Sugar accumulation in tomato and partial purification of buffer-insoluble invertase

Abstract

Sugar accumulation and invertase activity were studied during fruit development in tomatoes ( Lycopersicon esculentum) of two breeding lines. Tomatoes of breeding line A had substantially larger amounts of reducing sugars (38%), glucose (33%) and fructose (23%) at the ripe stage compared to breeding line B. Differences in sugar content between the two breeding lines were significant at all stages of maturation, and higher amounts of sugars were consistently linked to higher invertase activities. Buffer-insoluble acid invertase from pericarp was partially purified (12-fold) and consisted, under denaturing conditions (SDS-PAGE), of four distinct bands with estimated M r of 56, 32, 21 and 19 k, respectively. Gel filtration chromatography on Sephacryl S-200 and on FPLC Superose 12 indicated a M r of 50 k for the active invertase enzyme. Western blot analysis with an antibody to carrot cell wall invertase suggested that the two bands with M r of 56 and 21 k correspond to the invertase enzyme. IEF of purified invertase enzyme gave three major bands with estimated isoelectric points between 5.65 and 5.80. The pH optimum of invertase activity was 5.0, and the activity was stable from pH 3.5 to 7.0.