Plant Science 217–218 (2014) 78–86
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Changes in carbohydrate content in zucchini fruit (Cucurbita pepo L.) under low temperature stress Francisco Palma a,∗ , Fátima Carvajal a , Carmen Lluch a , Manuel Jamilena b , Dolores Garrido a a b
Department of Plant Physiology, Facultad de Ciencias, University of Granada, Fuentenueva s/n, 18071 Granada, Spain Department of Applied Biology, Escuela Superior de Ingeniería, University of Almería, La Ca˜ nada de San Urbano s/n, 04120 Almería, Spain
a r t i c l e
i n f o
Article history: Received 31 October 2013 Received in revised form 5 December 2013 Accepted 8 December 2013 Available online 14 December 2013 Keywords: Zucchini Postharvest Carbohydrate Raffinose Polyols Low-temperature
a b s t r a c t The postharvest handling of zucchini fruit includes low-temperature storage, making cold stress unavoidable. We have investigated the changes of soluble carbohydrates under this stress and its relation with weight loss and chilling injury in zucchini fruit during postharvest storage at 4 ◦ C and 20 ◦ C for up to 14 days. Two varieties with different degrees of chilling tolerance were compared: Natura, the more tolerant variety, and Sinatra, the variety that suffered more severe chilling-injury symptoms and weight loss. In both varieties, total soluble carbohydrates, reducing soluble carbohydrates and polyols content was generally higher during storage at 4 ◦ C than at 20 ◦ C, thus these parameters are related to the physiological response of zucchini fruit to cold stress. However, the raffinose content increased in Natura and Sinatra fruits during storage at 4 ◦ C and 20 ◦ C, although at 20 ◦ C the increase in raffinose was more remarkable than at 4 ◦ C in both varieties, so that the role of raffinose could be more likely related to dehydration than to chilling susceptibility of zucchini fruit. Glucose, fructose, pinitol, and acid invertase activity registered opposite trends in both varieties against chilling, increasing in Natura and decreasing in Sinatra. The increase in acid invertase activity in Natura fruit during cold storage could contribute in part to the increase of these reducing sugars, whose metabolism could be involved in the adaptation to postharvest cold storage. © 2013 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Stress in plants could be defined as any change in growth condition(s) that disrupts metabolic homeostasis and requires an adjustment of metabolic pathways in a process that is usually referred to as acclimation. Metabolomics could contribute significantly to the study of stress biology in plants by identifying different compounds, such as by-products of stress metabolism, stress-signal transduction molecules or molecules that are part of the acclimation response of plants [1]. From the standpoint of metabolomics, at least three different types of compounds are important for these processes: (1) signal-transduction molecules involved in mediating the acclimation response; (2) products of stress that appear in cells because of the disruption of normal homeostasis by the alteration(s) in growth conditions; and (3) compounds involved in the acclimation process such as antioxidants or osmoprotectants [1]. Some of these antioxidants or osmoprotectants are amino acids, sulfonium compounds, simple sugars, disaccharides, and sugar alcohols or polyols [2–4].
∗ Corresponding author. Tel.: +34 958 243159; fax: +34 958 248995. E-mail address: [email protected] (F. Palma). 0168-9452/$ – see front matter © 2013 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.plantsci.2013.12.004
Metabolomic studies have shown the prominent role of central carbohydrate metabolism during temperature stress [5]. Carbohydrates are a major category of compatible solutes that include hexoses (fructose and glucose), disaccharides (sucrose, trehalose), sugar alcohols (inositol, mannitol), and complex sugars (raffinose), all of which accumulate during stress [6,7], and play a major role in the structure and function of all cells. These compatible solutes, or osmoprotectants, can accumulate to high levels without disturbing intracellular biochemistry [8]. In previous studies, the involvement of soluble carbohydrate in the reactive oxygen species (ROS) balance and response to oxidative stress in plants has been thoroughly described [9,10]. In fact, different studies have demonstrated the importance of raffinose family oligosaccharides (RFOs) in stress, acting as osmoprotectants, cellmembrane stabilizers, or ROS scavengers [11–14]. Accumulation of polyols is also correlated with abiotic stress tolerance [4,6,15]. The raffinose family oligosaccharides (RFOs), such as raffinose, stachyose, and verbascose, are soluble galactosyl-sucrose carbohydrates. RFOs are synthesized from sucrose by the subsequent addition of activated galactose moieties donated by galactinol [16]. On the other hand, myoinositol is derived from glucose-6phosphate and can be further methylated to sequoyitol or ononitol, which are then epimerized to D-pinitol [17].
F. Palma et al. / Plant Science 217–218 (2014) 78–86
Refrigerated storage, considered to be the most effective method for preserving the quality of fruit and vegetables, allows long-distance transport and thus a more regulated supply of commodities in the market. However, this common storage practice can cause chilling injury in some fruit and consequently high economic losses. Chilling injury is a physiological disorder of fruits and vegetables caused by low temperatures above freezing. Zucchini (Cucurbita pepo L. morphotype Zucchini), an important greenhouse crop in south-eastern Spain, bears fruit that is marketed at an immature stage. Its subtropical origin makes zucchini fruit susceptible to chilling disorders when stored at low non-freezing temperatures (above 0 ◦ C). As with other types of environmental stress, chilling triggers ROS production, inducing damage at the cellular level [12]. In a previous work analysing the chilling resistance in fruit of different commercial varieties of C. pepo grown in the south-eastern Spain, we detected differences in chilling damage among varieties after 14 days of cold storage [18]. That is, some varieties registered a low chilling index with lower levels of accumulated reactive oxygen species (ROS) and malondialdehyde (MDA), while other varieties reached the maximum chilling index after 7 days of storage and accumulated greater amounts of ROS and MDA. The aim of this work was to evaluate the pattern and content of soluble carbohydrates in zucchini fruit in response to storage at two temperatures, and to compare two varieties with different responses to cold storage in order to relate specific sugars to adaptation low-temperature. 2. Materials and methods 2.1. Plant material and storage conditions Zucchini fruit (C. pepo L. morphotype Zucchini) of the commercial varieties Natura (Enza Zaden) and Sinatra (Clause-Tezier) were provided by Mayes Exportacion S.L. After harvest, fruit were stored in chambers at 4 ◦ C and 20 ◦ C. Three biological replicates were prepared per variety and storage period (0, 7 and 14 days), each consisting in 6 fruit of similar size and maturation time. After the storage period, weight loss and the chilling-injury index were determined. For each biological replicate, the exocarp of all fruits was separated, mixed and grinded in liquid nitrogen, and stored at −20 ◦ C. 2.2. Weight loss and chilling-injury index The fresh weight loss (18 fruit per variety, temperature and storage period) was determined after 7 and 14 days of storage at 4 ◦ C and 20 ◦ C and the percentage of weight loss of each fruit was calculated. The chilling-injury index (CI) was evaluated using a subjective scale of visual symptoms previously described MartínezTéllez et al. [19]: 0 = no pitting, 1 = slight (10% or less), 2 = medium (10–20%), and 3 = severe pitting (>20%). CI index was determined using the following formula: ˙ (pitting scale (0–3) × number of corresponding fruit within each class)/total number of fruit estimated (18 fruit). 2.3. Preparation of extracts Exocarp tissue (1 g) was homogenized in 8 ml of cold ethanol/chloroform/water (12/5/1, v/v/v) and the homogenate was centrifuged at 4 ◦ C and 3500 × g for 10 min [20]. The resulting insoluble residue was used to determine the starch, and the supernatant was separated into aqueous and chloroform phases by the addition of chloroform (5 ml) and water (3 ml). Soluble carbohydrates were determined from the aqueous phase.
