Pectin esterase, polygalacturonase and gel formation in peach pectin fractions

Peaches (Prunus persica cv. Hermoza) were stored at 0C in regular air (RA) or in controlled atmosphere (CA 10% CO2, 3% O2) for 4 weeks and then ripened for 4 days at 20 degrees C. Woolliness developed in the regular air stored fruit while the controlled atmosphere stored fruit ripened normally. In the woolly fruit symptoms of the disorder were greater in the inner mesocarp than in the outer. Polygalacturonase (PG) and pectin esterase (PE) activities differed in the outer and inner mesocarp of the affected fruit. PG activity was low and PE activity was high in the inner mesocarp of the woolly fruit during ripening relative to the outer mesocarp, while in the healthy fruit, activities were similar in both areas. Cell wall fractions of water-soluble, CDTA-soluble and carbonate-soluble pectins were prepared from freshly harvested peaches and incubated with PE and PG from ripe peaches at different ratios. Only the CDTA-soluble fraction formed a gel with peach enzymes, and the rate of gelation increased with increasing amounts of PE relative to PG. Both water-soluble and CDTA-soluble pectin fractions formed gels with commercial PE (extracted from orange peel). The PE extracted from peaches was stable when stored at 0 degrees C for 9 days, while PG activity was stable only for 1 day. We suggest that PE, acting on pectins in the cell wall in vivo may cause gel formation and that the CDTA-soluble polymers have the capacity to bind apoplastic water and create the dry appearance observed in woolly fruit.     

龙眼果实采后果肉自溶过程中细胞壁组分及其降解酶活性的变化

在(10±1)℃下贮藏的‘福眼’龙眼果实果肉自溶指数和自溶程度随着贮藏时间的延长而增加。果肉细胞壁干重、原果胶、纤维素、半纤维素和细胞壁蛋白含量不断减少。果肉果胶酯酶(PE)活性下降;多聚半乳糖醛酸酶(PG)活性在贮藏6～12d以及纤维素酶活性在贮藏0～12d期间均明显增强,到第12天达到活性高峰,之后下降。但在贮藏0～24d期间,PE、PG和纤维素酶仍然保持较高活性,贮藏24d之后快速下降。β-半乳糖苷酶活性在贮藏0～24d期间略有下降,而在贮藏24d后,活性增强,尤其是贮藏30d后,活性急剧升高。     

糯米糍荔枝裂果的生理机理与防裂效果研究

以果实易裂品种糯米糍荔枝为材料,对果皮内与裂果有关的一些生理指标以及两种处理硝酸钙(Ca(NO3)2)和赤霉素(GA)的防裂效果进行了探索性研究。结果表明:在果实发育初期,果皮内的水溶性果胶和原果胶的含量均升高,且原果胶含量的增长幅度大于水溶性果胶。除果实成熟期外,果胶脂酶(PE)和多聚半乳糖醛酸酶(PG)的活性一直呈上升趋势,且水溶性果胶的含量与PG的活性有显著正相关关系。纤维素酶(CX)在整个果实发育期都表现出较高的活性且有两次明显的活性高峰。同时还发现除果实成熟期外,Ca(NO3)2处理能增强PE和PG活性,但对CX则有抑制作用。此外,Ca(NO3)2处理和GA处理均能明显地改善糯米糍荔枝果皮质地的组成,使果实的裂果率明显降低,且GA处理的效果要明显好于Ca(NO3)2处理。     

Postharvest Variation in Cell Wall-Degrading Enzymes of Papaya (Carica papaya L.) during Fruit Ripening

Pectin methylesterase (PME), polygalacturonase (PG), xylanase, cellulase, and proteinase activity were determined and related to respiration, ethylene evolution, and changes in skin color of papaya (Carica papaya L.) fruit from harvest through to the start of fruit breakdown. PME gradually increased from the start of the climacteric rise reaching a peak 2 days after the respiratory peak. PG and xylanase were not detectable in the preclimacteric stage but increased during the climacteric: during the post climacteric stage, the PG declined to a level one-quarter of peak activity with xylanase activity returning to zero. Cellulase activity gradually increased 3-fold after harvest to peak at the same time as PME, 2 days after the edible stage. Proteinase declined throughout the climacteric and postclimacteric phases. A close relationship exists between PG and xylanase and the rise in respiration, ethylene evolution, and softening. Cultivar differences in postclimacteric levels of enzymic activity were not detected.

