Exposure of the shaded side of apple fruit to full sun leads to up-regulation of both the xanthophyll cycle and the ascorbate glutathione cycle

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1 Plant Science 1 (24) Exposure of the shaded side of apple fruit to full sun leads to up-regulation of both the xanthophyll cycle and the ascorbate glutathione cycle Fengwang Ma 1, Lailiang Cheng Department of Horticulture, Cornell University, 14A Plant Science, Ithaca, NY 1485, USA Received 12 December 2; received in revised form January 24; accepted 1 January 24 Abstract Approximately 8 days after full bloom, well-exposed fruit on the south part of the canopy of mature Liberty/M9 apple (Malus domestica Borkh.) trees were randomly assigned to one of the following two treatments. Some fruits were turned about 18 to expose the original shaded side to full sun, whereas the rest served as untreated controls. On days, 1, 2, 4, 7 and 1 after treatment, fruit peel samples were taken from the original shaded side of the treated fruit and both the sun-exposed side and the shaded side of the control fruit at midday, to determine photosynthetic pigments, enzymatic and non-enzymatic antioxidants. Maximum photosystem II (PSII) efficiency, measured as F v /F m (maximum fluorescence) of chlorophyll fluorescence at pre-dawn, was higher in the shaded side than the sun-exposed side of the control fruit. F v /F m of the original shaded side decreased sharply after 1 day exposure to full sun, and then gradually recovered to a similar value of the sun-exposed side of the control fruit by day 1. The shaded side of the control fruit had much lower xanthophyll cycle pool size, conversion and antioxidant enzymes and also soluble antioxidants of the ascorbate glutathione cycle than the sun-exposed side. Xanthophyll cycle pool size of the original shaded side increased in response to full sun exposure, reaching a similar value of the sun-exposed side by day 1. Almost all the xanthophyll cycle pool was converted to zeaxanthin and antheraxanthin starting from the first day of exposure to full sun. In contrast, chlorophyll content decreased whereas lutein or neoxanthin content remained unchanged. In response to full sun exposure, ascorbate peroxidase, monodehydroascorbate reductase, dehydroascorbate reductase and glutathione reductase and total pool size and reduction state of both ascorbate and glutathione, all increased to the corresponding values found in the sun-exposed side of the control fruit over a 1-day period. It is concluded that both xanthophyll cycle and the ascorbate glutathione cycle in the original shaded side are up-regulated in response to full sun exposure to minimize photo-oxidative damage and contributes to its acclimation to full sun. 24 Elsevier Ireland Ltd. All rights reserved. Keywords: Apple fruit; Ascorbate glutathione cycle; Malus domestica; Photoprotection; Acclimation; Xanthophyll cycle 1. Introduction Xanthophyll cycle-dependent thermal dissipation and detoxification of reactive oxygen species by the antioxidant system are the two photoprotective mechanisms which plants have evolved to cope with excess absorbed photon flux density (PFD). Xanthophyll cycle is a light-dependent cyclic conversion between three xanthophylls [1,2]. As PFD increases, the acidification of thylakoid lumen activates violaxanthin (V) de-epoxidase [,4], which converts V to zeaxanthin (Z) via antheraxanthin (A). Low lumen Corresponding author. Tel.: ; fax: address: lc89@cornell.edu (L. Cheng). 1 Present address: Department of Horticulture, Northwest Sci.