Photo-oxidative damage in the ripening tomato fruit: Protective role of superoxide dismutase

Factors relating to photo-oxidative damage in tomatoes were investigated during maturation of the fruit and upon induction of sunscald. Superoxide dismutase (SOD) activity passed through a minimum at the mature-green and breaker stages of ripening and availability of zinc and copper did not appear to be a limiting factor in the synthesis of the enzyme. Iron levels were maximal and total carotenoid concentrations were lowest during the same mature-green and breaker stages of maturation, while chlorophyll was starting to decrease but was still present in large amounts. Peroxidase activity decreased steadily during ripening. Artificial induction of tolerance to photodynamic damage by controlled heat treatment was accompanied by an increase in SOD activity, while carotenoid levels and peroxidase activity did not change. These findings support the thesis that the previously reported susceptibility of tomatoes to photodynamic damage, i.e. sunscald, during the mature-green and breaker stages of maturation is related to enhanced formation of superoxide ions, at a time when chloroplast structure begins to break down. SOD, by scavenging the superoxide, appears to supplement the protective action of carotenoids against photo-oxidative injury.     

Soperoxide dismutase activity in ripening cucumber and pepper fruit

Superoxide dismutase (SOD, EC 1.15.1.1) was identified from ripening cucumber ( Cucumis sativus L.) and pepper( Capsicum annuum L.) fruits, Gel filtration revealed the presence of two major peaks of SOD with molecular masses of about 32 000 and 40 000. Peroxidase activity was observed mainly in material with a molecular mass above 70 000. SOD activity was inhibited by cyanide but not by chloroform-ethanol, indicating a cupro-zinc enzyme. SOD activity levels were high in immature-green fruits of both species, declining in cucumbers to a minimum during the mature-green stage and in peppers during the mature-green and breaker stages. SOD levels increased again until peppers became orange and cucumbers turned yellow, and finally decreased with ripening. The possible connection between SOD activity and tolerance to sunscald is discussed.     

Effect of the Calcium on Polygalacturonase (PG) Activity and Synthesis at Different Ripening Stages of Tomato Fruits

It has been reported that PG is a key enzyme related to the tomato fruit ripening and that the application of calcium can dramatically decrease the PG activity and delay the ripening of fruits. In this paper the effects of calcium treament at various ripening stages on the transformation of absorbed calcium, PG activity and PG synthesis in tomato fruits were studicd. According to the analysis of calcium by atomic absorption spectroscopy, it was shown that the soluble and total calcium contents in pericarp of fruits treated with calcium at mature-green stage were increased significantly, and that more soluble calcium was transformed into bound calcium. Both the absorption and transformation of calcium decreased in fruits treated with calcium at later stage of ripening. The inhibition of calcium on PG activity was most effective by treatment at mature-green stage, but less effective at later stage of ripening. One reason for the decrease of calcium inhibition was probably due to the decline of calcium absorption as fruit ripening. The polyacrylamide gel electrophoresis of PG showed that PG with a molecular weight of 46.7 kD was absent in mature-green fruits, and PG synthesis occurred only at the later stage of ripening. It seems that the earlier the treatment was done the more effective of the calcium inhibition of PG synthesis. Based on the above results, it was concluded that the PG plays a major role in ripening and senescence of tomato fruits, and both PG synthesis and its activity were inhibited by calcium. In order to delay the ripening and senescence of tomato fruits, the treatment with calcium should be done at mature-green stage.     

钙对不同成熟期番茄果实的PG活性及其合成的影响

本文研究了钙处理不同成熟期番茄果实对果壁组织中钙含量与转化、多聚半乳糖醛酸酶(PG)活性与 PG 合成的影响。结果表明,钙处理绿熟期的番茄果实可使总钙和可溶性钙含量明显增加,并较多转化为结合钙;后期处理,进入和转化的钙都减少。同样,钙处理愈早,对果实 PG 活性的抑制愈强,绿熟期处理可完全抑制 PG 活性。凝胶电泳结合钌红染色,证明绿熟期果实无 PG,PG 是在果实成熟过程中新合成的。钙处理愈早,对 PG 合成的抑制愈强,绿熟期钙处理可完全抑制 PG 合成。     

