I
DEPARTEMENT OF AGRICULTURE, FOOD
AND ENVIRONMENT
PhD School in Agricultural and Veterinary Science
PhD program in “Crop Science”
(XXV cycle)
S.S.D.: AGR/13
A biochemical and molecular dissection of the
responses induced by postharvest UV-B radiation
on quality traits of peach fruits
(Prunus persica L. Batsch)
President: Prof. Alberto Pardossi
PhD candidate Supervisors
Claudia Scattino Prof. Annamaria Ranieri
II
“Adversity can be of tremendous opportunities”
III
Contents ... 1
1. Introduction ... 1
1.1 Origin of peach ... 1
1.2 Peach production and market ... 2
1.3 Peach botany ... 3
1.4 Peach fruit composition ... 6
1.4.1 Phenolic compounds ... 8
1.4.2 Regulation of flavonoid biosynthesis in peach fruit ... 12
1.4.3 Ascorbic acid ... 14
1.5 Peach quality ... 17
1.6 Maturity indices and quality ... 18
1.7 Peach flesh firmness and texture ... 20
1.8 Peach ripening and softening ... 23
1.9 Ethylene biosynthesis, perception and signal transduction ... 27
1.10 The use of postharvest abiotic stresses as a tool for enhancing the nutraceutical content of fruits ... 32
1.11 UV-B radiation ... 34
1.12 UV-B perception and signaling in plants ... 35
1.13 UV-B postharvest treatments on fruits ... 38
2. Purpose of the work... 41
3. Materials and methods ... 42
3.1 Fruit samples, treatment and storage ... 42
3.2 Quality parameters (FF, SSC, TA, ethylene) ... 44
3.3 Extraction and quantification of ascorbic acid ... 45
3.4 Phenolic compounds quantification ... 46
3.4.1 Analysis by spectrophotometric assays ... 46
3.4.2 Analysis by HPLC-DAD-ESI-MSn ... 47
3.5 Antioxidant activity (TEAC assay)... 48
IV
3.6.1 Static experiment ... 49
3.6.2 Dynamic experiment: trapping of free radicals ... 49
3.7 Cell wall enzymes extraction and activity assays ... 50
3.8 Analysis of Endo-PG and expansin proteins ... 52
3.8.1 Endo-PG extraction ... 52 3.8.2 Expansin extraction ... 53 3.8.3 SDS-PAGE ... 53 3.8.4 Western Blot ... 55 3.8.4.1 Anti-Endo-PG antibodies ... 56 3.8.4.2 Anti-LeExp1 antibodies ... 57
3.9 Gene expression analysis by qRT-PCR ... 57
3.10 Statistical analyses ... 59
4. Results ... 60
4.1 Experiment A ... 60
4.1.1 Soluble solid content, titratable acidity & flesh firmness…….60
4.1.2 Determination of ethylene evolution... 62
4.1.3 Concentration and redox state of ascorbic acid ... 63
4.1.4 Phenolic compound quantification ... 66
4.1.4.1 Spectrophotometric assays ... 66
4.1.4.2 HPLC-DAD-ESI-MSn ... 70
4.1.5 Phenylpropanoid pathway gene expression ... 78
4.1.6 Antioxidant activity ... 79
4.1.7 EPR measurements ... 80
4.1.8 Exo-PG, EGase, β-Gal and PME activities ... 88
4.1.9 Levels of Endo-PG- & expansin-like polypeptides in fruit flesh ... 90
4.1.10 Endo-PG and expansin gene expression ... 91
4.2 Experiment B ... 94
4.2.1 Soluble solid content, titratable acidity and flesh firmness ... 94
V
4.2.3 Concentration and redox state of ascorbic acid ... 97
4.2.4 Phenolic compound quantification ... 99
4.2.4.1 Spectrophotometric assays ... 99 4.2.4.2 HPLC-DAD-SEI-MSn ... 101 4.2.5 Antioxidant activity ... 109 4.2.6 EPR measurements ... 110 5. Discussion ... 111 6. Conclusions ... 130 7. References ... 132
VI
Abstract
In the present study the possibility of enhancing the content of health-promoting compounds of peaches and nectarines (Prunus persica L. Batsch) by post-harvest irradiation with B was assessed. Moreover, the impact of UV-B on the cell wall metabolism of peach fruits was also investigated.
Fruits of ‘Suncrest’ (Melting flesh-type, MF) and ‘Babygold 7’ (Non-Melting flesh-type, NMF) peach and ‘Big Top’ nectarine (Slow Melting flesh-type, SM) cultivars were firstly irradiated with UV-B (1.69 W/m2) for 12 h, 24 h and 36 h. A photosynthetic photon flux density of 500 µmol m-2 s-1 was provided. Control fruits underwent the same conditions but UV-B lamps were screened by benzophenone-treated polyethylene film. Subsequently, fruits of ‘Suncrest’ and ‘Big Top’ cultivars, which showed a positive response to the UV-B radiation, were subjected to a second different experiment providing 24 h of irradiation after which treated and control fruits were stored up to 36 hours at 10°C.
The effectiveness of the UV-B treatment in modulating the concentration of phenolic compounds and ascorbic acid was cultivar-dependent. ‘Big Top’ and ‘Suncrest’ fruits were generally affected by increasing health-promoting compounds whereas in ‘Babygold 7’levels of secondary metabolites decreased after UV-B irradiation. A corresponding trend was exhibited by most of phenylpropanoid biosynthetic genes which were tested by qRT-PCR. In MF-type fruits, UV-B radiation slowed down the softening process and reduced the flesh firmness loss. This evidence was attributed to the inhibition of expansin synthesis, regulated at gene level by UV-B. The UV-B treatment did not induce differences in flesh firmness between control and treated fruits of NMF and SM fruits.
After 36 h of storage at 10°C, fruits previously treated with UV-B exhibited generally higher contents of antioxidant compounds. In both experiments, the
VII concentration of phenolic compounds and ascorbic acid were correlated with the accumulation of radicals measured by EPR analysis.
Based on these results UV-B irradiation can be considered a promising technique to increase the health-promoting potential of peach fruits at the same time improving shelf-life and quality.
1
Contents
1.
Introduction
Origin of peach
1.1
Peach tree (Prunus persica L. Batch) originated in North-West China where it was first domesticated and cultivated. However, the botanical name wrongly suggests Persia (actual Iran) as putative country of peach tree origin (Bassi & Monet, 2008).
Alexander the Great (King of Macedonian Empire) introduced the peach into Europe after he conquered the middle East (Ensminger et al. 1994).
Peach tree was well known to the Romans in the I century AD. The oldest artistic representation of this fruit is in a fresco from the “Casa dei cervi” in Herculaneum, which was sealed by the Vesuvius eruption of 79 AD.
The historical sources (Pliny the Elder, Columella, Martial) refer to peach as mala persica (Persian apple) since the second half of I century AD, providing an age of its introduction into Italy (Pliny the Elder) that could be restricted to the last decade of the first half of the I century AD.
Peach continues to be appreciated in Italy also at the during Middle ages, while it disappear from central Europe since 250 AD (Sadori et al. 2009).
Peach could have reached France through the Balkan route almost simultaneously to its arrival in Italy. From the first half of the 16th century, peach was brought to the American continent for the first time by the Spanish, starting in Central America.
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Peach production and market
1.2
Nowadays, peaches are an important commodity widely distributed around the world. In 2012, FAO statistics estimated the worldwide production at about 21,083,151 tonnes. China, with 12,000,000 tonnes, is the 1st world producer, followed by Italy (1,331,621 tonnes) and USA (1,058,830 tonnes). The total European production estimated in 2012 was 3,394,641 tonnes. (http://faostat.fao.org).
