Pectate lyase activity during ripening of banana fruit

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

Compositional changes in cell wall polymers during mango fruit ripening

Softening of mango fruit has been investigated by analysis of ripening related changes in the composition of the fruit cell walls. There is an apparent overall loss of galactosyl and deoxyhexosyl residues during ripening, the latter indicating degradation of the pectin component of the wall. The loss of galactose appears to be restricted to the chelator soluble fraction of the wall pectin, whilst loss of deoxyhexose seems to be more evenly distributed amongst the pectin. The chelator soluble pectin fraction is progressively depolymerised and becomes more polydisperse during ripening. These changes are similar to those occurring in other fruit and are related to the action of wall hydrolases during ripening.     

Glycosyl-linkage composition of tomato fruit cell wall hemicellulosic fractions during ripening

Hemicelluloses were extracted from isolated tomato ( Lycopersicon esculentum Mill. cv. Rutgers) pericarp cell wall material at 3 different stages of ripeness with 4 M and 8 M KOH. Little change in molecular weight or composition of 4 M KOH-extracted material was observed during ripening. However, the composition of 8 M KOH-extracted material changed, and a relative increase in polymers of < 40 kDa was observed during ripening. Changes in glycosyl linkage composition of the 8 M KOH hemicellulosic material were detected, including increases in 4-linked mannosyl, 4,6-linked mannosyl, and 4-linked glucosyl, and decreases in 5-linked arabinosyl residues in polymers of < 40 kDa, and decreases in terminal glocosyl residues in polymers of > 40 kDa. These data may indicate that de novo hemicellulose synthesis occurs throughout tomato fruit ripening, even at the red ripe stage.     

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.     

Altered cell wall disassembly during ripening of Cnr tomato fruit: implications for cell adhesion and fruit softening

The Cnr ( C olourless n on- r ipening) tomato ( Lycopersicon esculentum Mill.) mutant has an aberrant fruit-ripening phenotype in which fruit do not soften and have reduced cell adhesion between pericarp cells. Cell walls from Cnr fruit were analysed in order to assess the possible contribution of pectic polysaccharides to the non-softening and altered cell adhesion phenotype. Cell wall material (CWM) and solubilised fractions of mature green and red ripe fruit were analysed by chemical, enzymatic and immunochemical techniques. No major differences in CWM sugar composition were detected although differences were found in the solubility and composition of the pectic polysaccharides extracted from the CWM at both stages of development. In comparison with the wild type, the ripening-associated solubilisation of homogalacturonan-rich pectic polysaccharides was reduced in Cnr. The proportion of carbohydrate that was chelator-soluble was 50% less in Cnr cell walls at both the mature green and red ripe stages. Chelator-soluble material from ripe-stage Cnr was more susceptible to endo-polygalacturonase degradation than the corresponding material from wild-type fruit. In addition, cell walls from Cnr fruit contained larger amounts of galactosyl- and arabinosyl-containing polysaccharides that were tightly bound in the cell wall and could only be extracted with 4 M KOH, or remained in the insoluble residue. The complexity of the cell wall alterations that occur during fruit ripening and the significance of different extractable polymer pools from cell walls are discussed in relation to the Cnr phenotype.     

Activity and expression of banana starch phosphorylases during fruit development and ripening

Two main forms of starch phosphorylase (EC 2.4.1.1) were identified and purified from banana (Musa acuminata Colla. cv. Nanic?o) fruit. One of them, designated phosphorylase I, had a native molecular weight of 155 kDa and subunit of 90 kDa, a high affinity towards branched glucans and an isoelectric point around 5.0. The other, phosphorylase II, eluted at a higher salt concentration from the anion exchanger, had a low affinity towards branched glucans, a native molecular weight of 290 kDa and subunit of 112 kDa. Kinetic studies showed that both forms had typical hyperbolic curves for orthophosphate (Pi) and glucose-1-phosphate, and that they could not react with substrates with a blocked reducing end or alpha-1,6 glucosidic bonds. Antibodies prepared against the purified type-II form and cross-reacting with the type-I form showed that there was an increase in protein content during development and ripening of the fruit. The changes in protein level were parallel to those of phosphorylase activity, in both the phosphorolytic and synthetic directions. Considering the kinetics, indicating that starch phosphorylases are not under allosteric control, it can be argued that protein synthesis makes a contribution to regulating phosphorylase activity in banana fruit and that hormones, like gibberellic acid and indole-3-acetic acid, may play a regulating role. For the first time, starch phosphorylases isoforms were detected as starch-granule-associated proteins by immunostaining of SDS-PAGE gels.     

