Metabolism of cell wall polysaccharides from persimmon fruit. Pectin solubilization during fruit ripening occurs in apparent absence of polygalacturonase activity

Pectins from persimmon ( Diospyros kaki L.) fruit pericarp were sequentially extracted with 0. 05 M trans -1,2-diaminocyclohexane-N,N, N', N'-tetraacetic acid (CDTA), 0. 05 M Na₂CO₃ (1°C) and Na₂CO₃ (20°C) and the carbohydrate composition and metabolism during development determined. Young persimmon fruits contained a large proportion of pectins, 46% by dry weight, that decreased to 20% with ripening. This decrease occurred in the CDTA and Na₂CO₃ (1°C) fractions, mainly composed of uronic acids, and represents a net loss of uronic acids, arabinose and galactose. The amount of non-cellulosic neutral sugars was especially high in the Na₂CO₃ (20°C) fraction. The loss of pectins was also accompanied by a depolymerisation of the polysaccharides extracted in the three pectic fractions. However, none of these changes can be attributed to the action of polygalacturonase activity. Proteins were extracted from the pericarp tissue, but endopolygalacturonase (EC 3. 2. 1. 15) activity, determined as a decrease in viscosity of polygalacturonic acid, was not observed in the extract. Determination of exopolygalacturonase (EC 3. 2. 1. 67) activity by measuring the release of reducing groups from polygalacturonic acid was also negative. The results presented indicate that polygalacturonase is not responsible for the metabolism of pectins during persimmon fruit ripening.     

Endogenous levels of polyamines and abscisic acid in pepper fruits during growth and ripening

The levels of polyamines (PA) and abscisic acid (ABA) in the pericarp of California variety pepper fruit ( Capsicum annuum L.) were analyzed during development and ripening. Putrescine level was 2.75 μmol g⁻¹ fresh weight 7 days after fruit set and fell during the exponential stage of growth to 1.05 μmol g⁻¹ fresh weight. During the second growth stage. PA and ABA levels remained stable and fell sharply at the beginning of maturation. The levels of spermidine and spermine decreased throughout fruit development and maturation from 0.61 to 0.05 and 0.31 to 0.02 μmol g⁻¹ fresh weight, respectively, but no changes were associated with the onset of maturation. ABA levels remained high (0.70-0.80 μg g⁻¹ fresh weight) during the stages of fruit growth and fell at the beginning of maturation to 0.12 μg g⁻¹ fresh weight, before rising again during the last stages of maturation and senescence. The decrease in putrescine and ABA levels and the subsequent increase in the latter may be responsible for controlling the processes of ripening in pepper fruit.     

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.     

Effect of tunicamycin on in vitro ripening of tomato pericarp tissue

Ripening of pericarp tissue from mature green, early breaker and late breaker stages of tomato ( Lycopersicon esculentum Mill. cv. Dombito) fruit development was inhibitied by tunicamycin. Ripening was evaluated by lycopene accumulation, chlorophyll degradation, rate of ethylene production and cell wall-bound polygalacturonase (EC 3.2.1.15) activity. Maximum inhibition of these ripening parameters occurred at a treatment of 240 μ M tunicamycin for 2 h except for cell wall-bound polygalacturonase activity, which was greatly inhibited by concentrations of 12 μ tunicamycin or higher. Tunicamycin treatment at 120 μ M for 2 h inhibited the incorporation of [³H]-mannose into macromolecules (about 70%) and pronase-sensitive material (about 65%) and the incorporation of [³H]-leucine into proteins (about 20%). Our results indicate that protein glycosylation plays an important role in the ripening of tomato pericarp tissue.     

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

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

Fractionation and partial characterization of cell walls from normal and non-ripening mutant tomato fruit

Cell walls extracted from cv. Rutgers, 7711 (ripening inhibited), and nor (non-ripening) tomato ( Lycopersicon eseulentum Mill.) pericarp tissue at various stages of post-maturation development have been separated into four distinct fractions and their carbohydrate composition characterized. The amount of ionically-associated, chelator-soluble (CDTA, cyclohexanediaminetetraacetic acid) uronic acid in 'Rutgers' fruit cell walls remained constant during ripening, whereas the amount of residual pectin, which was extracted with cold alkali (Na²CO³) and was apparently covalently bound, decreased. These changes did not occur in rin and nor mutant fruit at a similar chronological age. The galactose content in pectic polysaccharide preparations extracted from tomato cell walls with CDTA and Na²,CO³, decreased by 65% during ripening. A similar but diminished decrease also occurred in rin and nor fruit. A non-cellulosic polysaccharide(s) was present in walls which resisted extraction with Na-acetate/CDTA, Na²CO³, and 4 M KOH. In 'Rutgers' fruit, the content of galactose in this polysaccharide(s) decreased 44% during ripening, whereas little or no significant change was observed in rin or nor mutant fruit.     

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.     

