Pepper beta-galactosidase 1 (PBG1) plays a significant role in fruit ripening in bell pepper (Capsicum annuum)

During bell pepper (Capsicum annuum L.) fruit ripening, beta-galactosidase activity increased markedly as compared with other glycosidases. We purified 77.5 kDa exo-1,4-beta-D-galactanase from red bell pepper fruit classified as beta-galactosidase II. A marked decrease in galactose content appeared during fruit ripening, especially in the pectic fraction. The purified enzyme hydrolyzed a considerable amount of galactose residues in this fraction. We isolated bell pepper beta-galactosidase (PBG1) cDNA. This PBG1 protein contained the putative active site, G-G-P-[LIVM]-x-Q-x-E-N-E-[FY], belonging to glycosyl hydrolase family 35. Quantitative RT-PCR revealed that the expression of PBG1 in red fruit was significantly stronger than that from any other tissues. Moreover, expression of PBG1 occurred prior to that of pepper endo-polygalacturonase 1 (PPG1), the major fruit-ripening enzyme. Based on these results, it appears that the hydrolysis of galactose residues in pectic substances is the first event in the ripening process in bell pepper fruit.     

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。     

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.     

Action of exo- and endo-type cellulases from Irpex lacteus on Valonia cellulose

Cellulolytic mode of action of the two highly purified exo- and endo-type cellulases from Irpex lacteus on pure Valonia cellulose was investigated. Electron microscopy substantiated that both cellulases are adsorbed preferentially into the internal parts of microfibrils in the network structure of the cellulose at initial stages before enzymatic hydrolysis, and that the adsorption ratio of both cellulases onto the external surfaces of microfibrils increased with incubation time although this tendency was less remarkable with the exo-type cellulase than with the endo-type one. The exo-type cellulase exhibited relatively high activity producing cellobiose throughout 12-h incubation, while the endo-type cellulase produced small amounts of cellooligosaccharides. The degree of polymerization was far more suppressed by the endo-type cellulase than by the exo-type one. Degradation by the cellulases in typical exo- and endo-fashions yielded quite different morphological patterns in the microfibrils. Exo-type cellulase loosened the network structure of microfibrils and made them slightly thinner, while endo-type cellulase caused conspicuous swelling and dissolution of individual microfibrils.     

Down-regulation of a ripening-related beta-galactosidase gene (TBG1) in transgenic tomato fruits.

Exo-galactanase/beta-galactosidase (EC 3.2.1.23) activity is thought to be responsible for the loss of galactosyl residues from the cell walls of ripening tomatoes. Transgenic tomato plants (Lycopersicon esculentum Mill cv. Ailsa Craig) with reduced exo-galactanase/beta-galactosidase mRNA were generated to test this hypothesis and to investigate the role of the enzyme in fruit softening. A previously identified tomato beta-galactosidase cDNA clone, TBG1, was used in the experiments. Heterologous expression of the clone in yeast demonstrated that TBG1 could release galactosyl residues from tomato cell wall galactans. Transgenic plants showed a reduction in TBG1 mRNA to 10% of normal levels in the ripening fruits. However, despite the reduction in message, total beta-galactosidase and exo-galactanase activities were unaffected. Furthermore, there was no apparent effect on levels of cell wall galactosyl residues when compared with the control. It was concluded that during the ripening of tomato fruits a family of beta-galactosidases capable of degrading cell wall galactans are active and down-regulation of TBG1 message to 10% was insufficient to alter the degree of galactan degradation.     

