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.     

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.     

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.     

Physiology and firmness determination of ripening tomato fruit

Tomato ( Lycopersicon esculentum Mill.) genotypes varying in intrinsic firmness were examined to determine the quantitative relationships between polygalacturonase (EC 3.2.1.15) activity, firmness and other ripening parameters including rate (days from mature-green to full red) and intensity (rate of ethylene production at climacteric peak) of ripening. Texture, respiration and ethylene production were monitored in the immature-green through the red (ripe) stages of development. Polygalacturonase activity was measured by direct assay of salt-extractable wall protein or by monitoring the release of pectins from isolated, enzymically active wall. In all fruit, polygalacturonase activity was highly correlated with pericarp softening, but only moderately correlated with softening of whole fruit (r = 0.920 and 0.757, respectively). Polygalacturonase activity was positively correlated with cell-wall autolytic activity in pink (r = 0.969) and red (r = 0.900) fruit. Firmer genotypes exhibited lower rates of respiration and ethylene production during ripening. Polygalacturonase activity in isolates prepared from fruit at the climacteric peak was positively correlated with ethylene production and respiration, and negatively correlated with days to ripening (r = 0.929, 0.805, and -0.791, respectively). The data demonstrate the importance of selecting the appropriate method of firmness determination and are consistent with the hypothesis that pectin fragments released by polygalacturonase contribute to the production of autocatalytic (system II) ethylene.     

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.

     

Cell Wall Metabolism in Ripening Fruit (VII. Biologically Active Pectin Oligomers in Ripening Tomato (Lycopersicon esculentum Mill.) Fruits)

A water-soluble, ethanol-insoluble extract of autolytically inactive tomato (Lycopersicon esculentum Mill.) pericarp tissue contains a series of galacturonic acid-containing (pectic) oligosaccharides that will elicit a transient increase in ethylene biosynthesis when applied to pericarp discs cut from mature green fruit. The concentration of these oligosaccharides in extracts (2.2 [mu]g/g fresh weight) is in excess of that required to promote ethylene synthesis. Oligomers in extracts of ripening fruits were partially purified by preparative high-performance liquid chromatography, and their compositions are described. Pectins were extracted from cell walls prepared from mature green fruit using chelator and Na2CO3 solutions. These pectins are not active in eliciting ethylene synthesis. However, treatment of the Na2CO3-soluble, but not the chelator-soluble, pectin with pure tomato polygalacturonase 1 generates oligomers that are similar to those extracted from ripening fruit (according to high-performance liquid chromatography analysis) and are active as elicitors. The possibility that pectin-derived oligomers are endogenous regulators of ripening is discussed.     

Tissue Specific Localization of Pectin–Ca2+ Cross-Linkages and Pectin Methyl-Esterification during Fruit Ripening in Tomato (Solanum lycopersicum)

Fruit ripening is one of the developmental processes accompanying seed development. The tomato is a well-known model for studying fruit ripening and development, and the disassembly of primary cell walls and the middle lamella, such as through pectin de-methylesterified by pectin methylesterase (PE) and depolymerization by polygalacturonase (PG), is generally accepted to be one of the major changes that occur during ripening. Although many reports of the changes in pectin during tomato fruit ripening are focused on the relation to softening of the pericarp or the Blossom-end rot by calcium (Ca²⁺) deficiency disorder, the changes in pectin structure and localization in each tissues during tomato fruit ripening is not well known. In this study, to elucidate the tissue-specific role of pectin during fruit development and ripening, we examined gene expression, the enzymatic activities involved in pectin synthesis and depolymerisation in fruit using biochemical and immunohistochemical analyses, and uronic acids and calcium (Ca)-bound pectin were determined by secondary ion-microprobe mass spectrometry. These results show that changes in pectin properties during fruit development and ripening have tissue-specific patterns. In particular, differential control of pectin methyl-esterification occurs in each tissue. Variations in the cell walls of the pericarp are quite different from that of locular tissues. The Ca-binding pectin and hairy pectin in skin cell layers are important for intercellular and tissue–tissue adhesion. Maintenance of the globular form and softening of tomato fruit may be regulated by the arrangement of pectin structures in each tissue.     

