Potato SNF1-related protein kinase: molecular cloning, expression analysis and peptide kinase activity measurements

Abstract

A polymerase chain reaction product (PKIN503) was amplified from potato (Solanum tuberosum) cv. Désirée using oligonucleotide primers with sequences which are highly conserved in the plant sucrose non-fermenting 1 (SNF1)-related protein kinase gene family. Southern blot analysis showed the presence of 5-10 SNF1-related genes in the potato genome. PKIN503 was used to screen a tuber cDNA library and a genomic library, and one cDNA and five genomic clones were isolated. The nucleotide sequences of a portion of all five genomic clones were shown to be identical and only one, pgPKIN1, was analysed further. The cDNA was found to be truncated at the 5' end but the cDNA and genomic sequences contained only 15 substitutions, two of which resulted in changes in the derived amino acid sequence. PKIN1 was shown to encode an Mr 57,854 protein with 61-70% sequence similarity with other plant SNF1-related protein kinases. Northern blot analysis revealed some tissue-specific differences in PKIN1 transcript levels, the lowest being detected in leaves and the highest in stolons. However, much greater differences were found in SNF1-related activity, which was measured using a phosphorylation assay with a substrate peptide which has been shown previously to be phosphorylated by plant SNF1-related protein kinases. Activity decreased by almost 80% during development from stolons to mature tubers but it increased about seven-fold during the first seven days of storage after harvesting, before decreasing again. However, activity was highest in mini-tubers, where the levels were 37 times greater than those in mature tubers from a pot-grown plant. Transcript levels in these tissues were approximately equal, clear evidence that SNF1-related protein kinase activity in potato is regulated, in part, post-transcriptionally.