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Plant Science 217– 218 (2014) 78– 86

Contents lists available at ScienceDirect

Plant Science

jo u r n al homep age: www.elsev ier .com/ locate /p lantsc i

hanges in carbohydrate content in zucchini fruit (Cucurbita pepo L.)nder low temperature stress

rancisco Palmaa,∗, Fátima Carvajal a, Carmen Llucha, Manuel Jamilenab, Dolores Garridoa

Department of Plant Physiology, Facultad de Ciencias, University of Granada, Fuentenueva s/n, 18071 Granada, SpainDepartment of Applied Biology, Escuela Superior de Ingeniería, University of Almería, La Canada de San Urbano s/n, 04120 Almería, Spain

r t i c l e i n f o

rticle history:eceived 31 October 2013eceived in revised form 5 December 2013ccepted 8 December 2013vailable online 14 December 2013

eywords:ucchiniostharvestarbohydrate

a b s t r a c t

The postharvest handling of zucchini fruit includes low-temperature storage, making cold stress unavoid-able. We have investigated the changes of soluble carbohydrates under this stress and its relation withweight loss and chilling injury in zucchini fruit during postharvest storage at 4 ◦C and 20 ◦C for up to 14days. Two varieties with different degrees of chilling tolerance were compared: Natura, the more tolerantvariety, and Sinatra, the variety that suffered more severe chilling-injury symptoms and weight loss. Inboth varieties, total soluble carbohydrates, reducing soluble carbohydrates and polyols content was gen-erally higher during storage at 4 ◦C than at 20 ◦C, thus these parameters are related to the physiologicalresponse of zucchini fruit to cold stress. However, the raffinose content increased in Natura and Sinatrafruits during storage at 4 ◦C and 20 ◦C, although at 20 ◦C the increase in raffinose was more remarkable

◦

affinoseolyolsow-temperature

than at 4 C in both varieties, so that the role of raffinose could be more likely related to dehydrationthan to chilling susceptibility of zucchini fruit. Glucose, fructose, pinitol, and acid invertase activity reg-istered 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 theincrease of these reducing sugars, whose metabolism could be involved in the adaptation to postharvest

cold storage.

. Introduction

Stress in plants could be defined as any change in growthondition(s) that disrupts metabolic homeostasis and requires andjustment of metabolic pathways in a process that is usuallyeferred to as acclimation. Metabolomics could contribute signifi-antly to the study of stress biology in plants by identifying differentompounds, such as by-products of stress metabolism, stress-signalransduction molecules or molecules that are part of the acclima-ion response of plants [1]. From the standpoint of metabolomics,t least three different types of compounds are important for theserocesses: (1) signal-transduction molecules involved in mediat-

ng the acclimation response; (2) products of stress that appear inells because of the disruption of normal homeostasis by the alter-tion(s) in growth conditions; and (3) compounds involved in thecclimation process such as antioxidants or osmoprotectants [1].

ome of these antioxidants or osmoprotectants are amino acids,ulfonium compounds, simple sugars, disaccharides, and sugarlcohols or polyols [2–4].

∗ Corresponding author. Tel.: +34 958 243159; fax: +34 958 248995.E-mail address: fpalma@ugr.es (F. Palma).

168-9452/$ – see front matter © 2013 Elsevier Ireland Ltd. All rights reserved.ttp://dx.doi.org/10.1016/j.plantsci.2013.12.004

© 2013 Elsevier Ireland Ltd. All rights reserved.

Metabolomic studies have shown the prominent role of centralcarbohydrate metabolism during temperature stress [5]. Carbo-hydrates are a major category of compatible solutes that includehexoses (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 inthe structure and function of all cells. These compatible solutes, orosmoprotectants, can accumulate to high levels without disturbingintracellular biochemistry [8].

In previous studies, the involvement of soluble carbohydrate inthe reactive oxygen species (ROS) balance and response to oxidativestress in plants has been thoroughly described [9,10]. In fact, differ-ent studies have demonstrated the importance of raffinose familyoligosaccharides (RFOs) in stress, acting as osmoprotectants, cell-membrane stabilizers, or ROS scavengers [11–14]. Accumulationof 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 carbo-hydrates. 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-6-phosphate and can be further methylated to sequoyitol or ononitol,which are then epimerized to D-pinitol [17].

