Impact of Precooling and Controlled-Atmosphere Storage on γ

Jul 14, 2016 - Impact of Precooling and Controlled-Atmosphere Storage on ... However, GABA aminotransferase (GABA-T; EC 2.6.1.19) activities in elevat...
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Impact of Precooling and Controlled-Atmosphere Storage on γ‑Aminobutyric Acid (GABA) Accumulation in Longan (Dimocarpus longan Lour.) Fruit Molin Zhou, Kessy H. Ndeurumio, Lei Zhao,* and Zhuoyan Hu* College of Food Science, South China Agricultural University, Guangzhou 510642, People’s Republic of China S Supporting Information *

ABSTRACT: Longan (Dimocarpus longan Lour.) fruit cultivars ‘Chuliang’ and ‘Shixia’ were analyzed for γ-aminobutyric acid (GABA) accumulation after precooling and in controlled-atmosphere storage. Fruit were exposed to 5% O2 plus 3%, 5%, or 10% CO2 at 4 °C, and GABA and associated enzymes, aril firmness, and pericarp color were measured. Aril softening and pericarp browning were delayed by 5% CO2 + 5% O2. GABA concentrations and glutamate decarboxylase (GAD; EC 4.1.1.15) activities declined during storage at the higher-CO2 treatments. However, GABA aminotransferase (GABA-T; EC 2.6.1.19) activities in elevated CO2-treated fruit fluctuated during storage. GABA concentrations increased after precooling treatments. GAD activity and GABA-T activity were different between cultivars after precooling. GABA concentrations in fruit increased after 3 days of 10% CO2 + 5% O2 treatment and then declined as storage time increased. GABA accumulation was associated with stimulation of GAD activity rather than inhibition of GABA-T activity. KEYWORDS: longan fruit, Dimocarpus longan Lour., γ-aminobutyric acid (GABA), controlled-atmosphere storage, carbon dioxide, CO2, precooling

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salt stress, heat or cold shock, hypoxia, and drought.22,23 In addition, GABA can accumulate in response to elevated CO2 in some plants, including strawberry, tomato, and bean sprouts.24−26 Stress can promote GAD activity and decrease GABA-T activity, resulting in GABA accumulation.27,28 Limited information is available on GABA accumulation in CO2-treated horticultural products after harvest. Blanch et al. found that strawberry fruit stored with 40% CO2 at 0 °C for 3 days showed a sharp increase in GABA levels, coinciding with a rapid decline in glutamate levels, which suggests that elevated-CO2 storage increased glutamate conversion to GABA by the GABA shunt pathway.29 GABA accumulation was also found in Crisphead lettuce stored at 15% or 20% CO2, in which there was a higher incidence of CO2 injury,30 a physiological disorder that can cause serious losses of fruits and vegetables during controlledatmosphere storage.31 The injury is expressed as pericarp browning and aril softening.32 GABA accumulated in a linear fashion over the storage period in apple fruit, regardless of 1methylcyclopropene and CO2 treatment. However, apple fruit treated with 2.5 kPa CO 2 and 1-methylcyclopropene accumulated twice as much GABA as fruit receiving only 0.03 kPa CO2. This study attributed GABA accumulation to the enhancement of GAD activity via lower cytosolic pH or bound Ca2+−calmodulin and product inhibition of GABA-T activity due to the restricted activity of SSADH activity in elevated CO2 storage.33 GABA accumulated in CO2-treated tomato, but the

INTRODUCTION Longan (Dimocarpus longan Lour.) fruit has a short postharvest shelf life of 3 or 4 days at ambient temperature.1 However, the fruit is relatively tolerant of elevated CO2 (6%−8%) and reduced O2 (4%−6%) in controlled-atmosphere storage, and these concentrations are routinely used by the longan industry to reduce pericarp browning and maintain aril firmness.2 γ-Aminobutyric acid (GABA), a four-carbon nonprotein amino acid, is widely distributed in vertebrates, bacteria, and plants, although it is usually present only in small amounts. In plants and bacteria, it is a metabolite involved in bypassing two steps of the Krebs cycle3,4 and is synthesized by the irreversible α-decarboxylation of L-glutamic acid or its salts, catalyzed by glutamate decarboxylase (GAD; EC 4.1.1.15). GABA is catabolized by the irreversible transamination reaction of GABA to succinic semialdehyde (SSA), catalyzed by mitochondrial enzymes of GABA aminotransferase (GABA-T; EC 2.6.1.19). Subsequently, SSA is catabolized by the irreversible NADP-dependent oxidation of SSA to succinate, catalyzed by succinate−semialdehyde dehydrogenase (SSADH; EC 1.2.1.24). Alternatively, SSA is reduced by the irreversible NADPH-dependent reduction of SSA to γ-hydroxybutyric acid (GHB), catalyzed by succinic semialdehyde reductase (SSAR; EC 1.1.1.n11).5−9 In vertebrates, it acts as a central inhibitory transmitter in the central nervous system, acting by means of a hyperpolarization and shunting inhibition to reduce ongoing activity.10−12 In addition, GABA has been shown to affect anxiety, depression, anaphylaxis, cancer, and hypertension and to have effects on natriuresis and regulation of hormone secretion.13−21 Accumulation of GABA in plants is a mediating abiotic stress response to many factors, including acidosis, mechanical injury, © XXXX American Chemical Society

