The Tomato Fruit Cell Wall - NCBI

Received for publication July 5, 1989 and in revised form October 12, 1989

Plant Physiol. (1990) 92, 642-647 0032-0889/90/92/0642/06/$01 .00/0

The Tomato Fruit Cell Wall' II.

Polyuronide Metabolism in a Nonsoftening Tomato Mutant

James L. Koch2 and Donald J. Nevins* Department of Vegetable Crops, University of California, Davis, California 95616 by tomato PE, wall uronides from all stages of development were rendered equally susceptible to PG. Two hypotheses were considered as possible mechanisms to explain these observations: (a) PG activity is suppressed in the cell wall of these fruit during ripening, perhaps by hindrance of enzyme mobility, retention of the enzyme in the cytoplasm or by secondary regulation, and (b) cell wall synthesis maintains the net uronic acid content in the wall by incorporation of uronides sustained at a rate equal to their removal by in situ PG hydrolysis. Another variety was evaluated to compare the physiological characteristics leading to differences in softening during the ripening process. This variety, known as dg, has a background also derived from the 'manipal' line (I 1, 21), and while fruit undergo appropriate color changes and production of PG occurs, the tissues do not soften. This study was designed to assess whether changes in cell wall structure occur due to in situ PG activity during ripening. By comparing a normal softening cultivar with a variety which appears to ripen but does not soften, we have determined that the lack of in situ enzyme activity may lead to the inability to soften in dg. Two approaches for the determination of soluble components liberated from the tomato cell wall were selected. First, excised tomato pericarp segments were subjected to a low speed centrifugation by adapting a procedure used to evaluate polymer solubilization from pea stems (1618). Incubation of tomato pericarp sections in buffer and analysis of the bathing solution was selected as a second method to assess uronic acid solubilization in situ ( 12).

ABSTRACT 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.

MATERIALS AND METHODS

Developmental changes in the native substrate of tomato PG3 and PE have been examined in the tissues of tomato fruit (10). The results reveal no change in the total uronic acid content during ripening. Following uronide deesterification

Plant Material

Tomato fruit (Lycopersicon esculentum cv VF 145B-7879 and breeding line UCD 80007; dg) were harvested and visually sorted by ripening stage (10). Fruit were surface-sterilized for 10 min in 0.1I% hypochlorite, rinsed, and cut in half, the locular jelly was removed with a spatula, and the pericarp sections were rinsed in water.

'Supported in part by a gift from Chesebrough-Ponds. 2 Present address: ARS, Cereal Crops Research, 501 N. Walnut Street, Madison WI 53705. 3 Abbreviations: PG, polygalacturonase II, isoenzymes A and B; MG1, fruit with green exterior color, locule is solid and seeds are fully developed; MG2, one or two locules are fluid; MG3, all locules fluid; MG4, interior of fruit shows orange coloration; B, orange color visible on exterior of the blossom end of fruit; T, fruit are 10 to 30% red; P, fruit are 30 to 60% red; R, fruit are 100% red; UA, uronic acid; Gal A, galacturonic acid; HCW, heat-inactivated cell wall; Rha, rhamnose; PE, pectinmethylesterase.

Ethylene Evolution and Tissue Firmness

Ethylene evolution was measured by GLC with an alumina column and a flame ionization detector (20). Fruit were sorted visually, and individually placed in an unsealed 450 mL glass jar for 4 h. The jars were then sealed and the headspace was 642

643

POLYURONIDE METABOLISM IN NONSOFTENING TOMATO MUTANT

sampled with a mL syringe. Fruit firmness was assessed from the inner surface of the pericarp using a Hunter Spring Pressure Tester (model L-1ON) equipped with a 6.5 mm tip (23).

mers were converted to alditol acetates prior to analysis by GLC (7). Selected samples were subjected to analysis on a Bio-Rad HPX-87H organic acid analysis column using 0.005 M H2S04 as the mobile phase (0.5 mL/min, 30°C) to determine the level of citrate in the apoplastic solution.