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2.4. Soluble carbohydrate content To measure the content of total soluble carbohydrates, 0.1 ml of aqueous phase was mixed with 3 ml anthrone reagent (150 mg anthrone and 100 ml of 72% sulfuric acid). The samples were placed in a boiling water bath for 10 min. The light absorption of the samples was estimated at 625 nm. The contents in total soluble carbohydrates were determined using glucose as the standard. The aqueous phase was evaporated under a flow of nitrogen until dry, in order to measure the different soluble carbohydrates. Finally, dry residues were resolubilized in 0.5 ml of Milli-Q water at 40 ◦ C, centrifuged (10,000 × g 15 min at 4 ◦ C) and the supernatant was suitably diluted. Carbohydrates were separated and quantified by ion chromatography with pulsed amperometric detection according to Cataldi et al. [21] with modifications, conducted on a Dionex ICS-3000 chromatograph (Dionex Corp., Sunnyvale, CA, USA). The polyols, myoinositol, pinitol, galactinol, sorbitol and mannitol, and trehalose were separated in a CarboPac MA1 column and sucrose, glucose, fructose, galactose, raffinose, and stachyose were separated in a CarboPac PA20 column, plus precolumns (Dionex Corp., Sunnyvale, CA, USA) kept at 30 ◦ C, using water and NaOH (0.8 M) as eluents. Gradient conditions are shown in Table 1. 2.5. Starch content Insoluble residue was washed several times with 80% ethanol and dried. This residue was rehydrated in 1 ml of water and heated for 1 h at 90 ◦ C. The gelatinized starch sample was incubated with amyloglucosidase (10 units ml−1 0.1 M acetate buffer, pH 4.5) for 48 h at 40 ◦ C, following Holland et al. [3]. Starch content was determined as glucose produced. Glucose was measured using the Kit BioSystems (Ref. 11504). 2.6. Sucrose synthase-cleavage activity Extracts were prepared by homogenizing 0.5 g of exocarp in a mortar with 33% (w/w) polyvinyl-polypyrrolidone and 2 ml of 50 mM phosphate K buffer (pH 7.5) containing 1 mM EDTA, 5 mM -mercaptoethanol, 2.5 mM DTT, and 20% (v/v) glycerol. The homogenate was centrifuged at 4 ◦ C and 10,000 × g for 15 min, and the supernatant was desalted using Amicon Ultra 10K (Millipore). Sucrose synthase-cleavage activity was measured according to Morell and Copeland [22]. The production of UDP-glucose was coupled to the reduction of NAD+ in the presence of excess UDP-glucose dehydrogenase. Reaction mixtures contained 100 mM Hepes-KOH buffer (pH 7.5), extract, 100 mM sucrose, 2 mM UDP, 0.025 U UDPglucose dehydrogenase and 1.5 mM NAD+ . The enzyme activity was spectrophotometrically measured by following the NAD+ reduction at 340 nm. 2.7. Invertase activity Extracts were prepared by homogenizing 0.5 g of exocarp in a mortar with 33% (w/w) polyvinyl-polypyrrolidone and 2 ml of 50 mM Hepes-KOH buffer (pH 7.5) containing 1 mM EDTA, 10 mM MgCl2 , 2.5 mM DTT, and 10 mM ascorbic acid. The homogenate was centrifuged at 4 ◦ C and 10,000 × g for 15 min, and the supernatant was desalted using Amicon Ultra 10K (Millipore). Neutral invertase activity was assayed in an incubation mixture containing 100 mM phosphate K buffer (pH 7.5), extract, and 200 mM sucrose. The mixture was incubated during 30 min at 30 ◦ C. The assay was stopped by boiling, and the glucose generated was determined with the Kit BioSystems (Ref. 11504). Acid invertase activity was assayed in an incubation mixture containing 100 mM citrate buffer (pH 5), extract, and 200 mM
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Table 1 Gradient conditions for determination soluble carbohydrates using solvent A (water) and solvent B (NaOH 0.8 M). Column CarboPac MA1
Column CarboPac PA20 Eluent profile
Eluent profile
Time (min)
% Solvent A
% Solvent B
Time (min)
% Solvent A
% Solvent B
0 30 40 50
100 25 25 100
0 75 75 0
0 15 20 30 40 Flow rate 0.5 ml/min 15–20 min linear gradient 30–40 min linear gradient
98.5 98.5 70 70 98.5
1.5 1.5 30 30 1.5
Flow rate 0.4 ml/min 0–30 min linear gradient 40–50 min linear gradient
sucrose. The mixture was incubated during 30 min at 30 ◦ C. The assay was stopped by boiling, and neutralized with NaOH. Finally, the glucose generated was determined with the Kit BioSystems (Ref. 11504).
days a decrease of 55% and 77% was registered in fruit of Natura and Sinatra, respectively (Fig. 1).
3.3. Changes in soluble carbohydrates and starch content in zucchini fruit during storage
2.8. Statistical analysis Data were subjected to an analysis of variance (ANOVA) using the Statgraphics® Plus 5.1 software (Statistical Graphics Corp., Rockville, MD, USA). The data presented were means of three replicates of six fruit each with a standard error of means, except for the percentage of weight loss and chilling injury, which were means of 18 fruit. Means were compared by Fisher’s least significant difference test (LSD) and differences at p < 0.05 were considered significant. 3. Results 3.1. Weight loss and chilling-injury index in zucchini fruit during storage When fruit of both varieties was kept at 4 ◦ C, after 7 and 14 days, a significantly higher weight loss was recorded in Sinatra compared to Natura (Table 2). A similar result was found for the chilling-injury index, where Natura registered the lowest value even after 14 days of storage, a mean of 0.67 and 1.72 at days 7 and 14, respectively, while Sinatra reached an index of almost 3.0, the maximum, after 7 days of storage at 4 ◦ C (Table 2). In general, higher values of weight loss were observed in zucchini fruit stored at 20 ◦ C. 3.2. Changes in total soluble carbohydrates content in zucchini fruit during storage In the exocarp of Sinatra fruit stored at 4 ◦ C, the content in soluble carbohydrates did not significantly change, whereas this parameter increased in Natura fruit by about 1.3- and 2-fold during storage at 4 ◦ C after of 7 and 14 days, respectively (Fig. 1). On the contrary, during storage at 20 ◦ C the content in soluble carbohydrates diminished with storage time in both varieties; after 14
In the exocarp of Natura fruit, the galactose content did not significantly change at 4 ◦ C and it significantly diminished at 20 ◦ C. In this variety the reducing soluble carbohydrates glucose and fructose (Fig. 2) exhibited the same trend as the total soluble carbohydrates during the storage—in fact, after 14 days at 4 ◦ C, glucose and fructose increased around 2-fold, whereas at 20 ◦ C both carbohydrates declined. In general, these soluble carbohydrates (galactose, glucose, and fructose) did not increase significantly in fruit of the more susceptible cultivar (Sinatra) when stored at 4 ◦ C, and only after 7 days of storage did the galactose content increase 1.4-fold (Fig. 2). Similarly, as described in Natura, the galactose, glucose, and fructose content in Sinatra fruit also decreased during storage at 20 ◦ C (Fig. 2). In the case of both varieties (Natura and Sinatra) and storage temperatures (4 ◦ C and 20 ◦ C) the disaccharide trehalose was not detected, whereas sucrose showed a very low concentration (data not shown). In the present study, our results show that during storage at 4 ◦ C and 20 ◦ C, both Natura and Sinatra fruit accumulated raffinose; however, the Natura exhibited significantly higher levels of raffinose compared to Sinatra at 4 ◦ C (Fig. 3). The highest raffinose content was detected during storage at 20 ◦ C in both varieties, and, after 7 and 14 days, raffinose increased about 3.5-fold in Natura and Sinatra (Fig. 3). In Natura fruit stored at 4 ◦ C, the stachyose content fell significantly, about 2.5-fold and 3.5-fold after 7 and 14 days, respectively. In the more sensitive variety, the stachyose content did not change significantly at 4 ◦ C (Fig. 3). In general, in both varieties during storage at 20 ◦ C, a decrease in stachyose content was registered (Fig. 3). In Natura and Sinatra the starch content drastically fell (about 10-fold) by the storage of the fruit, at either 4 or 20 ◦ C (Fig. 4).
Table 2 Changes in percentage of weight loss and chilling injury index in zucchini fruit stored at 4 ◦ C and 20 ◦ C. Weight loss (%)
Chilling injury index
Temperature
Variety
7 days
4 ◦C
Natura Sinatra
3.46 ± 0.28bB 5.62 ± 0.41bA
14 days 6.50 ± 0.45aB 9.38 ± 0.38aA
20 ◦ C
Natura Sinatra
3.66 ± 0.30bB 7.97 ± 0.58bA
9.81 ± 0.68aA 10.61 ± 0.43aA
7 days
14 days
0.67 ± 0.18bB 2.81 ± 0.09aA
1.72 ± 0.27aB 2.82 ± 0.10aA
– –
– –
Values are means of 18 fruits ± SE. Different capital letters indicate significant differences among varieties (p < 0.05). Different lower case letters indicate significant differences among storage time (p < 0.05)
F. Palma et al. / Plant Science 217–218 (2014) 78–86
At harvest
7 days
Storage at 4ºC 32
14 days Storage at 20ºC
32
Total Soluble Carbohydrates
Total Soluble Carbohydrates
bA
16
mg glucose g-1FW
mg glucose g-1FW
aA
24 aA aA aB
cB
8
0
Natura
24 aA
16
aB bA bB
8
0
Sinatra
81
cA
Natura
cA
Sinatra
Fig. 1. Changes in total soluble carbohydrates content in the exocarp of zucchini fruit stored at 4 ◦ C and 20 ◦ C. Values are means ± SE of triplicate samples of six fruit each. Different capital letters indicate significant differences among varieties (p < 0.05). Different lower case letters indicate significant differences among storage time (p < 0.05).