An inhibitor of cellulase activity was detected in preclimacteric fruit. The inhibitor was not benzyl isothiocyanate (BITC). BITC did inhibit PG activity, though no inhibitor of PG activity was detected in preclimacteric homogenates when BITC was highest. The results indicate that inhibitors did not play a direct role in controlling wall softening.

     

三个脐橙品种果实主要细胞壁酶动态变化研究

以罗伯逊脐橙、丰脐、纽荷尔脐橙果实为试材,对其果皮和果肉的果胶甲酯酶(PE)、多聚半乳糖醛酸酶(PG)、β-葡萄糖苷酶和纤维素酶(CX)的动态变化进行研究。结果表明,三个品种PE活性均为果肉明显高于果皮,其中纽荷尔和罗脐分别在果实膨大期、转色期和成熟期出现三个峰值;PE活性变幅纽荷尔大于罗脐,丰脐变化较平稳。PG活性果肉略高果皮,且皆在转色期开始快速升高。首次对脐橙果实β-葡萄糖苷酶研究表明,该酶活性微弱,成熟期活性最高,纽荷尔高于丰脐和罗脐;动态变化中果皮和果肉无明显差异。酶活性高峰出现的时间为PE早于PG和β-葡萄糖苷酶;PE和PG是影响脐橙果实发育的主要酶,β-葡萄糖苷酶可能是PE和PG的补充。     

不同成熟度菠萝贮藏特性的差异

随着菠萝果皮由绿转黄,果肉硬度及可溶性固形物含量降低,可溶性糖醛酸含量增加,多聚半乳糖醛酸酶(PG)活性持续升高,而果胶脂酶(PE)活性在过熟期达到高峰并开始下降。研究表明,同一菠萝不同部位果肉硬度、PG活性及可溶性糖醛酸含量差异较大。     

Postharvest Variation in Cellulase, Polygalacturonase, and Pectinmethylesterase in Avocado (Persea americana Mill, cv. Fuerte) Fruits in Relation to Respiration and Ethylene Production

Cellulase, polygalacturonase (PG), pectinmethylesterase (PME), respiration, and ethylene production were determined in single “Fuerte” avocado fruits from the day of harvest through the start of fruit breakdown. PME declined from its maximum value at the time of picking to a low level early in the climacteric. PG activity was not detectable in the preclimacteric stage, increased during the climacteric, and continued to increase during the postclimacteric phase to a level three times greater than when the fruit reached the edible soft stage. Cellulase activity was low in the preclimacteric fruit, started to increase just as respiration increased, and reached a level two times greater than at the edible soft stage. Cellulase activity started to increase 3 days before PG activity could be detected. Increased production of ethylene followed the increase in respiration and cellulase activity by about 1.5 days. These results indicate that a close relation exists between the rapid increase in the cell wall-depolymerizing enzymes and the rise in respiration and ethylene production and refocused attention on the role of the cell wall and the associated plasma membrane in the early events of fruit ripening.     

Isolation and functional characterization of cold-regulated promoters, by digitally identifying peach fruit cold-induced genes from a large EST dataset

Background  

Cold acclimation is the process by which plants adapt to the low, non freezing temperatures that naturally occur during late autumn or early winter. This process enables the plants to resist the freezing temperatures of winter. Temperatures similar to those associated with cold acclimation are also used by the fruit industry to delay fruit ripening in peaches. However, peaches that are subjected to long periods of cold storage may develop chilling injury symptoms (woolliness and internal breakdown). In order to better understand the relationship between cold acclimation and chilling injury in peaches, we isolated and functionally characterized cold-regulated promoters from cold-inducible genes identified by digitally analyzing a large EST dataset.     

