Innovative Food Science and Emerging Technologies 30 (2015) 157–169

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Innovative Food Science and Emerging Technologies

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Nitric oxide prevents wound-induced browning and delays senescencethrough inhibition of hydrogen peroxide accumulation in fresh-cut lettuce

Elena T. Iakimova a,1, Ernst J. Woltering a,b,⁎a Wageningen University, Horticultural Supply Chains Group, Droevendaalsesteeg 1, P.O. Box 630, 6700 AP Wageningen, The Netherlandsb Wageningen University, Food and Biobased Research, Bornse Weilanden 9, P.O. Box 17, 6700 AA Wageningen, The Netherlands

⁎ Corresponding author at: Wageningen University,Bornse Weilanden 9, P.O. Box 17, 6700 AA Wageninge317480116, +31 317483527.

E-mail addresses: elena_iakimova@abv.bg (E.T. Iakimo(E.J. Woltering).

1 Permanent address: Institute of Ornamental Plants, 1

http://dx.doi.org/10.1016/j.ifset.2015.06.0011466-8564/© 2015 Elsevier Ltd. All rights reserved.

a b s t r a c t

a r t i c l e i n f o

Article history:Received 28 July 2014Received in revised form 27 March 2015Accepted 1 June 2015Available online 18 June 2015

Keywords:Fresh-cut lettuceWoundingBrowningNitric oxideHydrogen peroxideCell death

As a source of bioactive ingredients, lettuce is a preferable component of a healthy diet. In recent years the pro-duction of fresh-cut produce has become a fast growing business. However, the shreds are highly sensitive towound-induced browning and premature senescence that substantially reduces the visual and sensory qualitiesand shortens the shelf life. To improve the fresh-cut quality, in this work, short pre-storage exposure of shredsfrom butterhead and iceberg lettuce to nitric oxide (NO) gas was applied. It was found that fumigation with100 and 200 ppm NO for 1 or 2 h remarkably inhibited the browning of the cut surface and of other injuredleaf areas; NO treatment delayed the senescence and substantially prolonged the shelf life upon storage at 4 °Cand 12 °C. To obtain information on the physiological processes involved in the wound response, the generationof hydrogen peroxide (H2O2) and the occurrence of cell death were analyzed. The results revealed that thewounding stimulated the accumulation of H2O2 thus generating oxidative stress leading to cell death. A correla-tion between elevated H2O2 levels, cut surface browning, senescence and storability of the fresh-cuts wasestablished. In comparison to mature leaves, younger leaves expressed a lesser susceptibility to wound-induced browning and the associated oxidative stress. Applied NO strongly inhibited the H2O2 accumulationwhich may explain its beneficial effects.Industrial relevance: We demonstrate that short treatments with NO gas substantially inhibit wound-inducedbrowning and largely improve the storability of fresh-cut lettuce. This offers an option for adopting NO treat-ments for optimization of the post-processing conditions. Implementation of reported findings into practicewill offer innovative technological solutions for improvement of the post-harvest quality of fresh-cut lettuce ap-plicable for the industry, distributors and retailers. Moreover, our findings indicate that the browning disorder isto a large extent dependent on the severity of thewound-induced oxidative stress and cell death occurrence. Thisdiscovery opens a possibility for the development of metabolic and morphological markers appropriate for theprediction of the quality and shelf life of fresh-cut lettuce with perspective for expanding the research and intro-ducing the tests toward other perishable leafy vegetables.

© 2015 Elsevier Ltd. All rights reserved.

1. Introduction

The production of lightly processed (fresh-cut) leafy vegetables is afast growing business. These ready-to-use products save time andlabor which makes them preferred by the food service industry and byconsumers. Also, all waste material has been removed which reducesthe shipping costs (Bolin, Stafford, King, & Huxsoll, 1997). Butterhead,romaine and iceberg lettuce (Lactuca sativa L.) are recommended com-ponents of a healthy diet suggested to provide a source of antioxidants,

Food and Biobased Research,n, The Netherlands. Tel.: +31

va), ernst.woltering@wur.nl

222 Negovan, Sofia, Bulgaria.

phenolics, anthocyanins, carotenoids and vitamins C and E (Caldwell,2003; Llorach, Martínez-Sánchez, Tomás-Barberán, Gil, & Ferreres,2008). The consumption of lettuce may contribute to lowering of cho-lesterol, reducing the risks of coronary heart diseases, preventingsome types of cancer, slowing down aging and improving the overall vi-tality (Kaur & Kapoor, 2001; Nicolle, Cardinault, et al., 2004; Nicolle,Carnat, et al., 2004). However, the supply to consumers of fresh-cut let-tuce of good quality and high levels of health-promoting ingredients de-pends on a number of factors including genetic background, pre-harvestconditions, developmental stage at harvest, processing parameters andpostharvest handling conditions (Barrett, Beaulieu, & Shewfelt, 2010;Cantwell & Suslow, 2004; Gil, Tudela, Martínez-Sánchez, & Luna, 2012;Martínez-Sánchez et al., 2012; Saltveit, Ochoa, Campos-Vargas, &Michelmore, 2003; Witkowska & Woltering, 2010, 2013, 2014). Lightspectrumand intensity during the growingperiodhave been shown to af-fect the quality of lettuce at harvest and quality decay of fresh-cuts.

158 E.T. Iakimova, E.J. Woltering / Innovative Food Science and Emerging Technologies 30 (2015) 157–169

Combined lighting of hydroponically cultured lettuce with red (R), blue(B) and white (W) — RBW LEDs resulted in several positive effects ongrowth, development, appearance, marketable sensory characteristicsand nutritional quality of lettuce plants at harvest (Lin et al., 2013). In ad-dition, it was reported that exposure to high light intensities during thegrowing period is beneficial for inhibiting tissue browning (a strikingpost-harvest disorder), maintaining nutritional and health-promotingconstituents (e.g. soluble sugar and L-ascorbic acid (AsA)) and extendingthe postharvest life of the fresh-cut product (Witkowska & Woltering,2010; Zhan, Li, Hu, Pang, & Fan, 2012; Zhan et al., 2013). In addition,Woltering and Seifu (2015) established that continuous low light intensi-ty during storage of the fresh-cut product (5 μmol m−2 s−1) also consid-erably prolonged the shelf life of lettuce fresh-cuts.

During the postharvest period the whole-heads that are usually har-vested when they are immature, before growth has ceased, suffer froma sudden disruption of energy supply, depletion of nutrients, lack ofhormone supply, bacterial and fungal diseases and other stressful circum-stances that lead to substantial quality loss (Huber, 1987). Inappropriatedark/light regime, excessive water loss, inappropriate atmospheric com-position and stress-induced ethylene production during storage often re-sult in rapid deterioration of leaf tissues (Bolin et al., 1997; Saltveit, 2004).

The storage life of fresh-cuts is even shorter compared to whole-heads because during the processing (shredding, washing, drying) andpackaging they are additionally exposed to mechanical injury (King,Woollard, Irving, & Borst, 1990). Cutting, abrading, bruising or foldingof the detached leaves might stimulate the respiration and ethyleneproduction and sensitize the fresh-cuts to microbial spoilage thus to agreat extent playing a decisive role in quality and shelf life. The visibleand sensory quality declines appear as tissue discoloration, pinking,russet spotting, loss of crispness, formation of necrotic or water soakedlesions, undesirable flavor and texture, bitter taste and decay (Saltveitet al., 2003; Woltering & Iakimova, 2010). Among the physiological dis-orders detected in fresh-cut lettuce are also reduced photosynthetic ac-tivity, oxidative stress, including excessive production of reactiveoxygen species (ROS) and changes in antioxidant enzymatic and non-enzymatic systems and depletion of energy (e.g. sugar) sources (Gill &Tuteja, 2012 and references therein; Saltveit et al., 2003).

Presumably the tissue dismantlement might also be due to prema-ture (e.g. dark-, temperature-induced) senescence (a form of pro-grammed cell death, PCD) (Van Doorn & Woltering, 2004; Wagstaffet al., 2007; Woltering & Iakimova, 2010). According to the general def-inition, PCD is an active process of controlled cellular suicide that occursas a part of normal development of multicellular organisms (plants andanimals) and in response to stressful environmental cues. It is hypothe-sized that a number of postharvest disorders and the severe effects ofprocessing in leafy vegetables that are accompanied by macroscopicsigns of cell death (yellowing, internal browning, sunken and water-logged areas, disappearance of cells) are due to a form of PCD inducedby these specific circumstances (Woltering & Iakimova, 2010 and refer-ences therein).

