Flavedo Of Fruit Research Articles

Hot water and molybdenum dips: The case of antioxidant assays in lemon flavedo during cold storage

Antioxidants are part of the plant’s defense system, protecting tissue against oxidative stress. In citrus fruit, oxidative stress can occur during extended cold storage ultimately result in chilling injury. Citrus fruit contain certain rind antioxidants which occur in different forms (lipophilic and hydrophilic). Previous studies demonstrated a synergistic relationship between hot water dips (HWD) and molybdenum dips (Mo) in mitigating chilling injury in citrus fruit. Therefore, the aim of this study was to investigate the potential of HWD and Mo to alleviate chilling injury by enhancing antioxidant capacity in lemon flavedo of fruit stored at -0.5°C. Fruit from different sources, commonly displaying chilling injury and other seemingly resistant to the development of chilling symptoms, were preconditioned for 2 min with HWD (47 or 53°C) and thereafter soaked in 1 or 10 µM Na2MoO4 solution for 30 min. Fruit were subsequently stored at -0.5°C for 7 or 28 days, moved to ambient temperature for a week and then evaluated for chilling injury symptoms. Chilling susceptible lemon fruit showed low Trolox equivalent total antioxidant capacity (TEAC), phenolics, and flavonoids concentration and high lipid peroxidation. Hot water dips 53°C, as well as 1 and 10 µM Mo plus HWD 53°C treatments mitigated chilling injury by enhancing total antioxidant capacity, and total phenolics and flavonoids concentrations in the flavedo. However, the ability of these treatments to alter lipophilic and hydrophilic antioxidants was fruit source dependent. Key words: Lemon (Citrus limon), chilling injury, antioxidant assays, hot water dips, molybdenum, cold storage.

Canopy Position Affects Pigment Expression and Accumulation of Flavedo Carbohydrates of ‘Nules Clementine’ Mandarin Fruit, Thereby Affecting Rind Condition

Because photosynthesis provides the required carbohydrates for fruit development and respiration releases the stored energy from these carbon compounds, interalia during postharvest storage, it is therefore important that fruit tissues have an adequate carbohydrate concentration at the start of the postharvest period to ensure optimal storage life. In addition to photosynthate supply from leaves, the chlorophyll-containing flavedo of citrus (Citrus sp.) fruit (outer, colored part of the rind) has the ability to fix CO2 through its own photosynthetic system. In this experiment, spanning three seasons, the three main sugars (sucrose, glucose, and fructose) were quantified in the flavedo of ‘Nules Clementine’ mandarin (Citrus reticulata) fruit during Stages II and III of fruit development. Flavedo was sampled from fruit borne on the inside (low light intensity) or outside (high light intensity) of the tree’s canopy. In one season, the photosynthetic and respiration rates of fruit borne in the two canopy positions were measured pre- and post-color break (March and April, respectively). Sucrose concentration increased constantly from initial sampling in February until harvest (May), whereas glucose and fructose concentrations increased significantly only during the last month of fruit development. The flavedo of inside fruit, developing under low-light conditions, was less well colored (higher hue angle) and had a lower sugar concentration compared with outside fruit developing under conditions of high light levels. This response could be attributed to the higher pigment concentration leading to a higher photosynthetic rate as well as greater sink strength of the outside fruit. The inside fruit had an increased susceptibility to the progressive postharvest physiological disorder, rind breakdown. The lower carbohydrate and pigment concentrations of the rind from fruit borne inside the canopy compared with those from the outside of the canopy could be indicative of a weaker rind condition at the time of harvest.