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F. Palma et al. / Plant Scie

Refrigerated storage, considered to be the most effectiveethod for preserving the quality of fruit and vegetables, allows

ong-distance transport and thus a more regulated supply of com-odities in the market. However, this common storage practice

an cause chilling injury in some fruit and consequently high eco-omic losses. Chilling injury is a physiological disorder of fruits andegetables caused by low temperatures above freezing. ZucchiniCucurbita pepo L. morphotype Zucchini), an important greenhouserop in south-eastern Spain, bears fruit that is marketed at anmmature stage. Its subtropical origin makes zucchini fruit sus-eptible to chilling disorders when stored at low non-freezingemperatures (above 0 ◦C). As with other types of environmentaltress, chilling triggers ROS production, inducing damage at theellular level [12].

In a previous work analysing the chilling resistance in fruit ofifferent commercial varieties of C. pepo grown in the south-easternpain, we detected differences in chilling damage among varietiesfter 14 days of cold storage [18]. That is, some varieties registered aow chilling index with lower levels of accumulated reactive oxygenpecies (ROS) and malondialdehyde (MDA), while other varietieseached the maximum chilling index after 7 days of storage andccumulated greater amounts of ROS and MDA.

The aim of this work was to evaluate the pattern and contentf soluble carbohydrates in zucchini fruit in response to storaget two temperatures, and to compare two varieties with differ-nt responses to cold storage in order to relate specific sugars todaptation low-temperature.

. Materials and methods

.1. Plant material and storage conditions

Zucchini fruit (C. pepo L. morphotype Zucchini) of the com-ercial varieties Natura (Enza Zaden) and Sinatra (Clause-Tezier)ere provided by Mayes Exportacion S.L. After harvest, fruit were

tored in chambers at 4 ◦C and 20 ◦C. Three biological replicatesere prepared per variety and storage period (0, 7 and 14 days),

ach consisting in 6 fruit of similar size and maturation time. Afterhe storage period, weight loss and the chilling-injury index wereetermined. For each biological replicate, the exocarp of all fruitsas separated, mixed and grinded in liquid nitrogen, and stored at20 ◦C.

.2. Weight loss and chilling-injury index

The fresh weight loss (18 fruit per variety, temperature andtorage period) was determined after 7 and 14 days of storaget 4 ◦C and 20 ◦C and the percentage of weight loss of each fruitas calculated. The chilling-injury index (CI) was evaluated using a

ubjective scale of visual symptoms previously described Martínez-éllez et al. [19]: 0 = no pitting, 1 = slight (10% or less), 2 = medium10–20%), and 3 = severe pitting (>20%). CI index was determinedsing the following formula: (pitting scale (0–3) × number of cor-esponding fruit within each class)/total number of fruit estimated18 fruit).

.3. Preparation of extracts

Exocarp tissue (1 g) was homogenized in 8 ml of coldthanol/chloroform/water (12/5/1, v/v/v) and the homogenate was

entrifuged at 4 ◦C and 3500 × g for 10 min [20]. The resulting insol-ble residue was used to determine the starch, and the supernatantas separated into aqueous and chloroform phases by the addition

f chloroform (5 ml) and water (3 ml). Soluble carbohydrates wereetermined from the aqueous phase.

7– 218 (2014) 78– 86 79

2.4. Soluble carbohydrate content

To measure the content of total soluble carbohydrates, 0.1 mlof aqueous phase was mixed with 3 ml anthrone reagent (150 mganthrone and 100 ml of 72% sulfuric acid). The samples were placedin a boiling water bath for 10 min. The light absorption of thesamples was estimated at 625 nm. The contents in total solublecarbohydrates were determined using glucose as the standard.

The aqueous phase was evaporated under a flow of nitrogenuntil dry, in order to measure the different soluble carbohydrates.Finally, dry residues were resolubilized in 0.5 ml of Milli-Q water at40 ◦C, centrifuged (10,000 × g 15 min at 4 ◦C) and the supernatantwas suitably diluted.

