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A general feature of tomato fruit containing genetically reduced levels of polygalacmronase activity is decreased deterioration and cracking, particularly when handled at the ripe and over ripe stages. As fully ripe fruit are metabolically compromised and very prone to mechanical injuries, we investigated the influence of impact bruising on electrolyte leakage, pectin solubility, and depolymerization in ripening tomato fruit.
`Sunny' tomato fruit harvested at the mature-green, turning, and ripe stages of development and subjected to controlled impact injury exhibited elevated ethylene production at all developmental stages. Subsequent analyses were performed on discs prepared from bruised and uninjured pericarp tissue. Discs from bruised tissues exhibited enhanced electrolyte leakage and, in bruised tissues from ripe fruit, enhanced pectin efflux. Levels of soluble pectins derived from ethanol-insoluble powders were unaffected by bruising; however, pectins from bruised ripe fruit exhibited mol wt downshifts relative to those from nonbruised tissues.
Hydrolytic activities in liquefying locule tissue of mature-green tomato (Lycopersicon esculentum Mill. `Solar Set') fruit were studied in pursuing the understanding of mechanisms involved in the rheological changes occurring in this tissue. Ethanol-insoluble solids (EIS) were prepared with and without enzyme-inactivating treatment. The release of uronic acids from enzymically active EIS incubated under autolysic conditions was 5-fold higher than recoveries from inactive EIS. Uronic acid release was partially inhibited by 1 mm Hg2+. Cell-free proteins extracts from active EIS exhibited hydrolytic activity against inactive EIS. Pectins released from active EIS showed no evidence of main-chain hydrolysis. Neutral sugars recovered as 80% ethanol-soluble products of autolytic reactions included glc, gal, rha, ara, xyl, and man. Gal was recovered at significant higher levels in autolysates of active EIS. Glycosidases present at high activities in locule tissue included α- and β-galactosidases, β-mannosidase, β-arabinosidase, and β-glucosidase. The results confirm our earlier findings that the metabolism of water, chelator, and alkali-soluble pectins in tomato locule tissue involves deglycosylation with no apparent depolymeriation. These changes alone appear to be inadequate in explaining the unique rheological characteristics in locule gel tissue.
The high catalytic potential of PG evident in reactions with soluble pectic polymers is typically not expressed in vivo. In this study, the binding and catalytic properties of PG isozyme 2, and the influence of the B-subunit protein, were investigated using cell walls prepared from tomato fruit expressing the B-subunit antisense gene. Cell walls were prepared from mature-green fruit and treated to remove/inactivate endogenous enzymes. Walls were then preloaded with rate-limiting quantities of purified PG 2, and incubated under catalysis-promoting conditions over the range of pH from 4.5 to 6.0. Cell walls of both B-subunit antisense and wild-type fruit retained comparable quantities of loaded PG 2. The enzymic release of pectin from PG-loaded walls was proportional to the quantity of wall-bound PG 2. In walls lacking the B-subunit protein, the quantity of pectin released by a given dose of wall-associated PG was as much as 2-fold higher than from wild-type walls. The B-subunit protein also influenced the extent of pectin depolymerization during ripening. The release of pectin from cell walls during periods of catalysis was not the sole indicator of the range of pectins hydrolyzed. Treating postcatalytic loaded cell walls to inactivate PG, and subsequent extraction of residual wall pectins using 50 mm CDTA solutions solubilized polymers of significantly lower mol mass compared with pectins solubilized directly from nonloaded cell walls.
The absence of endo-α-1,4-d-galacturonanase (PG, EC 3.2.1.15) in some fruits and the molecular suppression of PG in tomato fruit have collectively provided evidence that this protein is neither required nor sufficient to achieve normal softening in fleshy fruits. On the other hand, initial claims that down-regulation of PG was without effect on tomato softening were overstated. The influence of PG on softening does appear to be minimal during the initial stages of ripening, during which time changes in the locule tissues can significantly alter texture as monitored in whole fruit. Enzymes, including pectinmethylesterases, cellulases, rhamnogalacturonanase, and glycosidases may also play pivotal roles in softening. β-galactosidases have attracted much attention as potential determinants of fruit texture; however, conclusive evidence for this role is lacking, and increased levels of β-galatosidase (and net cell wall galactosyl residue loss) have been noted in senescing vegetative and floral organs as well as in fruit. Apoplastic pH, ionic activity, and composition are likely to contribute to tissue and wholeorgan texture through weakening of polymer aggregates and/or through modulation of cell wall enzyme activity. During the latter stages of ripening and overripening, the role of PG is apparent from the persistent structural integrity of fruit transformed with PG antisense constructs. Patterns of softening and deterioration in normal tomato fruit suggest that the catalytic activity of PG in vivo is initially queued and does not parallel the accumulation of PG protein. Developmental changes in membrane permeability, physical injury, and other stress conditions can alter the apoplastic environment, releasing constraints on PG action.
