β-Galactosidase, polygalacturonase and pectinesterase in differential softening and cell wall modification during papaya fruit ripening

Papaya ( Carica papaya L. cv. Eksotika) fruit softens differentially in relation to position of the tissue. The inner mesocarp tissue is softer, and its firmness decreases more rapidly during ripening than that of the outer mesocarp tissue. As the fruit ripens, pectin solubility and depolymerisation increase. Hemicellulose, too, appears to be depolymerised but, unlike pectins, this apparent degradation of hemicellulose is associated with an increase rather than a decrease in its level. Pectin and hemicellulose depolymerisation began in the inner mesocarp tissue at about the same time as β-galactosidase (EC 3.2,1.23) activity started to increase and tissue firmness began to decrease more rapidly. In contrast, pectin solubilisation in both outer and inner mesocarp tissues occurred steadily throughout ripening at a comparable rate and paralleled closely the increase of polygalacturonase (PG; EC 3.2.1.67) and pectinesterase (EC 3.1.1.11). In general, irrespective of enzyme distribution, tissue softening during ripening was more closely related to changes in β-galactosidase activity than to PG or pectinesterase activity. Papaya, β-galactosidase appears to be an important wall degrading enzyme and may contribute significantly to differential softening, perhaps by complementing the action of polygalacturonase. Polygalacturonase activity increased with increasing depth of the mesocarp tissue, as did softening of the fruit.     

Metabolism of cell wall polysaccharides from persimmon fruit. Pectin solubilization during fruit ripening occurs in apparent absence of polygalacturonase activity

Pectins from persimmon ( Diospyros kaki L.) fruit pericarp were sequentially extracted with 0. 05 M trans -1,2-diaminocyclohexane-N,N, N', N'-tetraacetic acid (CDTA), 0. 05 M Na₂CO₃ (1°C) and Na₂CO₃ (20°C) and the carbohydrate composition and metabolism during development determined. Young persimmon fruits contained a large proportion of pectins, 46% by dry weight, that decreased to 20% with ripening. This decrease occurred in the CDTA and Na₂CO₃ (1°C) fractions, mainly composed of uronic acids, and represents a net loss of uronic acids, arabinose and galactose. The amount of non-cellulosic neutral sugars was especially high in the Na₂CO₃ (20°C) fraction. The loss of pectins was also accompanied by a depolymerisation of the polysaccharides extracted in the three pectic fractions. However, none of these changes can be attributed to the action of polygalacturonase activity. Proteins were extracted from the pericarp tissue, but endopolygalacturonase (EC 3. 2. 1. 15) activity, determined as a decrease in viscosity of polygalacturonic acid, was not observed in the extract. Determination of exopolygalacturonase (EC 3. 2. 1. 67) activity by measuring the release of reducing groups from polygalacturonic acid was also negative. The results presented indicate that polygalacturonase is not responsible for the metabolism of pectins during persimmon fruit ripening.     

Control and manipulation of gene expression during tomato fruit ripening

Ripening is a complex developmental process involving changes in the biochemistry, physiology and gene expression of the fruit. It is an active process characterised by changes in all cellular compartments. cDNA cloning has been used as an approach to analyse changes in gene expression during fruit ripening. This has revealed that several genes are switched on specifically during fruit ripening, including one encoding polygalacturonase (PG), a major cell wall protein. These cDNA clones have been used to study the expression of the genes in normal and ripening mutant fruits, and under environmental stress conditions.The PG gene has been isolated and it has been demonstrated that 1450 bases 5 of the coding region are sufficient for the tissue- and development-specific expression of a bacterial marker gene in transgenic tomatoes. Antisense RNA techniques have been developed to generate novel mutant tomatoes in which the biochemical function of this enzyme and its involvement in fruit softening has been tested.     