Emilia-Romagna represents the 1st Italianregion for peaches production, with about the 30% of the total, followed by Campania.
Nectarines are the type most produced in Italy with a share of 54%, followed by peaches with 40%, while the so called "clingstone" peaches, mainly used for canning, account for the remainder (www.ismeaservizi.it).
According to information from the last two censuses of agriculture (www.istat.it), the view of Italian peaches and nectarines cultivation denotes an important surface modification from 2000 to 2010. The last decade saw a decrease in the peach production of about 20%, corresponding to approximately 18,000 hectares less. This slowdown is more conspicuous in the regions of Northern Italy, such as Emilia-Romagna (-35%) and Veneto (-33%), while many southern regions show a stabilization of surfaces with positive peaks Puglia (+60%) and Sicily (+20%). The reduction in the area resulted in a decline in production (www.csoservizi.com).
The consumption of peaches and nectarines is very rooted in the food culture of Italian households and the internal market is for more than 70% of the national supply (www.csoservizi.com). In 2012, Spain was the 1st European country in quantity of peaches exported (650,000 tonnes) followed by Italy (350,000 tonnes) (ec.europa.eu/eurostat ). According to Eurostat data, in 2012, Germany spent 122 million € (about 288 million in total, of which 69.5% achieved by nectarines) to import Italian peaches and nectarines, a value slightly lower than the rest of the deliveries in other EU Member States (130
3 million €) and three times higher than the import of all other non-European countries taken together.
Peaches can be used for fresh consumption (mostly peaches and nectarines) or for canning purpose (entire fruit or slices with syrup, juices, dried or candies; usually Melting flesh peaches). In few country, such as Italy, Non-Melting flesh fruits are eaten as fresh as any other peach or nectarine.
Peach botany
1.3
Prunus persica (L.) Batsch belongs to the order of Rosales, family of Rosaceae, tribe of Amigdaleae, section of Prunoidee. It is a diploid species (2n = 16) with a medium tree height (up to 8 m). The tree can live for 20–30 years, but commercial plantings are limited to 12–15 years at most, because of either the cultivar becoming obsolete or the loss of productivity. Fruit production begins from the second or third year.
The root system develops within the more superficial layer (50-60 cm in depth), depending on soil type. Roots are orange-white while young turn dark orange when older with large lenticels.
The aereal system is usually not very large. One-year-old shoots are reddish-green, turning dark grey-silver since the second year. Buds are found at the base of the leaves.
Leaves are lance-shaped, glabrous, broadest near the middle, whit brighter green on the upper page. The stem is characterized by the presence of one or more glandular formations, that can be glabrous or kidney-shaped. However the leaf can completely lack of glands. The trunk is straight and smooth, with a reddish-greenish bark in the first year, later becoming dark grey-silver. Peach has hermaphroditic, perigynous flowers that are generally pink, but they vary from white to red, with normally five petals. Flowers can show two shapes of corolla: showy (large petals) or non-showy (with small petals). From twenty to
4
thirty stamens are attached to the calyx (Figure 1). The inner surface of the calyx vary according to the fruit flesh color: it is greenish in white-fleshed and yellow to deep orange in yellow-white-fleshed fruits. The female part of the flower is constituted by a pistil. The ovary possess two ovules but just one is usually fertilized.
Figure 1: Flower type showy
Peach fruit (Figure 2) development could be divided into 4 different phases. The first period is the first exponential growth phase, characterized by a rapid increase in cell division and elongation (S1). During the second period (S2) the endocarp hardens to form the stone (pit hardening) and there is hardly any increase in fruit size.
In the third stage (S3), known as the second exponential growth phase, a rapid increase in fruit size takes place, along with rapid cell division.
In the final stage (S4), the fruit reaches the final full size and enters the fruit ripening or climacteric stage. The S4 phase can be further subdivided into two phases: S4-1, in which the fruit arrives at its full size, and S4-2, during which the fruit continues to ripen in an ethylene-dependent manner. The S4-2 stage can also take place in peach fruit detached from the tree and usually occurs prior to human consumption (Lombardo et al. 2011)
The fruit is a drupe. Almost all commercial cultivars are globose or elongated (oval or oblong) fruits.
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Figure 2: Peach fruit terminology (http://waynesword.palomar.edu)
They are pubescent or glabrous, fleshy, and, in sound fruits, the mesocarp does not split. The stony endocarp is very deeply pitted and very hard. Fruit weight varies from less than 50 g over 680 g, although commercial standards require from 180 to 230 g. The apex and the suture can be sunken, flat or outcropping, rounded or pointed. Skin and flesh colour, that can be white or yellow, are useful for peach cultivar classification, as well. The flesh can show a considerable content of anthocyanins, mainly located under the skin and/or close to the pit.
The skin may exhibit many different colors due to presence of a red overcolor (anthocyanin accumulation) which cover the ground. The overcolor may present different extension and brightness depending on the cultivar and environmental factors. According to the flesh texture peach fruits may be classified into three groups: Melting, Non-Melting and Stony Hard (see paragraph 1.5.2). Considering the flesh acidity, acid or low-acid cultivars are known (Bassi & Monet, 2008).
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Peach fruit composition
1.4
A peach fruit consists of approximately 90% of water and the remaining part contains carbohydrates, organic acids, vitamins, minerals, fibers and trace amounts of proteins and lipids (www.inran.it).
Soluble sugars contribute approximately 7–18% of total weight and fibre contributes approximately 0.3% of fresh weight (FW) of total fruit. Sucrose, glucose and fructose represent about 75% of peach fruit soluble sugars (Crisosto & Valero, 2008). These sugars are followed by sorbitol, xylose, xylitol, mannose, maltose and inositole present in very low amount.(Bassi & Selli, 1990). Peach fruits deemed to be of good quality contain large amounts of fructose and low quantities of glucose and sorbitol (Brooks et al. 1993). Strong correlation between sugar composition and growth rate was found in peach fruit from Génard et al. (1999). SSC vary throughout fruit development according to the supply of phloem sugars, changes in fruit metabolism and dilution caused by increases in fruit volume.
In the fruit, during S1 high hexose concentrations are reported, when the energy requirement of the dividing cells is high. Sucrose accumulation during S3, toward fruit maturation, has been attributed to a de novo synthesis in fruit mesocarp cells as result of a rise in the activity of sucrose syntase and sucrose phosphate synthase or to a decrease in activity of the sucrose-cleaving enzymes (Morandi et al. 2008).
Malic acid, is the predominant organic acid in mature peach fruit followed by citric acid. Organic acids contribute for 0.4-1.2% FW (Crisosto & Valero, 2008). Malic acid and citric acid are the major contributors to the optimal degree of acidity, as well. Their ratio (malic acid/citric acid ratio) correlated with sensory evaluation of taste (Colaric et al. 2005). Malic acid provides a smooth, tart taste, persistent in the mouth. Compared to citric acid, malic acid has a much apparent acidic taste, and it also tastes better than citric acid (Dziezak, 2003).
7 A particular mention is due to “low acid” peach cultivars, in which the amount of malic and citric acids is poor. Those fruits exhibit a different taste and sweetness is enhanced (Byrne et al. 1991).
Despite peach fruit has a low protein content (0.5 to 0.8% FW), these small-size proteins have an important function as enzymes catalyzing the various chemical reactions. Lipids represent only 0.1 to 0.2% FW, but they are important because they make up surface wax and cuticle that protects fruit against water loss and pathogens. Lipids are also important because constitute cell membranes, which influence physiological activities of fruits.
The mineral composition presents in peaches include both base-forming elements (Ca, Mg, K, Na) and acid-forming elements (P, Cl, S). Potassium is the most abundant mineral in peach (Crisosto & Valero, 2008).