At harvest
7 days
Storage at 4ºC 0.6
0.4
aA
Galactose
aA bA
bB
0.2
mg g-1FW
mg g-1FW
aA
Storage at 20ºC
0.6
Galactose
aA
14 days
0.4
aA
aA bA
0.2
bA cA
cA
0.0
0.0 16
16
Glucose
Glucose
aA
12 aA abA
8
bA bB
bB
aA
mg g-1FW
mg g-1FW
12
4
8
aB
4
bA
bA cA
0
0
16 aA
bB
cB
4
8
4
Natura
Fructose
12
aA aA bA
0
16
Fructose
mg g-1FW
mg g-1FW
12
8
cA
Sinatra
0
aA
aB bA bA
Natura
bA cA
Sinatra
Fig. 2. Changes in galactose, glucose and fructose contents in the exocarp of zucchini fruit stored at 4 ◦ C and 20 ◦ C. Values are means ± SE of triplicate samples of six fruit each. Different capital letters indicate significant differences among varieties (p < 0.05). Different lower case letters indicate significant differences among storage time (p < 0.05).
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7 days
At harvest Storage at 4ºC 3.2
14 days Storage at 20ºC
3.2
Raffinose
Raffinose aA
aA
2.4 aA
1.6
bA bA
0.8
mg g-1FW
mg g-1FW
2.4
aA abB
cB
bA cB
0.0
0.4
0.4
Stachyose
0.3
aA
mg g-1FW
mg g-1FW
1.6
0.8
0.0
0.3
aA
bB
0.2 bA
0.1
aB aA aA
cA
Stachyose aA aA bA
0.2 aB
0.1
bB cB
0.0
Natura
0.0
Sinatra
Natura
Sinatra
Fig. 3. Changes in raffinose and stachyose contents in the exocarp of zucchini fruit stored at 4 ◦ C and 20 ◦ C. Values are means ± SE of triplicate samples of six fruit each. Different capital letters indicate significant differences among varieties (p < 0.05). Different lower case letters indicate significant differences among storage time (p < 0.05).
3.4. Changes in polyols content in zucchini fruit during storage
and sorbitol were not detected in either zucchini variety studied.
Galactinol and myoinositol increased in fruit stored at 4 ◦ C in Natura as well as Sinatra, so that after 14 days galactinol was about 10-fold and myoinositol was about 2.5-fold higher in both varieties. By contrast, during storage at 20 ◦ C these polyols showed no significant change or even decreased (Fig. 5). The pinitol content showed an opposite trend in the two varieties when stored at 4 ◦ C, increasing in the exocarp of Natura fruit after 14 days (1.5-fold), but decreasing with time in the exocarp of Sinatra fruit (Fig. 5). At 20 ◦ C this polyol decreased in both varieties, Sinatra exhibiting a greater reduction compared to Natura. The polyols mannitol
At harvest
3.5. Changes in sucrose synthase-cleavage, neutral invertase, and acid invertase activities in zucchini fruit during storage at 4 ◦ C The sucrose synthase-cleavage shows a very low activity in both varieties (Fig. 6). Invertase is the main enzyme responsible for the sucrose hydrolysing activity. In Natura fruit, acid and neutral invertase activities increased during storage at 4 ◦ C, both about 25% after 14 days (Fig. 6). Acid invertase showed an opposite trend in Sinatra fruit, its activity decreased about 10% after 14 days when the
7 days
Storage at 4ºC
0.45
Starch aA
aA
0.30
0.15 bB bA
bA
Storage at 20ºC
0.60
Starch mg glucose g-1FW
mg glucose g-1FW
0.60
14 days
0.45
aA
aA
0.30
0.15 bB bA
cA
bA bA
0.00
0.00
Natura
Sinatra
Natura
Sinatra
Fig. 4. Changes in starch content in the exocarp of zucchini fruit stored at 4 ◦ C and 20 ◦ C. Values are means ± SE of triplicate samples of six fruit each. Different capital letters indicate significant differences among varieties (p < 0.05). Different lower case letters indicate significant differences among storage time (p < 0.05).
F. Palma et al. / Plant Science 217–218 (2014) 78–86
At harvest
7 days
Storage at 4ºC 0.20
aA
bA
aB
0.10
bB
0.05
Galactinol
0.10
0.05 cA
cB
aB aA aB
aA aA aA
0.00
0.00 0.8
0.8
Myoinositol aA
aA aA
bB
0.4
bA
cA
0.4
0.2
0.0
0.0 aA
bA bB bB
1.6
Pinitol
aA
aA
aA
0.2
1.6
Myoinositol
0.6
mg g-1FW
0.6
mg g-1FW
Storage at 20ºC
0.15
mg g-1FW
mg g-1FW
0.15
14 days 0.20
Galactinol
83
aA
cA
Pinitol
bB bA
cA
0.8
0.8
aB
cA
bA cA
bA
0.4
0.4
0.0
1.2
bB
mg g-1FW
mg g-1FW
1.2
Natura
Sinatra
0.0
Natura
Sinatra
Fig. 5. Changes in galactinol, myoinositol and pinitol contents in the exocarp of zucchini fruit stored at 4 ◦ C and 20 ◦ C. Values are means ± SE of triplicate samples of six fruit each. Different capital letters indicate significant differences among varieties (p < 0.05). Different lower case letters indicate significant differences among storage time (p < 0.05).
fruit was kept at 4 ◦ C. On the other hand, neutral invertase activity showed a slight increase in Sinatra fruit (7%) after 14 days of storage at 4 ◦ C. Natura fruit exhibited significantly higher levels of both invertase activities compared to Sinatra fruit after 14 days of cold storage (Fig. 6).
4. Discussion The present study examines the importance of soluble carbohydrates in chilling adaptation of zucchini fruit during postharvest. For this, we used fruit of two varieties with different responses to cold storage: Natura, more tolerant to chilling storage; and Sinatra, more sensitive [18]. Our results show a greater weight loss in Sinatra compared to Natura during storage at 4 ◦ C and a similar result was found for the chilling-injury index. In both varieties and at both storage temperatures used, the fruit lost weight, the greatest losses being measured in fruit stored at 20 ◦ C, probably because at this temperature all the metabolic pathways were still active and
there was a higher respiration rate, as happens in citrus fruit at 12 ◦ C [3]. Soluble carbohydrates are considered important factors related to chilling adaptation in plants and fruits, and it has recently been proposed that sugars can act as real ROS scavengers in plants, especially when present at higher concentrations [11]. There is reportedly an increase in the content of reducing soluble carbohydrates with cold temperatures in grapefruit [23], red raspberry [24], squash [25], rice seedlings [26] and in cucumber [27]. In our experiment, fruit of both varieties was stored at 4 ◦ C and 20 ◦ C and samples were taken after 7 and 14 days. In the exocarp of Natura fruit the concentration of total soluble carbohydrates increased during storage at 4 ◦ C. By contrast, during storage at 20 ◦ C, this parameter diminished with storage time. This response could indicate that the accumulation of total soluble carbohydrates is related to adaptation to low-temperature in Natura, whereas in Sinatra kept at 4 ◦ C no significant changes were detected. High levels of total soluble carbohydrates were found under cold stress in a hardy raspberry cultivar [24,28], in cabbage [29], arabidopsis [30], olive [31], and cherry [32]. In our study, during storage at 20 ◦ C the reducing sugar
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F. Palma et al. / Plant Science 217–218 (2014) 78–86
At harvest
nmol NADH g-1FW min-1
160
7 days
14 days
Sucrose synthase-cleavage
120
80
aA aA bA
aB
aB
aA
40
0
Neutral invertase
900
600 bA aA 1
nmol glucose g-1FW min-1
1200
300
cA
bA bB aB
0
Acid invertase aA
900 bB bA
aA bA
cB
600 1
nmol glucose g-1FW min-1
1200
300
0
Natura
Sinatra
Fig. 6. Changes in sucrose synthase-cleavage, neutral invertase, and acid invertase activities in the exocarp of zucchini fruit stored at 4 ◦ C. Values are means ± SE of triplicate samples of six fruit each. Different capital letters indicate significant differences among varieties (p < 0.05). Different lower case letters indicate significant differences among storage time (p < 0.05).