Received: April 17, 2016 Revised: June 14, 2016 Accepted: July 14, 2016

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DOI: 10.1021/acs.jafc.6b01738 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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units of GABase/mL, 50 μL of 20 mM 2-oxoglutarate, and 550 μL of sample. GABase lyophilized powder was added to 0.1 M potassium phosphate buffer (pH 7.2) containing 5 mM 2-mercaptoethanol and 12.5% glycerol. The resulting solution was divided into 1 mL aliquots, each containing 2 units of activity. The reduction of NADP+ to NADPH was monitored at 340 nm before and after adding 2oxoglutarate for 10 min at 25 °C using a UV−vis spectrophotometer (UVmini-1240; Shimadzu, Kyoto, Japan). GABA concentration was calculated by NADPH produced (Mmol of GABA per kilogram of fresh weight). GAD and GABA-T Activity Determination. Enzyme was extracted in a buffer containing 1 mM dithiothreitol (DTT), 2 mM ethylenediaminetetraacetic acid (EDTA), 0.5 mM phenylmethylsulfonyl fluoride (PMSF),1 mM pyridoxal 5′-phosphate (PLP), 0.1 M Tris−HCl buffer (pH 9.1), and 10% (v/v) glycerol. Powder (2 g) was added to 10 mL of the precooled extraction buffer and shaken for 10 min. The mixture was filtered using Miracloth and centrifuged at 20000g at 4 °C for 20 min. The supernatant was used to analyze GAD and GABA-T activity. GAD activity was estimated according to Bartyzel et al.38 with some modifications. The activity was estimated by incubating at 30 °C in a 400 μL assay system containing 3 mM L-glutamate, 40 μM PLP, 0.25 M potassium phosphate buffer (pH 5.8), and 20 μL of crude extract. The enzyme was preincubated in the mixture without L-glutamate at 30 °C for 10 min. The reaction was started by adding L-glutamate and terminated after 60 min by adding 0.1 mL of 0.5 M muriatic acid. The mixture was centrifuged at 12500g at 4 °C for 10 min. The GABA concentration in supernatant was measured as described above. GAD activity was estimated in GABA produced (Mmol of GABA per kg of protein mass per s). GABA-T activity was estimated according to Ansari et al.39 with some modifications. The activity was assayed by incubating at 30 °C in a 500 μL assay system containing 4 mM pyruvate, 1.5 mM DTT, 0.1 mM PLP, 0.75 mM EDTA, 15 mM GABA, 50 mM Tris−HCl buffer (pH 8.2), 10% (v/v) glycerol, and 20 μL of crude extract. The enzyme was preincubated in the mixture without pyruvate at 30 °C for 10 min. The reaction was started by adding pyruvate and terminated after 60 min by adding 50 μL of 40 mM sulfosalicylic acid. The alanine (Ala) produced during the incubation was measured by enzymatic reaction with alanine dehydrogenase (ADH; EC 1.4.1.1). ADH assay was assayed in a 500 μL assay system containing 0.1 unit of ADH, 1.5 mM NAD+, 70 mM sodium carbonate buffer (pH 10), and 20 μL of terminated sample. The enzyme was preincubated in the mixture without terminated sample at 25 °C for 20 min. The reaction was started by adding terminated sample and was then incubated at 25 °C for 20 min. The increase in absorbance was then monitored at 340 nm by UV−vis spectrophotometer. GABA-T activity was calculated as Ala produced (Mmol of Ala per kilogram of protein mass per s.) Protein concentration in the enzyme extract was measured according to the method described by Bradford40 using bovine serum albumin (BSA) as the protein standard. Instrumental Texture Measurement. The fruit samples were equilibrated to ambient conditions for 1 h prior to the texture measurement. For the Kramer shear test, longan aril slices (approximately 20 mm × 20 mm × 5 mm) were tested using a texture analyzer (TA500; Lloyd Instruments, Fareham, UK) with a Kramer cell attachment. Aril firmness was measured at a cross-head speed of 100 mm·min−1. The data was expressed as maximum peak force (N peak force per g of aril sample). Pericarp Color Measurement. The color of longan pericarp was measured at three locations for each fruit by spectrocolorimeter (CM3500d; Minolta, Osaka, Japan). Statistical Analysis. Experimental design was a completely randomized design. All data were analyzed by one-way analysis of variance (ANOVA) using SPSS statistical software (version 19.0; SPSS Inc., Chicago, IL) and presented as the means ± standard deviation (SD) of three independent experiments. Mean separation was performed using Duncan’s multiple-range tests (P < 0.05).

GABA concentrations obtained were affected by the maturation stage.25 Postharvest precooling is important in the cold chain for longan fruit and can provide effective temperature management and remove field heat during subsequent storage or shipment.34 GABA accumulation in response to cold stress in soybean,35 barley, and wheat36 has been reported. There have been no studies reporting on GABA accumulation in longan fruit under high-CO2 controlled-atmosphere storage. Hence, the main objective of the present study was to analyze the GABA accumulation associated with the tolerance of fruit to high CO2 levels, as indicated by GABA metabolism in longan fruit. We examined GABA concentration and the enzymes responsible for GABA metabolism in two longan fruit cultivars exposed to high CO2. In addition, we compared aril firmness and pericarp color in response to high CO2. The possibility that postharvest precooling would accumulate GABA in longan fruit was also investigated. Our goal was to improve our understanding of GABA accumulation as affected by high CO2 concentrations during precooling and controlled-atmosphere, low-temperature storage.