Senescence (a form of PCD) is a natural process, inevitable last phaseof the life span of cells, organs and whole plants that culminates indeath. Apart from being a normal developmental event, in the presenceof stressful environmental factors of biotic and abiotic origin leaf senes-cence can be prematurely induced (Buchanan-Wollaston et al., 2003;Van Doorn & Woltering, 2004). Studies on postharvest disorders ofleafy vegetables have shown that the death of the cells might be dueto PCD. It is suggested that the wounding of lettuce leaf tissues mightalso trigger PCD (Wagstaff et al., 2007).

The reduced shelf life of harvested baby lettuce leaves was associat-ed with PCD features such as membrane disruption, plasmolysis andelectrolyte leakage. Ultrastructural cellular changes including the pres-ence of cytoplasmic fragments in the vacuole, increased appearance ofvesicles and microbodies in the mesophyll and epidermal cells and,disruption of the nuclear envelope and plastid membranes were ob-served (Wagstaff et al., 2007). Investigations on postharvest processes

in broccoli sepals demonstrated that the tissue collapse was accompa-nied with PCD markers such as laddering pattern of DNA degradationand changes in expression of cell death-related genes (Coupe, Sinclair,Watson, Heyes, & Eason, 2003; Eason, Ryan, Page, Watson, & Coupe,2007). These examples indicate that postharvest disorders and senes-cence can be considered as a form of PCD.

The most striking visual appearance of wound-induced damage infresh-cut lettuce is the brown coloration of the cut surface. Variousapproaches for delaying the postharvest senescence, preventing thebrowning and extending the storage life of lettuce have been proposed.Treatments with calcium salts (Perucka & Olszówka, 2011; Rico,Martín-Diana, Henehan, Frías, & Barry-Ryan, 2006) and antioxidants,AsA, green tea extract and hot water pulse treatments (Castañer, Gil,Asrtes, & Tomas-Barberan, 1996; Loaiza-Velarde, Tomás-Barberá, &Salveit, 1997; Martín-Diana, Rico, & Barry-Ryan, 2008) have been foundbeneficial for preventing thewound-induced browning of the cut surface.

An established practice is the shredded lettuce to be stored underquite severe conditions of low temperature and controlled or modifiedatmospheres (CA, MA) with low oxygen (b1%) and increased carbondioxide levels (up to 10–15%) (Ballantyne, Stark, & Selman, 1988;Fonseca, Oiveira, & Brecht, 2002; Kader & Saltveit, 2003). Althoughslowing down the aging of the product, such conditions may have sideeffects and may lead to “storage disorders” such as chilling-associatedinjuries and low oxygen or high carbon dioxide disorders (Saltveit,2002; Smyth, Song, & Cameron, 1998).

Thus, the search for alternative treatments to MA or CA packagingmight solve some of the problems. For example, UV-C radiation andozone have been found efficient for extending the lettuce shelf life(Allende & Artes, 2003; García, Mount, & Davison, 2003). Exposure offresh-cut lettuce to nitric oxide (NO) has been suggested as anotherpossibility for improving the storability of the product (Soegiarto,Wills, Seberry, & Leshem, 2003). It was reported that NO releasing com-pounds such as sodium nitroprusside (SNP) or fumigation with NO areeffective for reducing the browning (Hoque, Wills, & Golding, 2011;Wills, Pristijono, & Golding, 2008). However, the physiological and bio-chemicalmechanisms throughwhich exogenously appliedNO exerts itseffect in shredded lettuce are still poorly understood.

NO is a bioactive mobile gaseous signaling molecule that regulatesdiverse physiological processes and mediates various abiotic and bioticstress responses (Leshem, Wills, & Ku, 1998; Neill, Desikan, Clarke,Hurst, &Hancock, 2002 and references therein). As a free radical species,the effects of endogenous NO are mainly executed through oxidativedamage, caused by reactive nitrogen species (RNS). In planta NO can in-teract with cysteine–thiol groups and inactivate proteins through S-nitrosylation or through inactivating enzyme co-factors such as ferrousion. NO operates in crosstalk with hydrogen peroxide (H2O2), otherROS, salicylic acid (SA), methyl jasmonate (MeJa), abscisic acid (ABA)and ethylene and can directly interact with transcription factors (Neill,Desikan, & Hancock, 2003 and references therein). Although generallyconsidered as a highly damaging molecule, the studies have demon-strated that endogenous NO has also beneficial effects. It can act asprotector against various stressful impacts, scavenge free oxygen radi-cals and counteract oxidative damage, inhibit ethylene biosynthesis,delay ripening and senescence and enhance the resistance to diseases(Beckman & Koppenol, 1996; Leshem et al., 1998). In cross-talk withROS, NO is an important mediator in leaf cell death (Wang, Lin, Loake,& Chu, 2013). Depending on the physiological state of the plant cellsand the nature and severity of applied stress, NO can play a dual roleas a potent pro- or anti-cell death agent (Delledonne, Xia, Dixon, &Lamb, 1998; Neill et al., 2003; Wendehenne, Durner, & Klessig, 2004).

In variousworks thewound-induced browning of lettuce is attribut-ed to accumulation of pink and brown colored substances (o-quinones)that are the result of oxidation of phenols. The production of thesecompounds is thought to be associated with stimulation of activities ofenzymes involved in phenolicmetabolism— such as phenylalanine am-monia lyase (PAL), polyphenol oxidase (PPO) and peroxidase (POD)

Final NO concentrations 50, 100, 200, 400 ppm

Step 5. The shreds are transferred to plastic boxes and stored: at 4oC or 12oC

Step1. Lettuce shreds(15 g) are placed in 500 ml

tightly closed jar

Step 2. Nitric oxidestock 5000 ppm

Step 6. Shelf life scoringH2O2 determinationcell death markers

Step 3. Exposure times0.5, 1, 2, 3 h

at 21oC in the dark

Step 4. Jars are opened and left in fume hood to vent out the gas

Fig. 1. Schematic presentation of the experimental procedure. Freshly prepared fresh-cutlettuce (butterhead and iceberg)was treatedwith a fixed amount of gaseous NO for shortperiod of time in darkness. Thereafter vessels were aerated and the fresh-cut producttransferred to well aerated plastic boxes (16 small holes in the lid) and stored at either4 or 12 °C in darkness and product quality features were regularly analyzed. The parame-ters investigated for determination of quality and biochemical characteristics of storedshreds are indicated. In some experiments, this procedurewas applied to fresh-cut lettuce24 h after preparation.

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(Amiot, Fleuriet, Veronique, & Nicolas, 1997; Couture, Cantwell, Ke, &Saltveit, 1993; Degl'Innocenti, Guidi, Pardossi, & Tognoni, 2005;Degl'Innocenti, Pardossi, Tognoni, & Guidi, 2007; López-Gálvez,Saltveit, & Cantwell, 1996a; Pereyra, Roura, & del Valle, 2005). Shorttreatments of fresh-cuts with hypertonic mannitol solution oraromatic- and di-carboxylates were shown to reduce PAL activity andbrowning-associated accumulation of phenols (Kang & Saltveit, 2003;Saltveit & Choi, 2007).

The effects of exogenous NO on enzymatic browning and phenolicmetabolism in fresh-cut lettuce are not known and scarce information isavailable on such interaction in other products exposed to NO treatment.It has been shown that NO can react directlywith various phenols (e.g.α-tocopherol, butylated hydroxytoluene, 2,4,6-tri-tert-butylphenol andothers used as antioxidants) to produce phenoxyl radicals that furthercan bind the excess of NO followed by slow dissociation of the adductback to phenoxyl radical and NO but the extent of reversibility of the re-action may depend on the structure and stability of the phenoxyl radical.Phenoxyl radicals such as those derived from α-tocopherol have beensuggested to function as NO carriers in biological systems (Janzen,Wilcox, & Manoharant, 1993). In harvested longan fruit, treatment withthe NO donor SNP inhibited the activities of PPO, POD and PAL, main-tained high total phenol content and relatively high level of AsA whichwas associated with delayed pericarp browning during storage (Duanet al., 2007). The exposure of mushrooms to the NO releasing chemicaldiethylene triamine-nitric oxide (DETANO)maintained the phenolic con-tent and reduced browning (Jiang et al., 2011).

In the present work, the effect of exogenously applied NO gas onwound-induced browning of fresh-cut butterhead lettuce was studied.In order to elucidate the physiological processes underlying the actionof exogenousNO, the hydrogen peroxide (H2O2) production in responseto wounding and during senescence of the lettuce shreds was assessed.Further, the occurrence of cell death as a part of wound-triggered disor-ders was analyzed.