-Tech. University of Agriculture and Forestry, Yangling, Shaanxi 7121, PR China. ph also results in protonation of proteins in the PSII lightharvesting complexes. The interaction of Z and/or A with the specific sites of the protonated PSII proteins induces a conformational change that leads to increased rate constant of heat dissipation for excitation energy in the PSII antenna [5,]. In addition to thermal dissipation, Z and A are capable of de-exciting singlet oxygen [7,8], minimizing the damage of singlet oxygen on photosynthetic apparatus and cell structure. The reactive oxygen species formed via direct photoreduction of oxygen at PSI under excess PFD is detoxified by an integrated antioxidant system consisting of enzymatic and non-enzymatic antioxidants [9]. The initial product of photoreduction of oxygen, superoxide, is dismuted to hydrogen peroxide, which is then converted to water and oxygen via the ascorbate glutathione cycle [1] in the chloroplast and cytosol. The ascorbate glutathione cycle involves ascorbate peroxidase (EC , APX), monodehydroascorbate /$ see front matter 24 Elsevier Ireland Ltd. All rights reserved. doi:1.11/j.plantsci

2 148 F. Ma, L. Cheng / Plant Science 1 (24) reductase (EC , MDAR), dehydroascorbate reductase (EC , DHAR), glutathione reductase (EC , GR), ascorbate (AsA), glutathione (GSH), and NADPH in a series of cyclic reactions to detoxify H 2 O 2 and also regenerate AsA and GSH [1]. Apple fruit forms both a sun-exposed side and a shaded side as a result of self-shading during its development. The shaded side of apple fruit receives much less PFD than the sun-exposed side. Under low incident PFD, there is not much need to dissipate excess absorbed PFD and to scavenge reactive oxygen species generated via photoreduction of oxygen. Correspondingly, the shaded side of apple fruit acclimates to this low PFD [11]. Compared with the sun-exposed side, the shaded side of apple fruit has a smaller xanthophyll cycle pool size and a lower de-epoxidation state, and lower activities of the antioxidant enzymes (APX, MDAR, DHAR and GR) and lower levels of soluble antioxidants (AsA and GSH) in the ascorbate glutathione cycle [11]. How shade leaves respond to a sudden exposure to a high PFD depends on species and the degree of shading they have experienced during their development [12]. For obligate shade species that can only grow under low PFD, sudden exposure to a high PFD may cause severe photoinhibition, leading to photo-bleaching and even death of the leaves or the whole plant. For shade leaves of obligate or facultative sun species, as long as the PFD in which they grow is not too low, they may suffer from the sudden exposure of full sun initially, then gradually recover from photoinhibition and eventually acclimate to the new PFD environment [1 15]. During the recovery and acclimation process, photoprotective mechanisms are up-regulated to cope with the excess absorbed PFD, which include xanthophyll cycle dependent on thermal dissipation and the antioxidant system [14,1]. It is not known, however, how the xanthophyll cycle and the ascorbate glutathione pathway in the shaded peel of apple fruit respond to a sudden exposure to a much higher PFD than the one it has been adapted to. Re-exposure of shaded fruit inside the canopy or the shaded side of well-exposed fruit to full sun often occurs during summer pruning or fruit turning to enhance the red color formation of apple fruit. We hypothesized that both the up-stream xanthophyll cycle and the down-stream ascorbate glutathione cycle of the shaded side of apple fruit are enhanced in response to a sudden increase of incident PFD to provide photoprotection against the excess PFD. 2. Materials and methods 