Carbohydrates were separated and quantified by ion chro-matography with pulsed amperometric detection according toCataldi et al. [21] with modifications, conducted on a DionexICS-3000 chromatograph (Dionex Corp., Sunnyvale, CA, USA). Thepolyols, myoinositol, pinitol, galactinol, sorbitol and mannitol, andtrehalose were separated in a CarboPac MA1 column and sucrose,glucose, fructose, galactose, raffinose, and stachyose were sepa-rated in a CarboPac PA20 column, plus precolumns (Dionex Corp.,Sunnyvale, CA, USA) kept at 30 ◦C, using water and NaOH (0.8 M) aseluents. Gradient conditions are shown in Table 1.

2.5. Starch content

Insoluble residue was washed several times with 80% ethanoland dried. This residue was rehydrated in 1 ml of water and heatedfor 1 h at 90 ◦C. The gelatinized starch sample was incubated withamyloglucosidase (10 units ml−1 0.1 M acetate buffer, pH 4.5) for48 h at 40 ◦C, following Holland et al. [3]. Starch content was deter-mined as glucose produced. Glucose was measured using the KitBioSystems (Ref. 11504).

2.6. Sucrose synthase-cleavage activity

Extracts were prepared by homogenizing 0.5 g of exocarp ina mortar with 33% (w/w) polyvinyl-polypyrrolidone and 2 mlof 50 mM phosphate K buffer (pH 7.5) containing 1 mM EDTA,5 mM �-mercaptoethanol, 2.5 mM DTT, and 20% (v/v) glycerol. Thehomogenate was centrifuged at 4 ◦C and 10,000 × g for 15 min, andthe supernatant was desalted using Amicon Ultra 10K (Millipore).

Sucrose synthase-cleavage activity was measured according toMorell and Copeland [22]. The production of UDP-glucose was cou-pled to the reduction of NAD+ in the presence of excess UDP-glucosedehydrogenase. Reaction mixtures contained 100 mM Hepes-KOHbuffer (pH 7.5), extract, 100 mM sucrose, 2 mM UDP, 0.025 U UDP-glucose dehydrogenase and 1.5 mM NAD+. The enzyme activity wasspectrophotometrically measured by following the NAD+ reductionat 340 nm.

2.7. Invertase activity

Extracts were prepared by homogenizing 0.5 g of exocarp ina mortar with 33% (w/w) polyvinyl-polypyrrolidone and 2 ml of50 mM Hepes-KOH buffer (pH 7.5) containing 1 mM EDTA, 10 mMMgCl2, 2.5 mM DTT, and 10 mM ascorbic acid. The homogenate wascentrifuged at 4 ◦C and 10,000 × g for 15 min, and the supernatantwas desalted using Amicon Ultra 10K (Millipore).

Neutral invertase activity was assayed in an incubation mix-ture containing 100 mM phosphate K buffer (pH 7.5), extract, and200 mM sucrose. The mixture was incubated during 30 min at 30 ◦C.

The assay was stopped by boiling, and the glucose generated wasdetermined with the Kit BioSystems (Ref. 11504).

Acid invertase activity was assayed in an incubation mixturecontaining 100 mM citrate buffer (pH 5), extract, and 200 mM

80 F. Palma et al. / Plant Science 217– 218 (2014) 78– 86

Table 1Gradient 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

0 100 0 0 98.5 1.530 25 75 15 98.5 1.540 25 75 20 70 3050 100 0 30 70 30

40 98.5 1.5

sat(

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3

3s

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3f

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TC

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Flow rate 0.4 ml/min

0–30 min linear gradient

40–50 min linear gradient

ucrose. The mixture was incubated during 30 min at 30 ◦C. Thessay was stopped by boiling, and neutralized with NaOH. Finally,he glucose generated was determined with the Kit BioSystemsRef. 11504).

.8. Statistical analysis

Data were subjected to an analysis of variance (ANOVA) usinghe Statgraphics®Plus 5.1 software (Statistical Graphics Corp.,ockville, MD, USA). The data presented were means of three repli-ates of six fruit each with a standard error of means, exceptor the percentage of weight loss and chilling injury, which were

eans of 18 fruit. Means were compared by Fisher’s least significantifference test (LSD) and differences at p < 0.05 were consideredignificant.

