Changes in Soluble Sugar Accumulation and Activities of Sucrose-Metabolizing Enzymes during Fruit Ripening of Jackfruit

Jackfruit (Artocarpus heterophyllus Lam.) is an important food crop widely grown in the tropical region. However, little is known about sugar metabolism during fruit ripening of jackfruit. Here we examined sugar profiles (sucrose, glucose and fructose) and corresponding enzyme activities (SPS, E.C.2.4.1.14; SuSy, EC 2.4.1.13; IV, EC 3.2.1.26) of four soft type and four firm type varieties of jackfruit during four stages of fruit ripening. We found that during fruit ripening, there was a rapid increase in contents of total soluble sugar and sucrose, whereas increases in glucose and fructose contents were much slower. Ratios of glucose versus fructose varied among different varieties and ripening stages but within the range of 0.9 to 1.2 in the ripe fruits. Five of these varieties exhibited markedly high levels of SuSy activity for sucrose synthesis at early ripening stage, and then decreased towards fully ripe stage. All soft type varieties exhibited a conspicuous peak of AIV activity and had overall higher AIV activities than NIV during ripening. The changing patterns for other enzymes varied among varieties. Our studies support the notion that sucrose was the major sugar species contributing to the fruit sweetness, followed by fructose and glucose. We also demonstrated that AIV and NIV were probably the primary enzymes responsible for sucrose hydrolysis during ripening, while SPS and SuSy were responsible for sucrose synthesis. We propose that during fruit ripening of jackfruit, glucose is released from starch hydrolysis, followed by sucrose hydrolysis leading to increase in both glucose and fructose contents.


Introduction
Jackfruit (Artocarpus heterophyllus Lam.) is a medium-sized evergreen tree belonging to the family Moraceae.It is reported to have originated in the Western Ghats region of India (Purseglove, 1968) and the rainforests of Malaysia (Ruehle, 1967).Due to its easy growth and tolerance to pests, diseases, high temperature and drought, jackfruit has been cultivated for centuries, e.g.3000 to 6000 years in India (Preedy, Watson, & Patel, 2011), and is mostly grown in tropical or close to tropical climates, especially throughout south-east Asia where it is mainly consumed as fresh fruit or vegetable (Soepadmo, 1991;Wu et al., 2013).It has been predicted that jackfruit may play an increasingly prominent role in achieving food security in the populous Asia under future challenges from climate change.ripe, rich in carbohydrates (starch and various sugars), vitamins, dietary fiber and minerals, and can be eaten fresh when ripe or cooked as vegetable.Its large starchy seeds can also be cooked as food.Its fibrous fruit rind can be used as livestock feed.
In spite of its obvious potential as a nutritious and healthy food staple, the popularity of jackfruit as a commercial crop is still lackluster, mainly due to its wide variation in fruit quality (Samaddar, 1985).Fruit ripening is one of the most important processes of fruit quality development.Previous studies have examined the free sugar distribution and compositions of fatty acids (Chowdhury, Raman, & Mian, 1997), carbohydrates (Rahman, Nahar, Jabbar, & Mosihuzzaman, 1999), aroma volatiles (Swords, Bobbio, & Hunter, 1978;Rasmussen, 1983;K. Wong, Lim, & L. Wong, 1992;Maia, Andrade, & Zoghbi, 2004), carotenoids and phenolics (Chandrika, Jansz, & Warnasuriya, 2005;Jayasinghe, Rupasinghe, Hara, & Fujimoto, 2006;Faria, Rosso, & Mercadante, 2009) from fruits of different jackfruit varieties, as well as antioxidant capacity and phenol content in jackfruit fruit pulp (Jagtap, Panaskar, & Bapat, 2010).For example, an early study found little changes in fruit total acidity during ripening (Bhatia, Siddapa, & Lal, 1955).A more recent study reported significant changes in acidity, color, total soluble solids and total sugars in ripening jackfruit (Ong et al., 2006).The inhibitor of the plant hormone ethylene, 1-methylcyclopropene, was found to significantly delay the ripening process and extend the shelf life for 8-12 days (Mata-Montes, Oca, Osuna- Garcia, & Hemandez-Estrada, 2007), implicating the role of ethylene in fruit ripening in jackfruit.Study on soft and hard types of jackfruit suggested that while both fruit types shared a similar ripening pattern, the soft type exhibited a greater extent of microscopic and chemical changes, which may contribute to its unique textural features (Rahman, Huq, Mian, & Chesson, 1995).
Sweetness is a major determinant of edible quality of fruits, and depends largely on the type, composition and amount of sugars in fruit.In jackfruit, sucrose, glucose and fructose constitute the major proportion of free sugars (Chowdhury et al., 1997).It is well known that sucrose synthase (SuSy, EC 2.4.1.13)is a bi-functional enzyme that involves both sucrose synthesis and sucrose hydrolysis to glucose and fructose.Sucrose phosphate synthase (SPS,EC 2.4.1.14)and sucrose synthetic SuSy are generally considered the major players in sucrose synthesis using glucose-phosphate and triose-phosphate released from starch breakdown during fruit ripening.On the other hand, both SuSy and invertases (IV, EC 3.2.1.26)can catalyze sucrose hydrolysis to glucose and fructose.
Although previous studies have examined changes in sugar contents during ripening of jackfruit (Chowdhury et al., 1997;Rahman et al., 1999;Ong et al., 2006), there is no published study that correlates sugar contents with responsible sugar metabolic enzymes during ripening process.In order to better understand the mechanisms underlying sucrose metabolism during jackfruit ripening, we investigated changes in enzyme activities of SPS, SuSy and IV along with sucrose, glucose, fructose and total soluble sugar accumulation during fruit ripening in both soft and firm varieties of jackfruit.Understanding of the biochemical kinetics associated with sugar accumulation during fruit ripening will help select, cultivate and breed better varieties of jackfruit to achieve its potential as a valuable major food and fruit crop for the near future.