Pre-ripe `Booth 7' avocado (Persea americana Mill.) fruit, a cross of West Indian and Guatemalan strains, were treated with 0.9 μL·L-1 1-methylcyclopropene (1-MCP) for 12 hours at 20 °C. After storage for 18 days in air at 13 °C, at which time whole fruit firmness values averaged about 83 N, half of the 1-MCP-treated fruit were treated with 100 μL·L-1 ethylene for 12 hours and then transferred to 20 °C. 1-MCP delayed softening, and fruit treated with 1-MCP retained more green color than air-treated fruit when full ripe (firmness 10 to 15 N). 1-MCP affected the activities of pectinmethylesterase (EC 3.2.1.11), α-(EC 3.2.1.22) and β-galactosidases (EC 3.2.1.23), and endo-β-1,4-glucanase (EC 3.2.1.4). The appearance of polygalacturonase (EC 3.2.1.15) activity was completely suppressed in 1-MCP-treated fruit for up to 24 days, at which time the firmness of 1-MCP-treated fruit had declined nearly 80% compared with initial values. The effect of exogenous ethylene applied to partially ripened 1-MCP-treated fruit differed for different ripening parameters. Ethylene applied to mid-ripe avocado exerted no effect on the on-going rate or final extent of softening of 1-MCP-treated fruit, even though polygalacturonase and endo-1,4-β-glucanase activities increased in response to ethylene. β-galactosidase decreased in 1-MCP-treated fruit in response to ethylene treatment. 1-MCP delayed the increase in solubility and depolymerization of water- and CDTA (1,2-cyclohexylenedinitrilotetraacetic acid)-soluble polyuronides, likely due to reduced polygalacturonase activity. At the full-ripe stage, the levels of arabinose, galactose, glucose, mannose, rhamnose, and xylose associated with the CDTA-soluble polyuronide fraction were similar among all treatments. In contrast, the galactose levels of water-soluble polyuronides declined 40% and 17% in control and 1-MCP treated fruit, respectively. Hemicellulose neutral sugar composition was unaffected by 1-MCP or ethylene treatment. The data indicate that the capacity of avocado fruit to recover from 1-MCP-mediated suppression of ripening can be only partially amended through short-term ethylene application and differs significantly for different ripening parameters.
Enzymically active cell wall isolated from mature-green and ripening tomato (Lycopersicon esculentum Mill cv. `Rutgers') fruit was employed to investigate the mobility of the enzyme polygalacturonase (PG, EC 3.2.1.15). Cell walls from mature-green `Rutgers' fruit or from the ripening mutant rin, which alone exhibits little or no release of pectin, were unaffected by the addition of enzymically active cell wall from ripening `Rutgers' fruit, indicating that PG is either not transferred at all or is not transferred to sites of pectin hydrolysis. The quantity of pectin released by the addition of soluble PG to enzymically active wall depended on the quantity of enzyme added. Similar data were obtained using purified PG2. Pectin solubilization from all wall isolates exhibiting enzymically mediated pectin release diminished with time; however, transfer to fresh buffer initiated a resumption of autolytic activity, indicating that an inhibitor is released during the course of pectin hydrolysis.
Abstract
A vacuum infiltration technique that allowed precise control of both infiltration rate and amount of solution administered to whole tomato (Lycopersicon esculentum) fruit was developed. Controlled volumes of 5 mm solutions of CuSO4, Cu(NO3)2, HgCl2, CaSO4, KNO3, (NH4)2C2O4, Na2HPO4, citrate and 1 mm EDTA or EGTA were infiltrated into intact, mature-green tomato fruit and evaluated with regard to their effect on the pattern of tomato ripening. Copper significantly accelerated lycopene accumulation and influenced both the timing and magnitude of climacteric ethylene production. Infiltration with HgCl2 elicited similar effects as copper, but severe phytotoxicity was observed. In contrast, CaSO4, KNO3, and chelators had no significant effect on the pattern of ripening. Copper initiated wound ethylene production in the ripening mutant rin that reached up to 50% of the wound levels observed in normal fruit, but rin was not induced to ripen.
