinsed thoroughly, followed by seed removal, dicing or slicing and frozen for 48 hours at ­20°C. Freeze‐drying Prior to the extraction of the profiled sugars and organic acids, samples were freeze­dried in a Labconco freeze­drier (Labconco Corp., Kansas, MO, USA). After six days and complete drying, samples were sealed in plastic bags under vacuum using a MULTIVAC C100 vacuum packer (MULTIVAC, Wolfertschwenden, Germany) and stored under dry­ ness in a desiccator until further use. Extraction of sugars and organic acids metabolites Sugars and organic metabolites were extracted by the method described by Broeckling et al. (2005) with some modifications. In an Eppendorf tube, 300 mg of freeze­dried samples were mixed with 0.75 mL HPLC grade water containing 26 μg/mL Ribitol was added as internal standard and the tubes vortexed. After equilibrating to room temperature. The tubes were incubated in a shaker for 10 min at 80˚C, fol­ Fig. 1 ­ Carambola (A), June plum (B) and otaheite (C) at diffe­ rent development and ripening stages. Adv. Hort. Sci., 2022 36(1): 13­26 16 lowed by incubation at room temperature for c.a. 45 minutes. Afterwards, the tubes were cooled to 4˚C and centrifuged at 10 000 rpm for 15 minutes, the supernatant collected, and the pellet discarded. To the collected supernatants, 250 µL were mixed with 100 mL absolute EtOH and the samples dried under vacuum until dryness and stored at ­20°C until GC­MS analysis. Derivatization Prior to GC­MS analysis, samples were derivatised as described by Broeckling et al. (2005) with some minor modifications. The dry residues were mixed with 80 μL of BSTFA+1% TMCS (Sigma Aldrich, St Louis, MI, USA) and 20 µL pyridine, vortexed and cen­ trifuged for 10 seconds at 10 000 rpm. Afterwards, the mixtures were incubated for 20 minutes at 85˚C. After incubation and equilibrating to room tempera­ ture, 200 μL isooctane (2,2,4 trimethylpentane) (Sigma Aldrich, St Louis, MI, USA) were added to the mixture, vortexed and followed by a centrifugation at 10 000 rpm for 10 seconds. From the mixtures, 100 μL were transferred to a 300 μL glass insert for the GC­MS analysis. GC‐MS analysis of sugars and organic acids The samples were analysed by GC­MS using an Agilent 7890B gas chromatograph coupled to an Agilent 5977A mass spectrometer scanning in the m/z range from 40 to 550. The column used was an HP 5MS (5% Phenyl Methyl Polysiloxane, 30 m × 250 µm × 0.25 μm) with helium as the gas carrier at a constant flow rate of 1.0 mL/min. The samples were injected at a 15:1 split ratio. Initially, the inlet line was held at 260˚C and the transfer line was held at 280˚C. Separation was achieved with an initial tem­ perature program of 40˚C for 2 min, then ramped up at 4˚C per minute to 240˚C and held for 1 minute. The temperature was then increased to 10˚C per minute to 315˚C. In order to produce the concentration curve, a mixture containing 250 µL of 26 µg/mL of ribitol was used. To the mixture, 20 μL of pyridine and 80 μL of BSTFA containing 1% TMCS was added, vortexed and injected into the GC­MS. Prior to the injection, 50 µL of the mixture were taken and 50 μL of isooctane were added. This was repeated by adding each time 50 μL of isooctane until 6 concentration curves were produced. From the curve a scatter plot with a trend line was generated. The concentrations of the differ­ ent profiled sugars and organic acids metabolites were calculated from the generated trend line. The generated MS files were extracted, and the deconvo­ lution and identification of the metabolites was car­ ried out by using Agilent MSD Chem Station (Version F.01.01.2017) along with NIST library (Version 11 MS Mass Spectral Library) and AMDIS (Version 2.66) soft­ ware. Statistical analyses For the analysis of each sample (three fruits for each ripening stage and for each species), six samples were analysed, and the data were averaged. The data were analysed and compared by running analysis of variance (ANOVA), Tukey’s Honestly Significant Difference (HSD) Post Hoc test using SPSS software package (version 22.0, (IBM Corp., New York, USA). The significance level of all statistical hypotheses testing procedures was predetermined at P < 0.05 and 0.01. For the classification, clustering, and regression, PCA (principal component analysis) and HCA (hierarchical cluster analysis) were performed using SPSS software package (version 22.0, IBM Corp., New York, USA), while the clustered heatmap was generated using ClustVis free software (https://biit.cs.ut.ee/clustvis/). 3. Results Profile and sugar contents of carambola, June plum and otaheite fruits The profiling of sugars showed that seven saccha­ rides and one sugars alcohol were detected in caram­ bola, June plum and otaheite fruits (Table 1). Glucose, fructose, sucrose, galactose, arabinose, and myo­Inositol have been detected in carambola, June plum and otaheite, however, mannose and xylose were not detected in June plum while sucrose and mannose were not detected in otaheite. Overall, fructose, sucrose, galactose, in carambola and June plum, and glucose, fructose and galactose in otaheite increased during maturation and ripening stages, while the other sugars varied differently in the three fruits. Interestingly the highest levels of glucose, fruc­ tose and sucrose were observed in June plum, high­ est levels of