Characterization, Purification of Poncirin from Edible Citrus Ougan (Citrus reticulate cv. Suavissima) and Its Growth Inhibitory Effect on Human Gastric Cancer Cells SGC-7901

Poncirin is a bitter flavanone glycoside with various biological activities. Poncirin was isolated from four different tissues (flavedo, albedo, segment membrane, and juice sac) of Ougan fruit (Citrus reticulate cv. Suavissima). The highest content of poncirin was found in the albedo of Ougan fruit (1.37 mg/g DW). High speed counter-current chromatography (HSCCC) combined with D101 resin chromatography was utilized for the separation and purification of poncirin from the albedo of Ougan fruit. After this two-step purification, poncirin purity increased from 0.14% to 96.56%. The chemical structure of the purified poncirin was identified by both HPLC-PDA and LC-MS. Poncirin showed a significant in vitro inhibitory effect on the growth of the human gastric cancer cells, SGC-7901, in a dose-dependent manner. Thus, poncirin from Ougan fruit, may be beneficial for gastric cancer prevention. The purification method demonstrated here will be useful for further studies on the pharmacological mechanism of poncirin activity, as well as for guiding the consumption of Ougan fruit.

Modification of Carotenoid Levels by Abscission Agents and Expression of Carotenoid Biosynthetic Genes in ‘Valencia’ Sweet Orange

The effect of 5-chloro-3-methyl-4-nitro-1H-pyrazole (CMNP) and ethephon on peel color, flavedo carotenoid gene expression, and carotenoid accumulation was investigated in mature 'Valencia' orange ( Citrus sinensis L. Osbeck) fruit flavedo at three maturation stages. Abscission agent application altered peel color. CMNP was more effective than ethephon in promoting green-to-red (a) and blue-to-yellow (b) color at the middle and late maturation stages and total carotenoid changes at all maturation stages. Altered flow of carotenoid precursors during maturation due to abscission agents was suggested by changes in phytoene desaturase (Pds) and ζ-carotene desaturase (Zds) gene expression. However, each abscission agent affected downstream expression differentially. Ethephon application increased β-carotene hydroxilase (β-Chx) transcript accumulation 12-fold as maturation advanced from the early to middle and late stages. CMNP markedly increased β- and ε-lycopene cyclase (Lcy) transcript accumulation 45- and 15-fold, respectively, at midmaturation. Patterns of carotenoid accumulation in flavedo were supported in part by gene expression changes. CMNP caused greater accumulation of total flavedo carotenoids at all maturation stages when compared with ethephon or controls. In general, CMNP treatment increased total red carotenoids more than ethephon or the control but decreased total yellow carotenoids at each maturation stage. In control fruit flavedo, total red carotenoids increased and yellow carotenoids decreased as maturation progressed. Trends in total red carotenoids during maturation were consistent with measured a values. Changes in carotenoid accumulation and expression patterns in flavedo suggest that regulation of carotenoid accumulation is under transcriptional, translational, and post-translational control.

Effect of Naringin Radical Formed by Peroxidase on Chlorophyll Degradation during Storage of Citrus nagato-yuzukichi Fruit

Free radical formation from flavonoids by peroxidase action and changes in flavonoid levels and peroxidase activity during storage were determined to clarify the involvement of naringin radical in chlorophyll (Chl) degradation in stored green Nagato-yuzukichi (Citrus nagato-yuzukichi hort. ex Y. Tanaka) fruit. Chl a was degraded by peroxidase-hydrogen peroxide system not in the presence of hesperidin but with naringin. However, radical formation of both naringin and hesperidin, which were the main flavonoids in the flavedo of Nagato-yuzukichi fruit, with peroxidase-mediated oxidation was found by electron spin resonance analysis. The naringin radical, which was formed by a radical forming reagent, potassium ferricyanide, was related to the formation of 132-hydroxychlorophyll (OHChl) with Chl a degradation, whereas the hesperidin radical hardly affected OHChl a formation. In addition, superoxide dismutase did not inhibit Chl a degradation by naringin radical formed by potassium ferricyanide. In Nagato-yuzukichi fruit, Chl-degrading peroxidase activity increased considerably during storage at 20°C, with a peak value on day 6 of storage in accordance with decrease in Chl content, after which it decreased. The contents of naringin and hesperidin, especially the former, decreased during storage at 20°C, but afterwards increased slightly. These findings suggest that the naringin radical formed by peroxidase could be partly involved in Chl degradation during storage in Nagato-yuzukichi fruit.

