In vitro characterization of tomato fruit softening : the use of enzymically active cell walls

Cell wall isolated from pericarp of normal tomato (Lycopersicon esculentum Mill. cv `Rutgers') fruit released pectic polymers in a reaction apparently mediated by wall-bound polygalacturonase that appears with the onset of ripening. Release was negligible in wall preparations from normal green and the ripening mutant rin fruit. Pectin solubilization was most extensive at pH 2.5 with a less significant peak at 5.5. Brief exposure to low (1.5) or high (7.5) pH resulted in reduction of autolytic activity, which was also inhibited by high temperature, Ca²⁺, and treatments employed to dissociate protein from cell wall. Uronic acid solubilization was significantly enhanced by 150 millimolar NaCl and by increasing temperature within the physiological range. These data indicate that the release of polyuronide from isolated cell walls is enzymic and may provide a convenient and reliable system for the study of softening metabolism.     

Degradation of Cell Wall Polysaccharides during Tomato Fruit Ripening

Changes in neutral sugar, uronic acid, and protein content of tomato (Lycopersicon esculentum Mill) cell walls during ripening were characterized. The only components to decline in amount were galactose, arabinose, and galacturonic acid. Isolated cell walls of ripening fruit contained a water-soluble polyuronide, possibly a product of in vivo polygalacturonase action. This polyuronide and the one obtained by incubating walls from mature green fruit with tomato polygalacturonase contained relatively much less neutral sugar than did intact cell walls. The ripening-related decline in galactose and arabinose content appeared to be separate from polyuronide solubilization. In the rin mutant, the postharvest loss of these neutral sugars occurred in the absence of polygalacturonase and polyuronide solubilization. The enzyme(s) responsible for the removal of galactose and arabinose was not identified; a tomato cell wall polysaccharide containing galactose and arabinose (6:1) was not hydrolyzed by tomato β-galactosidase.     

The Tomato Fruit Cell Wall : II. Polyuronide Metabolism in a Nonsoftening Tomato Mutant

A nonsoftening tomato (Lycopersicon esculentum L.) variety, dg, was examined to assess the physiological basis for its inability to soften during ripening. Total uronic acid levels, 18 milligrams uronic acid/100 milligrams wall, and the extent of pectin esterification, 60 mole%, remained constant throughout fruit development in this mutant. The proportion of uronic acid susceptible to polygalacturonase in vitro also remained constant. Pretreatment of heat-inactivated dg fruit cell walls with tomato pectinmethylesterase enhances polygalacturonase susceptibility at all ripening stages. Pectinesterase activity of cell wall protein extracts from red ripe dg fruit was half that in extracts from analogous tissue of VF145B. Polygalacturonase activities of cell wall extracts, however, were similar in both varieties. Diffusion of uronic acid from tissue discs of both varieties increased beginning at the turning stage to a maximum of 2.0 milligrams uronic acid released/gram fresh weight at the ripe stage. The increased quantity of hydrolytic products released during ripening suggests the presence of in situ polygalacturonase activity. Low speed centrifugation was employed to induce efflux of uronide components from the cell wall tree space. In normal fruit, at the turning stage, 2.1 micrograms uronic acid/gram fresh weight was present in the eluant after 1 hour, and this value increased to a maximum of 8.2 micrograms uronic acid/gram fresh weight at the red ripe stage. However, centrifuge-aided extraction of hydrolytic products failed to provide evidence for in situ polygalacturonase activity in dg fruit. We conclude that pectinesterase and polygalacturonase enzymes are not active in situ during the ripening of dg fruit. This could account for the maintenance of firmness in ripe fruit tissue.     

Tomato fruit cell wall : I. Use of purified tomato polygalacturonase and pectinmethylesterase to identify developmental changes in pectins