galactose and xylose in otaheite, and the highest levels of arabinose and myo­inositol in carambola. Results also showed that glucose and fructose were the most predominant monosaccha­ rides in carambola and June plum, while in otaheite glucose, fructose and galactoses were predominant. https://biit.cs.ut.ee/clustvis/ Smith et al. ‐ Ripening metabolome of tropical fruits 17 On the other hand, a significant increase in total sug­ ars was noted in carambola and otaheite, while in June plum sugars content increased slightly. The total increase of sugars averaged 38% and 45% in caram­ bola and otaheite, respectively, but in June plum increases averaged 9%. Statistically, total sugar contents in carambola and otaheite were significantly different and their con­ tents in immature fruits were significantly different in comparison with mature and ripe carambola. Immature carambola and otaheite had 35% and 36%, and 28% and 46% less total sugars than the mature and ripe stages, respectively. However, statistical analysis showed no significant difference in sugar contents of June plum during the three maturation and ripening stages. Profile and organic acids contents of carambola, June plum and otaheite fruits With ten different acids identified in carambola, June plum and otaheite fruits, eight were detected in June plum, six in carambola and four in Otaheite (Table 2). Overall, malic acid, oxalic acid, propionic acid and acetic acids were the most abundant organic Table 1 ­ Profiled sugars and sugar alcohols and their contents (mg/g dry weight) in carambola, June plum and Otaheite during the three development and ripening stages Different letters of the same row indicate significant difference at P= 0.05. n.d. = not detected. Metabolites Immature Mature Ripe Carambola Glucose 51.40 ± 3.27 a 68.20 ± 5.18 b 56.36 ±2 .99 ab Fructose 30.69 ± 2.71 a 28.87 ±1.23 a 57.79 ± 7.98 b Sucrose 1.97 ± 0.08 a 13.73 ± 2.05 b 3.59a ± 0.34 a Galactose 9.50 ± 0.45 a 14.14 ± 0.52 b 11.11 ± 0.77 a Mannose n.d. n.d. 1.23 ± 0.53 Arabinose 2.18 ± 0.42 a 1.86 ± 0.01 a 1.90 ± 0.03 a Xylose n.d 11.62 ± 1.85 a 6.28 ± 1 .11 b Myo­Inositol 2.16 ± 0.11 a 3.30 ± 1.28 a 2.20 ± 0.08 a Total 97.9 a 133.2 b 134.1 b June plum Glucose 124.11 ± 7.62 a 112.38 ± 12.84 a 90.63 ± 18.41 b Fructose 79.43 ± 3.26 a 72.68 ± 14.12 a 94.38 ± 18.01 b Sucrose 7.46 ± 2.36 a 11.33 ± 1.76 a 35.03 ± 10.06 a Galactose 13.01 ± 0.61 a 12.36 ± 1.77 a 23.67 ± 5.75 b Mannose n.d. n.d. n.d. Arabinose n.d. 1.28 ± 0.09 a 1.26 ± 0.56 a Xylose n.d. n.d. n.d. Myo­Inositol 1.88 ± 0.08 a 1.99 ± 0.11 a 1.55 ± 0.61 a Total 225.9 a 210.8 a 245.8 b Otaheite Glucose 51.88 ± 3.12 a 103.40 ± 10.2 b 111.86 ± 6.59 b Fructose 43.97 ± 2.75 a 62.40 ± 1.24 b 68.87 ± 9.78 b Sucrose n.d. n.d. n.d. Galactose 34.33 ± 1.66 b 12.44 ± 2.45 a 10.79 ± 2.57 a Mannose n.d. n.d. n.d. Arabinose 1.67 ± 0.77 a n.d. 2.13 ± 0.61 a Xylose 6.34 ± 2.58 a n.d. 9.31 ± 1.44 a Myo­Inositol 1.07 ± 0.81 a n.d. n.d. Total 139.3 a 178.4 ab 202.9 b 18 Adv. Hort. Sci., 2022 36(1): 13­26 acids. In carambola and otaheite, malic and tartaric acids were the most abundant, while in June plum malic and acetic acids were predominant. Malic, cit­ ric, propionic and acetic acids have been detected in carambola, June plum and Otaheite, while tartaric acid in carambola and otaheite only. On the other hand, ascorbic acid was detected in carambola only, and succinic, oxalic, threonic and gluconic acids were detected in June plum only. Results also showed that organic acids content decreased with development and ripening of carambola and otaheite, while a steady state was observed in June plum. Total organ­ ic acids decreased by 20% and 49% in carambola and otaheite, respectively, while in June plum variation of organic acids during the three stages averaged only 3%. Different letters of the same row indicate significant difference at P= 0.05. n.d. = not detected. Table 2 ­ Profiled organic acids and their contents (mg/g dry weight) in carambola, June plum and otaheite during the three develop­ ment and ripening stages Metabolites Immature Mature Ripe Carambola Malic Acid 22.11 ± 4.87 b 13.68 ± 2.4 ab 6.73 ± 0.88 a Citric Acid 2.30 ± 0.2 a 2.20 ± 0.07 a 1.77 ± 0.34 a Propionic Acid 2.23 ± 0.01 a 2.15 ± 0.04 a 2.19 ± 0.12 a Acetic Acid 2.01 ± 0.02 a 1.78 ± 0.39 a 1.84 ± 0.24 a Tartaric Acid 16.67 ± 3.45 a 10.24 ± 2.44 a 10.65 ± 2.65 a Ascorbic Acid 1.74 ± 0.34 a 1.80 ± 0.3 a 2.12 ± 0.02 a Succinic Acid n.d. n.d. n.d. Oxalic Acid n.d. n.d. n.d. Threonic Acid n.d. n.d. n.d. Gluconic Acid n.d. n.d. n.d. Total 14.73 a 14.14 ab 11.71 b June plum Malic Acid 7.34 ± 0.43 a 6.33 ± 0.67 a 6.45 ± 1.45 a Citric Acid 2.12 ± 0.09 a 2.13 ± .009 a 2.13 ± 0.12 a Propionic Acid 1.89 ± 0.05 a 3.47 ± 0.09 b 3.45 ± 0.41 b Acetic Acid 3.24 ± 0.36 a 3.64 ± 0.25 a 3.62 ± 0.44 a Tartaric Acid n.d. n.d. n.d. Ascorbic Acid n.d. n.d. n.d. Succinic Acid 1.82 ± 0.03 a 1.90 ± 0.1 a 1.88 ± 0.07 a Oxalic Acid 4.52 ± 0.3 a 4.32 ± 0.18 a 4.03 ± 0.2 a Threonic Acid 1.97 ± 0.05 a 2.26 ± 0.02 ab 2.34 ± 0.19 b Gluconic Acid 1.84 ± 0.03 a 1.74 ± 0.24 a 1.73 ± 0.28 a Total 24.74 a 25.79 a 25.63 a Otaheite Malic Acid 22.11 ± 4.87 b 13.68 ± 2.4 ab 6.73 ± 0.88 a Citric Acid 2.30 ± 0.2 a 2.20 ± 0.07 a 1.77 ± 0.34 a Propionic Acid 2.23 ± 0.01 a 2.15 ± 0.04 a 2.19 ± 0.12 a Acetic Acid 2.01 ± 0.02 a 1.78 ± 0.39 a 1.84 ± 0.24 a Tartaric Acid 16.67 ± 