Excessive softening is the main factor limiting fruit shelf life and storage. Transgenic plants modified in the expression of cell wall modifying proteins have been used to investigate the role of particular activities in fruit softening during ripening, and in the manufacture of processed fruit products. Transgenic experiments show that polygalacturonase (PG) activity is largely responsible for pectin depolymerization and solubilization, but that PG-mediated pectin depolymerization requires pectin to be de-methyl-esterified by pectin methylesterase (PME), and that the PG β-subunit protein plays a role in limiting pectin solubilization. Suppression of PG activity only slightly reduces fruit softening (but extends fruit shelf life), suppression of PME activity does not affect firmness during normal ripening, and suppression of β-subunit protein accumulation increases softening. All these pectin-modifying proteins affect the integrity of the middle lamella, which controls cell-to-cell adhesion and thus influences fruit texture. Diminished accumulation of either PG or PME activity considerably increases the viscosity of tomato juice or paste, which is correlated with reduced polyuronide depolymerization during processing. In contrast, suppression of β-galactosidase activity early in ripening significantly reduces fruit softening, suggesting that the removal of pectic galactan side-chains is an important factor in the cell wall changes leading to ripening-related firmness loss. Suppression or overexpression of endo-(1\to4)β-d-glucanase activity has no detectable effect on fruit softening or the depolymerization of matrix glycans, and neither the substrate nor the function for this enzyme has been determined. The role of xyloglucan endotransglycosylase activity in softening is also obscure, and the activity responsible for xyloglucan depolymerization during ripening, a major contributor to softening, has not yet been identified. However, ripening-related expansin protein abundance is directly correlated with fruit softening and has additional indirect effects on pectin depolymerization, showing that this protein is intimately involved in the softening process. Transgenic work has shown that the cell wall changes leading to fruit softening and textural changes are complex, and involve the coordinated and interdependent activities of a range of cell wall-modifying proteins. It is suggested that the cell wall changes caused early in ripening by the activities of some enzymes, notably β-galactosidase and ripening-related expansin, may restrict or control the activities of other ripening-related enzymes necessary for the fruit softening process.

Cell wall metabolism in fruit softening and quality and its manipulation in transgenic plants

CELL WALL STRUCTURE AND RIPENING-ASSOCIATED CHANGES .. " ......... 676 Cellulose 677 Hemicellulose 678 Pectin . .... ........ . 679 Other Cell Wall Components " . . . . . . " . . . . . . " . . . . . . " . . . . . . " . . . . . . . . . . . . . . . . . . . . . . . 681 CELL WALL HYDRO LASES . ....... . . ....... . . 681 Polygalacturonase . .. , . . . . . . . . . .. .. . . . ..... . . . . . . . . . . . . . . . . . . ... .. ... .. . ...... .. . . . . . 682 Pectinmethylesterase..... ......... . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . 686 Cx-Cellulase . ......... 687 Other Cell Wall Hydrolases 688 CELL WALL HYDROLASE FUNCTION DURING FRUIT RIPENING 689 Physical. Genetic. and Biochemical Modification of Cell Wall Hydrolase Activity 689 Molecular Genetic Modification of Cell Wall Hydrolase Activity ". . . . . . . . . 690 FUTURE PROSPECTS . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . .. ... .. . . . .. . . .. ........ .. . . ...... . .. 695

Role of cell wall hydrolases in fruit ripening

It is proposed to describe briefly the rise in popularity of the tomato during the 20th century to become the world's fourth most significant fruit, making an important contribution to human nutrition. The grading standards for fruit used in various major tomato-producing countries will be outlined. A major part of the review will be devoted to describing changes in composition during the maturation, ripening, preservation, and storage of good quality tomatoes especially as regards carbohydrates, organic and amino acids, proteins, steroids, pigments, minerals, and the lipids, volatiles, and other minor constituents. A range within which composition should normally fall will be given. Additionally, the effects on composition of environment, cultivar, nutrition, and physiological disorders inter alia will be described. How new growing methods and genetic manipulation could influence the tomato of the future will also be considered.

The constituents of tomato fruit — the influence of environment, nutrition, and genotype

Tomato fruit ripening is accompanied by extensive degradation of pectic cell wall components. This is thought to be due to the action of a single enzyme, polygalacturonase, whose activity is controlled, at least in part, at the level of gene expression. At the onset of tomato fruit ripening, polygalacturonase enzyme activity, mRNA levels, and relative rate of gene transcription all increase dramatically. To elucidate the role of polygalacturonase during tomato fruit ripening, we utilized a pleiotropic genetic mutation, rin, that blocks many aspects of ripening, including the activation of polygalacturonase gene transcription. The polygalacturonase structural gene was ligated to a promoter that is inducible in mature rin fruit and inserted into the fruit genome, and plants were regenerated. This allowed expression of the polygalacturonase gene in transgenic rin fruit at a time corresponding to ripening in wild-type fruit. Expression of this gene resulted in the accumulation of active polygalacturonase enzyme and the degradation of cell wall polyuronides in transgenic rin fruit. However, no significant effect on fruit softening, ethylene evolution, or color development was detected. These results indicate that polygalacturonase is the primary determinant of cell wall polyuronide degradation, but suggest that this degradation is not sufficient for the induction of softening, elevated rates of ethylene biosynthesis, or lycopene accumulation in rin fruit.

Expression of a chimeric polygalacturonase gene in transgenic rin (ripening inhibitor) tomato fruit results in polyuronide degradation but not fruit softening.

The role of the ripening-specific expansin Exp1 protein in fruit softening and cell wall metabolism was investigated by suppression and overexpression of Exp1 in transgenic tomato plants. Fruit in which Exp1 protein accumulation was suppressed to 3% that of wild-type levels were firmer than controls throughout ripening. Suppression of Exp1 protein also substantially inhibited polyuronide depolymerization late in ripening but did not prevent the breakdown of structurally important hemicelluloses, a major contributor to softening. In contrast, fruit overexpressing high levels of recombinant Exp1 protein were much softer than controls, even in mature green fruit before ripening commenced. This softening was correlated with the precocious and extensive depolymerization of structural hemicelluloses, whereas polyuronide depolymerization was not altered. These data are consistent with there being at least three components to fruit softening and textural changes. One component is a relaxation of the wall directly mediated by Exp1, which indirectly limits part of a second component due to polyuronide depolymerization late in ripening, perhaps by controlling access of a pectinase to its substrate. The third component is caused by depolymerization of hemicelluloses, which occurs independently of or requires only very small amounts of Exp1 protein.

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Modification of Expansin Protein Abundance in Tomato Fruit Alters Softening and Cell Wall Polymer Metabolism during Ripening

SUMMARY

An enzyme that attacks carboxymethyl cellulose may be extracted efficiently from tomato fruit by salt solutions. From a high initial value in small green fruit, activity fell gradually during fruit swelling. With incipient ripeness, the activity increased again and continued to rise to the full red condition. The green areas of “blotchy” ripened fruit showed 40% less activity than the adjacent red tissue. Fruit of tomato species in the sub-genus Eriopersicon contained considerably more activity than examples from the sub-genus Eulycopersicon. Firmness measurements on fruit from both sub-genera were not significantly correlated with the cellulase activities, and from this and other evidence it is concluded that is is not a major factor controlling the softening of at fruit, at least during the ripening period.

G. E. Hobson

Papers

Cellulase Activity during the Maturation and Ripening of Tomato Fruit