Cell wall isolation procedures were evaluated to determine their effect on the total pectin content and the degree of methylesterification of tomato (Lycopersicon esculentum L.) fruit cell walls. Water homogenates liberate substantial amounts of buffer soluble uronic acid, 5.2 milligrams uronic acid/100 milligrams wall. Solubilization appears to be a consequence of autohydrolysis mediated by polygalacturonase II, isoenzymes A and B, since the uronic acid release from the wall residue can be suppressed by homogenization in the presence of 50% ethanol followed by heating. The extent of methylesterification in heat-inactivated cell walls, 94 mole%, was significantly greater than with water homogenates, 56 mole%. The results suggest that autohydrolysis, mediated by cell wall-associated enzymes, accounts for the solubilization of tomato fruit pectin in vitro. Endogenous enzymes also account for a decrease in the methylesterification during the cell wall preparation. The heat-inactivated cell wall preparation was superior to the other methods studied since it reduces β-elimination during heating and inactivates constitutive enzymes that may modify pectin structure. This heat-inactivated cell wall preparation was used in subsequent enzymatic analysis of the pectin structure. Purified tomato fruit polygalacturonase and partially purified pectinmethylesterase were used to assess changes in constitutive substrates during tomato fruit ripening. Polygalacturonase treatment of heat-inactivated cell walls from mature green and breaker stages released 14% of the uronic acid. The extent of the release of polyuronides by polygalacturonase was fruit development stage dependent. At the turning stage, 21% of the pectin fraction was released, a value which increased to a maximum of 28% of the uronides at the red ripe stage. Pretreatment of the walls with purified tomato pectinesterase rendered walls from all ripening stages equally susceptible to polygalacturonase. Quantitatively, the release of uronides by polygalacturonase from all pectinesterase treated cell walls was equivalent to polygalacturonase treatment of walls at the ripe stage. Uronide polymers released by polygalacturonase contain galacturonic acid, rhamnose, galactose, arabinose, xylose, and glucose. As a function of development, an increase in the release of galacturonic acid and rhamnose was observed (40 and 6% of these polymers at the mature green stage to 54 and 15% at the red ripe stage, respectively). The amount of galactose and arabinose released by exogenous polygalacturonase decreased during development (41 and 11% from walls of mature green fruit to 11 and 6% at the red ripe stage, respectively). Minor amounts of glucose and xylose released from the wall by exogenous polygalacturonase (4-7%) remained constant throughout fruit development.     

Ultrastructure of tomato fruit ripening and the role of polygalacturonase isoenzymes in cell wall degradation

Ultrastructural changes in the pericarp of tomato (Lycopersicon esculentum Mill) fruit were followed during ripening. Ethylene production was monitored by gas chromatography and samples analyzed at successive stages of the ripening process.

Changes in the cytoplasmic ultrastructure were not consistent with the suggestion that ripening is a `senescence' phenomenon. A large degree of ultrastructural organization, especially of the mitochondria, chromoplasts, and rough endoplasmic reticulum, was retained by ripe fruit.

Striking changes in the structure of the cell wall were noted, beginning with dissolution of the middle lamella and eventual disruption of the primary cell wall. These changes were correlated with appearance of polygalacturonase (EC 3.2.1.15) isoenzymes. Application of purified tomato polygalacturonase isoenzymes to mature green fruit tissue duplicated the changes in the cell wall noted during normal ripening. Possible roles of the polygalacturonase isoenzymes in cell wall disorganization are discussed.

     

An Antisense Pectin Methylesterase Gene Alters Pectin Chemistry and Soluble Solids in Tomato Fruit

Pectin methylesterase (PME, EC 3.1.11) demethoxylates pectins and is believed to be involved in degradation of pectic cell wall components by polygalacturonase in ripening tomato fruit. We have introduced antisense and sense chimeric PME genes into tomato to elucidate the role of PME in fruit development and ripening. Fruits from transgenic plants expressing high levels of antisense PME RNA showed <10% of wild-type PME enzyme activity and undetectable levels of PME protein and mRNA. Lower PME enzyme activity in fruits from transgenic plants was associated with an increased molecular weight and methylesterification of pectins and decreased levels of total and chelator soluble polyuronides in cell walls. The fruits of transgenic plants also contained higher levels of soluble solids than wild-type fruits. This trait was maintained in subsequent generations and segregated in normal Mendelian fashion with the antisense PME gene. These results indicate that reduction in PME enzyme activity in ripening tomato fruits had a marked influence on fruit pectin metabolism and increased the soluble solids content of fruits, but did not interfere with the ripening process.     

Degradation of isolated tomato cell walls by purified polygalacturonase in vitro

Cell wall preparations from green pericarp of normal and mutant Neverripe (Nr) and ripening inhibitor (rin) tomato (Lycopersicon esculentum Mill.) fruit were all equally degraded in vitro by a cell wall-bound protein extract from ripe normal tomatoes.     