The Role of Polygalacturonase (PG) in Tomato Fruit Ripening and Effects of Divalent Metal Ions and Ethylene on PG Activity

It has been reported that PG is a key enzyme related to the tomato fruit ripening. In this study tomato fruits were harvested at the mature-green stage and stored at room temperature. The cell ultrastructure of pericarp tissue was observed at different ripening stages, and the effects of treatments with ethylene and calcium on PG activity and fruit ripening were examined. The object of this study is to elucidate the role of PG in regulation of tomato fruit ripening by ethylene and calcium. PG activity, was undetectable at mature-green stage, but it rose rapidly as fruif ripening. The rise in PG activity was coincided with the dechnmg of fruit firmness during ripening of tomato fruits. The observation of cell ultrastructure showed that the most of grana in chloroplast were lost and the mitochondrial cristae decreased as fruit ripening. Striking changes of cell wall structure was most noted, beginning with dissolution of the middle lamella and eventual disruption of primary cell wall. A similar pattern of changes of cell wall and chloroplast have been observed in pericarp tissue treated with PG extract. In fruits treated with calcium and other divalent metal ions atmature-green stage, the lycopene content and PG activity decreased dramatically. Ethylene application enhanced the formation of lycopene and PG activity. The inhibition of Ca2+ on PG ac ivity was removed by ethylene. Based on the above results, it was demonstrated that PG played a major role in ripening of tomato fruits, and suggested that the regulation of fruit ripening by ethylene and Ca2+ was all mediated by PG. PG induced the hydrolysis of cell wall and released the other hydrolytic enzymes, then effected the ripening processes follow up.     

The appearance of polygalacturonase mRNA in tomatoes: one of a series of changes in gene expression during development and ripening

Tomato mRNA was extracted from individual fruits at different stages of development and ripening, translated in a rabbit reticulocyte lysate and the protein products analysed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis. The results indicate that there are at least two classes of mRNA under separate developmental control. One group of approximately six mRNAs is present during fruit growth and then declines at the mature-green stage. Another group of between four and eight mRNAs increases substantially in amount at the onset of ripening, after the start of enhanced ethylene synthesis by the fruit, and continues to accumulate as ripening progresses. Studies of protein synthesis in vivo show that several new proteins are synthesised by ripening fruits including the fruit-softening enzyme polygalacturonase. One of the ripening-related mRNAs is shown to code for polygalacturonase, by immunoprecipitation with serum from rabbits immunised against the purified tomato enzyme. Polygalacturonase mRNA is not detectable in green fruit but accumulates during ripening. It is proposed that the ripening-related mRNAs are the products of a group of genes that code for enzymes important in the ripening process.Abbreviation SDS sodium dodecyl sulfate     

PG在番茄果实成熟中的作用及二价金属离子与乙烯对PG活性的影响

对采后番茄果实的电镜观察表明:当果实成熟衰老时,叶绿体数量减少,多数基粒结构丧失;成熟果实胞壁中胶层水解成中空的电子透明区,初生壁的纤丝也发生一定程度的水解,相邻细胞分离;外源 PG(多聚半乳糖醛酸酶)提取物处理绿熟期果实组织,也可引起胞壁结构和叶绿体发生与正常衰老相同的变化。Ca~(2+)、Mg~(2+)、Co~(2+)二价金属离子处理果实,可明显降低番茄红素含量和 PG 活性,延缓果实软化。外源乙烯处理果实,可促进番茄红素的形成,提高 PG活性,并能解除钙对 PG 活性的抑制。本文也对 PG 在乙烯和 Ca~(2+)调节果实成熟中的作用进行了讨论。     

Effect of silver ions on ethylene biosynthesis by tomato fruit tissue

Mature-green tomato fruit (Lycopersicon esculentum Mill.) were treated asymmetrically with 2 millimolar silver thiosulfate (STS) through a cut portion of the peduncle while still attached to the plant. One-half of the fruit received silver and remained green while the other half ripened normally and was silver-free (less than 0.01 parts per billion). Harvested mature-green fruit were also treated with STS through the cut pedicel. Green tissue from silver-treated fruit had levels of 1-aminocyclopropane-1-carboxylic acid (ACC, the immediate ethylene precursor) slightly less or similar to that of turning or red-ripe tissue from the same fruit, and similar to that of mature-green tissue from control fruit. Ethylene production was higher in green tissue from silver-treated fruit than from either red tissue from the same fruit, or mature-green tissue from control fruit. By inhibiting ACC synthesis with aminoethoxyvinyl glycine, and by applying ACC ± silver to excised disks of pericarp tissue from control or silver-treated tomatoes, we showed that short-term silver treatment did not affect the biological conversion of ACC to ethylene, while long-term treatment stimulated both the conversion of ACC to ethylene and the synthesis of ACC.     

