Oxidized Polymeric Phenolics - American Chemical Society

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OXIDIZED POLYMERIC PHENOLICS: COULD THEY BE CONSIDERED PHOTOPROTECTORS? Laura Rustioni J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b03704 • Publication Date (Web): 18 Aug 2017 Downloaded from http://pubs.acs.org on August 18, 2017

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Journal of Agricultural and Food Chemistry

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Oxidized Polymeric Phenolics: Could They Be

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Considered Photoprotectors?

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Laura Rustioni*

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DISAA – Dipartimento di Scienze Agrarie e Ambientali - Università degli Studi di Milano; via Celoria 2, 20133

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Milano – Italia

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*Corresponding author (Tel: 0039 02 50316556; Fax: 0039 02 50316553; E-mail: [email protected])

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ABSTRACT

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Photooxidative sunburn is the consequence of photosystem overexcitations. It results in tissue color

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changes due to chlorophyll degradation and accumulation of oxidized polymeric phenolics (OPPs) resulting

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from scavenging of reactive oxygen species (ROS). From a productive point of view, OPPs should be

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considered as damages, decreasing the economical and esthetical values of plants and crops. However,

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from a physiological perspective, OPPs could be also play a screening role against excessive

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photosynthetically active radiation (PAR), as they follow the criteria proposed for the identification of

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photoprotectors, as follows: i) Due to the complex conjugated double bond systems, OPPs absorb and,

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thus, screen the visible photosynthetically active radiation; ii) The accumulation of brown OPPs is well-

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known to be stimulated by light exposure resulting in sunburn symptoms; iii) OPPs induce PAR resistance:

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for example, the sunburned brown skin allows the fruit ripening to proceed without further interferences;

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iv) the screen provided by the accumulated OPPs in death cells protect underlying tissues, demonstrating

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an increased resistance to radiation when other physiological processes are not functioning.

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KEYWORDS: Photooxidation; sunburn; brown pigments; PAR screen; radiative excess


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OXIDIZED POLYMERIC PHENOLICS AND PHOTOOXIDATIVE SUNBURN

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Plants are photoautotrophs and light is the basis of their life. However, excessive radiation, often coupled

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by thermal stresses (due to radiative heating), could produce serious damages to the exposed tissues. In

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their extensive review, Racsko and Schrader1 reported a clear classification of sunburn effects on apples,

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discriminated by characteristic symptoms and originating causes. The first sunburn type, sunburn necrosis,

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is the result of excessively high temperatures (higher than 52 °C for at least 10 min), independent of light

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stimuli. These lethal conditions result in loss of membrane integrity in correspondence of symptomatic

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necrotic areas. 2 The second sunburn type, sunburn browning, results from the combination of excesses in

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temperature (cultivar specific threshold ranging between 46 and 49 °C) and UV-B radiation. 2 These sub-

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lethal conditions do not affect the membrane integrity; however, they produce tan areas on the sun

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exposed side of the fruits. 1 Although the above described symptoms could represent a major injury in

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agricultural productions, they are expected to afflict mainly organs with an inadequate thermal regulation

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due to stomata degeneration. For example, considering grapevines, the berry latent heat flow is considered

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nearly null, due to the extremely limited evapotranspiration, 3 while, in leaves, transpiration significantly

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decreases the organ temperature. 4 In this case, also the water status could affect the plant susceptibility. 4

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This Perspective focuses on the third type of sunburn: photooxidative sunburn. In this case, the triggering

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cause is not heat nor UV exposure, in fact the sole visible radiation is able to produce the symptomatic

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pigmentations in both apples and grapes. 1, 5, 6 Excessive photosynthetically active radiation could produce

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serious damages to photosystems, due to the production of reactive oxygen species (ROS).7 In steady state

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conditions, ROS are scavenged by various anti-oxidative defense mechanisms, but stress factors (such as

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radiative excesses) could perturb the equilibrium between the production and the scavenging of ROS.

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These free radicals can act as damaging, protective or signaling factors. 8-11

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The physiological response to photodamage is preserved among plants (it appears in different species,

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organs and tissues), and it could be experienced by exposing non-acclimated plants to excessive radiation



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(e.g.: an indoor plant, adapted to low photosynthetically active radiation (PAR) input, suddenly moved to

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direct sunlight). The first photooxidation symptoms appear as tissue color changes. The green color

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decreases as a consequence of chlorophyll degradation and brown pigments are accumulated as a results

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of ROS scavenging by phenolic compounds. 5, 6, 12 ROS scavenging requires phenolic hydrogen atoms, and

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the obtained phenolic free radicals are then deactivated by polymerizations which result in brown pigment