Comparison of ripening processes in intact tomato fruit and excised pericarp discs

Physiological processes characteristic of ripening in tissues of intact tomato fruit (Lycopersicon esculentum Mill.) were examined in excised pericarp discs. Pericarp discs were prepared from mature-green tomato fruit and stored in 24-well culture plates, in which individual discs could be monitored for color change, ethylene biosynthesis, and respiration, and selected for cell wall analysis. Within the context of these preparation and handling procedures, most whole fruit ripening processes were maintained in pericarp discs. Pericarp discs and matched intact fruit passed through the same skin color stages at similar rates, as expressed in the L^*a^*b^* color space, changing from green (a^* < −5) to red (a^* > 15) in about 6 days. Individual tissues of the pericarp discs changed color in the same sequence seen in intact fruit (exocarp, endocarp, then vascular parenchyma). Discs from different areas changed in the same spatial sequence seen in intact fruit (bottom, middle, top). Pericarp discs exhibited climacteric increases in ethylene biosynthesis and CO₂ production comparable with those seen in intact fruit, but these were more tightly linked to rate of color change, reaching a peak around a^* = 5. Tomato pericarp discs decreased in firmness as color changed. Cell wall carbohydrate composition changed with color as in intact fruit: the quantity of water-soluble pectin eluted from the starch-free alcohol insoluble substances steadily increased and more tightly bound, water-insoluble, pectin decreased in inverse relationship. The cell wall content of the neutral sugars arabinose, rhamnose, and galactose steadily decreased as color changed. The extractable activity of specific cell wall hydrolases changed as in intact fruit: polygalacturonase activity, not detectable in green discs (a^* = −5), appeared as discs turned yellow-red (a^* = 5), and increased another eight-fold as discs became full red (a^* value +20). Carboxymethyl-cellulase activity, low in extracts from green discs, increased about six-fold as discs changed from yellow (a^* = 0) to red.     

Activity and expression of polygalacturonase vary at different fruit ripening stages of sweet pepper cultivars

Activity and expression of polygalacturonase (PG), a hydrolytic enzyme involved in ultrastructural changes in the pericarp of sweet pepper (Capsicum annaum), were investigated at different ripening stages of the pepper cultivars Mandi and Talanduo. Molecular cloning of CaPG was carried out by constructing a cDNA library from three stages of fruit ripening. Morphological determination, PG assay, RT-PCR, and ultrastructural studies were used to quantify changes in CaPG gene expression in the pericarp from green, color change and fully ripened stages. We found that CaPG gene expression, PG activity and striking changes in the structure of the cell wall occurred with the transition of ripening stages. CaPG gene expression was high (obvious PCR products) in mature and ripened stages of both cultivars; however, the CaPG gene was not expressed in preclimacteric fruits or vegetative tissues. We conclude that developmental regulation of CaPG gene expression is instrumental for sweet pepper fruit ripening; its expression during development leads to dissolution of middle lamella and eventually disruption of the fully ripened cell wall.     

Polygalacturonase Activity in Ripening Tomato Fruit Determined using Pericarp Discs

Discs prepared from the outer pericarp of tomato (Lycopersiconesculentum Mill. cv. Sunny) and placed in buffer exhibit anenzymic release of pectin fragments. Over a 2.5 h period at34 °C, discs from mature-green, 4 d and 10 d postbreakerfruit released approximately 90, 440 and 675 µg galacturonicacid equivalents (g^–1 disc fr. wt.), respectively. Bio-GelP-2 chromatography of the products revealed the presence ofpolymeric, oligomeric and monomeric uronic acids. The similarityof these products to those released from isolated, enzymatically-activecell walls and from enzymatically-inactive walls treated withpurified PG 2 provides evidence for the participation of polygalacturonase(PG, E. C. 3.2.1.15 [EC] ) in the release of pectin from disc tissue. The magnitude of pectin release from external pericarp discswas found to parallel ripening and increase progressively indiscs from the blossom, equatorial and shoulder regions, respectively.The use of discs and other systems to estimate in vivo PG activitywill be discussed. Key words: Cell wall, polyuronides     

PG在番茄果实成熟中的作用及二价金属离子与乙烯对PG活性的影响

对采后番茄果实的电镜观察表明:当果实成熟衰老时,叶绿体数量减少,多数基粒结构丧失;成熟果实胞壁中胶层水解成中空的电子透明区,初生壁的纤丝也发生一定程度的水解,相邻细胞分离;外源 PG(多聚半乳糖醛酸酶)提取物处理绿熟期果实组织,也可引起胞壁结构和叶绿体发生与正常衰老相同的变化。Ca~(2+)、Mg~(2+)、Co~(2+)二价金属离子处理果实,可明显降低番茄红素含量和 PG 活性,延缓果实软化。外源乙烯处理果实,可促进番茄红素的形成,提高 PG活性,并能解除钙对 PG 活性的抑制。本文也对 PG 在乙烯和 Ca~(2+)调节果实成熟中的作用进行了讨论。     