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MATERIALS AND METHODS

Plant Material and Treatments. Longan (D. longan Lour. cultivars ‘Chuliang’ and ‘Shixia’) fruit were obtained from a commercial market in Conghua, China. Fruits were manually harvested on August 2014 at the maturity stage and immediately transported to the laboratory in Guangzhou within 2 h. On arrival, fruits were sorted to ensure uniformity in color and size and absence of any damaged fruits and then hydro-cooled in iced water at approximately 4 °C for 2 h. Longan fruits from two cultivars (12 kg of ‘Chuliang’ and 12 kg of ‘Shixia’) were randomly divided into eight groups of 3 kg (with no mixing between the cultivars), comprising three replicate samples of 1 kg. To study the effect of controlledatmosphere storage, the fruits were placed in 24 single controlledatmosphere chambers (∼8 L each). The chambers were connected to a continuous flow of gas system with humidified air or a gas mixture containing 3% CO2 + 5% O2 + 92% N2, 5% CO2 + 5% O2 + 90% N2, or 10% CO2 + 5% O2 + 85% N2 at a flow rate of 100 mL·min−1 and monitored daily by headspace gas analyzer (CheckMate 9900; PBIDansensor, Ringsted, Denmark). All of the chambers were stored in a refrigerated warehouse at 4 °C for 18 days; it was connected via rubber catheter to the gas system, located outside the refrigerated warehouse. To avoid the impact of modified atmosphere on the fruits, the gas was kept as a continuous flow in all chambers during the storage. Samples were collected from nine fruits at a period of 3 days, and each fruit was cut in half vertically. The entire first half was immediately ground in liquid nitrogen to a fine powder and analyzed for GABA and enzymes responsible for GABA metabolism. All of the second half of the fruit was taken for instrumental texture measurement and pericarp-color measurement. A total of three independent replicate experiments were conducted. GABA Determination. GABA concentration in longan aril was determined using the method described by Zhang and Bown37 with some modifications. Powder (0.1 g) was added to a 1.5 mL Eppendorf tube containing 400 μL of methanol, mixed by vortex mixer, and then extracted at 25 °C for 10 min. The extract was vacuum freeze-dried with a lyophilizer (Heto PowerDry PL3000; Thermo Fisher Scientific, Waltham, MA). The dried sample was added to 1 mL of 70 mM lanthanum chloride, followed by shaking for 15 min and centrifugation at 13000g for 5 min. The supernatant (0.8 mL) was removed to a second Eppendorf tube and mixed with 160 μL of 1 M potassium hydroxide. The mixture was then shaken for 3 min and centrifuged as before. The supernatant was used in the spectrophotometric GABA determination using a GABase (G7509; Sigma-Aldrich, St. Louis, MO). The 1 mL assay system contained 150 μL of 4 mM NADP+, 200 μL of 0.5 M potassium pyrophosphate buffer (pH 8.6), 50 μL of 2 B

DOI: 10.1021/acs.jafc.6b01738 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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RESULTS Precooling Treatment. GABA concentrations increased in both cultivars after precooling treatment of longan fruits and to a greater extent in ‘Chuliang’ than in ‘Shixia’ fruits (Table 1). A

slight increase occurred in 3% CO2 + 5% O2 by day 9. For ‘Shixia’ fruit, GABA concentration increased in 10% CO2 + 5% O2 during the first 3 days, being 19.72 ± 0.64 mmol·kg−1 by day 3, and then decreased sharply until day 9 but to a relatively smaller extent after day 9 than before. The concentrations in three other treatments decreased smoothly during storage. These data suggested that, using controlled-atmosphere storage to obtain the largest GABA concentration of fresh longan aril, the optimized parameter was 10% CO2 + 5% O2 in ‘Shixia’ fruit. GABA Shunt Enzyme Activities. GAD activity had different changes in the various controlled-atmosphere storage of both cultivars (Figure 2). For ‘Chuliang’ fruit, GAD activity increased in 5% CO2 + 5% O2 and 10% CO2 + 5% O2 during the first 3 days and up to the height of activity, being 17.13 ± 0.59 and 19.14 ± 0.32 mmol·kg−1·s−1 by day 3, respectively, and then sharply decreased from day 3 to day 6, but a relatively slow decline occurred after day 6. The activity in 3% CO2 + 5% O2 and air decreased sharply during the first 3 days, followed by a relatively slow decline, and a slight increase occurred in air from day 9 to day 15. For ‘Shixia’ fruit, GAD activity in all treatments had increased during the first 3 days and showed the largest activity in 10% CO2 + 5% O2 by day 3, being 14.83 ± 0.61 mmol·kg−1·s−1. After that, the activity in all treatments generally decreased in the rest of the storage period. These data suggested that GAD activity was approximately the same as the change trend of GABA concentration, perhaps because GAD catalyzes the synthesis of GABA as the most important synthetase by the irreversible α-decarboxylation of L-glutamic acid or its salts. GABA-T activity in all treatments of both cultivars fluctuated during storage. The activity in 3% CO2 + 5% O2 was the largest activity in all treatments, while the activity in 10% CO2 + 5% O2 remained at generally lower levels during storage compared with those of three other treatments. These suggested there was a correlation between GABA-T activity and GABA concentration. The higher CO2 concentration helped to improve GAD activity and inhibit GABA-T activity to increase GABA concentration. Texture Analysis. Aril firmness generally declined during storage of both cultivars, though with some fluctuations, and a relatively slow decline occurred in 5% CO2 + 5% O2 treated fruit (Figure 3). For ‘Chuliang’ fruit, the firmness had the largest decline in 10% CO2 + 5% O2 treated fruit, from 18.86 ± 0.31 to 15.78 ± 0.26 N·g−1. For ‘Shixia’ fruit, the firmness was higher than for ‘Chuliang’ at the start or end of the storage. The