Cell Wall Preparation

HCW were prepared and analyzed as described previously (10). Briefly, frozen fruit were peeled, homogenized in 50% ethanol in a Waring blender, and heated to 80°C for 20 min. The tomato homogenates were then washed with water, acetone, and rinsed with water. PG and PE enzymes used to treat HCW were prepared from fruit of VF145B (10). Protein extracts were prepared from ripe fruit according to DellaPenna et al. (4). PG activity was measured by observing the increase in reducing groups (13) in the presence of 0.5% citrus pectin as substrate (Sigma Chemical Co.) in 20 mm acetate buffer (pH 5.0) at 22°C. PE activity was estimated from the release of methanol from 0.1% DD Slow Set, 66% methylesterified pectin (Hercules Chemical Co.) in 20 mm acetate buffer (pH 5.0) at 22°C (22). Evaluation of Tomato Fruit Cell Wall Free Space

Two rectangular segments of tissue excised from the pericarp, cut between the radial arms of the fruit (1.5 x 1.0 cm), were rinsed three times in water and placed in a 20 mL syringe barrel adapted for tissue (14, 16). The syringe was modified by placing a circular piece of fine stainless steel mesh into the syringe barrel for support of a circle of 30 um Spectra Mesh. Two tissue pieces were placed in the syringe barrel to maintain

the vascular elements in vertical alignment and provide stability for the tissue. The syringe containing the tissue was placed in a 50 mL tube and centrifuged at 400g for 15 min at 22°C. Following centrifugation, the syringe barrel containing the pericarp tissue was placed in a 50 mL centrifuge tube containing 10 mL water for 5 min. Excess surface water was removed by tapping the syringe barrel tip onto a layer of paper towels. The centrifugation sequence was repeated two more times. The efflux solution after each centrifugation was collected separately, and buffered with 10 100 mm sodium acetate (pH 5.0). Thimerosal was added at a final concentration of 0.02% to retard microbial growth. Samples were frozen (-20°C) until assayed.

Analysis of Tissue Disruption

Tissue damage was assessed by visual inspection (waterlogging of tissue) and by measuring the citrate content of the eluant. The amount of citrate was compared to the amount of citrate/g fresh weight found in either crushed tissue or pericarp sections which had been frozen (-20°C), thawed, and centrifuged as described above. Tissue eluants containing greater than 2% of the total tissue citrate were not used for evaluation.

RESULTS Ripening Characteristics Increases in ethylene evolution begin with the MG3 stage in fruit of cultivar VF145B, 0.23 nL ethylene fresh weightr'h-', and continues to increase, reaching a maximum rate at the turning stage, 4.18 nL ethylene g fresh weightr'h-' (Fig 1). The evolution of ethylene then decreases to a stable level at the pink and ripe stages, 1.80 nl ethylene g fresh weight-lh-'. The dg variety exhibits a similar pattern of ethylene evolution, but ethylene evolution is at a lower rate than observed for VF145B (Fig. 1). The initial increase in ethylene by dg fruit at the MG4 stage, 0.69 nL ethylene g fresh weightr'h-' is also somewhat delayed. The rate of evolution increases to a maximum of 1.85 nL ethylene g fresh weight-'h-' at the pink stage. Firmness of tomatoes was determined using the same fruit 5

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Diffusion Assay of Apoplast Components

Five mm cores were cut from pericarp tissue and rinsed four times in water. In each experiment, five discs were incubated in 1 mL 20 mm acetate buffer (pH 5.0) for 1 h with a complete change of buffer at 30 min. Thimerosal was added at a final concentration of 0.02% to retard microbial growth. Analysis of Solubilized Components

The solution containing the soluble cell wall components assayed for uronic acid content according to Blumenkrantz and Asboe-Hansen (2). Released polysaccharides were hydrolyzed in the presence of 2 M TFA, 100°C for 1 h, to determine neutral sugars composition. Carbohydrate mono-

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Development Stage Figure 1. Ethylene evolution of tomato fruit during ripening. Ethylene evolution was measured by GLC after sampling the headspace of single tomato fruit incubated in a glass jar for 1 h. Developmental stages are as follows: MG1, fruit with green exterior color, locule is solid and seeds are fully developed; MG2, one or two locules are fluid; MG3, all locules fluid; MG4, interior of fruit shows orange coloration; B, orange color visible on exterior of the blossom end of the fruit; T, fruit are 10 to 30% red; P, fruit are 30 to 60% red; R, fruit are 100% red. Ethylene evolution of VF1 45B, *. Ethylene evolution of dg fruit, 0.