Upsurge of particulate peroxidase in ripening banana fruit

Peroxidase was isolated from the pulp of ripening banana fruit and assayed with o-dianisidine as hydrogen-donor. Cell macerates contained soluble and particle-bound peroxidase. Soluble peroxidase levels did not appreciably differ in pre-climacteric, climacteric and post-climacteric fruit. Particulate peroxidase levels increased 3-fold with the initiation of the respiration climacteric and gradually declined with the onset of senescence. Bound peroxidase was released from cell wall and membrane fractions with washing in 0–8 M CaCl₂.     

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.     

Expression of multiple forms of polygalacturonase gene during ripening in banana fruit.

The activity of polygalacturonase (PG, E.C 3.2.1.15) during ripening in climacteric fruits has been positively correlated with softening of the fruit tissue and differential expression of its gene is suspected to be regulated by the plant hormone ethylene. We have cloned four partial cDNAs, MAPG1 (acc. no. AF311881), MAPG2 (acc. no. AF311882), MAPG3 (acc. no. AF542382) and MAPG4 (acc. no. AY603341) for PG genes and studied their differential expression during ripening in banana. MAPG3 and MAPG4 are believed to be ripening related and regulated by ethylene whereas MAPG2 is associated more with senescence. MAPG1 shows constitutive expression and is not significantly expressed in fruit tissue. The genomic clone MAGPG (acc. No. AY603340) includes the complete MAPG3 gene, which consists of four exons and three introns. The structure of the gene has more similarity to tomato abscission PG rather than tomato fruit PG. It is concluded that softening during ripening in banana fruit results from the concerted action of at least four PG genes, which are differentially expressed during ripening.     

Changes in cell wall polysaccharides and monosaccharides during development and ripening of Japanese pear fruit

1. Changes in polysaccharide and monosaccharide components in thecell wall were studied during cell division, cell enlargmementand softening in Japanese pear fruit. Wall polysaccharides werefractionated into water soluble carbohydrate, NaClO₂ solublecarbohydrate, EDTA soluble carbohydrate, acid soluble hemicellulose,alkali soluble hemicellulose and cellulose. These polysaccharideswere composed of glucose, uronic acid, xylose, arabinose, galactose,rhamnose, mannose and fucose.
2. The total polysaccharide contentof the cell wall per cell (DNAcontent basis) remained constantduring the cell division period(S1). But during the pre-enlargementperiod (S2) it began toincrease rapidly in spite of the slightnessof cell enlargement.Thereafter, during the enlargement period(S3) the polysaccharidesremained almost constant although thefruits enlarged dramatically,and the polysaccharides increasedsomewhat with ripening. Thequality of the polysaccharides,however, seemed to change activelyat each stage. This suggestedthat the extensive fruit enlargementdid not require an increasein polysaccharide content, and wasrather accompanied by thepartial breakdown or partial interconversionof polysaccharidecomponents already present.
3. The loss of arabinose and galactosein acid soluble hemicellulosewas prominant in fruit softeningoccurring in the ripening stage.The cellulose component decreasedwith overripening. Water solublepectin increased parallel tothe increase in total pectin withripening. On the other hand,xylose and non-cellulosic glucoseresidues did not alter withripening or overripening. Non-cellulosicglucose continued toaccumulate during cell enlargement.

¹ This paper is Contribution A-88, Fruit Tree Research Station. (Received August 4, 1978; )     

Fruit softening in banana: correlation among stress-relaxation parameters, cell wall components and starch during ripening

Bananas, Musa (AAA group, Cavendish subgroup) 'Giant Cavendish', were ripened in a biotron at 25°C with ethylene during 4 days. Changes in mechanical properties of pulp were detected by a stress-relaxation technique. The decrease in T₀, the parameter for minimum stress-relaxation time, began between day 0.5 and 1, while the decrease in initial stress began between day 0 and 0.5, suggesting that the decrease in elasticity and viscosity of pulp is a crucial physical event of pulp softening. Cellulose and moisture contents were about 3 and 780 mg (g fresh weight)⁻¹, respectively, which were unchanged during ripening. The decrease in starch content of cell materials and in uronic acid content of the pectic polysaccharides of the cell walls began between day 0.5 and 1. As regards the sugar composition of the hemicellulose fraction, decreases in arabinose, mannose and galactose contents began between day 0 and 0.5. The results show that the partial decrease in hemicelluloses preceded the breakdown of starch and suggest that the coordinated degradation of pectic and hemicellulosic polysaccharides and starch is the main cause for the pulp softening process.