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.     

Content of RNA polymerase in haploid and merodiploid strains of Escherichia coli

Summary In cell-free extracts of E. coli merodiploids carrying F-factor with ilv-thi chromosome fragment the activity of RNA polymerase is not increased, and there is no excess of free active core-enzyme or sigma-factor. Only immunochemical analysis reveals 25% excess of RNA polymerase material in some merodiploids as compared to a haploid. However, neither the amount of + relative to total protein nor : ratio does not differ in haploid and merodiploids.     

Accumulation of the β-subunit of polygalacturonase 1 in normal and mutant tomato fruit

Polyclonal antiserum raised against the native PG1 isoform of tomato fruit (Lycopersicon esculentum Mill.) polygalacturonase [poly(1,4--d-galacturonide) glycanohydrolase, EC 3.2.1.15] bound to each of the subunits of the protein and also to a range of other fruit proteins. Affinity purification was used to remove antibody molecules that bound to the native form of the PG2 isoform. The resulting serum bound to native PG1, denatured PG2 and -subunits of PG1 but not to native PG2 or other fruit proteins. This anti-PG1 serum was used to monitor the occurrence of the PG1 -subunit and PG2 in detergent extracts of tomato tissues. The -subunit polypeptide was detected in pericarp but not locule tissue of fruit, including fruit of the rin and nor mutants. It increased in amount in the pericarp tissues from an early stage to the mature green stage, clearly prior to any appreciable accumulation of the PG2 subunit. The -subunit polypeptide was not detected in stem or leaf tissues. A PG2-specific antiserum was used to study the interaction of PG2 with the isolated -subunit. The PG2 isoform was bound to the -subunit over a wide range of salt concentrations and pH; the interaction was independent of the presence of reducing agents. It is concluded that strong non-covalent forces are involved in the interaction. The results are consistent with a model in which the -subunit is positioned in the cell wall structure and provides a specific binding site for the active PG2 subunit when this is synthesised during ripening.Abbreviations B breaker - MG mature green - M_r relative molecular mass - nor non-ripening mutant - PAGE polyacrylamide gel electrophoresis - PG polygalacturonase - rin ripening inhibitor mutant - SDS sodium dodecyl sulphate     

Kiwifruit β-galactosidase: Isolation and activity against specific fruit cell-wall polysaccharides

A -galactosidase (EC 3.2.1.23) capable of degrading a number of fruit cell-wall polysaccharides in vitro, was isolated from ripening kiwifruit (Actinidia deliciosa [A. Chev.] C.F. Liang et A.R. Ferguson cv. Hayward). The enzyme has a molecular weight of approximately 60 kDa by gel permeation and consists of several basic isoforms. Several polypeptides were enriched during purification, with 33-, 46- and 67-kDa bands being predominant after sodium dodecyl sulphate-polyacrylamide gel electrophoresis. The optimum activity of the enzyme against p-nitrophenyl--d-galactopyranoside was at pH 3.2, but against a galactan purified from kiwifruit cell walls, it was at pH 4.9. The enzyme was specific for galactosyl residues in the -configuration, releasing galactose from a variety of kiwifruit cell-wall polysaccharide fractions including cell wall material, Na₂CO₃-soluble pectin, high-molecular-weight galactan, xyloglucan, and galactoglucomannan. A galactosylated glucuronomannan found throughout the kiwifruit plant was also a substrate for the enzyme. The results indicate that the enzyme attacks the non-reducing end of galactose side chains, cleaving single galactose residues which may be attached to the 2, 3, 4, or 6 position of the aglycone. Activity of the enzyme in-vitro was too low to account for the total loss of galactose from the cell walls during ripening. If the -galactosidase of this study is solely responsible for the removal of galactose from the cell wall during ripening then its in-vivo activity must be much greater than that observed in-vitro.Abbreviations CWM cell wall material - SDS-PAGE sodium dodecyl sulphate-polyacrylamide gel electrophoresis We thank Bronwyn Culling and Teresa Wegrzyn for assistance and acknowledge a contribution towards the cost of the research from the New Zealand Kiwifruit Marketing Board.     

Microbial transformation of ginsenoside Rb1 by Rhizopus stolonifer and Curvularia lunata

Of 49 microbial strains screened for their capabilities to transform ginsenoside Rb₁, Rhizopus stolonifer and Curvularia lunata produced four key metabolites: 3-O-[-d-glucopyranosyl-(1,2)--d-glucopyranosyl]- 20-O-[-d-glucopyranosyl]-3,12, 20(S)-trihydroxydammar-24-ene (1), 3-O-[-d-glucopyranosyl-(1,2)--d- glucopyranosyl]-20-O-[-d-glucopyranosyl]-3,12, 20(S)-trihydroxydammar-24-ol (2), 3-O-[-d-gluco- pyranosyl-(1,2)--d-glucopyranosyl]-3, 12, 20(S)-trihydroxydammar-24-ene (3), and 3-O--d-glucopyranosyl-3, 12, 20(S)-trihydroxydammar-24-ene (4), identified by TOF-MS, ¹H- and ¹³C-NMR spectral data. Metabolites 1, 3 and 4 were from the incubation with R. stolonifer, and 1 and 2 from the incubation with C. lunata. Compound 2 was identified as a new compound.     

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.     

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.     

Anti-diabetic activity of <Emphasis Type="Italic">β</Emphasis>-glucans and their enzymatically hydrolyzed oligosaccharides from <Emphasis Type="Italic">Agaricus blazei</Emphasis>

-Glucans were prepared from Agaricus blazei Murill by repeated extraction with hot water. The average molecular weights of -glucans were 30–50 kDa by gel filtration chromatography. Oligosaccharides (AO), derived from hydrolyzing -glucans with an endo--(16)-glucanase from Bacillus megaterium, were mainly di- and tri-saccharides. Though -glucans and AO both showed anti-hyperglycemic, anti-hypertriglyceridemic, anti-hypercholesterolemic, and anti-arteriosclerotic activity indicating overall anti-diabetic activity in diabetic rats, AO had about twice the activity of -glucans with respect to anti-diabetic activity.     

Cell wall metabolism in gibberellin-treated persimmon fruits

The application of gibberellin [GA₃] to persimmon fruits as an orchard spray, at least 2 weeks prior to harvest, has been shown to delay ripening of the fruit on the tree and its rate of softening after harvest. This effect persisted during and after cold storage. The delay in softening has been attributed to the effect of the phytohormone on cell wall metabolism. To examine this hypothesis, cell walls of GA₃-treated fruit were compared to those of non-treated fruit. Comparison between fruit was from harvest till the termination of post-storage softening. The study included TEM examinations, assay of certain hydrolase activities and determination of compositional changes occurring in the various cell-wall carbohydrate polymers. Our findings indicate that GA₃ either delays or inhibits all of the cell wall changes that were found to accompany fruit softening, including dissolution of the middle lamella, separation of the plasmalemma from the cell-wall, mitigation of the structural coherence and density of the primary cell wall, increases solubilization of pectic polymers, loss of neutral sugars, predominantly arabinose and galactose, and increased activities of exo-polygalacturonase [PG] and endo-1,4--glucanase [EGase]. The principal discernible compositional difference between GA₃-treated fruit and control fruit at harvest was a higher total carbohydrate content in the cell wall material extracted from GA₃-treated fruit, which was due chiefly to an increased amount of cellulose.