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

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

Changes in cell wall neutral sugar composition during fruit ripening: a species survey

Non-cellulosic neutral sugar composition of cell walls from seventeen fruit types were analysed during ripening. Galactose was the major non-cellulosic neutral sugar in cell walls of cucurbit and solanaceous fruit, xylose was the predominant non-cellulosic neutral component of berries, and arabinose was the major non-cellulosic component of pome fruits. The major non-cellulosic neutral sugar residue in cell walls of stone fruits varied. In nectarine and peach, plum, and apricot, the major sugar was arabinose, galactose, and xylose, respectively. In 15 of the 17 types of fruit, a net loss of non-cellulosic neutral sugar residues occurred during ripening. No net loss occurred in plums and cucumbers. A net loss of cell wall galactose and/or arabinose occurred in 14 of the types of fruit. Xylose was the major neutral sugar residue lost from walls of apricot during ripening. In general, berry cell walls were comparatively low in galactose and arabinose content.     

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.     

Enzymic analysis of cell wall structure in apple fruit cortical tissue

Galactanase from Phytophthora infestans and an arabinosidase isoenzyme from Sclerotinia fructigena attacked the cortical cell walls of apple fruits liberating galactose and arabinose residues, respectively. Other arabinosidase isoenzymes from S. fructigena attacked cell walls very slowly. A S. fructigena polygalacturonase isoenzyme liberated half of the uronic acid residues with few associated neutral residues, while a second polygalacturonase isoenzyme released more uronic acid with a substantial proportion of arabinose and galactose and lesser amounts of xylose, rhamnose and glucose; reaction products of this enzyme could be further degraded by the first isoenzyme to give high MW fragments, rich in arabinose with most of the xylose, rhamnose and glucose, and low MW fragments rich in galactose and uronic acid. Endoglucanase from Trichoderma viride released a small proportion of the glucose residues from cell walls together with uronic acid, arabinose, xylose and galactose; more extensive degradation occurred if walls were pre-treated with the second polygalacturonase isoenzyme. Endoglucanase reaction products were separated into a high MW fraction, rich in arabinose, and lower MW fractions rich in galactose and glucose residues. The high MW polygalacturonase and endoglucanase products could be degraded with an arabinosidase isoenzyme to release about 75% of their arabinose. Cell walls from ripe fruit showed similar susceptibility to arabinosidase and galactanase to those from unripe apples. Cell walls from fruit, ripened detached from the tree were more susceptible to degradation by polygalacturonase than walls from unripe fruit or fruit ripened on the tree. Endoglucanase released less carbohydrate from ripe fruit cell walls than from unripe fruit cell walls.     

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.     

Effects of NaCl, pH and Ca2+ on autolysis of isolated tomato fruit cell walls

Cell walls isolated from ripening tomato ( Lycopersicon esculentum Mill. cv. Rutgers) fruit released pectic polymers when incubated under conditions that allow activity of wall-bound polygalacturonase (EC 3.2.1.15). Autolysis was optimally stimulated by 150–300 m M NaCl at either pH 2.5 or 4.5. This stimulation was negated by exposure to pH 6.5 or higher and by pretreatment of walls with boiling 80% ethanol. Five m M CaCl₂ did not affect autolysis at pH 2.5, but significantly inhibited at pH 4.5 or higher. Inclusion of 1 M NaCl at selected steps in the extraction scheme did not inhibit subsequent autolysis of isolated walls. Exposure of isolated walls to 1 M NaCl at pH 2.5–8.5 also did not inhibit autolytic activity compared to walls that received no ionic treatment. These data support the concept that cell wall hydrolysis during tomato fruit softening is regulated by pH, Ca²⁺ levels and ionic strength of the apoplast.     

The role of ethylene and cell wall modifying enzymes in raspberry (Rubus idaeus) fruit ripening

This study focuses on four raspberry ( Rubus idaeus ) genotypes from two different genetic backgrounds: cvs Glen Prosen and Glen Clova, bred at the Scottish Crop Research Institute (SCRI) and genotypes bred at Horticulture Research International (HRI), East Malling (EM), EM 4997 and EM 5007. The ripe fruit of each genotype pair were characterised subjectively by raspberry breeders as relatively firm or soft, respectively. Different stages of fruit development from each genotype were used to quantify fruit firmness, rates of ethylene evolution and ripening rate. Penetrometry data confirmed suspected firmness differences. Firmness correlated with rates of ethylene evolution. Rates of ethylene production also correlated with receptacle size. Storage of green fruits in 20 μl l⁻¹ ethylene reduced fruit firmness, enhanced respiration rate and colour (anthocyanin) development and stimulated the development of cell wall hydrolase activities. However, during natural ripening in the field, fruit respiration rate declined, which indicates a non-climacteric ripening pattern. In drupelets, the activities of polygalacturonase (PG), pectin methylesterase (PME), C x -cellulase (C x ) and β -galactosidase ( β -gal.) increased substantially as ripening progressed. More detailed studies with ripe fruit of cv. Glen Clova indicated major isoforms of PG at pIs 3.3, 8.6 and 10.1; of PME at pIs 7.2, 8.5, 8.7, 8.8; of C x at pI 2.4; and of β -gal. at pIs 6.3 and 6.7.     