Isolation, characterization and significance of papaya β‐galactanases to cell wall modification and fruit softening during ripening

β‐Galactosidases (EC 3.2.1.23) from ripe papaya ( Carica papaya L. cv. Eksotika) fruits having galactanase activities were fractionated by a combination of cation exchange and gel‐filtration chromatography into three isoforms, viz., β‐galactosidase I, II and III. The native proteins of the respective isoforms have apparent molecular masses of 67, 67 and 55 kDa, each showing one predominant polypeptide upon SDS‐PAGE of about 31 and 33 kDa for β‐galactosidases I and III, respectively, and of 67 kDa for β‐galactosidase II. The β‐galactosidase I protein, which was undetectable in immature fruits, appeared to be specifically accumulated during ripening. The β‐galactosidase II protein was present in developing fruits, but its level seemed to decrease with ripening. β‐Galactosidase I seemed to be an important softening enzyme; its activity increased dramatically (4‐ to 8‐fold) to a peak early during ripening and correlated closely with differential softening as related to position in the fruit tissue. The inner mesocarp tissue was softer, and its wall pectins were modified earlier and firmness decreased more rapidly during ripening compared to the outer mesocarp tissue. β‐Galactosidase II also may contribute significantly to softening because of its ability to catalyse increased solubility and depolymerization of pectins as well as through its ability to modify the alkali‐soluble hemicellulose fraction of the cell wall. The physiological significance of both β‐galactosidase isoforms may partly be attributed to their functional capacity as β‐(1,4)‐galactanases.     

Polygalacturonase: a candidate gene for the soft flesh and deciduous fruit mutation in Capsicum

The soft flesh and deciduous fruit of pepper (Capsicum spp.) originated from the wild C. frutescens BG 2816 accession is a complete dominant trait controlled by the S gene. We constructed an F₂ population from a cross of BG 2816 (SS) and the bell-type C. annuum cultivar Maor (ss) and determined that S cosegregated with the tomato fruit-specific endo-polygalacturonase (PG) gene. The soft flesh and deciduous fruit phenotypes were observed together in all F₂ individuals, indicating a pleiotropic effect of PG on the two traits. We mapped S to pepper chromosome 10 in the region corresponding to that in which PG was previously mapped in tomato. Northern, RT-PCR and western analyses and enzyme activity assays, collectively, indicated that PG is not detected in green, breaker or red fruits of Maor, nor in green fruits of BG 2816. Accumulation of PG mRNA and protein was detected in the fruits of BG 2816, and it increased during ripening from breaker to red stages. The sequence analysis of partial PG cDNA isolated from BG 2816 revealed high homology (87% identity) with the tomato PG. The resemblance of the soft flesh and deciduous fruit phenotypes to PG-associated phenotypes in other fruit crops, the complete linkage between Sand PG, and the greater expression of PG in the fruits of BG 2816 than in those of Maor, all strongly indicate that PG is a candidate gene for S.     

Function of the polygalacturonase convertor in ripening tomato fruit

In extracts from pericarp tissue of ripening tomato ( Lycopersicon esculentum Mill. cv, Sonato) fruits, two isoenzymes of polygalacturonase (E.C. 3.2.1.15), PG1 and PG2, are usually found. Also in such extracts, or as part of PG1, a convertor (CV) occurs. Incubation of PG2 with this CV gives rise to PG1 or a different isoenzyme, PGx, that is also stable at 65°C but differs in _pH optimum and size from PG1. It appears that CV has two affinity sites that can bind with PG2 or with a polydextran. PG1 is an extraction artifact, consisting of one molecule of CV and two molecules of PG2. PGx is made up of one molecule of CV and one molecule of PG2. It is the CV part of PGx that binds to polydextrans such as Blue Dextran 2000, Sephadex G-100, and cell wall preparations. In this last form PGx is the physiologically active form of the enzyme, solubilizing demethylated pectin.
On Sephacryl S-300, CV appears to have a molecular weight of 81 kDa, but because of its heat stability and partial leakage through a 10 kDa cut-off membrane, it might be a much smaller, rod-like molecule. The polygalacturonase convertor might be a lectin without intrinsic enzyme activity, with a function to immobilize, stabilize and activate enzymic proteins in the cell wall.     