. Results

.1. Weight loss and chilling-injury index in zucchini fruit duringtorage

When fruit of both varieties was kept at 4 ◦C, after 7 and 14 days, significantly higher weight loss was recorded in Sinatra comparedo Natura (Table 2). A similar result was found for the chilling-injuryndex, where Natura registered the lowest value even after 14 daysf storage, a mean of 0.67 and 1.72 at days 7 and 14, respectively,hile Sinatra reached an index of almost 3.0, the maximum, after 7ays of storage at 4 ◦C (Table 2). In general, higher values of weight

oss were observed in zucchini fruit stored at 20 ◦C.

.2. Changes in total soluble carbohydrates content in zucchiniruit during storage

In the exocarp of Sinatra fruit stored at 4 ◦C, the content inoluble carbohydrates did not significantly change, whereas this

arameter increased in Natura fruit by about 1.3- and 2-fold dur-

ng storage at 4 ◦C after of 7 and 14 days, respectively (Fig. 1). Onhe contrary, during storage at 20 ◦C the content in soluble carbo-ydrates diminished with storage time in both varieties; after 14

able 2hanges in percentage of weight loss and chilling injury index in zucchini fruit stored at 4

Weight loss (%)

Temperature Variety 7 days

4 ◦C Natura 3.46 ± 0.28bB

Sinatra 5.62 ± 0.41bA

20 ◦C Natura 3.66 ± 0.30bB

Sinatra 7.97 ± 0.58bA

alues are means of 18 fruits ± SE. Different capital letters indicate significant differences among storage time (p < 0.05)

Flow rate 0.5 ml/min15–20 min linear gradient30–40 min linear gradient

days a decrease of 55% and 77% was registered in fruit of Naturaand Sinatra, respectively (Fig. 1).

3.3. Changes in soluble carbohydrates and starch content inzucchini fruit during storage

In the exocarp of Natura fruit, the galactose content did not sig-nificantly change at 4 ◦C and it significantly diminished at 20 ◦C.In this variety the reducing soluble carbohydrates glucose andfructose (Fig. 2) exhibited the same trend as the total solublecarbohydrates during the storage—in fact, after 14 days at 4 ◦C, glu-cose and fructose increased around 2-fold, whereas at 20 ◦C bothcarbohydrates declined. In general, these soluble carbohydrates(galactose, glucose, and fructose) did not increase significantly infruit of the more susceptible cultivar (Sinatra) when stored at 4 ◦C,and only after 7 days of storage did the galactose content increase1.4-fold (Fig. 2). Similarly, as described in Natura, the galactose,glucose, and fructose content in Sinatra fruit also decreased duringstorage at 20 ◦C (Fig. 2).

In the case of both varieties (Natura and Sinatra) and storagetemperatures (4 ◦C and 20 ◦C) the disaccharide trehalose was notdetected, whereas sucrose showed a very low concentration (datanot shown).

In the present study, our results show that during storage at4 ◦C and 20 ◦C, both Natura and Sinatra fruit accumulated raffi-nose; however, the Natura exhibited significantly higher levels ofraffinose compared to Sinatra at 4 ◦C (Fig. 3). The highest raffinosecontent was detected during storage at 20 ◦C in both varieties, and,after 7 and 14 days, raffinose increased about 3.5-fold in Natura andSinatra (Fig. 3).

In Natura fruit stored at 4 ◦C, the stachyose content fell signifi-cantly, about 2.5-fold and 3.5-fold after 7 and 14 days, respectively.In the more sensitive variety, the stachyose content did not changesignificantly 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 (about10-fold) by the storage of the fruit, at either 4 or 20 ◦C (Fig. 4).

◦C and 20 ◦C.

Chilling injury index

14 days 7 days 14 days

6.50 ± 0.45aB 0.67 ± 0.18bB 1.72 ± 0.27aB

9.38 ± 0.38aA 2.81 ± 0.09aA 2.82 ± 0.10aA

9.81 ± 0.68aA – –10.61 ± 0.43aA – –

mong varieties (p < 0.05). Different lower case letters indicate significant differences

F. Palma et al. / Plant Science 217– 218 (2014) 78– 86 81

At harvest 7 da ys 14 da ys

0

8

16

24

32Total Soluble C arb ohydrates

aA

cB

bA aA aBaA

mg

gluc

ose

g-1FW

Sinatra Natura

Stora ge at 4ºC

0

8

16

24

32Total Soluble Carbo hydrates

aB

cAbB

aA

cA

bA

mg

gluc

ose

g-1FW

Sinatra Natura

Stora ge at 20ºC

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).

