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  • Author or Editor: Russell Pressey x
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A new procedure has been developed for separating the 2 polygalacturonases (PG I and PG II) in ripe tomatoes (Lycopersicon esculentum Mills cv. Better Boy). It involves high performance liquid chromatography of tomato extracts on a short column of a strong cation exchanger and elution with a salt gradient. The advantages of this procedure are that resolution of the polygalacturonases is achieved in only 20 min, and the extracts containing low levels of activity can be analyzed.

Open Access
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Abstract

The conditions for extracting and assaying tomato (Lycopersicon esculentum Mill.) polygalacturonases (PG I and PG II) have been re-examined. The enzymes were not extracted by water at pH 3, which allowed washing of the cell wall fraction to remove the soluble components that interfere in the PG assay. The extractability of PG in water increased as the pH was lowered or raised from 3, with optima near pH 1.8 and 6.5. Only PG II was extracted by water at pH 1.8, whereas both isoenzymes were extracted at pH 6.5. The extractabilities of the PGs were increased by NaCl, but the amount of total activity extracted by 1.2 m NaCl was independent of pH between 2 and 9. Extracts in 1.2 m NaCl of pH 3 washed cell walls from ripe tomatoes could be assayed without concentration or dialysis. Higher PG activity was recovered when extracts were concentrated by ultrafiltration than by precipitation with (NH4)2SO4. The results indicate that the isoenzyme composition and recovery of PG from tomatoes were dependent on extraction and concentration procedures.

Open Access
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Tomatoes (Lycopersicon esculentum Mill. ‘Tropic’) were examined for changes during ripening in fruit firmness, water-soluble pectin, the 2 polygalacturonases (PG I and PG II), and polygalacturonase converter. The loss of fruit firmness and increase in water-soluble pectin at the turning stage of ripeness coincided with the appearance of PG activity. The initial activity was due exclusively to PG I, which continued to increase during ripening. PG I was the major polygalacturonase isoenzyme in extracts of tomatoes at all stages of ripeness, contrary to previous reports. PG II was first detected in pink fruit and increased markedly with ripening. Polygalacturonase converter was present in green tomatoes, but also began to increase at the turning stage. The results indicate that PG I may be formed during extraction of tomatoes from PG II and the converter as they are solubilized.

Open Access
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Chinese water chestnuts retain crispness during heating much better than most vegetables. To help explain this unusual property of water chestnuts, a study was conducted to determine their cell wall composition and to assay some of the enzymes that may be involved in hydrolysis of cell walls and starch. Water chestnuts were found to contain high levels of β-1,3-glucanase and β-glucosidase but low cellulase. A number of other enzymes were detected including invertase, α- and β-galactosidases and α-mannosidase. A rather high level of amylase is present in water chestnuts and most of the activity appears to be due to β-amylase. Water chestnuts contain low pectinesterase but a moderate amount of polygalacturonase which was purified and characterized. It is an exoenzyme that does not require Ca2+ for activity in contrast to most other exopolygalacturonases. An unusual property of the water chestnut polygalacturonase is its stability to heat, with retention of most of its activity after heating at 80°C for 5 min. The cell walls of water chestnuts contain low pectin which is solubilized slowly by pectic enzymes.

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Polygalacturonase (PG) in higher plants has been considered to be associated with ripening fruits although it is now known to be present in foliage and storage organs. We recently found very high levels of PG in some grass pollens (Plant Science 59, 57-62, 1989). This prompted an examination of other pollens for PG activity. All of the pollens analyzed contained PG but the range of activities was great. Eastern cottonwood pollen contained the most PG, with a level about 12 times higher than that usually found in ripe tomato fruit. Pollens from the other members of Populus were generally high in PG. Pollens from the oak family also contained very high PG, with the highest amount in white oak pollen. Pollens from pecan, English walnut, willows, birch and hickories contained moderate levels of PG. The lowest amounts of PG were found in pollens from beech, sycamore and conifers. The PG's from the two richest sources (eastern cottonwood and white oak pollens) were partially purified and characterized. Both enzymes were found to be exopolygalacturonases that require Ca2+ for activity. PG may be involved in some function related to pollination but an explanation for the wide range of activities indifferent pollen is not obvious.

