Influence of light on ascorbate formation and metabolism in apple fruits

To further understand the regulatory mechanism of light on the formation of ascorbic acid (AsA) in the sink organs of plants, a systematical investigation on AsA levels, activities of two key biosynthsis enzymes and their mRNA expression as well as the recycling was performed in the fruits of apple (Malus domestica Borkh), under different levels of shade. After the whole trees were shaded with the sun-light about 50–55% for 20 days, AsA levels were significantly decreased in fruit peel, flesh and leaves, while mRNA expression levels and activities of l-galactose dehydrogenase (l-GalDH, EC 1.1.1.117) and l-galactono-1,4-lactone dehydrogenase (l-GalLDH, EC 1.3.2.3) as well as activities of recycling enzymes was clearly declined in the leaf and peel but not in the flesh. By shading fruits only for 20 days, AsA levels, relative mRNA levels and activities of l-GalDH and l-GalLDH as well as activities of recycling enzymes all showed obvious decrease in the peel, but not in the flesh. However, their levels in the peel were markedly increased after the full shade was removed and re-exposed these fruits on natural light for 5 days. It is concluded that light affects AsA biosynthesis and recycling in the peel and leaf, but did not in the fresh. Results also suggest that apple fruit is potential to biosynthesize AsA via the l-galactose pathway, and AsA content in the fruits may depend partly on levels of AsA or other photochemistry controlled by light in the leaves.     

The shaded side of apple fruit becomes more sensitive to photoinhibition with fruit development

Developmental changes of photochemical and non-photochemical processes and the antioxidant system in the shaded peel vs the sun-exposed peel of 'Gala' apple and their responses to sudden exposure of high light were determined to understand the susceptibility of the shaded peel to high light damage with fruit development. As fruit developed, actual PSII efficiency of the shaded peel decreased, whereas non-photochemical quenching (mainly the slow component) increased at any given PFD. Photochemical quenching coefficient of the shaded peel decreased at any given PFD with fruit development. As fruit developed, the activity of superoxide dismutase, ascorbate peroxidase and dehydroascorbate reductase and the level of reduced ascorbate and total ascorbate decreased; the activity of monodehydroascorbate reductase and glutathione reductase remained low, whereas catalase activity and the level of reduced glutathione and total glutathione increased in the shaded peel. Exposure to high light (1500 micromol m(-2) s(-1)) for 2 h significantly decreased the maximum quantum efficiency of PSII (F(V)/F(M)) in the shaded peel at each developmental stage, with the decrease being larger with fruit development. The F(V)/F(M) of the sun-exposed peel was also decreased by the high light treatment, but the decrease was much smaller than that in the shaded peel at each developmental stage. We conclude that the shaded peel of apple fruit becomes more sensitive to photoinhibition with fruit development, and this increased sensitivity is apparently related to the decease in the overall capacity for photosynthesis and photoprotection of the shaded peel with fruit development.     

Ascorbic acid formation and profiling of genes expressed in its synthesis and recycling in apple leaves of different ages

Ascorbic acid (AsA), as a unique antioxidant and enzyme cofactor, has multiple roles in plants. However, there is very limited information on the mechanism of AsA accumulation and controlling in leaves. In this study, we determined AsA accumulation levels, analyzed expression patterns of the genes involved in synthesizing via l-galactose pathway and recycling as well as enzyme activities in apple (Malus domestica Borkh) leaves with different age. AsA content was found to increase with leaf development, reaching the highest level in 20-day-old leaves. This level was maintained in mature leaves until the dropping in senescent leaves. Comparing with young and senescent leaves, mature leaves had higher capability for AsA synthesis with high expression levels and activity of l-galactose dehydrogenase and l-galactono-1,4-lactone dehydrogenase. The mRNA expression of genes involved in AsA synthesis also showed highest abundance in 20-day-old leaves, though GDP-mannose-3′,5′-epimerase and l-galactose-1-phosphate phosphatase expression reached the highest levels before 20 days old. These results suggest that AsA accumulation in apple leaves mainly occurs during the transition phase from young to mature leaves with high rates of synthesis and recycling, and that l-galactose-1-phosphate phosphatase could play an important role in regulating AsA biosynthesis via the l-galactose pathway.     