Ultrastructural changes in the cell walls of ripening apple and pear fruit

Ultrastructural changes in the cell walls of “Calville de San Sauveur” apples (Malus sylvestris Mill) and “Spadona” pear (Pyrus communis L.) fruit were followed during ripening. In apple, structural alterations in cell walls became apparent at advanced stages of softening and showed predominantly dissolution of the middle lamella. In pears softening was also associated with the dissolution of the middle lamella, and in addition a gradual disintegration of fibrillar material throughout the cell wall. In fully ripe fruit almost all of the fibrillar arrangement in the cell wall was lost. Application of enzyme solutions containing polygalacturonase and cellulase to tissue discs from firm pear fruit led to ultrastructural changes observed in naturally ripening pears. In apple polygalacturonase alone was sufficient to dissolve the middle lamella region of the cell walls, as was also found to occur in naturally ripening fruit. In both apple and pear the cell wall areas containing plasmodesmata maintained their structural integrity throughout the ripening process. At advanced stages of ripening vesicles appeared in the vicinity of plasmodesmata.     

Glycosidases in Cell Wall-degrading Extracts of Ripening Tomato Fruits

Enzyme preparations were obtained from cell wall debris of tomato (Lycopersicon esculentum L. cv. Tropic) fruits at various stages of ripeness and were assayed for glycosidase and polysaccharidase activities. In addition to polygalacturonase (mol wt 40,000), ripening fruits contain β-galactosidase (mol wt 63,000) and β-1, 3-glucanase (mol wt 12,000). The β-glycosidases, unlike polygalacturonase, are active in extracts of green fruits. Placental tissue shows very low polygalacturonase but increasing β-galactosidase and β-1, 3-glucanase activities as ripening proceeds. A large change in the susceptibility of the walls to hydrolase action occurs before the stage in which the greatest polygalacturonase activity occurs. The possibility that the β-glycosidases contribute to the wall modifications that lead to fruit softening is discussed.     

Analysis of tomato polygalacturonase expression in transgenic tobacco.

Tomato polygalacturonase is a cell wall enzyme secreted in large amounts during tomato fruit ripening. Polygalacturonase is synthesized as a glycoprotein precursor that undergoes numerous cotranslational and post-translational processing steps during its maturation, yielding three isozymes in tomato fruit, PG1, PG2A, and PG2B. To investigate the physiological roles of the three isozymes and the functional significance of the polygalacturonase processing domains in its intracellular transport and activity, we have examined polygalacturonase expression in transgenic tobacco plants. A full-length polygalacturonase cDNA was placed under control of the cauliflower mosaic virus 35S promoter and introduced into tobacco by way of Agrobacterium-mediated transformation. Analysis of transgenic tobacco plants indicated that (1) immunologically detectable polygalacturonase can be extracted from leaves, roots, and stems of transgenic tobacco plants; (2) only PG2A and PG2B were detectable in transgenic tobacco; (3) the polygalacturonase isozymes present in transgenic tobacco were electrophoretically indistinguishable from the tomato isozymes; (4) the N-terminal sequence, degree of N-linked glycosylation, and extent of oligosaccharide processing were similar in polygalacturonase from transgenic tobacco and tomato; (5) polygalacturonase was properly localized in cell walls of transgenic tissue; (6) the protein was enzymatically active in vitro; however, (7) accumulation of PG2A and PG2B in cell walls of transgenic tobacco did not result in pectin degradation in vivo. These results indicated that tomato polygalacturonase was properly processed and transported to the cell wall of tobacco. However, accumulation of the two polygalacturonase isozymes expressed in this heterologous host was insufficient to promote polyuronide degradation in tobacco leaf tissue.     

Loss of tomato cell wall galactan may involve reduced rate of synthesis

Changes in the galactose content of the noncellulosic polysaccharides of tomato (Mill) fruit cell walls were analyzed under various conditions. On the plant, galactan decreased gradually during fruit growth. As normal fruits ripened, the loss of galactan increased sharply; this was not observed in attached rin fruits beyond the fully mature stage. The ability to produce new wall galactan in vitro was retained in mature fruit tissue but declined with ripening. Normal tomatoes ripening on the plant showed a transient increase in galactan content at the climacteric. It is suggested that the decline in wall galactan is partly due to reduced synthesis in senescing, normal fruits and in detached rin tomatoes.     