3.45 a 10.24 ± 2.44 a 10.65 ± 2.65 a Ascorbic Acid n.d. n.d. n.d. Succinic Acid n.d. n.d. n.d. Oxalic Acid n.d. n.d. n.d. Threonic Acid n.d. n.d. n.d. Gluconic Acid n.d. n.d. n.d. Total 45.31 b 30.05 ab 23.19 a Smith et al. ‐ Ripening metabolome of tropical fruits 19 On the other hand, the ratio of sugars/organic acids plays an important role that can characterise the ripe stage of fruits. During the different stages, the ratio of sugars/organic acids maintained a signifi­ cant rising trend especially in carambola and ota­ heite. In carambola, the ratio was 6.47, 9.42 and 11.45 in immature, mature and ripe, respectively. In June plum, the ration was 9.13, 8.17 and 9.59, in immature, mature and ripe, respectively. In otaheite, the ratio was 3.07, 5.93 and 8.75, in immature, mature and ripe, respectively. Statistical analysis showed that total organic acid contents of carambola was not significantly different between either immature and mature, or mature and ripe stages. Malic acid content varied significantly during the development and ripening of carambola and otaheite, but not significantly in June plum. Malic acid was also the main organic acid accumulating in carambola and otaheite and its level was significantly different among the three stages of the maturation and ripening of the two fruit. The statistical analysis also showed that citric acid and acetic acid contents did not show significant difference among the three developmental stages of carambola. Comparatively, total sugars and total organic acids during the three stages showed different correlations. In carambola and June plum, weak correlation (R2 =0.18 and R2 = 0.33, respectively) was observed between sugars and organic acids contents, however, a moderate correla­ tion (R2 = 0.56) was observed between sugars and organic acids contents of otaheite during the three stages. Factoring and clustering of the profiled metabolites The principal component analysis of the data sets revealed two individual clusters that seem to be gov­ erned by the developmental and the ripening stages of the fruits (Fig. 2). The analysis showed that in PC 1, the metabolites in carambola with the highest load­ ing scores were glucose (0.99), galactose (0.99), xylose (0.97), sucrose (0.96), myoinositol (0.93) and mannose (0.92), while in PC 2 ascorbic acid (0.99), fructose (0.98), citrate (0.95) and acetic acid (0.98) had the highest loading scores. In June plum, the highest loading sores metabolites in PC 1 and PC 2 were oxalic acid (0.90), acetic acid (0.85), sucrose (0.84) and gluconic acid (0.80), and malic acid (0.89), fructose (0.89), arabinose (0.82, galactose (0.82) and sucrose (0.77), respectively. In otaheite, the metabo­ lites with the highest scores were galactose (0.98), myoinositol (0.98), malic acid (0.96), tartaric acid (0.96), acetic acid (0.89), citric acid (0.79) and propanoic acid (0.74), and arabinose (0.99) and xylose (0.99), respectively. Overall, principal compo­ nents analysis (PCA) of samples based on the devel­ opment and ripening stages revealed a difference between grouped metabolites. As suggested by the PCA in the figure 2, profiled metabolites were then divided into two classes, and loading values of sugars Fig. 2 ­ Principal component analysis (PCA) of the profiled and identified metabolites in carambola, June plum and otaheite fruits through different development and ripe­ ning stages. For PCA analysis, data sets were normalised for better comparison of the variable levels of the diffe­ rent metabolites. Adv. Hort. Sci., 2022 36(1): 13­26 20 and organic acids of fruits samples were found most­ ly in quadrant PC2+, illustrating the discriminated metabolites reflecting the development and ripening of fruits. Hierarchical cluster analysis (HCA) was applied to a data set of the profiled and detected metabolites during the three stages of the three fruits. The den­ drograms (Fig. 3) show that the profiled metabolites were quite homogeneous and tend to be distributed into three groups. According to the dendrograms of the HCA, at the distance of three, the metabolites can be grouped as shown in Table 3. Interestingly, three metabolites have been classified within the same groups of the three fruits. Myoinositol, citric acid and arabinose were classified in group 1, group 2 and group 3, respectively. The clusters of the differ­ ent metabolites in the three fruits showed that the metabolites were quite clearly hierarchically separat­ ed, and these results were also clearly depicted by the PCA (Fig. 2) and the HCA (Fig. 3) which show the distribution of the metabolites into three main clus­ ters. Furthermore, the heatmap (Fig. 4) also shows that June plum concentrates the highest levels of ten metabolites, while carambola and otaheite concen­ trate the highest levels of four and six other metabo­ lites, respectively. Indeed, primary metabolites profiling led to the identification 8, 6 and 5 sugars, and 6, 8 and 5 organ­ ic acids in carambola, June plum and otaheite, respectively. On the other hand, our results showed that the key marker metabolites of the maturation and ripening of the three fruits are glucose and fruc­ tose in carambola and otaheite, while in June plum glucose, fructose, galactose and sucrose were the key marker metabolites of the three different stages. Similarly, malic and tartaric acids