Table 1. GABA Concentration, GAD Activity, GABA-T Activity, and Aril Firmness of ‘Chuliang’ and ‘Shixia’ Longan Fruit Precooled in Iced Water at Approximately 4 °C for 2 h GABA (mmol·kg−1) Chuliang Shixia GAD (mmol·kg−1·s−1) Chuliang Shixia GABA-T (mmol·kg−1·s−1) Chuliang Shixia firmness (N·g−1) Chuliang Shixia

untreated

precooling

7.18 ± 1.02 b 11.59 ± 0.80 b

13.58 ± 0.81 a 14.01 ± 0.65 a

6.47 ± 0.56 b 11.06 ± 0.49 a

16.23 ± 0.63 a 7.07 ± 0.53 b

49.57 ± 2.29 a 47.27 ± 1.98 a

48.51 ± 2.13 a 28.73 ± 1.83 b

18.20 ± 0.36 a 19.03 ± 0.40 a

18.86 ± 0.31 a 19.82 ± 0.35 a

The data are presented as the means ± SD of the three replicates, and the different letters within rows indicate significant differences at P < 0.05.

a

pronounced increase in GAD activity occurred in ‘Chuliang’; the GAD activity of precooling treated fruit was 2.51-fold higher than of untreated fruit in ‘Chuliang’, whereas the GAD activity of ‘Shixia’ had decreased markedly by 1.56-fold. GABAT activity remained unchanged in ‘Chuliang’, while GABA-T activity in precooling-treatment ‘Shixia’ fruit was 1.65-fold lower than in untreated fruit. There were no significant changes in aril firmness in both cultivars. These data suggested the influence of precooling on GABA metabolism related to the longan cultivars. GABA Concentration. GABA concentration had different change in the various controlled atmosphere stored of both cultivars (Figure 1). For ‘Chuliang’ fruit, GABA concentration increased in 5% CO2 + 5% O2 and 10% CO2 + 5% O2 during the first 3 days of storage and up to the height concentration, being 15.70 ± 0.49 and 18.53 ± 0.92 mmol·kg−1 by day 3, respectively, and then decreased after day 3 until day 12 but remained relatively stable after day 12. The concentrations decreased in 3% CO2 + 5% O2 and in air during storage, when a

Figure 1. GABA concentration of ‘Chuliang’ and ‘Shixia’ longan fruit stored in 3%, 5%, or 10% CO2 in 5% O2 or in air at 4 °C for 18 d. C

DOI: 10.1021/acs.jafc.6b01738 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Figure 2. GAD activity (based on GABA production per kg of protein per second) and GABA-T activity (based on alanine production per kg of protein per second) of ‘Chuliang’ and ‘Shixia’ longan fruit stored in 3%, 5%, or 10% CO2 in 5% O2 or in air at 4 °C for 18 d.

Figure 3. Aril firmness of ‘Chuliang’ and ‘Shixia’ longan fruit stored in 3%, 5%, or 10% CO2 in 5% O2 or in air at 4 °C for 18 d.

firmness was the largest decline in 10% CO2 + 5% O2 treated fruit, from 19.82 ± 0.35 to 16.50 ± 0.30 N·g−1. These data suggested that using the controlled-atmosphere storage can delay longan aril softening and that the optimized parameter was 5% CO2 + 5% O2. However, a higher CO2 level would cause CO2 injury. Pericarp Color. The overall lightness (L*), yellowness (b*), and hue angle (ho) of both cultivars decreased during controlled-atmosphere storage and, to a relatively smaller extent, in fruit with 5% CO2 + 5% O2 treatment than in the three other treated fruits (Table 2). These data suggested that using the controlled-atmosphere storage in 5% CO2 + 5% O2 can delay longan pericarp browning, and higher CO2 level would cause a higher incidence of CO2 injury.

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oxidative stress, regulation of osmotic balance, maintenance of cytosolic pH, and transient nitrogen storage.6,26 We studied the effects of controlled-atmosphere storage on longan cultivars ‘Chuliang’ and ‘Shixia’. Decay of 5% CO2 + 5% O2 treated fruit was inhibited to a greater extent than in fruit exposed to the higher CO2 concentration and the lower CO2 concentration and air-treated fruit, as judged by CIE L*, b*, and ho values. CO2 injury of 10% CO2 + 5% O2 treated fruit was more serious to longan aril than to 5% CO2 + 5% O2 treated fruit, as judged by measurement of aril firmness. GABA concentrations generally decreased during storage in all treatments in both ‘Chuliang’ and ‘Shixia’ but only following a large transient increase during the first 3 days in 10% CO2 + 5% O2 for both cultivars. The higher CO2 concentration coincided with the larger accumulation of GABA, the pattern of which could be attributed to the higher CO2 concentration lowering the cytosolic pH and then stimulating GAD activity and inhibiting GABA-T activity, thereby resulting in accumulated GABA. However, these findings on GABA accumulation are at odds with previous studies of other horticultural

DISCUSSION

GABA is an important amino acid in longan fruit during development and reaches peak values at the third day of highCO2 controlled-atmosphere storage before declining as storage time is extended. GABA may be involved in protection against D

DOI: 10.1021/acs.jafc.6b01738 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Table 2. Lightness (L*), Yellowness (b*), and Hue Angle (ho) of ‘Chuliang’ and ‘Shixia’ Longan Fruit Stored in 3%, 5%, or 10% CO2 in 5% O2 or in Air at 4 °C for 18 d Chuliang storage time (day) lightness (L*) 0 3 6 9 12 15 18 yellowness (b*) 0 3 6 9 12 15 18 hue angle (ho) 0 3 6 9 12 15 18 a