2.1. Plant material, treatments, and sampling Nine-year-old apple (Malus domestica Borkh. cv. Liberty) trees on M9 rootstock were grown at a spacing of 1.52 m 4.27 m in the field at Cornell Orchards, in Ithaca (42 2 N, 7 29 W; elevation 5 m), New York, USA. The trees were trained as a central leader system. They were approximately.5 m tall. They received standard horticultural practices, diseases and pest control. On 2 August 22, approximately 8 days after full bloom, well-exposed fruit that were on the south part of the canopy were randomly assigned to one of the following two treatments. Some fruit were turned about 18 after sunset to expose the shaded side to full sun starting the next day (day 1), whereas the rest served as untreated controls. There were four replications for each treatment with six trees per replicate. On day, 1, 2, 4, 7 and 1 after treatment, fruit peel discs (1 cm 2 in size, 1 mm thick) were taken from the original shaded side of the treated fruit and both the sun-exposed side and the shaded side of the control fruit at midday, frozen in liquid nitrogen, and stored at 8 C until analysis of pigments, enzymatic and non-enzymatic antioxidants. At midday sampling on day, 1, 2, 4, 7 and 1, PFD (measured with an LI-19SA quantum sensor) and air temperature were 192, 195, 188, 18, 17, and 185 mol m 2 s 1, and 1.1, 1.4, 1.8, 29.4, 2., and. C, respectively Measurements of reflectance and chlorophyll fluorescence Reflectance of fruit peel was measured with an LI-18 spectroradiometer and an 18-12S integrating sphere attachment (LI-COR Inc., Lincoln, NE, USA). A piece of peel (1 1.5 mm thick) was cut from the sun-exposed side or the shaded side of the fruit with a razor blade, and both the reference and the sample scan of reflectance were made from 4 to 7 nm at 1 nm interval. The sample scan was divided by its corresponding reference scan, and integrated over the wavelength range to obtain the average reflectance. Maximum PSII efficiency of fruit peel was measured with a pulse-modulated fluorometer, FMS2 (Hansatech, Norfolk, UK). A custom-made device was used to hold the fruit and keep the fiber optic of the FMS2 at approximately angle relative to the fruit surface. Maximum fluorescence (F m ) and minimum fluorescence (F ) of dark-adapted fruit were measured at pre-dawn. The maximum PSII efficiency was calculated as: F v /F m = (F m F )/F m [17]. Because it was almost impossible to keep the distance between the end of the fiber optic of FMS2 and the surface of attached fruit exactly the same between pre-dawn and noon measurements, we were unable to estimate non-photochemical quenching. The significant decrease in pre-dawn F v /F m of the original shaded side upon exposure to full sun (Fig. 1) prevented us from using efficiency of excitation transfer, F v /F m = (F m F o )/F m, to monitor thermal dissipation of the original shaded side in response to full sun exposure in this study. 2.. Analysis of pigments Chlorophyll and xanthophyll pigments were extracted from two fruit peel discs (total of 2 cm 2 ) and analyzed by using an HPLC procedure as described previously [18].

