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Summary

This report describes the analysis of transgenic potato plants stably transformed with chimeric genes comprising either a patatin (tuber-specific) or a ST-LS1 (leaf- and stem-specific) gene promoter and a potato sucrose non-fermenting-1 (SNF1)-related protein kinase gene (PKIN1) sequence in the antisense orientation. Presence of the transgene was confirmed by Southern analysis. The transformants were screened using a peptide kinase activity assay for SNF1-related activity and/or Northern blot analysis, and two independent transgenic lines from each transformation, PAT 1.3, PAT 1.10, LS 1.4 and LS 1.11, were selected. Antisense PKIN1 transcripts were detectable in all four of the selected lines, and measurements made using the specific peptide phosphorylation assay showed that SNF1-related protein kinase activity had decreased in both PAT 1.3 and PAT 1.10 compared with controls. SNF1 regulates the expression of many genes encoding enzymes of carbohydrate metabolism in yeast. In order to investigate an analogous role for PKIN1, the activities of fructokinase, glucokinase, neutral and acid invertase and sucrose synthase in the tubers of PAT 1.3 and PAT 1.10 were compared with those in wild-type controls. Sucrose synthase activity was decreased to 36% of wild-type activity in tubers of PAT 1.10, and sucrose synthase transcript levels were decreased in tubers of both PAT 1.3 and PAT 1.10. Activities of the other enzymes were unaffected. Leaves of lines LS 1.4, LS 1.11 and controls were then excised and cultured on a medium containing 250 mM sucrose. This treatment induced sucrose synthase gene expression in the control leaves but not those of the transgenic lines. This finding is the first demonstration of a role for SNF1-related protein kinases in the regulation of carbohydrate metabolism of higher plants.