The results demonstrate that wounding induces excessive H2O2 ac-cumulation that was associated with browning of the cut surface anddeath of the cells at the sites of physical damage. The detected oxidativestress appeared similar to that occurring later during tissue senescenceupon storage of the fresh cuts. Pre-storage exposure of the shreds to NOgas remarkably reduced the level of wound associated H2O2, preventedthe browning and delayed the senescence, resulting in extended shelflife. The findings reveal still unknown mechanisms of NO effects inshredded lettuce and are a step toward development of innovative in-dustrial technologies based on the efficiency of NO for controlling thebrowning and improving the storability of this highly perishableproduct.

2. Material and methods

2.1. Plant material

Butterhead lettuce (L. sativa L.), cv. Cosmopolia, was obtained from acommercial grower in The Netherlands. Immediately after harvest theheads were transported to the laboratory, plastic covered and storedfor 12 h in a cold room (4 °C and 96% relative humidity). For experi-ments with fresh-cuts the most outer leaves of the heads werediscarded. In order to study the dependence of the wound responseon leaf age, the shredswere prepared from leaves taken from twodiffer-ent positions of the heads: outer (mature) leaves (detached from the2nd and 3rd whorls inward from discarded leaves) and inner (young)leaves (from 6th and 7th whorls). In experiments with leaf disks(Section 2.5) alsomiddle (middle-aged) leaves (from4th and5thwhorls)were used. Midribs and major veins were removed and the leaves werecut with a sharp stainless steel knife into strips of approximately8 × 2 cm. Shreds prepared from leaves excised from different headsweremixed and samples of themixed batch were exposed to treatmentswith NO. Apart from the wounding caused by the cutting, to additionally

assess the effect of wounding on browning, slight bruising of leaf tissue,limited to an area of approximately 1 mm2 was also done in selectedshreds by gently pressing with the tip of a blunt knife. In one experimenticeberg lettuce from a local supermarket was obtained to validate the ef-fect of NO on a different type of lettuce.

2.2. NO treatment

The treatment procedure was performed as described byWills et al.(2008) and Hoque et al. (2011) with slight modifications. Fifteen gramsof fresh-cuts made from leaves of different ages (an average sample foreach set of young ormature leaves) were transferred to 500ml jars andthe jars were closed tightly without removing the air. A fixed amount ofNO gas was taken from a concentrated 5000 ppm stock in nitrogen(Linde Gas Benelux B.V., Schiedam, The Netherlands). Using a syringewith hypodermic needle a fixed amount of NO gas was injected intothe head space of the jars through a rubber septum. This yielded finalconcentrations of 50, 100, 200 and 400 ppm NO. The lettuce sampleswere exposed to NO treatment for 0.5, 1.0, 2.0 or 3.0 h, in the dark, at21 °C. Following the NO treatment, the jars were opened for 30 min ina fume hood to vent out the gas from the head space. Then the sampleswere subjected to cold storage. Control shreds were treated in a similarway but without NO application (Fig. 1).

To assess whether to be effective NO has to be applied immediatelyafter preparation of the fresh cuts, the fumigation (100 and 200 ppmNOfor 1 h or 2 h) was done 24 h post-processing. Meanwhile the shredswere kept in well ventilated conditions (plastic boxes covered withpunctured lids), in the dark, at 4 °C, as described in Section 2.3.

2.3. Storage conditions

The NO-treated fresh-cuts were transferred to white plastic boxeslinedwithmoist filter paper (Whatman grade no. 3) to prevent desicca-tion and a layer of maze (for preventing the direct contact of plant

160 E.T. Iakimova, E.J. Woltering / Innovative Food Science and Emerging Technologies 30 (2015) 157–169

tissueswith themoist paper). The boxeswere coveredwith transparentplastic lids that were previously punctured at 16 points (approximately0.5 mm diameter) to allow a gas exchange with the environment andprevent accumulation of CO2, ethylene and other gasses released fromthe plant material. The in this way prepared samples were stored in cli-mate rooms in the dark at 4 °C or 12 °C and approximately 90–95% rel-ative humidity (RH) inside the boxes.

2.4. Assessment of quality and shelf life

The shelf life of fresh-cuts was evaluated by combining overall visualquality (OVQ) and appearance of browning at the cut surface. OVQ wasassessed by using a 1 to 9 (2 increments) scale as described in Kader,Lipton, and Morris (1973): 9 — excellent, essentially free of defects;7 — good, minor defects, not objectionable; 5 — fair, slightly to moder-ately objectionable defects; lower limit of sales appeal; 3— poor, exces-sive defects, limit of salability; and 1 — extremely poor, not usable.Browning was ranked in 5 level scale as follows: 5 — none; 4 — slight;3 — moderate; 2 — severe; and 1 — extreme. The shelf life was consid-ered terminated when the shreds reached OVQ level 5 and/or the cutedge browning reached level 3.

2.5. Histochemical detection of hydrogen peroxide

H2O2 was visualized in leaf tissues by using 3,3′-diaminobenzidine(DAB) staining as described by Thordal-Christensen, Zhang, Wei, andCollinge (1997) and Iriti et al. (2006). Leaf disks (0.5 cm diameter) in-cluding the cut surface or taken from other injured places of the shredswere excised with a cork borer; the disks were placed in 50 ml tubes,covered with 0.1% (w/v) DAB and placed on an orbital shaker for 18 hin darkness. The dyewas poured off and chlorophyll removed by boilingthe leaf disks in 96% (v/v) ethanol for about 30 min. DAB is rapidlyabsorbed by the plant tissues and is polymerized locally in the presenceof H2O2 giving a visible brown stain with intensity corresponding to theamount of H2O2.

To assess the timing of H2O2 production and the leaf age-dependenceof wound-induced H2O2, a separate experiment was done. In this experi-ment H2O2was analyzed in leaf disks (diameter 1.8 cm) excised from let-tuce leaves of three different positions in the head (inner, middle andouter whorls). The wounding in this case was executed at the excisionof the disks (containing the cut surface) with a cork borer and by addi-tional dot bruising (as described in Section 2.1). The disks were kept incovered Petri dishes lined with moist paper for up to 96 h. The sampleswere examined microscopically and imaged using a light microscopeLeitz Aristoplan equipped with Nikon Digital camera DMX 1200. The im-ages are representative from 3 independent sets of experiments.

2.6. H2O2 quantification

H2O2 amount in leaf tissue was quantified by pixel intensity of DABdeposits measured with ImageJ (Image Processing & Analysis Applica-tion in Java, National Institute of Health (NIH), USA). The pixel intensi-ties of DAB images (in gray values, background subtracted) rangefrom 0 to 255. Value 0 represents the darkest shade of the color and255 represents the lightest shade of the color in the image. Higher inten-sity corresponds to lower H2O2 amount. Presented values are average ofat least 5 spots containing H2O2 deposits per sample (3 samples pertreatment, experiments were repeated at least 3 times with differentlettuce heads).

2.7. Cell death determination

Cell deathwasmeasured using Evans Blue staining and by determin-ing the ion leakage.

Evans Blue staining of the dead cells (the dye is permeable in thedead cells only), according to Keogh, Deverall, and Mcleod (1980) was

carried out by boiling leaf disks for 1 min in a mixture of equal amountsof phenol, lactic acid, glycerol and distilled water containing 20 mg l−1

Evans Blue, prepared immediately before use. Tissues were then clari-fied in 96% ethanol for 30–60 min at room temperature. The sampleswere examined and imaged using the light microscope and the cameradescribed in Section 2.5. The images are representative from 3 indepen-dent sets of experiments.

Tissue deterioration in stored fresh-cuts was estimated by electro-lyte leakage, determined as described by Song et al. (2012). The lettuceshreds were washed in ultrapure (Milli-Q) water and gently dried outwith miracloth. Leaf disks (diameter 0.5 cm) were excised from lettuceshreds by using a cork borer. Five disks (approximately 0.2 g FW) werefloated on with their abaxial part up in 10 ml deionized water andincubated at room temperature (21 °C). The electrical conductivity ofthe solution (microsiemens (μS))wasmeasured after 2 h (this value de-termined as EC1) using conductivity meter (pH/Cond 340i Set,manufactured by WTW, Germany). Following this measurement, thesamples were boiled for 15 min to completely destroy the tissue thusallowing a release of all electrolytes. After cooling the samples to roomtemperature, the electrical conductivity was measured again anddetermined as total conductivity (EC2). The electrolyte leakage wasexpressed as a percentage, calculated using the formula: electrolyteleakage (EL) % = EC1/EC2 × 100.