3% CO2 + 5% O2

5% CO2 + 5% O2

Shixia

10% CO2 + 5% O2

3% CO2 + 5% O2

air

5% CO2 + 5% O2

10% CO2 + 5% O2

air

39.8 37.1 36.7 35.1 34.6 33.2

± ± ± ± ± ±

1.2 1.9 1.9 1.3 1.1 0.9

a a a ab ab b

39.1 38.4 37.3 36.9 35.9 35.5

± ± ± ± ± ±

41.6 ± 0.9 2.1 a 39.5 ± 2.0 a 1.3 a 38.2 ± 1.2 a 1.3 a 36.7 ± 1.4 a 2.0 a 36.1 ± 1.1 a 1.6 a 34.7 ± 1.0 ab 1.0 a 33.6 ± 1.0 ab

38.9 36.3 35.4 33.2 33.1 32.2

± ± ± ± ± ±

1.6 1.4 1.5 0.9 1.3 1.5

a a a b b b

43.6 41.1 40.6 38.0 36.3 34.5

± ± ± ± ± ±

0.1 1.8 1.6 1.9 1.1 1.3

a a a a ab ab

43.4 42.1 40.4 39.1 38.0 37.1

± ± ± ± ± ±

45.1 ± 0.7 2.0 a 43.1 ± 1.1 a 2.1 a 42.6 ± 1.2 a 1.6 a 40.2 ± 1.4 a 0.9 a 38.6 ± 1.1 a 1.2 a 36.8 ± 0.8 ab 1.3 a 35.1 ± 1.4 ab

43.1 41.0 38.7 37.6 35.0 34.2

± ± ± ± ± ±

0.8 1.0 2.3 1.4 1.7 1.9

a a a a b b

25.6 24.5 22.6 20.3 20.9 19.4

± ± ± ± ± ±

1.3 0.8 1.2 1.3 1.1 0.5

a ab b b bc bc

26.6 26.1 25.0 24.1 23.8 23.2

± ± ± ± ± ±

27.6 ± 1.4 0.8 a 25.8 ± 0.6 a 0.7 a 23.9 ± 1.2 b 1.1 a 22.2 ± 0.8 b 0.8 a 21.9 ± 0.8 b 0.8 a 21.7 ± 1.2 b 0.8 a 20.8 ± 0.5 b

25.3 24.5 22.4 20.2 19.3 18.4

± ± ± ± ± ±

1.0 0.7 1.0 1.2 0.8 1.3

a ab b b c c

29.0 27.0 25.8 23.8 23.7 21.8

± ± ± ± ± ±

1.9 2.4 1.0 0.7 0.9 1.2

a a ab b b b

29.4 28.8 27.7 26.4 25.9 25.2

± ± ± ± ± ±

32.2 ± 1.0 1.4 a 28.5 ± 1.4 a 1.6 a 28.0 ± 1.3 a 0.8 a 26.4 ± 0.8 ab 1.3 a 25.2 ± 0.6 ab 1.1 a 23.8 ± 1.6 b 0.5 a 22.2 ± 1.1 b

28.1 25.7 25.2 23.4 22.6 19.1

± ± ± ± ± ±

1.3 1.3 2.1 1.9 0.3 1.2

a a b b b c

63.4 60.3 59.4 57.8 54.2 52.1

± ± ± ± ± ±

0.6 2.5 1.3 2.2 2.1 0.2

a a ab ab b c

64.7 63.0 62.3 60.5 59.2 57.6

± ± ± ± ± ±

65.6 ± 2.5 1.5 a 63.0 ± 2.4 a 2.7 a 61.0 ± 1.9 a 1.7 a 60.4 ± 2.2 ab 1.7 a 57.2 ± 2.1 ab 1.9 a 55.6 ± 1.1 b 1.5 a 54.7 ± 0.3 b

62.1 60.4 58.3 56.7 53.3 52.8

± ± ± ± ± ±

1.9 2.8 1.5 1.0 1.7 1.9

a a b b b bc

67.4 66.1 62.3 60.5 58.5 57.0

± ± ± ± ± ±

1.3 1.1 1.8 2.4 2.0 1.6

a a a b b bc

68.8 67.1 65.2 66.0 64.5 63.4

± ± ± ± ± ±

71.8 ± 0.4 1.1 a 67.3 ± 0.5 a 1.1 a 66.2 ± 1.4 a 2.1 a 64.6 ± 1.9 a 0.8 a 63.1 ± 2.7 ab 1.4 a 60.5 ± 1.6 b 0.7 a 58.8 ± 1.6 b

67.1 65.3 62.7 60.3 57.8 55.6

± ± ± ± ± ±

2.0 1.8 1.6 1.8 0.5 1.6

a a a b b c

The data are presented as the means ± SD of the three replicates, and the different letters within rows indicate significant differences at P < 0.05.

accumulation in longan fruit increased initially and then decreased, while the pattern showed continued decline in low-CO2 and ambient-air atmospheres. Importantly, the general trend of GABA accumulation in longan fruit was not influenced by cultivar. These findings were inconsistent with previous studies of strawberries (relatively stable or slowly increasing, depending on cultivar), tomato (continuously decreasing or generally increasing, depending on maturity stages), and apple (continuously increasing),24,25,33 which indicates that the pattern of GABA accumulation is different in each of the species in response to elevated-CO2 storage. This suggests that there may be an unknown pathway of GABA accumulation that may be species-dependent. However, this suggestion is inconsistent with the generally accepted view that GABA accumulates when plants respond to abiotic stress. GABA accumulation could be a transient mediating abiotic stress response in plants, and it continuously decreases after transiently accumulating; the time of transient accumulation may be a few minutes or several weeks, depending on the type or intensity of abiotic stress and the abiotic-stress-resistance of the species. Regarding the increased GABA concentrations in fruits, in longan, it increased from 14.01 to 19.72 mmol·kg−1 (1.4-fold) and was much lower than in strawberry, cherimoya and apple, from 0.2 to 1.7 mmol·kg−1 (8.5-fold), from 0.05 to 0.45 mmol· kg−1 (9-fold), and from 0.03 to 0.3 mmol·kg−1 (10-fold), respectively.24,41,43 These result in longan might be attributed to the higher initial GABA concentration and the lower concentration of the precursor to GABA (i.e., concentration of L-glutamic acid or its salts; data not shown) at harvest.

