Cell wall composition and structure were examined in visually normal (N), granulated (G), and collapsed (VC) juice vesicles of `Marsh Seedless' grapefruit (Citrus paradisi) Macf.). According to gel-filtration data, VC appeared to be associated with a modification of water-soluble (WSP) and chelate-soluble (CSP) pectin molecular weight (Mr); small-Mr pectins increased, whereas large-J4. pectins decreased. The difference in M = of pectins did not appear to be mediated by polygalacturonases. Molecular weight of hemicelluloses did not differ. Granulated vesicles contained about two times more structural polysaccharides (pectins, hemicelhdose, and cellulose) than N vesicles, although hemicellulose and pectin M = modification were absent. Ion-exchange profiles of WSP, CSP, and hemicelhrlose fractions of VC and G vesicles were not different from those of N vesicles. Individual cells in vesicles with G and these vesicles themselves were much larger than those of N vesicles, whereas cells in VC were partially or completely collapsed.
Pawpaw fruit were harvested at the advent of the ripening process and were ripened at room temperature. Based on fruit firmness and respiration and ethylene production rates at harvest and during ripening, fruit were classified into one of four categories: preripening (no to very slight loss of firmness; preclimacteric), early ripening (some softening; increasing rates of ethylene and CO2 production), mid-ripening (soft; at or just past climacteric), and late ripening (very soft; postclimacteric). The activities of the cell-wall degrading enzymes polygalacturonase (PG), endo-(1→4)ß-D-glucanase (EGase), and endo-ß-1,4-mannanase (MAN) were low in the preripening and early ripening stages, increased dramatically by mid-ripening coincident with the respiratory and ethylene climacterics, and decreased at late ripening. However, pectin methylesterase (PME) activity per milligram protein was highest at the green stage when the fruit firmness was high and decreased as ripening progressed. Tissue prints indicated both EGase and MAN increased as ripening proceeded. The EGase activity was evident near the seeds and the surface of the fruit at preripening and eventually spread throughout, while MAN activity was evident near the fruit surface at preripening and was progressively expressed throughout the flesh as fruit ripened. The greatest decline in fruit firmness occurred between pre- and early ripening, before the peak activities of PG, EGase, and MAN, although MAN exhibited the greatest relative increase of the three enzymes in this period. The data suggest that PME may act first to demethylate polygalacturonate and may be followed by the action of the other enzymes resulting in cell wall disassembly and fruit softening in pawpaw.
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.
The Stonyhard peach fruit mutation has been used to study softening and textural changes during ripening. Without ethylene exposure, firmness of Stonyhard remains fairly constant at room temperature. When exposed to 1 or 100 ppm C2H2 for 48 hours, fruits soften at a rate consistent with control fruit (`Cresthaven') to a similar firmness. However, 1 ppm—treated fruit attains a normal juicy texture, while 100 ppm—treated fruit attains a pasty texture. Control fruit softened to a normal juicy texture with either ethylene treatment. Cell wall endopolygalacturonase (endo-PG) was not detectable in Stonyhard fruit without C2H2 exposure; it increased at a rate similar to control fruit when exposed to 1 ppm C2H2, and was double that of 1 ppm for fruit exposed to 100 ppm for up to 48 hours. Low levels of endo-PG were detected in control fruit not exposed to C2H2; 1 ppm treatment led to a normal increase, which was comparable to that in Stonyhard. However, endo-PG in 100 ppm—treated fruit was very similar to that of 1 ppm for up to 24 hours, though high levels of endo-PG were observed at 48 hours. Attainment of the pasty texture in 100 ppm—treated Stonyhard fruit may have been related to release of large quantities of pectic polysaccharides as a result of the sudden increase in endo-PG activity. Work was supported by USDA grant 96-34150-2540 and the Oklahoma Agricultural Experiment Station.
The high catalytic potential of PG evident in reactions with soluble pectic polymers is typically not expressed in vivo. In this study, the binding and catalytic properties of PG isozyme 2, and the influence of the B-subunit protein, were investigated using cell walls prepared from tomato fruit expressing the B-subunit antisense gene. Cell walls were prepared from mature-green fruit and treated to remove/inactivate endogenous enzymes. Walls were then preloaded with rate-limiting quantities of purified PG 2, and incubated under catalysis-promoting conditions over the range of pH from 4.5 to 6.0. Cell walls of both B-subunit antisense and wild-type fruit retained comparable quantities of loaded PG 2. The enzymic release of pectin from PG-loaded walls was proportional to the quantity of wall-bound PG 2. In walls lacking the B-subunit protein, the quantity of pectin released by a given dose of wall-associated PG was as much as 2-fold higher than from wild-type walls. The B-subunit protein also influenced the extent of pectin depolymerization during ripening. The release of pectin from cell walls during periods of catalysis was not the sole indicator of the range of pectins hydrolyzed. Treating postcatalytic loaded cell walls to inactivate PG, and subsequent extraction of residual wall pectins using 50 mm CDTA solutions solubilized polymers of significantly lower mol mass compared with pectins solubilized directly from nonloaded cell walls.