2.8. Imaging of chlorophyll fluorescence

In separate samples of wounded green fresh-cuts the chlorophyllfluorescence was qualitatively assessed and imaged by Zeiss Axioskopmicroscope (filter excitation/emission wavelength 490/525), equippedwith Nikon Digital camera DMX 1200.

2.9. Data analyses

Presented values are average of at least three independent experi-ments (5 lettuce heads per experiment, each variant in 3 replicates).Data were subjected to one-way analysis of variance (ANOVA) andDuncan's multiple range test at probability level p b 0.05 (SPSS, IBM)was used to separate treatment means. The presented values aremeans of all experiments ± SEM (n − 1) or ± SD.

3. Results and discussion

This study was based on the hypothesis that in lightly processed let-tuce the wounding might induce excessive ROS accumulation and celldeath being associated with the browning and tissue deterioration. Inaddition it was hypothesized that treatments reducing the severity ofthe oxidative stress could prevent the browning, retard the senescenceand extend the shelf-life. To verify the hypothesis, the following ques-tions with respect to the postharvest physiological and quality perfor-mance of the fresh-cuts were addressed: a) does the wounding oflettuce shreds stimulate elevated levels of H2O2 corresponding to thebrowning of damaged tissue; b) does the browning-associated woundresponse involves cell death events; c) is the severity of wound-induced browning dependent on leaf age and d) is there an effect ofexogenous NO on browning, H2O2 accumulation, senescence andstorability of the shreds.

3.1. Effect ofwounding onROS production and cell death in fresh-cut lettuce

Thewoundingwas caused by cutting of lettuce leaves into shreds orby bruising the leaf tissue in specified areas. In these experiments thesamples were kept at 12 °C, in the dark (as described in Section 2.3)and microscopically examined 3 days post-wounding. No structuralchanges in leaf tissue close to the cut edge or at other wounded placeswere observed and no weakening of chlorophyll fluorescence was de-tected (Fig. 2A, B, E, F). However, DAB staining revealed accumulation

161E.T. Iakimova, E.J. Woltering / Innovative Food Science and Emerging Technologies 30 (2015) 157–169

of H2O2 at the cut surface and in a narrow strip of cells in proximity tothe edge (Fig. 2C). H2O2 was also detected in the bruised leaf areaswhere it appeared in single or in a limited number of epidermal cells di-rectly exposed tomechanical damage (Fig. 2G). For the time of observa-tions (every 3 days until termination of shelf life, images not shown)H2O2 was not spreading toward non-wounded leaf parts. A similar pat-tern of localization of H2O2 within the zone of browning/woundingwasobserved in the shreds stored at 4 °C. The H2O2 amount (estimated bythe visual intensity of DAB brown staining) at the cut surface or in theother damaged parts of the shreds corresponded to the severity ofwound-induced browning as visually judged (Fig. 2I).

Evans Blue staining revealed dead cells at the places of wounding.The cell death was restricted mainly to epidermal cells at, and in prox-imity, to the cut edge and in the cells subjected to bruising in whichhigh levels of H2O2 were detected (Fig. 2D, H).

It has been suggested that a wound signal produced at the cut sur-face might act locally and/or be transmitted and cause further damagein the remaining part of the leaf (Campos-Vargas & Saltveit, 2002;Wildon et al., 1992). In fresh-cut lettuce, candidates for woundmessen-gers are ABA, jasmonic acid, SA and ethylene. However, the exogenousapplication of suggested chemicals did not confirm a crucial involve-ment of these molecules in wound-induced browning as a result ofaltering the phenol metabolism (Campos-Vargas & Saltveit, 2002).Here we show that H2O2 is produced in response to wounding andstays restricted to the primary site of injury. The intensity of DAB stain-ing corresponded to the advancement of tissue deterioration and devel-opment of browning within the wounded areas. This suggests that infresh-cut lettuce, H2O2 might not be a transmittable wound signal andmight act locally contributing to oxidative stress associated tissue col-lapse. This is in accordance with the findings in other plant modelsthat H2O2 is synthesized in response to wounding and acts as a localsignal which, together with other ROS, is responsible for cell damage(León, Rojo, & Sánchez-Serrano, 2001). Excessive generation of H2O2

at the site of physical impact might trigger further pathways resultingin accumulation of brown compounds.

The oxidative stress is the resultant of the overproduction of ROS(superoxide, hydroxyl, perhydroxyl and alkoxy radicals, H2O2 and sin-glet oxygen) that are highly toxic and cause damage to proteins, lipids,carbohydrates and DNA and its severity is controlled by the activity ofthe antioxidant system. The antioxidant system consists of enzymaticand non-enzymatic factors. Among the enzymatic factors are superox-ide dismutase, catalase, ascorbate peroxidase, glutathione reductase,

A B

E F

Fig. 2.Microscopic analyses representing the effect of wounding on H2O2 production and cell dethe wounding executed by cutting the leaves into shreds and by bruising. Images were taken 3chlorophyll fluorescence in image A (B); H2O2 accumulation at the cut surface (C); dead cells at(F); H2O2 accumulation in single cells exposed to bruising (G); a group of dead cells in bruised aleaf disk with a cork borer and at bruised places— indicatedwith arrows (I). H2O2 (C and G) isafter Evans Blue staining. Images A, C, D, E, G and H— light microscopy; images B and F— fluorebars = 10 μm.

monodehydroascorbate reductase, dehydroascorbate reductase, gluta-thione peroxidase, guaiacol peroxidase and glutathione-S-transferase;among the non-enzymatic factors are AsA, glutathione, phenolic com-pounds, alkaloids, non-protein amino acids and α-tocopherols. The ac-tivity of the scavenging of system determines the oxidation status andprotects plant cells from oxidative damage by scavenging or detoxifyingthe ROS (Gill & Tuteja, 2012; Neill et al., 2002).

In the process of PCD including senescence, plant–pathogen interac-tions (hypersensitive response, HR) and at conditions of abiotic (e.g.desiccation, wounding) stress, oxidative stress is a common physiolog-ical response (Jabs, 1999; Neill et al., 2002). PCD is also distinguished byother features such as protoplast shrinkage, nucleus compaction, activa-tion of specific cysteine and serine proteases and expression of celldeath associated genes, DNA fragmentation, increased membranepermeability and tonoplast rupture, followed by autolysis of cellularcontent (van Doorn & Woltering, 2005). Our observations that the celldeath was detected only in wounded cells in which H2O2 accumulatesprovide evidence that the wound-induced collapse of the tissues infresh-cut lettuce involves PCD events. In wounded and senescing cells,morphological hallmarks of PCD such as protoplast shrinkage anddisintegrated nucleiwere also detected (datawill be reported elsewhere).

3.2.Wound-induced H2O2 production in excised leaf disks is age dependent

The experimentswith leaf disks excised from leaves of different ages(young, middle-aged and mature leaves) showed that 24 h post-wounding H2O2 accumulation at the cut edge of the young leaves wasmuch less expressed or even lacking in comparison to middle-agedand mature leaves (Fig. 3A, B, C). Within the time interval 24–96 h theintensity of H2O2 deposits in cells at the cut surface of mature leaveswas increasing (Fig. 3D, E, F) but the staining was not spreading awayfrom thewounded areas. The increase of H2O2 level correlated to the ad-vancement of tissue browning at the cut edge and in bruised parts of theleaf disks (Fig. 3D, E). Until 96 h the advancement in H2O2 accumulationwith the timewasmuch less pronounced in the young andmiddle-agedleaves (images not shown). In the leaf disks at final stage of senescenceH2O2 still remained within the zone of wounded dead cells (Fig. 3D).This additionally shows that H2O2 is not transmitted away from thewound site.