products. Deewatthanawong et al. found that GABA accumulation occurred in CO2-treated strawberry fruit of all cultivars but with significant differences among cultivars.24 In apple fruit, regardless of 0.03 and 2.5 kPa CO2 treatment, GABA concentration increased steadily during the 48 day storage period but to a much greater extent under 2.5 kPa CO2 than under 0.03 kPa CO2 treatment.33 Blanch et al. found that GABA accumulation in strawberries treated with 40% CO2 had the highest levels, but differences in GABA accumulation between 20% CO2-treated and air-treated fruit were not statistically significant. 29 Ke et al. found that GABA accumulated in 20% CO2-treated Crisphead lettuce, probably due to partially inhibited succinate dehydrogenase (SDH; EC 1.3.5.1) and substantially activated GAD via lower cytosolic pH.30 GABA concentration in 20% CO2 + 20% O2 treated cherimoya fruit was observed to be higher than air-treated fruit and decreased after transfer to air.41 These findings show that the pattern of GABA accumulation in response to elevated-CO2 storage exhibits a huge difference among various horticultural products. Interestingly, longan fruits contained the highest GABA concentrations among common fruits and vegetables. GABA concentrations in longan fruits reached 1998 mg·kg−1 at harvest, whereas other fruits contained much-lower GABA concentrations, such as litchi (1390 mg·kg−1), tomato (1054 mg·kg−1), and cantaloupe (745 mg·kg−1).42 Few data sources on the pattern of GABA accumulation in longan fruit during storage were available, but reported accumulations in other fruits during storage were much lower compared with longan fruit. Longan fruit also accumulated GABA to a higher level when exposed to elevated-CO2 atmosphere. In high-CO2 atmosphere, the pattern of GABA E

DOI: 10.1021/acs.jafc.6b01738 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Journal of Agricultural and Food Chemistry

optimum pH of cytosolic Ca2+−calmodulin-dependent GAD activity depend on the species; for example, the optimum pHs for Escherichia coli, Lactobacillus brevis, and Neurospora crassa were 3.8, 4.2, and 5.0, respectively.50,56 GAD activity in longan fruit has full activity at pH 5.8 and only around 35% of full activity at pH 7.0. However, slightly increased GAD activities were observed while the incubation time was prolonged to 2 h from 1 h (data not shown). Gut et al. and Shelp et al. found that plant GAD exhibited two relatively independent mechanisms of plant GAD regulation in response to different cellular stresses occurring at different cytosolic pHs: when at neutral pH, the release of Ca2+ induced conformational changes in calmodulin, leading to the binding of GAD and the calmodulin-binding domain and then activated GAD. When at acidic pH, GAD activity was pH-dependent, but release of the calmodulin-binding domain from the active sites was also possible where additional stimulation of GAD activity by Ca2+− calmodulin had a negligible effect.48,49 Recent studies of plant GAD in response to elevated-CO2 storage shed light on the possible regulation of GABA accumulation; elevated-CO2 storage leads to cytoplasm acidification in plant tissues, which is presumably due to the differential disorganization of cellular compartments and the consequent release of apoplastic and vacuolar protons into cytoplasm.57 The pHs of the cytosolic, vacuolar, and apoplastic compartments of the plant cell are 7.2−7.6, 5.8−5.9, and 5.6−6.0, respectively.58 Then, cytoplasm acidification leads to GABA accumulation by activating GAD and simultaneously pH increases as the reaction proceeds. When cytosolic pH reaches neutral, GAD is Ca2+-dependent and mediated by calmodulin binding. GABA-T from various sources is specific for GABA as an amino donor and is a member of the PLP-dependent enzyme family, maximally active at pH 7.8−9.5, and appears to be a homodimer.49,59 GABA-T activity under high-CO2 conditions was generally lower than under low-CO2 conditions, especially in ‘Chuliang’ fruit, suggesting that elevated-CO2 storage in longan fruit could result in decreased GABA-T activity. However, we found no significant statistical correlation between GABA-T activity and GABA catabolism in our current study. This could be due to only measured pyruvate-dependent GABA-T (GABA-TP) activity in this study. Akihiro et al. found that 2-oxoglutarate-dependent GABA-T (GABA-TK) activity was significantly higher than GABA-TP activity, and a significant positive correlation existed between GABA-TK activity and GABA catabolism.60 These results suggested that GABA-TK, rather than GABA-TP, was crucial to GABA catabolism. Shear stress at mechanical fracture was the fundamental texture measurement used to determine aril firmness. In general, 5% CO2 + 5% O2 treatment had the best effect for maintaining aril firmness. In addition, a high CO2 concentration might cause CO2 injury to the longan aril, resulting in morerapid softening. The results for pericarp color were similar to those for aril firmness, i.e., 5% CO2 + 5% O2 treatment had the best effect for delaying longan pericarp browning. Cold stress is a kind of abiotic stress associated with the enrichment of GABA in some plants.61 As a kind of cold stress, precooling increased GABA concentration in both cultivars and, to a greater extent, in ‘Chuliang’ than in ‘Shixia’ fruits. These findings on GABA accumulation in response to cold stress were consistent with previous studies of soybean,35 barley, and wheat.36 The difference in GABA accumulation between cultivars was attributed to the low temperature