Effect of Blue and Red LED Light Irradiation on β-Cryptoxanthin Accumulation in the Flavedo of Citrus Fruits

β-Cryptoxanthin (β-cry), an antioxidant abundant in citrus fruits, plays an important role in the prevention and treatment of certain diseases, especially cancers. In the present study, to increase the content of β-cry in citrus flavedo, the effects of blue (470 nm) and red (660 nm) light-emitting diode (LED) lights on the accumulation of carotenoids and expression of genes related to carotenoid biosynthesis were investigated in the flavedo of Satsuma mandarin. The results showed that accumulation of β-cry was induced by red light, while it was not affected by blue light. The accumulation of β-cry under red light was attributed to simultaneous increases in the expression of CitPSY, CitPDS, CitZDS, CitLCYb1, CitLCYb2, CitHYb, and CitZEP. The results presented herein might provide new strategies to enhance the commercial and nutritional value of citrus fruits.

Fruiting position during development of ‘Nules Clementine’ mandarin affects the concentration of K, Mg and Ca in the flavedo

Adequate K, Mg and Ca supply is important to develop well-structured and functional cell walls and membranes in fruit, and insufficient levels or imbalances of these minerals are known to be involved in various postharvest disorders. Microclimatic variation exists in the ‘Nules Clementine’ mandarin tree canopy and results in lower photosynthetically active radiation (PAR) levels and temperature as well as a higher humidity inside the tree canopy. The aim of this experiment was to determine the impact of this variation in microclimate on accumulation patterns of K, Ca and Mg in the flavedo of the fruit rind during stages II and III of fruit development. Fruit mass, dimensions, rind colour development and mineral composition of the flavedo were measured to describe the condition of fruit borne on the outside and inside of the tree's canopy. The data revealed that canopy position influenced mineral nutrient accumulation patterns in the flavedo. Outside fruit flavedo accumulated significantly higher concentrations of Ca and Mg in all three seasons (2005–2007). In contrast, inside fruit flavedo (shaded fruit) accumulated significantly higher levels of K compared with outside fruit flavedo. The accumulation of K and Ca differed from that of kiwifruit and apple in that Ca concentration increased and K decreased towards maturity. These results suggest that xylem, as in citrus leaves, is the main vasculature supply conduit to the citrus fruit flavedo for mineral nutrients. The reduction of transpiration potential by lower temperatures and higher humidity inside the canopy could be responsible for the reduced accumulation of Ca and Mg. The high K concentration of inside fruit flavedo is suggested to be a stress response, due to the low light levels, to maintain osmotic potential in the shaded rind tissue, and this imbalance could possibly lead to a reduction in rind condition, which manifests through rind breakdown symptom development.

Effects of low UV-B doses on the accumulation of UV-B absorbing compounds and total phenolics and carbohydrate metabolism in the peel of harvested lemons

This paper examined the peel (albedo and flavedo) of postharvest lemon fruits after UV-B exposure in order to analyze relationships between soluble carbohydrate metabolism and secondary metabolite accumulation. Lemons ( Citrus limon, cv. Limoneira 8A) were harvested in winter months (June to August), treated with 0.43 W m −2 (22 kJ m −2 d −1 UV-BBE) of UV-B radiation during 0 (control), 0.5, 1.0, 2.0, 3.0, and 5.0 min, and then stored at 25 °C for 24 h. Peel samples from irradiated areas were obtained with a razor blade and frozen in liquid nitrogen until use for measurements. Data obtained showed that 2 and 3 min of UV-B exposure effectively increased the level of UV-B absorbing compounds and total phenolics in flavedo without causing visual alterations of the peel colour as compared with non-irradiated lemons. By contrast, there were no significant changes in albedo secondary metabolite accumulation. The amount of secondary metabolites was depending upon UV-B time–dose. Exposure over 3.0 min did not further improve the accumulation of UV-B absorbing and phenolic compounds. Soluble sugars (sucrose, glucose and fructose) also accumulated in the lemon peel after UV-B exposure, but the distribution patterns were different. After 3 min time–dose, sucrose and hexoses increased in flavedo, whereas in albedo only increased the sucrose and glucose. This effect was related to UVB-induced changes in the activity of sucrose-hydrolyzing and sucrose-synthesizing enzymes: soluble and cell-bound invertase, sucrose synthase (SS) and sucrose phosphate synthase (SPS). Data indicate that lemon peel retains the capacity to modify the enzyme activity of sucrose metabolism in response to UV-B exposure. Our results also suggest that the exposure of postharvest lemons to low supplemental UV-B doses produces changes in the carbon allocation of peel tissues including synthesis, but probably not only limited to them, of UV-B absorbing and phenolic compounds.