The Role of Polygalacturonase (PG) in Tomato Fruit Ripening and Effects of Divalent Metal Ions and Ethylene on PG Activity

It has been reported that PG is a key enzyme related to the tomato fruit ripening. In this study tomato fruits were harvested at the mature-green stage and stored at room temperature. The cell ultrastructure of pericarp tissue was observed at different ripening stages, and the effects of treatments with ethylene and calcium on PG activity and fruit ripening were examined. The object of this study is to elucidate the role of PG in regulation of tomato fruit ripening by ethylene and calcium. PG activity, was undetectable at mature-green stage, but it rose rapidly as fruif ripening. The rise in PG activity was coincided with the dechnmg of fruit firmness during ripening of tomato fruits. The observation of cell ultrastructure showed that the most of grana in chloroplast were lost and the mitochondrial cristae decreased as fruit ripening. Striking changes of cell wall structure was most noted, beginning with dissolution of the middle lamella and eventual disruption of primary cell wall. A similar pattern of changes of cell wall and chloroplast have been observed in pericarp tissue treated with PG extract. In fruits treated with calcium and other divalent metal ions atmature-green stage, the lycopene content and PG activity decreased dramatically. Ethylene application enhanced the formation of lycopene and PG activity. The inhibition of Ca2+ on PG ac ivity was removed by ethylene. Based on the above results, it was demonstrated that PG played a major role in ripening of tomato fruits, and suggested that the regulation of fruit ripening by ethylene and Ca2+ was all mediated by PG. PG induced the hydrolysis of cell wall and released the other hydrolytic enzymes, then effected the ripening processes follow up.     

The inactivation of pectin depolymerase associated with isolated tomato fruit cell wall: implications for the analysis of pectin solubility and molecular weight

An approach commonly employed to assess the potential role of the enzyme polygalacturonase (PG, EC 3.2.1.15) in tomato fruit cell-wall pectin metabolism includes correlating levels of extractable PG with changes in specific characteristics of cell wall pectins, most notably solubility and molecular weight. Since information on these features of pectins is generally derived from analyses of subfractions of isolated cell wall, assurance of inactivation of the various isoforms of wall-associated PG is imperative. In the present study, cell wall prepared from ripe tomato (Lycopersicon esculentum Mill. cv. Rutgers) fruit was examined for the presence of active PG and for the ability of phenolic solvents to inactivate the enzyme. Using pectin solubility and M_r (relative molecular mass) changes as criteria for the presence of wall-associated PG activity, pectins from phenol-treated and nonphenol-treated (enzymically active) cell wall from ripe fruit incubated in 50 mM Na-acetate, 50 mM cyclohexanetrans-1,2-diamine tetraacetic acid (CDTA), pH 6.5 (outside the catalytic range of PG), were of similar M_r and exhibited no change in size with incubation time. Wall prepared without exposure to the phenolic protein-denaturants exhibited extensive pectin solubilization and depolymerization when incubated in 50 mM Na-acetate, 50 mM CDTA at pH 4.5, indicating the presence of active PG. Based on the changes in the M_r of pectins solubilized in 50 mM Na-acetate, 50 mM CDTA, pH 4.5, active PG was also detected in wall exposed during isolation to phenolacetic acid-water (PAW, 2:1:1, w/v/v), a solvent commonly employed as an enzyme denaturant. Although the depolymerization of pectins in PAW-treated wall was extensive, oligouronides constituted minor reaction products. Interestingly, PAW-treated wall did not exhibit PG-mediated pectin release when incubated under conditions (30 mM Na-acetate, 150 mM NaCl, pH 4.5) in which nonphenol-treated cell wall exhibited high autolytic activity. In an alternative protocol designed to inactivate PG, cell wall was exposed to Tris-buffered phenol (BP). In contrast to pectins released from PAW-treated wall, pectins solubilized from BP-treated wall at pH 4.5 were indistinguishable in M_r from those recovered from BP-treated wall at pH 6.5 Even when incubated at pH 4.5 at 34°C, conditions under which pectins from PAW-treated wall underwent more rapid and extensive depolymerization, pectins from BP-treated wall exhibited no change in M_r, providing evidence that active PG was not present in these wall preparations. The implications of this study in interpreting the solubility and M_r of pectin in cell wall from ripening fruit are discussed.     