galactose, glucose, and fructose diminished in both varieties. However, at 4 ◦ C, glucose and fructose showed opposite responses in the zucchini varieties studied. That is, in the exocarp of Natura fruit the content of these carbohydrates increased dramatically, whereas in Sinatra fruit that started with a higher amount in these sugars, no increase was detected after 14 days, and a small reduction in glucose was found, but values were in all cases greater than the amount in the fruit kept at 20 ◦ C. The results for Natura imply that reducing soluble carbohydrates (glucose and fructose) could be involved in the cold-storage adaptation in zucchini fruit. However, there is some controversy in the literature about the role of these reducing sugars (glucose and fructose). Purvis and Grierson [23] showed that glucose and fructose are involved in the increased chilling tolerance in grapefruit, whereas Holland et al. [3] concluded that the changes in carbohydrate levels in citrus pretreated with heat and stored at 2 ◦ C and 12 ◦ C are related mainly to the consumption of reserves in these types of fruit for respiration and not to chilling tolerance. On the other hand, many fruit species reportedly undergo an increase in the soluble carbohydrates due to ripening when stored at room
temperature [33–35]. Zucchini fruit is marketed at an immature stage, but we nevertheless detected a decrease in the content of total soluble carbohydrates, galactose, glucose, and fructose when the fruit was kept at 20 ◦ C, a finding that could be due to an increase in carbon consumption required to sustain fruit respiration at a higher temperature, as happens in mandarin fruit (Holland et al., 2002). During the exposure of plants to chilling temperatures, the most abundant soluble sugar that accumulates is usually sucrose [24,36]; however, we detected very low concentrations of sucrose (data not shown). Raffinose may increase chilling tolerance via its role in stabilization of membranes by interacting with phospholipid headgroups [37,38]. Recently, it has been reported that the accumulation of galactinol, raffinose, and other RFOs in plant cells is closely associated with responses to different types of environmental stress, including chilling [12,26,27,39]. Nishizawa et al. [12] suggested the possibility that raffinose acts not only as an osmoprotectant and stabilizer of cell membranes but also as ROS scavenger, thus playing a role in protecting the cell metabolism from oxidative damage caused by salinity, drought or chilling. Our results show that during storage at 4 ◦ C and 20 ◦ C both Natura and Sinatra fruit accumulated raffinose; however, Natura exhibited significantly higher levels of raffinose compared to Sinatra at 4 ◦ C. It is noteworthy that during storage at 20 ◦ C the increase in raffinose was more remarkable than at 4 ◦ C in both varieties, and this increase correlates with the greater weight loss detected at 20 ◦ C, so that in zucchini fruit the role of raffinose may be to help protect tissues against dehydration, perhaps acting as an osmoprotectant and stabilizer of cell membranes. Other studies have reported that the acquisition of desiccation tolerance is associated with an accumulation of raffinose and stachyose [40]. The accumulation of raffinose, galactinol, galactose, glucose, and fructose could limit stachyose accumulation, since these sugars act as a substrate for the stachyose synthesis, and low levels of these soluble carbohydrates were detected when raffinose increased. In both varieties the starch content was drastically reduced by the fruit storage, at either 4 or 20 ◦ C. Respiration is an ongoing process in fruit that cannot be stopped after harvest, and the continuation of respiration could be responsible for starch hydrolysis during storage. According to our results, Palonen et al. [24] have been reported that starch contents in container-grown plants declined during cold acclimation and were lowest when plants achieved maximum hardiness, while Kami et al. [25] observed that during storage of squash fruits the starch contents were significantly lower at 15 ◦ C than at the other temperatures, although concentrations decreased throughout the storage period at all temperatures. Polyols, like raffinose, appear to protect against stress with osmotic adjustment and with osmoprotection, acting as osmolytes to facilitate water retention in the cytoplasm and to protect cellular structures, possibly by scavenging ROS [6]. Palma et al. [4] recently reported that augmented pinitol synthesis under salt stress could be one of the adaptive features used by the plant, and Nishizawa et al. [12] have suggested that galactinol protects plants from oxidative damage. Furthermore, myoinositol and galactinol are required for the raffinose synthesis, raffinose synthase being one of the key enzymes regulating soluble carbohydrate metabolism in cucumber [27]. In the present study, we detected increases in myoinositol and galactinol in fruit stored at 4 ◦ C in both zucchini varieties; by contrast at 20 ◦ C these polyols showed no significant change or even decreased. The response observed for these polyols in both varieties supports the hypothesis ascribing an important role to galactinol and myoinositol in the adaptation to low-temperature of zucchini fruit. Transgenic Arabidopsis plants overexpressing galactinol synthase and raffinose synthase accumulated high levels of galactinol and raffinose, and these plants were more tolerant to oxidative damage, salinity, chilling, and drought [12]. In this study, mannitol and sorbitol were not detected, although these polyols may
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participate in a wide range of physiological processes in plants, and especially serving as an osmotically active compatible solutes [41]. Sorbitol can be converted both to fructose by sorbitol dehydrogenase and to glucose by sorbitol oxidase [42]. Once unloaded, sorbitol is immediately transformed to other compounds and does not accumulate in fruit tissues, regardless of whether the assimilate availability is high or low [43]. In fruit kept at 4 ◦ C, we found opposite response in the pinitol content in the two varieties, i.e. increasing in Natura fruit after 14 days, but decreasing in Sinatra fruit after 7 and 14 days. At 20 ◦ C this polyol decreased in both varieties. These results suggest that pinitol metabolism is important for cold protection of the stressed fruit tissues, since the accumulation was higher in Natura, although the fruit of Sinatra showed a higher amount in pinitol before storage. It has been reported that pinitol is related to different types of stress, including cold [6]. Pinitol augments in stressed ice plant (Mesembryanthemum crystallinum) to become the major low-molecular-weight carbon compound, with concentrations exceeding 700 mM in cytosol and chloroplasts [44,45]. The levels of galactinol, myoinositol, and pinitol rose in the more tolerant cultivar; this might indicate that such increases are fruit responses to cope with chilling stress, since in fruit kept at 20 ◦ C all three sugar alcohols declined with storage time. In order to investigate the origin of the reducing soluble sugars, sucrose hydrolysing enzymes were measured in both varieties. The highest activity corresponded to acid and neutral invertase, and the sucrose synthase activity was much lower in all the fruit exocarp measured. During storage at 4 ◦ C, Natura fruit showed an increase of the levels of glucose and fructose and this increase was concomitant with higher activities of the sucrose hydrolysing enzymes, both acid and neutral invertase. In Sinatra, neutral invertase shows a slight increase during cold storage, but acid invertase decreases after 14 days at 4 ◦ C. These results suggest that the differences in hexose content in Natura after 14 days of cold storage is related in part to an increase in acid invertase activity in this variety. Thus, induction of acid invertase activity in zucchini fruit might be a factor of adaptation to cold storage. Changes in invertase activity under cold stress have been reported in many plant species. In wheat, a stimulation of acid invertase activity in leaves by cold acclimation of winter wheat, but not spring wheat has been detected [46]. In potato tubers, low temperature storage induces acid invertase activity [47]. Also, potato plants transformed with a yeast-invertase gene were more resistant to chilling than control plants [48]. In view of the above, the reducing soluble carbohydrates and polyols are related to the physiological response of zucchini fruit to cold stress, whereas the role of raffinose is more likely related to dehydration than to chilling susceptibility of zucchini fruit. Glucose, fructose, and pinitol responded oppositely against chilling in the two varieties, so that the metabolism of these sugars could be involved in the adaptation to chilling of zucchini fruit, with an induction of acid invertase in the resistant variety. Therefore, further research studying changes in metabolism carbohydrates in the fruit of different cultivars showing different susceptibility to chilling would shed more light on these findings. Acknowledgments This research has been funded by the Ministerio de Educación y Ciencia (Project AGL2011-30568-C02-01). F. Carvajal was supported with a grant FPU (Ministerio de Educación, Spain). We are grateful to Mayes Exportacion S.L. for supplying the fruit. References [1] V. Shulaev, D. Cortes, G. Miller, R. Mittler, Metabolomics for plant stress response, Physiol. Plant. 132 (2008) 199–208.