Proteome from Lemon Fruit Flavedo Reveals That This Tissue Produces High Amounts of the Cit s1 Germin-like Isoforms

A multistep procedure has been developed and applied to extract and purify proteins from lemon fruit flavedo. 2DE, LC-ESI-MS/MS, and bioinformatics were used to detect the high abundance of the germin-like glycoprotein Cit s1, a powerful allergen in humans. Peptide alignments against Citrus EST repositories gave the best scores with the C. sinensis cDNA (gi|188354270/EY710037), annotated as unknown sweet orange fruit protein; additional BLAST of peptides against NCBI databases gave high sequence identities with sequence of orange Cit s1 (gi|52782810/P84159), suggesting that the unknown sweet orange fruit protein is consistent with the Cit s1 protein. Peptides of Cit s1 were detected in 17 spots ranging from 120 to 20 kDa, pointing out that in the flavedo of lemon the Cit s1 may be expressed as several isoforms of which the 120 kDa isoform is the largest monomer and the 20 kDa is the smallest one. This finding adds information about the features of Cit s1, because it has been previously reported as a unique monomeric glycoprotein of 24 kDa.

Preharvest Foliar Sprays of Prohexadione–calcium, a Gibberellin-biosynthesis Inhibitor, Induce Chlorophyll Degradation and Carotenoid Synthesis in Citrus Rinds

Rind color is an important cosmetic preference of consumers when purchasing citrus fruit. As citrus fruit mature, a decrease in chlorophyll concentration unmasks the presence of carotenoid pigments followed by further synthesis of carotenoids, resulting in the first appearance of the characteristic orange color of mandarins and sweet oranges. Factors contributing to invigorating growing conditions are antagonistic to optimal rind color development and tree vegetative vigor as well as high gibberellin and cytokinin levels are also thought to adversely affect rind color. Thus, a method to increase preharvest rind color by moderating vegetative vigor using a growth retardant was investigated. Prohexadione–calcium (ProCa; Regalis®), a gibberellin-biosynthesis inhibitor with growth retardant activity, was applied to ‘Nules Clementine’ mandarin (Citrus reticulata Blanco), ‘Navelina Navel’ orange [C. sinensis (L.) Osbeck], and ‘Eureka’ lemon [C. limon (L.) Burm. f.] during the 2005 and 2006 seasons at 200 and 400 mg·L−1 a.i. Rind color rating, colorimeter measurements, and pigment analyses were conducted directly after harvest, after ethylene degreening, and 3 weeks after cold storage. In the 2005 season, ProCa significantly increased rind color of ‘Nules Clementine’ mandarin and ‘Navelina Navel’ orange directly after harvest and after ethylene degreening by decreasing chlorophyll and increasing carotenoid concentrations in the flavedo of fruit but did not affect the pigment concentration of ‘Eureka’ lemon despite an improvement in rind color rating. After cold storage, however, rind color was not significantly different among treatments. In the 2006 season, rind color was significantly increased directly after harvest, and chlorophyll degradation together with carotenoid synthesis of all Citrus spp. tested were stimulated by the late 400 mg·L−1 ProCa application. Therefore, foliar spray application of ProCa at a concentration of 400 mg·L−1 applied 6 plus 3 weeks before anticipated harvest has the potential to increase preharvest rind color of early-maturing citrus cultivars as a result of increased carotenoid-to-chlorophyll ratio. This treatment provides a novel approach to manipulate chlorophyll degradation and carotenoid synthesis in citrus fruit, and these results support the hypothesis that there may be an inverse relationship between vegetative vigor and rind color development of citrus fruit. Therefore, by moderating vegetative vigor through the use of growth retardants, rind color of citrus fruit can be enhanced.