3 1. F. Ma, L. Cheng / Plant Science 1 (24) Extraction and analysis of antioxidant metabolites Fv/Fm shade sun shade to sun Fig. 1. Maximum quantum efficiency of photosystem II of apple fruit peel, measured as F v /F m of chlorophyll fluorescence at pre-dawn, in response to sudden exposure to full sun. Open circle, closed circle, and triangle represent the sun-exposed side of control fruit, the shaded side of control fruit, and the original shaded side of the treated fruit, respectively. Each point was the mean of four replicates with standard error Extraction and assay of antioxidant enzymes Antioxidant enzymes were extracted according to Grace and Logan [19]. Briefly, two fruit peel discs (total of 2cm 2 ) were ground with a pre-cooled mortar and pestle in ml ice-cold extraction buffer containing 5 mm KH 2 PO 4 KOH (ph 7.5),.1 mm ethylenediaminetetraacetic acid (EDTA),.% (w/v) Triton X1, and 4% (w/v) insoluble polyvinylpolypyrrolidone (PVPP). The extract was kept on ice for 1 min, and then centrifuged at 1, g for 1 min in a microcentrifuge at 2 C. The supernatant was used immediately for measuring the following antioxidant enzymes. SOD activity was assayed at 55 nm by using the cytochrome c method [2]. One unit SOD activity is the amount required to cause a 5% inhibition of cytochrome c reduction. APX activity was measured by monitoring the decrease in absorbance at 29 nm (extinction coefficient of 2.8 mm 1 cm 1 ) [21]. The assay mixture (1 ml) contained 5 mm Hepes KOH (ph 7.),.1 mm EDTA,.2 mm H 2 O 2,.5 mm AsA and enzyme extract. The reaction was initiated by adding H 2 O 2. MDAR activity was assayed at 4 nm (extinction coefficient of.2 mm 1 cm 1 ) in 1 ml reaction mixture containing 5 mm Hepes KOH (ph 7.),.1 mm NADH,.25 mm AsA,.25 units AsA oxidase (EC 1.1..). The reaction was initiated by adding AsA oxidase [22]. DHAR activity was measured at 25 nm (extinction coefficient of 14 mm 1 cm 1 ) in 1 ml assay solution containing 1 mm Hepes KOH (ph 7.), 1 mm EDTA, 2.5 mm GSH,.2 mm dehydroascorbate (DAsA). The reaction was initiated by adding DAsA [2]. GR activity was monitored at 4 nm (extinction coefficient of.2 mm 1 cm 1 ) in 1 ml reaction mixture containing 1 mm Tris HCl (ph 8.), 1 mm EDTA, 1 mm oxidized glutathione (GSSG),.2 mm NADPH. The reaction was initiated by adding NADPH [19]. Two peel discs (total of 2 cm 2 ) were ground in 1.5 ml of ice-cold 7% (w/v) sulfosalicylic acid. GSH and GSSG were measured according to Griffith [24]. AsA and DAsA were measured according to Logan et al. [25] with minor modifications. Briefly, fruit peel discs were ground in 1.5 ml ice-cold of % (v/v) HClO 4. The extract was centrifuged at 1, g for 1 min at 2 C and then the supernatant was immediately used for the measurements. One hundred microliter of extract was neutralized with l 1.5 M Na 2 CO to raise the ph to 1 2. AsA was assayed spectrophotometrically at 25 nm in 1 mm potassium phosphate buffer (ph 5.), before and after 15 min incubation with 5 units AsA oxidase. For total ascorbate, 1 l extract was neutralized with l 1.82 M Na 2 CO to raise the ph to 7 and incubated for min at room temperature with equal volume (1 l) of 2 mm GSH in 1 mm Tricine KOH (ph 8.5). Total ascorbate was assayed as above. DAsA was calculated as the difference between total ascorbate and AsA.. Results.1. Maximum PSII efficiency (F v /F m ) Pre-dawn F v /F m was higher in the shaded side than in the sun-exposed side (Fig. 1). One day after exposure to full sun, the F v /F m of the shaded side decreased from.85 to Fig. 2. (A) Reflectance of apple fruit peel, averaged over 4 7 nm, in response to sudden exposure to full sun. Open circle, closed circle, and triangle represent the sun-exposed side of control fruit, the shaded side of control fruit, and the original shaded side of the treated fruit, respectively. Each point was the mean of four replicates with standard error; (B) Representative reflectance spectrum (4 7 nm) of the original shade peel in response to sudden exposure to full sun. From top to bottom: lines 1 is the original shaded peel of the treated fruit on day, 1, 2, 4, 7, and 1, respectively; line 7 is the sun-exposed peel of the control fruit.