Polygalacturonase gene expression in kiwifruit: relationship to fruit softening and ethylene production

In kiwifruit, much of the softening process occurs prior to the respiratory climacteric and production of ethylene. This fruit therefore represents an excellent model system for dissecting the process of softening in the absence of endogenous ethylene production. We have characterized the expression of three polygalacturonase (PG) cDNA clones (CkPGA, B and C) isolated from fruit of Actinidia chinensis. Expression of CkPGA and B was detected by northern analysis only in fruit producing endogenous ethylene, and by RT-PCR in other tissues including flower buds, petals at anthesis, and senescent petals. CkPGA promoter fragments of 1296, 860 and 467 bp fused to the -glucuronidase (uidA) reporter gene directed fruit-specific gene expression during the climacteric in transgenic tomato. CkPGC gene expression was observed in softening fruit, and reached maximum levels (50-fold higher than for CkPGA and B) as fruit passed through the climacteric. However, expression of this gene was also readily detected during fruit development and in fruit harvested prior to the onset of softening. Using RT-PCR, expression of CkPGC was also detected at low levels in root tips and in senescent petals. These results suggest that PG expression is required not only during periods of cell wall degeneration, but also during periods of cell wall turnover and expansion.     

Identification of mRNAs with enhanced expression in ripening strawberry fruit using polymerase chain reaction differential display

Fruit ripening is a complex developmental process that involves specific changes in gene expression and cellular metabolism. In climateric fruits these events are coordinated by the gaseous hormone ethylene, which is synthesized autocatalytically in the early stages of ripening. Nonclimacteric fruits do not synthesize or respond to ethylene in this manner, yet undergo many of the same physiological and biochemical changes associated with the production of a ripe fruit. To gain insight into the molecular determinants associated with nonclimacteric fruit ripening, we examined mRNA populations in ripening strawberry fruit using polymerase chain reaction (PCR) differential display. Five mRNAs with ripening-enhanced expression were identified using this approach. Three of the mRNAs appear to be fruit-specific, with little or no expression detected in vegetative tissues. Sequence analysis of cDNA clones revealed positive identities for three of the five mRNAs based on homology to known proteins. These results indicate that the differential display technique can be a useful tool to study fruit ripening and other developmental processes in plants at the RNA level.     

Calcium in plant senescence and fruit ripening

Abstract. Calcium has long been associated with regulation of the ripening process of fruit and post-harvest storage life. Specifically, maintenance of relatively high calcium concentrations in fruit tissue results in a slower rate of ripening, as seen in lower respiration rates, reduced ethylene production, and slower softening of fruit flesh. There are also some specific fruit disorders such as bitter pit, which can be prevented if sufficient calcium is present. Senescence of other plant tissues such as leaves and flowers has also been shown to be retarded by the application of calcium.
Work leading to the above information is reviewed and discussed in the context of what is currently known of cellular regulation of calcium in plants. The major sites for the action of calcium in senescence and ripening are suggested to be in membrane structure and function, and in cell wall structure. Although high external concentrations of calcium are an advantage in reducing the rate of senescence or ripening, it is emphasized that normal cell function requires the maintenance of low concentrations of free calcium in the cell cytosol. It is suggested that one possible feature of senescence is a breakdown in such cellular regulation.     

β-Galactosidase activity in ripening apples

β-Galactosidase activity has been identified in soluble and cell wall preparations from apple cortex tissue. The enzyme degrades pectin galactan and has a pH optimum of 4·0 with p-nitrophenyl-β, d-galactopyranoside as substrate. Soluble polygalacturonide increased as the applies softened with ripening and these changes were preceded by loss of galactose residues from the cell wall and an increase in β-galactosidase activity.     