As mentioned above, it has been hypothesized that wound effects inyoung and mature lettuce tissues might involve different chemical sig-nals. Studies on the biochemical and physiological mechanisms of the

C D

HG

I

ath in fresh-cut butterhead lettuce. The fresh-cuts were prepared frommature leaves anddays post-wounding after maintaining the shreds on 12 °C, in the dark. Cutting edge (A);and in vicinity of the cut surface (D); bruised area (E); chlorophyll fluorescence in image Erea (H); a shredwith browning at the cutting edge, at the place of injury after excision of avisualized in brown following DAB staining; the dead cells (D and H) are visualized in bluescence microscopy–filter combination excitation/emission wavelength 495/520 nm. Scale

A B C

D E F

Fig. 3. Timing and age dependency of wound-induced H2O2 accumulation in leaf disks excised from butterhead lettuce leaves. The wounding was executed at the isolation of the shredswith cork borer (cutting) and by bruising of leaf tissue in specified areas. A–C — 24 h post-wounding. Young leaves from 6th and 7th whorls (A); middle-aged leaves from 4th and 5thwhorls (B); mature leaves from 2nd and 3rd whorls (C); D–F — mature leaves: 48 h (D), 72 h (E), and 96 h (F) post-wounding. Arrows indicate DAB stained brown H2O2 deposits atthe cut surface and in bruised tissue. For details on the experimental procedure refer to the Material and methods section. Scale bars = 500 μm.

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wound response in postharvest baby lettuce crop and in young andma-ture leaves of romaine lettuce suggested that thewound stress signalingmight be under developmental control and the involvement of hormon-al factors might be dependent on the stage of tissue maturity(Campos-Vargas & Saltveit, 2002; Pereyra et al., 2005; Wagstaff et al.,2007). So far, the primary wound-induced signal remains uncovered.An interesting suggestion is that wounding might trigger electricwaves that can function as a putative long-distance wound messenger.Wildon et al. (1992) demonstrated the existence of propagated electri-cal wound-induced signal that might transfer the information of localphysical damage in tomato to distant unwounded plant parts and in-duce systemic defence wound-related response such as accumulationof transcripts and activation of a proteinase inhibitor.

Here we show that wound-induced H2O2 accumulation occurs to alesser extent in the young leaves which indicates age dependency ofthe capacity to produce and/or scavenge wound H2O2. Theoretically,the reduced H2O2 accumulation in younger lettuce leaves might be at-tributed to their higher ROS scavenging capacity but the available infor-mation is controversial and does not provide a clear answer about thismechanism. Generally, the antioxidant capacity of lettuce leaves isconsidered dependent on the levels of endogenous compounds withfree-radical-scavenging activity such as polyphenols, carotenoids andAsA. It has been shown that increased amounts of these substances cor-respond to reduced browning in packed shreds of iceberg lettuce(Martín-Diana et al., 2008). Kang and Saltveit (2002) reported elevatedantioxidant capacity of the wounded leaf tissue of romaine and iceberglettuce, measured as DPPH (α,α-diphenyl-β-picrylhydrazyl)-radicalscavenging activity or as ferric-reducing antioxidant power (FRAP).The higher antioxidant potential was associated with accumulation ofphenolic compounds. However these authors also established that al-though beneficial for improving the antioxidant capacity of fresh-cutlettuce, the accumulation of chlorogenic, isochlorogenic, caffeoyltartaricand dicaffeoyltartaric acids that are induced by woundingwas associat-ed with subsequent tissue browning resulting in reduced quality andshelf life. In fresh-cuts of rocket salad, which is resistant to browning,a higher level of AsA (a powerful antioxidant) as compared to lettuceand escarole was found. In rocket salad the level of AsA decreased

upon storage whereas in escarole it increased and in lettuce remainedconstant. The authors proposed that the resistance of rocket salad tobrowning disorder might be due to inhibition of PPO activity and/orthe reduction of quinones to phenols, both of which may be inducedby AsA (Degl'Innocenti et al., 2007). Observations on green salad bowl(GSB) and red salad bowl (RSB) lettuce (L. sativa L. var. acephala)showed that the antioxidant capacity (determined by FRAP analysis) di-minished significantly during storage in the “red” lettuce, whereas astrong increase was determined in the “green” one. Based on theestablished lack of substantial browning until 10 days of storage, RSBwas considered as resistant to browning whereas GSB was consideredsusceptible as the browning in this variety started already after 48 h. Al-though FRAP assay detected in GSB an increase in antioxidant capacityduring storage, the amount of AsA inGSBdiminishedwhichhas been at-tributed to its conversion to inactive form. These findings suggest thatAsA might not be the main compound responsible for antioxidant ca-pacity of lettuce leaves and may indicate that AsA levels may be moreimportant in preventing browning than the total antioxidant capacity(Degl'Innocenti et al., 2005). Martínez-Sánchez et al. (2012) analyzedthe content of vitamin C during the shelf-life of stored (in MA packages,at 4 °C) fresh-cuts from green and red baby-sized lettuce varieties:baby-leaf (young), multi-leaf (mature) and whole-heads. Initially theshreds from multi-leaf of the green variety showed the highest contentof vitamin C, the samples from baby-leaf contained an intermediate andthe fresh cuts from the whole-heads the lowest amounts. In the shredsfrom the red variety no significant differences among the three types ofrawmaterial were detected. In all fresh-cuts (of the green and red vari-eties) the level of vitamin C decreased during the storage periodreaching similar values of AsA and dehydroascorbate at the end ofshelf life (9–11 days). The shredded product from baby-sized leaveshad higher phenolic contents than the whole-heads which was sug-gested to contribute to the longer shelf life (11 days) of baby-sizedleaves. These findings suggested that age-dependency of shelf lifemight be related on the content of phenolics.

In a study performed in our laboratory in parallel to the here report-ed one, Witkowska and Woltering (2013) found that younger leaves ofboth green and red butterhead lettuce expressed better capacity to cope

C

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Fig. 4. Effect of short NO treatment on shelf life, cell death and H2O2 production in freshcuts prepared from mature butterhead lettuce leaves and maintained at 4 °C. Shelf life(A). Electrolyte leakage (B). H2O2 accumulation (C). Shelf life is determined as the numberof days till OVQ= 5 and/or the browning occurred at level 3. Cell death was estimated byelectrolyte leakage (EL). H2O2 accumulation was quantified by pixel intensity of DAB de-posits. Presented data in B and C were recorded at the termination of shelf life. Errorbars represent SEM(n − 1) (A and B) and SD (C). Values annotated by different letters arestatistically significant (p b 0.05). For details on the experimental procedures see theMaterial and methods section.

Table 1Summary of the effect of NO treatment (100 ppm, 1 h) on the shelf life of fresh-cut butter-head and iceberg lettuce. Shelf life is determined as the number of days until OVQ = 5and/or the browning occurred at level 3. Fresh-cuts (shreds of approximately 2 × 8 cm)of butterhead and iceberg lettuce were prepared from leaves of different developmentalstages (mature and young). Data are means ± SEM(n − 1) of 3 experiments Values withdifferent letters in the same column are significantly different from each other at p b 0.05.

Lettucevariety

Developmentalstage

Storagetemp.(°C)

Shelf life (days ± SEM) % increaseover control

Control NO treated

Butterhead Mature 4 7.2 ± 2.9b 22.2 ± 1.2c 208.3e

12 3.6 ± 0.4a 8.4 ± 1.2a 133.3c

Young 4 12.5 ± 1.2c 26.0 ± 0.4d 108.0b

12 5.6 ± 0.9a 14.2 ± 1.1b 153.5d

Iceberg Mature 4 10.6 ± 1.5b 19.3 ± 1.8c 82.1a

12 4.3 ± 0.8a 9.8 ± 2.1a 127.9c

163E.T. Iakimova, E.J. Woltering / Innovative Food Science and Emerging Technologies 30 (2015) 157–169

withwound-induced browning and senescence than older leaves. How-ever, there was not a clear relation between plant age and either the vi-tamin C or total antioxidant levels. Taken together, the existing findingscannot claim the total antioxidant capacity or the vitamin C content oflettuce leaves as a determining factor responsible for age-related differ-ences in browning response and shelf life between shreds from youngand older leaves.

Regarding the possible existence of diverse wound signals in theyoung and older leaves our findings suggest that in mature leaves theprimary wound response might involve generation of H2O2 or otherROS.

Altogether, the results reported in Sections 3.1 and 3.2 demonstratethat: 1) the wounding of fresh-cut lettuce induces H2O2 production;2) the level of H2O2 corresponds to severity of tissue browning; 3) thewounding induces cell death which is associated with H2O2 accumula-tion, and 4) in comparison to mature leaves, the browning response ofyoung leaves to wound stress is less pronounced and not associatedwith excessive H2O2 accumulation.

3.3. Effect of NO on browning and storability of fresh-cut lettuce

Wehave tested the effect of short-term (0.5–3.0 h) NO treatment onbrowning and shelf life of shredded lettuce upon storage. Fresh-cutsprepared from leaves of different ages were exposed to a range of NOconcentrations and following the treatment subjected to storage at4 °C or 12 °C in darkness. Shelf life was assessed by OVQ and severityof browning.