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accumulations6, 12-14 (Figure 1). It is interesting to note that the central role of phenolic compounds as

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oxidized polymeric phenolic (OPP) precursors in early plant adaptation to terrestrial stress conditions have

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been proposed.15 Close and McArthur16 demonstrated the main function of plant phenolics as defense

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against photodamage, suggesting their role in plant ecology and evolution related to different risks of

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photodamage. They stated that “differences in phenolic levels at any level (within a plant, between

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individuals within a species, and between species) are a response to oxidative pressure and risk of

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photodamage”. In their review, the authors limited the phenolic role to their antioxidant capacity. It is

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worth to notice that OPPs undergo a phenolic regeneration process during polymerization14 and the

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regenerated phenols are more readily oxidized than their corresponding original ones13 resulting in an

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increased antioxidant capacity of the oxidized compounds. In addition to this evidence, a possible

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photoprotective screening role of oxidized phenolics against PAR excesses is proposed in this Perspective.

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Different photoprotective mechanisms have been described. Mostly of them dedicated to the reparation or

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mitigation of radiative damages. Nevertheless, the importance of photoprotective mechanisms based on

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the attenuation of harmful excessive PAR has also been underlined: 17 plants can respond to excessive

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radiation by the synthesis and accumulation of pigments able to selectively absorb specific regions of the

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visible spectrum. 17

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Cockell and Knowland18 proposed four criteria to define the compounds with a UV-screening role, and in

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accordance with Solovchenko and Merzlyak, 17 these principles will be generalized to excessive PAR-

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screening.


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1. OPPs must screen the PAR radiation. Phenolic compounds are characterized by the presence of one

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or more aromatic rings substituted by hydroxyl group(s). The presence of conjugated double bond

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systems favors the absorption of low energetic radiation. The absorbed radiative energy allows the

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excitation of π-electrons, which make a transition from π orbitals to π* orbitals. 18 A UV absorption

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band peaking around 280 nm appears in all the phenolic spectra, due to the presence of aromatic

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ring(s). A second long-wave band is generally situated in the 300–360 nm range, but it could also

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extend into the blue-green spectral region, and the exact position varies depending on the phenol

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classes and structure (Figure 2).17 Generally, an increase in the molecular complexity (number of

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conjugated bonds and substituents) results in a bathochromic shift of the absorption bands. 18, 19

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The heterogeneous polymerization characteristic of OPP formation, results in an increase of the

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molecular complexity, 13, 14, 20 including the number of conjugated aromatic rings. The absorption

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band obtained shows a maximum around 500 nm,12 overlapping the spectral region of highest

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sunlight irradiance. 21 Furthermore, due to the heterogeneous molecular structures of OPPs, the

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main absorption band of oxidized tissues appears broad, and, thus, able to absorb radiations in a

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wide range of the PAR wavelengths. 12

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2. OPPs accumulation should be induced in the living cell by PAR radiation. Recently, grape sunburn

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symptoms were characterized by reflectance spectroscopy. The accumulation of OPPs in

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consequence of excessive visible radiation was described both in field condition (solar radiation) 12

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and under controlled conditions (LED light sources). 6 Moreover, the biosynthesis of phenolic

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compounds (OPP precursors) is well-known to be stimulated by light exposure. 16, 22, 23 Jakopic et

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al.24 observed significant increases in flavonoid concentrations due to PAR exposure in apple skins.

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Concerning grapes, light exposure anticipates the beginning of berry anthocyanin accumulation at

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veraison, and, thus, in susceptible green berries. 25 Furthermore, an increased proportion of ortho-

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diphenols (highly oxidizable) has been reported in the anthocyanin profile of sunlight exposed

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berries. 25 Finally, it has been observed that grape sunlight exposure obtained by leaf removal,



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results in an increased accumulation of both OPPs (quantified as sunburn symptoms) and flavonols (phenolic precursors) in Raboso Piave and Sangiovese berries. 26

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3. The accumulation of OPPs should induce the resistance to the radiation in the spectral range of their

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absorption. Considering viticulture, defoliation is a common and well-studied practice and, thus,

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grapevines (Vitis vinifera L.) will be considered as a reference plant in this section. Defoliation

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results in an increase of berry radiative exposure and, at least in some days or portion of them, in

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photosystem overexcitations. In some cases, bunches directly exposed to solar radiation could

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produce evident sunburn symptoms. 12, 26, 27 Green berries are more susceptible than ripened fruits,

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due to the higher concentration of chlorophylls.6 Nevertheless, early defoliations (around

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flowering) cause a decrease in the susceptibility to evident sunburn symptoms, when compared to

