Sink Metabolism in Tomato Fruit

Abstract

In developing tomato (Lycopersicon esculentum Mill.) fruit, starch levels reach a peak early in development with soluble sugars (hexoses) gradually increasing in concert with starch degradation. To determine the enzymic basis of this transient partitioning of carbon to starch, the activities of key carbohydrate-metabolizing enzymes were investigated in extracts from developing fruits of three varieties (cv VF145-7879, cv LA1563, and cv UC82B), differing in final soluble sugar accumulation. Of the enzymes analyzed, ADPglucose pyrophosphorylase and sucrose synthase levels were temporally correlated with the transient accumulation of starch, having highest activities in cv LA1563, the high soluble sugar accumulator. Of the starch-degrading enzymes, phosphorylase levels were fivefold higher than those of amylase, and these activities did not increase during the period of starch degradation. Fiften percent of the amylase activity and 45 to 60% of the phosphorylase activity was localized in the chloroplast in cv VF145-7879. These results suggest that starch degradation in tomato fruit is predominantly phosphorolytic. The results suggest that starch biosynthetic capacity, as determined by levels of ADPglucose pyrophosphorylase rather than starch degradative capacity , regulate the transient accumulation of starch that occurs early in tomato fruit development. The results also suggest that ADPglucose pyrophosphorylase and sucrose synthase levels correlated positively with soluble sugar accumulation in the three varieties examined. In developing tomato fruit, starch transiently accumulates early in development with soluble sugars gradually increasing as starch levels decrease later in development (3). A comparison of tomato genotypes differing in levels of soluble sugars in ripe fruit indicated a positive correlation between peak levels of starch early in development and final levels of soluble sugars (4). These results suggest that starch biosynthesis may provide an additional sink for carbon during early fruit development when carbon import is greatest. Starch accumulation and degradation occurs on a diurnal basis in many green tissues and this regulation can be accounted for by photosynthetically mediated fluctuations in metabolites that regulate ADPG4 pyrophosphorylase (11). In tomato fruit, however , a major shift in starch metabolism occurs over a period of ' 4Abbreviations: ADPG, ADPglucose; SS, sucrose synthase; Pi, ortho-phosphate. weeks and it seemed possible that this regulation could occur through regulation of levels of enzymes contributing to either starch biosynthesis or degradation. To assess this possibility, carbohydrate-metabolizing enzymes were assayed in tomato fruit extracts from 20 to 40 d after anthesis. The enzymes most closely associated with starch metabolism, ADPG pyrophosphorylase, amylase, and phosphorylase were of special interest. Other enzymes potentially important in sucrose assimilation, invertase and SS, were also assayed throughout development. This survey ofenzyme activities provides information on pathways ofcarbon assimilation in developing tomato fruit and which steps are developmentally regulated. The enzymes were assayed in three tomato varieties differing in their final soluble solids accumulation. The varieties were cv LA1563, a high soluble sugar accu-mulator (6.3 'Brix); cv VF145-7879, an intermediate soluble sugar accumulator (5.5 'Brix); and cv UC82B, a low soluble sugar accumulator (4.6 'Brix). By analyzing three varieties differing in soluble sugar accumulation, the enzymic basis for this difference in accumulation could be investigated. This information provides a basis for identifying important metabolic steps in tomato fruit carbohydrate assimilation and may assist in selecting molecular genetic targets to enhance carbohydrate accumulation in tomato fruit. MATERIALS AND METHODS Plant Material. Seeds of tomato (Lycopersicon esculentum Mill.) varieties cv LA1563 (1563), cv VF145-7879 (7879), and cv UC82B (UC82) were sown in growing trays (Growing Systems, Inc.) and transplanted to the field approximately 6 weeks later. The plants were accorded normal cultural practices used for processing tomatoes in California. Extraction of Tomato Fruit. Fruit age was determined by tagging the truss when the two proximal flowers were open. All fruit subsequently used were tagged on June 25, 1986. This meant that all fruit experienced the same environmental conditions until harvested, reducing one experimental variable. On the harvest date the fruit were brought into the laboratory, fresh weight was determined, and pericarp tissue was obtained. The following steps were done at 0°C. The tissue, 10 g, was cut into small pieces and ground with a Tissuemizer (Tekmar) in 10 ml homogenization buffer for approximately 20 s. The homogeni-zation buffer contained 50 mM Hepes-KOH (pH 8.3), 2 mM EDTA, 1 mM MgCl2, 1 mm MnCI2, 2 mm EGTA, and 2 mM DTE. After homogenization, 1 ml aliquots were removed for determining starch and sugar levels. The remaining extract was filtered through Miracloth and divided into 0.5 ml aliquots and frozen in liquid N2. These aliquots were used for enzyme assays. Freezing in liquid N2 had no effect on enzyme activity (N Robinson, unpublished results). The results are an average of three fruits per time point. Chloroplast Isolation. Chloroplasts were isolated from 20 and 30 d old 7879 fruit as outlined by Fish and Jagendorf (5). The 727