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

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

Isolation of β-galactosidase fractions from Japanese pear: Activity against native cell wall polysaccharides

β-Galactosidase (EC 3.2.1.23) was purified from the cell wall of the fruit of Japanese pear ( Pyrus serotina Rehder var. culta Rehder cv. Hosui) and characterized. Five peaks of β-galactosidase activity, designated as Gal I to V, were separated by hydrophobic chromatography on butyl toyopearl and ion exchange chromatography on Mono S. These isolated β-galactosidases were investigated with regard to their abilities to release monomeric galactose from the fractionated polymers of native cell wall (cyclo-hexane-trans-1,2-diamine tetraacetic acid-, Na₂CO₃-, guanidine thiocyanate- and KOH-soluble fractions) and arabinogalactan (from larch wood). All the β-galactosidase fractions were active against native cell wall polysaccharides although to varying degrees. Gal I reacted to all fractions of native cell wall polysaccharides although to varying degrees. Gal I reacted to all fractions of native cell wall and arabinogalactan. Gal II released much galactose only from KOH-soluble polymers and arabinogalactan. Gal III released the most galactose. from cyclohexane- trans -1,2-diamine tetraacetic acid-, Na₂CO₃- and guanidine thiocyanate-soluble cell wall polymers which probably contained galactosyl side chains of pectic polymers, although it did not react much to arabinogalactan. In addition, the activity of Gal Ill dramatically increased as ripening proceeded. Furthermore, Gal III was purified to homogeneity by gel filtration on TSKgel 3000SW and the size of a polypeptide was 80 kDa on SDS-PAGE.     

Stimulation of polysaccharide formation by 2,4-dichlorophenoxyacetic acid in callus tissues of Arabidopsis thaliana

A relatively high concentration of 2,4-dichlorophenoxyacetic acid (45 μ M ) in solid culture medium stimulated the formation and secretion of mucilage polysaccharides by callus tissues of Arabidopsis thaliana L. Heynh. (line Estland). The mucilage was composed of at least two polysaccharides as revealed by gel chromatography on Sepharose 4B: the major component (87%) eluted in the void volume (molecular weight 2 × 10⁶ or greater) and the minor component (13%) eluted in the molecular weight range from 2 × 10⁴ to 4 × 10⁵. Both polysaccharide components contained small amounts of uronic acids. The major polysaccharide consisted mostly of galactose (49%), arabinose (28%) and fucose (10%), whereas the minor one consisted of galactose (44%), xylose (18%), arabinose (14%) and rhamnose (14%). One of the components of the secreted mucilage seems to be an arabinogalactan.     

Alteration of pectic polysaccharides in cell walls, extracellular polysaccharides, and glycan-hydrolytic enzymes of growth-restricted carrot cells under calcium deficiency

Suspension-cultured carrot ( Daucus carota L. cv. Kintoki) cells were grown in calcium (Ca²⁺)-deficient and normal liquid media. Cell growth was limited by the Ca²⁺ deficiency. Similar amounts of pectic fractions were extracted from the walls of control and Ca²⁺-deprived cells, but the fractions from the walls of Ca²⁺-deprived cells showed a substantial decrease in galacturonic acid content. However, after 15 days of culture, Ca²⁺-deprived cells released galacturonic acid-rich extracellular polysaccharides at twice the rate of control cells. The polysaccharides consisted of a mixture of several polymers containing predominantly arabinose, galactose and galacturonic acid. Ca²⁺-deprived cells also secreted three times more extracellular proteins, containing many glycan-hydrolytic enzymes, into the medium than did normal cells. SDS-PAGE analysis revealed several distinct changes in the polypeptide pattern in the medium of control and Ca²⁺-deprived cells. Activities of α -galactosidase, β -glucosidase and exo- polygalacturonase increased considerably during Ca²⁺ deficiency, whereas α - l -arabinofuranosidase and β -galactosidase activities were much reduced.     

Do tomatoes on the plant behave as climacteric fruits?

I considered the possibility that changes in fruit photosynthesis obscure the occurrence of the climacteric rise in respiration in tomato fruits attached to the plant. Internal CO₂ and ethylene concentrations in tomatoes ( Lycopersicon esculentum Mill. cv. OH 7814) were analyzed after direct sampling through polyethylene tubes implanted in the external pericarp. Fruits which were shaded with aluminium foil contained up to 60 ml 1⁻¹ CO₂, until the internal ethylene concentration exceeded 1 μl l⁻¹, when CO₂ concentration declined to below 40 ml l⁻¹; the CO₂ concentration in fruits exposed to light only occasionally exceeded 40 ml 1⁻¹. The internal CO₂ concentration of detached fruits first declined and then increased along with ethylene concentration, as expected for the climacteric. Detached green fruits under continuous low photosynthetic photon flux density (100 μmol m⁻² s⁻¹) contained almost no internal CO₂ and produced no CO₂. Changes in photosynthesis and an associated CO₂-generating system in green fruits are thought to obscure the climacteric rise in tomato fruits developing on the plant.     

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.