were the key organ­ ic acids metabolites of the maturation and ripening Fig. 3 ­ Dendrogram showing the Hierarchical Cluster Analysis (HCA) of profiled and identified metabolites in Carambola, June plum and Otaheite fruits through diffe­ rent development and ripening stages. To run HCA, data sets were normalised to reduce of the original data sets and hence allowing better clustering. Table 3 ­ The hierarchical clusters distribution of the profiled metabolites of carambola, June plum and otaheite during the three stages (underlined metabolites are classified within the same groups of the three fruits) Group 1 Group 2 Group 3 Carambola Mannose Fructose Arabinose Succinate Citric acid Malic acid Myo­inositol Acetic acid Propionic acid Glucose Ascorbic acid Galactose Xylose June plum Succinic acid Glucose Succinic acid Acetic acid Threonic Galactose Gluconic acid Citric acid Fructose Oxalic acid Propionic Arabinose Myo­inositol Malic acid Otaheite Galactose Malic acid Arabinose Myo­inositol Citric acid Xylose Tartaric acid Glucose Acetic acid Fructose Propionic acid Smith et al. ‐ Ripening metabolome of tropical fruits 21 of carambola and otaheite fruits, while malic acid was the key marker metabolite of June plum matura­ tion and ripening. 4. Discussion and Conclusions Although extensive literature is readily available, the variation of sugars and organic acids of many fruits including carambola and June plum at ripe stage, less and scattered work was done on the varia­ tion of the metabolites including sugars and organic acids in carambola, June plum and otaheite fruits during the development and ripening stages. On the other hand, most of the work carried out on caram­ bola targeted the postharvest physiology and bio­ chemistry of the fruit during storage. In carambola, Campbell and Koch (1989) found that total soluble sugars concentration, mainly glu­ cose and fructose, increased during ripening and var­ ied between 22 and 27 mg/g fresh weight depending on the varieties, while Narain et al. (2001) investigat­ ed the variation of the chemical composition of carambola at three different ripening stages and found that total sugars increased from 2.91 to 5.60 g/100 g fresh weight. Later, Patil et al. (2010) report­ ed the composition of the fruit at three stages of maturity (young, half­ripe and ripe), and they noted a tremendous increase of total sugars, oxalic acid and ascorbic acid by 100%, 89% and 65%, respectively. Similar increase by 33% and 90% of total sugars and ascorbic acid respectively were also reported by Ali and Jaafar (2012). Glucose, fructose and sucrose were reported to be the most predominant sugars in carambola (Mohd Zainudin et al., 2014; Benkeblia and López, 2015), however, Benkeblia and López (2015) reported an increase of glucose and fructose, but a slight decrease of sucrose in the ripe fruit com­ pared to the green one, while Mohd Zainudin et al. (2014) noted an increase of the three sugars. There is almost no work reporting on the compo­ sition of June plum fruit during maturation and ripen­ ing except from the one of Benkeblia and López (2015). The authors investigated the variation of glu­ cose, fructose and sucrose in green and ripe June plum and found that in ripe fruit glucose and fructose increased in ripe fruit, while sucrose decreased signif­ icantly. Other scattered studies reported on the vari­ ation of sugars and organic acids in June plum fruit but at a specific stage. In a study carried out on immature green June plum, Franquin et al. (2005) investigated the composition at this early stage and found the concentrations of glucose, fructose, sucrose, citric acid, malic acid, oxalic acid and ascor­ bic acid were 1.5 (± 0.2), 1.2 (± 0.2), 3.1 (± 0.3), 0.9 (± 0.1), 0.2 (± 0.02,) 0.03 (± 0.01), and 52.0 (± 4.9) g/100 g fresh weight, respectively. In his study, Nahar et al. (1990) reported that 0.3% of fresh weight of the pulp is composed by free sugars, where glucose, fructose and sucrose were the most predominant (Nahar et al., 1990; Mahmood et al., 2012). Similarly to June plum, few studies investigated the composition of otaheite during the maturation and ripening, however, few studies reported on ripe otaheite. Lu and Lin (2011) investigated the sugars in ripe otaheite and found that fructose yielded the highest content compared to glucose and sucrose which were detected at this ripe stage. The untargeted profiling of primary metabolites during the maturation and ripening of fruits is a good approach to provide better insight into their metabolome changes during these stages. Different studies on metabolite analyses of fruits have focused on temperate and stone fruits such as tomato, peach, strawberry, and grape among many others, but scarce studies focused on tropical fruits. However, these studies revealed similar dynamic variations in the levels of sugars and organic acids, as well as many other primary and secondary metabolites dur­ ing fruits maturation and ripening (Oikawa et al., Fig. 4 ­ Heatmap of the profiled and identified metabolites in Carambola, June plum and Otaheite fruits through diffe­ rent development and ripening stages. The color scale represents the relative concentration of each metaboli­ tes. Adv. Hort. Sci., 2022 36(1): 13­26 22 2015). Our results showed patterns of variation in sugars and organic acids