Activity of cell wall-associated enzymes in ripening olive fruit

Enzymatically active cell wall isolaled from olive (Olea europaea) fruit was employed Hi investigate some hydrolytic enzymes bound to the cell wall and the changes in these during ripening. Seven glycosidases. β-glucosidase (EC 3.2.1.21) α-galactosidase (EC 3.2.1.22). β-galactosidase (EC 3.2.1.23). α-arabinosidase (EC 3.2.1.55), α-mannosidase (EC 3.2.1,24). β-xylosidase (EC 3.2.1.37) and β-N-acetylglucosamidase (EC 3.2.1.30). as well as Cx-cellulase (EC 3.2.1.4) and endo-polygalacturonase (EC 3.2.1.15). were identified in the cell wall preparation, at four stages of ripeness (mature green. changing colour, black and black-ripe). Activities of all these cell wall-associated enzymes fionicallv and covalently linked) were determined either by cell wall incubation with artificial substrate or after extraction from the cell wall with buffers of high salt concentration (Cx-cellulase). and were compared to those of forms solubilized from acetone powders with 500 nM citrate buffer (cytoplasmic and/or apoplastic plus ionically hound to cell wall) In general, the activities of low ionic strength buffer-soluble enzymes were found to be much higher than those of the bound enzymes. The bound enzymes are present in the fruit at the green colour stage, whereas the activities of the soluble enzymes only increased from the changing colour stage onwards. The tenacity of binding of enzymes to the wall was investigated by treating the walls with high salt and measuring residual activity. The nature of the ionic and covalent binding and the changes during ripening were also established for wall-hound glycosidase During ripening there was a marked change in the percentages of covalently- and tonically linked activities of β-glucosidase and β-galaclosidase: al the changing colour stages about 75–80% of the bound active in was present in high ionic strength buffer while al the black-ripe stage it was only 15–20. A possible role for these cell wall degradative enzymes in olive softening is discussed.     

番茄果实中乙烯与多聚半乳糖醛酸酶的关系

乙烯与多聚半乳糖醛酸酶(PG)都是果实成熟过程中关键的调节因子.一方面,在有乙烯合成缺陷的转反义ACS番茄和乙烯感受缺陷的Nr突变体番茄果实中PG基因表达量都明显下降,PG酶活性明显降低;用外源乙烯(100 μL/L)处理绿熟期番茄果实使PG基因的表达明显增强,而1-甲基环丙烯(1-MCP,1 μL/L)处理转色期番茄果实明显抑制PG基因表达.另一方面,转反义PG基因番茄果实乙烯释放量在授粉后低于其野生型,番茄乙烯受体基因LeETR4和乙烯反应因子LeERF2基因表达量比野生种低.PG降解果胶的产物D-GA(100 mg/L)促进未熟期番茄果实中的乙烯生成和LeETR4、LeERF2基因的表达.     

Cloning and characterization of avocado fruit mRNAs and their expression during ripening and low-temperature storage

Differential sereening of a cDNA library made from RNA extracted from avocado (Persea americana Mill cv. Hass) fruit stored at low temperature (7°C) gave 23 cDNA clones grouped into 10 families, 6 of which showed increased expression during cold storage and normal ripening. Partial DNA sequencing was carried out for representative clones. Database searches found homologies with a polygalacturonase (PG), endochitinase, cysteine proteinase inhibitor and several stress-related proteins. No homologies were detected for clones from six families and their biological role remains to be elucidated. A full-length cDNA sequence for avocado PG was obtained and the predicted amino acid sequence compared with those from other PGs. mRNA encoding PG increased markedly during normal ripening, slightly later than mRNAs for cellulase and ethylene-forming enzyme (EFE). Low-temperature storage delayed ripening and retarded the appearance of mRNAs for enzymes known to be involved in cell wall metabolism and ethylene synthesis, such as cellulase, PG and EFE, and also other mRNAs of unknown function. The removal of ethylene from the atmosphere surrounding stored fruit delayed the appearance of the mRNAs encoding cellulase and PG more than the cold storage itself, although it hardly affected the expression of the EFE mRNA or the accumulation of mRNAs homologous to some other unidentified clones.AFRC Research Group in Plant Gene Regulation     