Peach fruit is a good source of antioxidants such as ascorbic acid (vitamin C), carotenoids and phenolic compounds (Tomás-Barberán et al. 2001; Byrne, 2002; Remorini et al. 2008). All these molecules are mainly located in the peel, which constitutes only about 15% of total fruit FW.
The total ascorbic acid (vitamin C; see paragraph 1.4.2) content in Californian peaches ranged from 6 to 9 mg/100 g in white flesh and from 4 to 13 mg/100 g in yellow flesh, as reported by Gil et al. (2002). Similar concentrations of ascorbic acid (5–6 mg/100 g) were found in European peach cultivars (Carbonaro et al. 2002; Proteggente et al. 2002).
Total carotenoids concentration has been described in the range of 71–210 mg/100 g FW for yellow-fleshed and 7–20 mg/100 g FW for white-fleshed peach cultivars by Gil et al. (2002). Much higher content of carotenoids were measured in yellow-fleshed than in white-fleshed peach cultivars (approximately ten times higher). The main carotenoid detected was β-carotene (provitamin A), but also amounts of phytoene, β-criptoxanthin, zeaxanthin, luteoxanthin, and violaxanthin were reported (Caprioli et al. 2009). However, carotenoids content in ripe peaches may greatly vary among cultivars and fruit harvested from different areas (Di Vaio et al. 2008).
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Phenolic compounds are the major sources of antioxidant capacity in peaches (Gil et al. 2002) also involved in fruit visual appearance (pigmentation and browning) and taste (astringency) (Tomás-Barberán et al. 2001). Peaches have been reported to contain flavonols, anthocyanins, flavan-3-ols and hydroxycinnamates (Tomás-Barberán et al. 2001) but their contents vary in relation to different influencing factors, such as cultivar, rootstock, water supply and ripening stage at harvest (Tavarini et al. 2011).
The total phenolics concentration expressed as mg/100 g FW varied from 28 to 111 for white-fleshed and from 21 to 61 for yellow-fleshed Californian cultivars (Gil et al. 2002). Other European cultivars had values of 38 mg/100 g (Proteggente et al. 2002), while the Spanish cultivar ‘Caterina’ showed values of 240 and 470 mg/100 g for pulp and peel, respectively (Goristein et al. 2002). The predominant hydroxycinnamic acid is chlorogenic acid. Catechin and epicatechin are the main flavan-3-ols identified and their concentrations are higher in white-fleshed than in yellow-fleshed peaches. Concentrations of flavonols are higher in yellow-fleshed than in white-fleshed peaches. Cyanidin-3-glucoside is the main anthocyanin present, mostly in the skin (Gil et al. 2002).
1.4.1 Phenolic compounds
Phenolic compounds are secondary metabolites deriving from pentose phosphate, shikimate, and phenylpropanoid pathways in plants (Randhir et al. 2004). The phenylpropanoid pathway starts from phenylalanine to produce an activated (hydroxy)cinnamic acid derivative via the actions of phenylalanine ammonialyase (PAL), cinnamate 4-hydroxylase (C4H), and 4-coumarate:coenzyme A ligase (4CL). Specific branch pathways lead to the formation of monolignols/lignin, coumarins, benzoic acids, stilbenes, and flavonoids/isoflavonoids (Figure 3) (Dixon et al. 2002).
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Figure 3: Phenylpropanoid pathway (Dixon et al. 2002)
The contents of phenolic compounds are closely associated with the sensory and nutritional quality of foods, contributing directly or indirectly to desirable or undesirable aroma and taste. In low concentrations, phenolics may protect food from oxidative deterioration; however, at high concentration, they (or their oxidation products) may participate in discoloration of foods, and interact with proteins, carbohydrates, and minerals (Imeh & Khokhar, 2002).
Moreover, phenolics exhibit a wide range of physiological properties, such as anti-allergenic, anti-artherogenic, anti-inflammatory, anti-microbial, antioxidant, anti-thrombotic, cardioprotective and vasodilatory effects
10
(Balasundram et al. 2006). Structurally, phenolic compounds comprise an aromatic ring, bearing one or more hydroxyl substituents, and range from simple molecules to highly polymerized compounds.
Their activity seems to be related to the molecular structure, more precisely to the presence and number of hydroxyl groups, and to conjugation and resonance effects (Leopoldini et al. 2004).
Most naturally occurring phenolic compounds are present as conjugates with mono- and polysaccharides, linked to one or more of the phenolic groups, and may also occur as functional derivatives such as esters and methyl esters. Polyphenols can be categorized into several classes (Figure 4).
Figure 4: Classes of phenolic compounds in plants (Balasundram et al. 2006)
Flavonoid derivatives constitute the largest group of plant phenolics, accounting for over half of the eight thousand naturally occurring phenolic compounds. Variations in substitution patterns to ring C result in the major flavonoid classes (Figure 5). Substitutions to rings A and B (oxygenation, alkylation, glycosylation, acylation, and sulfation) give rise to the different compounds within each class of flavonoids (Balasundram et al. 2006). These compounds are powerful free radical scavengers acting by H-atom transfer from their OH groups to the free radicals (R•):
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Figure 5: Flavonoid structures (Crozier et al. 2009)
Structure–activity relationships (SAR) of polyphenol have been established as determinant of their radical scavenging and metal chelating activity and has been defined: a) the important role of the 3-OH group of the quercetin derivatives (flavonols); b) the important role of the 2,3 double bond; c) the catechol moiety in the B-ring and the passive role of the 5-OH group, because it is engaged in a strong H-bond with the keto group at C4; d) the minor direct role of the 7-OH group (see Figure 6 for numbering); and e) the important role of intra-molecular H-bonding.
To be fully active, a polyphenol must react by H-atom transfer faster than at least one of the reactions of free-radical-production cascades (e.g., the limiting propagation step in lipid peroxidation) (Di Meo et al. 2013).
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The possible health benefits derived from dietary phenolic compounds depend on their absorption and metabolism (Parr & Bolwell, 2000). The involvement of lactase phlorizin hydrolase (LPH), active sugar transporter (SGIT1), carrier-mediated transport process and the possible role of multidrug-resistant associated proteins (MRP) has been postulated from many authors (Yang et al. 2001; Clifford, 2004; Murota & Terao, 2003). Phenolic compounds that are unabsorbed in the small intestines, as well as compounds absorbed and metabolized in the liver and excreted in bile, enter the colon (Scalbert & Williamson, 2000; Yang et al. 2001). Enzymes secreted by colonic microflora hydrolyse the unabsorbed glycosides, strip the conjugates of their attached moieties, break the larger phenolic compounds to simpler molecules such as phenolic acids, or split the heterocyclic oxygen-containing ring (Hollman, 2001; Scalbert & Williamson, 2000). The primary site of metabolism depends on the dose, smaller doses being metabolized in the intestinal mucosa with the liver playing a secondary role, while larger doses are metabolized in the liver (Scalbert & Williamson, 2000).
However, phenolic should be considered in relation to the perceived role as “antinutrients”, particularly due to their ability to reduce digestibility of proteins, either by direct precipitation or by inhibition of enzyme activity (tannins for example) (Balasundram et al. 2006).
1.4.2
Regulation of flavonoid biosynthesis in peach fruit
Flavonoids play a central role in fruit colour, flavour and health attributes. Flavonoids are produced by a well-studied pathway (Figure 7) which is mainly regulated at the level of transcription of genes encoding the enzymes of the pathway.Six enzymes are generally involved in the anthocyanin biosynthesis pathway: calcone synthase (CHS), calcone isomerase (CHI), flavanone-3-hydroxylase
13 (F3H), dihydroflavonol 4-reductase (DFR), anthocyanidin synthase (ANS) and UDP-glucose:flavonoid-3-O-glycosyltransferase (UFGT) (Tsouda et al. 2004). Genes encoding enzymes specific for the proanthocyanidins (ANR: anthocyanidin reductase; LAR: leucoanthocyanidin reductase) have been isolated. Flavonols are synthesized from the dihydroflavonols by flavonol synthase (FLS) enzymes (Ravaglia et al. 2013).