Down-regulation of two non-homologous endogenous tomato genes with a single chimaeric sense gene construct

Tomatoes (Lycopersicon esculentum Mill cv. Ailsa Craig) were transformed with a gene construct having 244 bp of the 5 end of a polygalacturonase (PG) cDNA, coding for a 71 amino acid N-terminal extension to the mature protein, fused to 1320 bp of a pectinesterase (PE) cDNA encoding the full sequence of the mature PE protein. This chimaeric gene was inserted in a sense orientation between a CaMV 35S promoter and terminator for constitutive expression. In transformed tomato plants expression of the endogenous PG and PE genes in the fruit was inhibited; there was little or no observable PG and PE mRNA and a substantial reduction in the level of PG and PE enzyme activity. The transgene was expressed in the leaves of the transformed plants as demonstrated by the accumulation of mRNA, but no protein product could be identified. However, no transgene mRNA or protein were observed in the transgenic fruit.This paper represents the first report of the down-regulation of two non-homologous endogenous genes using a single gene construct. A sense gene construct was responsible for these effects. These findings are discussed in relation to possible mechanisms of action of co-suppression.     

细胞壁代谢与琯溪蜜柚果实成熟过程汁胞粒化的关系

以易发生汁胞粒化的老龄树和不易发生汁胞粒化的适龄树的琯溪蜜柚（Citrus grandis（L．）Osbeck‘Guanximiyou’1果实为材料,研究了果实成熟过程中汁胞粒化发生与细胞壁代谢的关系。结果表明：老龄树果实的汁胞粒化指数随着果实成熟而上升。在汁胞粒化发生过程中,汁胞维持较低的细胞壁降解酶[果胶甲酯酶（PE）、多聚半乳糖醛酸酶（PG）、纤维素酶（Cx）]活性,保持较高的细胞壁物质（原果胶、纤维素、半纤维素）含量;尤其在汁胞粒化的起动阶段和加快阶段,纤维素、半纤维素含量极显著增加。相反,适龄树果实的汁胞粒化指数在果实成熟过程中变化不大,汁胞中细胞壁降解酶活性较高,促进原果胶、纤维素、半纤维素等细胞壁物质的降解,保持较低的细胞壁物质含量,使汁胞发育正常、柔软多汁。这说明PE、PG、Cx活性和原果胶、纤维素、半纤维素含量与錧溪蜜柚汁胞粒化密切相关。     

Reduction of tomato polygalacturonase beta subunit expression affects pectin solubilization and degradation during fruit ripening.

The developmental changes that accompany tomato fruit ripening include increased solubilization and depolymerization of pectins due to the action of polygalacturonase (PG). Two PG isoenzymes can be extracted from ripe fruit: PG2, which is a single catalytic PG polypeptide, and PG1, which is composed of PG2 tightly associated with a second noncatalytic protein, the beta subunit. Previous studies have correlated ripening-associated increases in pectin solubilization and depolymerization with the presence of extractable PG1 activity, prior to the appearance of PG2, suggesting a functional role for the beta subunit and PG1 in pectin metabolism. To assess the function of the beta subunit, we produced and characterized transgenic tomatoes constitutively expressing a beta subunit antisense gene. Fruit from antisense lines had greatly reduced levels of beta subunit mRNA and protein and accumulated < 1% of their total extractable PG activity in ripe fruit as PG1, as compared with 25% for wild type. Inhibition of beta subunit expression resulted in significantly elevated levels of EDTA-soluble polyuronides at all stages of fruit ripening and a significantly higher degree of depolymerization at later ripening stages. Decreased beta subunit protein and extractable PG1 enzyme activity and increased pectin solubility and depolymerization all cosegregated with the beta subunit antisense transgene in T2 progeny. These results indicate (1) that PG2 is responsible for pectin solubilization and depolymerization in vivo and (2) that the beta subunit protein is not required for PG2 activity in vivo but (3) does play a significant role in regulating pectin metabolism in wild-type fruit by limiting the extent of pectin solubilization and depolymerization that can occur during ripening. Whether this occurs by direct interaction of the beta subunit with PG2 or indirectly by interaction of the beta subunit with the pectic substrate remains to be determined.     