Regulation of carotenoid biosynthesis during fruit maturation in the red-fleshed orange mutant Cara Cara

Cara Cara is a spontaneous bud mutation of Navel orange ( Citrus. sinensis L. Osbeck) characterized by developing fruits with a pulp of bright red coloration due to the presence of lycopene. Peel of mutant fruits is however orange and indistinguishable from its parental. To elucidate the basis of lycopene accumulation in Cara Cara, we analyzed carotenoid profile and expression of three isoprenoid and nine carotenoid genes in flavedo and pulp of Cara Cara and Navel fruits throughout development and maturation. The pulp of the mutant accumulated high amounts of lycopene, but also phytoene and phytofluene, from early developmental stages. The peel of Cara Cara also accumulated phytoene and phytofluene. The expression of isoprenoid genes and of carotenoid biosynthetic genes downstream PDS (phytoene desaturase) was higher in the pulp of Cara Cara than in Navel. Not important differences in the expression of these genes were observed between the peel of both oranges. Moreover, the content of the plant hormone ABA (abscisic acid) was lower in the pulp of Cara Cara, but the expression of two genes involved in its biosynthesis was higher. The results suggest that an altered carotenoid composition may conduct to a positive feedback regulatory mechanism of carotenoid biosynthesis in citrus fruits. Increased levels of isoprenoid precursors in the mutant that could be channeled to carotenoid biosynthesis may be related to the red-fleshed phenotype of Cara Cara.

Extraction of valuable compounds from the flavedo of Citrus junos using subcritical water

Pectin contained in the flavedo of citrus fruits was sequentially extracted with water, a chelating agent, and hydrochloric acid. The amount of pectin obtained was quantified as the amount of galacturonic acid according to the dimethylphenol method and found to be 21%. Next, pectin was separated from the flavedo of Citrus junos with hot compressed water (HCW) using a semi-continuous flow reactor. HCW was continuously delivered into the reactor while gradually increasing the temperature from 60 to 160 °C at 20 MPa with flow rates of 2.1, 3.5, and 7.0 mL/min. The extract from the reactor was cooled and 7-mL fractions were collected. From the analysis of each fraction, it was found that pectin was rapidly extracted when the temperature of the reactor reached 160 °C. Yield of the pectin reached as high as 80%. From cation analysis, a large amount of potassium was detected in the leading extraction peak.

Analysis of Alternariol and Alternariol Monomethyl Ether on Flavedo and Albedo Tissues of Tangerines (Citrus reticulata) with Symptoms of Alternaria Brown Spot

A method was developed for the quantification of alternariol and alternariol monomethyl ether on tangerines with and without symptoms of Alternaria brown spot disease. The method employs solid-phase extraction for cleanup, followed by high-performance liquid chromatography coupled with tandem mass spectrometry (HPLC-MS/MS) for detection. This method was validated on flavedo (exocarp or epicarp, exterior yellow peel) and on albedo tissue (mesocarp, interior white peel). An excellent linearity over a range of 0.50-20.0 mg/kg was achieved, with r2 >or= 0.997. The limits of detection (LOD) and quantification (LOQ) were fewer than 0.13 and 0.50 microg/kg, respectively. The relative standard deviations (RSDs) were <or=14.4% during the validation. The levels of these mycotoxins on flavedo of fruits with symptoms vary from 2.54 +/- 0.24 to 17.40 +/- 1.05 mug/kg. Surprisingly, neither alternariol nor altenariol monomethyl ether was detected on albedo tissues, suggesting that flavedo works as a barrier for such substances.

