Effect of the Colorless non-ripening mutation on cell wall biochemistry and gene expression during tomato fruit development and ripening.

The Colorless non-ripening (Cnr) mutation in tomato (Solanum lycopersicum) results in mature fruits with colorless pericarp tissue showing an excessive loss of cell adhesion (A.J. Thompson, M. Tor, C.S. Barry, J. Vrebalov, C. Orfila, M.C. Jarvis, J.J. Giovannoni, D. Grierson, G.B. Seymour [1999] Plant Physiol 120: 383-390). This pleiotropic mutation is an important tool for investigating the biochemical and molecular basis of cell separation during ripening. This study reports on the changes in enzyme activity associated with cell wall disassembly in Cnr and the effect of the mutation on the program of ripening-related gene expression. Real-time PCR and biochemical analysis demonstrated that the expression and activity of a range of cell wall-degrading enzymes was altered in Cnr during both development and ripening. These enzymes included polygalacturonase, pectinesterase (PE), galactanase, and xyloglucan endotransglycosylase. In the case of PE, the protein product of the ripening-related isoform PE2 was not detected in the mutant. In contrast with wild type, Cnr fruits were rich in basic chitinase and peroxidase activity. A microarray and differential screen were used to profile the pattern of gene expression in wild-type and Cnr fruits. They revealed a picture of the gene expression in the mutant that was largely consistent with the real-time PCR and biochemical experiments. Additionally, these experiments demonstrated that the Cnr mutation had a profound effect on many aspects of ripening-related gene expression. This included a severe reduction in the expression of ripening-related genes in mature fruits and indications of premature expression of some of these genes in immature fruits. The program of gene expression in Cnr resembles to some degree that found in dehiscence or abscission zones. We speculate that there is a link between events controlling cell separation in tomato, a fleshy fruit, and those involved in the formation of dehiscence zones in dry fruits.

Mechanical effects of plant cell wall enzymes on cellulose/xyloglucan composites.

Xyloglucan-acting enzymes are believed to have effects on type I primary plant cell wall mechanical properties. In order to get a better understanding of these effects, a range of enzymes with different in vitro modes of action were tested against cell wall analogues (bio-composite materials based on Acetobacter xylinus cellulose and xyloglucan). Tomato pericarp xyloglucan endo transglycosylase (tXET) and nasturtium seed xyloglucanase (nXGase) were produced heterologously in Pichia pastoris. Their action against the cell wall analogues was compared with that of a commercial preparation of Trichoderma endo-glucanase (EndoGase). Both 'hydrolytic' enzymes (nXGase and EndoGase) were able to depolymerise not only the cross-link xyloglucan fraction but also the surface-bound fraction. Consequent major changes in cellulose fibril architecture were observed. In mechanical terms, removal of xyloglucan cross-links from composites resulted in increased stiffness (at high strain) and decreased visco-elasticity with similar extensibility. On the other hand, true transglycosylase activity (tXET) did not affect the cellulose/xyloglucan ratio. No change in composite stiffness or extensibility resulted, but a significant increase in creep behaviour was observed in the presence of active tXET. These results provide direct in vitro evidence for the involvement of cell wall xyloglucan-specific enzymes in mechanical changes underlying plant cell wall re-modelling and growth processes. Mechanical consequences of tXET action are shown to be complimentary to those of cucumber expansin.