levels in carambola, June plum and otaheite during maturation and ripening, and provided fundamental metabolomic data that is useful for understanding fruits physiology. Our reported results are in agreement with those report­ ed by Ramadan et al. (2020) who used metabolomics approach carambola fruit of different origins and at two different ripening stages. Mamat et al. (2019) and Parijadi et al. (2018) analysed the distribution of primary metabolites in mangosteen (Garcinia man‐ gostana Linn.) fruit during ripening, and their results showed that fructose was the kye marker metabolite of mangosteen ripening. Similar results were report­ ed by Ogawa et al. (2018) who noted that the increasing sucrose level might be a key marker metabolite of pineapple ripening. Beside metabolomics, Pandit et al. (2010) used transcriptomics markers to understand the matura­ tion and ripening programmes in mango (Mangifera indica L.) fruit. Among eighteen genes related to the fruit physiology and biochemistry, genes related to primary metabolism showed higher expression in comparison to that of the genes related to flavour production. However, regardless of the origin and environ­ mental zones, the maturation and ripening of fruits are complex and highly coordinated processes. Globally, the increase in sugar and decline in organic acids are one of the main changes associated with these processes (Giovannoni, 2001; Klee and Giovannoni, 2011; Osorio et al., 2013; Batista­Silva et al., 2018). During maturation and ripening of fruits, organic acids contents are inversely related to sugar contents. The rising trend of sugars is due to photo­ synthates import or starch degradation, while organ­ ic acids that accumulated in young fruits strongly decrease by being converted to other organic acids (Carrari et al., 2006; Beauvoit et al., 2018). Although environmentally different from tropical fruits, there have been a number of different studies reporting similar metabolic changes that occur in temperate fruits during maturation and ripening stages (Fait et al., 2008; Osorio et al., 2011, 2012). Sugars accumu­ lation and organics acids decrease trend were report­ ed in blueberries (Vaccinium arctostaphylos and Vaccinium myrtillus) (Ayaz et al., 2001), apple (Malus domestica) (Liu et al., 2016; Yang et al., 2021), apri­ cot (Prunus armeniaca L.), plumcot (Prunus armenia‐ ca x Prunus salicina L.), plum (Prunus salicina Lindl.), and peach (Prunus persica L.) (Bae et al., 2014), Damson plum (Prunus domestica) (García­Mariño et al., 2008), different citrus cultivars (Bermejo and Cano, 2012), grapes (Kurt et al., 2017; Liang et al., 2011; Muñoz­Robredo et al. , 2011), loquat (Eriobotrya japonica Lindl.) (Amorós et al., 2003), mango (Mangifera indica L.) (Mokhtar et al., 2014), melon (Cucumis melo L.) (Wang et al., 1996), pome­ granate (Punica granatum L.) (Nuncio­Jáuregui et al., 2014) and wolfberry (Lycium barbarum L.) (Zhao et al., 2015) among other reported fruits. Furthermore and in agreement with our finding, glucose, fructose, and sucrose were found to be the most predominant among mono and disaccharides, while malic, citric and tartaric acids were predominant organic acids (Wang et al., 1996; Liang et al., 2011; Mahmood et al., 2012; Kurt et al., 2017; Yang et al., 2021). Indeed, the relative levels of sugars and organic acids in fruits are of great importance for harvesting time and are one of the determinants of the organoleptic quality attributes of fruits particularly sweetness (Itai and Tanahashi, 2008). Furthermore, the postharvest quality attributes of fruits, their shelf­life and even processed products are strongly associated to their sugars and organic acids levels (Matsumoto and Ikoma, 2012; Aprea et al., 2017). In order to preserve freshness and reduce economic losses, it is of great importance to understand the metabolic changes occurring during maturation and ripening which might contribute to accelerate fruits senescence and perishability after harvesting. In this sense, metabolomic profiling of key metabolites responsible for quality attributes such as sugars and organic acids can be a powerful tool for further understanding the biochemical basis of pre­ and postharvest physiology and have the potential to play a critical role in the identification of the path­ ways affected by fruit maturation and ripening (Allwood et al., 2021; Pott et al., 2020; Tian et al., 2021). The data presented here indicates that the pro­ filed metabolites varied significantly during the matu­ ration and ripening of the fruits. Glucose, fructose, galactose, sucrose and myoinositol were found pre­ dominantly in all the fruits and during the three stages, except sucrose in otaheite. Comparatively, June plum showed the highest content of total sugars and carambola the lowest, while the highest total in organic acids content was noted in otaheite and the lowest in carambola. On the other hand, most sugars increased during ripening of the three fruits, while organic acids decreased. Interestingly, the multi­vari­ Smith et al. ‐ Ripening metabolome of tropical fruits 23 able analysis showed than all the metabolites were clustered into three main clusters, and myoinositol, citric acid and arabinose shared group one, group two and group three, respectively. From the different profiled sugars and organic