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Changes in carbohydrate content in zucchini fruit (Cucurbita pepo L.) under low temperature stress Francisco Palma a,∗ , Fátima Carvajal a , Carmen Lluch a , Manuel Jamilena b , Dolores Garrido a a b
Department of Plant Physiology, Facultad de Ciencias, University of Granada, Fuentenueva s/n, 18071 Granada, Spain Department of Applied Biology, Escuela Superior de Ingeniería, University of Almería, La Ca˜ nada de San Urbano s/n, 04120 Almería, Spain
a r t i c l e
i n f o
Article history: Received 31 October 2013 Received in revised form 5 December 2013 Accepted 8 December 2013 Available online 14 December 2013 Keywords: Zucchini Postharvest Carbohydrate Raffinose Polyols Low-temperature
a b s t r a c t The postharvest handling of zucchini fruit includes low-temperature storage, making cold stress unavoidable. We have investigated the changes of soluble carbohydrates under this stress and its relation with weight loss and chilling injury in zucchini fruit during postharvest storage at 4 ◦ C and 20 ◦ C for up to 14 days. Two varieties with different degrees of chilling tolerance were compared: Natura, the more tolerant variety, and Sinatra, the variety that suffered more severe chilling-injury symptoms and weight loss. In both varieties, total soluble carbohydrates, reducing soluble carbohydrates and polyols content was generally higher during storage at 4 ◦ C than at 20 ◦ C, thus these parameters are related to the physiological response of zucchini fruit to cold stress. However, the raffinose content increased in Natura and Sinatra fruits during storage at 4 ◦ C and 20 ◦ C, although at 20 ◦ C the increase in raffinose was more remarkable than at 4 ◦ C in both varieties, so that the role of raffinose could be more likely related to dehydration than to chilling susceptibility of zucchini fruit. Glucose, fructose, pinitol, and acid invertase activity registered opposite trends in both varieties against chilling, increasing in Natura and decreasing in Sinatra. The increase in acid invertase activity in Natura fruit during cold storage could contribute in part to the increase of these reducing sugars, whose metabolism could be involved in the adaptation to postharvest cold storage. © 2013 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Stress in plants could be defined as any change in growth condition(s) that disrupts metabolic homeostasis and requires an adjustment of metabolic pathways in a process that is usually referred to as acclimation. Metabolomics could contribute significantly to the study of stress biology in plants by identifying different compounds, such as by-products of stress metabolism, stress-signal transduction molecules or molecules that are part of the acclimation response of plants [1]. From the standpoint of metabolomics, at least three different types of compounds are important for these processes: (1) signal-transduction molecules involved in mediating the acclimation response; (2) products of stress that appear in cells because of the disruption of normal homeostasis by the alteration(s) in growth conditions; and (3) compounds involved in the acclimation process such as antioxidants or osmoprotectants [1]. Some of these antioxidants or osmoprotectants are amino acids, sulfonium compounds, simple sugars, disaccharides, and sugar alcohols or polyols [2–4].
∗ Corresponding author. Tel.: +34 958 243159; fax: +34 958 248995. E-mail address: [email protected] (F. Palma). 0168-9452/$ – see front matter © 2013 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.plantsci.2013.12.004
Metabolomic studies have shown the prominent role of central carbohydrate metabolism during temperature stress [5]. Carbohydrates are a major category of compatible solutes that include hexoses (fructose and glucose), disaccharides (sucrose, trehalose), sugar alcohols (inositol, mannitol), and complex sugars (raffinose), all of which accumulate during stress [6,7], and play a major role in the structure and function of all cells. These compatible solutes, or osmoprotectants, can accumulate to high levels without disturbing intracellular biochemistry [8]. In previous studies, the involvement of soluble carbohydrate in the reactive oxygen species (ROS) balance and response to oxidative stress in plants has been thoroughly described [9,10]. In fact, different studies have demonstrated the importance of raffinose family oligosaccharides (RFOs) in stress, acting as osmoprotectants, cellmembrane stabilizers, or ROS scavengers [11–14]. Accumulation of polyols is also correlated with abiotic stress tolerance [4,6,15]. The raffinose family oligosaccharides (RFOs), such as raffinose, stachyose, and verbascose, are soluble galactosyl-sucrose carbohydrates. RFOs are synthesized from sucrose by the subsequent addition of activated galactose moieties donated by galactinol [16]. On the other hand, myoinositol is derived from glucose-6phosphate and can be further methylated to sequoyitol or ononitol, which are then epimerized to D-pinitol [17].
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Refrigerated storage, considered to be the most effective method for preserving the quality of fruit and vegetables, allows long-distance transport and thus a more regulated supply of commodities in the market. However, this common storage practice can cause chilling injury in some fruit and consequently high economic losses. Chilling injury is a physiological disorder of fruits and vegetables caused by low temperatures above freezing. Zucchini (Cucurbita pepo L. morphotype Zucchini), an important greenhouse crop in south-eastern Spain, bears fruit that is marketed at an immature stage. Its subtropical origin makes zucchini fruit susceptible to chilling disorders when stored at low non-freezing temperatures (above 0 ◦ C). As with other types of environmental stress, chilling triggers ROS production, inducing damage at the cellular level [12]. In a previous work analysing the chilling resistance in fruit of different commercial varieties of C. pepo grown in the south-eastern Spain, we detected differences in chilling damage among varieties after 14 days of cold storage [18]. That is, some varieties registered a low chilling index with lower levels of accumulated reactive oxygen species (ROS) and malondialdehyde (MDA), while other varieties reached the maximum chilling index after 7 days of storage and accumulated greater amounts of ROS and MDA. The aim of this work was to evaluate the pattern and content of soluble carbohydrates in zucchini fruit in response to storage at two temperatures, and to compare two varieties with different responses to cold storage in order to relate specific sugars to adaptation low-temperature. 2. Materials and methods 2.1. Plant material and storage conditions Zucchini fruit (C. pepo L. morphotype Zucchini) of the commercial varieties Natura (Enza Zaden) and Sinatra (Clause-Tezier) were provided by Mayes Exportacion S.L. After harvest, fruit were stored in chambers at 4 ◦ C and 20 ◦ C. Three biological replicates were prepared per variety and storage period (0, 7 and 14 days), each consisting in 6 fruit of similar size and maturation time. After the storage period, weight loss and the chilling-injury index were determined. For each biological replicate, the exocarp of all fruits was separated, mixed and grinded in liquid nitrogen, and stored at −20 ◦ C. 2.2. Weight loss and chilling-injury index The fresh weight loss (18 fruit per variety, temperature and storage period) was determined after 7 and 14 days of storage at 4 ◦ C and 20 ◦ C and the percentage of weight loss of each fruit was calculated. The chilling-injury index (CI) was evaluated using a subjective scale of visual symptoms previously described MartínezTéllez et al. [19]: 0 = no pitting, 1 = slight (10% or less), 2 = medium (10–20%), and 3 = severe pitting (>20%). CI index was determined using the following formula: ˙ (pitting scale (0–3) × number of corresponding fruit within each class)/total number of fruit estimated (18 fruit). 2.3. Preparation of extracts Exocarp tissue (1 g) was homogenized in 8 ml of cold ethanol/chloroform/water (12/5/1, v/v/v) and the homogenate was centrifuged at 4 ◦ C and 3500 × g for 10 min [20]. The resulting insoluble residue was used to determine the starch, and the supernatant was separated into aqueous and chloroform phases by the addition of chloroform (5 ml) and water (3 ml). Soluble carbohydrates were determined from the aqueous phase.