It was established that treatments with 100 or 200 ppm NO for 1 or2 h substantially prolonged the shelf life (more than2 times the control)of fresh-cuts prepared from mature leaves and stored at 4 °C (Fig. 4A).These concentrationswere also very efficient for delaying the browningof cut surface until 20–22 days of storage. In control shreds frommatureleaves OVQ and browning reached the limit of saleability after 7 days.The beneficial effect of NO treatmentswas dependent on the concentra-tion and exposure time. No significant improvement of storability wasobserved at shorter (30 min) exposure to 100 and 200 ppm NO. Inde-pendent on treatment duration, 50 ppm NO did not exert a positive ef-fect on shelf life and browning (data not shown). Exposure to 400 ppmNO also did not greatly improve the postharvest performance (Fig. 4A).The application of all tested NO concentrations for 3 h failed to preventthe browning and did not improve shelf life over the control (data notshown). The same NO concentrations and treatment times applied toshreds from mature leaves were also applied to shreds prepared fromyoung leaves. Again 100 and 200 ppm NO remarkably delayed thebrowning and sustained high OVQ of the shreds for up to 26 daysagainst 12.5 days in control non-treated shreds from young leaves(Table 1, Fig. 5C and D). The control shreds from young leaves showed5 days longer storability than the control shreds from mature leaves(Table 1). These results indicated an age-related response to woundingand confirm the above reported data (Section 3.2) for lesser sensitivityof younger leaves to wound-induced browning.

The effect of 100 and 200 ppm NO on storability of shreds preparedfrommature leaveswas also tested upon storage at 12 °C. In reference tostorage at 4 °C, the shelf life of both control and NO-treated shreds wasalmost halved at 12 °C. However, the effect of NO was still well pro-nounced by increasing the shelf life approximately two fold over non-NO-treated fresh-cuts (Fig. 6A). In a similar experiment at 12 °C withshreds prepared from young leaves, the NO treatment as well morethan doubled the shelf life (Table 1).

Browning inhibition and prolongation of shelf life were alsoestablished when the shreds were exposed for 1 h to 100 and200 ppm NO, at 24 h post-cutting and storage at 4 °C. Following100 ppm NO treatment, the shelf life of mature butterhead shreds wasapproximately 21–22 days and that of young shreds approximately25–26 days. Following treatment with 200 ppm NO the shelf life of ma-ture butterhead shreds was approximately 17–18 days and that of

young shreds approximately 20–21 days. These results are consistentwith the results obtained for the fresh-cuts exposed to NO treatmentimmediately after preparation of the shreds. This indicates that a 24 hdelay of fumigation is not critical for the positive effect of NO onstorability. Generally, at low temperature, no browning was observedwithin one day after cutting. This might be at least in part attributedto the effect of the low temperature onpreventing the immediate occur-rence of wound-stimulated oxidative stress, on sustaining low respira-tion rate and suppressing other unfavorable physiological processesthat are associated with initiation of early senescence. In practical as-pect, the possibility for postponement of NO application may be of

A B

C D

E

F

Fig. 5. Effect of 1 h exposure to 100 ppm NO on browning of cut surface of butterhead and iceberg lettuce shreds stored at 4 °C in the dark. Butterhead lettuce (A–D). Control shred frommature leaf, day 5 (A); NO-treated shred frommature leaf, day 20 (B); control shred from young leaf, day 12 (C); NO-treated shred from young leaf, day 25 (D). Senescent area of controlshred frommature leaf of iceberg lettuce, day 10 (E). NO-treated shred frommature leaf of iceberg lettuce, day 19 (F). Control leaves were not treatedwith NO but otherwise handled andstored under similar condition. The indicated storage times correspond to the last day of shelf life. Scale bars (E and F)= 1 cm. For details on the experimental procedure see theMaterialand methods section.

164 E.T. Iakimova, E.J. Woltering / Innovative Food Science and Emerging Technologies 30 (2015) 157–169

importance for providing more flexibility at the industrial sites in termsof managing the time between the physical preparation of the shredsand the NO processing.

The above discussed experiments were carried out with butterheadlettuce. As a comparison an experiment was done with fresh-cutsmadefrom mature (from 2nd and 3rd whorls) iceberg lettuce leaves. Also inthis variety a treatment with 100 ppm NO for 1 h was highly efficientin suppressing the pinking and extending the postharvest life of theshreds. Both at 4 °C and at 12 °C, the shelf life of NO-treated shredswas doubled; from 11 to 19 days at 4 °C and from 4 to 10 days at12 °C (Table 1, Fig. 5E and F). In none of the NO-treated fresh-cuts badodor or decay was detected.

The obtained results clearly show that the fumigation of fresh-cutlettuce with NO remarkably delays the tissue deterioration and inhibitsthe browning of wounded leaf areas. In all of the cases, the most effec-tive treatments were comprised of 100–200 ppm NO during 1–2 h.Lower or higher levels of NO and shorter or longer exposure times hadsignificantly less beneficial effects.

Our data substantiate the findings of Wills et al. (2008) andHoque et al. (2011) for NO effect on preventing the browning of let-tuce shreds. However, these authors reported that 500 ppm NO pro-vided the highest inhibition, whereas we found that 400 ppm wasnot very effective. The difference might be due to differences in let-tuce type, amount of treated material, absorption of the gas by theproduct or other conditions of the experimental procedure. Here itis of importance to consider the stability of NO. Soegiarto et al.(2003) studied the rate of degradation of NO in atmospheres con-taining 0.3, 5, 10 and 21% O2. These atmospheres were obtained byflushing a sealed container with nitrogen until the desired O2 levelwas achieved. Final concentrations of NO (range 30–100 ppm) inthe container were established by injecting NO gas from a concen-trated stock of approximately 4500 ppm NO in nitrogen. The authorsfound that at 21% O2 in the absence of plant material it takes approx-imately 3 h for 30 ppmNO to degrade by 50%. In the presence of plantmaterial (cut carnation and lily flowers, strawberry, banana, apple,lime, lettuce, tomato) the rate of loss of NO in air is higher, occursquicker and to a larger extent when the initial concentration of thegas is higher. The authors conclude that NO loss largely depends onthe absorption, weight, surface and the physical characteristics ofthe material (Soegiarto et al., 2003). These findings suggested that

low NO concentrations could be applied for post-harvest treatmentsof flowers, fruits and vegetables.

It should be taken into account that in air NO can be converted to thetoxic oxidant nitric dioxide (NO2) which in the presence of water canform nitric (HNO3) and nitrous (HNO2) acids (Beckman & Koppenol,1996; Lamattina, Garcỉa-Mata, Graziano, & Pagnussat, 2003; Neillet al., 2003). These compounds might negatively affect the quality ofstored products. In our experiments the air was not removed from theheadspace of the jars in which the fresh-cuts were exposed to NO andwater from the shreds was released, which does not exclude the forma-tion of the above chemical compounds. However, no symptoms of tox-icity on NO treated shreds were observed. Theoretically, this might inpart be due to the half-life of NO which is longer than that of NO2 andalso is inversely proportional to the concentration meaning that half-time is longer at lower concentration of NO (Beckman & Koppenol,1996). The finding of Soegiarto et al. (2003) demonstrated that incontainers containing various post-harvest plant materials, without re-moving the air from the headspace, the rate of aerial oxidation of NOwas lower than expected and the rate of absorption by produce wasmuch greater than that of aerial oxidation. Hypothetically, this suggeststhat at the conditions of our experiments, the amount of formed NO2,HNO3 and HNO2 might be too low for exerting harmful effects.

Different effective NO concentrations are reported to inhibit thebrowning of cut slices from apple and peach, to sustain the bioactive in-gredients and to prolong the storage life of strawberry and mushrooms(Dong, Zhang, Lu, Sun, & Xu, 2012; Pristijono, Wills, & Golding, 2006;Zhu & Zhou, 2007; Zhu, Zhou, Zhu, & Guo, 2009). Hoque et al. (2011)tested NO gas and NO donors SNP and DETANO and found that thethree compounds were efficient in inhibiting the browning and extend-ing the post-harvest life of fresh-cut lettuce. These authors suggestedthat lettuce cells are able to metabolize RNS such as NO+ cation andNO− free radical. The NO action is thought to be related to NO abilityto scavenge ROS and in this way interrupting the oxidation processesleading to the formation of browning compounds (Hoque et al., 2011).However, the mechanism of NO action on reducing the severity of oxi-dative stress in lettuce is still largely unknown.