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leaf removal in late green phenological stages (pea-size, veraison).28,29 These results indicate the

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existence of adaptive mechanisms able to limit the photooxidation susceptibility. Among these, the

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accumulation of photoprotective OPPs should be considered: during ripening, at least in white

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berried grapes, beside the decrease in chlorophyll content, catabolic processes favor the

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accumulation of OPPs, 30 which could have an additional protective effect against radiative

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excesses. Finally, also in concomitance with sunburn evidence, resulting from oxidative stresses in

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the skin (the external and protective fruit tissue), the berry pulp keeps ripening, as well as seeds. In

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fact, the protective brown skin generally allows the berry ripening to proceed without further

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interferences from the increased radiative stress, when compared to fruits shaded during all of the

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ripening phase. The results reported by Mosetti et al.27 show that leaf removal applied after fruit

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set (and the consequent direct exposition to sunlight) did not affect yield and fruit technological

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composition at harvest despite the presence of sunburn symptoms. Also, Pastore et al.26 showed

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that the berry exposure to excessive radiation in concomitance with significant sunburn symptom

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observations, did not prejudice the fruit development of exposed bunches in comparison to shaded

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grapes.


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4. OPPs must provide an increased resistance to radiation when other physiological processes are not

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functioning. Fruits have developed complex defense mechanisms against excessive sunlight. These

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systems can safely scavenge free radicals to prevent or minimize sunburn damages. However, the

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high incidence of sunburn under field conditions suggests that these mechanisms are often

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inadequate. 1 It is worth noticing that sunburn symptoms are considered as damages in fruit

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production fields. However, from a physiological point of view, the symptomatic brown areas

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related to the accumulation of OPPs could be considered as an extreme defense mechanism: a sort

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of “parasol” for the underlying plant reproductive organs. The photooxidative sunburn in apples is

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caused by excess of photosynthetically active radiation in non-acclimated fruits. The symptoms

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appear first as photobleached areas, that often turn brown. In this case, the accumulation of brown

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pigments could be associated with cell necrosis. 1, 5 In these death cells of damaged tissues, it is

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difficult to imagine other functioning physiological processes except the screen provided by the

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accumulated pigments, nevertheless the ripening of the underlying rest of the fruit (and of seeds) is

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not blocked by these symptoms. Piskolczi et al.31 state that sunburn does not usually cause serious

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damage to the epidermal tissue, and rarely affects the subepidermal tissue at all, despite the

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golden-bronze discolorations on the sunlit side of the fruit, detracting from its appearance.

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A photoprotective screen is considered as a long-term protection against photodamages. It is characterized

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by highly photostable pigments and it also protects the plant independently from environmental stresses

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(e.g. high temperature) which could disadvantage protective mechanisms based on enzymes. However,

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generally, the initial buildup of photoprotective compounds is considered as a high cost and slow plant

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activity. 17 Considering the OPPs, the accumulation of their precursors is cost and time consuming for the

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plant. It is generally stimulated by high irradiance; 16 however, it commonly occurs in all the organs also

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without specific stimuli. 14, 30 Furthermore, phenolic compounds, the OPP precursors, play different roles

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(e.g. antioxidants, biotic and abiotic stress protection, etc.) in plants and, thus, their accumulation costs are

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split up by the numerous functions. Nevertheless, the oxidative burst could occur in a very short time6, 12, 13

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and, thus, the transformation of molecules unable to absorb the visible radiation in pigments able to screen 7 ACS Paragon Plus Environment


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the PAR excesses is extremely fast. Thus, the OPPs could represent the most extreme PAR screen against

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photodamages.

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The accumulation of brown pigments in response to radiative excess in plants is a ubiquitous mechanism.

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This process is generally considered a damage, as it usually causes a decrease in the economic and

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aesthetic values of plants and crops. However, in plants, the sacrifice of the functionality, and, in extreme

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conditions, also of the life of cells, tissues or organs is a common adaptation strategy of biotic and abiotic

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stress response. For example, defense mechanisms of resistant grapevines against Plasmopara viticola

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include the necrosis of stomata and cells surrounding the pathogen invasion point. 32 Considering abiotic

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stresses, the selective abscission of leaves has been demonstrated to participate in the drought adaptation

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mechanisms of Jatropha curcas. 33

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In conclusion, different physiological roles have been proposed for OPPs, 14 and a photoprotective activity

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should also be considered.