Inheritance and effect on ripening of antisense polygalacturonase genes in transgenic tomatoes

The role of the cell wall hydrolase polygalacturonase (PG) during fruit ripening was investigated using novel mutant tomato lines in which expression of the PG gene has been down regulated by antisense RNA. Tomato plants were transformed with chimaeric genes designed to express anti-PG RNA constitutively. Thirteen transformed lines were obtained of which five were analysed in detail. All contained a single PG antisense gene, the expression of which led to a reduction in PG enzyme activity in ripe fruit to between 5% and 50% that of normal. One line, GR16, showed a reduction to 10% of normal PG activity. The reduction in activity segregated with the PG antisense gene in selfed progeny of GR16. Plants homozygous for the antisense gene showed a reduction of PG enzyme expression of greater than 99%. The PG antisense gene was inherited stably through two generations. In tomato fruit with a residual 1% PG enzyme activity pectin depolymerisation was inhibited, indicating that PG is involved in pectin degradation in vivo. Other ripening parameters, such as ethylene production, lycopene accumulation, polyuronide solubilisation, and invertase activity, together with pectinesterase activity were not affected by the expression of the antisense gene.     

Apoplastic pH and inorganic ion levels in tomato fruit: A potential means for regulation of cell wall metabolism during ripening

Apoplastic pH and ionic conditions exert strong influence on cell wall metabolism of many plant tissues; however, the nature of the apoplastic environment of ripening fruit has been the subject of relatively few studies. In this report, a pressure-bomb technique was used to extract apoplastic fluid from tomato fruit ( Lycopersicon esculentum Mill.) pericarp at several developmental stages. pH and the levels of K⁺, Na⁺, Ca²⁺, Mg²⁺, Cl⁻ and P were determined and compared with the values for the bulk pericarp and locule tissues. The pH of the apoplastic fluid from pericarp tissue decreased from 6.7 in immature and mature-green fruits to 4.4 in fully-ripe fruit. During the same period, the K⁺ concentration increased from 13 to 37 m M . The levels of Na⁺ and divalent cations did not change, whereas the anions P and Cl⁻ increased in ripe fruit. Ca²⁺ levels remained relatively constant during ripening at 4–5 m M , concentrations that effectively limit pectin solubilization. The electrical conductivity of the apoplastic liquid increased 3-fold during ripening, whereas osmotically active solutes increased 2-fold. Pressure-treated fruit retained the capacity to ripen. The decline in apoplastic pH and increase in ionic strength during tomato fruit ripening may regulate the activity of cell wall hydrolases. The potential role of apoplastic changes in fruit ripening and softening is discussed.     

Pectate lyase activity during ripening of banana fruit

Pectate lyase (PEL) activity was demonstrated in ripe banana fruits on supplementing the homogenizing medium with cysteine and Triton X-100. The enzyme was characterized on the basis of alkaline pH optimum, elimination of the activity by EDTA and activation by Ca(2+). PEL activity was not detected in preclimacteric banana fruits. PEL activity increased progressively from early climacteric and reached maximum level at climacteric peak and declined in post climacteric and over ripened fruits. Replacing pectate with pectin in PEL assay manifested enzyme activity even in preclimacteric fruits. In contrast to PEL, polygalacturonase activity progressively increased during fruit ripening even in postclimacteric fruits.     

Tomato Fruit Cell Wall Synthesis during Development and Senescence : In Vivo Radiolabeling of Wall Fractions Using [C]Sucrose

The pedicel of tomato fruit (Lycopersicon esculentum Mill., cv `Rutgers') of different developmental stages from immature-green (IG) to red was injected on the vine with 7 microcuries [¹⁴C(U)]sucrose and harvested after 18 hours. Cell walls were isolated from outer pericarp and further fractionated yielding ionically associated pectin, covalently bound pectin, hemicellulosic fraction I, hemicellulosic fraction II, and cellulosic fraction II. The dry weight of the total cell wall and of each cell wall fraction per gram fresh weight of pericarp tissue decreased after the mature-green (MG) stage of development. Incorporation of radiolabeled sugars into each fraction decreased from the IG to MG3 (locules jellied but still green) stage. Incorporation in all fractions increased from MG3 to breaker and turning (T) and then decreased from T to red. Data indicate that cell wall synthesis continues throughout ripening and increases transiently from MG4 (locules jellied and yellow to pink in color) to T, corresponding to the peak in respiration and ethylene synthesis during the climacteric. Synthesis continued at a time when total cell wall fraction dry weight decreased indicating the occurrence of cell wall turnover. Synthesis and insertion of a modified polymer with removal of other polymers may produce a less rigid cell wall and allow softening of the tissue integrity during ripening.     

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.