Plant Materials
Eight varieties of jackfruit (Artocarpus heterophyllus Lam.) were obtained from a commercial farm in Zhanjiang, Guangdong province, China, including 4 varieties of soft type (12As, 12Cs, 13Bs and 13Ls) and 4 varieties of firm type (12E, 13D, 13K and 148-4).Mature fruits that produced a dull, hollow sound when tapped were collected from each variety and immediately transported to the laboratory at Guangdong Ocean University.Fruits were then allowed to ripen at ambient temperature (±28 C; 70-75% RH).
For sampling, fruits were first grouped into four ripening stages, ripening stage I (mature fruit), ripening stage II (fruit pulp started to soften), ripening stage III (fruit pulp started to form aroma substances) and ripening stage IV (fully ripened), and then were cleaned and cut into two halves.The fruit bulbs from middle portion were deseeded and 10 samples weighing about 1 g each were collected.Three fruits at each stage were sampled for each variety.The sampled fruit pulps were stored in a freezer (-80 C) until further analysis.

Determination of Sugar Contents
Soluble sugars were measured according to Li, Liu, Zhu and Yang (2014b).Approximately 3 g pulp randomly mixed from 3 samples of each fruit was extracted three times in 4 ml 80% ethanol (v:v).Each time, homogenate was centrifuged at 4 C and 15,000×g for 10 min and supernatant was collected.Supernatant was then evaporated using a rotary vacuum evaporator (RE-2000A, PUNA, China) at 90 C to a final volume of about 1 ml, and then was added ultrapure water to make the final volume to 30 ml.Five milliliters of the solution were filtered through 0.45 μm filter membrane and stored in vials for further analysis.
Contents of soluble sugars were analyzed by PerkinElmer Series 200 HPLC Systems equipped with a PE200 refractive index detector and an AT-130 column oven (AUTO SCIENCE, China), using the CARBOSep CHO-820 Calcium column (Transgenomic, USA).Ultrapure water was used as a mobile phase and the flow rate was 0.5 ml/min.Ten microliters of the extracted sample were injected and the temperature of the column was 90 C.Sucrose, glucose and fructose were identified by their retention times and quantified by comparing peak areas of the samples with standards.Data were expressed in g/100 g of pulp fresh weight.
Total soluble sugar content was measured based on the anthrone method (Irigoyen, Emerich, & Sanchez-Diaz, 1992).Five milliliters of 0.2% anthrone were added to 1 ml of the sample solution.The reaction was carried out in boiling water for 10 min, and terminated by incubating the mixture on ice for 5 min.Total soluble content was calculated by creating a standard curve using a standard glucose and was expressed in g/100 g fresh weight.
SuSy sucrose synthesis activity was measured in 1 ml reaction mixture containing 0.4 ml reaction buffer [100 mM Tris-HCl (pH 7.0), 10 mM fructose, 5 mM magnesium acetate, 5 mM DTT], 0.1 ml 10 mM UDP-glucose, 0.05 ml freshly desalted extract and 0.45 ml ultrapure water.Measurement for SPS activity in saturated conditions was carried out in 1 ml reaction mixture containing 0.4 ml reaction buffer [100 mM Tris-HCl (pH 7.0), 10 mM fructose-6-P, 5 mM magnesium acetate, 5 mM DTT], 0.1 ml 10 mM UDP-glucose and 0.05 ml freshly desalted extract.Reaction mixtures were incubated at 30 C for 10 min and then terminated by placing reaction tubes in boiling water for 3 min.The released sucrose was measured based on the anthrone method (Irigoyen et al., 1992).Data were expressed in mg sucrose produced per hour per gram of fresh pulp.
The SuSy sucrose hydrolytic activity was measured in 1 ml reaction mixture containing 0.4 ml reaction buffer [100 mM Tris-HCl (pH 7.0), 100 mM sucrose, 10 mM UDP], 0.05 ml freshly desalted extract and 0.55 ml ultrapure water.The neutral IV activity was measured by adding 0.2 ml desalted extract to 0.8 ml reaction buffer made of 80 mM potassium acetate buffer (pH 7.0) containing 100 mM sucrose.The acid IV activity was measured by adding 0.2 ml desalted extract to 0.8 ml reaction buffer made of 80 mM potassium acetate buffer (pH 4.5) containing 100 mM sucrose.Reaction mixtures were incubated at 37 C for 1 hr and stopped by boiling for 3 min.The amount of reducing sugars produced from sucrose was determined using 3,5-dinitrosalicylic acid method (Luchsinger & Cornesky, 1962).Data were expressed in g reducing sugar produced per hour per gram of fresh pulp.