Our data show that NOpre-storage treatment can be successfully ex-ecuted at room temperature and in normal air and thereafter the stor-age of the product can be successfully performed in non-MA/CAconditions. We believe that NO processing of fresh-cut lettuce is of

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Fig. 6. Effect of short NO treatment on shelf life, cell death and H2O2 production of fresh-cuts prepared from mature butterhead lettuce leaves and maintained at 12 °C. Shelf life(A). Electrolyte leakage (B). H2O2 accumulation (C). Shelf life is determined as the numberof days till OVQ drops below 5 and/or the browning occurred at level 3. Cell death was es-timated by electrolyte leakage (EL). H2O2 accumulation was quantified by pixel intensityof DAB deposits. Presented data in B and C were recorded at the termination of shelf life.Error bars represent SEM(n− 1) (A and B) and SD (C). Values annotated by different lettersare statistically significant (p b 0.05). For details on the experimental procedures see theMaterial and methods section.

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importance for the industry in terms of cost saving and achieving excel-lent postharvest quality. Furthermore, it might be recommended aspostharvest practice alternative or supplementary to MA/CA packaging.In the closed packages the atmosphere is the resultant of the starting at-mosphere and the metabolic activity of the packed product. MA is ap-propriate for short-term storage but in longer term it may stimulatetissue deterioration due to excess CO2 (N15%), lack of sufficient oxygen(b0.1%) and high air humidity (near 100% RH) in the packages that inturn, may promote anaerobic respiration and microbial growth (Bolinet al., 1997; Fonseca et al., 2002; Smyth et al., 1998).

In our experiments the 1 h pre-treatment with 100 ppm NO pre-served the post-harvest quality of the butterhead shreds stored at 4 °Cfor 22 days (mature fresh-cuts) and 26 days for young fresh-cuts and,for 19 days for iceberg lettuce (Table 1). This differs from the earlier re-ported average shelf life of 8.5–9.5 days of iceberg lettuce following fu-migation with 100 ppm NO for 2 h and storage at 0 °C or 5 °C (Hoqueet al., 2011; Wills et al., 2008). The reason behind these differencesmight be due to the above suggested specificity of the type and amountof used lettucematerial, respiration rate and absorption of the gas by the

product, among others (Fonseca et al., 2002; Kader & Saltveit, 2003;Soegiarto et al., 2003). Although suggested that NO could be injectedinto MA packages (Wills et al., 2008) the short-term stability of NO(Soegiarto et al., 2003) and its possible conversion to highly cell damag-ing RNS such as peroxynitrite (Delledonne et al., 1998; Neill et al., 2003;Wendehenne et al., 2004) should be taken into consideration as poten-tial risk for using the gas as a component of MA for long-term handling.

Storage of various lettuce types in CA (3% O2 + 10% CO2) has beenfound beneficial for retarding the browning of romaine and iceberg let-tuce as compared to storage in air Ballantyne et al. (1988), but not forbutterhead lettuce (López-Gálvez, Salveit, & Cantwell, 1996b). Thesedata indicate that for butterhead lettuce MA/CA storage might not al-ways be efficient. In our experiments NO treatment was generallymore effective in prolonging the shelf life in air than the reported effectsof MA/CA storage which suggests that short NO treatments might indeedbe an appropriate pre-storage processing, alternative to MA packaging.

3.4. Effect of NO on senescence of fresh-cuts

To investigate whether the protective effect of NO on tissue disman-tlement in fresh-cut lettuce is due to an effect on senescence, cell dam-agewas evaluated bymeasuring electrolyte leakage, a cell deathmarkerwhich indicates changes in permeability of the cellular membranes. Itwas found that treatments with 100 and 200 ppm NO for 1 h and 2 hsubstantially reduced the electrolyte leakage in stored shreds and thiscorresponded to an extended storage life both at 4 °C and 12 °C(Figs. 4B, 5B). The senescence delaying effect of NO in fresh-cuts frommature leaves was also observed in fresh-cuts from young leaves(Table 1). These results indicate that the effect of NO on prolongationof shelf life of lettuce shreds ismost probably exerted through inhibitionof cell death-related processes and support the suggestion that NOmayact as negative regulator of leaf senescence (Mishina, Lamb, & Zeier,2007).

Exogenously applied NO gas possibly penetrates the leaf tissue viathe damaged cells at the cut surface and bruised areas. Our data suggestthat it acts as antioxidant agent with local effect on preventing the ex-cess accumulation of H2O2 and cell death at the wounded sites. Thedata also suggest that in non-wounded leaf areas the gas may act bysuppressing the occurrence of oxidative stress associated with later se-nescence. A reasonable question is whether the exogenous NO mayexert physiological effects andmediatemetabolic reactions in amannersimilar to NO generated in planta. A way to approach this issue is to es-tablish what amount of exogenous NO has been taken up by the planttissue and whether this provides endogenous levels comparable to thelevels produced in plant cells. Depending on the plant, light/dark regimeand themethod of NO detection, at normal physiological conditions themeasured rates of NO emission from leaves may vary, for example 0.2–20 nmol h−1 g−1 FW in cut leaves of Arabidopsis thaliana and Vicia faba,0.02–0.32 nmol h−1 g−1 FW in detached sunflower leaves, 5–10 nmol h−1 g−1 FW in intact tobacco leaves. Under stressful circum-stances of abiotic and biotic origin the endogenous level might be aug-mented several hundred times (Mur et al., 2005; Rockel, Strube,Rockel, Wildt, & Kaiser, 2002; Vanin et al., 2004; Zeier et al., 2004). Byusing a laser photoacoustic detector, Mur et al. (2005) found that fol-lowing a treatment of attached tobacco leaves with the NO donor SNP(introduced into intercellular leaf spaces) the emission of NO from theleaves increased proportionally to the concentration of SNP until 6 hafter application, thereafter the emission decreased. The authorsestablished that SNP stimulated the occurrence of HR (a formof PCD en-gaged as a defence reaction at plant–microbe interactions, expressed bydevelopment of confined necrotic lesions) induced by Pseudomonassyringae pv. tabaci and reduced the severity of disease symptoms. Thissuggested that the NO released from SNP has been sufficiently utilizedby plant cells and incorporated in the HR signaling pathways. In a sim-ilar elegant experimental design the question whether exogenous NOcan properlymimic the functions of endogenousNOhas been addressed

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by Floryszak-Wieczorek, Milczarek, Arasimowicz, and Ciszewski (2006).They used pelargonium leaf disks exposed to treatments with NO donorsSNP, S-nitroso-N-acetyl-D-penicillamine (SNAP) and S-nitrosoglutathione(GSNO) and measured NO generation capacity and the half-life of thechemicals in light and dark and in the presence of reducing agents suchasAsA and reduced glutathione (GSH). The usually useddonor concentra-tions range from 10 to 500 μM. The kinetics of NO release from the donoris dependent on the factors related to stability of the compound such astemperature and light sensitivity. In the mentioned work the presenceof NO in epidermal cells following the treatment of leaf material withNO releasing compounds has been evidenced by staining with 5,6-diaminofluorescein diacetate (DAF-2DA) — a fluorescent probe specificfor labeling of NO. In the presence of the NO scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (CPTIO)the level offluorescencewas substantially reduced.Moreover, in responseto application of NOdonors the activity of PALwas substantially increasedand suppressed in thepresence of CPTIO. Thesefindings indicated thatNOfrom exogenous sources at certain levels might mimic the effects of en-dogenously produced NO.

In our experiments the effective concentrations of applied NO gaswere 100 and 200 ppm but it is hard to estimate what the final levelsin the tissue would have been as it is not known how much is takenup and what the half-life has been. Assuming that some NO gas has en-tered thewound surfaces, itmay be expected that NO could have a com-parable effect as NO produced endogenously either naturally or from anappliedNOdonor. As shownby Soegiarto et al. (2003) dependingon thephysical characteristics of the plant material a high percentage of exog-enously applied NO gas is absorbed by the plant tissue. Assuming that50% of the NO gas applied to the jars in our experiments is taken upby the lettuce tissue, this would amount to NO concentrations in therange of 50–100 μM, concentrations that are expected to be physiolog-ically effective. The determination of NO levels in wounded and NO-treated lettuce leaf tissue would provide more insight into the modeof action of exogenously administrated NO.