Products Released from Enzymically Active Cell Wall Stimulate Ethylene Production and Ripening in Preclimacteric Tomato (Lycopersicon esculentum Mill.) Fruit

Enzymically active cell wall from ripe tomato (Lycopersicon esculentum Mill.) fruit pericarp release uronic acids through the action of wall-bound polygalacturonase. The potential involvement of products of wall hydrolysis in the induction of ethylene synthesis during tomato ripening was investigated by vacuum infiltrating preclimacteric (green) fruit with solutions containing pectin fragments enzymically released from cell wall from ripe fruit. Ripening initiation was accelerated in pectin-infiltrated fruit compared to control (buffer-infiltrated) fruit as measured by initiation of climacteric CO₂ and ethylene production and appearance of red color. The response to infiltration was maximum at a concentration of 25 micrograms pectin per fruit; higher concentrations (up to 125 micrograms per fruit) had no additional effect. When products released from isolated cell wall from ripe pericarp were separated on Bio-Gel P-2 and specific size classes infiltrated into preclimacteric fruit, ripening-promotive activity was found only in the larger (degree of polymerization >8) fragments. Products released from pectin derived from preclimacteric pericarp upon treatment with polygalacturonase from ripe pericarp did not stimulate ripening when infiltrated into preclimacteric fruit.     

Uronic Acid products release from enzymically active cell wall from tomato fruit and its dependency on enzyme quantity and distribution

Isolated cell wall from tomato (Lycopersicon esculentum Mill. cv Rutgers) fruit released polymeric (degree of polymerization [DP] > 8), oligomeric, and monomeric uronic acids in a reaction mediated by bound polygalacturonase (PG) (EC 3.2.1.15). Wall autolytic capacity increased with ripening, reflecting increased levels of bound PG; however, characteristic oligomeric and monomeric products were recovered from all wall isolates exhibiting net pectin release. The capacity of wall from fruit at early ripening (breaker, turning) to generate oligomeric and monomeric uronic acids was attributed to the nonuniform ripening pattern of the tomato fruit and, consequently, a locally dense distribution of enzyme in wall originating from those fruit portions at more temporally advanced stages of ripening. Artificial autolytically active wall, prepared by permitting solubilized PG to bind to enzymically inactive wall from maturegreen fruit, released products which were similar in size characteristics to those recovered from active wall isolates. Extraction of wall-bound PG using high concentrations of NaCl (1.2 molar) did not attenuate subsequent autolytic activity but greatly suppressed the production of oligomeric and monomeric products. An examination of water-soluble uronic acids recovered from ripe pericarp tissue disclosed the presence of polymeric and monomeric uronic acids but only trace quantities of oligomers. The significance in autolytic reactions of enzyme quantity and distribution and their possible relevance to in vivo pectin degradation will be discussed.     

Flavor volatilcs, sugars and color development in ripening in vitro-cultured tomato fruit and calyx

Previous studies showed that the developmental program of calyces of a tomato cultivar ( Lycopersicon esculentum , cv. VFNT Cherry) changed in many aspects to that of fruit when cultured in vitro. The calyces turned red, produced ethylene, had increased tissue content of 1-aminocyclopropane-1-carboxylic acid, had increased levels of the mRNA of polygalacturonase and developed ultrastructural changes in their cell walls that were indistinguishable from those of ripe tomato fruit tissue. We report in the present study the synthesis of volatile flavor compounds, changes in sugar concentrations and color development in cultured calyces that are characteristic of ripening tomato fruit. These ripening parameters of in vitro-cultured tomato fruit were also compared to those of fruit grown in the greenhouse.     

Developmental Regulation Is Altered in the Calyx during in Vitro Ovary Culture of Tomato

To develop a system with which to study fruit ripening, in vitro ovary cultures were initiated from tomato flowers. As reported previously [Nitsch, J.P. (1951). Am. J. Bot. 38, 566-577], tomato fruit ripened after 6 to 7 weeks, but calyces swelled unexpectedly, lost their green color, and gradually became red and succulent. Investigations were conducted, therefore, to verify the occurrence of the ripening process in the calyx. Ethylene production increased in both ripening fruit and red calyx, as did tissue contents of its immediate precursor, 1-aminocyclopropane-1-carboxylic acid. In addition, an increase in the mRNA of polygalacturonase [poly(1,4-[alpha]-D-galacturonide) glucanohydrolase, EC 3.2.1.15], an enzyme that in tomato is present in large amounts only in ripening fruit, was established in both ripe fruit and red calyx by RNA gel blot analysis. Ultrastructural studies showed that the disruption of cell walls in red calyx was indistinguishable from that occurring in ripe tomato fruit. Thus, the developmental program of the calyx changed in several aspects to resemble that of tomato fruit.     