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evaluated for ethylene evolution. VF145B fruit remain firm, 4.1 kg (Fig. 2), through the MG and B stages and begin to soften by the turning stage, 3.3 kg. Fruit achieve greatest reduction in firmness at the pink stage, 2.1 kg, but they remain at this level through the remainder of the ripening process. In contrast, fruit from dg have an initial firmness of 5.1 kg at the MG stage. These fruit remain firm throughout development with only a slight decrease in firmness between the MG I stage and the B stages. The firmness of dg fruit at the B stage corresponds to that of the VF145B fruit at the MG through B stages, 3.7 kg; however, the values for dg fruit remain at this relatively firm level throughout the remaining

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development. Cell Wall Composition of Tomato Fruit Cell wall extraction yields 15 g cell wall/kg fruit at all ripening stages in both varieties. The UA content remains constant in both cultivars, 18 to 19 mg/ 100 mg wall, throughout fruit development. Figure 3, A and B, displays values from dg fruit for comparison with VF145B data reported in a previous paper in this series (10). Most of the uronic acid, 16.8 mg UA/100 mg wall, of VF145B is esterified during the MG through the B stages (10). Esterification decreases to 6.8 mg UA/100 mg wall by the pink stage (10). The walls of fruit from dg have lower initial levels of esterified pectin at the MG stage than observed for VF145B, 10.6 mg UA/100 mg wall; however, this degree of esterification is maintained throughout ripening (Fig. 3A). A relatively small amount of uronic acid is released from HCW by PG treatment of VF145B fruit, 2.4 mg UA/100 mg wall at the MG through B stages (10). The quantity of pectin released increases to 4.2 mg UA/100 mg wall beginning with the turning stage, and reaches a maximum susceptibility to PG at the ripe stage, with 5.2 mg UA released/100 mg wall (10). In contrast, HCW from dg fruit at all ripening stages show a low susceptibility to PG, ca. 2.1 mg UA/100 mg wall c]

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Plant Physiol. Vol. 92, 1990

KOCH AND NEVINS

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Development Stage Figure 3. Cell wall UA composition of dg fruit. (A) Total UA content measured according to Ahmed and Labavitch (1), 0. Methyl-esterified uronic acid content was determined according to Wood and Siddiqui (22), 0. (B) UA solubilized by treatment of HCW from dg fruit with PG from VF145B. UA released from HCW by 24 h incubation of walls with 5 Ag PG (800 ,ug GalA reducing equivalents h-1) in 20 mm acetate buffer (pH 5.0) at 220C, 0. Uronic acid released by PG following pretreatment of HCW with 5 Mg tomato fruit PE (1.5 ,ug methanol released min-'), 0. Values represent the mean ± SE from three separate extractions.

(Fig. 3B), corresponding to the amount of PG susceptible uronic acid present at the MG stage of VF145B. When the cell wall residue is pretreated with PE to deesterify the wall pectin, both varieties show an equal susceptibility to PG at all developmental stages, 5.2 mg UA/100 mg wall (Fig. 3B) (10).

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To evaluate whether differences in softening behavior were a function of the presence of hydrolytic enzymes, protein was extracted from ripe fruit tissue of each variety. The 1 M NaCl extract of cell walls revealed that PG activity was similar for both varieties, 284 mg GalA reducing equivalents released h-' kg-' fruit. However, PE activity of dg, 25.4 mg methanol released min-' kg-' fruit, was less than half the activity observed in extracts of VF145B, 59.4 mg methanol released min-' kg-'. When MG fruit cell walls are allowed to autohy-

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Development Stage Figure 2. Changes in the firmness of tomato fruit during ripening. Firmness was determined using a Hunter Spring Pressure Tester equipped with a 6.5 mm tip. The pressure required to depress tissue was measured from the locular face of the pericarp to avoid the effects of skin toughness on the appraisal. Firmness of VF145B pericarp tissue, *. Firmness of dg fruit pericarp, 0. Each value is the mean

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of measurements from at least five different fruit.

drolyze, both varieties exhibit maximum deesterification of the cell wall pectin by wall-bound PE within 10 min incubation (data not shown).


