Extensive solubilization and depolymerization of cell wall polysaccharides during avocado (Perseaamericana) ripening involves concerted action of polygalacturonase and pectinmethylesterase

During the ripening of avocado ( Persea americana Mill.) fruit, water-soluble polyuronides increased dramatically, concomitant with marked downshifts in molecular mass. Treatment of cell walls from pre-ripe fruit with purified avocado polygalacturonase (PG, EC 3.2.1.15) promoted the release and molecular mass downshift of polyuronides. The polyuronides released by PG were similar in size distribution to water-soluble polyuronides from fruit at intermediate stages of ripening. Polyuronides released from pre-ripe fruit by PG, although of relatively high molecular mass, were not further degraded upon additional incubation with fresh enzyme. Similarly, water-soluble polyuronides prepared from fruit at intermediate stages of ripening were largely resistant to the action of purified PG in vitro. When polyuronides derived from fruit at intermediate stages of ripening were treated with weak alkali or pectinmethylesterase (PME, EC 3.1.1.11), extensive molecular mass downshifts occurred in response to incubation with PG. These results suggest that PG plays the central role in polyuronide degradation in ripening avocado fruit cell walls and that partial de-esterification is necessary for the increase in the susceptibility of polyuronides to PG. Differences in the patterns of polyuronide depolymerization in avocado fruit compared with the more thoroughly characterized tomato fruit are discussed.     

Polyuronides in Avocado (Persea americana) and Tomato (Lycopersicon esculentum) Fruits Exhibit Markedly Different Patterns of Molecular Weight Downshifts during Ripening

Avocado (Persea americana) fruit experience a rapid and extensive loss of firmness during ripening. In this study, we examined whether the chelator solubility and molecular weight of avocado polyuronides paralleled the accumulation of polygalacturonase (PG) activity and loss in fruit firmness. Polyuronides were derived from ethanolic precipitates of avocado mesocarp prepared using a procedure to rapidly inactivate endogenous enzymes. During ripening, chelator (cyclohexane-trans-1,2-diamine tetraacetic acid [CDTA])-soluble polyuronides increased from approximately 30 to 40 [mu]g of galacturonic acid equivalents (mg alcohol-insoluble solids)-1 in preripe fruit to 150 to 170 [mu]g mg-1 in postclimacteric fruit. In preripe fruit, chelator-extractable polyuronides were of high molecular weight and were partially excluded from Sepharose CL- 2B-300 gel filtration media. Avocado polyuronides exhibited marked downshifts in molecular weight during ripening. At the postclimacteric stage, nearly all chelator-extractable polyuronides, which constituted from 75 to 90% of total cell wall uronic acid content, eluted near the total volume of the filtration media. Rechromatography of low molecular weight polyuronides on Bio-Gel P-4 disclosed that oligomeric uronic acids are produced in vivo during avocado ripening. The gel filtration behavior and pattern of depolymerization of avocado polyuronides were not influenced by the polyuronide extraction protocol (imidazole versus CDTA) or by chromatographic conditions designed to minimize interpolymeric aggregation. Polyuronides from ripening tomato (Lycopersicon esculentum) fruit extracted and chromatographed under conditions identical with those used for avocado polyuronides exhibited markedly less rapid and less extensive downshifts in molecular weight during the transition from mature-green to fully ripe. Even during a 9-d period beyond the fully ripe stage, tomato fruit polyuronides exhibited limited additional depolymerization and did not include oligomeric species. A comparison of the data for the avocado and tomato fruit indicates that downshifts in polyuronide molecular weight are a prominent feature of avocado ripening and may also explain why molecular down-regulation of PG (EC 3.2.1.15) in tomato fruit has resulted in minimal effects on fruit performance until the terminal stages of ripening.     

Analysis of the molecular size of tomato (Lycopersicon esculentum Mill) fruit polyuronides by gel filtration and low-speed sedimentation equilibrium.

The cell-wall structures of tomato (Lycopersicon esculentum Mill) and other fruit are intimately linked with the nature of their polyuronides. Cell-wall polyuronides from unripe and ripe tomato fruit were isolated and purified and their molecular size and molecular-size distributions were compared. It was demonstrated that there is a considerable decrease in the weight-average Mr upon ripening (from 160,000 +/- 10,000 to 96,000 +/- 4000) and a corresponding increase in polydispersity, particularly at the low-Mr end of the distribution. The estimates of polyuronide molecular size and molecular-size distribution were obtained without the need for polyuronide standards of known Mr by using gel-filtration chromatography combined with the absolute method of low-speed sedimentation equilibrium.     