Red 'Anjou' pear has a higher photoprotective capacity than green 'Anjou'

Photoprotective function of anthocyanins along with xanthophyll cycle and antioxidant system in fruit peel was investigated in red 'Anjou' vs green 'Anjou' pear (Pyrus communis) during fruit development and in response to short-term exposure to high light. The sun-exposed peel of red 'Anjou' had higher maximum quantum yield of photosystem II (F(V)/F(M)) than that of green 'Anjou' and both the sun-exposed peel and the shaded peel of red 'Anjou' had smaller decreases in F(V)/F(M) after 2-h high light (photon flux density of 1500 mumol m(-2) s(-1)) treatment than those of green 'Anjou'. At the middle and late developmental stages, the xanthophyll cycle pool size on a chlorophyll basis, the activity of superoxide dismutase, ascorbate peroxidase (APX), monodehydroascorbate reductase (MDAR), dehydroascorbate reductase (DHAR) and glutathione reductase (GR) and the level of reduced ascorbate and total ascorbate pool in the sun-exposed peel were either the same or lower in red 'Anjou' than in green 'Anjou', whereas the xanthophyll cycle pool size on a chlorophyll basis and the activity of APX, catalase, MDAR, DHAR and GR in the shaded peel were higher in red 'Anjou' than in green 'Anjou'. It is concluded that red 'Anjou' has a higher photoprotective capacity in both the sun-exposed peel and the shaded peel than green 'Anjou'. While the higher anthocyanin concentration along with the larger xanthophyll cycle pool size and the higher activity of some antioxidant enzymes may collectively contribute to the higher photoprotective capacity in the shaded peel of red 'Anjou', the higher photoprotective capacity in the sun-exposed peel of red 'Anjou' is mainly attributed to its higher anthocyanin concentration.     

Comparison of thermotolerance of sun-exposed peel and shaded peel of ‘Fuji’ apple

The thermotolerance of the sun-exposed peel and the shaded peel of ‘Fuji’ apple (Malus domestica Borkh.) fruit was evaluated by measuring pigments, chlorophyll a fluorescence transients and O₂ evolution or uptake after exposure to 25, 35, 40, 42, 44, 46 or 48 °C for 30 min in the dark. A major effect of heat stress at 46–48 °C on the chlorophyll a fluorescence transients was the appearance of a very clear K step at 200–300 μs for both peel types. The K step was slightly more pronounced in the sun-exposed peel than in the shaded peel, suggesting that the resistance of oxygen-evolving complex to heat stress is slightly lower in the sun-exposed peel than in the shaded peel. Minimal fluorescence (F_O), relative to the value at 25 °C, increased to a greater extent in the shaded peel than in the sun-exposed peel after exposure to 46–48 °C, but the temperature dependencies of F_O changes were similar for both peel types. Maximum quantum yield of PSII (F_V/F_M) decreased to a similar extent in the sun-exposed peel and the shaded peel as temperature rose from 25 to 44 °C, but the sun-exposed peel reached slightly lower values at 46–48 °C. Correspondingly, gross O₂ evolution rate, relative to that at 25 °C, was also slightly lower in the sun-exposed peel than in the shaded peel at 46–48 °C. In response to heat stress, the ratio of Q_A-reducing reaction centers (RCs) to total RCs and the ratio of Q_B-reducing RCs to Q_A-reducing RCs decreased, but both of them decreased to lower values in the sun-exposed peel than in the shaded peel at 46–48 °C, indicating that the capacity of electron transfer between P₆₈₀⁺ and Q_B via Q_A was damaged to a greater extent in the sun-exposed peel than in the shaded peel. At each given temperature, dark respiration was similar between the two peel types. Overall, it appears that the exposure to higher surface temperature under high light does not make the sun-exposed peel more tolerant of heat stress than the shaded peel of apple fruit.     