葡萄贮藏过程中落粒与离区酶活性变化及植物生长调节物质的关系

研究了无核白葡萄(Vitis vinifera L.)采后贮藏过程中离区纤维素酶、果胶酯酶(Pectinesterase,PE)、多聚半乳糖醛酸酶(polygalacturonase,PG)、脂氧合酶(lipoxygenase,LOX)和过氧化物酶(peroxidase,POD)活性的变化与落粒的关系及植物生长调节物质对其的影响。结果表明,葡萄在贮藏过程中,伴随浆果落粒的增加,离区纤维素酶、PG、LOX、POD活性升高,PE活性下降。离区纤维素酶、PG、LOX等酶的活性与葡萄落粒程度之间呈显著正相关。外源ABA和CEPA处理能增强离区纤维素酶、PG、LOX活性,促进落粒;GA3,IAA处理则能抑制离区纤维素酶、PG、LOX活性,减轻落粒。ABA对落粒的促进效应及GA3对纤维素酶活性和落粒的抑制效应尤为明显,表明GA3与ABA比值在葡萄采后落粒过程中起重要的作用。     

Isolation and characterization of a tomato non-specific lipid transfer protein involved in polygalacturonase-mediated pectin degradation

An important aspect of the ripening process of tomato fruit is softening. Softening is accompanied by hydrolysis of the pectin in the cell wall by pectinases, causing loss of cell adhesion in the middle lamella. One of the most significant pectin-degrading enzymes is polygalacturonase (PG). Previous reports have shown that PG in tomato may exist in different forms (PG1, PG2a, PG2b, and PGx) commonly referred to as PG isoenzymes. The gene product PG2 is differentially glycosylated and is thought to associate with other proteins to form PG1 and PGx. This association is thought to modulate its pectin-degrading activity in planta. An 8 kDa protein that is part of the tomato PG1 multiprotein complex has been isolated, purified, and functionally characterized. This protein, designated 'activator' (ACT), belongs to the class of non-specific lipid transfer proteins (nsLTPs). ACT is capable of 'converting' the gene product PG2 into a more active and heat-stable form, which increases PG-mediated pectin degradation in vitro and stimulates PG-mediated tissue breakdown in planta. This finding suggests a new, not previously identified, function for nsLTPs in the modification of hydrolytic enzyme activity. It is proposed that ACT plays a role in the modulation of PG activity during tomato fruit softening.     

利用电子鼻评价桃果实香气

为了明确不同桃品种资源果实香气差异,对桃果实香气评价和品质改良提供参考,本研究利用电子鼻系统对桃品种资源果实整果香气进行测定和区分.通过PEN 3.5电子鼻系统采集74份不同品种资源桃果实芳香成分并得到了不同传感器的响应值,采用主成分(PCA)、线性判别法(LDA)和负荷加载(LO)方法分析数据.LO分析结果显示,硫化...     