4 1482 F. Ma, L. Cheng / Plant Science 1 (24) , and then gradually increased to the same value of the sun-exposed side (.725) over a course of 1 days..2. Reflectance The shaded side of the fruit had higher reflectance averaged over 4 7 nm than the sun-exposed side (Fig. 2A). Reflectance spectrum showed that the shaded side reflected less light in the 5 nm region (Fig. 2B). When exposed to full sunlight, the reflectance of the shaded side decreased gradually to a similar value of the sun-exposed side (Fig. 2A and B)... Xanthophyll cycle pigments, carotenoids and chlorophylls The xanthophyll cycle pool size (V + A + Z) was much smaller in the shaded side than in the sun-exposed side (Fig. A). In response to full sun exposure, the xanthophyll cycle pool size of the original shaded side increased gradually, reaching a similar value of the sun-exposed side on day 1. The conversion state of the xanthophyll cycle at noon was much lower in the shaded side than in the sun-exposed side (Fig. B). Almost all the xanthophyll cycle pool was converted to Z + A in the sun-exposed side. Exposure of the original shaded side to full sun increased the conversion state to a similar level as in the sun-exposed side from day 1 upon exposure to full sun (Fig. B). There were no significant differences in lutein or neoxanthin content between the sun-exposed side and the shaded side (Fig. C). Exposure to full sunlight did not significantly alter lutein or neoxanthin level of the original shaded side. Total carotenoids showed a similar trend as the xanthophyll cycle pool size (Fig. D). The shaded side had lower carotenoids than the sun-exposed side. Exposure of the original shaded side to full sunlight increased the total carotenoids. Chlorophyll content was higher in the shaded side than in the sun-exposed side (Fig. E). In response to full sun exposure, chlorophyll content of the original shaded side decreased to the level of the sun-exposed side. There was no significant difference in chlorophyll a/b ratio between the shaded side and the sun-exposed side (Fig. F). It appeared that chlorophyll a/b ratio of the original shaded side increased slightly first in response to full sun exposure, then decreased to a similar level as in the sun-exposed and shaded sides..4. Antioxidant enzymes There was no difference in SOD activity between the shaded side and the sun-exposed side of control fruit V+A+Z ( µ mol m -2 ) A+Z (% of V+A+Z) (A) (B) (D) (E) shade sun shade to sun Total carotenoids ( mol m -2 ) µ µ Chl (a+b) ( mol m -2 ) Lutein or neoxanthin ( µ mol m -2 ) (C) Lutein Neoxanthin (F) Chl a/b Fig.. Xanthophyll cycle pool size (A), and conversion state (B), lutein and neoxanthin (C), total carotenoids (D), chlorophyll (a + b) (E) and chlorophyll a/b (F) of apple fruit peel in response to a sudden exposure to full sun. Open circle, closed circle, and triangle represent the sun-exposed side of control fruit, the shaded side of control fruit, and the original shaded side of the treated fruit, respectively. Each point was the mean of four replicates with standard error.

5 F. Ma, L. Cheng / Plant Science 1 (24) SOD activity (1 units) (A) shade sun shade to sun (D) DHAR activity (µmol m -2 s -1 ) 7 (B) (E).5. MDAR activity APX activity -2-2 ( s -1 (µmol m s -1 ) µmol m ) (C) (F) GR activity ( µmol m -2 s -1 ) Proteins (g m -2 ) Fig. 4. Superoxide dismutase, SOD (A), ascorbate peroxidase, APX (B), monodehydroascorbate reductase, MDAR (C), dehydroascorbate reductase, DHAR (D), glutathione reductase, GR (E), and soluble proteins (F) of apple fruit peel in response to a sudden exposure to full sun. Open circle, closed circle, and triangle represent the sun-exposed side of control fruit, the shaded side of control fruit, and the original shaded side of the treated fruit, respectively. Each point was the mean of four replicates with standard error. (Fig. 4A). Exposure of the original shaded side to full sun did not alter SOD activity. The shaded side had lower activities of APX, MDAR, DHAR and GR than the sun-exposed side (Fig. 4B E). APX, MDAR, DHAR and GR activities in the original shaded side increased in response to full sun exposure, reaching similar values of those in the sun-exposed side by the end of the 1-day period. Total protein concentration was lower in the shaded side than in the sun-exposed side (Fig. 4F). Exposure to full sunlight increased the total protein concentration of the original shaded side to a similar level as in the sun-exposed