Isolation and purification of isoenzymes of cellobiohydrolase I and II of trichoderma reesei using LPLC methods

Five cellulases were fractionated from a commercial cellulase preparation (Celluclast^TM) Two isoenzymes of cellobiohydrolase I (CBHI)(pI = 4.1) could be proved to be real exo-glucanases due to their activity towards MU (=methylumbelliferyl)-lactoside being inhibited by cellobiose (5 mM) and due to production of cellobiose from carboxymethylcellulose (CMC) as the sole final product.Two isoenzymes of CBHII (pI=6.15, 6.0) were shown to act as endo-glucanases because they produced glucose, cellobiose and cellotetraose from CMC and because they were not inhibited by cellobiose when decomposing MU-lactoside. Results confirm recent reports in the literature classifying CBHI and CBHII as exo-type and endo-type cellulases, respectively. Both the CBHI and the CBHII isoenzymes were shown to be active towards CMC and amorphous cellulose.CBHI and CBHII reactions could be differentiated from one another by the velocities of decomposition of CMC: CBHI acts slowly and linearly whereas CBHII acts strongly and exponentially.The fifth of the purified enzymes must be classed as a conventional endoglucanase which exhibits activity towards CMC but fails to be active towards MU-lactoside and amorphous cellulose.     

Ripening-related occurrence of phosphoenolpyruvate carboxykinase in tomato fruit

Phosphoenolpyruvate carboxykinase (PEPCK) is present in ripening tomato fruits. A cDNA encoding PEPCK was identified from a PCR-based screen of a cDNA library from ripe tomato fruit. The sequence of the tomato PEPCK cDNA and a cloned portion of the genomic DNA shows that the complete cDNA sequence contains an open reading frame encoding a peptide of 662 amino acid residues in length and predicts a polypeptide with a molecular mass of 73.5 kDa, which corresponds to that detected by western blotting. Only one PEPCK gene was identified in the tomato genome. PEPCK is shown to be present in the pericarp of ripening tomato fruits by activity measurements, western blotting and mRNA analysis. PEPCK abundance and activity both increased during fruit ripening, from an undetectable amount in immature green fruit to a high amount in ripening fruit. PEPCK mRNA, protein and activity were also detected in germinating seeds and, in lower amounts, in roots and stems of tomato. The possible role of PEPCK in the pericarp of tomato fruit during ripening is discussed.     

香蕉果实成熟软化时果皮和果肉中α-L-阿拉伯呋喃糖苷酶活性的变化

阿拉伯糖是果实软化过程中变化最明显的细胞壁糖残基之一,α-L-阿拉伯呋喃糖苷酶是导致细胞壁多糖中阿拉伯糖残基降解的主要糖苷酶。为阐明该酶在香蕉果实成熟软化中的作用,实验对香蕉贮藏过程中果皮和果肉中该酶活性以及果实硬度、呼吸强度和乙烯释放量的变化进行了研究。结果表明:α-L-阿拉伯呋喃糖苷酶在果实初期的变化很小,到果实硬度开始急剧下降时达到最大,增加量达10倍以上,且果肉中的酶活性大于果皮中;乙烯吸收剂处理延缓了香蕉果实呼吸和乙烯高峰的出现时间,降低了果实硬度、果皮和果肉中α-L-阿拉伯呋喃糖苷酶活性变化的速度和幅度。以上结果表明α-L-阿拉伯呋喃糖苷酶起诱导香蕉果实成熟的作用,在果实的软化中起着十分重要的作用,且其活性受乙烯的调节。     

Effect of phytohormones on pectate lyase activity in ripening Musa acuminata.

A differential activity peak of pectate lyase (PEL) was observed during ripening of banana fruits (Musa acuminata Harichhal) receiving different hormone treatments. Exposure of fruits to 25 ppm ethylene for 24 h, as well as dipping of M. acuminata fruits in 1 mM 2,4-dichlorophenoxy acetic acid (2,4-D) for 4 h, hastened fruit ripening. Both PEL activity peak and climacteric peak were observed on the 4th and 10th days of treatment with ethylene and 2,4-D, respectively, compared to the 16th day in control fruits. Gibberellic acid (GA) treatment retarded fruit ripening and both PEL activity and climacteric peaks were observed on the 19th day. Treatment of fruits with ethylene or 2,4-D also advanced the appearance of a polygalacturonase (PG) peak and GA delayed its appearance, but the activity peaks always appeared in post-climacteric fruits, in contrast to PEL activity peaks coinciding with the respiratory peaks.     