Differential Expression of the Two Subunits of Tomato Polygalacturonase Isoenzyme 1 in Wild-Type and rin Tomato Fruit

The [beta] subunit of tomato (Lycopersicon esculentum Mill.) fruit polygalacturonase 1 is a cell wall glycoprotein that binds to and apparently regulates the catalytic PG2 polypeptide in vivo. [beta] Subunit and polygalacturonase 2 (PG2) expression have been investigated in both wild-type and ripening inhibitor (rin) mutant fruit. During fruit development and ripening, [beta] subunit expression was unrelated to expression of the catalytic PG2 protein. In wild-type fruit, [beta] subunit mRNA and protein were first detected early in development and increased to maximal levels before PG2 mRNA and protein were detected. At the onset of ripening [beta] subunit mRNA decreased dramatically, but [beta] subunit protein levels remained stable. In rin fruit, which fail to ripen, [beta] subunit expression was similar to that in wild type, although PG2 mRNA and protein were not detected. These data suggest that [beta] subunit expression is ethylene independent and regulated primarily by developmental cues. This conclusion is supported by results from ethylene-treated immature (20 days after pollination) wild-type and rin fruit in which no significant differences were observed in [beta] subunit expression patterns in response to ethylene treatment. Surprisingly, RNA blot analysis indicated that catalytic PG2 mRNA was induced in immature rin fruit after 3 d of exogenous ethylene treatment. In addition, [beta] subunit mRNA and protein were also detected at lower levels in root, leaf, and flower tissues of both genotypes, suggesting a broader functional role for the protein.     

Polygalacturonase and Cellulase Enzymes in the Normal Rutgers and Mutant rin Tomato Fruits and Their Relationship to the Respiratory Climacteric

Cell wall enzymes at different stages of fruit development were compared between the normal Rutgers and the isogenic nonripening rin tomato. In Rutgers, a detectable increase in polygalacturonase (PG) activity was observed 6 days prior to the respiratory climacteric (43 days postanthesis). The maximum increase in PG activity occurred after C₂H₂ and CO₂ production reached their peak. However, in the rin tomato, no change in PG activity was noted up to 100 days postanthesis. Cellulase activity increased in Rutgers fruits prior to the respiratory climacteric and continued to increase thereafter. Similar changes in cellulase activity were also observed in the nonclimacteric rin fruits. Short term ethylene treatment (2 days) of 36-day-old rin fruits increased cellulase activity, but had no effect on PG activity. Detectable changes in other parameters of ripening, such as chlorophyll loss and softening, also occurred prior to the respiratory climacteric. These results suggest that the failure of rin fruits to ripen is related to their low PG activity during maturity as compared with normal fruits.     

Cell Wall Metabolism in Ripening Fruit (VI. Effect of the Antisense Polygalacturonase Gene on Cell Wall Changes Accompanying Ripening in Transgenic Tomatoes)

Cell walls of tomato (Lycopersicon esculentum Mill.) fruit, prepared so as to minimize residual hydrolytic activity and autolysis, exhibit increasing solubilization of pectins as ripening proceeds, and this process is not evident in fruit from transgenic plants with the antisense gene for polygalacturonase (PG). A comparison of activities of a number of possible cell wall hydrolases indicated that antisense fruit differ from control fruit specifically in their low PG activity. The composition of cell wall fractions of mature green fruit from transgenic and control (wild-type) plants were indistinguishable except for trans-1,2-diaminocyclohexane-N,N,N[prime],N[prime]-tetraacetic acid (CDTA)-soluble pectins of transgenic fruit, which had elevated levels of arabinose and galactose. Neutral polysaccharides and polyuronides increased in the water-soluble fraction of wild-type fruit during ripening, and this was matched by a decline in Na2CO3-soluble pectins, equal in magnitude and timing. This, together with compositional analysis showing increasing galactose, arabinose, and rhamnose in the water-soluble fraction, mirrored by a decline of these same residues in the Na2CO3-soluble pectins, suggests that the polyuronides and neutral polysaccharides solubilized by PG come from the Na2CO3-soluble fraction of the tomato cell wall. In addition to the loss of galactose from the cell wall as a result of PG activity, both antisense and control fruit exhibit an independent decline in galactose in both the CDTA-soluble and Na2CO3-soluble fractions, which may play a role in fruit softening.