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ABBREVIATIONS

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OPP = Oxidized Polymeric Phenolic; PAR = Photosynthetically Active Radiation; ROS = Reactive Oxygen

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Species

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REFERENCES

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1. Racsko, J.; Schrader, L.E. Sunburn of Apple Fruit: Historical Background, Recent Advances and Future Perspectives. Crit. Rev. Plant Sci. 2012, 31, 455–504. 2. Schrader, L. E.; Zhang, J.; and Duplaga, W. K. Two types of sunburn in apple caused by high fruit surface (peel) temperature. Plant Health Prog. 2001 doi:10.1094/PHP-2001-1004-01-RS.


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3. Cola, G.; Failla, O.; Mariani, L. BerryTone—A simulation model for the daily course of grape berry temperature. Agricultural and Forest Meteorology 2009, 149, 1215–1228. 4. Jones, H.G.; Stoll, M.; Santos, T.; de Sousa, C.; Chaves, M.M.; Grant, O.M. Use of infrared

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thermography for monitoring stomatal closure in the field: application to grapevine. J. Exp. Bot.,

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2002, 53(378), 2249-2260.

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5. Felicetti, D.A.; Schrader, L.E. Photooxidative sunburn of apples: characterization of a third type of apple sunburn. International Journal of Fruit Science 2008, 8(3), 160-172. 6. Rustioni, L.; Milani, C.; Parisi, S.; Failla, O. Chlorophyll role in berry sunburn symptoms studied in different grape (Vitis vinifera L.) cultivars. Sci. Hortic. 2015a, 185, 145–150. 7. Müller, P.; Li, X.P.; Niyogi, K.K. Non-photochemical quenching. A response to excess light energy. Plant Physiol. 2001, 125, 1558-1566. 8. Gill, S.S.; Tuteja, N. Reactive oxygen species and antioxidant machinery in abiotic stress tolerance in crop plants. Plant Physiol. Biochem. 2010, 48(12), 909-930. 9. Foyer, C.H.; Shigeoka, S. Understanding oxidative stress and antioxidant functions to enhance photosynthesis. Plant. Physiol. 2011, 155, 93–100. 10. Kreslavski, V.D.; Los, D.A.; Allakhverdiev, S.I.; Kuznetsov, V.V. Signaling role of reactive oxygen species in plants under stress. Russ. J. Plant Physiol. 2012, 59(2), 141–154. 11. Mullineaux, P.M.; Karpinski, S.; Baker, N.R. Spatial dependence for hydrogen peroxide-directed signaling in light-stressed plants. Plant Physiol. 2006, 141, 346–350. 12. Rustioni, L.; Rocchi, L.; Guffanti, E.; Cola, G.; Failla, O. Characterization of grape (Vitis vinifera L.) berry sunburn symptoms by reflectance. J. Agric. Food Chem. 2014, 62, 3043–3046.

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13. Li, H.; Guo, A.; Wang, H. Mechanisms of oxidative browning of wine. Food Chem. 2008, 108, 1–13.

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14. Pourcel, L.; Routaboul, J.M.; Cheynier, V.; Lepiniec, L.; Debeaujon, I. Flavonoid oxidation in plants:

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from biochemical properties to physiological functions. Trends Plant Sci. 2007, 12(1), 29–36.

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15. Graham, L.E.; Kodner, R.B.; Fisher, M.M.; Graham, J.M.; Wilcox, L.W.; Hackney, J.M.; Obst, J.; Bilkey, P.C.; Hanson, D.T.; Cook, M.E. Early land plant adaptations to terrestrial stress: A focus on 9 ACS Paragon Plus Environment


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phenolics, In: The Evolution of Plant Physiology. From whole plants to ecosystems. Edited by:Alan R.

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Hemsley and Imogen Poole. ISBN: 978-0-12-339552-8. 2004; pp. 155-169.

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16. Close, D.C.; McArthur, C. Rethinking the role of many plant phenolics – protection from photodamage not herbivores? OIKOS 2002, 99, 166–172. 17. Solovchenko, A.E.; Merzlyak, M.N. Screening of visible and uv radiation as a photoprotective mechanism in plants. Russ. J. Plant Physiol. 2008, 55(6), 719–737.

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18. Cockell, C.S.; Knowland, J. Ultraviolet radiation screening compounds. Biol. Rev. 1999, 74, 311-345.