Pectolytic and Cellulolytic Activity of Botrytis cinerea Pers. Related to Infection of Non-ripening Tomato Mutants

Enzymes of Botrytis cinerea were detected in vitro using various carbon sources. Pectin-pectate as a sole carbon source induced both polygalacturonase (PG) and pectin lyase (PL) activity, whereas carboxymethylcellulose served as an inducer for cellulase (C_x) activity. PG activity appeared earlier than C_x activity when induced by their respective sources. Both PG and PL activities were detected earlier and their level was higher on cell walls of the normal tomato fruit, than of the nor mutant, and in each case activity was higher on cell walls of the mature fruits than of the mature-green ones. Whereas relatively high rates of PG and PL activity were recorded on autoclaved tomato homogenate (TH) of both the normal and the nor fruits, only trace levels of PG activity were recorded on unautoclaved media, except for those prepared from ripe normal fruits, and no PL activity was detected on either of the unsterilized media. Botrytis-infection resulted in PG activity in the enzyme-less rin and nor mutant fruits at both stages of maturity and in the normal and hybrid fruits at their mature-green stage. In the ripe normal and hybrid fruits, infection increased the level of PG activity recorded prior to inoculation. An association was drawn between the low PG activity recorded in the nor mutant and its hybrid at initial stages of invasion and their resistance to infection. Following infection an increase in the level of C_x activity over that recorded in healthy fruits was found in all the tomato genotypes, whereas no PL was recorded in either healthy or infected fruits.     

Antioxidant System Differential Regulation is Involved in Coffee Ripening Time at Different Altitudes

Fruit ripening can be seen as an oxidative phenomenon that, depending on its intensity, may directly influence fruit quality. At relatively higher altitudes, coffee fruit ripening takes place through an extended period of time, which positively affects coffee quality. However, little is known about the oxidative processes and antioxidant metabolism of coffee fruits grown at these altitudes. Thus, this study aimed to characterise coffee fruit development from trees grown at two contrasting altitudes (965 m and 1310 m) through phenological analysis and antioxidant metabolism evaluation (Hydrogen peroxide (H₂O₂) and malondialdehyde (MDA) contents; superoxide dismutase (SOD), catalase (CAT), and ascorbate peroxidase (APX) activity and gene expression). Phenological analysis showed that altitude extended coffee reproductive cycle by a month and promoted a higher ripening uniformity, with 100% of fruits at the ideal ripening stage for harvest (cherry stage) in the last evaluation time. H₂O₂ and malondialdehyde contents revealed that in both altitudes fruits went through oxidative damage, though in an early manner at the lower altitude. Although gene expression and enzyme activity did not well correlate, the delay in the oxidative damage in fruits of the higher altitude was probably a result of an increased efficiency in H₂O₂ neutralisation due to the higher activity levels of the APX and CAT enzymes, mainly in green fruits. Thus, a better removal of reactive oxygen species in coffee fruits from plants grown at higher altitudes is involved in the extension of the coffee reproductive cycle, contributing to the production of a higher cup quality coffee.     

冷激处理对油桃贮藏品质和抗氧化酶活性的影响

以‘秦光2号’油桃为材料。研究了冷激处理对果实冷藏中品质及相关酶活性的影响。结果表明,0℃冷空气．处理3．5h可明显延迟油桃的后熟衰老;同时有推迟乙烯释放高峰和呼吸高峰。提高膜脂过氧化保护酶SOD、CAT、POD的活性,保持果肉硬度。减轻冷害发生的作用。但对可溶性固形物和可滴定酸含量无明显影响。     

Effects of ethylene on the susceptibility to Botrytis cinerea infection of different tomato genotypes