宁夏枸杞果实内源激素的变化及其与细胞壁成分和相关酶的关系

采用高效液相色谱技术, 研究了不同发育时期宁杞1号和宁杞5号枸杞(Lycium barbarum)果皮和种子内源激素(玉米素(ZT)、赤霉素(GA₃)、生长素(IAA)和脱落酶(ABA))含量与果实生长发育的关系。结果表明, 宁杞5号果实横纵径和单粒重均大于宁杞1号, 果实发育前期, 尤其是缓慢生长期, 是宁杞5号果实大小和重量积累的关键时期。宁杞1号种子中的生长素含量与果实横径和果实单粒重均呈极显著正相关, 与果实纵径呈显著正相关。宁杞5号果皮中玉米素含量与果实横径和单粒重均呈显著正相关, 种子中生长素含量与果实横径和单粒重均呈显著正相关。玉米素促进细胞的分裂, 而细胞数目比细胞体积对决定果实大小的作用更大; 在缓慢生长期(开花后8–25天), 宁杞5号果皮和种子中的脱落酸含量均显著小于宁杞1号。以上两点可能是宁杞5号的横纵径和单粒重均大于宁杞1号的主要原因。宁杞1号果皮中的GA₃与半纤维素酶(Cx)活性的变化相反, 说明宁杞1号果实发育前期和中期果皮中高含量的GA₃对细胞壁中Cx活性有一定的抑制作用, 从而表现出果实膨大。宁杞5号果皮中的IAA与多聚半乳糖醛酸酶(PG)和果胶酯酶(PE), ZT与PE, ABA与Cx, 种子中的ZT与PE的变化均相反, 说明宁杞5号果实发育前期和中期果皮中高含量的IAA、ZT、ABA及种子中的ZT可促进果实的膨大。推测这可能是宁杞5号果实单粒重大于宁杞1号的主要原因之一。宁杞5号果皮中的ZT和种子中的IAA可以增强Cx的活性; 宁杞1号果皮中的ABA可以增强PE的活性, 进而促进果实的成熟。     

Cell Wall Metabolism in Ripening Fruit (VI. Effect of the Antisense Polygalacturonase Gene on Cell Wall Changes Accompanying Ripening in Transgenic Tomatoes)

Cell walls of tomato (Lycopersicon esculentum Mill.) fruit, prepared so as to minimize residual hydrolytic activity and autolysis, exhibit increasing solubilization of pectins as ripening proceeds, and this process is not evident in fruit from transgenic plants with the antisense gene for polygalacturonase (PG). A comparison of activities of a number of possible cell wall hydrolases indicated that antisense fruit differ from control fruit specifically in their low PG activity. The composition of cell wall fractions of mature green fruit from transgenic and control (wild-type) plants were indistinguishable except for trans-1,2-diaminocyclohexane-N,N,N[prime],N[prime]-tetraacetic acid (CDTA)-soluble pectins of transgenic fruit, which had elevated levels of arabinose and galactose. Neutral polysaccharides and polyuronides increased in the water-soluble fraction of wild-type fruit during ripening, and this was matched by a decline in Na2CO3-soluble pectins, equal in magnitude and timing. This, together with compositional analysis showing increasing galactose, arabinose, and rhamnose in the water-soluble fraction, mirrored by a decline of these same residues in the Na2CO3-soluble pectins, suggests that the polyuronides and neutral polysaccharides solubilized by PG come from the Na2CO3-soluble fraction of the tomato cell wall. In addition to the loss of galactose from the cell wall as a result of PG activity, both antisense and control fruit exhibit an independent decline in galactose in both the CDTA-soluble and Na2CO3-soluble fractions, which may play a role in fruit softening.     

Reevaluation of the changes in polygalacturonases in tomatoes during ripening

A procedure was developed for the differential extraction of polygalacturonases (PG) I and II from tomatoes (Lycopersicon esculentum Mill.). Extraction of pericarp tissue from ripe fruit at conventional conditions of 1.0 M NaCl and pH 6.0 yielded nearly equal amounts of the two enzymes. However, most of the PG activity could be extracted also with water at pH 1.6, and the water extract contained only PG II. Subsequent extraction of the pellet with 1.0 M NaCl at pH 6.0 and 10.0 yielded some PG I and high levels of PG converter, the protein in tomatoes that reacts with PG II to form PG I. Application of this procedure to tomatoes at different stages of ripening showed that PG II appeared as ripening began and then increased during ripening. Much lower levels of PG I than of PG II were extracted at all stages of ripeness. The PG converter was present in unripe fruit and increased during ripening. The results demonstrate that PG I is formed when PG II and PG converter are solubilized simultaneously and that PG II is the only endogenous PG in tomatoes.Abbreviation PG polygalacturonase