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2.4. Soluble carbohydrate content To measure the content of total soluble carbohydrates, 0.1 ml of aqueous phase was mixed with 3 ml anthrone reagent (150 mg anthrone and 100 ml of 72% sulfuric acid). The samples were placed in a boiling water bath for 10 min. The light absorption of the samples was estimated at 625 nm. The contents in total soluble carbohydrates were determined using glucose as the standard. The aqueous phase was evaporated under a flow of nitrogen until dry, in order to measure the different soluble carbohydrates. Finally, dry residues were resolubilized in 0.5 ml of Milli-Q water at 40 ◦ C, centrifuged (10,000 × g 15 min at 4 ◦ C) and the supernatant was suitably diluted. Carbohydrates were separated and quantified by ion chromatography with pulsed amperometric detection according to Cataldi et al. [21] with modifications, conducted on a Dionex ICS-3000 chromatograph (Dionex Corp., Sunnyvale, CA, USA). The polyols, myoinositol, pinitol, galactinol, sorbitol and mannitol, and trehalose were separated in a CarboPac MA1 column and sucrose, glucose, fructose, galactose, raffinose, and stachyose were separated in a CarboPac PA20 column, plus precolumns (Dionex Corp., Sunnyvale, CA, USA) kept at 30 ◦ C, using water and NaOH (0.8 M) as eluents. Gradient conditions are shown in Table 1. 2.5. Starch content Insoluble residue was washed several times with 80% ethanol and dried. This residue was rehydrated in 1 ml of water and heated for 1 h at 90 ◦ C. The gelatinized starch sample was incubated with amyloglucosidase (10 units ml−1 0.1 M acetate buffer, pH 4.5) for 48 h at 40 ◦ C, following Holland et al. [3]. Starch content was determined as glucose produced. Glucose was measured using the Kit BioSystems (Ref. 11504). 2.6. Sucrose synthase-cleavage activity Extracts were prepared by homogenizing 0.5 g of exocarp in a mortar with 33% (w/w) polyvinyl-polypyrrolidone and 2 ml of 50 mM phosphate K buffer (pH 7.5) containing 1 mM EDTA, 5 mM -mercaptoethanol, 2.5 mM DTT, and 20% (v/v) glycerol. The homogenate was centrifuged at 4 ◦ C and 10,000 × g for 15 min, and the supernatant was desalted using Amicon Ultra 10K (Millipore). Sucrose synthase-cleavage activity was measured according to Morell and Copeland [22]. The production of UDP-glucose was coupled to the reduction of NAD+ in the presence of excess UDP-glucose dehydrogenase. Reaction mixtures contained 100 mM Hepes-KOH buffer (pH 7.5), extract, 100 mM sucrose, 2 mM UDP, 0.025 U UDPglucose dehydrogenase and 1.5 mM NAD+ . The enzyme activity was spectrophotometrically measured by following the NAD+ reduction at 340 nm. 2.7. Invertase activity Extracts were prepared by homogenizing 0.5 g of exocarp in a mortar with 33% (w/w) polyvinyl-polypyrrolidone and 2 ml of 50 mM Hepes-KOH buffer (pH 7.5) containing 1 mM EDTA, 10 mM MgCl2 , 2.5 mM DTT, and 10 mM ascorbic acid. The homogenate was centrifuged at 4 ◦ C and 10,000 × g for 15 min, and the supernatant was desalted using Amicon Ultra 10K (Millipore). Neutral invertase activity was assayed in an incubation mixture containing 100 mM phosphate K buffer (pH 7.5), extract, and 200 mM sucrose. The mixture was incubated during 30 min at 30 ◦ C. The assay was stopped by boiling, and the glucose generated was determined with the Kit BioSystems (Ref. 11504). Acid invertase activity was assayed in an incubation mixture containing 100 mM citrate buffer (pH 5), extract, and 200 mM
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Table 1 Gradient conditions for determination soluble carbohydrates using solvent A (water) and solvent B (NaOH 0.8 M). Column CarboPac MA1
Column CarboPac PA20 Eluent profile
Eluent profile
Time (min)
% Solvent A
% Solvent B
Time (min)
% Solvent A
% Solvent B
0 30 40 50
100 25 25 100
0 75 75 0
0 15 20 30 40 Flow rate 0.5 ml/min 15–20 min linear gradient 30–40 min linear gradient
98.5 98.5 70 70 98.5
1.5 1.5 30 30 1.5
Flow rate 0.4 ml/min 0–30 min linear gradient 40–50 min linear gradient
sucrose. The mixture was incubated during 30 min at 30 ◦ C. The assay was stopped by boiling, and neutralized with NaOH. Finally, the glucose generated was determined with the Kit BioSystems (Ref. 11504).
days a decrease of 55% and 77% was registered in fruit of Natura and Sinatra, respectively (Fig. 1).
3.3. Changes in soluble carbohydrates and starch content in zucchini fruit during storage
2.8. Statistical analysis Data were subjected to an analysis of variance (ANOVA) using the Statgraphics® Plus 5.1 software (Statistical Graphics Corp., Rockville, MD, USA). The data presented were means of three replicates of six fruit each with a standard error of means, except for the percentage of weight loss and chilling injury, which were means of 18 fruit. Means were compared by Fisher’s least significant difference test (LSD) and differences at p < 0.05 were considered significant. 3. Results 3.1. Weight loss and chilling-injury index in zucchini fruit during storage When fruit of both varieties was kept at 4 ◦ C, after 7 and 14 days, a significantly higher weight loss was recorded in Sinatra compared to Natura (Table 2). A similar result was found for the chilling-injury index, where Natura registered the lowest value even after 14 days of storage, a mean of 0.67 and 1.72 at days 7 and 14, respectively, while Sinatra reached an index of almost 3.0, the maximum, after 7 days of storage at 4 ◦ C (Table 2). In general, higher values of weight loss were observed in zucchini fruit stored at 20 ◦ C. 3.2. Changes in total soluble carbohydrates content in zucchini fruit during storage In the exocarp of Sinatra fruit stored at 4 ◦ C, the content in soluble carbohydrates did not significantly change, whereas this parameter increased in Natura fruit by about 1.3- and 2-fold during storage at 4 ◦ C after of 7 and 14 days, respectively (Fig. 1). On the contrary, during storage at 20 ◦ C the content in soluble carbohydrates diminished with storage time in both varieties; after 14
In the exocarp of Natura fruit, the galactose content did not significantly change at 4 ◦ C and it significantly diminished at 20 ◦ C. In this variety the reducing soluble carbohydrates glucose and fructose (Fig. 2) exhibited the same trend as the total soluble carbohydrates during the storage—in fact, after 14 days at 4 ◦ C, glucose and fructose increased around 2-fold, whereas at 20 ◦ C both carbohydrates declined. In general, these soluble carbohydrates (galactose, glucose, and fructose) did not increase significantly in fruit of the more susceptible cultivar (Sinatra) when stored at 4 ◦ C, and only after 7 days of storage did the galactose content increase 1.4-fold (Fig. 2). Similarly, as described in Natura, the galactose, glucose, and fructose content in Sinatra fruit also decreased during storage at 20 ◦ C (Fig. 2). In the case of both varieties (Natura and Sinatra) and storage temperatures (4 ◦ C and 20 ◦ C) the disaccharide trehalose was not detected, whereas sucrose showed a very low concentration (data not shown). In the present study, our results show that during storage at 4 ◦ C and 20 ◦ C, both Natura and Sinatra fruit accumulated raffinose; however, the Natura exhibited significantly higher levels of raffinose compared to Sinatra at 4 ◦ C (Fig. 3). The highest raffinose content was detected during storage at 20 ◦ C in both varieties, and, after 7 and 14 days, raffinose increased about 3.5-fold in Natura and Sinatra (Fig. 3). In Natura fruit stored at 4 ◦ C, the stachyose content fell significantly, about 2.5-fold and 3.5-fold after 7 and 14 days, respectively. In the more sensitive variety, the stachyose content did not change significantly at 4 ◦ C (Fig. 3). In general, in both varieties during storage at 20 ◦ C, a decrease in stachyose content was registered (Fig. 3). In Natura and Sinatra the starch content drastically fell (about 10-fold) by the storage of the fruit, at either 4 or 20 ◦ C (Fig. 4).
Table 2 Changes in percentage of weight loss and chilling injury index in zucchini fruit stored at 4 ◦ C and 20 ◦ C. Weight loss (%)
Chilling injury index
Temperature
Variety
7 days
4 ◦C
Natura Sinatra
3.46 ± 0.28bB 5.62 ± 0.41bA
14 days 6.50 ± 0.45aB 9.38 ± 0.38aA
20 ◦ C
Natura Sinatra
3.66 ± 0.30bB 7.97 ± 0.58bA
9.81 ± 0.68aA 10.61 ± 0.43aA
7 days
14 days
0.67 ± 0.18bB 2.81 ± 0.09aA
1.72 ± 0.27aB 2.82 ± 0.10aA
– –
– –
Values are means of 18 fruits ± SE. Different capital letters indicate significant differences among varieties (p < 0.05). Different lower case letters indicate significant differences among storage time (p < 0.05)
F. Palma et al. / Plant Science 217–218 (2014) 78–86
At harvest
7 days
Storage at 4ºC 32
14 days Storage at 20ºC
32
Total Soluble Carbohydrates
Total Soluble Carbohydrates
bA
16
mg glucose g-1FW
mg glucose g-1FW
aA
24 aA aA aB
cB
8
0
Natura
24 aA
16
aB bA bB
8
0
Sinatra
81
cA
Natura
cA
Sinatra
Fig. 1. Changes in total soluble carbohydrates content in the exocarp of zucchini fruit stored at 4 ◦ C and 20 ◦ C. Values are means ± SE of triplicate samples of six fruit each. Different capital letters indicate significant differences among varieties (p < 0.05). Different lower case letters indicate significant differences among storage time (p < 0.05).