Variation and genetic parameters of fruit colour and polyphenol composition in an apple seedling population segregating for red leaf

Red flesh colour is a relatively new target for apple breeding programmes and understanding genetic relationships between this trait and other fruit characters, including polyphenol compounds, is important for both breeders and marketers of new red flesh cultivars. In this study, fruit peel and flesh colours and concentrations of up to 20 individual fruit polyphenols within each tissue were examined in fruit harvested from a 14-family apple seedling population segregating for red and green leaf. Red leaf seedlings always produced red flesh fruit that varied from pale red to complete dark red cortical tissue (type 1 red flesh). Some (20 %) of green leaf seedlings also produced fruit with red flesh, albeit at low intensity (type 2 red flesh). Cyanidin 3-O-galactoside was the dominant anthocyanin in both fruit tissues, with concentrations being 1,900 times higher in the flesh and 2.5 times higher in the peel of fruit from red than from green leaf seedlings. Red leaf seedlings also had 59 % more flesh epicatechin and 17 % less total peel flavonols, but other polyphenols were not associated with leaf colour. Heritability estimates for red flesh colour, flesh cyanidin 3-O-galactoside, flesh and peel catechins were high in red leaf and low in green leaf seedlings. Conversely, estimates for red peel coverage and two peel anthocyanins were higher in green compared to those from red leaf seedlings. Other than these, heritability estimates were high only for dihydrochalcones and hydroxycinnamic acids from each tissue for both leaf colours but low for all other flesh and peel flavan-3-ols, procyanidins and most peel flavonols irrespective of leaf colour. Genetic correlations between polyphenol compounds varied considerably, but were broadly similar for red and green leaf seedlings. Genetic correlations were mostly moderate to high between compounds of the same metabolic group, but low between compounds from different groups. These results are discussed in relation to the genetic control of flesh colour and polyphenol accumulation in apple, as well as to implications for breeding red flesh apples with altered polyphenol composition.     

晚熟脐橙果实发育过程中抗坏血酸含量及相关酶活性的变化

以‘鲍威尔’脐橙为试材, 研究了果实发育期间果皮和果肉中抗坏血酸（AsA）含量及相关酶活性的变化。结果表明, 果皮中总抗坏血酸（T-AsA）和AsA含量显著高于果肉, 且在发育期间T-AsA和AsA的变化趋势一致; 果皮中L-半乳糖内酯脱氢酶（g·LLDH）活性与T-AsA和AsA积累速率的变化趋势基本一致, 呈显著正相关关系, 而在果肉中的变化趋势却不明显; 在发育过程各阶段中果皮的抗坏血酸氧化酶（AAO）、抗坏血酸过氧化物酶（APX）、脱氢抗坏血酸还原酶（DHAR）和单脱氢抗坏血酸还原酶（MDHAR）活性均高于果肉; 相关性分析显示, 果皮中AsA含量积累主要取决于g·LLDH活性, 而果肉中AsA含量水平可能取决于AsA的再生循环系统。     

Patterns of pigment changes in apple fruits during adaptation to high sunlight and sunscald development

Reflectance spectra of four apple (Malus domestica Borkh.) cultivars were studied and chlorophyll, carotenoid, anthocyanin and flavonoid content in sunlit and shaded peel was determined. In all cases sunlit peel accumulated high amounts of phenolics (flavonoid glycosides). Adaptation to strong sunlight of an apple cultivar with limited potential for anthocyanin biosynthesis (Antonovka) was accompanied by a decrease in chlorophyll and a significant increase in total carotenoid content. The increase in carotenoids also took place in sunlit sides of the Zhigulevskoye fruits, accumulating high amounts of anthocyanins, but chlorophyll content in sunlit peel was higher than that in shaded peel. Significant increases in carotenoids and anthocyanins were detected during fruit ripening when chlorophyll content fell below 1.5–1.8 nmol cm^–2. Chlorophyll in sunlit fruit surfaces of both cultivars was considerably more resistant to photobleaching than in shaded (especially of Zhigulevskoye) sides. Induced by sun irradiation, the photoadaptive responses were cultivar-dependent and expressed at different stages of fruit ripening even after storage in darkness. The development of sunscald symptoms in susceptible apple cultivars (Granny Smith and Renet Simirenko) led to a dramatic loss of chlorophylls and carotenoids, which was similar to that observed during artificial photobleaching. The results suggest that apple fruits exhibit a genetically determined strategy of adaptation of their photoprotective pigments to cope with mediated by reactive oxygen species photodynamic activity of chlorophyll under strong solar irradiation. This includes induction of synthesis and accumulation of flavonoids, anthocyanins and carotenoids that could be expressed, if necessary, at different stages of fruit development     

