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- Author or Editor: Donald J. Huber x
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.
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.
Chilling injury limits the postharvest handling of many fruit and vegetables. In low-temperature storage trials, control treatments typically consist of fruit stored above the injury threshold. Since chilling exposures for tomato fruit often exceed 2 weeks, controls stored above the threshold continue to ripen, confounding comparisons with fruit maintained at low temperatures. In this study, the ethylene action inhibitor 1-MCP was used to arrest ripening to permit more valid comparisons between fruit stored under the two temperature regimes. Mature-green tomatoes were treated with EthylBloc and then stored at 5 or 15 °C for 2 or 3 weeks after which time the fruit stored at 5 °C were transferred to 15 °C to allow the expression of injury symptoms. 1-MCP inhibited ripening of fruit stored at 15 °C for 2 to 3 weeks. Color, pericarp firmness, and pectin solubilization of MCP-treated fruit stored at 15 °C remained at the values of mature-green fruit, validating their use as controls for these physiological characteristics. After 2 to 3 weeks at 15 °C, MCP-treated fruit resumed normal ripening. Comparing the fruit removed from low-temperature storage with nonripening controls at 15 °C revealed that storage at 5 °C for 2 to 3 weeks decreased the hue (yellowing) but did not affect chroma or lightness, maintained firmness, and did not affect pectin metabolism. Electrolyte leakage increased or remained unaffected by cold storage. MCP-treated fruit had slightly higher electrolyte leakage than non-MCP-treated fruit after storage at either 5 or 15 °C. We conclude that MCP-treated fruit provide adequate controls in experiments designed to study many aspects of low-temperature storage.
This study was performed to characterize the physiological responses of tomato (Lycopersicon esculentum L.) fruit harvested at either 10% to 30% or 30% to 60% color change and treated with two forms of 1-methylcyclopropene (1-MCP). Tomato fruit were treated either by submersion for 1 min in 1-MCP aqueous solution at the ambient temperature or by exposure for 12 h at 20 °C in air with 1-MCP gas, then stored at 20 °C. The concentrations (1.0, 5.0, or 10.0 μL·L-1) in 1-MCP aqueous solution were achieved through addition of 0.5, 2.5, or 5.0 g of AFxRD-300 powder (2.0% formulation, Agro-Fresh, Inc.) to 10 L of the de-ionized water, following manufacturer's instructions. 1-MCP (0.5 μL·L-1) gas in a 174-L container was achieved through addition of 0.22 g of SmartFresh® powder (0.14% formulation, Agro-Fresh, Inc.) to 100 mL of tap water. Both forms of 1-MCP significantly delayed ripening of fruit at the two initial ripeness stages, as noted by a significant delay in fruit softening and peel color change. The firmness of 30% to 60% color change tomatoes was significantly retained in response to gaseous or aqueous 1-MCP. Control fruit softened rapidly and reached the minimum marketable firmness value (about 5 N) within 8 days of storage at 20 °C, whereas fruit treated with gaseous 1-MCP (0.5 μL·L-1) or aqueous 1-MCP (1.0 or 5.0 μL·L-1) reached the same stage after 16, 20, or 24 days, respectively. Firmness retention was also highly significant for 10% to 30% color change tomatoes treated with both forms of 1-MCP. The highest concentration of aqueous 1-MCP (10.0 μL·L-1) did not result in a further delay in ripening compared with treatment at 5.0 or 1.0 μL·L-1 1-MCP.
Abstract
Studies were conducted to investigate the influence of 50 μl·liter−1 ethylene on the cell wall, polygalacturonase (PG) activity, and electrolyte leakage of harvested watermelon [Citrullus lanatus (thunb) Matsum and Nakai] fruit. Electrolyte leakage was significantly increased in tissues from ethylene-treated fruit. The highest leakage occurred in distilled water, although the net effect of ethylene was less dramatic due to high leakage from control fruit. Leakage was greatly reduced but the ethylene effect more apparent compared to the control when tissues were incubated in an isotonic medium of mannitol or in isotonic medium containing CaCl2. Polygalacturonase activity increased markedly in ethylene-treated fruit, showing a > 10-fold rise during the first 6 days of treatment. Little change in PG activity occurred in melons stored in air, even in fruit stored for as long as 120 days. Cell walls of fruit exposed to ethylene exhibited acute ultrastructural damage. The decline in placental tissue firmness and the development of watersoaking symptoms observed by the third day of 50 μl·liter−1 ethylene treatment were apparently due, in part, to the PG-mediated cell wall breakdown resulting in cell rupture. Additionally, ethylene appeared to enhance membrane permeability.
The dramatic softening of avocado fruit is frequently attributed to endo-B-l,4-glucanase, an enzyme which accumulates to high levels during ripening. Definitive proof is lacking, however, and the in vivo function of this protein remains to be determined. In this study, we examined the potential involvement of pectic polysaccharides in avocado fruit ripening. Soluble uronic acids increased dramatically during avocado ripening, ranging from 30 ug/mg (15% of total uronic acids) in preripe fruit to 180 ug/mg (90% of total) in ripe fruit. During this period, soluble pectins exhibited dramatic downshifts in mol wt along with a loss in associated neutral sugars, primarily GAL and ARA. Quantities of oligomeric uronic acids were also recovered, but only during the latter stages of ripening. The degree of depolymerization observed with avocado fruit far exceeds that reported for tomato fruit; however, differences can not be explained on the basis of the activity of extractable polygalacturonase.