At harvest
7 days
Storage at 4ºC 0.6
0.4
aA
Galactose
aA bA
bB
0.2
mg g-1FW
mg g-1FW
aA
Storage at 20ºC
0.6
Galactose
aA
14 days
0.4
aA
aA bA
0.2
bA cA
cA
0.0
0.0 16
16
Glucose
Glucose
aA
12 aA abA
8
bA bB
bB
aA
mg g-1FW
mg g-1FW
12
4
8
aB
4
bA
bA cA
0
0
16 aA
bB
cB
4
8
4
Natura
Fructose
12
aA aA bA
0
16
Fructose
mg g-1FW
mg g-1FW
12
8
cA
Sinatra
0
aA
aB bA bA
Natura
bA cA
Sinatra
Fig. 2. Changes in galactose, glucose and fructose contents in the exocarp of zucchini fruit stored at 4 ◦ C and 20 ◦ C. Values are means ± SE of triplicate samples of six fruit each. Different capital letters indicate significant differences among varieties (p < 0.05). Different lower case letters indicate significant differences among storage time (p < 0.05).
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7 days
At harvest Storage at 4ºC 3.2
14 days Storage at 20ºC
3.2
Raffinose
Raffinose aA
aA
2.4 aA
1.6
bA bA
0.8
mg g-1FW
mg g-1FW
2.4
aA abB
cB
bA cB
0.0
0.4
0.4
Stachyose
0.3
aA
mg g-1FW
mg g-1FW
1.6
0.8
0.0
0.3
aA
bB
0.2 bA
0.1
aB aA aA
cA
Stachyose aA aA bA
0.2 aB
0.1
bB cB
0.0
Natura
0.0
Sinatra
Natura
Sinatra
Fig. 3. Changes in raffinose and stachyose contents in the exocarp of zucchini fruit stored at 4 ◦ C and 20 ◦ C. Values are means ± SE of triplicate samples of six fruit each. Different capital letters indicate significant differences among varieties (p < 0.05). Different lower case letters indicate significant differences among storage time (p < 0.05).
3.4. Changes in polyols content in zucchini fruit during storage
and sorbitol were not detected in either zucchini variety studied.
Galactinol and myoinositol increased in fruit stored at 4 ◦ C in Natura as well as Sinatra, so that after 14 days galactinol was about 10-fold and myoinositol was about 2.5-fold higher in both varieties. By contrast, during storage at 20 ◦ C these polyols showed no significant change or even decreased (Fig. 5). The pinitol content showed an opposite trend in the two varieties when stored at 4 ◦ C, increasing in the exocarp of Natura fruit after 14 days (1.5-fold), but decreasing with time in the exocarp of Sinatra fruit (Fig. 5). At 20 ◦ C this polyol decreased in both varieties, Sinatra exhibiting a greater reduction compared to Natura. The polyols mannitol
At harvest
3.5. Changes in sucrose synthase-cleavage, neutral invertase, and acid invertase activities in zucchini fruit during storage at 4 ◦ C The sucrose synthase-cleavage shows a very low activity in both varieties (Fig. 6). Invertase is the main enzyme responsible for the sucrose hydrolysing activity. In Natura fruit, acid and neutral invertase activities increased during storage at 4 ◦ C, both about 25% after 14 days (Fig. 6). Acid invertase showed an opposite trend in Sinatra fruit, its activity decreased about 10% after 14 days when the
7 days
Storage at 4ºC
0.45
Starch aA
aA
0.30
0.15 bB bA
bA
Storage at 20ºC
0.60
Starch mg glucose g-1FW
mg glucose g-1FW
0.60
14 days
0.45
aA
aA
0.30
0.15 bB bA
cA
bA bA
0.00
0.00
Natura
Sinatra
Natura
Sinatra
Fig. 4. Changes in starch content in the exocarp of zucchini fruit stored at 4 ◦ C and 20 ◦ C. Values are means ± SE of triplicate samples of six fruit each. Different capital letters indicate significant differences among varieties (p < 0.05). Different lower case letters indicate significant differences among storage time (p < 0.05).
F. Palma et al. / Plant Science 217–218 (2014) 78–86
At harvest
7 days
Storage at 4ºC 0.20
aA
bA
aB
0.10
bB
0.05
Galactinol
0.10
0.05 cA
cB
aB aA aB
aA aA aA
0.00
0.00 0.8
0.8
Myoinositol aA
aA aA
bB
0.4
bA
cA
0.4
0.2
0.0
0.0 aA
bA bB bB
1.6
Pinitol
aA
aA
aA
0.2
1.6
Myoinositol
0.6
mg g-1FW
0.6
mg g-1FW
Storage at 20ºC
0.15
mg g-1FW
mg g-1FW
0.15
14 days 0.20
Galactinol
83
aA
cA
Pinitol
bB bA
cA
0.8
0.8
aB
cA
bA cA
bA
0.4
0.4
0.0
1.2
bB
mg g-1FW
mg g-1FW
1.2
Natura
Sinatra
0.0
Natura
Sinatra
Fig. 5. Changes in galactinol, myoinositol and pinitol contents in the exocarp of zucchini fruit stored at 4 ◦ C and 20 ◦ C. Values are means ± SE of triplicate samples of six fruit each. Different capital letters indicate significant differences among varieties (p < 0.05). Different lower case letters indicate significant differences among storage time (p < 0.05).
fruit was kept at 4 ◦ C. On the other hand, neutral invertase activity showed a slight increase in Sinatra fruit (7%) after 14 days of storage at 4 ◦ C. Natura fruit exhibited significantly higher levels of both invertase activities compared to Sinatra fruit after 14 days of cold storage (Fig. 6).
4. Discussion The present study examines the importance of soluble carbohydrates in chilling adaptation of zucchini fruit during postharvest. For this, we used fruit of two varieties with different responses to cold storage: Natura, more tolerant to chilling storage; and Sinatra, more sensitive [18]. Our results show a greater weight loss in Sinatra compared to Natura during storage at 4 ◦ C and a similar result was found for the chilling-injury index. In both varieties and at both storage temperatures used, the fruit lost weight, the greatest losses being measured in fruit stored at 20 ◦ C, probably because at this temperature all the metabolic pathways were still active and
there was a higher respiration rate, as happens in citrus fruit at 12 ◦ C [3]. Soluble carbohydrates are considered important factors related to chilling adaptation in plants and fruits, and it has recently been proposed that sugars can act as real ROS scavengers in plants, especially when present at higher concentrations [11]. There is reportedly an increase in the content of reducing soluble carbohydrates with cold temperatures in grapefruit [23], red raspberry [24], squash [25], rice seedlings [26] and in cucumber [27]. In our experiment, fruit of both varieties was stored at 4 ◦ C and 20 ◦ C and samples were taken after 7 and 14 days. In the exocarp of Natura fruit the concentration of total soluble carbohydrates increased during storage at 4 ◦ C. By contrast, during storage at 20 ◦ C, this parameter diminished with storage time. This response could indicate that the accumulation of total soluble carbohydrates is related to adaptation to low-temperature in Natura, whereas in Sinatra kept at 4 ◦ C no significant changes were detected. High levels of total soluble carbohydrates were found under cold stress in a hardy raspberry cultivar [24,28], in cabbage [29], arabidopsis [30], olive [31], and cherry [32]. In our study, during storage at 20 ◦ C the reducing sugar
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F. Palma et al. / Plant Science 217–218 (2014) 78–86
At harvest
nmol NADH g-1FW min-1
160
7 days
14 days
Sucrose synthase-cleavage
120
80
aA aA bA
aB
aB
aA
40
0
Neutral invertase
900
600 bA aA 1
nmol glucose g-1FW min-1
1200
300
cA
bA bB aB
0
Acid invertase aA
900 bB bA
aA bA
cB
600 1
nmol glucose g-1FW min-1
1200
300
0
Natura
Sinatra
Fig. 6. Changes in sucrose synthase-cleavage, neutral invertase, and acid invertase activities in the exocarp of zucchini fruit stored at 4 ◦ C. Values are means ± SE of triplicate samples of six fruit each. Different capital letters indicate significant differences among varieties (p < 0.05). Different lower case letters indicate significant differences among storage time (p < 0.05).