Significance of skin flavonoids for UV-B-protection in apple fruits

An attempt has been made to assess the UV-B-protective capacity of phenolic compounds accumulated in superficial structures of plants using apple fruit as a model. Two apple (Malus domestica Borkh.) cultivars (Braeburn and Granny Smith) differing in response to high fluxes of solar radiation were selected and exposed to increasing doses of UV-B radiation. The extent of UV-B-induced damage to photosystem II of apple skin correlated with its quercetin glycoside (but not anthocyanin) content. Granny Smith apples did not demonstrate a pronounced response to high sunlight in terms of the accumulation of phenolic substances in the skin and exhibited similar patterns of Fo, Fm, and Fv/Fm changes in the course of UV-B irradiation both on sun-exposed and shaded surfaces of a fruit. Unlike Granny Smith, Braeburn fruits were characterized by a significant accumulation of quercetin glycosides in sun-exposed skin, however, shaded skin contained these compounds in similar amounts to those in Granny Smith. Accordingly, photosystem II in sun-exposed skin of Braeburn apples was resistant to high doses of UV-B radiation (up to 97 kJ m-2), whereas the susceptibility of the photosynthetic apparatus in shaded skin of Braeburn to UV-B-induced damage was much higher and similar to that of both sun-exposed and shaded skin of Granny Smith fruits. Anthocyanins, at least in the amounts found in Braeburn, did not show an additional effect in UV-B protection.     

Relationships between fruit exocarp antioxidants in the tomato (Lycopersicon esculentum) high pigment-1 mutant during development

Development-dependent changes in fruit antioxidants were examined in the exocarp (epidermal and hypodermal tissues) of the monogenic recessive tomato (Lycopersicon esculentum L.) mutant high pigment (hp-1) and its wild-type parent 'Rutgers' grown under non-stress conditions in a greenhouse. The hp-1 mutant was chosen for this study because the reportedly higher lycopene and ascorbic acid (AsA) contents of the fruit may alter its tolerance to photooxidative stress. Throughout most of fruit development, reduced AsA concentrations in the exocarp of hp-1 were 1.5 to 2.0 times higher than in 'Rutgers', but total glutathione concentrations were similar in both genotypes. Only in ripe red fruit were reduced AsA and total glutathione concentrations lower in hp-1 than in 'Rutgers'. The redox ratios (reduced : reduced + oxidized) of AsA in hp-1 and 'Rutgers' exocarps were similar and usually > 0.9, however, the redox ratio of glutathione was lower in hp-1 than in 'Rutgers' throughout development. Lycopene concentrations in ripe red fruit were about 5 times higher in hp-1 than in 'Rutgers'. Large increases in the specific enzyme activities of superoxide dismutase (EC 1.15.1.1), ascorbate peroxidase (EC 1.11.1.11), and monodehydroascorbate reductase (MDHAR; EC 1.6.5.4) occurred during fruit development in both genotypes, with an inverse relationship between the activities of these enzymes and chlorophyll content. Glutathione reductase (EC 1.6.4.2) and MDHAR-specific activities were higher in hp-1 than 'Rutgers' only at the later stages of fruit development. Dehydroascorbate reductase (EC 1.8.5.1) activities, however, were usually higher in 'Rugters' than in hp-1. Catalase (CAT, EC 1.11.1.6) activities increased with fruit development until the fruit were orange/light red, when CAT was higher in 'Rutgers' than in hp-1, but then declined in the ripe red fruit of both genotypes. These results suggest that elevated AsA in the exocarp of hp-1 fruit early in fruit development may increase the tolerance of hp-1 fruit to photooxidative injury at that time, but the increasing activities of antioxidant enzymes appear to be developmentally associated with fruit ripening.     