A preliminary understanding of developmental processes among divergent species is essential to evaluate the applicability of information from model species to plants of agricultural importance. In tomato (Lycopersicon esculentum Mill.), where the molecular biology associated with fruit ripening has been studied most extensively, tissue softening is due at least in part to the activity of proteins called expansins, in concert with enzymatic activities that modify the pectin and xyloglucan components of the cell wall. We evaluated the potential for the concerted action of expansins and other cell wall-modifying enzymes during ripening in a highly divergent fruit species, sour cherry (Prunus cerasus L.). We identified a family of four expansin genes that was strongly upregulated at the advent of ripening. Activation of these genes was accompanied by strong upregulation of gene(s) encoding potential pectin methylesterases, pectate lyase(s), and xyloglucan endotransglycosylase(s). Initiation of ripening and gene induction were also associated with a rapid decrease in cell wall weight. These results suggest that expansin and several other distinct activities could be involved in ripening-associated cell wall modification in cherries.
Abstract
The capacity of ‘Eldorado’ pears to ripen increased dramatically after 4 weeks of exposure to 0°C and was associated with the synthesis of ethylene by pear tissue. Endogenous levels of ACC and internal ethylene were low after harvest, but increased rapidly after 4 weeks at 0°. Exposure to 0° for 4 weeks also resulted in an increase in soluble polyuronide during subsequent ripening at 20°. In contrast, after 9 months at 0°, soluble polyuronide content showed little increase when pears were transferred to 20°, and fruit failed to soften normally even though ACC content, internal ethylene concentration, ethylene evolution, and respiration remained relatively high. The content of arabinose, galactose, and rhamnose residues in cell walls decreased substantially during the ripening period after 4 weeks or longer at 0°. These cell wall neutral sugars decreased during ripening, even after 9 months of storage at 0°, while firmness and soluble polyuronide showed little change after fruit were transferred to 20°. These data indicate that the failure of pears to soften normally at 20° after prolonged storage at 0° is not related to ethylene synthesis or to changes in cell wall noncellulosic neutral sugar content, but is probably associated with mechanisms of polyuronide solubilization. Chemical name used: 1-aminocyclopropane-1-carboxylic acid (ACC).
Fruit softening occurs by several mechanisms, including modifications of cell wall structure by wall degrading enzymes. The most prominent change in tomato fruit pericarp wall composition is the loss of galactosyl residues throughout development and especially during ripening. In order to understand the role of galactosyl turnover in fruit softening, we successfully produced three recombinant tomato β-galactosidase/exo-galactanase (TBG) fusion proteins in yeast. TBG1, 4 and 5 enzyme properties and substrate specificities were assessed. Optimum pH of TBG1, 4 and 5 was 5.0, 4.0, and 4.5 and optimum temperature was 40∼50, 40, and 40 °C, respectively. The K ms for TBG1, 4 and 5 were 7.99, 0.09, and 2.42 mm, respectively, using p-nitrophenyl-β-D-galactopyranoside as substrate. Using synthetic and plant-derived substrates, TBG1 and 5 released galactosyl residues from 1 → 4 linkages. TBG4 released galactosyl residues from a wide range of plant-derived oligosaccharides and polysaccharides. Using tomato fruit cell wall material, TBG1, TBG4 and TBG5 released galactosyl residues from a variety of fruit stages and cell wall fractions. TBG4 released the most galactosyl residues from the ASP fraction and especially the ASP fraction from fruit at the turning stage. Interestingly, even though walls from Turning fruit stage contain less total galactosyl residues than at the Mature Green stage, TBG4 released 3–4 fold more galactose from the CSP and ASP fractions from Turning fruit. These results suggest that changes in structure of wall pectic polysaccharides leading up to the Turning stage may cause the wall to become more susceptible to hydrolysis by the TBG4 product.
Abstract
‘Valencia’ oranges (Citrus sinensis (L.) Osbeck) with attached pedicels, harvested in April, May, and June, were exposed to 1 ppm ethylene or air for 0, 1, 3, 5, or 7 days. Ethylene lowered fruit removal force but the effect was less pronounced on fruit harvested in May than on fruit harvested in April or June. Endogenous gibberellins (GA) increased prior to regreening and development of the low-response period, and cell-wall hydrolase activity was lower in May than in April or June.
`Anna' and `Granny Smith' apples (Malus domestics Borkh.) that were held at 38C for 4 days before storage at 0C not only were firmer than controls upon removal from storage, but also softened more slowly during shelf life at 17C. Skin yellowing and loss of acidity attendant upon the heat treatment were not prevented by dipping fruit in 2% CaCl2 before heating. Both heat-treated and control fruit softened at the same rate upon exposure to ethylene at 100 μl·liter-1 upon removal from storage. The insoluble pectin content of cortical tissues was higher in heat-treated fruit than in controls after 10 days at 17C, while soluble pectin levels were lower. Arabinose and xylose levels were lower in cell walls from heat-treated cortical tissue, but the treatment had no effect on loss of galactose residues during shelf life.