Analysis of intermediate carbon metabolism in strawberry plants

[摘要] Strawberry (Fragaria x ananassa) fruit quality is largely determined by the relative amounts ofsugars and organic acids present, as well as soluble solid content. This study had three components:1) Characterisation of cytosolic carbohydrate metabolism and carbon partitioning to sugars andorganic acids in two commercial varieties, 2) analysis of transgenic strawberry fruit with increasedpyrophosphate: D-fructose-6-phosphate 1-phosphotransferase (PFP) activity and 3) analysis oftransgenic strawberry fruit with increased ß-fructosidase (invertase) activity in either cytosol orapoplast. Analyses of transgenic strawberry may inform similar attempts in grape berries.Festival and Ventana, two popular commercial strawberry cultivars in South Africa, were fairlysimilar with respect to sugar and organic acid content. Twelve cytosolic enzymes wereinvestigated. Temporal differences in maximum catalytic activity were observed for invertase, PFP,pyruvate kinase and ADP-glucose pyrophosphorylase (AGPase). Invertase, PFP and AGPaseactivity also differed between the cultivars. One enzyme, SuSy, could not be analysed effectively,due to the purification method employed. These analyses established methodology for the analysisof transgenic berries.Constructs were designed to constituitively express Giardia lamblia PFP (GL-PFP), or toexpress Saccharomyces cerevisiae invertase (SCI) in a fruit-specific manner. A second invertaseconstruct was designed to target SCI to the apoplast. Strawberry (cv. Selekta) was transformed andthe presence of each transgene confirmed by PCR. Untransformed Selekta was used as control inboth transgenic studies.Transgenic lines were selected based on GL-PFP activity in leaves and total PFP activity in ripefruit. Sugar and organic acid content of ripe berries with high PFP activity was determined.Although berries displayed marked changes in sugar composition, the total sugar content wassimilar to controls, in all except one line. Organic acid content was decreased, leading to a clearreduction in organic acid-to-sugar ratio. This points to a gluconeogenic role for PFP in strawberryfruit.Transgenic berries were screened for SCI activity. Berries containing untargeted SCI exhibitedtotal invertase activity similar to controls and were not analysed further. Berries with apoplasttargetedSCI displayed three-fold increases in invertase activity compared to controls. Total sugarcontent was reduced and exhibited reduced sucrose content relative to hexoses. Despite the effectof increased invertase activity on metabolites, maximum catalytic activity of enzymes involved incytosolic sucrose, hexose and organic acid metabolism were unchanged. Transgenic plants selectedin these studies were subsequently vegetatively replicated and future work will include immaturefruit.

[发布机构] Stellenbosch University