Changes in the gel filtration behavior (apparent mol mass) of cell wall pectic polymers have been observed in a number of ripening fruits, including some that express little or no detectable polygalacturonase (PG). Pectins from tomato (Lycopersicon esculentum, Mill. v. Solar Set) fruit locule tissue show limited depolymerization during ripening, although alkali-soluble polymers are of reduced mol mass relative to water- and chelator-soluble polymers (Plant Physiol. 111:447). This study addressed whether the lower mol mass of alkali-soluble polymers was a consequence of extraction or specific metabolism of these wall polymers. Pectins from sequential water and chelator extractions of ethanol-insoluble solids from mature green tomato locule tissue were subjected to alkaline conditions. The size distribution of both water- and CDTA-soluble pectins treated with weak alkali were downshifted and similar to those extracted directly by weak alkali, indicating structural similarities of the three pectin fractions. Spectrophotometric analysis showed no involvement of β-elimination hydrolysis in the apparent mol mass reduction. The alkali-treated polymers were of greatly enhanced susceptibility to PG-mediated degradation. The alkali-associated changes also occurred in response to pectinmethylesterase hydrolysis. The results indicate that deesterification can strongly influence gel filtration behavior of pectins and may explain the apparent mol mass decreases of pectins in fruits not containing PG.
A study was conducted to determine the influence of the ethylene action inhibitor, 1-methylcyclopropene (1-MCP) on the shelf life and deterioration of fresh-cut cantaloupe (Cucumis melo var. reticulatus) during storage at 5 °C. Intact cantaloupe fruit, cv. Durango (3/4 to full-slip stage) were treated with 1-MCP (1 μL·L-1) for 24 h at 20 °C. Following cooling to 5 °C, the fruit were processed into ≈2.5-cm cubes and subsequently dipped in 1.34 mm sodium hypochlorite solution for 20 s. Fresh-cut cubes were stored in 1.7-L vented plastic containers for 12 d at 5 °C (85% RH). Intact fruit treated and stored under identical conditions were also examined. While 1-MCP-treated cantaloupe cubes were about 35% firmer than control cubes after the 24-h at 20 °C 1-MCP treatment, little softening occurred in either treatment during the subsequent 12-d at 5 °C storage period. In contrast, control and 1-MCP-treated intact fruit softened nearly 40% and 15%, respectively. 1-MCP did not significantly influence flesh color and soluble solid contents of either intact cantaloupe or fresh-cut cubes during storage at 5 °C. Increased decay incidence was observed in 1-MCP-treated fresh-cut cantaloupe cubes.
Pectin solubility in ripening tomato fruit is typically studied in vitro, employing isolated cell walls; however, it is unknown whether in vitro studies address the actual changes in the status of pectins in the fruit in situ. In vivo pectin solubilization was examined in a pressure-extracted apoplastic fluid obtained from ripening and chill-injured tomato fruit with down-regulated polygalacturonase (PG) activity and untransformed wild-type. Pectin levels in apoplastic fluid increased 3-fold during ripening and were not affected by PG levels. In contrast, PG strongly affected pectin levels in bulk, enzymically active pericarp fluid. There was a 14-fold increase in bulk pectin levels during ripening of PG-antisense fruit and a 36-fold increase in wild-type fruit. Pectin levels in the apoplastic fluid of fruit stored at 5 °C for 14 days were 40% lower than that of freshly harvested mature-green fruit, but increased significantly upon transfer of fruit to 15 °C. Monomeric galactose in the apoplastic fluid increased from 41 mg·mL–1 at the mature-green stage to 67 mg·mL–1 in ripe fruit. Bulk levels of galactose were 3- to 4-fold higher than apoplastic levels. After low-temperature storage galactose levels were 50% and 20% lower than in freshly harvested fruit for the bulk and apoplastic fluids, respectively. These results indicate that in vivo pectin solubilization is restricted and largely independent of PG. Low-temperature storage reduces in vivo pectin solubilization, an effect that is reversed upon transfer of fruit to higher temperature following cold storage.