Methyl de-esterification as a major factor regulating the extent of pectin depolymerization during fruit ripening: a comparison of the action of avocado (Persea americana) and tomato (Lycopersicon esculentum) polygalacturonases

Pectinmethylesterase (PME, EC 3.2.1.11) and polygalacturonase (PG, EC 3.2.1.15) are known to operate in tandem to degrade methylesterified polyuronides. In this study, PGs purified from tomato and avocado fruit were compared in terms of their capacity to hydrolyze water-soluble polyuronides from avocado before and following enzymic or chemical de-esterification. When assayed using polygalacturonic acid or polyuronides from avocado fruit, the activity of PG from tomato fruit was 3-4 times higher than that from avocado fruit. High molecular mass, low methylesterified (33%) water-soluble polyuronides (WSP) from pre-ripe avocado fruit (day 0) were partially depolymerized upon incubation with purified avocado and tomato PGs. In contrast, middle molecular mass, highly methylesterified (74%) WSP from day 2 fruit were largely resistant to the action of both PGs. PME or weak alkali treatment of highly methylesterified WSP decreased the methylesterification values to 11 and 4.5%, respectively. Treatment of de-esterified WSP with either avocado or tomato PGs caused extensive molecular mass downshifts, paralleling those observed during avocado fruit ripening. Although PME and PG are found in many fruits, the pattern of depolymerization of native polyuronides indicates that the degree of cooperativity between these enzymes in vivo differs dramatically among fruits. The contribution of PME to patterns of polyuronide depolymerization observed during ripening compared with physically compromised fruit tissues is discussed.     

Molecular size and separability features of pea cell wall polysaccharides : implications for models of primary wall structure

Relative molecular size distributions of pectic and hemicellulosic polysaccharides of pea (Pisum sativum cv Alaska) third internode primary walls were determined by gel filtration chromatography. Pectic polyuronides have a peak molecular mass of about 1100 kilodaltons, relative to dextran standards. This peak may be partly an aggregate of smaller molecular units, because demonstrable aggregation occurred when samples were concentrated by evaporation. About 86% of the neutral sugars (mostly arabinose and galactose) in the pectin cofractionate with polyuronide in gel filtration chromatography and diethylaminoethyl-cellulose chromatography and appear to be attached covalently to polyuronide chains, probably as constituents of rhamnogalacturonans. However, at least 60% of the wall's arabinan/galactan is not linked covalently to the bulk of its rhamnogalacturonan, either glycosidically or by ester links, but occurs in the hemicellulose fraction, accompanied by negligible uronic acid, and has a peak molecular mass of about 1000 kilodaltons. Xyloglucan, the other principal hemicellulosic polymer, has a peak molecular mass of about 30 kilodaltons (with a secondary, usually minor, peak of approximately 300 kilodaltons) and is mostly not linked glycosidically either to pectic polyuronides or to arabinogalactan. The relatively narrow molecular mass distributions of these polymers suggest mechanisms of co- or postsynthetic control of hemicellulose chain length by the cell. Although the macromolecular features of the mentioned polymers individually agree generally with those shown in the widely disseminated sycamore cell primary wall model, the matrix polymers seem to be associated mostly noncovalently rather than in the covalently interlinked meshwork postulated by that model. Xyloglucan and arabinan/galactan may form tightly and more loosely bound layers, respectively, around the cellulose microfibrils, the outer layer interacting with pectic rhamnogalacturonans that occupy interstices between the hemicellulose-coated microfibrils.     

Effect of Antisense Suppression of Endopolygalacturonase Activity on Polyuronide Molecular Weight in Ripening Tomato Fruit and in Fruit Homogenates