0

4

8

12

16

mg

g-1FW

Glucos e

aB

cAbA

aA

cAbA

0

4

8

12

16Glucos e

bAbB

aA

abA

bB

aA

mg

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At harvest 7 da ys 14 da ys

0.0

0.2

0.4

0.6GalactoseaA

aA

aA

bA bB

aA

mg

g-1FW

Storage at 4ºC

0.0

0.2

0.4

0.6m

g g-1

FWGalactose

aA

cA

bA

aA

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Stora ge at 20ºC

0

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mg

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Fructos e

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Sinatra Natura 0

4

8

12

16Fructose

mg

g-1FW

bA

cB

aA

aA

bB

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Sinatra Natura

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).

82 F. Palma et al. / Plant Science 217– 218 (2014) 78– 86

0.0

0.8

1.6

2.4

3.2Raffinose

bA

cB

aA

bA abBaA

mg

g-1FW

Stora ge at 4ºC

0.0

0.8

1.6

2.4

3.2

mg

g-1FW

Raffinose

aA

cB

bB

aA

bA

aA

Stora ge at 20ºC

0.0

0.1

0.2

0.3

0.4

mg

g-1FW

Stachyos e

aA

bA

aA

aB

cBbB

Sinatra Natura 0.0

0.1

0.2

0.3

0.4Stach yos e

bA

cA

aA

aA aAaB

mg

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Sinatra Natura

7 days At harvest 14 da ys

F it storD ifferen

3

N1essibAi

Fi

ig. 3. Changes in raffinose and stachyose contents in the exocarp of zucchini fruifferent capital letters indicate significant differences among varieties (p < 0.05). D

.4. Changes in polyols content in zucchini fruit during storage

Galactinol and myoinositol increased in fruit stored at 4 ◦C inatura as well as Sinatra, so that after 14 days galactinol was about0-fold and myoinositol was about 2.5-fold higher in both vari-ties. By contrast, during storage at 20 ◦C these polyols showed noignificant change or even decreased (Fig. 5). The pinitol contenthowed an opposite trend in the two varieties when stored at 4 ◦C,

ncreasing in the exocarp of Natura fruit after 14 days (1.5-fold),ut decreasing with time in the exocarp of Sinatra fruit (Fig. 5).t 20 ◦C this polyol decreased in both varieties, Sinatra exhibit-

ng a greater reduction compared to Natura. The polyols mannitol

0.00

0.15

0.30

0.45

0.60

At ha rve st 7 day

Starch

mg

gluc

ose

g-1FW

bB

aA

bAbA

aA

cA

Storage at 4ºC

Natura Sinatra

ig. 4. Changes in starch content in the exocarp of zucchini fruit stored at 4 ◦C and 20 ◦C. Vndicate significant differences among varieties (p < 0.05). Different lower case letters ind

ed at 4 ◦C and 20 ◦C. Values are means ± SE of triplicate samples of six fruit each.t lower case letters indicate significant differences among storage time (p < 0.05).

and sorbitol were not detected in either zucchini variety stud-ied.

3.5. Changes in sucrose synthase-cleavage, neutral invertase, andacid invertase activities in zucchini fruit during storage at 4 ◦C

The sucrose synthase-cleavage shows a very low activity in bothvarieties (Fig. 6). Invertase is the main enzyme responsible for the

sucrose hydrolysing activity. In Natura fruit, acid and neutral inver-tase activities increased during storage at 4 ◦C, both about 25% after14 days (Fig. 6). Acid invertase showed an opposite trend in Sina-tra fruit, its activity decreased about 10% after 14 days when the

s 14 days

0.00

0.15

0.30

0.45

0.60Starch

bB

aA

bA

mg

gluc

ose

g-1FW

bA

aA

bA

Storage at 20ºC

Natura Sinatra

alues are means ± SE of triplicate samples of six fruit each. Different capital lettersicate significant differences among storage time (p < 0.05).

