Purification and characterization of two ribonucleases from developing tomato fruit

Area of Science:

• Plant biochemistry
• Molecular biology
• Enzymology

Background:

• Ribonucleases (RNases) play crucial roles in RNA metabolism and plant defense.
• Understanding RNase activity during fruit development is key to unraveling ripening processes.
• Specific RNases in Solanaceae plants are linked to self-incompatibility.

Purpose of the Study:

• To purify and characterize neutral ribonucleases from developing tomato fruit.
• To investigate the enzymatic properties and developmental regulation of these RNases.
• To compare these tomato RNases with known S-glycoprotein ribonucleases.

Main Methods:

• Purification of ribonucleases using chromatography.
• Enzyme activity assays at different developmental stages.
• Molecular weight determination via SDS-PAGE.
• Subunit analysis and renaturation studies.
• Immunological cross-reactivity tests.
• Characterization of substrate specificity and cleavage products.

Main Results:

• Two neutral ribonucleases, Tf1 (59 K) and Tf2 (29 K), were purified.
• RNase activity peaked 5 days post-anthesis, decreasing during maturation.
• Tf1 has two subunits (30 K and 29 K), with only the 30-K subunit being active and cross-reactive with Tf2.
• Both are endoribonucleases with pyrimidine preference, producing 2',3'-cyclic phosphate products.
• Tf2 is a single, renaturable 29-K polypeptide.

Conclusions:

• The characterized tomato fruit ribonucleases (Tf1 and Tf2) exhibit distinct molecular properties and developmental expression patterns.
• These enzymes are glycoproteins with specific cleavage preferences, suggesting roles in RNA processing during fruit development.
• Shared properties with S-glycoprotein ribonucleases hint at potential conserved functions in Solanaceae plants.