Ethylene-induced tolerance to non-chilling peel pitting as related to phenolic metabolism and lignin content in ‘Navelate’ fruit

The involvement of phenylpropanoid metabolism in the beneficial effect of ethylene conditioning in reducing postharvest non-chilling peel pitting occurring in ‘Navelate’ orange fruit has been investigated. We examined changes in the activities of the enzymes phenylalanine ammonia-lyase (PAL; EC 4.3.1.5), soluble and ionically bound peroxidase (POD, EC 1.11.1.7) and on phenolic and lignin contents in the flavedo and albedo tissues of fruit stored in air at 22 °C and constant high (90–95%) RH (control fruit), and in fruit conditioned for 4 days with 2 or 10 μL L −1 ethylene at 22 °C and 90–95% RH, and then transferred to air at the same temperature and RH. Non-chilling peel pitting was visible in control fruit after 7 days and sharply increased for up to 14 days, to remain nearly constant thereafter; while conditioning the fruit with both ethylene concentrations considerably reduced it. Soluble and cell wall-related POD activities decreased, and PAL transiently increased, in the albedo and flavedo of control fruit with the appearance of damage. Phenolic and lignin contents slightly increased in the flavedo, while in the albedo, which had a lower PAL activity than the flavedo, phenolic contents barely changed and lignin decreased. Thus, the activation of PAL in both tissues and the slight rise in lignin and phenols in the flavedo may reflect a demand for phenylpropanoid products to reduce non-chilling peel pitting. Furthermore, the decline in POD in both tissues and also the lack of the ability of the albedo to increase phenolic and lignin contents might be related to the low ability of air-treated ‘Navelate’ fruit to overcome this physiological disorder. Fruit conditioned with 10 μL L −1 ethylene maintained higher phenol and lignin levels up to 14 days and higher soluble and ionically bound POD levels up to 21 days in the flavedo, while applying 2 μL L −1 ethylene was less effective. In the albedo, both ethylene concentrations activated soluble POD after transfer of fruit to air and PAL also, though to a lower extent than in the flavedo. Such an increase had a low impact on the phenolic content. Likewise, both ethylene treatments delayed the decline in lignin in the albedo, but the most important differences between control and ethylene-treated fruit occurred when peel damage was very evident in control fruit. The overall results indicate, therefore, that phenolic metabolism may be required for building protecting barriers that would help ‘Navelate’ fruit to reduce non-chilling peel pitting, although additional defense mechanisms which would assist in reducing non-chilling peel pitting in ‘Navelate’ fruit appear to be induced by ethylene pretreatment.

Ethylene modulates transcription of the phosphoenolpyruvate carboxykinase gene in grapefruit flavedo

SummaryThe role of ethylene in citrus fruit physiology is poorly understood. Gene expression studies with ethylene and ethylene inhibitors suggest a regulatory role for the low basal levels of endogenous ethylene in citrus fruits; however, only a few ethylene-modulated genes have been reported. 1-Methylcyclopropene (1-MCP), an ethylene antagonist that inhibits ethylene signal transduction by preventing ethylene from binding to its receptors, is a useful tool to further identify genes associated with the regulatory roles of ethylene. We determined the effects of ethylene and 1-MCP on gene expression in grapefruit flavedo. Total RNA from fruit treated with air, 5 µl l–l ethylene, or 0.3 µl l–l 1-MCP for 48 h was used in differential-display reverse transcription-polymerase chain reaction (DD-RT-PCR) analysis. Our hypothesis was that genes whose expression is induced by ethylene would be up-regulated in response to ethylene, whereas genes whose expression is inhibited by ethylene would be up-regulated in response to 1-MCP. A 440 bp cDNA, induced by ethylene, was used as a probe to screen a cDNA library of flavedo, and obtain a clone with a deduced amino acid sequence 88% similar to the enzyme phosphoenolpyruvate carboxykinase (PEPCK, EC 4.1.49) from both Arabidopsis and Flaveria trinervia. We designated this gene CpPEPCK (Citrus paradisi phosphoenolpyruvate carboxykinase). Northern blot and RT-PCR analyses revealed increased expression of CpPEPCK in grapefruit flavedo after exposure to ethylene, and with increased exposure time. In contrast, treatment with 1-MCP inhibited the accumulation of CpPEPCK transcripts in the same tissue. Although CpPEPCK is expressed in leaves, roots, and petals, higher transcript abundance was found in the flavedo of ethylene-treated fruit than in other tissues. This study provides additional evidence for a role for ethylene in citrus fruit physiology.