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Polygalacturonase (PG) in higher plants has been considered to be associated with ripening fruits although it is now known to be present in foliage and storage organs. We recently found very high levels of PG in some grass pollens (Plant Science 59, 57-62, 1989). This prompted an examination of other pollens for PG activity. All of the pollens analyzed contained PG but the range of activities was great. Eastern cottonwood pollen contained the most PG, with a level about 12 times higher than that usually found in ripe tomato fruit. Pollens from the other members of Populus were generally high in PG. Pollens from the oak family also contained very high PG, with the highest amount in white oak pollen. Pollens from pecan, English walnut, willows, birch and hickories contained moderate levels of PG. The lowest amounts of PG were found in pollens from beech, sycamore and conifers. The PG's from the two richest sources (eastern cottonwood and white oak pollens) were partially purified and characterized. Both enzymes were found to be exopolygalacturonases that require Ca2+ for activity. PG may be involved in some function related to pollination but an explanation for the wide range of activities indifferent pollen is not obvious.

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Tomatoes contain a high level of the enzýme invertase which hydrolyzes sucrose to glucose and fructose. A protein that inhibits invertase has been found in tomatoes, suggesting that it may be involved in the regulation of invertase activity. A procedure was developed for the separation of the inhibitor from invertase based on the higher stability of the inhibitor in acid solutions. Residual invertase in acid extracts of pericarp tissue was removed by anion exchange chromatography. The inhibitor has been purified to homogeneity by subsequent gel filtration and cation exchange chromatography. It is a heat-stable protein with a mol wt of 18,000. The reactivity of the inhibitor with tomato invertase is pH dependent with maximum inhibition at pH 4.7 and no inhibition at pH 3.5 and 6.2. The result is double pH optima for invertase activity in the presence of the inhibitor. The inhibitor is effective on invertase from potatoes and may be similar to the invertase inhibitor found in potatoes.

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Polygalacturonase inhibitors have been reported in a number of dicotyledonous plant tissues including pear and raspberry fruits and bean seedlings. These proteins inhibit fungal polygalacturonases and thus have been implicated in disease resistance in plants. The earlier work on the inhibitor from bean plants was conducted with hypocotyls as the source. We have found that immature bean pods contain much more inhibitor than other parts of the plant and developed a procedure for purification of this inhibitor. Fresh bean pods were extracted with 1.0 M NaCl at pH 7 and the proteins were precipitated with ammonium sulfate. The proteins were dissolved, dialyzed and chromatographed on a column of S-Sepharose. The inhibitor from this step was then chromatographed on a Mono Q column at high pH. Yields of the inhibitor varied somewhat with bean cultivar and pod maturity but were about ten times higher than from hypocotyls. The purified inhibitor reacted optimally with Aspergillus niger endopolygalacturonase at pH 4.3 and appeared to be similar to the inhibitor from hypocotyls. Bean pods thus are a convenient source of polygalacturonase inhibitor for studies on fruit maturation and disease resistance in plants.

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Exopolygalacturonase (exo-PG) (EC 3.2.1.67) was investigated for ability to induce ethylene production in green cherry tomatoes (Lycopersicon esculentum Mill.). The fruit were vacuum-infiltrated with various levels of exo-PG from green tomato fruit, squash flower, or oak pollen and compared to boiled enzyme or salt controls for ethylene production. In all cases, fruit treated with active enzymes produced significantly higher levels of ethylene than did control fruit. The ethylene response was evident 2 hours after treatment and was transient in nature, returning to basal levels by 22 hours. The amount of ethylene produced did not appear to be influenced by the source of exo-PG.

Free access

Abstract

The changes in pectinesterase (PE) activity during maturation and ripening of ‘Long Keeper’ tomatoes (Lycopersicon esculentum Mill.) were similar to those of ‘Marion’ tomatoes. The pectinesterase activity was due to similar forms of this enzyme in both cultivars. In contrast, the polygalacturonase (PG) activity in ripe ‘Long Keeper’ tomatoes was much lower than that in ripe ‘Marion’ fruit. The activity in ‘Long Keeper’ fruit was due to 2 enzymes, one of which was similar to that found in ‘Marion’ fruit, but most of the activity appeared to be due to a new polygalacturonase. These differences may to a large extent account for the postharvest quality retention of the ‘Long Keeper’ cultivar.

Open Access