Leona Claire Fitzmaurice

Isolation and characterization of fruit vacuolar invertase genes from two tomato species and temporal differences in mRNA levels during fruit ripening.

To determine the relationship between invertase gene expression and glucose and fructose accumulation in ripening tomato fruit, fruit vacuolar invertase cDNA and genomic clones from the cultivated species, Lycopersicon esculentum cv. UC82B, and a wild species, Lycopersicon pimpinellifolium, were isolated and characterized. The coding sequences of all cDNA clones examined are identical. By comparison to the known amino acid sequence of mature L. esculentum fruit vacuolar invertase, a putative signal sequence and putative amino-terminal and carboxy-terminal propeptides were identified in the derived amino acid sequence. Of the residues 42% are identical with those of carrot cell wall invertase. A putative catalytic site and a five-residue motif found in carrot, yeast, and bacterial invertases are also present in the tomato sequence. Minor differences between the nucleotide sequences of the genomic clones from the two tomato species were found in one intron and in the putative regulatory region. The gene appears to be present in one copy per haploid genome. Northern analysis suggests a different temporal pattern of vacuolar invertase mRNA levels during fruit development in the two species, with the invertase mRNA appearing at an earlier stage of fruit development in the wild species. Nucleotide differences found in the putative regulatory regions may be involved in species differences in temporal regulation of this gene, which in turn may contribute to observed differences in hexose accumulation in ripening fruit.

Purification, Characterization, and Subcellular Localization of Soluble Invertase from Tomato Fruit

Summary Soluble invertase (β-fructosidase EC 3.2.1.256) was purified to apparent homogeneity from ripe tomato ( Lycopersicon esculentum Mill.) fruit using conventional procedures. Soluble tomato invertase is a glycoprotein of 52 kDa with a polypeptide moiety of 45 kDa. This purified enzyme hydrolyzes sucrose to fructose and glucose and raffinose to fructose and melibiose. Lycopersicon pimpinellifolium (Jusl.) Mill. fruits also contain an immunologically cross-reactive protein with the same molecular weight as L. esculentum invertase. Antibodies to deglycosylated carrot cell-wall invertase recognized two polypeptides in both tomato species: 52 kDa and 68 kDa. The 52 kDa polypeptides were abundant in fruit extracts, and the 68 kDa polypeptides were abundant in extracts of suspension culture cells. The 52 kDa invertase is localized in vacuoles of the tomato fruit cells. Amino acid sequence data obtained from the 52 kDa polypeptide, two smaller polypeptides (33 kDa and 24 kDa) believed to be degradation products of the intact protein, and three cyanogen bromide cleavage fragments reveal significant amino acid sequence similarity with carrot, yeast, and bacterial invertases.