Response of the physiological parameters of mango fruit (transpiration,water relations and antioxidant system) to its light and temperature environment

Depending on the position of the fruit in the tree, mango fruit may be exposed to high temperature and intense light conditions that may lead to metabolic and physiological disorders and affect yield and quality. The present study aimed to determine how mango fruit adapted its functioning in terms of fruit water relations, epicarp characteristics and the antioxidant defence system in peel, to environmental conditions. The effect of contrasted temperature and light conditions was evaluated under natural solar radiation and temperature by comparing well-exposed and shaded fruit at three stages of fruit development. The sun-exposed and shaded peels of the two sides of the well-exposed fruit were also compared.     

秦冠苹果品种对苹果黑星病的组织学抗性研究

为揭示苹果抗病品种秦冠在组织细胞学水平上抗苹果黑星病的特征,本研究采用扫描和透射电镜技术,将苹果黑星菌Venturia inaequalis接种侵染寄主后,系统观察抗病品种秦冠和感病品种嘎啦的叶片组织和细胞结构变化。扫描电镜观察结果表明,黑星菌分生孢子悬浮液接种秦冠和嘎啦叶片48 h后,病菌沿叶脉生长扩展,其菌丝可从叶片气孔或直接侵入。透射电镜观察结果表明,秦冠叶片的角质层厚度明显高于嘎啦,其中秦冠角质层平均厚度为1.75 μm,嘎啦为1.06 μm。透射电镜观察结果表明,黑星菌菌丝在寄主叶肉细胞间扩展,导致嘎啦栅栏组织细胞萎缩,排列松散,叶绿体变形受损,细胞内出现较大淀粉粒和胞内物质外渗流失,并在后期发展成大量细胞坏死;而秦冠虽症状类似,但受损程度明显小于嘎啦。以上结果表明,秦冠在组织细胞学上对苹果黑星病具有抗侵染、抗扩展和延缓病程发展的作用,可作为苹果黑星病抗性育种材料加以利用。     

High Temperature Inhibits Ascorbate Recycling and Light Stimulation of the Ascorbate Pool in Tomato despite Increased Expression of Biosynthesis Genes

Understanding how the fruit microclimate affects ascorbate (AsA) biosynthesis, oxidation and recycling is a great challenge in improving fruit nutritional quality. For this purpose, tomatoes at breaker stage were harvested and placed in controlled environment conditions at different temperatures (12, 17, 23, 27 and 31°C) and irradiance regimes (darkness or 150 µmol m^-2 s^-1). Fruit pericarp tissue was used to assay ascorbate, glutathione, enzymes related to oxidative stress and the AsA/glutathione cycle and follow the expression of genes coding for 5 enzymes of the AsA biosynthesis pathway (GME, VTC2, GPP, L-GalDH, GLDH). The AsA pool size in pericarp tissue was significantly higher under light at temperatures below 27°C. In addition, light promoted glutathione accumulation at low and high temperatures. At 12°C, increased AsA content was correlated with the enhanced expression of all genes of the biosynthesis pathway studied, combined with higher DHAR and MDHAR activities and increased enzymatic activities related to oxidative stress (CAT and APX). In contrast, at 31°C, MDHAR and GR activities were significantly reduced under light indicating that enzymes of the AsA/glutathione cycle may limit AsA recycling and pool size in fruit pericarp, despite enhanced expression of genes coding for AsA biosynthesis enzymes. In conclusion, this study confirms the important role of fruit microclimate in the regulation of fruit pericarp AsA content, as under oxidative conditions (12°C, light) total fruit pericarp AsA content increased up to 71%. Moreover, it reveals that light and temperature interact to regulate both AsA biosynthesis gene expression in tomato fruits and AsA oxidation and recycling.     

Ascorbate biosynthesis during early fruit development is the main reason for its accumulation in kiwi

Background

Ascorbic acid (AsA) is a unique antioxidant as well as an enzyme cofactor. Although it has multiple roles in plants, it is unclear how its accumulation is controlled at the expression level, especially in sink tissues. Kiwifruit (Actinidia) is well-known for its high ascorbate content. Our objective was to determine whether AsA accumulates in the fruits primarily through biosynthesis or because it is imported from the foliage.

Methodology/Principal Findings

We systematically investigated AsA levels, biosynthetic capacity, and mRNA expression of genes involved in AsA biosynthesis in kiwi (A. deliciosa cv. Qinmei). Recycling and AsA localization were also monitored during fruit development and among different tissue types. Over time, the amount of AsA, with its capacity for higher biosynthesis and lower recycling, peaked at 30 days after anthesis (DAA), and then decreased markedly up to 60 DAA before declining more slowly. Expression of key genes showed similar patterns of change, except for L-galactono-1,4-lactone dehydrogenase and L-galactose-1-phosphate phosphatase (GPP). However, GPP had good correlation with the rate of AsA accumulation. The expression of these genes could be detected in phloem of stem as well as petiole of leaf and fruit. Additionally, fruit petioles had greater ascorbate amounts, although that was the site of lowest expression by most genes. Fruit microtubule tissues also had higher AsA. However, exogenous applications of AsA to those petioles did not lead to its transport into fruits, and distribution of ascorbate was cell-specific in the fruits, with more accumulation occurring in larger cells.

Conclusions

These results suggest that AsA biosynthesis in kiwi during early fruit development is the main reason for its accumulation in the fruits. We also postulate here that GPP is a good candidate for regulating AsA biosynthesis whereas GDP-L-galactose-1-phosphate phosphorylase is not.     

Contents of Sugars, Fruit Acids, Amino Acids and Phenolic Compounds of Apple Fruits i n Relation to their Susceptibility to Botryosphaeria ribis

Five apple cultivars showing different levels of susceptibility to Botryosphaeria ribis were used to study the relationship between susceptibility and contents of sugars, fruit acids, amino acids and phenolic compounds in the fruits at various stages of maturity. Immature fruits of all the cultivars tested were completely resistant until 10 July. The fungus grew better on the yellow-coloured cultivars than the red-coloured cultivars. The low amount of reducing sugars and also the high levels of fruit acids, amino acids and total phenols were found in immature apple fruits, irrespective of cultivar. No consistent differences between the cultivars were recognized with regard to the contents of soluble sugars, fruit acids and phenols in fruit flesh during the maturity of fruits. However, only the levels of total soluble amino acids in fruit flesh and of total phenols in fruit peel at late stage of maturity consistently varied between the cultivars. The dark red-coloured, resistant cultivars Miller spur and Starkrimson showed higher contents of amino acids and phenols in fruit tissues than did the yellow-coloured, susceptible cultivars.     

Individual phenolic response and peroxidase activity in peel of differently sun-exposed apples in the period favorable for sunburn occurrence

Extreme weather events like high solar radiation can cause stress in apple fruits (Malus domestica Borkh.). The aim of the study was to make a screening of individual phenols and peroxidase activity in apple peel as a response to sunburn and different sun-exposures in the period when weather conditions are suitable for sunburn occurrence. Apple fruits of ‘Golden Delicious’ and ‘Braeburn’ were sampled. Fruit temperature and color were measured prior HPLC–MS² and peroxidase activity analyses. Sunburned peel was darker and more yellow-red in comparison to healthy peel, which appeared yellow-green. Fruit temperature, total as well as individual flavonols and dihydrochalcones, total hydroxycinnamics and perixodase activity were highest in sunburned peel in comparison with healthy sun-exposed peel, furthermore both were different than shaded sides of both fruits and peel of apples inside the tree crown; moreover in sunburned peel dihydrochalcones were determined for the first time. Chlorogenic acid was up to 2.5 times higher, 3-hydroxy-phloretin-2′-O-xyloglucoside was up to 10 times higher and quercetin-3-galactoside was up to 33 times higher in sunburned peel, comparing to shaded sided peels. Flavanols did not show a distinct pattern. A deeper insight in phenolic response against environmental stress caused by high solar radiation and high air temperatures has been made.