GAD activity was higher under high-CO2 than low-CO2 conditions during the first 6 days of storage, but it was not significantly different among treatments during the rest of storage. GAD activity under high-CO2-treated ‘Chuliang’ fruit increased initially and then decreased, while it was in generally steady decline under low-CO2 and ambient-air treatments. In contrast, GAD activity in ‘Shixia’ fruit were increased initially before declining under all of the atmosphere treatments. This result was inconsistent with previous reports on other species, in which GAD activity had no obvious regularity in either strawberry24 or tomato.25 Higher CO2 treatment could result in higher GAD activity, and GAD activity had a significant positive correlation with GABA accumulation. However, the effects of atmosphere treatment on GAD activity exhibited significant differences between cultivars, which lends itself to further research on gene expression. GABA-T activity had no significant correlation with atmosphere treatment, which might indicate that CO2 did not affect GABA-T activity. These findings suggest that higher GABA accumulation in longan fruit may be attributed to higher GABA production associated with increased GAD activity rather than the lower GABA degradation associated with decreased GABA-T activity. GAD activity is dependent upon cytosolic pH and Ca2+− calmodulin activity in response to cytosolic acidification. GAD activity, in turn, could prevent an excessive increase in cytosolic pH while keeping its resistance toward cytosolic acidification.44,45 Plant GAD polypeptides exist as 43−62 kDa subunits, in which their native conformation appears to be dimeric or hexameric; thus, the plant protein is more similar to the bacterial protein than the vertebrate protein in terms of both sequence identity and conformation.46,47 In addition, calmodulin is apparently essential for the creation of larger GAD complexes because calmodulin contributes to the dimerization of GAD subunits and facilitates the formation of high-molecular-mass calmodulin-activated GAD complexes.48 Further research showed that most plant GAD, unlike bacterial GAD, are activated more effectively by Ca2+−calmodulin at neutral pH than at optimum pH.49 Moreover, GAD is a PLPdependent enzyme that is able to serve as a coenzyme to increased GAD activity, resulting in enhanced GABA accumulation.50 The influence of L-glutamic acid or its salts for GABA accumulation is controversial. In soybean (Glycine max L.) seeds, GABA accumulation is regulated by the changes in GAD and GABA-T activities rather than requiring enough glutamic acid resulting from the degradation of protein.51 However, Shelp et al. suggested that GABA accumulation was enhanced by increasing glutamic acid concentration in the vicinity of cytosolic Ca2+−calmodulin-dependent GAD under long-term conditions by declining protein synthesis, elevated protein degradation, and limited glutamine synthesis.52 Although the polyamine-degradation pathway in angiosperms, especially dicotyledons, participates in GABA formation via γaminobutyraldehyde,6 the GABA shunt, mediated by GAD activity, is recognized as the primary pathway for GABA accumulation.53 Therefore, information generated by studying the pattern of GAD activity will provide insight into their functional regulatory role in GABA accumulation in response to elevated-CO2 storage. The biochemical properties of GAD include sensitivity to pH, and it is active only in acidic cytoplasm, even though the medium pH inevitably increases as the reaction proceeds.54 For example, GAD in cherry tomato shows only 15% of the full activity at pH 7.0 and its full activity at pH 6.0.55 Moreover, the F

DOI: 10.1021/acs.jafc.6b01738 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Journal of Agricultural and Food Chemistry tolerance and the severity of cold stress.36 However, an increase in GAD activity occurred in ‘Chuliang’, whereas the GAD activity of ‘Shixia’ had decreased. GABA-T activity remained unchanged in ‘Chuliang’, while GABA-T activity in precooling treatment of ‘Shixia’ fruit had decreased. These results may be related to the longan cultivars. Further research from the perspective of gene expression is ongoing.