Comparative Study of Scoparone Accumulation in Various Citrus Species after Inoculation with Gray Mold

Gray mold (Botrytis cinerea Persoon) induced the accumulation of scoparone (6,7-dimethoxycoumarin) in the flavedo of fruit in all citrus species used in this study. The time-course of the accumulation of this compound was species dependent. In the fruit of ‘Kiyomi’ and Meiwa kumquat, scoparone accumulated gradually over several days after inoculation. No accumulation occurred in the non-inoculated citrus fruit. In several citrus species, including Tankan, ‘Seminole’, and ‘Ellendale’, Scoparone concentration increased up to 4 days after treatment. In ‘Murcott’, ‘Triumph’, and Yuzu, the maximum concentration was observed within 7 days of treatment. On the other hand, in ‘Imamura-unshu’, ‘Kawano Natsudaidai’, Ponkan, ‘Morita Navel’, Kinukawa, ‘Eureka’ lemon, kumquats, calamondin, and citrangequat, the increase in scoparone was much lower. These results suggest that scoparone is a phytoalexin induced by gray mold, accumulating in a broad range of citrus species.

Spatial study of antioxidant enzymes, peroxidase and phenylalanine ammonia-lyase in the citrus fruit– Penicilliumdigitatum interaction

Changes in the activities of the antioxidant enzymes, superoxide dismutase (SOD; EC 1.15.1.1), catalase (CAT; EC 1.11.1.6), ascorbate peroxidase (APX; EC 1.11.1.11) and glutathione reductase (GR; EC 1.6.4.2), as well as in soluble and insoluble peroxidase (POD; EC 1.11.1.7) and in phenylalanine ammonia-lyase (PAL; EC 4.3.1.5) activity and Pal transcript levels in flavedo and albedo tissues of citrus fruit infected with Penicillium digitatum have been examined. Specific probes corresponding to a fragment of the large subunit of the nuclear rRNA from both organisms have been cloned and used to determine the advance of the fungus through the fruit. The activities of all the enzymes were higher in non-infected areas of the flavedo than in the albedo, a fact that may be related to the higher resistance of the flavedo to infection by the pathogen. In both tissues, the activity of the antioxidant enzymes decreased at different rates with the advance of the fungus, except for CAT and soluble POD activities that increased in the area of the flavedo completely colonized by the fungus. On the other hand, the flavedo, but not the albedo, was able to respond to pathogen attack by increasing insoluble POD and PAL activities and Pal transcript levels. However, discrepancies between PAL activity and mRNA levels suggest the ability of P. digitatum to suppress this defense response. Thus, the present study shows that in the interaction between citrus– P. digitatum both host defense responses and pathogen virulence mechanisms are taking place.