Fruit of tomato (Lycopersicon esculentum Mill.) in which endopolygalacturonase (PG) activity had been suppressed to <1% of wild-type levels were slightly firmer than nontransgenic controls later in ripening. Enzymically inactive cell walls were prepared from these ripening fruit using Tris-buffered phenol. When extracted with chelator followed by Na2CO3, the amounts of pectin solubilized from cell walls of nontransgenic control or from transgenic antisense PG fruit were similar. Size-exclusion chromatography analysis showed that, relative to controls, in antisense PG fruit polyuronide depolymerization was delayed in the chelator-soluble fraction throughout ripening and reduced in the Na2CO3-soluble fraction at the overripe stage. Reduced pectin depolymerization rather than altered extractability thus may have contributed to enhanced fruit firmness. Substantially larger effects of suppressed PG activity were detected in tomato fruit homogenates processed to paste. In control paste the majority of the polyuronide was readily soluble in water and was very highly depolymerized. In antisense PG paste the proportion of polyuronide solubilized by water was reduced, and polyuronides retained a high degree of polymerization. The suppression of fruit PG activity thus has a small effect on polyuronide depolymerization in the fruit but a much larger effect in paste derived from these fruit. This indicates that in the cell wall PG-mediated degradation of polyuronide is normally restricted but that in tissue homogenates or in isolated cell walls this restriction is removed and extensive pectin disassembly results unless PG is inactivated.     

Cell Wall Metabolism in Ripening Fruit: IV. Characterization of the Pectic Polysaccharides Solubilized during Softening of ;Bartlett' Pear Fruit

Fractionation of pectic polysaccharides from the juice of ripening `Bartlett' pears (Pyrus communis) gave two general types of polyuronides. The major type was a homogalacturonan (HGA) whose molecular weight decreased upon ripening. The other type comprised heteropolymers composed of various amounts of arabinose, rhamnose, and galactose. Treatment of the major arabinose-containing heteropolymeric fraction of high molecular weight (400,000) with a pear exo-polygalacturonase to degrade contaminating HGA gave a polyuronide which was inert to tomato endopolygalacturonase. Glycosyl-linkage analysis of this arabinosyl-polyuronide gave results expected from a rhamnogalacturonan I-like polysaccharide with large, highly branched araban side chains (RG-I). A linkage between HGA and RG-I was not found. RG-I, in ripening pears, appeared to be degraded with the initial loss of much of its arabinose.     

Polyuronide solubilization during ripening of normal and mutant tomato fruit

The amount and molecular size of soluble polyuronide extractable from ripening tomatoes is markedly affected by residual enzyme activity. The efficacy of phenol-acetic acid-water treatment to remove this residual activity is demonstrated. Data obtained using treated wall preparations confirms that there is an increase in soluble polyuronide during normal ripening and that this also occurs in the ‘Never-ripe’ mutant, and to a lesser degree in the ‘ripening-inhibitor’ mutant. However, changes in the molecular size of this polyuronide during normal ripening were not as extensive as previously reported and few changes were apparent in either of the mutants.Measurements were also made of polygalacturonase (EC 3.2.1.15) and pectinesterase (EC 3.1.1.11) activity during ripening. The level of polygalacturonase activity does not appear to correlate with the amount of soluble polyuronide released, but may be related to the extent of depolymerisation. No relationship was apparent between the level of pectinesterase and either soluble polyuronide released or depolymerization.     

Soluble and wall-bound glycoproteins of apple fruit tissue

Apple fruit tissue contains small amounts of readily soluble glycoproteins, rich in hydroxyproline; polymethylgalacturonide is not covalently bound to the soluble glycoproteins. Barium hydroxide hydrolysis of apple fruit cell walls liberated glycopeptides containing 4 arabinosyl residues per hydroxyprolyl residue, which were attacked very slowly by α-l-arabinofuranosidase. Hydrazinolysis liberated similar glycopeptides, which were difficult to separate from a polysaccharide containing galactose residues. Protease treatment of walls also released glycopeptides containing hydroxyproline, and a small proportion of these were associated with polyuronide. Polygalacturonase pretreatment of walls led to increased release of hydroxyprolyl residues by protease. Susceptibility of the hydroxyproline containing glycoprotein in the cell wall to attack by protease and arabinosidase did not change during fruit ripening. The amount of an unknown hexosamine associated with the wall was less in ripethan in unripe fruit.     

ESR evidence for sequential divalent cation binding in higher plant cell walls

Electron spin resonance linewidth measurements have been made on intact cell walls exchanged with various combinations of Mn²⁺ and Ca²⁺. These experiments were performed to find the Mn²⁺ nearest-neighbor distance and thereby determine whether carboxylate-Mn²⁺ complexes potentiate ion association at adjacent sites on cell wall polyuronides. Our results show that as the fraction of available binding sites occupied by Mn²⁺ increased from 2% to 27%, the nearest-neighbor distance parameter decreased only from 14 to 11 Å. These distances are close to polyuronide interanionic spacings. The small change in the distance parameter with concentration is evidence for sequential rather than random binding. Competitive ion-exchange with Ca²⁺ was found to reduce the Mn²⁺ spin-spin line broadening at similar total bound Mn²⁺ concentrations. This is expected only if Ca²⁺ competes at adjacent sites. The data presented offer strong support for the hypothesis that carboxylate groups near already occupied sites have a greater affinity for divalent cations than other sites along the polyuronide main chain.     