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AUTHOR INFORMATION

(5) Allan, W. L.; Peiris, C.; Bown, A. W.; Shelp, B. J. Gammahydroxybutyrate accumulates in green tea and soybean sprouts in response to oxygen deficiency. Can. J. Plant Sci. 2003, 83, 951−953. (6) Fait, A.; Fromm, H.; Walter, D.; Galili, G.; Fernie, A. R. Highway or byway: the metabolic role of the GABA shunt in plants. Trends Plant Sci. 2008, 13, 14−19. (7) Lamberts, L.; Joye, I. J.; Beliën, T.; Delcour, J. A. Dynamics of γaminobutyric acid in wheat flour bread making. Food Chem. 2012, 130, 896−901. (8) Nomura, M.; Nakajima, I.; Fujita, Y.; Kobayashi, M.; Kimoto, H.; Suzuki, I.; Aso, H. Lactococcus lactis contains only one glutamate decarboxylase gene. Microbiology 1999, 145, 1375−1380. (9) Trobacher, C. P.; Clark, S. M.; Bozzo, G. G.; Mullen, R. T.; DeEll, J. R.; Shelp, B. J. Catabolism of GABA in apple fruit: subcellular localization and biochemical characterization of two γ-aminobutyrate transaminases. Postharvest Biol. Technol. 2013, 75, 106−113. (10) Ben-Ari, Y. The GABA excitatory/inhibitory developmental sequence: a personal journey. Neuroscience 2014, 279, 187−219. (11) Matsuki, T.; Takasu, M.; Hirose, Y.; Murakoshi, N.; Sinton, C. M.; Motoike, T.; Yanagisawa, M. GABAA receptor-mediated input change on orexin neurons following sleep deprivation in mice. Neuroscience 2015, 284, 217−224. (12) Wu, Q.; Shah, N. P. Gas release-based prescreening combined with reversed-phase HPLC quantitation for efficient selection of highγ-aminobutyric acid (GABA)-producing lactic acid bacteria. J. Dairy Sci. 2015, 98, 790−797. (13) Abe, Y.; Umemura, S.; Sugimoto, K.; Hirawa, N.; Kato, Y.; Yokoyama, N.; Yokoyama, T.; Iwai, J.; Ishii, M. Effect of green tea rich in γ-aminobutyric acid on blood pressure of Dahl salt-sensitive rats. Am. J. Hypertens. 1995, 8, 74−79. (14) Chuang, C.; Shi, Y.; You, H.; Lo, Y.; Pan, T. Antidepressant effect of GABA-rich monascus-fermented product on forced swimming rat model. J. Agric. Food Chem. 2011, 59, 3027−3034. (15) Constantin, S.; Jasoni, C. L.; Wadas, B.; Herbison, A. E. γAminobutyric acid and glutamate differentially regulate intracellular calcium concentrations in mouse gonadotropin-releasing hormone neurons. Endocrinology 2010, 151, 262−270. (16) Cryan, J. F.; Kaupmann, K. Don’t worry ‘B’ happy!: a role for GABAB receptors in anxiety and depression. Trends Pharmacol. Sci. 2005, 26, 36−43. (17) Krystal, J. H.; Sanacora, G.; Blumberg, H.; Anand, A.; Charney, D. S.; Marek, G.; Epperson, C. N.; Goddard, A.; Mason, G. F. Glutamate and GABA systems as targets for novel antidepressant and mood-stabilizing treatments. Mol. Psychiatry 2002, 7 (1), S71−80. (18) Oh, S.; Oh, C. Effects of germinated brown rice extracts with enhanced levels of GABA on cancer cell proliferation and apoptosis. J. Med. Food 2004, 7, 19−23. (19) Shimada, M.; Hasegawa, T.; Nishimura, C.; Kan, H.; Kanno, T.; Nakamura, T.; Matsubayashi, T. Anti-hypertensive effect of γaminobutyric acid (GABA)-rich chlorella on high-normal blood pressure and borderline hypertension in placebo-controlled double blind study. Clin. Exp. Hypertens. 2009, 31, 342−354. (20) Yang, N.; Jhou, K.; Tseng, C. Antihypertensive effect of mulberry leaf aqueous extract containing γ-aminobutyric acid in spontaneously hypertensive rats. Food Chem. 2012, 132, 1796−1801. (21) Yoshimura, M.; Toyoshi, T.; Sano, A.; Izumi, T.; Fujii, T.; Konishi, C.; Inai, S.; Matsukura, C.; Fukuda, N.; Ezura, H.; Obata, A. Antihypertensive effect of a gamma-aminobutyric acid rich tomato cultivar ‘DG03−9’ in spontaneously hypertensive rats. J. Agric. Food Chem. 2010, 58, 615−619. (22) Bouché, N.; Lacombe, B.; Fromm, H. GABA signaling: a conserved and ubiquitous mechanism. Trends Cell Biol. 2003, 13, 607− 610. (23) Kinnersley, A. M.; Turano, F. J. γ-Aminobutyric acid (GABA) and plant responses to stress. Crit. Rev. Plant Sci. 2000, 19, 479−509. (24) Deewatthanawong, R.; Nock, J. F.; Watkins, C. B. γAminobutyric acid (GABA) accumulation in four strawberry cultivars in response to elevated CO2 storage. Postharvest Biol. Technol. 2010, 57, 92−96.

S Supporting Information *

; These two files are available free of charge via the Internet at http://pubs.acs.org. The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jafc.6b01738. L-Glutamic acid concentration of ‘Chuliang’ and ‘Shixia’ longan fruit at harvest (PDF) The effect of pH and incubation time on GAD activity (PDF)

Corresponding Authors

*Z.H. telephone: +86-20-8528-0266; fax: +86-20-8528-0270; email: [email protected]. *L.Z. e-mail: [email protected]. Funding

This work was supported by the Earmarked Fund for China Agriculture Research System (CARS-33), the Yan Fan Innovative and Entrepreneurial Research Team Project (2014YT02H013), and the Science and Technology Planning Project of Guangdong Province of China (2013B020502012 and 2015A020209143). Notes

The authors declare no competing financial interest.

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ABBREVIATIONS USED GABA, γ-aminobutyric acid; GAD, glutamate decarboxylase; GABA-T, GABA aminotransferase; CO2, carbon dioxide; SSA, succinic semialdehyde; NADP, nicotinamide adenine dinucleotide phosphate; NADPH, triphosphopyridine nucleotide; NAD+, nicotinamide adenine dinucleotide; SSADH, succinate−semialdehyde dehydrogenase; GHB, γ-hydroxybutyric acid; SSAR, succinic semialdehyde reductase; DTT, dithiothreitol; EDTA, ethylenediaminetetraacetic acid; PLP, pyridoxal 5′-phosphate; PMSF, phenylmethylsulfonyl fluoride; Ala, alanine; ADH, alanine dehydrogenase; BSA, bovine serum albumin; SD, standard deviation; SDH, succinate dehydrogenase; GABA-TP, pyruvate-dependent γ-aminobutyric acid aminotransferase; GABA-TK, 2-oxoglutarate-dependent γ-aminobutyric acid aminotransferase

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DOI: 10.1021/acs.jafc.6b01738 J. Agric. Food Chem. XXXX, XXX, XXX−XXX