香蕉果实软化相关β-半乳糖苷酶基因的克隆及其表达分析

β-半乳糖苷酶（β-galactosidase）通过分解细胞壁半纤维素切除半乳糖键而参与果实软化。为了阐明香蕉（Musasp．）果实成熟过程中的软化与细胞壁代谢酶β-半乳糖苷酶基因表达之间的关系,采用RT-PCR方法,从成熟香蕉果实果肉中分离了编码β-半乳糖苷酶基因的部分cDNA（MA-Gal）,序列分析表明,MA-Gal包含927bp,编码309个氨基酸,包含5个β-半乳糖苷酶结构域（典型真核生物中β-半乳糖苷酶包含7个结构域）,推导的MA-Gal蛋白质中有β-半乳糖苷酶蛋白的催化活性部位GGPIILSQIENEY（F）;系统进化树分析结果表明MA-Gal属于第一类β-半乳糖苷酶基因（该类主要在果实中表达）;β-半乳糖苷酶活性和硬度的变化表明其与香蕉果实硬度变化密切相关;Northern分析显示,跃变前期的果肉中,MA-Gal基因的表达量很低,后随着果实的软化表达量不断增加,并在呼吸跃变后达到最高。所有结果表明,MA-Gal参与香蕉果实成熟过程中的软化。     

Cell wall metabolism during the development of chilling injury in cold-stored peach fruit: association of mealiness with arrested disassembly of cell wall pectins

Partially tree-ripened ripe fruit of peach (Prunus persica L.) were stored for 1-4 weeks at 5 degrees C and then ripened at 20 degrees C for 3 d to induce chilling injury. With increasing cold storage the incidence and severity of mealiness symptoms increased progressively, manifested as reduced quantities of free juice and internal flesh browning. Relative to juicy fruit, tissue of mealy fruit showed altered intercellular adhesion when examined by microscopy and, upon crushing, a higher proportion of cells remained intact and did not release cellular contents. Substantial alterations in the metabolism of cell wall polysaccharides were observed. Chelator-soluble polyuronides from mealy fruit were partially depolymerized during cold storage in a manner dissimilar to that in unripe or ripe juicy fruit, and were not depolymerized further during the ripening period. The solubility of these high molecular weight pectins remained low, and did not show the increase characteristic of juicy fruit. Furthermore, in mealy fruit the dramatic decline in the polymeric Ara content of base-soluble, matrix glycan-enriched fractions occurring during normal ripening was absent, indicating diminished disassembly of an arabinan-rich polysaccharide firmly attached to cellulose. A corresponding rise in the polymeric Ara content of the most soluble pectin fraction was also absent, as was a decline in the Gal content of this extract. The depolymerization of matrix glycans showed only minor differences between juicy and mealy fruit. After cold storage and ripening, the activities of endo-1,4-beta-glucanase (EC 3.2.1.4), endo-1,4-beta-mannanase (EC 3.2.1.78), beta-galactosidase (EC 3.2.1.23), alpha-arabinosidase (EC 3.2.1.55), and particularly endo-polygalacturonase (EC 3.2.1.15) were lower in mealy fruit than in juicy fruit, whereas pectin methylesterase activity (EC 3.1.1.11) was lower in slightly mealy and higher in very mealy fruit. The data suggest that cold storage affects the activities of numerous cell wall-modifying enzymes, with important consequences for pectin metabolism. These changes alter the properties of the primary wall and middle lamella, resulting in tissue breakage along enlarged air spaces, rather than across cells, which reduces the amount and availability of free juice upon tissue fragmentation.     

草莓果实发育成熟过程中糖苷酶和纤维素酶活性及细胞壁组成变化

以丰香和红丰草莓为试材,对果实发育成熟过程中细胞壁水解酶活性和细胞壁成份变化进行了研究．结果表明：半乳糖苷酶和α-甘露糖苷酶活性随草莓果实成熟而提高,葡萄糖苷酶活性不随草莓果实成熟而提高．随着果实发育成熟,纤维素酶活性、果胶酶活性不断提高．果实中未检测到内切多聚半乳糖醛酸酶活性,外切多聚半乳糖醛酸酶活性变化不随果实成熟软化而提高．随果实发育成熟,细胞壁中可溶性果胶和半纤维素增加,而离子结合果胶和共价结合果胶及纤维素减少．     

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。     

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.     