F. Palma et al. / Plant Science 217– 218 (2014) 78– 86 83

0.0

0.2

0.4

0.6

0.8

mg

g-1FW

Myoinosit ol

aA

bBbB

aA

cAbA

0.0

0.2

0.4

0.6

0.8Myoinosit ol

bB

cA

aA aA

bA

aA

mg

g-1FW

At harvest 7 da ys 14 da ys

0.00

0.05

0.10

0.15

0.20Galacti nol

bB

cB

aB bA

cA

aA

mg

g-1FW

Stora ge at 4ºC

0.00

0.05

0.10

0.15

0.20

mg

g-1FW

Galactino l

aB aBaA aA aAaA

Stora ge at 20ºC

Natura Sinatra 0.0

0.4

0.8

1.2

1.6

mg

g-1FW

Pinit ol

aB

bAcA

aA

cAbA

0.0

0.4

0.8

1.2

1.6Pinit ol

mg

g-1FW

bAbB

aA aA

bBcA

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 sixf p < 0.0(

fsais

4

dFcmtwblt

ruit each. Different capital letters indicate significant differences among varieties (p < 0.05).

ruit was kept at 4 ◦C. On the other hand, neutral invertase activityhowed a slight increase in Sinatra fruit (7%) after 14 days of stor-ge at 4 ◦C. Natura fruit exhibited significantly higher levels of bothnvertase activities compared to Sinatra fruit after 14 days of coldtorage (Fig. 6).

. Discussion

The present study examines the importance of soluble carbohy-rates in chilling adaptation of zucchini fruit during postharvest.or this, we used fruit of two varieties with different responses toold storage: Natura, more tolerant to chilling storage; and Sinatra,ore sensitive [18]. Our results show a greater weight loss in Sina-

ra compared to Natura during storage at 4 ◦C and a similar result

as found for the chilling-injury index. In both varieties and at

oth storage temperatures used, the fruit lost weight, the greatestosses being measured in fruit stored at 20 ◦C, probably because athis temperature all the metabolic pathways were still active and

5). Different lower case letters indicate significant differences among storage time

there was a higher respiration rate, as happens in citrus fruit at12 ◦C [3].

Soluble carbohydrates are considered important factors relatedto chilling adaptation in plants and fruits, and it has recently beenproposed that sugars can act as real ROS scavengers in plants,especially when present at higher concentrations [11]. There isreportedly an increase in the content of reducing soluble carbohy-drates with cold temperatures in grapefruit [23], red raspberry [24],squash [25], rice seedlings [26] and in cucumber [27]. In our experi-ment, fruit of both varieties was stored at 4 ◦C and 20 ◦C and sampleswere taken after 7 and 14 days. In the exocarp of Natura fruit theconcentration of total soluble carbohydrates increased during stor-age at 4 ◦C. By contrast, during storage at 20 ◦C, this parameterdiminished with storage time. This response could indicate thatthe accumulation of total soluble carbohydrates is related to adap-tation to low-temperature in Natura, whereas in Sinatra kept at 4 ◦C

no significant changes were detected. High levels of total solublecarbohydrates were found under cold stress in a hardy raspberrycultivar [24,28], in cabbage [29], arabidopsis [30], olive [31], andcherry [32]. In our study, during storage at 20 ◦C the reducing sugar

84 F. Palma et al. / Plant Science 21

At harvest 7 da ys 14 days

0

40

80

120

160Sucro se synthase-c leav age

bA

aA aA

aBaAaB

nmol

NA

DH

g-1

FW m

in-1

0

300

600

900

1200Neu tral invert ase

bAcA

aA

bBbA aB

nmol

glu

cose

g-1

FW m

in-1

1

0

300

600

900

1200Acid inv ertas e

bAbB

aA

aA bAcB

nmol

glu

cose

g-1

FW m

in-1

1

Sinatra Natura

Fig. 6. Changes in sucrose synthase-cleavage, neutral invertase, and acid invertaseactivities in the exocarp of zucchini fruit stored at 4 ◦C. Values are means ± SE oftriplicate samples of six fruit each. Different capital letters indicate significant dif-fd

gezcSiciiissgii2tti

thase and raffinose synthase accumulated high levels of galactinol

erences among varieties (p < 0.05). Different lower case letters indicate significantifferences among storage time (p < 0.05).