Figure 7: Scheme of the flavonoid biosynthetic pathway in plants. Genes encoding enzymes for each step are indicated as follows: PAL, phenylalanine ammonia-lyase; C4H, cinnamate
4-hydroxylase; 4CL, 4-coumarate-CoA ligase; CHS, chalcone synthase; CHI, chalcone isomerase; F3H, flavanone 3-hydroxylase; DFR, dihydroflavonol 4-reductase; LDOX, leucoanthocyanidin dioxygenase; FLS, flavonol synthase; LAR, leucoanthocyanidin reductase; ANR, anthocyanidin reductase; UFGT, UDP-glucose:flavonoid-3-O-glycosyltransferase
(Ravaglia et al. 2013)
It has been shown by Ravaglia and collaborators (2013) that a regulatory complex, the MYB-bHLH-WD40 "MBW" complex, composed of a MYB, bHLH3 (basic helix-loop-helix) and WD40 controls promoters of polyphenolic biosynthetic steps in peach fruit. Usually, the MBW complex regulates groups of flavonoid biosynthetic genes, varying between different species.
The pattern of expression of the candidate transcription factor (TF) genes assayed in developing ‘Stark Red Gold’ nectarines suggests a tight regulation
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of UFGT expression, which controls accumulation of anthocyanin in the fruit peel. LAR and ANR may be regulated by MYBPA1 in peach, while MYB15 and MYB123 are both good candidates for controlling FLS expression.
Peach MYBPA1 was shown to be able to activate the DFR and LAR promoters. The MYB10 gene was the most strongly responsive to the light, with transcripts not detectable in the fruit kept in the dark and 30-fold more abundant in the light-exposed fruit compared to the fruit at harvest time.
MYB111 and MYB16 instead repress the transcription of the flavonoid biosynthetic genes.
The characterization of these transcription factors involved flavonoid regulation provides information for peach fruit breeders who have the final aim of selecting new cultivars with improved aesthetical and nutritional properties.
1.4.3 Ascorbic acid
L-ascorbic acid (ASA) is one of the simplest vitamins. It is related to the C6 sugars, being the aldono-1,4-lactone of a hexonic acid (galactonic or L-gulonic acid), and contains an enediol group on carbons 2 and 3 (Figure 8).
Figure 8: Chemistry of ascorbic acid
Delocalization of the p-electrons over the C2 -C3 conjugated enediol system stabilizes the molecule and causes the hydrogen of the C3 hydroxyl to become highly acidic, and to dissociate with a pKa of 4.13. At physiological pH, ASA exists as a monovalent anion (L-ascorbate). ASA is stable when dry, but
15 solutions readily oxidize, especially in the presence of trace amounts of copper, iron and alkali.
In plants, at least three distinct pathways for ASA biosynthesis have been described (Figure 9).
Figure 9: The L-ascorbic acid biosynthesis pathways in plants (Cruz-.Rus et al. 2010)
A key intermediate in the first pathway is L-galactose (L-Gal). The L-Gal pathway involves conversion of D-glucose to GDP-D-mannose, GDP-L-galactose, L-Gal, L-galactono-1,4-lacton and ASA. GDP-D-mannose pyrophosphorylase (GMPase: EC 2.7.7.22) is an enzyme catalyzing the tagging
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of the hexose with UDP. The conversion of GDP-D-mannose to GDP-L-Galactose requires a double epimerization, which is catalyzed by GDP-d-mannose-3′,5′-epimerase (GDP-D-man-3′,5′-epimerase: E.C. 5.1.3.18). Biochemical studies also pointed to GDP-L-gulose as an alternative product of this epimerization step. L-Gal is produced from GDP-L-Gal by two enzymes: GDP-L-Gal pyrophosphatase/VTC2 and L-Gal-L-P phosphatase (L-Gal-L-Pase). The oxidation of L-Gal to L-GalL, as the penultimate step in the synthesis of ascorbate in this pathway, is catalyzed by L-GalL dehydrogenase (L-GaLDH). The last enzyme in this putative ASA biosynthetic pathway of plants, L-galactono-1,4-lactone dehydrogenase (L-GalLDH) (EC 1.3.2.3), is the only enzyme of the L-Gal pathway that has been shown to be associated with organelles, and catalyzes the oxidation of L-Gal to ASA.
A second pathway starts from D-galacturonic acid (D-GalUA), which leads to ASA through L-galactonic acid. A third pathway was suggested to be initiated from myo-inositol (Cruz-Rus et al. 2010).
Three main kind of biological activity can be attributed to ASA: its function as enzyme cofactor, as radical scavenger, and as donor/acceptor in electron transport.
One of the more clearly defined functions of ASA is to modulate enzymatic reactions. These enzymes are mono- or dioxygenases, containing iron or copper at the active site and which require ASA for maximal activity.
In plants, the ability of ASA to interact with ROS implicates it in the modulation of processes such as lignification, cell division and the hypersensitive response. The biological importance of the antioxidant behavior of ASA is to terminate radical chain reactions by disproportionation to non-toxic, non-radical products. Further, since ASA is only mildly electronegative, it can donate electrons to a wide range of substrates. Moreover, one of the most important features of the non-enzymatic antioxidant activity of ASA is its involvement in the regeneration of the α-tocopherol radical.
17 ASA is essential for chloroplast activity by serving as a substrate for ascorbate peroxidase (APX), to scavenge peroxide formed in the thylakoids. In the chloroplasts, malondehydroascorbate generated in the reaction catalysed by APX, is reduced back to ASA either by acting as a direct electron acceptor to PSI at the reduced ferredoxin on the outside of the thylakoid membrane, or alternatively by the ascorbate-glutathione cycle, a coupled series of enzymatic reactions which links H2O2 to the oxidation of light-generated NADPH (Davey et al. 2001)
Humans, primates, and a few other animals depend on the diet as a source of vitamin C to prevent the vitamin C deficiency disease, known as scurvy, and to maintain general health. Many authors have recently discussed the effects of vitamin C on cancer chemoprevention (Gann, 2009; Gaziano et al. 2009) and in the treatment of cancer (Padayatty et al. 2010), sepsis (Wilson, 2009), and neurodegenerative diseases (Bowman et al. 2009).
Peach quality
1.5
Fruit ‘quality’ is a concept comprehensive of sensory properties (appearance, texture, taste and aroma), nutritive value, mechanical properties, safety and defects. Altogether, these attributes give the fruit a degree of excellence and an economic value (Abbott, 1999). The term ‘quality’ takes different meanings along the chain from the grower to the consumer. Yield, size and resistance to diseases are considerably important parameters for growers, while flesh firmness mainly interests packers, shippers, distributors and wholesalers. It is known that flesh firmness gives a good indication of the storage potential and market life. The consumer, instead, is attracted by red color, size, firmness, sugar content, acidity and aroma. On the contrary, some of the most frequent consumer complaints about peach and nectarine cultivars are hard fruit at consumption and a lack of flavour, and both of these problems are caused by
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harvesting fruit at immature stages in order to avoid product wastage on the supermarket shelves and to allow for long distance shipment (Della Cara, 2005). indicated that hard fruit, mealiness, lack of taste, and failure to ripen are the main reasons of Californian consumers do not eat more stone fruits.
It is evident at this point that growers and individuals in the delivery chain should pay attention to fruit quality looking from the consumer’s perspective. An emerging trend in consumer preferences includes in fruit ‘quality’ also nutritional characteristics (determined by vitamins, minerals and dietary fibres) and fruit health benefits linked mainly to antioxidant contents.