Ethylene at 10 and 100 μl/litre stimulated germ-tube elongation of Botrytis cinerea spores incubated within normal and non-ripening nor tomato fruits, but had little influence on the total percent of germination. Values of germ-tube length within the mature-green normal fruits and the mature-green or mature nor fruits were similar to those recorded within the normal mature fruits when held in air. Exposure of the normal and the mutant fruits to 100 μl/litre ethylene immediately after inoculation with B. cinerea insignificantly increased lesion development, but resulted in increased sporulation. When tomato fruits were exposed to ethylene for 3 days before inoculation a marked stimulatory effect on rot development was exhibited on the mature-green normal fruits but not on the nor mutant fruits. The results indicate that exogenous ethylene may directly stimulate germ tube growth of B. cinerea in both normal and mutant fruit, but that it may affect subsequent fungal growth indirectly, via stimulation of the ripening process, only in preclimacteric normal tomato fruit.     

Relationships between the occurrence of misshapen fruit on late-season strawberry in the United Kingdom and infestation by insects, particularly the European tarnished plant bug, Lygus rugulipennis

In experiments in which the European tarnished plant bug, Lygus rugulipennis, was caged on the developing flowers or young fruits of strawberry, the insects caused malformation of the fruits. Another species of capsid, Plagiognathus chrysanthemi, caused similar damage; this species is less numerous than L. rugulipennis on late-season crops of strawberry in UK. Other insects which sometimes occur in large numbers in the flowers of late-season strawberry, i.e. various species of thrips and pollen beetles, did not cause fruit malformation in caging experiments, though thrips sometimes caused discoloration of the fruit. In field experiments where numbers of L. rugulipennis were reduced by the use of insecticides, the amount of misshapen fruit was reduced greatly compared to untreated plots. Correlations between the numbers of L. rugulipennis present at the early stages of fruit development and damage scores for fruit deformity were highly significant. This capsid is likely to be the major cause of fruit malformation in late-season crops of strawberry in the UK.     

Pectin methylesterase activity in vivo differs from activity in vitro and enhances polygalacturonase-mediated pectin degradation in tabasco pepper

Polygalacturonase (PG) and pectin methylesterase (PME) activities were analyzed in ripening fruits of two tabasco pepper (Capsicum frutescens) lines that differ in the extent of pectin degradation (depolymerization and dissolution). Ripe 'Easy Pick' fruit is characterized by pectin ultra-degradation and easy fruit detachment from the calyx (deciduous trait), while pectin depolymerization and dissolution in ripe 'Hard Pick' fruit is limited. PG activity in protein extracts increased similarly in both lines during fruit ripening. PME activity in vivo assessed by methanol production, however, was detected only in fruit of the 'Easy Pick' line and was associated with decreased pectin methyl-esterification. In contrast, methanol production in vivo was not detected in fruits of the 'Hard Pick' line and the degree of pectin esterification remained the same throughout ripening. Consequently, a ripening specific PME that is active in vivo appears to enhance PG-mediated pectin ultra-degradation resulting in cell wall dissolution and the deciduous fruit trait. PME activity in vitro, however, was detected in protein extracts from both lines at all ripening stages. This indicates that some PME isozymes are apparently inactive in vivo, particularly in green fruit and throughout ripening in the 'Hard Pick' line, limiting PG-mediated pectin depolymerization which results in moderately difficult fruit separation from the calyx.     

Inhibition of chloroplastic fructose 1,6-bisphosphatase in tomato fruits leads to decreased fruit size,but only small changes in carbohydrate metabolism