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19. Rustioni, L.; Di Meo, F.; Guillaume, M.; Failla, O.; Trouillas, P. Tuning color variation in grape

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anthocyanins at the molecular scale. Food Chem. 2013, 141, 4349–4357. 20. Waterhouse, A.L.; Laurie, V.F. Oxidation of wine phenolics: a critical evaluation and hypotheses. Am. J. Enol. Vitic. 2006, 57(3), 306-313. 21. Leckner, B. The spectral distribution of solar radiation at the earth's surface—elements of a model. Solar Energy 1978, 20(2), 143-150. 22. Dixon, R.A.; Paiva, N.L. Stress-induced phenilpropanoid metabolism. The plant cell 1995, 7, 10851097. 23. Downey, M.O.; Harvey, J.S.; Robinson, S.P. The effect of bunch shading on berry development and flavonoid accumulation in Shiraz grapes. Aust. J. Grape Wine Res. 2004, 10, 55-73. 24. Jakopic, J.; Stampar, F.; Veberic, R. The influence of exposure to light on the phenolic content of ‘Fuji’ apple. Sci. Hortic. 2009, 123, 234–239.

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25. Rustioni, L.; Rossoni, M.; Cola, G.; Mariani, L.; Failla, O. Bunch exposure to direct solar radiation

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increases ortho-diphenol anthocyanins in northern italy climatic condition. J. Int. Sci. Vigne Vin,

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2011, 45(2), 85-99.

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26. Pastore, C.; Allegro, G.; Valentini, G.; Muzzi, E.; Filippetti, I. Anthocyanin and flavonol composition

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response to veraison leafremoval on Cabernet Sauvignon, Nero d’Avola, Raboso Piave

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andSangiovese Vitis vinifera L. cultivars. Sci. Hortic., 2017, 218, 147–155.


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27. Mosetti, D.; Herrera, J.C.; Sabbatini, P.; Green, A.; Alberti, G.; Peterlunger, E.; Lisiak, K.; Castellarin,

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S.D. Impact of leaf removal after berry set on fruit composition and bunch rot in 'Sauvignon blanc'.

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Vitis 2016, 55, 57–64.

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28. Molitor, D.; Behr, M.; Fischer, S.; Hoffmann, L.; Evers, D. Timing of cluster-zone leaf removal and its

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impact on canopy morphology, cluster structure and bunch rot susceptibility of grapes. J. Int. Sci.

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Vigne Vin. 2011, 45(3), 149-159.

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29. Pastore, C.; Zenoni, S.; Fasoli, M.; Pezzotti, M.; Tornielli, G.B.; Filippetti, I. Selective defoliation

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affects plant growth, fruit transcriptional ripening program and flavonoid metabolism in grapevine

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BMC Plant Biol. 2013, 13:30. https://doi.org/10.1186/1471-2229-13-30

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30. Rustioni, L.; Rocchi, L.; Failla, O. Effect of anthocyanin absence on white berry grape (Vitis vinifera L.) Vitis 2015b, 54, 239–242.

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31. Piskolczi, M.; Varga, C.; Racskó, J. A Review of the meteorological causes of sunburn injury on the

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surface of apple fruit (Malus domestica BORKH). J. Fruit Ornamental Plant Res. 2004, 12, 245-252.

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32. Toffolatti, S.L.; Venturini, G.; Maffi, D.; Vercesi, A. Phenotypic and histochemical traits of the

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interaction between Plasmopara viticola and resistant or susceptible grapevine varieties. BMC Plant

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Biology 2012, 12:124.

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33. Fini, A.; Bellasio, C.; Pollastri, S.; Tattini, M.; Ferrini, F. Water relations, growth, and leaf gas

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exchange as affected by water stress in Jatropha curcas. Journal of Arid Environments 2013, 89, 21-

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34. Allen, M. Phenolics demystified. In: Proceedings ASVO oenology seminar. Phenolics and extraction. Adelaide, 9 October 1997. Australian Society of Viticulture and Oenology. 1998

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FIGURE CAPTIONS

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Figure 1: Example of possible flavan-3-ol oxidative dimerization mechanism, in consequence of photo-

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oxidative stress and ROS formation (adapted from Waterhouse and Laurie). 20



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Figure 2: Examples of phenolic structures and their approximative longwave absorption regions: (A) flavan-

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3-ols; (B) hydroxycinnamic acids; (C) OPP dimers (structure proposed by Allen); 34 (D) anthocyanins.


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Figure 1: Example of possible flavan-3-ol oxidative dimerization mechanism, in consequence of photooxidative stress and ROS formation (adapted from Waterhouse and Laurie). 20 220x279mm (96 x 96 DPI)

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Figure 2: Examples of phenolic structures and their approximative longwave absorption regions: (A) flavan3-ols; (B) hydroxycinnamic acids; (C) OPP dimers (structure proposed by Allen); 34 (D) anthocyanins. 500x279mm (96 x 96 DPI)


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Table of Contents Graphic 84x47mm (96 x 96 DPI)


Oxidized Polymeric Phenolics - American Chemical Society

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