3.5. Effect of NO on H2O2 production in senescing fresh-cuts

Cell death and senescence are marked by the generation of oxidativestress (Jabs, 1999; Neill et al., 2002). To obtain more information on themechanism of action of NO on senescence in fresh-cut lettuce, weassessed the effect of NO on H2O2 production. Under storage at 4 °C,excessive H2O2 accumulation in the epidermal and mesophyll cells ofsenescing control shreds from mature leaves (day 7) was observed(Fig. 7A). In the senescing fresh-cuts from young leaves (day 12) loweramounts of H2O2 in mesophyll cells and xylem vessels were detected(Fig. 7B). In shreds from mature leaves, treated for 1 and 2 h with 100and 200 ppmNO no H2O2 production was observed until 22 days of stor-age (Fig. 7C). These results indicate that NO efficiently prevents the latersenescence that is associated with oxidative stress. The quantification ofH2O2 by pixel intensity of DAB staining revealed that NO treatments

BA

Fig. 7. Effect of NO treatment onH2O2 accumulation in fresh-cut lettucemaintained at 4 °C. The i(A). Untreated shreds fromyoung leaves— day 12 (B). 100 ppmNO for 1 h treated shreds frommmethods section. Scale bars = 10 μm.

(100 and 200 ppm for 1 and 2 h) significantly reduced the H2O2 amountwhich remained low until the end of storage period (Figs. 4C, 5C). Thelower levels of H2O2 were in line with the extended shelf life and the re-duced electrolyte leakage of treated fresh-cuts (Figs. 4, 6).

The interaction between NO and H2O2 during senescence and celldeath is complicated and still not fully understood. These moleculesare suggested to operate in cross talk acting synergistically or indepen-dently. NO and H2O2 can chemically react to produce singlet oxygen orhydroxyl radicals, which lead to cell death. However, NO can also scav-enge H2O2 and potentially counteract the oxidative stress and cell death(Delledonne et al., 1998; Neill et al., 2003; Wang et al., 2013;Wendehenne et al., 2004). Another suggested mechanism for antioxi-dant activity of NO is that it may S-nitrosylate the NADPH oxidasethus affecting its ability to synthesize ROS (Yun et al., 2011). InArabidopsis challenged with P. syringae pv. tomato, these authorsfound that S-nitrosylation of proteins stimulates the hypersensitivecell death, but when concentrations of S-nitrosothiols are high, NOmay also operate through a negative feedback loop limiting the HRme-diated by S-nitrosylation of the NADPH oxidase. The latter abolishes theability of the enzyme to synthesize reactive oxygen intermediates. InArabidopsis, bean, cucumber and maize exposed to ABA, H2O2, UV andbrassinosteroids, it has been demonstrated that H2O2 stimulates theproduction of NO. Contrary, NO supplied by SNP did not induce H2O2

synthesis and inhibition of NO synthesis did not affect the levels ofH2O2 produced (Bright, Desikan, Hancock, Weir, & Neill, 2006; Cuiet al., 2011; He, Xu, She, Song, & Zhao, 2005; Lum, Butt, & Lo, 2002;Zhang et al., 2007).

Using DAF-2DA staining, in senescing lettuce fresh-cuts we have de-tected NO accumulation in xylem vessels, in the guard cells and in theneighboring epidermal cells concomitantwith the increase of H2O2 pro-duction (data not shown). This supports the suggestion that these twocell death messengers might function in interrelation.

The here presented results provide sound indication that NO actsthrough interacting with wound- and senescence-associated H2O2 andsubstantiate the understanding about its ROS scavenging potential.However, a better elucidation of intimate biochemical and molecularmechanisms of NO effects in fresh-cut lettuce requires additional stud-ies addressing NO interaction with ROS detoxifying and phenol synthe-sizing enzymes andwith other pathways related towound-induced andsenescence-associated cellular processes. The physiological effects ofNO on fresh-cuts maintained under different light/dark regimes alsohave to be addressed. Further studies regarding the possible influenceof NO treatment on the nutritional constituents such as carbohydratesand bioactive substances such as vitamins and antioxidant compoundsshould also be investigated.

4. Concluding remarks

The browning of fresh-cut lettuce, which mainly is a resultant of themechanical damage (wounding) duringprocessing, represents a serious

C

mageswere taken on the last day of shelf life. Untreated shreds frommature leaves— day 7ature leaves— day 22 (C). For details on the experimental procedure see theMaterial and

167E.T. Iakimova, E.J. Woltering / Innovative Food Science and Emerging Technologies 30 (2015) 157–169

post-harvest disorder responsible for quality loss. The understanding ofthe physiological mechanisms underlying the wound response is of im-portance for the development of techniques for preventing the wound-induced browning and sustaining a longer shelf life of this perishableproduct.

In various works, the highly dynamic response of plant tissue todiverse stressful abiotic and biotic challenges has been studied in dif-ferent plant models. Generation and accumulation of H2O2 beinglimited to cells in proximity to the cut edges, to bruised leaf areasand surrounding veins directly exposed to wounding have been de-scribed (Guan & Scandalios, 2000; Karpinski et al., 1999; Lamb &Dixon, 1997; Orozco-Cárdenas, Narváez-Vásquez, & Ryan, 2001;Prasad, Anderson, Martin, & Stewart, 1994). It has been shown thatH2O2 is a diffusible signal mediating localized death of cells duringstress response (Levine, Pennell, Alvarez, Palmer, & Lamb, 1996;Levine, Tenhaken, Dixon, & Lamb, 1994), thus providing evidencefor a role of H2O2 in cell death (Mittler et al., 1999). The here report-ed findings substantiate the previously established contribution ofH2O2 and cell death to wound- and stress-induced tissue damages.However, little is known about the effect of exogenously appliedNO on oxidative stress signaling in relation to the occurrence ofwound-induced browning and the associated cell death eventsupon storage of the fresh-cut lettuce.

In this study we focused on the application of short-term pre-storage exposure of lettuce shreds to NO. The findings demonstratethat treatment with 100 and 200 ppm NO for 1 or 2 h remarkablyinhibited the browning of the cut surface and the browning of other-wise injured (bruised) leaf tissue. Nitric oxide treatment, in addition,delayed the senescence and substantially prolonged the shelf lifeupon storage at 4 °C and 12 °C. To obtain information on the mecha-nisms of NO action, the generation of H2O2 and the occurrence of celldeath in response to wounding and senescence of the shreds wereanalyzed. The results showed that the wounding stimulates the syn-thesis of H2O2 and causes cell death as evidenced by e.g. increasedion leakage. A correlation between augmented H2O2 levels, brow-ning, senescence and storability of fresh-cuts was established. Exog-enous NO appeared to inhibit H2O2 accumulation and to prevent thestored fresh-cuts from browning and premature senescence. Incomparison to shreds from mature leaves, shreds from young leavesexhibited reduced levels of H2O2 in response to wounding, less pro-nounced browning and a longer shelf life.

The reported data provide novel information of industrial relevance.Short NOpre-storage treatment prevents thewound-induced browningand sustains the post-harvest quality for an extended storage period.Fumigation with NOmight be recommended as an option for appropri-ate postharvest processing of fresh-cut lettuce, potentially supplemen-tary or as an alternative to MA packaging. The application of NOrequires the relevant infrastructure, but once established, the equip-ment and treatment procedures are easy to manage. NO treatment offresh-cuts is an innovative approach for post-harvest handling offresh-cut lettuce. The introduction of this technology to fresh-cut indus-try might have economic impact by substantially reducing the posthar-vest losses. From a fundamental point of view the present studydisclosed novel elements of the mechanism of NO action, namely thata single NO treatment within 24 h of fresh-cut processing alleviatesthe wounding and senescence-associated oxidative stress and delaysthe tissue deterioration due to delaying the occurrence of premature se-nescence. The data show that shreds prepared from young leaves areless prone to browning and show suppressed wound-related H2O2 pro-duction than shreds frommature leaves.We also demonstrate that PCDis a part of browning associated wound response. The idea that storagedisorders are a form of PCDmay direct research activities to treatmentsthat specifically tackle cell death as away to optimize storage conditionsand to increase quality of stored fresh-cuts. In addition, early detectionof cell death eventsmay serve as qualitymarkers for stored lightly proc-essed vegetables.

Acknowledgments

This research was financially supported by a visitor travel grant(project no. 040.11.306) from the Netherlands Organisation for Scientif-ic Research (NWO), by project no. 08013 from the C.T. de Wit GraduateSchool Production Ecology & Resource Conservation, Wageningen, TheNetherlands and partially by project no. 158/2014 from the AgriculturalAcademy of Bulgaria. The logistics support and collegiality provided byHorticultural Supply Chains Group, Department of Plant Science,Wageningen University, The Netherlands is highly appreciated.

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