alactose, glucose, and fructose diminished in both varieties. How-ver, at 4 ◦C, glucose and fructose showed opposite responses in theucchini varieties studied. That is, in the exocarp of Natura fruit theontent of these carbohydrates increased dramatically, whereas ininatra fruit that started with a higher amount in these sugars, noncrease was detected after 14 days, and a small reduction in glu-ose was found, but values were in all cases greater than the amountn the fruit kept at 20 ◦C. The results for Natura imply that reduc-ng soluble carbohydrates (glucose and fructose) could be involvedn the cold-storage adaptation in zucchini fruit. However, there isome controversy in the literature about the role of these reducingugars (glucose and fructose). Purvis and Grierson [23] showed thatlucose and fructose are involved in the increased chilling tolerancen grapefruit, whereas Holland et al. [3] concluded that the changesn carbohydrate levels in citrus pretreated with heat and stored at◦C and 12 ◦C are related mainly to the consumption of reserves in

hese types of fruit for respiration and not to chilling tolerance. Onhe other hand, many fruit species reportedly undergo an increasen the soluble carbohydrates due to ripening when stored at room

7– 218 (2014) 78– 86

temperature [33–35]. Zucchini fruit is marketed at an immaturestage, but we nevertheless detected a decrease in the content oftotal soluble carbohydrates, galactose, glucose, and fructose whenthe fruit was kept at 20 ◦C, a finding that could be due to an increasein carbon consumption required to sustain fruit respiration at ahigher temperature, as happens in mandarin fruit (Holland et al.,2002). During the exposure of plants to chilling temperatures, themost 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 stabiliza-tion of membranes by interacting with phospholipid headgroups[37,38]. Recently, it has been reported that the accumulation ofgalactinol, raffinose, and other RFOs in plant cells is closely asso-ciated with responses to different types of environmental stress,including chilling [12,26,27,39]. Nishizawa et al. [12] suggested thepossibility that raffinose acts not only as an osmoprotectant andstabilizer of cell membranes but also as ROS scavenger, thus play-ing a role in protecting the cell metabolism from oxidative damagecaused by salinity, drought or chilling. Our results show that duringstorage at 4 ◦C and 20 ◦C both Natura and Sinatra fruit accumulatedraffinose; however, Natura exhibited significantly higher levels ofraffinose compared to Sinatra at 4 ◦C. It is noteworthy that duringstorage at 20 ◦C the increase in raffinose was more remarkable thanat 4 ◦C in both varieties, and this increase correlates with the greaterweight loss detected at 20 ◦C, so that in zucchini fruit the role of raf-finose may be to help protect tissues against dehydration, perhapsacting as an osmoprotectant and stabilizer of cell membranes. Otherstudies have reported that the acquisition of desiccation toleranceis associated with an accumulation of raffinose and stachyose [40].The accumulation of raffinose, galactinol, galactose, glucose, andfructose could limit stachyose accumulation, since these sugars actas a substrate for the stachyose synthesis, and low levels of thesesoluble carbohydrates were detected when raffinose increased. Inboth varieties the starch content was drastically reduced by thefruit storage, at either 4 or 20 ◦C. Respiration is an ongoing processin fruit that cannot be stopped after harvest, and the continuation ofrespiration could be responsible for starch hydrolysis during stor-age. According to our results, Palonen et al. [24] have been reportedthat starch contents in container-grown plants declined duringcold acclimation and were lowest when plants achieved maximumhardiness, while Kami et al. [25] observed that during storage ofsquash fruits the starch contents were significantly lower at 15 ◦Cthan at the other temperatures, although concentrations decreasedthroughout the storage period at all temperatures.