side..5. Soluble antioxidants Total ascorbate pool (Fig. 5A), reduced ascorbate (Fig. 5B), the reduction state of the ascorbate pool (Fig. 5C), total glutathione (Fig. 5D), reduced glutathione (Fig. 5E), and the reduction state of the glutathione pool (Fig. 5F) were lower in the shaded side than in the sun-exposed side. They all increased in the original shaded side in response to full sun exposure, reaching a similar level as in the sun-exposed side within 1 days. 4. Discussion The shaded side of Liberty apple fruit had both lower xanthophyll cycle pool size and conversion state and lower antioxidant enzymes and soluble antioxidants of the ascorbate glutathione cycle than the sun-exposed side (Figs. 5). This is similar to the result previously obtained on two other apple varieties, Gala and Smoothee [11]. When suddenly exposed to full sun, the original shaded side of apple fruit received a much higher incident PFD than it used to recieve, resulting in much more excess absorbed PFD in the peel. Because the shaded peel had a low capacity for photoprotection, the excess absorbed PFD caused a sharp decrease in the maximum PSII efficiency 1 day after full sun exposure (Fig. 1), indicating that photo-oxidative damage occurred in the original shaded peel. However, both the xanthophyll cycle and the ascorbate glutathione cycle were up-regulated in response to full sun exposure (Figs. 5), which counteracted the photooxidation and helped the original shaded side to acclimate to the much higher PFD. Thermal dissipation of the excess absorbed PFD is dependent on the xanthophyll cycle pool size and the conversion of V to Z and A [2,2]. Although we were unable to directly measure non-photochemical quenching, the increase

6 1484 F. Ma, L. Cheng / Plant Science 1 (24) AsA + DAsA (mmol m -2 ) (A) shade sun shade to sun (D) GSH + GSSG (mmol m -2 ). (B) (E) 2. AsA (mmol m -2 ) GSH (mmol m -2 ) AsA (% of AsA + DAsA) (C) (F) GSH (% of GSH + GSSG) Fig. 5. Total ascorbate pool, including reduced ascorbate, AsA and oxidized ascorbate, DAsA (A), reduced ascorbate (B), reduction state of the ascorbate pool (C), total glutathione pool, including reduced glutathione, GSH and oxidized glutathione, GSSG (D), reduced glutathione (E), and reduction state of the glutathione pool (F) of apple fruit peel in response to a sudden exposure to full sun. Open circle, closed circle, and triangle represent the sun-exposed side of control fruit, the shaded side of control fruit, and the original shaded side of the treated fruit, respectively. Each point was the mean of four replicates with standard error. in xanthophyll cycle pool size in the original shaded side in response to full sun exposure indicates that the capacity for thermal dissipation increased to meet the greater need for dissipating excess absorbed PFD. This acclimation response of apple fruit peel is similar to that found in leaves of Cucurbita pepo and Vinca major [1]. The conversion state of xanthophyll cycle in the original shaded side rose sharply to the same level (almost 1%) as that in the sun-exposed side (Fig. B). This indicates that xanthophyll cycle operates at its full capacity to dissipate excess excitation energy and the xanthophyll cycle pool size may become limiting. Z plays an important role in quenching singlet oxygen as well as in thermal dissipation [7,8]. The increase in xanthophyll cycle pool size and conversion state in the original shade side in response to full sun exposure may also indicate an increased capacity for quenching singlet oxygen. Full operation of the xanthophyll cycle in the original shaded side upon exposure to full sun suggests that there was a strong need to dissipate the excess absorbed PFD and the resulting excess electrons. Although we did not measure the flux of direct photoreduction of oxygen, the increase in the down-stream antioxidant enzymes (APX, MDAR, DHAR and GR) of the ascorbate glutathione cycle (Fig. 4B E) is consistent with the idea that direct electron transfer from PSI to oxygen was enhanced. Among the antioxidant enzymes examined, SOD was the only one that did not show any response to full sun exposure (Fig. 4A). This contrasts with the results obtained in C. pepo and V. major, where SOD activity increased significantly when shade leaves were suddenly exposed to full sun [1]. As suggested before [11], the SOD activity of apple fruit peel is very high compared with other species and the SOD-catalyzed reaction may not be a limiting