Browsing by Author "Huber, Donald J."
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- Autolysis of cell walls from polygalacturonase-antisense tomato fruit in simulated apoplastic solutionsPublication . Almeida, Domingos P. F.; Huber, Donald J.Autolysis of cell walls from polygalacturonase (PG)-antisense tomato fruit was studied in a conventional buffer designed to maximize the catalytic activity of PG (30 mM sodium acetate, 150 mM NaCl, pH 4.5), and in solutions mimicking the pH and mineral composition of the fruit apoplast at the mature-green and ripe stages. Autolytic release of uronic acids was very limited under simulated apoplastic conditions compared with the conventional buffer, but minimal differences in the release of reducing groups were observed among the incubation conditions. Autolytic release of uronic acids from active walls was lower than solubilization from enzymically inactive walls. Uronic acids that remained ionically bound to the cell walls during autolysis were subsequently extracted and analyzed by size exclusion chromatography. The elution profiles of ionically bound uronic acids from cell walls incubated under optimal conditions were similar for all ripening stages. In solutions mimicking the pH and mineral composition of the apoplast of mature-green and ripe fruit, uronic acids extracted from pink and ripe fruit cell walls showed a decrease in average molecular mass compared with polymers from mature-green cell walls. The results suggest that the composition of the incubation solution exert strong influence on PG-independent cell wall autolysis and that enzymically active walls restrain PG-independent pectin solubilization.
- In vivo pectin solubility in ripening and chill-injured tomato fruitPublication . Almeida, Domingos P. F.; Huber, Donald J.In vivo pectin solubility was examined in ripening and chill-injured tomato fruit with down-regulated polygalacturonase (PG, EC 3.2.1.15) activity and untransformed wild-type fruit by analyzing a pressure-extracted fluid of apoplastic origin. Pectin concentration in the apoplastic fluid increased threefold during ripening and was not affected by endogenous PG. In contrast, PG strongly affected pectin concentration in a bulk pericarp fluid obtained after tissue disruption. There was a 14-fold increase in bulk pectin levels during ripening of PG-antisense fruit and a 36-fold increase in wild-type. Pectins soluble in the apoplastic fluid decreased slightly during storage of fruit at 5ºC for 14 days but increased considerably upon subsequent transfer to 15ºC. Concentration of monomeric galactose in the apoplastic fluid increased during ripening from 41 to 67 µg mL⁻¹. Galactose levels were threefold to fourfold higher in the bulk than in the apoplastic fluid. Low-temperature storage caused a 50% reduction in the galactose present in the bulk fluid and a 20% reduction in apoplastic concentration of galactose. These results indicate that pectin dissolution in ripening tomato fruit is PG-independent even though the enzyme is catalytically active in ripe fruit. Low-temperature storage reduces in vivo pectin solubility, an effect that is reversed upon transfer to higher temperature following cold storage
- Polygalacturonase-mediated dissolution and depolymerization of pectins in solutions mimicking the pH and mineral composition of tomato fruit apoplastPublication . Almeida, Domingos P. F.; Huber, Donald J.The effects of polygalacturonase (PG) on pectin dissolution and depolymerization were examined in cell walls from mature-green tomato fruit incubated in a conventional (C) buffer (30 mMsodium acetate, 150 mMNaCl, pH 4.5) and in buffers mimicking the apoplastic solution of maturegreen (MG) and ripe fruit (R). Pectin dissolution from cell walls was much higher in C-buffer than in MG- or R-buffers. Buffered phenol inactivated cell walls incubated in C-buffer released 4.9 mg mg 1 pectin, which increased to 86.4 mg mg 1 when PG was added. In the R-buffer, PG increased the pectin dissolution from inactive cell walls from 0.5 to 18.3 mg mg 1. However, when the assay was conducted in buffer mimicking maturegreen fruits, added PG did not increase pectin dissolution. The release of uronic acids from active cell walls in C-buffer and R-buffer was consistently lower than that from inactive walls due to the activity of pectinmethylesterase. Gel filtration profiles of CDTA-soluble pectins extracted from cell walls previously incubated in C-buffer or R-buffer with PG reveal that the enzyme is capable of hydrolyzing insoluble, ionically bound, pectins. These data support the idea that pH and mineral composition of the fruit apoplast provide a means for biochemical regulation of cell wall metabolism.