Adsorption mode of exo- and endo-cellulases from Irpex lacteus (Polyporus tulipiferae) on cellulose with different crystallinities.

The adsorption mode of two highly purified cellulases, exo- and endo-type cellulases, from Irpex lacteus (Polyporus tulipiferae) was investigated by using pure cellulosic materials with different crystallinity as substrates. Adsorption of the two enzymes on the substrates was found to fit the Langmuir-type adsorption isotherm. Maximum amount of adsorbed enzyme obtained from the Langmuir plots showed an inverse correlation to the crystallinity of the substrate with both enzymes, and this value of endo-type cellulase was less dependent on the degree of crystallinity of substrates than that of exo-type cellulase, whose isotherms reached saturation in the range of low enzyme concentrations. The two enzymes showed relatively high affinities for all the substrates and their affinities increased with increasing crystallinity, but this tendency was less marked with endo-type cellulase than with exo-type one. In addition, large negative values of free energy change were observed on the adsorption of both enzymes, and the values became more negative with increasing crystallinity. Consequently, both cellulases showed high adsorption on crystalline cellulose and the adsorption process became smoother with increasing crystallinity. The adsorption of the two types of cellulases was endothermic with an increase in entropy, especially for amorphous cellulose, suggesting the occurrence of water release from the substrates during enzyme adsorption. In addition, the changes in thermodynamic parameters (delta H, delta S, and delta G) in adsorption of exo-type cellulase were larger than in that of endo-type enzyme.     

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.     

Changes in structural features of free N-glycan and endoglycosidase activity during tomato fruit ripening

In developing plants, free N-glycans occur ubiquitously at micromolar concentrations. Such oligosaccharides have been proposed to be signaling molecules in plant development. As a part of a study to elucidate the physiological roles of de-N-glycosylation machinery involved in fruit ripening, we analyzed changes in the amounts and structural features of free N-glycans in tomato fruits at four ripening stages. The amount of high-mannose type free N-glycans increased significantly in accordance with fruit ripening, and the relative amounts of high-molecular size N-glycans, such as Man(8-9)GlcNAc(1), became predominant. These observations suggest that the de-N-glycosylation machinery, including endo-beta-N-acetylglucosaminidase (ENGase) activity, is stimulated in the later stages of fruit ripening. But contrary to expectation, we found that total ENGase activities in the tomato fruits did not vary significantly with the ripening process, suggesting that ENGase activity must be maintained at a certain level, and that the expression of alpha-mannosidase involved in the clearance of free N-glycans decreases during tomato fruit ripening.     

Effects of seasonal temperature changes on <Emphasis Type="Italic">DkMyb4</Emphasis> expression involved in proanthocyanidin regulation in two genotypes of persimmon (<Emphasis Type="Italic">Diospyros kaki</Emphasis> Thunb.) fruit

Persimmon fruits accumulate a large amount of proanthocyanidin (PA). Fruits of the mutant non-astringent (NA) type lose their ability to accumulate PA at an early stage of fruit development, whereas fruits of the normal astringent (A) type sustain PA accumulation until ripening. This allelotype is determined by the genotype of a single ASTRINGENCY (AST) locus. It is possible that the reduction in PA accumulation in NA-type fruits is due to phenological down-regulation of DkMyb4 (a PA regulator) and the resultant down-regulation of structural genes in the PA pathway. In this study, attempts were made to identify the regulatory mechanisms of phenological PA accumulation in A- and NA-type fruits, focusing particularly on the effects of ambient temperature. Continuous cool temperature conditions caused sustained expression of DkMyb4 in NA-type fruits, as well as in A-type fruits, resulting in increased expression of PA pathway genes and PA accumulation. However, the expression of some A/NA phenotypic marker genes was not significantly affected by the cool temperature conditions. In addition, PA composition in NA-type fruits exposed to cool temperatures differed from that in A-type fruits. These results indicate that a cool ambient temperature may have induced DkMyb4 expression and resultant PA accumulation, but did not directly affect the expression of the AST gene.