Carbohydrate Composition of Cell Wall Free Space Measured by Diffusion After 1 h, the UA fragments of the cell wall free space remained constant at 700 ,ug/g fresh weight from the mature green to turning stages of development in VF145B (Fig. 4A). Beginning at the pink stage, the appearance of soluble UA containing material in VF145B increases, 1.8 mg/g fresh weight, to a maximum of 2.0 mg/g fresh weight at the ripe stage. Extended incubations in buffer (up to 3 h) show significant signs of tissue damage (data not shown), assessed both visually and as measured by increased citrate in the buffer (greater than 5% of the total tissue citrate levels were released into the bathing medium after 1 h). UA that diffuses from the cell wall free space of dg fruit remains low in the MG through T stages, 800 ,tg/g fresh weight (Fig. 4A). Efflux of UA increased beginning with the pink stage, 1.7 mg/g fresh weight, and reached a maximum at the ripe stage, 2.0 mg/g fresh weight. There is no apparent quantitative difference in the rate that hydrolytic products appear in the bathing solution of the normal and nonsoftening cultivars (Fig. 4A).

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Ara and Gal released from the apoplast are low in fruit from the mature green and breaker stages, 25 and 48 ,Ag/g fresh weight, respectively, in both varieties (Table I). These components increase, beginning with the turning stage and reach a maximum at the ripe stage, 130 ,ug/g fresh weight for each sugar. The increase in Gal and Ara appears to occur at the turning stage and precedes the appearance of UA. Rha and Xyl in the apoplast eluant do not change as development proceeds from the MG to T stages, about 30 and 60 ng/g fresh weight, respectively. At the pink stage, there is an increase in polymers containing Rha and Xyl. The values for Rha and Xyl reach a maximum at the ripe stage, 130 and 90 ng/g fresh weight, respectively. There were no quantitative differences between polymers released from dg and VF145B (data not shown).

Carbohydrate Composition of Cell Wall Free Space Extracted by Centrifugation Solutions collected from MG and B stages contained 0.30 ,ug UA/g fresh weight of VF145B fruit tissue (Fig. 4B). The estimated amount of UA in the apoplast appeared to increase during development, beginning at the turning stage, to a maximum of 8.23 ,ug UA/g fresh weight at the ripe stage. In each of the three centrifugation sequences, for a given set of tissue, the amount of uronic acid and citrate in the extract was similar. This pattern was observed for all stages of development. When the tissue free space is subjected to centrifugation, walls derived from fruit of the dg cultivar fail to relinquish soluble uronic acid fragments at any stage throughout development (Fig. 4B). Extended centrifugation sequences extracted significantly greater amounts of UA, 36 ,ug UA/g fresh weight from ripe fruit after six centrifugations. Enhanced release of UA may be due to continued autohydrolysis of the cell wall as a response to tissue wounding. Increases in the citrate leakage from the tissue (up to 10% of the tissue citrate) was also noted in these treatments. There were no significant differences in the citrate content of the eluate between the two varieties. Gal-, Rha-, Ara-, and Xyl-containing materials are all present in the cell wall free space of VF145B (Table II). The presence of neutral sugars and UA suggests that the products are derived from the same substrate affected by in vitro

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Development Stage Figure 4. UA composition of the cell wall free space. (A) UA diffusion from the cell wall of fruit pericarp tissue. UA was released from five 5 mm pericarp discs into 20 mm acetate buffer (pH 5.0), at 220C for 1 h. UA released from VF1 45B pericarp tissue, *. UA released from dg pericarp tissue, 0. (B) UA in fluid expressed from the cell wall of fruit pericarp tissue when subjected to centrifugation. UA is the sum of the UA eluted by three 15 min extractions at 400g at 220C. UA expressed from VF145B fruit, *. UA expressed from dg fruit, 0. Values represent the mean ± SE from at least six extractions.

Table I. Neutral Sugar Composition of Polysaccharide Fragments from Tomato Fruit Cell Wall Polysaccharide released during a 1 h incubation of pericarp discs in 20 mm acetate buffer (pH 5.0) at 220C. There were no significant differences in the composition of released polysacchandes between the two varieties. Values represent the mean ± 5 gg of at least six measurements.

Development Stage

Rha

MG

24 28 37 83 128

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Xyl

gg released/g fresh wt 25 53 31 60 80 59 117 63 127 90

Gal

48 62 144 122 132

KOCH AND NEVINS

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Table II. Neutral Sugar Composition of Polysaccharide Fragments Released from Tomato Fruit Cell Wall Total carbohydrate was evaluated following three extractions of VF1 45B fruit sections at 400g for 15 min. Values represent mean + 50 ng/g fresh wt of at least six measurements.