Polysaccharide changes in cell walls of ripening apples

Changes in the polysaccharide composition of apple fruits ripening on and off the tree were compared. Polysaccharide fractions defined by their method of extraction were analysed colorimetrically, and the monosaccharide composition of total acetone insoluble material was analysed. Neutral carbohydrate associated with pectic extractives decreased; correspondingly galactose residues were lost in detached fruit, while galactose and arabinose residues were lost in fruit on the tree. Decreases in hemicellulose were correlated with losses of wall glucan; xylose contents did not change. Soluble polyuronide increased especially in detached fruit. DEAE-cellulose chromatography showed that this polyuronide was free from neutral sugar residues. Amounts of soluble neutral polysaccharides and glycoproteins did not change during fruit ripening.     

Composition of the cell wall formed by protoplasts isolated from cell suspension cultures of Vinca rosea

The biochemistry of cell-wall regeneration in protoplasts obtained from Vinca rosea L. (Catharanthus roseus (L.) G. Don) cells grown in suspension culture by isolating the regenerated wall and the extracellular polysaccharides of protoplasts cultured for various periods, and investigating their composition. Gas-liquid chromatography and tracer studies with D-[U-¹⁴C]glucose showed that the sugar composition of the extracellular polysaccharides was similar to that of the original cell culture, consisting mainly of polyuronide and 3,6-linked arabinogalactan. the regenerated cell wall was composed of non-cellulosic glucans having 1,3- and 1,4-linkages, while its content in pectic and hemicellulosic components was very low.     

Changes in Cell Wall Composition and Water-soluble Polysaccharides During Kiwifruit Development

Changes in both cell wall and water-soluble polysaccharide compositionduring the growth of kiwifruits [Actinidia deliciosa (A. chev.) C. F. Liang and A. R. Ferguson var. deliciosa ‘Hayward’]were investigated. Cellulose was the major wall polysaccharide,with galactose and uronics the main non-cellulosic sugars. Muchsolubilization of cell wall pectic polysaccharides was detected.While wall-galactose solubilization started 3 months after anthesis,polyuronide degradation did not start until the fifth month,1 month prior to the harvest date. Parallel to these processes,a linear increase in water-soluble polysaccharides was detected.These mainly comprised galactose-rich polymers in the first3 months and little-branched polyuronides after the fifth month.Two different mechanisms for galactose and uronic acid solubilizationfrom kiwifruit cell walls during fruit development are proposed. Actinidia deliciosa ; cell wall; fruit growth; kiwifruit; water-soluble polysaccharides     

Cell wall modification in 1-methylcyclopropene-treated post-climacteric fresh-cut and intact papaya fruit

Papaya is a climacteric fruit in which ripening is greatly regulated by ethylene often associated with stress responses such as wounding. The changes in cell wall compositions in papaya fruit at an advanced stage of ripening under stress conditions including chilling temperature of 5°C and wounding employed as fresh-cut and how these changes were affected by an ethylene action inhibitor of 1-methylcyclopropopene (1-MCP) were examined in the study. The recovery of ethanol-insoluble solids, total soluble sugars, water-soluble polyuronides, neutral hemicelluloses, and neutral sugars of rhamnose, arabinose, mannose and glucose were not affected by 1-MCP or fresh-cut processing. The fresh-cut processing, however, caused a higher loss of total polyuronides and the neutral sugar galactose while increasing the recovery of chelator-soluble polyuronides. Few significant differences due to 1-MCP application were recorded in the recoveries of alkali-soluble polyuronides, hemicellulosic polyuronides extracted with 4% KOH, and the neutral sugar xylose. Modifications of cell wall polyuronides and hemicelluloses in ripe fresh-cut papaya fruit exhibited mostly similar patterns to those in intact ripe papaya fruit under the chilling temperature of 5°C while minimally affected by 1-MCP.     

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.     