acids, our results are sug­ gesting that glucose and fructose are the marker metabolites of the maturation and ripening of caram­ bola and otaheite, while the ripening marker metabolites in June plum are glucose, fructose, galac­ tose and sucrose. Because this study represents the first report on the profiling of sugars and organic acids in carambola, June plum and otaheite, it might be interesting to profile the secondary metabolites mainly phenolics and volatiles, and their variation during the maturation and ripening of these fruits. Acknowledgements This work is a part of a research project supported by the Graduate Studies and Research, University of West Indies, Mona Campus, under ‘‘New Initiative Research Grant’’, and partly by Research & Publications and Graduate Award to Tracy­Ann Smith for her travel to Mexico. References ABDULLAH A.G., NIK SULAIMAN N.M., AROUA M.K., MEGAT MOHD NOOR M.J., 2007 ­ Response surface optimization of conditions for clarification of carambola fruit juice using a commercial enzyme. ­ J. Food Eng., 81(1): 65­71. ADMAS­PHILLIPS L., BARRY B., GIOVANNONI J., 2004 ­ Signal transduction systems regulating fruit ripening. ­ Trends Plant Sci., 9(7): 331­338. ALI S.H., JAAFAR M.D., 2012 ­ Effect of harvest maturity on physical and chemical characteristics of carambola (Averrhoa carambola L.). ­ NZ. J. Crop Hort. Sci., 20(2): 133­136. ALLWOOD J.W., GIBON Y., OSORIO S., ARAÚJO W.L., VAL­ LARINO J.G., PÉTRIACQ P., MOING A., 2021 ­ Developmental metabolomics to decipher and improve fleshy fruit quality, pp. 3­34. ­ In: PÉTRIACQ, P., and A. BOUCHEREAU (eds.) Advances in botanical research. Academic Press, London, pp. 372. AMOROS A., ZAPATA P., PRETEL M.T., BOTELLA M.A., SER­ RANO M., 2003 ­ Physico‐chemical and physiological changes during fruit development and ripening of five loquat (Eriobotrya japonica Lindl.) cultivars. ­ Food Sci. Technol. Int., 9(1): 43­51. APREA E., CHARLES M., ENDRIZZI I., COROLLARO M.L., BETTA E., BIASIOLI F., GASPERI F., 2017 ­ Sweet taste in apple: the role of sorbitol, individual sugars, organic acids and volatile compounds. ­ Sci. Rep. 7: 44950. AYAZ F.A., KADIOGLU A., BERTOFT E., ACAR C., TURNA I., 2001 ­ Effect of fruit maturation on sugar and organic acid composition in two blueberries (Vaccinium arc‐ tostaphylos and V. myrtillus) native to Turkey. ­ NZ J. Crop Hort. Sci., 29(2): 137­141. BAE H., YUN S.K., JUN J.H., YOON I.K., NAN E.Y., KWON J.H., 2014 ­ Assessment of organic acid and sugar com‐ position in apricot, plumcot, plum, and peach during fruit development. ­ J. Appl. Bot. Food Qual., 87: 24­29. BASANTA A., 1998 ­ The postharvest storage of pomerac (Syzygium malaccense) under refrigerated and con‐ trolled atmosphere conditions. – M.Phil. Thesis, University of the West Indies, St. Augustine, Trinidad and Tobago. BATISTA Â.G., DA SILVA J.K., BETIM CAZARIN C.B., TELLES BIASOTO A.C., FRANKLAND SAWAYA A.C.H., PRADO M.A., MARÓSTICA M.R. Jr., 2017 ­ Red‐jambo (Syzygium malaccense): Bioactive compounds in fruits and leaves. ­ LWT­Food Sci. Technol., 76: 284­291. BATISTA­SILVA W., NASCIMENTAO V.L., MEDEIROS D.B, NUNES­NESI A., RIBEIRO D.M., ZSÖGÖN A., ARAÚJO W.L., 2018 ­ Modifications in organic acid profiles dur‐ ing fruit development and ripening: Correlation or cau‐ sation? ­ Front. Plant Sci., 9: 1­20. BEAUVOIT B., BELOUAH I., BERTIN N., CAKPO C.B., COLOM­ BIÉ S., DAI Z., GAUTIER H., GÉNARD M., MOING A., ROCH L., VERCAMBRE G., GIBON Y., 2018 ­ Putting pri‐ mary metabolism into perspective to obtain better fruits. ­ Ann. Bot., 122(1): 1­21. BENKEBLIA N., 2016 ­ Application of metabolomics to postharvest science of fresh crops. ­ CAB Rev., 11: 046. BENKEBLIA N., LÓPEZ M.G., 2015 ­ Saccharides and fruc‐ tooligosaccharides composition of green and ripe Averrhoa carambola, Blighia sapida and Spondias dulcis fruits. ­ Food Chem., 176: 314­318. BERMEJO A., CANO A., 2012 ­ Analysis of nutritional con‐ stituents in twenty citrus cultivars from the Mediterranean area at different stages of ripening. ­ Food Nutr. Sci., 3(5): 639­650. BROECKLING C.D., HUHMAN D.V., FARAG M.A., SMITH J.T., MAY G.D., MENDES P., DIXON R.A., SUMNER L.W., 2005 ­ Metabolic profiling of Medicago truncatula cell cultures reveals the effects of biotic and abiotic elicitors on metabolism. ­ J. Exp. Bot., 56(410): 323­336. CAMPBELL C., KOCH E.K., 1989 ­ Sugar and acid composi‐ tion and development of the sweet and tart carambola fruit. ­ J. Am. Soc. Hort. Sci., 114(3): 455­456. CARRARI F., BAXTER C., USADEL B., URBANCZYK­WOCHNI­ AK E., ZANOR M.I., NUNES­NESI A., NIKIFOROVA V., CENTERO D., RATZKA A., PAULY M., SWEETLOVE L.J., FERNIE A.R., 2006 ­ Integrated analysis of metabolite and transcript levels reveals the metabolic shifts that underlie tomato fruit development and highlight regu‐ Adv. Hort. Sci., 2022 36(1): 13­26 24 latory aspects of metabolic network behavior. ­ Plant Physiol., 142(4): 1380­1396. DAULMERIE S., 1994 ­ Golden apple (Spondias Cytherea) production with particular reference to post‐harvest technology and processing. ­ M.Sc. Thesis, École Nationale Supérieure des Industries Agricoles et Alimentaires, Paris, France, pp. 112. ETIENNE A., GENARD M., LOBIT P., MBEGUIE­A­MBEGUIE D., BUGAUD C., 2013 ­ What controls fleshy fruit acidi‐ ty? A review of malate and citrate accumulation in fruit cells. ­ J. Exp. Bot., 64(6): 1451­1469. FABI