Physiological responses of leaves, tree growth and fruit yield of grapefruit trees under reflective shade screens

Understanding the influence of reductions in solar radiation on crop physiology, growth and yield is important because of the widespread practice of protected cultivation under various light reducing covers. Parts of a commercial bearing grapefruit orchard ( Citrus paradisi L.) were covered with aluminized reflective screens during three consecutive summers in southern Israel. Screens were installed shortly after fruit set and removed a week or 2 before harvest. Screens were of two types, 30 and 60% shade, erected horizontally over the rows. Shading reduced mid-day direct radiation reaching the trees and reduced exposed leaf temperatures, but increased their net CO 2 uptake ( A) and conductance ( g l). The increased g l was reflected by decreases in carbon isotope ratios ( δ 13C) of leaves from under both shades and of fruit flavedo from the 60% shade. Final leaf area index (LAI), determined by gap fraction inversion, was 10% higher in 30% shade than in the control and canopy volume increased similarly. Trunk cross-section, total yield and size distribution of fruit were little affected by shade. During the second year, however, early shading presumably induced more than normal fruit abscission, leading to fewer but larger fruit. Fruit in the middle of the canopy of shaded trees were consistently heavier and also larger in the first 2 years, but peel width was not affected by shade. Juice content was reduced in the second and third years, but the ratio of sugar to acid was not affected. Mid-day leaf water potentials in August and September under 60% shade increased by 13%. Shading did not significantly reduce fruit yield.

PHENYLALANINE AMMONIA-LYASE AND ACCUMULATION OF PHENOLIC COMPOUNDS IN THE ETHYLENE-INDUCED TOLERANCE TO RINDSTAINING IN ´NAVELATE´ FRUIT

The orange fruit cultivar 'Navelate' is prone to develop postharvest rindstaining (RS) during storage at non-chilling temperatures. Ethylene has been shown to protect citrus fruit against RS but the mechanisms associated with the ethylene-induced tolerance to this disorder are unknown. In this study we have examined the effect of applying ethylene, before or during holding of 'Navelate' fruit under postharvest conditions inducing RS (22°C, 90-95% RH), on changes in the activity of the enzyme phenylalanine ammonia-lyase (PAL), total phenolic content and fruit quality. PAL activity increased with the development of damage in the albedo and flavedo of fruit maintained under air. Exogenous ethylene treatments, which efficiently reduced RS incidence, increased PAL activity as well as the levels of total phenolic content in the flavedo and in the pulp. However, these levels were not maintained after prolonged storage, either in fruit kept continuously under ethylene or in those transferred to air. The global results indicate that the induction of the enzyme PAL and of subsequent phenylpropanoid compounds may play a role in reducing the development of RS symptoms and might be a part of the mechanisms involved in the beneficial effect of ethylene in protecting citrus fruit against this physiological disorder.

Biochemical and Molecular Analysis of Carotenoid Biosynthesis in Flavedo of Orange (Citrus sinensis L.) during Fruit Development and Maturation

Changes in carotenoid content and composition and expression of carotenoid biosynthetic genes were analyzed in the flavedo of sweet orange (Citrus sinensis L. Osbeck, cv. Navelate) fruit during development and maturation. Lutein and all-E-violaxanthin were the major carotenoids in chloroplast-containing tissues. During fruit coloration, phytoene, beta-cryptoxanthin, zeaxanthin, and mainly (9Z)-violaxanthin progressively accumulated, and a large proportion of apocarotenoids was also found in the flavedo of full-colored fruits. We have cloned partial and full-length cDNAs corresponding to genes involved in early condensation and desaturase reactions [phytoene synthase (PSY), phytoene desaturase (PDS), and zeta-carotene desaturase (ZDS)], coupled redox reaction (plastid terminal oxidase), cyclizations [beta-lycopene cyclase (beta-LCY) and epsilon-lycopene cyclase (epsilon-LCY)], hydroxylation [beta-carotene hydroxylase (beta-CHX)], and epoxidation [zeaxanthin epoxidase (ZEP)] and analyzed their mRNA accumulation in the flavedo of fruits during development and ripening as compared with those of leaves. Collectively, the results indicated that PDS gene expression correlated with carotenoid content in developing fruit and that up-regulation of PSY and ZDS genes at the onset of fruit coloration would enhance the production of linear carotenes and the flux into the pathway. The shift from the beta,epsilon-branch to the beta,beta-branch of the pathway that originates the changes in carotenoid composition during fruit coloration may be explained by a down-regulation of epsilon-LCY and by the increase of the beta-CHX transcript.