step in detoxifying active oxygen species generated via photoreduction of oxygen in apple fruit peel. The total pool size and reduction state of both AsA and GSH in the original shaded side showed a similar response to full sun exposure as did the antioxidant enzymes in the ascorbate glutathione cycle (Figs. 4B E and 5). The synchronous increases in both soluble antioxidants and antioxidant enzymes in the pathway optimize its total capacity for detoxifying active oxygen species. In addition to detoxifying active oxygen species, the up-regulation of the ascorbate glutathione cycle helps to support the full operation of the xanthophyll cycle by: (1) generating a transthylakoid ph difference [27], which activates violaxanthin de-epoxidase and (2) providing the reduced ascorbate required for the de-epoxidation of violaxanthin to zeaxanthin []. When the total ascorbate pool or the regeneration

7 F. Ma, L. Cheng / Plant Science 1 (24) of reduced ascorbate is limiting, xanthophyll cycle will be compromised as detoxification of hydrogen peroxide by APX competes for the reduced ascorbate [28]. Recently, it has been demonstrated in an Arabidopsis mutant that ascorbate deficiency can limit violaxanthin de-epoxidase activity in vivo, lowering non-photochemical quenching [29]. One of the most obvious responses observed in the original shaded side after being exposed to full sun was the induction of anthocyanin synthesis. This was demonstrated clearly in the reflectance spectrum (Fig. 2B) although we did not chemically determine the anthocyanin content. It has been proposed that anthocyanins may function as an internal light trap to reduce absorption of PFD by the light-harvesting pigments for photosynthesis [,1]. However, the absorption spectrum of anthocyanin is mainly in the green between 5 and nm [,2], which just falls between the absorption spectra of chlorophylls and carotenoids. Because anthocyanins have very low absorption in the blue-orange region, the effect of anthocyanin formation on reducing PFD absorption by chlorophylls and carotenoids would be small. The apparent total absorption of PFD of fruit peel, however, was increased by the formation of anthocyanins, as shown in the decrease of reflectance averaged over 4 7 nm in the original shaded side. By comparing two apple varieties that have different levels of anthocyanin synthesis in response to light exposure and photoinhibitory treatments, Merzlyak and Chivkunova [] suggested that the high resistance to photooxidation observed in the red peel was due to the presence of anthocyanins. However, other photoprotective mechanisms were not examined in the study, which may operate differentially between varieties and between red and green parts of the same fruit as well. The role of anthocyanin in photoprotection of apple fruit, therefore, can only be resolved by specifically altering its synthesis. The current study is focused on both the xanthophyll cycle and the ascorbate glutathione cycle. It should be pointed out, however, that other photoprotective mechanisms in the original shaded side may also have been up-regulated in response to a sudden exposure to full sun to cope with the large amount of excess PFD [], such as the D1 protein repair process [4]. Collectively, upregulation of all these photoprotective mechanisms enabled the PSII reaction centers of the original shaded side to recover from the initial photo-oxidative damage and eventually acclimate to the much higher PFD environment. To conclude, in addition to confirming our previous finding that the sun-exposed peel of apple fruit has both a larger xanthophyll cycle pool size and conversion state and higher levels of antioxidants in the ascorbate glutathione pathway, the current study clearly demonstrated that both xanthophyll cycle and the ascorbate glutathione pathway in the original shaded side are up-regulated in response to a sudden exposure to full sun to minimize the photooxidative damage that the excess absorbed PFD causes on the original shaded peel. Acknowledgements This work was supported in part by Washington Tree Fruit Research Commission. The authors would like to thank Drs. Chris Watkins and Lisong Chen for helpful discussions on antioxidant measurements and Mr. Richard Raba for technical assistance with HPLC. References [1] H.Y. Yamamoto, Biochemistry of violaxanthin cycle in higher plants, Pure Appl. Chem. 51 (1979) [2] B. Demmig-Adams, W.W. 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