In fact, it has been demonstrated by epidemiological and clinical studies that the consumption of fresh fruit and vegetables exerts an important role in preventing chronic and degenerative diseases such as tumors, cardiovascular diseases and atherosclerosis (Boeing et al. 2012; Bazzano, 2006; Gundgaard et al. 2003).
Enhancing the health benefit properties of produce will add value and create new opportunities for growers and processors by reaching health-oriented markets. To achieve this goal, technologies that can ensure the delivery of high quality products with high levels of desired compounds are needed.
To increase fruit consumption, marketing promotion and education programs are useful.
An efficient management of both preharvest and postharvest activities may considerably affect the quality of fruits.
Maturity indices and quality
1.6
Studies have associated high consumer acceptance of peaches with high soluble solids concentration (SSC) (Mitchell et al. 1990; Parker et al. 1991; Kader, 1995; Ravaglia, 1996).
19 A minimum percentage of 10% SSC was proposed in California as a quality standard for yellow-fleshed peaches and nectarines (Kader, 1995). In France, a minimum value of 10% SSC for low-acidity (TA <0.9%) and 11% SSC for high-acidity (TA ≥0.9%) peaches was proposed (Hilaire, 2003). In Italy, for yellow-fleshed peaches was suggested a minimum of 10% SSC for early-season, 11% for mid-season and 12% for late-season cultivars (Testoni, 1995; Ventura et al. 2000). Other reports have pointed out that there are more factors involved in consumer acceptance apart SSC such as titratable acidity (TA) (Peterson & Ivans, 1988), volatiles (Romani and Jennings, 1971) and the sugar-to-acid ratio (SSC:TA) (Crisosto et al. 2006). However, on very low TA cultivars the relationship between consumer acceptance and SSC:TA ratio is not a full positive linear relationship. Thus, it appears that consumer acceptance may be more sensitive to the SSC:TA than SSC alone, but only within an acceptable TA range (Iglesias & Echeverría, 2009).
A decrease of consumer acceptance associated to an increase of flesh firmness was reported by Crisosto et al. (1997). These authors however observed at the same time that an adequate fruit firmness is essential for ease of handling and marketing.
Crisosto (2002) reported that peach fruits reaching 26.5–35.3 N flesh firmness values are considered “ready to buy” whereas, fruit between 8.8–13.2 N are considered ripe (‘‘ready to eat’’). Maximum levels of fruit firmness for marketing fresh peaches and nectarines are set by the EU at a 6.5 kg (=63.7 N)/0.5 cm2 (8 mm diameter probe) [Commission Regulation (EC) No. 1861/2004 of 28 October 2004].
These parameters can be assessed by simple devices such as penetrometers, refractometers and titrators. However, in recent years, extensive research has been focused on the development of non-destructive techniques for assessing several internal fruit quality attributes at the same time on a large number of fruits.
20
Peach flesh firmness and texture
1.7
There is wide variability in texture among the peach grown around the world. Different regional preferences explain the diversity of peach texture in commerce. Firmness is one of the most important characteristic in terms of cultivar development and selection for breeders who have been selecting for increased fruit firmness over time. The first two phenotypes described according to the characteristics of textural changes and softening during ripening by Bailey and French on 1932 are the melting (MF) and the non-melting (NMF). The MF texture shows a prominent softening in the last stage of ripening, until a complete melting in correspondence to the peak of ethylene evolution (Tonutti et al. 1996; Morgutti et al. 2006). The NMF phenotype shows a firm texture even when the fruit is fully ripe, it softens slowly but never melts (Bailey and French, 1949; Bassi and Monet, 2008). The NMF peaches present a higher concentration of Ca2+ than MF (Bassi et al. 1998). Melting flesh trait is controlled by “F locus”, a gene also responsible for pit adherence. Studies on progenies segregating for endocarp adherence to the pit and flesh texture (MF and NMF), four alleles for the endoPG enzyme were found responsible for three flesh phenotypes at the “F locus”: freestone-MF (FF, Ff or Ff1; flesh do not adhere to the pit) and clingstone-MF (ff or ff1), and
clingstone-NMF (f1f1). The fourth allele is a null-allele (absence) that has the
same phenotypic effect as the f1 allele (Peace et al. 2005).
MF fruits are usually grown for the fresh market and allowed to ripen on the tree in order to achieve maximum quality. They show a propensity to mechanical damage and decay during shipping and handling. NMF cultivars, instead, are traditionally grown for canning purposes.
Flesh texture in these phenotypes is affected by cell wall composition (see paragraph 1.8). The MF phenotype is associated with a large increase in the amount of soluble pectin and progressive pectin depolymerization. In contrast,
21 NMF peaches lack the final melting phase of softening and pectins undergo just little solubilization or depolymerization (Brummel et al., 2004).
The melting phase of MF peaches is associated with marked increases in gene expression and enzymatic activity of Endo-PG (Pressey and Avants, 1978; Orr and Brady, 1993; Lester et al. 1994; Trainotti et al. 2003), conversely of NMF, which have less ripening-related Endo-PG expression and activity (Pressey and Avants, 1978; Lester et al. 1994).
Morgutti et al. (2006) detected an Endo-PG protein not only in MF but also in NMF varieties of peaches, contradicting the previous hypothesis affirming that the NMF phenotype could be caused by a partial or complete deletion of the Endo-PG gene (Lester et al. 1996; Callahan et al. 2004). The same authors showed that the levels of this Endo-PG protein were higher and increased with softening in MF fruit, but remained lower and were constant in NMF fruit. Moreover, they proposed that different levels of Endo-PG are caused by the differential expression of an Endo-PG gene and suggested a regulation of Endo-PG production at the transcriptional level, but the authors did not exclude an additional regulation at the translational or post-translational level.
Ghiani and co-authors (2011a) suggested that Endo-PG activity was necessary to achieve melting flesh texture, which was characterized by wide apoplastic spaces and partially deflated mesocarp cells, but they observed that Endo-PG activity had no critical influence on the reduction of fruit firmness. In their studies was observed a large numbers of Endo-PG isoforms highly expressed and mainly localized on middle lamella of MF peaches that produced, as a consequence, wide apoplastic spaces in the pericarp of ripe MF fruit. In contrast, no loss of cell adhesion was observed in NMF cultivars or in unripe MF peaches. Endo-PG was not detected in unripe NMF fruit, whereas in ripe NMF and in unripe MF peaches were detected few and poorly expressed enzyme isoforms localized mainly in vesicles within the cytoplasm and inner primary cell wall.
22
The same authors observed in the exocarp of NMF ripe fruit an increase in cell size while in both MF and NMF cultivar types, mesocarp cells lose their turgidity. From these evidences emerges that the change in cell turgidity is a process common to MF and NMF fruit but it was speculated that a less permeable cuticle should decrease transpirational water loss, reducing the softening in NMF. In peaches as in tomato (Vicente et al. 2007; Saladié et al. 2007), the loss of firmness and the textural changes seem to be related to different but interconnected mechanisms: the cell turgor variation and the cell wall disassembly.