Statistical Analysis
Data collected was subjected to one-way analysis of variance (ANOVA) using the SPSS statistical software (version 13.0, USA).
The difference between contents of total soluble sugar and free sugar (sucrose, glucose and fructose) among different varieties may be related to the process of cell wall decomposition during fruit ripening, which causes the release of saccharides (Rees, Dixon, Pollock, & Franks, 1981;Rahman et al., 1995) as well as residual soluble starch stored in vacuole and plastids.Previous studies reported a wide range of glucose vs fructose ratio, from 0.5 to 42, in ripe fruits for both soft and firm type varieties (Rahman et al., 1995;Chowdhury et al., 1997;Rahman et al., 1999;Ong et al., 2006).We found that the glucose:fructose ratio for the 8 jackfruit varieties tested ranged from 0.9 to 1.2, suggesting a similar content for these two sugars.Therefore, we believe that the glucose:fructose ratio is probably ripening stage-and variety-specific rather than type (soft or firm) specific.
Sugar accumulation during fruit ripening probably results from increased starch hydrolysis and decreased respiration-associated sugar breakdown, as reported in guava (Bashir & Abu-Goukh, 2003), jackfruit (Li et al., 2014a;Rahman et al., 1995), mangoes (Simäo et al., 2008;Castillo, Kruger, & Whatley, 1992) and banana (S.Chacon, Viquez, G. Chacon, 1987;Garcia & Lajalo, 1988).A likely scenario during fruit ripening of jackfruit is that glucose is first accumulated from starch hydrolysis, followed by glucose and fructose accumulation as a result of sucrose hydrolysis.This is supported by the facts that more glucose than fructose was measured at ripening stage I in all varieties we tested (Figure 2B).When fruits reached the stage of fully ripe, starch hydrolysis finished and sucrose hydrolysis caused the molar ratio of glucose versus fructose to be close to one as observed in our study.
A direct correlation between sucrose accumulation and increased enzyme activity of SPS has been reported for typical climacteric fruits tomato (Yelle, Chetelat, Dorais, De Verna, & Bennett, 1991;Dali, Michaud, & Yelle, 1992) and muskmelon (Hubbard, Pharr, & Huber, 1989), but not for banana (Fils-Lycaon et al., 2011).Our study did not find such a clear correlation for most of the jackfruit varieties tested (Figures 1 and 3).Considering that SPS activity in most of the jackfruit varieties was lower than SuSy sucrose synthetic activity (Figure 3), we propose that SuSy is the major enzyme contributing to sucrose synthesis and accumulation during fruit ripening, whereas SPS may play a minor role, at least for the jackfruit varieties we have tested.However, the precise biochemical mechanism for sucrose accumulation in jackfruits is still unknown.
Among the three enzymes responsible for sucrose hydrolysis, AIV was more active than NIV and NIV was more active than SuSy throughout the four ripening stages (Figures 3 and 4), suggesting AIV probably serving a primary role, followed by NIV, in hydrolyzing sucrose in jackfruit during fruit ripening.One interesting observation was a conspicuous peak AIV activity during fruit ripening of soft varieties, but not so obvious for firm varieties (Figure 4).Since the AIV enzymes in our study were extracted using the gentle conditions without adding salt and thus were most likely a vacuolar form, we speculate that the AIV peak activity observed only in soft type jackfruit (Figure 4) could be the cell wall bounded AIV form, released by cell wall degradation during fruit ripening of soft type jackfruit (Li et al., 2014a;Rahman et al., 1995), which is not likely to occur actively in the firm type jackfruits.
In conclusion, the results of this study support the conclusions that sucrose is the major sugar contributing to sweetness of jackfruit, followed by fructose and glucose.Although the glucose:fructose ratio varies during ripening process, it reaches close to one in ripe fruits.Our study also reveals that among the enzymes examined, AIV is more active than NIV for sucrose hydrolysis, while SuSy probably plays a more important role than SPS in sucrose synthesis and accumulation during fruit ripening.We propose that in early stages of fruit ripening, glucose is released from starch hydrolysis followed by sucrose hydrolysis that contributes to increase in levels of both glucose and fructose.In ripe fruit, while sucrose is still the dominant form of the free sugar, its elevated hydrolysis results in almost equal amount of glucose and fructose.
Figure underlined II: fruit pu