Isolation and partial characterization of uronic acid-containing glycoproteins from Mucor rouxii

Five different fractions containing uronic acids associated with protein were isolated from the cytoplasm of the filamentous form of Mucor rouxii. A signle fraction was isolated from the cell wall by hot sodium dodecyl sulfate followed by ion exchange column chromatography. Two cytoplasmic entities (peaks I and II) were not adsorbed to DEAE Bio-Gel A. The molecular mass of peaks I to V ranged from 16.5 to 210 kDa. The protein-uronic acid ratios were different for each fraction. The cell wall fraction showed a molecular mass of 16.5 kDa, similar to that of peak II but with differences in chromatographic behavior and protein-uronic acid ratio. The possible role of these molecules as acceptors of sugar residues during polyuronide chain growth is discussed.     

Polysaccharides and glycoproteins of apple fruit cell walls

A proportion of the polysaccharides and glycoproteins of apple fruit cell walls can be readily extracted in neutral buffer at or below 20°. Removal of more material was not achieved with a wide range of dissociative aqueous reagents or non-aqueous solvents. Thus traditional degradative extractants were used to obtain soluble components for further characterization. Polysaccharides and glycoproteins were separated and purified by chromatography on DEAE-cellulose columns and by gel filtration. Purified components were hydrolysed and analysed for neutral sugar and uronic acid content and for their amino acid and hydroxyproline content. The possibility of linkages existing in the cell wall between polyuronide and glycoproteins containing hydroxyproline, arabinose and galactose residues is discussed. Because of aggregation between these components, which occurs after extraction, the presence of such linkages in vivo is difficult to establish. Other cell wall glycoproteins containing xylose and glucose residues are thought to have a possible role in stabilizing hemicellulose structure.     

Modifications of cell wall polysaccharides during auxin-induced growth in azuki bean epicotyl segments

Changes in sugar compositions and the distribution pattern ofthe molecular weight of cell wall polysaccharides during indole-3-aceticacid (IAA)-induced cell elongation were investigated. Differentialextraction of the cell wall and gel permeation chroma-tographyof wall polysaccharides indicated that galactans, polyuronides,xylans, glucans and cellulose were present in the azuki beanepicotyl cell wall. When segments were incubated in the absence of sucrose, IAAenhanced the degradation of galactans in both the pectin andhemicellulose fractions, whereas to some extent it enhancedthe polymerization of xylans and glucans in the hemicellulosefraction and an increase in the amounts of polyuronides in thepectin fraction and of -cellulose. In the presence of 50 mMsucrose, IAA caused large increases in the amounts of all thewall polysaccharides, and enhanced the polymerization of galactans,xylans and glucans in the hemicellulose fraction. These results and an important role of galactan turnover incell wall extension are discussed. (Received December 11, 1979; )     

Cell wall-degrading enzymes and pectin solubility and depolymerization in immature and ripe watermelon (Citrullus lanatus) fruit in response to exogenous ethylene

Watermelon fruit have been shown to be extremely sensitive to exogenous ethylene, exhibiting acute symptoms of whole-fruit softening and placental-tissue water-soaking following short periods of exposure to the gas. This study addressed the firmness, specific activities of cell wall hydrolases, and solubility and molecular mass properties of polyuronides in placental tissue in response to treatment of intact fruit with ethylene. Watermelon fruit were harvested at the immature and full-ripe stages and exposed to 50 µl l⁻¹ ethylene for 6 days at 20°C. The firmness of placental tissue from ethylene-treated ripe and immature fruit decreased nearly 80% during 6 days of ethylene exposure, whereas the firmness of placental tissue from fruit maintained in air remained relatively constant up to day 3 and then decreased slightly (12%) during the following 3 days of storage. Although ethylene treatment in some cases influenced the levels of extractable placental-tissue polygalacturonase (EC 3.2.1.15), pectinmethylesterase (EC 3.2.1.11), and α -(EC 3.2.1.22) and β -galactosidase (EC 3.2.1.23) specific activities, these effects were not observed for fruit of both developmental stages and appeared not to be directly involved in the water–soaking syndrome. Symptoms of water-soaking were accompanied by increases in the levels of water- and CDTA ( trans -1,2-cyclohexanediamine- N,N,N',N' -tetraacetic acid)-soluble polyuronides and significant molecular mass downshifts in polyuronides in both immature and ripe watermelon fruit. Polyuronide depolymerization in ethylene-treated ripe fruit was extensive. The parallel trends of enzyme changes in ethylene- compared with air-treated fruit indicate that extractable enzyme levels are not associated with development of the water-soaking disorder. The potential involvement of membrane dysfunction in the water-soaking phenomenon is discussed.