A third phenotype of is represented by “Stony hard-flesh” (SH). This kind of fruit are characterized by a reduced level of ethylene production due to low expression of PpACS1, by crispy fruit flesh and a lack of softening during fruit ripening (Hayama et al. 2000; Haji et al. 2003 and 2004). The stony hard trait is controlled by a single recessive gene (hd) which is inherited independently of the M (MF/NMF) trait (Yoshida, 1976; Haji et al. 2005). The cells of these fruits contain higher levels of Ca2+ than MF phenotype but lower of NMF ones (Bassi et al. 1998). When the stony hard is combined with Melting, the fruit reduce their firmness through continuous exposure to ethylene (Hayama et al. 2003; Haji et al. 2005). Therefore, the softening process in stony hard peaches appears to be blocked by a lack of ethylene, and not by mutations of cell wall-modifying enzymes. The stony hard mutation does not seem related to fruit softening enzymes, but to the control of ethylene levels in the ripening fruit (Hayama et al. 2006). Recent studies showed that PpACS1 expression at the late-ripening stage of stony hard peach may result from a low level of indole-3-acetic acid (IAA) and that a high concentration of IAA is required to generate a large amount of system 2 ethylene in peaches (Tatsuki et al. 2013)
23
Peach ripening and softening
1.8
Ripening of fleshy fruit could be considered as a syndrome that comprises a complex series of genetically programmed events leading to edible fruit with desirable aspects (Brummell & Harpster, 2001).
Softening is one of the major ripening-related phenomena. Large interest has been dedicated over the years upon the study of fleshy fruits ripening and softening since this process possess relevant economic implications. It is largely responsible for the length of its post-harvest life, for the chances of escaping a successful pathogen attack and for transportation and storage expenses (Trainotti et al. 2003).
Previous studies well established that texture changes in fruits are largely determined by modification of the cell wall and middle lamella polysaccharides (Manrique & Laiolo, 2004; De Roeck et al. 2008).
Lamella is the outer layer of the cell wall made by pectin, controlling the cell-to-cell adhesion.
The primary cell wall is a complex structure composed of various polysaccharides, structural proteins and some phenolics. Secondary cell wall, leaning against the the inner face of primary wall, is highly hydrated (~65% water) and in the aqueous component are dissolved various solutes, ions and enzymes (Brummel, 2006). The secondary wall may contain molecules that confer impermeability such as suberin and cutin, mechanical resistance like lignin and mineral salts.
The polymers constituting the primary cell vary in structure between species, but few components (cellulose, the matrix glycans composed of neutral sugars, pectins rich in D-galacturonic acid, and structural proteins) are usually present (Brummel & Hapster, 2001).
Cellulose is composed of 1,4-β-D-glucan chains assembled into very long crystalline microfibrils. The matrix glycans (known as hemicelluloses, as well) are neutral or weakly acidic, composed predominantly of neutral sugars and do
24
not contain galacturonic acid. The most abundant is xyloglucan, which possesses a 1,4-β-D-glucan backbone (like cellulose), but has numerous regularly spaced xylose side chains, some extended with either galactose-fucose or with arabinose. The two other major matrix glycans are glucuronoarabinoxylan (a 1,4-β-D-xylan backbone with occasional single glucuronic acid and arabinose side chains), and glucomannan (alternating regions of 1,4-β-D-glucan and 1,4-β-D-mannan, sometimes with single galactose side chains). Pectins are characterised by a high content of galacturonic acid residues, and can be linear and unbranched (poly 1,4-α-D-galacturonic acid, homogalacturonan), or with a homogalacturonan backbone and single xilose side chains (xylogalacturonan) or complex, highly conserved side chains composed of numerous neutral sugars (rhamnogalacturonan II, or RG-II). Rhamnogalacturonan I (RG-I) has a backbone of alternating galacturonic acid and rhamnose residues, possessing large linear or branched arabinan and galactan side chains.
Several types of structural proteins are present, some of which are heavily glycosylated. The primary cell wall ratio between components varies between species, but generally equal amounts (one-third of the dry weight each) of cellulose, matrix glycans and pectins are present even if fruit cell walls are generally enriched in pectins, which can form up to half of the polymeric content of the wall. Structural proteins are much less abundant, comprising only 1–10% of the dry weight (Brummel, 2006).
It seems probable that softening involves the action of several hydrolytic enzymes on carbohydrate polymers. The most important enzymes include pectinmethylesterase (PME) (EC 3.1.1.15), polygalacturonase (PG) (EC 3.2.1.15), β-(1,4)-glucanase (EGase) (EC 3.2.1.4) and β-galactosidase (β-Gal) (EC 3.2.1.23). Among them, pectin methylestherase and β-galactosidase activities are present during the development of the fruit. Both polygalacturonase and glucanase tend to be absent in mature green fruit but
25 their activities become measurable only with the onset of ripening and increase dramatically afterwards (Ramina et al. 2008).
PG are directly involved in pectin galacturonic binds degradation. Two classes of PG were previously described in peaches: Endo-PG are the predominant form in freestone type, whereas Exo-PG were observed in the mesocarp of both freestone and clingstone varieties. As the name suggests, Exo-PG remove galacturonic acid moieties of pectin from the terminal reducing end of the chain. Endo-PG can randomly cleave the pectin chain.
Of the two forms, Endo-PG appeared to be more influenced by ethylene concentration of peaches and more related to ripening than Exo-PG. EndoPG and exoPG activity increase gradually as fruit soften, but the rate of increase in activity accelerates when the fruit are very soft (<20 N) (Ramina et al. 2008) EGase (also known as cellulase) hydrolyses the β-1,4-glucan linkages of plant cell wall polymers, contributing to the wall structure weakening during ripening. A relationship between ethylene and EGase was proved. EGase is mainly involved in the initial phase of peach fruit softening, before the action of PME and PG (Ramina et al. 2008).
PME catalyses the hydrolysis of galactosyluronate methyl esters that results in the deesterification of pectins. This enzyme in involved in the degradation of cell wall by lowering the degree of methoxylation of fruit pectins, making galacturonoside linkages in the pectin backbones available for cleavage by PG (Dris & Jain, 2004).
The removal of sugar residues that are parts of side-chains appended to the backbones of different matrix polysaccharides is attributable to glycosidases action (Dris & Jain, 2004). β-Gal was reported as involved in the degradation of galactosyl residue of polyuronides and other cell wall polysaccharides during softening. β -Gal activity was high in unripe peach fruit and it an overall decline during peach ripening was reported by Jin et al. (2006).
Expansins are cell wall proteins that have been shown to contribute to fruit softening. Expansins have been related to a loss of fruit firmness in peach.
26
They act by disrupting non-covalent linkages (H-bridges) at the cellulose-hemicellulose interface, allowing sliding between each other with a “inchworm progress” or “raptation” and do not act as hydrolases (Cosgrove, 2000a) (Figure 10).
Although expansin lacks hydrolytic activity by itself, it does enhance the hydrolysis of crystalline cellulose by cellulases. This synergistic action may indicate that expansin makes glucans on the surface of the microfibril more accessible to enzymatic attack by cellulases (Cosgrove, 2000b).
Expansins are encoded by a multigene family and have been divided into α- and β-expansins.
Figure 10: A model of expansin’s wall-loosening action. Cellulose microfibrils are connected to each other by glycans (thin yellow and red strands) that can stick to the microfibrilsurface and to each other. The expansin protein (blue) is hypothesized to disrupt the bonding of the glycans to the microfibril surface (a) or to each other (b). Under the mechanical stress arising from turgor, expansin action results in a displacement of the wall polymers (c) and slippage in the points of polymer adhesion (compare b and c) (Cosgrove, 2000b)
Three expansins, Pp-Exp1, Pp-Exp2 and Pp-Exp3, have been isolated from ripe peach fruit, each showing a different pattern of expression during fruit development. Pp-Exp2 RNA was expressed constitutively throughout fruit development but was more abundant in stage III, during exponential growth and maturation. Pp-Exp1 and Pp-Exp3 were up-regulated at the onset of ripening but Pp-Exp1 was induced at an earlier stage. However, by comparing cultivars with different rates of softening, Pp-Exp3 shows a closer association with softening (Hayama et al. 2003; Ramina et al. 2008).