Polyols, like raffinose, appear to protect against stress withosmotic adjustment and with osmoprotection, acting as osmolytesto facilitate water retention in the cytoplasm and to protect cellularstructures, possibly by scavenging ROS [6]. Palma et al. [4] recentlyreported that augmented pinitol synthesis under salt stress couldbe one of the adaptive features used by the plant, and Nishizawaet al. [12] have suggested that galactinol protects plants from oxida-tive damage. Furthermore, myoinositol and galactinol are requiredfor the raffinose synthesis, raffinose synthase being one of the keyenzymes regulating soluble carbohydrate metabolism in cucumber[27]. In the present study, we detected increases in myoinositol andgalactinol in fruit stored at 4 ◦C in both zucchini varieties; by con-trast at 20 ◦C these polyols showed no significant change or evendecreased. The response observed for these polyols in both varietiessupports the hypothesis ascribing an important role to galactinoland myoinositol in the adaptation to low-temperature of zucchinifruit. Transgenic Arabidopsis plants overexpressing galactinol syn-

and raffinose, and these plants were more tolerant to oxidativedamage, salinity, chilling, and drought [12]. In this study, man-nitol and sorbitol were not detected, although these polyols may

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articipate in a wide range of physiological processes in plants,nd especially serving as an osmotically active compatible solutes41]. Sorbitol can be converted both to fructose by sorbitol dehy-rogenase and to glucose by sorbitol oxidase [42]. Once unloaded,orbitol is immediately transformed to other compounds and doesot accumulate in fruit tissues, regardless of whether the assimilatevailability is high or low [43].

In fruit kept at 4 ◦C, we found opposite response in the pini-ol content in the two varieties, i.e. increasing in Natura fruit after4 days, but decreasing in Sinatra fruit after 7 and 14 days. At0 ◦C this polyol decreased in both varieties. These results sug-est that pinitol metabolism is important for cold protection ofhe stressed fruit tissues, since the accumulation was higher inatura, although the fruit of Sinatra showed a higher amount ininitol before storage. It has been reported that pinitol is relatedo different types of stress, including cold [6]. Pinitol augments intressed ice plant (Mesembryanthemum crystallinum) to become theajor low-molecular-weight carbon compound, with concentra-

ions exceeding 700 mM in cytosol and chloroplasts [44,45]. Theevels of galactinol, myoinositol, and pinitol rose in the more tol-rant cultivar; this might indicate that such increases are fruitesponses to cope with chilling stress, since in fruit kept at 20 ◦Cll three sugar alcohols declined with storage time.

In order to investigate the origin of the reducing soluble sugars,ucrose hydrolysing enzymes were measured in both varieties. Theighest activity corresponded to acid and neutral invertase, and theucrose synthase activity was much lower in all the fruit exocarpeasured. During storage at 4 ◦C, Natura fruit showed an increase of

he levels of glucose and fructose and this increase was concomitantith higher activities of the sucrose hydrolysing enzymes, both acid

nd neutral invertase. In Sinatra, neutral invertase shows a slightncrease during cold storage, but acid invertase decreases after 14ays at 4 ◦C. These results suggest that the differences in hexoseontent in Natura after 14 days of cold storage is related in part ton increase in acid invertase activity in this variety. Thus, induc-ion of acid invertase activity in zucchini fruit might be a factorf adaptation to cold storage. Changes in invertase activity underold stress have been reported in many plant species. In wheat,

stimulation of acid invertase activity in leaves by cold acclima-ion of winter wheat, but not spring wheat has been detected [46].n potato tubers, low temperature storage induces acid invertasectivity [47]. Also, potato plants transformed with a yeast-invertaseene were more resistant to chilling than control plants [48].

In view of the above, the reducing soluble carbohydrates andolyols are related to the physiological response of zucchini fruito cold stress, whereas the role of raffinose is more likely relatedo dehydration than to chilling susceptibility of zucchini fruit. Glu-ose, fructose, and pinitol responded oppositely against chilling inhe two varieties, so that the metabolism of these sugars coulde involved in the adaptation to chilling of zucchini fruit, withn induction of acid invertase in the resistant variety. Therefore,urther research studying changes in metabolism carbohydrates inhe fruit of different cultivars showing different susceptibility tohilling would shed more light on these findings.

cknowledgments

This research has been funded by the Ministerio de Educación Ciencia (Project AGL2011-30568-C02-01). F. Carvajal was sup-orted with a grant FPU (Ministerio de Educación, Spain). We arerateful to Mayes Exportacion S.L. for supplying the fruit.

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