A potato (Solanum tuberosum L. ) cDNA coding for the chloroplastic isoform of fructose 1,6-bisphosphatase (cp-FBPase) was utilized to repress its activity in tomatoes (Lycopersicon esculentum Mill.) using antisense techniques. The patatin B33 promoter was used to ensure fruit specificity of the antisense effect. Transgenic plants were isolated in which fructose 1,6-bisphosphatase activity was reduced by more than 50% of the control in green fruits. Immunoblots indicated that the plastidial isoform was almost completely eliminated in the most strongly inhibited lines. Fruits of the transgenic plants were analyzed for levels of metabolites during fruit development. Glucose and fructose concentrations were increased in green fruits in the transgenic lines, but unchanged at later stages of development. The sucrose concentration was low, and was not significantly altered in the transgenic lines. There was net degradation of starch over the developmental period, but the starch content was not decreased. In green fruit the levels of hexose phosphates were unchanged, whilst the level of 3-phosphoglyceric acid was significantly increased in one line. Most importantly the deduced ratio of hexose phosphate to 3-phosphoglyceric acid decreased, consistent with an in vivo inhibition of fructose 1,6-bisphosphatase activity. One consequence of this reduction of in vivo activity of cp-FBPase was that the average weight of fully ripe fruits was significantly decreased by up to 20% in all transgenic lines in comparison with the control.Abbreviations AGPase ADP-glucose pyrophosphorylase - cp-FBPase Chloroplastidic fructose 1,6-bisphosphatase - cy-FBPase Cytoplasmic fructose 1,6-bisphosphatase - DAF Days after flowering     

Carbohydrate solutilization of tomato locule tissue cell walls: Parallels with locule tissue liquefaction during ripening

Carbohydrate solubilization and glycosidase activities were investigated in tomato ( Lycopersicon esculentum Mill. cv. Solar Set) locule cell walls to identity processes involved in the liquefaction of this tissue. Cell walls were prepared from the locule tissue of fruit at the immature green, mature green, and breaker stages of development. Enzymically active walls incubated in dilute buffer released high molecular mass pectins, oligomeric carbohydrate, and the neutral sugars rhamnose, glucose, galactose, arabinose, xylose, and mannose. The release was sustained for at least 50 h at 34°C and was inhibited more than 50% by 1 m M Hg²⁺. Pectins released from the cell walls of locule tissue at progressive stages of liquefaction were similar in molecular mass and showed no evidence of downshifts on a Sepharose CL–2B–300 column during prolonged incubation. A cell-free protein extract prepared from the locule tissue of mature-green fruit promoted a net release of polymeric and monomeric carbohydrates from high-temperature inactivated cell walls. Polygalacturonase activity was not detected in locule protein although glycosidases including β-mannosidase (EC 3.2.1.25), α- and β-galactosidases (EC 3.2.1.22–23), β-arabinosidase (EC 3.2.1.56) and β-glucosidase (EC 3.2.1.21) were present. Pectinmethylesterase (EC 3.1.1.1 1) activity was detected at the immature-green stage but declined to negligible levels in mature-green and breaker locule tissue. Parallels between the in vitro solubilization of carbohydrate from locule tissue cell walls and the changes occurring during locule liquefaction are discussed.     

Photosynthetic Activity of Ripening Tomato Fruit

Gas exchanges, chlorophyll (Chl) a fluorescence and carboxylation activities of ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBPCO) and phosphoenolpyruvate carboxylase (PEPC) were determined in tomato (Lycopersicon esculentum Mill.) fruits picked at different developmental stages (immature, red-turning, mature, and over-ripe). The fruits did not show signs of CO₂ fixation. However, photochemical activity was detectable and an effective electron transport was observed, the values of Chl fluorescence parameters in green fruits being similar to those determined in the leaves. The RuBPCO activity, which was similar to those recorded in the leaves at the immature stage of the fruit, decreased as the fruit ripened. PEPC activity was always higher than RuBPCO activity.     

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     

Transient increase in locular pressure and occlusion of endocarpic apertures in ripening tomato fruit

Locular pressure was monitored during ripening of tomato (Lycopersicon esculentum Mill.) fruit and the anatomy of the endocarp surface examined using scanning electron microscopy. The manometric pressure of the locule tissue increased from 0 in mature-green fruit to 10 to 50 Pa at the turning or pink stages, and then subsided in ripe fruit. Nonclimacteric fruit containing the ripening inhibitor (rin) mutation showed a similar pattern of internal pressure accumulation during senescence. Build-up of locular tissue pressure occurred in fruit ripening, on or off the plant, as well as in fruit with different susceptibility to cuticle cracking. Apertures ranging from 18-31 μm in width and 33-41 μm in length, with densities ranging from 6.7 to 47.9 apertures · mm⁻² were observed in the endocarp of mature-green fruit. These apertures were progressively occluded during early ripening and were absent in late ripening fruit. Aperture occlusion might result in reduced gas exchange between the locule and external fruit atmosphere, resulting in modification of the locular gas composition.