27
Ethylene
biosynthesis,
perception
and
signal
1.9
transduction
Ethylene (C2H4) is a gaseous natural hormone, biologically active also at very low concentrations. The diffusion of ethylene through intracellular spaces is extremely fast and uniform differently from other plant hormones, since not require active transport from its production site to its active site.
Hormone action is complex and varies according to the tissue and the organ considered, the developmental stage and interactions with many other regulatory factors.
In plants, ethylene is involved not only in the ripening process but it affects many aspects of the plant life cycle, including seed germination, root hair development, root nodulation, flower senescence and abscission. The production of ethylene is regulated by internal signals during development and in response to environmental stimuli from biotic (e.g., pathogen attack) and abiotic stresses, such as wounding, hypoxia, ozone, chilling, or freezing (Wang et al. 2002 )
Fruits have classically been categorized based upon their abilities to undergo a program of enhanced ethylene production and an associated increase in respiration rate at the onset of ripening. Fruits that undergo this transition are referred to as climacteric and include tomato, apple, peach, and banana, whereas fruits that do not produce elevated levels of ethylene are known as non-climacteric and include citrus, grape, and strawberry. However, these distinctions are not absolute, as closely related melon and capsicum species can be both climacteric and non-climacteric and some non-climacteric fruits display enhanced ripening phenotypes in response to exogenous ethylene. (Barry & Giovannoni, 2007). The key features of ethylene biosynthesis were reviewed by Yang and Hoffmann (1984).
28
Figure 11: Biosynthetic pathway and regulation of ethylene (Wang et al. 2002)
Three pathways are involved in ethylene production (Figure 11): 1) the activated methyl cycle, 2) the S-methylmethionine cycle and 3) the methyonin or Yang cycle.
The formation of S-adenosylmethionine (S-AdoMet) is catalyzed by SAM synthetase from the methionine at the expense of one molecule of ATP per molecule of S-AdoMet synthesized. 1-aminocyclopropane-1-carboxylic acid (ACC) is the immediate precursor of ethylene. The rate-limiting step of ethylene synthesis is the conversion of S-AdoMet to ACC by ACC synthase under most conditions. 5′-methylthioadenosine (MTA) is the by-product generated along with ACC production by ACC synthase. Recycling of MTA back to methionine conserves the methylthio group and is able to maintain a
29 constant concentration of cellular methionine even when ethylene is rapidly synthesized. Malonylation of ACC to malonyl-ACC (MACC) deprives the ACC pool and reduces the ethylene production. ACC oxidase catalyses the final step of ethylene synthesis using ACC as substrate and generates carbon dioxide and cyanide. Reversible phosphorylation of ACC synthase is hypothesized and may be induced by unknown phosphatases (Ptase) and kinases, the latter presumably activated by stresses. Cyanide is detoxified to β-cyanoalanine by β-β-cyanoalanine synthase (β-CAS) to prevent toxicity of accumulated cyanide during high rates of ethylene synthesis (Wang et al. 2002 ) (ACS and ACO are encoded by multigene families in higher plants. Three ACS isogenes (Pp-ACS1, Pp-ACS2 and Pp-ACS3) have been reported in peach but only Pp-ACS1 is associated with ripening (Mathooko et al. 2001; Tatsuki et al. 2006).
Ruperti et al. (2001) isolated and characterized two members of the peach ACO gene family, named Pp-ACO1 and Pp-ACO2. Pp-ACO1 transcripts accumulated late strongly in ripe mesocarp as well as in abscising fruitlets and senescing leaves, and are enhanced by ethylene. Pp-ACO2 mRNA is detected in fruits only during early development and is unaffected by ethylene.
Two systems of ethylene production have been defined in plants. System 1 functions during normal growth and during stress responses, whereas system 2 operates during floral senescence and fruit ripening. System 1 is autoinhibitory, such that exogenous ethylene inhibits synthesis while inhibitors of ethylene can stimulate hormone production. In contrast, system 2 is stimulated by ethylene and is therefore autocatalytic, and inhibitors of ethylene action inhibit ethylene production (Barry & Giovannoni, 2007). At stage IV ethylene evolution begins to increase, reaching a peak usually associated with the last stage of ripening (Ramina et al. 2008). After its synthesis, ethylene is perceived and its signal transduced through transduction machinery to trigger specific biological responses.
30
Receptors can be structurally separated into three domains briefly summarized as follows: the “sensor” domain contains three hydrophobic, putative transmembrane stretches, The “kinase” domain has extensive sequence homology to His kinases (HK), the “receiver” domain has sequence identity to the output portion of bacterial two-component systems and contains aspartate (Klee 2004).
The ethylene receptors are derived from the family of two-component Histidine protein kinase receptors, which transmit their signal through Histidine autophosphorylation on the Histidine kinase domain, followed by transfer of the phosphate to a conserved aspartate residue in the “receiver” domain (Chang & Bleecker, 2004).
There are five ethylene receptors in Arabidopsis, ETR1, ETR2, EIN4, ERS1, and ERS2. ETR1 and ERS1 (Wang et al. 2002). Six ethylene receptors have been identified in tomato: LeETR1, LeETR2, NR (also referred to as LeETR3), and LeETR4, 5, and 6 (Barry & Giovannoni, 2007).
In peach, ethylene signal perception and transduction are regulated by the homologous to the Arabidopsis ethylene receptor genes ETR1 (Ethylene Responsive) and ERS1 (Ethylene Responsive Sensor), named Pp
‐
ETR1 and Pp‐
ERS1 respectively, by Pp-ETR2, which shows increased expression during the transition from pre-climacteric to climacteric stage, and CTR1 (Constitutive Triple Response) which negatively regulates the downstream ethylene response pathway (Rasori et al. 2002; Dal Cin et al. 2006). Ethylene binding occurs in presence of a copper co-factor (Wang et al. 2002).Pp-ETR1 appears to be constitutive and ethylene-independent during fruit development and ripening, whereas Pp-ERS1 transcripts increase during fruit ripening and its expression appears to be up-regulated by ethylene (Rasori et al. 2002).
After CTR1 action is inactivated by ethylene the ethylene signal is allowed to reach EIN genes (Ethylene INsensitive). In peach fruit, an induction of a
31 putative ortholog of EIN2 was observed during the transition from immature to mature stage (Trainotti et al. 2006)
Ethylene receptors bind the hormone molecules in a more or less irreversible way, self-inactivating, functioning as “negative regulators”. Ethylene effects become visible when certain number of receptors are inactivated. When receptors bind the hormone, they are not able anymore to down-regulate the expression of genes induced by ethylene and the synthesis of new receptors is necessary.
However, the amount of ethylene evolved during the ripening is overabundant if compared to tissue capacity of synthetize new receptors to block the hormone effect (Figure 12) (Klee, 2004).
Some chemicals are available for fruit management during the postharvest, able to slow down or inhibit ethylene effect on tissues, prolonging the storability. 1-methylcyclopropane (1-MCP, a competitor for ethylene binding sites), poliamines (i.e. putrescine, spermidine and spermine) and aminoethoxyvinylglycine (AVG, inhibits ethylene synthesis by preventing ACS action) can inhibit the ripening process (Bregoli et al. 2002).
Figure 12: Model for ethylene receptor action. A, In the absence of ethylene, receptors (AR) are actively suppressing ethylene responses such as fruit ripening. Upon ethylene binding, receptors become inactive (IR) and ethylene responses can proceed. Mutant receptors (M) cannot bind ethylene and continue to actively suppress downstream ethylene responses (Klee, 2004)
32
The use of postharvest abiotic stresses as a tool for
1.10
enhancing the nutraceutical content of fruits
In the last decades consumers have become more aware of the relationships between diet and diseases. High quality products which associate health, safety and convenience accomplish consumer preferences.