Development Stage

Rha

MG B T P R

546 668 1234 1200 1909

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320 290 340 661 2296

116 294 315 494 722

405 471 906 1046 4021

treatment of cell walls with PG (1O). The Rha and Gal content in the apoplast of pericarp tissue from mature green and breaker stage fruit can be estimated as 600 and 450 ng/g fresh

weight, respectively. These values increased with fruit development. Rha and Gal release was maximum, 19 10 and 4020 ng/g fresh weight, respectively, at the ripe stage. Ara in released products remains constant ca. 300 ng/g fresh weight from the MG to T stages but increase beginning with the pink stage, reaching a maximum in ripe fruit, 2300 ng/g fresh weight. Xylose increased with development from 120 ng/g FW at the MG stage to 722 ng/g fresh weight at the ripe stage. Neutral sugars other than glucose were present in trace amounts in dg fruit eluates. DISCUSSION

The ripening process in tomato fruit has been extensively studied. It has been documented that although PG is induced during development and its induction correlates with softening of fruit tissue, there is no change in the uronic acid content of the cell wall (10). If in situ PG hydrolysis occurs in fruit tissue, it must coincide with pectin synthesis since the pectin content of the cell wall remains constant throughout ripening. Therefore, the fact that soluble pectins are present in normal softening tomato fruit tissue, indicates PG is active in situ. The physiological changes in fruit of the breeding line dg, color development, ethylene evolution and synthesis of PG and PE, indicate that this variety exhibits developmental behavior similar to VF145B. The ripening behavior of the mutant lines rin, nor, and Nr have been extensively studied in an effort to link hydrolytic enzymes in fruit ripening to differences in softening behavior (3, 5, 19). Two of these ripening mutants, rin and nor, fail to exhibit enhanced ethylene evolution during development and all three differ in the expression of pigmentation when compared to typical ripening behavior (19). These mutations appear to affect physiological events at the onset ofthe ripening process, confounding their use for the evaluation of the physiological consequences of specific softening behavior. Transformation of dg with genes for PG and/or PE may allow a direct comparison of cell wall hydrolytic activity with fruit softening. Fruit from the dg line exhibit ethylene evolution at a lower rate than normal fruit, but appropriate color changes corresponding to normal ripening do occur (1 1, 21), and the tissues produce specific cell wall hydrolases thought to be involved

Plant Physiol. Vol. 92, 1990

in softening events as defined by this study. The two genotypes investigated have similar levels of soluble solids and citric acid as well as comparable tissue pH (8). Thus, events normally associated with tomato fruit are present in the dg breeding line. Since dg cell wall composition indicates no change in the degree of esterification throughout development, while VF145B decreases its degree of esterification during development, the resident PE in dg fruit appears to be incapable of acting on pectins. Subjecting the intact pericarp tissue to centrifugation, followed by analysis of efflux products, also suggests that little UA solubilization occurs during the ripening process in dg fruit. Therefore PG may also be inactive in situ. Inactivity may be due to metabolic regulation, differences in enzyme mobility conditioned by physical restriction within the wall matrix, failure of PE to properly modify the pectin for subsequent digestion by PG, or a failure of the secretory mechanisms for cell wall proteins. The neutral sugar composition of the cell wall free space indicates that hydrolases capable of releasing galactose and arabinose may appear prior to PG in VF145B. An increase in soluble Gal polymers is seen in the breaker stage (Table I) while Ara solubilization is delayed until the turning stage. Both of these increases precede the uronic acid increase observed after collection of solubilized polymers by diffusion. Gross and Wallner (6) have documented, using fruit from rin tomatoes, that changes in Ara and Gal content of the pectin component can occur independent of PG mediated changes in pectins. The two approaches used to measure in situ activity reveal differences in (a) the developmental stage in which uronides are observed in the cell wall free space and (b) the quantity of the UA containing polymers present in the cell wall. The latter observation may be attributed to the relatively greater tissue damage incurred when pericarp discs are prepared for diffusion analysis than when tissue is prepared for centrifugation. The surface area to volume ratio is much greater for the pericarp discs than in the sections excised for centrifugation. The greater degree of wounding would presumably cause a release of hydrolytic enzymes and reduce cytoplasmic control of cell wall events, which in turn would increase the amount of UA solubilized by the tissue. The rate of UA efflux during diffusion is similar to the rate observed when cell wall homogenates are allowed to autolyze (9, 15). This relationship corroborates evidence for the disruption of tissue metabolism in the pericarp discs. These results are consistent with the interpretation that the breeding line, dg, may be a useful mutant for the evaluation of softening behavior in tomato fruit. Since induction of ripening occurs in this variety and cell wall hydrolytic enzymes are induced to activity levels similar to those found in normal fruit, dg appears to express a narrower range of physiological consequences as a result of the single mutation (3, 19). It is appropriate in subsequent studies to resolve the mechanism which accounts for the apparent inactivity of PE and PG in this mutant to determine the role these cell wall hydrolases play in the softening of tomato fruit.