J.P., PERONI F.H.G., ARUJO GOMEZ M.L.P., 2010 ­ Papaya, mango and guava fruit metabolism during ripening: Postharvest changes affecting tropical fruit nutritional content and quality. ­ Fresh Produce, 4(1): 56­66. FAIT A., HANHINEVA K., BELEGGIA R., DAI N., ROGACHEV I., NIKIFOROVA i V.J., FERNIE A.R., AHARONI A., 2008 ­ Reconfiguration of the achene and receptacle metabol‐ ic networks during strawberry fruit development. ­ Plant Physiol., 148(2): 730­750. FRANQUIN S., MARCELLIN O., BRILLOUET J.M., 2005 ­ Optimising quality of a mature‐green golden apple (Spondias cytherea) drink using amyloglucosidase treatment: Information and technology for sustainable fruit and vegetable production. ­ Oral Presentation at the 7th Fruit, nut and vegetable production engineering symposium, Frutic 05, Montpellier, France. GARCÍA­MARIÑO N., DE LA TORRE F., MATILLA A.J., 2008 ­ Organic acids and soluble sugars in edible and nonedi‐ ble parts of Damson plum (Prunus domestica L. subsp. insititia cv. Syriaca) fruits during development and ripening. ­ Food Sci. Technol. Int., 14(2): 187­193. GIOVANNONI J., 2001 ­ Molecular biology of fruit matura‐ tion and ripening. ­ Annu. Rev. Plant Biol., 52: 725­749. GRAHAM O.S., MOHAMMED M., WICKHAM L.D., 2004 b ­ Effects of heat treatments on the quality of miniature golden applies (Spondias cytherea Sonn) during low temperature storage. ­ Int. J. Food Agric. Environ., 2(2): 48­53. GRAHAM O.S., WICKHAM L.D., MOHAMMAD M., 2004 a ­ Growth, development and quality attributes of minia‐ ture golden apple fruit (Spondias cytherea Sonn) part I: Fruit growth and development to maturity. ­ J. Food Agric. Environ., 2(1): 90­94. HARUENKIT R., 2004 ­ Analysis of sugars and organic acids in pineapple, papaya and star fruit by HPLC using an Aminex HPx‐87 H column. ­ Agritech J., 22: 11­18. ITAI A., TANAHASHI T., 2008 ­ Inhibition of sucrose loss dur‐ ing cold storage in Japanese pear (Pyrus pyrifolia Nakai) by 1‐MCP. ­ Postharvest Biol. Technol., 48(3): 355­363. KLEE H.J., GIOVANNONI J.J., 2011 ­ Genetics and control of tomato fruit ripening and quality attributes. ­ Annu. Rev. Genet., 45: 41­59. KURT A., TORUN H., COLAK N., SEILER G., HAYIRLIOGLU­ AYAZ S., AYAZ F.A., 2017 ­ Nutrient profiles of the hybrid grape cultivar ‘Isabel’ during berry maturation and ripening. ­ J. Sci. Food Agric., 9(8): 2468­2479. LIANG Z., SANG M., FAN P., WU B., WANG L., DUAN W., LI S., 2011 ­ Changes of polyphenols, sugars, and organic acid in 5 Vitis genotypes during berry ripening. ­ J. Food Sci., 76(9): C1231­C1238. LIU Y., CHEN N., MA Z., CHE F., MAO J., CHEN B., 2016 ­ The changes in color, soluble sugars, organic acids, antho‐ cyanins and aroma components in “Starkrimson” dur‐ ing the ripening period in China. ­ Molecules, 21(6): 812. LOMBARDO V.A., ODORIO S., BORSANI J., LAUXMANN M.A., BUSTAMANTE C.A., BUDDE C.O., ANDREO C.S., LARA M.V., FERNIE A.R., DRINCOVICH M.F., 2011 ­ Metabolic profiling during peach fruit development and ripening reveals the metabolic networks that underpin each developmental stage. ­ Plant Physiol., 157: 1696­ 1710. LU P.L., LIN C.H., 2011 ­ Physiology of fruit cracking in wax apple (Syzygium samarangense). ­Bot. Orientalis J. Plant Sci., 8: 70­76. MAHMOOD T., ANWAR F., ABBAS M., BOYCE M.C., SAARI N., 2012 ‐ Compositional variation in sugars and organ‐ ic acids at different maturity stages in selected small fruits from Pakistan. ­ Int. J. Mol. Sci., 13(2): 380­1392. MAMAT S.F., AZIZAN K.A., BAHARUM S.N., NOOR N.M., AIZAT W.M., 2019 ­ GC‐MS and LC‐MS analyses reveal the distribution of primary and secondary metabolites in mangosteen (Garcinia mangostana Linn.) fruit during ripening. ­ Sci. Hortic., 262: 109004. MATSUMOTO H., IKOMA Y., 2012 ­ Effect of different postharvest temperatures on the accumulation of sug‐ ars, organic acids, and amino acids in the juice sacs of Satsuma mandarin (Citrus unshiu Marc.) fruit. ­ J. Agric. Food Chem., 60(39): 9900­9909. MOHD ZAINUDIN M.A. ABDUL HAMID A., ANWAR F., OSMANA A., SAARI N., 2014 ­ Variation of bioactive compounds and antioxidant activity of carambola (Averrhoa carambola L.) fruit at different ripening stages. ­ Sci. Hortic., 172: 325­331. MOKHTAR S.I., LEONG P.C., VEN L.E., ABD AZIA N.A., 2014 ­ Total phenolic contents, antioxidant activities and organic acids composition of three selected fruit extracts at different maturity stages. ­ J. Trop. Resour. Sustain. Sci., 2: 40­46. MUÑOZ­ROBREDO P., ROBLEDO P., MANRÍQUEZ D., MOLI­ NA R., DEFILIPPI B.G., 2011 ­ Characterization of sugars and organic acids in commercial varieties of table grapes. ­ Chilean J. Agric. Res., 71(3): 452­458. NAHAR N., RAHMAN S., MOSIHUZZAMAN M., 1990 ­ Analysis of carbohydrates in seven edible fruits of Bangladesh. ­ J. Sci. Food Agric., 51(2): 185­192. NARAIN P., BORA P.S., HOLSCHUH H.J., VASCONCELOS M.D.S., 2001 ­ Physical and chemical composition of carambola fruit (Averrhoa carambola L.) at three Smith et al. ‐ Ripening metabolome of tropical fruits 25 stages of maturity. ­ Cien. Tecnol. Alim., 3(3): 144­148. NETO M.M., SILVA G.E.B., COSTA R.S., VIEIRA­NETO O.M., GARCIA­CAIRASCO N., LOPES N.P., HAENDCHEN P.F., SILVEIRA C., MENDES A.R., FILHO R.R., DANTAS M., 2009 ­ Star fruit: Simultaneous neurotoxic and nephro‐ toxic effects in people with previously normal renal function. ­ NDT Plus 2(6): 485­488. NUNCIO­JÁUREGUI N., CALÍN­SÁNCHEZ A., CARBONELL­ BARRACHINA A., HERNÁNDEZ F., 2014 ­ Changes in quality parameters, proline, antioxidant activity and color of pomegranate (Punica granatum L.) as affected by fruit position within tree, cultivar and ripening stage. ­ Sci. Hortic., 165: 181­189. OGAWA E.M., COSTA H.B., VENTURA J.A., CAETANO L.C., PINTO F.E., OLIVEIRA B.G., BARROSO M.E.S., SCHERER R., ENDRINGER D.C., ROMÃO W., 2018 ­ Chemical pro‐ file of pineapple cv. Vitória in different maturation stages using electrospray ionization mass spectrome‐ try. ­ J. Sci. Food Agric., 98(3): 1105­1116. OIKAWA A., OTSUKA T., NAKABAYASHI R., JIKUMARU Y., ISUZUGAWA K., MURAYAMA H., SAITO K., SHIRATAKE K., 2015 ­ Metabolic profiling of developing pear fruits reveals dynamic variation in primary and secondary metabolites, including plant hormones. ­PLOS ONE 10(7): e0131408. OLIVEIRA F.I.P., GALLÃO M.I., RODRIGUES S., FERNANDES F.A.N., 2011­ Dehydration of Malay apple (Syzygium malaccense L.) using ultrasound as pre‐treatment. ­ Food Bioproc. Technol., 4: 610­615. OSORIO S., AALBA R., NIKOLOSKI Z., KOCHEVENKO A., FER­ NIE A.R., GIOVANNONI J.J., 2012 ­ Integrative compara‐ tive analyses of transcript and metabolite profiles from pepper and tomato ripening and development stages uncovers species‐specific patterns of network regulato‐ ry behavior. ­ Plant Physiol., 159(4): 1713­1729. OSORIO S., ALBA R., DAMASCENO C.M.B., LOPEZ­CASADO G., LOHSE M., ZANOR M.I., TOHGE T., USADEL B., ROSE J.C.K., FEI Z., GIOVANNONI J.J., FERNIE A.R., 2011 ­ Systems biology of tomato fruit development: com‐ bined transcript, protein, and metabolite analysis of tomato transcription factor (nor, rin) and ethylene receptor (Nr) mutants reveals novel regulatory interac‐ tions. ­ Plant Physiol. 157(1): 405­425. OSORIO S., SCOSSA F., FERNIE, A.R., 2013 ­ Molecular regu‐ lation of fruit ripening. ­ Front. Plant Sci., 4: 198. PANDIT S.S., KULKARNI R.S., GIRI A.P., KÖLLNER T.G., DEGENHARDT J., GERSHENZON J., GUPTA V.S., 2010 ­ Expression profiling of various genes during the fruit development and ripening of mango. ­ Plant Physiol. Biochem., 48(6): 426­433. PARIJADI A.A.R., PUTRI S.P., RIDWANI S., DWIVANY F.M., FUKUSAKI E., 2018 ­ Metabolic profiling of Garcinia mangostana (mangosteen) based on ripening stages. ­ J. Biosci. Bioeng., 125(2): 238­244. PATIL A.G., PATIL D.A., PHATAK A.V., CHANDRA N., 2010 ­ Physical and chemical characteristics of carambola (Averrhoa carambola L.) fruit at three stages of maturi‐ ty. ­ Int. J. Appl. Biol. Pharma. Technol., 1(2): 624­629. PECH J.C., PURGATTO E., GIRARDI C.L., ROMBALDI C.V., LATCHÉ A., 2013 ­ Current challenges in postharvest biology of fruit ripening. ­ Curr. Agric. Sci. Technol., 19(1): 1­18. POTT D.M., VALLARINO J.G., OSORIO S., 2020 ­ Metabolite changes during postharvest storage: Effects on fruit quality traits. ­ Metabolites, 10(5): 187. RAMADAN N.S., WESSJOHANN L.A., MOCAN A., C VODNAR D.C., El­Sayed N.H., El­Toumy S.A., ABDOU MOHAMED D., ABDEL AZZIZ Z., EHRLICH A., FARAG M.A., 2020 ­ Nutrient and sensory metabolites profiling of Averrhoa carambola L. (Starfruit) in the context of its origin and ripening stage by GC/MS and chemometric analysis. ­ Molecules, 25(10): 2423. SHUI G., LEONG L.P., 2006 ­Residue from star fruit as valu‐ able source for functional food ingredients and antioxi‐ dant nutraceuticals. ­ Food Chem., 97(2): 277­284. THOMPSON A.K., 2003 ­ Fruit and vegetables: Harvesting, handling and storage. ­ Chapter 1. Blackwell Publishing, Oxford, UK, pp. 465. TIAN X., ZHU L., YANG N., SONG J., ZHAO H., ZHANG J., MA F., LI M., 2021 ­ Proteomics and metabolomics reveal the regulatory pathways of ripening and quality in post‐harvest kiwifruits. J. Agric. Food Chem., 69(2): 824­835. TUCKER G.A., 2012 ­ Introduction, pp. 1­51. ­ In: SEYMOUR G.B., J.E. TAYLOR, and G.A. TUCKER (eds.) Biochemistry of fruit ripening. Springer, Berlin, Germany, pp. 454. WANG Y., WYLLIE S.G., LEACH D.N., 1996 ­ Chemical changes during the development and ripening of the fruit of Cucumis melo (Cv. Makdimon). ­ J. Agric. Food Chem., 44(1): 210­216. WARREN.O., 2009 ­ Quality of carambola fruit (Averrhoa carambola L.) as affected by harvest maturity, posthar‐ vest wax coating, ethylene, and 1‐methylcyclopropene. ­ MSc Thesis, University of Florida, Horticultural Sciences Department, Gainesville, FL, USA. WHISTLER W.A., ELEVITCH C.R., 2006 ­ Syzygium malac­ cense (Malay Apple). ­ Species Profiles Pacific Island Agroforest., 2(4): 1­13. WU Y., XU J., HE Y., SHI M., HAN X., LI W., ZHANG X., WEN X., 2019 ­ Metabolic profiling of pitaya (Hylocereus polyrhizus) during fruit development and maturation. ­ Molecules, 24(6): 1114. YANG S., MENG Z., LI Y., CHEN R., YANG Y., ZHAO Z., 2021 ­ Evaluation of physiological characteristics, soluble sug‐ ars, organic acids and volatile vompounds in ‘Orin’ apples (Malus domestica) at different ripening stages. ­ Molecules, 26: 807. ZHANG J., WANG X., YU O., TANG J., GU X., WAN X., FANG C., 2011 ­ Metabolic profiling of strawberry (Fragaria × ananassa Duch.) during fruit development and matura‐ tion. ­ J. Exp. Bot., 62(3): 1103­1118. ZHAO J., LI H., XI W., AN W., NIU L., CAO Y., WANG H., Adv. Hort. Sci., 2022 36(1): 13­26 26 WANG Y., YIN Y., 2015 ­ Changes in sugars and organic acids in wolfberry (Lycium barbarum L.) fruit during development and maturation. ­ Food Chem., 173: 718­ 724.