For satisfying this current demand, increasing attention for functional foods is growing up in fruit and vegetable market (Schreiner et al. 2013). Technologies able to ensure high quality products with high levels of the desired compounds are needed in order to enhance the health benefits and create new opportunities for growers and processors (Cisneros-Zevallos, 2003). The health-promoting properties of fruits and vegetables are due to the presence of some vitamins, dietary fibers and secondary plant metabolites. Polyphenols, which are prevailed by flavonoids, are one of the main group of secondary plant metabolites. Flavonoids are the most common phenolics obtained from the everyday plant-source diet (Chun et al. 2007) and have aroused substantial attention due to their protective potential against chronic diseases (Weng & Yen, 2013). Although plant materials do not contain vitamin A, they provide carotenoids that are converted to vitamin A after ingestion. Provitamin A carotenoids found in significant quantities in fruits include carotene, β-cryptoxanthin and α-carotene. Other carotenoids, such as lycopene, may have a role in cancer prevention by acting as free radical scavengers or antioxidants (Wright & Kader, 1997)
Postharvest abiotic stresses including storage/processing temperature, wounding, phytohormones such as ethylene or methyl jasmonate and light condition, may affect the levels of secondary metabolites in crop tissues by inducing an increase or reduction in key enzyme activities of secondary metabolic pathways (Figure 13). Previous studies indicate that there is a potential in using stresses to induce accumulation of targeted phytochemicals,
33 thereby enhancing the genetic potential of fruits and vegetables and yielding products with increased health benefit properties. However, information in the literature reporting the use of controlled stresses to enhance the accumulation of nutraceuticals is scanty.
An interesting possibility to exploit is the use during postharvest of UV-B radiation as tool for inducing metabolic changes in fruit and vegetables and satisfy consumer demand for natural health-promoting food products.
Figure 13: Proposed concept for using controlled postharvest abiotic stresses to enhance the nutraceutical content to fruits and vegetables (Cisneros-Zevallos, 2003)
34
UV-B radiation
1.11
In recent years, increasing UV-B radiation by air pollution-induced ozone depletion by man-made halogenated chemicals, including chlorofluorocarbons , has raised awareness of the effects of UV-B on the ecosystem. Increases in levels of Eart surface UV-B have been estimated to be in the range of 2–5% per decade for Central Europe (McKenzie et al. 2007)
UV-B (280–315 nm) (Figure 14) is the band of lowest wavelength and highest energy that penetrates the ozone layer of the stratosphere. It comprises about 5% of the whole UV or 0.5% of the total energy of solar radiation. In spite of the relatively low irradiance, UV-B radiation could induce severe damage to plants via direct and indirect effects on nucleic acids, proteins and cell membranes (Solovchenko & Schmitz-Eiberger, 2003).
The UV-screening stratospheric ozone layer is relatively thin at low latitudes and this, in combination with a steep solar angle, results in relatively high B levels in the tropics, compared to mid and high latitudes. High levels of UV-B also occur at high altitudes. Temporal variation due to by changes in the position of the sun, as well as seasonal changes and in general meteorological conditions can occur. (Jansen et al. 2008).
Hideg and co-authors (2013) hypothesized that low UV-B doses cause ‘eustress’ in plants (good stress, not inducing permanent plant damage but rather promoting health and growth) and that stimuli specific signaling pathways pre-dispose plants to a state of low alert that includes activation of antioxidant defenses. On the contrary, early studies showed extensive “distress” on plants associated with high levels of UV-B (Caldwell & Flint, 1994; Caldwell et al. 1998).
It is well known that living organisms have evolved mechanisms to protect against UV-B and to repair UV damage. Among the protective mechanisms implemented by higher plants there is the deposition of UV-absorbing phenolic
35 compounds in epidermal tissues, the production of cuticular waxes and hairs and the enhancement of cellular antioxidant systems (Jenkins 2009).
Figure 14: The spectrum of light
UV-B perception and signaling in plants
1.12
Plants are able to specifically perceive UV-B photons. The existence of a specific UV-B photoreceptor, which detects UV-B radiation and initiates a signaling cascade, was proposed many years ago but only recently it has been possible to identify this photoreceptor and the components of the downstream signaling pathway (Jenkins, 2009).
Plants have at least five types of sensory photoreceptors. The UV-B photoreceptor UV RESPONSE LOCUS 8 (UVR8) UVR8 is a β-propeller protein of 440 amino acids (Christie et al. 2012, Wu et al. 2012) originally identified in Arabidopsis mutants hypersensitive to UV-B.
UVR8 is expressed throughout plant bodies, which technically gives any plant organ the ability to respond to UV-B (Rizzini et al. 2011). The majority of UVR8 protein is located in the cytoplasm but a small portion is also detectable in the nucleus, even in the absence of UV-B.
Upon UV-B exposure, UVR8 accumulates within minutes in the nucleus, although not exclusively, as the majority of UVR8 remains cytoplasmic (Kaiserli & Jenkins, 2007). UV-B absorption by UVR8 is mediated by tryptophan residues, at least tryptophan-285 and tryptophan-233 (Trp-285 or Trp-233), which act as chromophore and are excited by UV-B (Tilbrook et al.
36
2013. In Arabidopsis, after the monomerization, the receptor interacts in a UV-B dependent manner with the E3 ubiquitin ligase CONSTITUTIVELY PHOTOMORPHOGENIC 1 (COP1), a multifunctional protein known for its role as a repressor of photomorphogenesis (Favory et al. 2009).
UVR8 monomer is redimerized through the action of REPRESSOR OF UV-B PHOTOMORPHOGENESIS1 (RUP1) and RUP2, which disrupts the UVR8-COP1 interaction, inactivates the signaling pathway and regenerates the UVR8 homodimer again ready for UV-B perception (Figure 15) (Tilbrook et al. 2013).
Figure 15: The UVR8 photocycle (Tilbrook et al. 2013)
Both UVR8 and COP1 are required for UV-B-mediated activation of HY5 (ELONGATED HYPOCOTYL5) a bZIP transcription factor which is important in the de-etiolation of plants. HYH (HY5 HOMOLOG) functions in a partially redundant manner with HY5, as effector of the UVR8-mediated UV-B signaling pathway. HY5 and HYH control expression of a range of key elements for UV-B acclimation, including genes encoding enzymes of the phenylpropanoid pathway (Brown et al., 2005).
COP1 represses photomorphogenesis by promoting degradation of HY5 (and other promotive transcription factors), but is under the negative control of light-activated phytochromes and cryptochromes (Heijde & Ulm, 2012).
The UVR8-COP1-HY5 pathway (Figure 16) is the most extensively analyzed UV-signaling pathway but the existence of a further UVR8 independent
37 pathway, operating under low levels of UV-B, has also been supposed (Jenkins, 2009).
Differently, under high UV-B fluence rates, UV-B specific signaling pathways are complemented by non-specific damage response pathways (Figure 17). High UV-B levels in fact, have been demonstrated to cause damage to macromolecules (DNA, proteins and lipids), to induce impairment of gene-transcription, DNA-replication and photosynthesis (Jansen et al. 1998; Kunz et al. 2007; Schreiner et al. 2012). The nonspecific pathways affects the amounts of plant hormones and grow regulators (e.g., ethylene, jasmonic acid, salicylic acid) (Mackerness et al. 1999; Predieri et al. 1993).
Figure 16: Model of UVR8-mediated signaling (Heijde & Ulm, 2012)
Both low and high levels of UV-B radiation can change antioxidant metabolism and the expression of genes that impact on the cellular redox state (i.e., change the size and/or oxidation–reduction state of the ascorbate, glutathione, and tocopherol pools, and induce accumulation of flavonols and related phenolics, which are strong cellular antioxidants).
Changes in ROS and antioxidant metabolism are an intrinsic part of both low or high UV-B levels (Hideg et al. 2013).