Biochemical changes associated with the ripening of hot pepper fruit

Hot pepper ( Capsicum annuum L. cv. Chooraehong) fruit underwent a respiratory climacteric during ripening. However, the rate of ethylene production was low, reaching a maximum of approximately 0.7 μl kg⁻¹ h⁻¹ at the climacteric peak when the surface color was 30 to 40% red. Ripening was accompanied by a loss of galactose and arabinose residues from the cell wall. The content of uronic acid and cellulose in the wall changed only slightly during ripening. The average molecular weight of a cell wall hemicellulosic fraction shifted progressively toward a lower molecular weight during ripening. Total β-galactosidase (EC 3.2.1.23) activity increased 50-fold from the immature green to the red ripe stage. No polygalacturonase (EC 3.2.1.15) activity was detected at any stage of ripeness. Thus, the loss of galactose and arabinose residues from the cell wall, as well as the observed modification of hemicelluloses during ripening, seem to be unrelated to active polygalacturonase. Soluble polyuronide content remained relatively constant at approximately 60 μg (g fresh weight)⁻¹ as fruit ripended.     

T4转基因番木瓜遗传性和果实品质分析

对T 4代转基因番木瓜进行了分子生物学和果实品质分析,结果表明,筛选获得的转基因番木瓜均为转番木瓜环斑病毒(PRV)复制酶突变体基因(RP),且对PRV抗性达到了高抗或免疫,RP基因在转基因植物中能稳定遗传至后代并在RNA水平上表达。在田间种植时,转基因木瓜的生长状况普遍好于普通番木瓜,尤其在生长后期(10月以后),普通番木瓜100%发病(大规模种植时),而大部分(约91.8%)转基因植株生长良好,果实较多且表面光洁、基本上无环斑。与非转基因亲本相比,T 4代转基因番木瓜的果实长度增加2.6%~5%,果实直径变小0.6%~1.5%,果肉厚度增加了12%~15%,因而果实形状与亲本相近或更好,且信用价值更高。转基因番木瓜果实中水分、蛋白质、氮、脂肪、还原性糖、维生素A、维生素C和类胡萝卜素的含量与对照都无显著性差异,即转基因番木瓜与亲本具有实质等同性,这表明转入的外源基因对番木瓜果实品质没有不良影响。     

Analysis on virus resistance and fruit quality for T4 generation of transgenic papaya

Molecular biological characterization,fruit characters,and nutrients were analyzed for T4 generation of transgenic papaya.All transgenic papaya plants with the mutated replicase (RP) gene from papaya ringspot virus (PRSV) showed high resistance or immunity against PRSV in the field.The RP transgene can be steadily inherited to,and expressed at RNA level,the progenies.The growth characteristics of transgenic papaya were much better than nontransgenic papaya in the field.The non-transgenic papaya seedlings began to show typical symptoms caused by PRSV after being inoculated with PRSV.They died quickly and never grew to produce fruit.The adult trees developed yellow leaves and produced smaller fruits and were doomed to a slow death after some time,while most oftransgenic papaya plants (about 91.8%) did not show any symptoms caused by PRSV,and produced more,bigger,and high quality fruits.Compared with non-transgenic plants,the fresh fruit length of T4 generation of transgenic papaya increased 2.6%-5%,and the diameter decreased 0.6%-1.5%.The flesh thickness of fresh fruit increased 12%-15%,which made it fitter for eating.Although the fresh fruit quality changed,there was no significant difference between transgenic and non-transgenic papaya.The quality characteristics of dry fruit including the contents of water,lipid,N,protein,reduced sugar,vitamin A,vitamin C,and carotene in the T4 generation of transgenic papaya were all the same as their non-transgenic parents.This means that transgenic plants and non-transgenic plants are substantially equivalent,and the transgene has no effect on dry fruit quality.In this study,we found that vitamin A and vitamin C in red-fleshed papaya were 1.4-1.8 and 1.78-2.07 times more than the yellow-fleshed ones,respectively,while N and protein were only 84.2%-92.1% and 82.1%-98.9% of the yellow-fleshed ones.