A novel biotechnological approach to modify the viscosity of tomato fruit pulp: Ribozymes active against endo-1,4-β-glucanase mRNAs

The endo-1,4-β-glucanases (EGases, cellulases) are enzymes active in virtually all phases of plant development where a modification of the cell wall occurs, such as cell expansion and differentiation, fruit ripening and abscission. Tomato genes coding for EGases (tcels) constitute a gene family with at least eight members. Two of these genes, tcell and tcel2, exhibit increased transcription during fruit ripening, but the suppression of mRNA accumulation of tcell alone or tcel2 alone did not detectably alter fruit softening or pulp viscosity. We therefore decided to explore the possibility of down regulating the expression in planta of several EGases using the ribozyme strategy. Specifically we constructed two ribozymes: the first one with a catalytic activity against the mRNA of the tcel3, tcel5, and tcel8 genes, and the second one active against tcell, tcel2, tcel4, tcel7 gene transcripts. Tomato cotyledons were transformed with both ribozymes cloned in two different transgenes under the control of 35S promoter. TO transgenic plants detected by Southern analysis were self-fertilised and T1 seeds were planted in soil inside a greenhouse. Fruits were harvested at the red ripe stage and were used to determine rheological (viscosity and firmness) and chemical parameters (acidity, pH, Brix degrees, reducing sugars). These determinations were conducted on fruits from 85 different transgenic plants. Analysis of viscosity measurement permitted the identification of 9 plants with a very high pulp viscosity, derived from five different T0 transgenic genotypes. The average viscosity of this group was 236.7 cPoise significantly higher than 57.7 cPoise which was the mean of the remaining 76 plants. Interestingly, the high viscosity was not correlated to the glucose and fructose content, as it was instead shown for the pulp with normal viscosity. Additional investigations are undertaken on these 9 transgenic plants to explain the transgene effects.