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LITERATURE CITED Ahmed A, Labavitch JM (1977) A simplified method for accurate determination of cell wall uronide content. J Food Biochem 1: 361-365 Blumenkrantz N, Asboe-Hansen G (1973) New method for quantitative determination of uronic acids. Anal Biochem 54: 484489 Brady CJ (1987) Fruit ripening. Annu Rev Plant Physiol 38: 155-178 DellaPenna D, Alexander DC, Bennett AB (1986) Molecular cloning of tomato fruit polygalacturonase: analysis of polygalacturonase mRNA levels during ripening. Proc Natl Acad Sci USA 83: 6420-6424 DellaPenna D, Kates DS, Bennett AB (1987) Polygalacturonase gene expression in rutgers, rin, nor, and Nr tomato fruits. Plant Physiol 85: 502-507 Gross KC, Wallner S.J (1979) Degradation of cell wall polysaccharides during tomato fruit ripening. Plant Physiol 63: 117120 Hatfield R, Nevins DJ (1986) Characterization of the hydrolytic activity of avocado cellulose. Plant Cell Physiol 27: 541-552 Hewitt JD, Dickinson GL, Aguirre MS, Blaker NS (1985) Breeding tomatoes for processing. Progress Report, Vegetable Crops Series 222, Department of Vegetable Crops, University of California, Davis Koch JL, Nevins DJ (1986) Characterization of tomato fruit cell walls: the nature ofpolysaccharides released during autohydrolysis. HortScience 21: 837 Koch JL, Nevins DJ (1989) The tomato fruit cell wall. I. The use of purified tomato polygalacturonase and pectinmethylesterase to identify developmental changes in endogenous substrates. Plant Physiol 91: 816-822 Konsler TR (1973) Three mutants appearing in 'manapal' tomato. HortScience 8: 331-333

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12. Maynard JW, Lucas WJ (1982) Sucrose and glucose uptake into Beta vulgaris leaf tissue; a case for general apoplastic retrieval systems. Plant Physiol 70: 1436-1443 13. Milner Y, Avigad G (1967) A copper reagent for the determination of hexuronic acids and certain ketohexoses. Carbohydr Res 4: 359-361 14. Morrison JC, Greve LC, Labavitch JM (1987) The role of cell wall-degrading enzymes in the formation of gum ducts in almond fruit. J Am Soc Hort Sci 112: 367-372 15. Rushing JW, Huber DJ (1984) In vitro characterization of tomato fruit softening. The use of enzymatically active cell walls. Plant Physiol 75: 891-894 16. Terry ME, Bonner BA (1980) An examination of centrifugation as a method of extracting an extracellular solution from peas, and its use for the study of indolacetic acid-induced growth. Plant Physiol 66: 321-335 17. Terry ME, Rubinstein B, Jones RL (1981) Soluble cell wall polysaccharides released from pea stems by centrifugation. I. Effect of auxin. Plant Physiol 68: 531-537 18. Terry ME, Rubinstein B, Jones RL (1981) Soluble cell wall polysaccharides released from pea stems by centrifugation. II. Effect of ethylene. Plant Physiol 68: 538-542 19. Tigchelaar EC, McGlasson WB, Buescher RW (1978) Genetic regulation of tomato fruit ripening. HortScience 13: 508-513 20. Ursin VM (1987) Morphogenetic and physiological analysis of two developmental mutants of tomato, Epinastic and Diageotropica, PhD thesis, University of California, Davis 21. Wann EV, Jourdain EL, Pressey R, Lyon BG (1982) Effect of mutant genotypes hp ogc and dg ogc on tomato fruit quality. J Am Sco Hort Sci 110: 212-215 22. Wood PJ, Siddiqui IR (1971) Determination of methanol and its application to measurement of pectin ester content and pectin methylesterase activity. Anal Biochem 39: 418-428 23. Yamaguchi M, Hughes DL, Yabumoto K, Jennings WG (1977) Quality of cantaloupe muskmelons: variability and attributes. Sci Hortic 6: 59-70