galactose, glucose, and fructose diminished in both varieties. However, at 4 ◦ C, glucose and fructose showed opposite responses in the zucchini varieties studied. That is, in the exocarp of Natura fruit the content of these carbohydrates increased dramatically, whereas in Sinatra fruit that started with a higher amount in these sugars, no increase was detected after 14 days, and a small reduction in glucose was found, but values were in all cases greater than the amount in the fruit kept at 20 ◦ C. The results for Natura imply that reducing soluble carbohydrates (glucose and fructose) could be involved in the cold-storage adaptation in zucchini fruit. However, there is some controversy in the literature about the role of these reducing sugars (glucose and fructose). Purvis and Grierson [23] showed that glucose and fructose are involved in the increased chilling tolerance in grapefruit, whereas Holland et al. [3] concluded that the changes in carbohydrate levels in citrus pretreated with heat and stored at 2 ◦ C and 12 ◦ C are related mainly to the consumption of reserves in these types of fruit for respiration and not to chilling tolerance. On the other hand, many fruit species reportedly undergo an increase in the soluble carbohydrates due to ripening when stored at room
temperature [33–35]. Zucchini fruit is marketed at an immature stage, but we nevertheless detected a decrease in the content of total soluble carbohydrates, galactose, glucose, and fructose when the fruit was kept at 20 ◦ C, a finding that could be due to an increase in carbon consumption required to sustain fruit respiration at a higher temperature, as happens in mandarin fruit (Holland et al., 2002). During the exposure of plants to chilling temperatures, the most abundant soluble sugar that accumulates is usually sucrose [24,36]; however, we detected very low concentrations of sucrose (data not shown). Raffinose may increase chilling tolerance via its role in stabilization of membranes by interacting with phospholipid headgroups [37,38]. Recently, it has been reported that the accumulation of galactinol, raffinose, and other RFOs in plant cells is closely associated with responses to different types of environmental stress, including chilling [12,26,27,39]. Nishizawa et al. [12] suggested the possibility that raffinose acts not only as an osmoprotectant and stabilizer of cell membranes but also as ROS scavenger, thus playing a role in protecting the cell metabolism from oxidative damage caused by salinity, drought or chilling. Our results show that during storage at 4 ◦ C and 20 ◦ C both Natura and Sinatra fruit accumulated raffinose; however, Natura exhibited significantly higher levels of raffinose compared to Sinatra at 4 ◦ C. It is noteworthy that during storage at 20 ◦ C the increase in raffinose was more remarkable than at 4 ◦ C in both varieties, and this increase correlates with the greater weight loss detected at 20 ◦ C, so that in zucchini fruit the role of raffinose may be to help protect tissues against dehydration, perhaps acting as an osmoprotectant and stabilizer of cell membranes. Other studies have reported that the acquisition of desiccation tolerance is associated with an accumulation of raffinose and stachyose [40]. The accumulation of raffinose, galactinol, galactose, glucose, and fructose could limit stachyose accumulation, since these sugars act as a substrate for the stachyose synthesis, and low levels of these soluble carbohydrates were detected when raffinose increased. In both varieties the starch content was drastically reduced by the fruit storage, at either 4 or 20 ◦ C. Respiration is an ongoing process in fruit that cannot be stopped after harvest, and the continuation of respiration could be responsible for starch hydrolysis during storage. According to our results, Palonen et al. [24] have been reported that starch contents in container-grown plants declined during cold acclimation and were lowest when plants achieved maximum hardiness, while Kami et al. [25] observed that during storage of squash fruits the starch contents were significantly lower at 15 ◦ C than at the other temperatures, although concentrations decreased throughout the storage period at all temperatures. Polyols, like raffinose, appear to protect against stress with osmotic adjustment and with osmoprotection, acting as osmolytes to facilitate water retention in the cytoplasm and to protect cellular structures, possibly by scavenging ROS [6]. Palma et al. [4] recently reported that augmented pinitol synthesis under salt stress could be one of the adaptive features used by the plant, and Nishizawa et al. [12] have suggested that galactinol protects plants from oxidative damage. Furthermore, myoinositol and galactinol are required for the raffinose synthesis, raffinose synthase being one of the key enzymes regulating soluble carbohydrate metabolism in cucumber [27]. In the present study, we detected increases in myoinositol and galactinol in fruit stored at 4 ◦ C in both zucchini varieties; by contrast at 20 ◦ C these polyols showed no significant change or even decreased. The response observed for these polyols in both varieties supports the hypothesis ascribing an important role to galactinol and myoinositol in the adaptation to low-temperature of zucchini fruit. Transgenic Arabidopsis plants overexpressing galactinol synthase and raffinose synthase accumulated high levels of galactinol and raffinose, and these plants were more tolerant to oxidative damage, salinity, chilling, and drought [12]. In this study, mannitol and sorbitol were not detected, although these polyols may
F. Palma et al. / Plant Science 217–218 (2014) 78–86
participate in a wide range of physiological processes in plants, and especially serving as an osmotically active compatible solutes [41]. Sorbitol can be converted both to fructose by sorbitol dehydrogenase and to glucose by sorbitol oxidase [42]. Once unloaded, sorbitol is immediately transformed to other compounds and does not accumulate in fruit tissues, regardless of whether the assimilate availability is high or low [43]. In fruit kept at 4 ◦ C, we found opposite response in the pinitol content in the two varieties, i.e. increasing in Natura fruit after 14 days, but decreasing in Sinatra fruit after 7 and 14 days. At 20 ◦ C this polyol decreased in both varieties. These results suggest that pinitol metabolism is important for cold protection of the stressed fruit tissues, since the accumulation was higher in Natura, although the fruit of Sinatra showed a higher amount in pinitol before storage. It has been reported that pinitol is related to different types of stress, including cold [6]. Pinitol augments in stressed ice plant (Mesembryanthemum crystallinum) to become the major low-molecular-weight carbon compound, with concentrations exceeding 700 mM in cytosol and chloroplasts [44,45]. The levels of galactinol, myoinositol, and pinitol rose in the more tolerant cultivar; this might indicate that such increases are fruit responses to cope with chilling stress, since in fruit kept at 20 ◦ C all three sugar alcohols declined with storage time. In order to investigate the origin of the reducing soluble sugars, sucrose hydrolysing enzymes were measured in both varieties. The highest activity corresponded to acid and neutral invertase, and the sucrose synthase activity was much lower in all the fruit exocarp measured. During storage at 4 ◦ C, Natura fruit showed an increase of the levels of glucose and fructose and this increase was concomitant with higher activities of the sucrose hydrolysing enzymes, both acid and neutral invertase. In Sinatra, neutral invertase shows a slight increase during cold storage, but acid invertase decreases after 14 days at 4 ◦ C. These results suggest that the differences in hexose content in Natura after 14 days of cold storage is related in part to an increase in acid invertase activity in this variety. Thus, induction of acid invertase activity in zucchini fruit might be a factor of adaptation to cold storage. Changes in invertase activity under cold stress have been reported in many plant species. In wheat, a stimulation of acid invertase activity in leaves by cold acclimation of winter wheat, but not spring wheat has been detected [46]. In potato tubers, low temperature storage induces acid invertase activity [47]. Also, potato plants transformed with a yeast-invertase gene were more resistant to chilling than control plants [48]. In view of the above, the reducing soluble carbohydrates and polyols are related to the physiological response of zucchini fruit to cold stress, whereas the role of raffinose is more likely related to dehydration than to chilling susceptibility of zucchini fruit. Glucose, fructose, and pinitol responded oppositely against chilling in the two varieties, so that the metabolism of these sugars could be involved in the adaptation to chilling of zucchini fruit, with an induction of acid invertase in the resistant variety. Therefore, further research studying changes in metabolism carbohydrates in the fruit of different cultivars showing different susceptibility to chilling would shed more light on these findings. Acknowledgments This research has been funded by the Ministerio de Educación y Ciencia (Project AGL2011-30568-C02-01). F. Carvajal was supported with a grant FPU (Ministerio de Educación, Spain). We are grateful to Mayes Exportacion S.L. for supplying the fruit. References [1] V. Shulaev, D. Cortes, G. Miller, R. Mittler, Metabolomics for plant stress response, Physiol. Plant. 132 (2008) 199–208.
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