D. Donna, R. Jona, B. Dore, P. Pattono, E. Ruffa
Ripening of fruit pulp is a complex process, which is involving many components of the pulp. The processes of fruit ripening and their regulation have been extensively studied, but the approaches have been almost invariably biochemical. These processes have seldom been approached histologically. Jona et al. (1977) started the histochemical analysis of fruit pulp tissues, which later (Jona et al., 1997) became more detailed. The main objective of these studies is to elucidate the factors which influence the texture of the fruit pulp, i.e., the cell-wall polysaccharides. These have been extensively studied by Jona et al. (1983, 1988) from the point of view of changes of levels of various polysaccharides in the cell wall.

Instrumental in these changes is a complex enzymatic mechanism in which polygalacturonase plays a pivotal role: levels of polygalacturonase activity are positively correlated with fruit ripening and softening. The enzyme catalyses the hydrolytic cleavage of &alfa 1 &rarrow; 4 galacturonan linkage and its presence increases in parallel with the ripening of the fruit. For this reason our methodology now involves the use of histological procedures which keep the enzymes active in the tissue, even after inclusion, cutting and affixing on the slide.

Chemical fixation and paraffin embedding have been replaced with a glycol-methacrylate resin (Tecnovit 7100) after an appropriate cycle of cryostabilization (Dore et al., 1992; Dore et al., 1993; Jona et al., 1998). This procedure is far superior to classical fixation and embedding, in maintaining cell structures and biological activity. Very thin sections (1-4 µm) can be cut, with optimal preservation of cell morphology (Fig.1).

Specific stains have been employed to reveal the various cell structures and organelles: nuclei (nuclear red; Feulgen; 4,6-diamino-2-phenylindole fluorochrome (DAPI)), cell walls (periodic acid Schiff; periodic acid silver methenamine) and lipid components (Nile red). The lytic action of the endogenous polygalacturonase on pectins was revealed by a new tetrazolium salt method.

Two outstanding features of these preliminary experiments are: 1 - The method can be applied to the in situ study of the enzymatic activity of the cell organelles. Various enzymes, the products of their activities, and the resulting changes to the cellular structures can all be studied together, on the same slice of tissue. 2 - The water-soluble fluorochrome, Nile red, reveals the location of lipid components with both resolution and sensitivity. It exhibits optimal lipochrome properties: being water soluble, it does not dissolve the lipids, so that the thin sections, which are a prerequisite to fluorochrome revelation, can be used. This is an important step forward in comparison with embedding in paraffin, which requires ingredients that dissolve the lipids.

Donna, D., Jona, R., Dore, B., Pattono, P. and Ruffa, E. (2001). IMPROVED HISTOCHEMISTRY OF RIPENING FRUIT PULP CELLS. Acta Hortic. 553, 211-212
DOI: 10.17660/ActaHortic.2001.553.46
Cryostabilization, enzymes, glycol-methachrylate resin, polygalacturonase, semi-thin slices.

Acta Horticulturae