Keith Stuart Blundy

Genetic manipulation of 6-phosphofructokinase in potato tubers

The aim of this work was to discover whether genetic manipulation of 6-phosphofructokinase [EC 2.7.1.11; PFK(ATP)] influenced the rate of respiration of tuber tissue of Solanum tuberosum L. Transgenic plants were produced that contained the coding sequence of the Escherichia coli pfkA gene linked to a patatin promoter. Expression of this chimaeric gene in tubers resulted in a 14to 21-fold increase in the maximum catalytic activity of PFK(ATP) without affecting the activities of the other glycolytic enzymes. Tubers, and ‘aged’ disks of tuber tissue, from transformed plants showed no more than a 30% fall in the content of hexose 6-monophosphates; the other intermediates of glycolysis increased threeto eightfold. Fructose-2,6-bisphosphate was barely detectable in aged disks of transformed tubers. The relative rates of 14CO2 production from [1-14C]-and [6-14C]-glucose supplied to disks of transformed and control tubers were similar. Oxygen uptake and CO2 production by aged disks of transformed tubers did not differ significantly from those from control tubers. The same was true of CO2 production, in air, and in nitrogen, for tuber tissue. It is concluded that PFK(ATP) does not dominate the control of respiration in potato tubers.

The expression of class I patatin gene fusions in transgenic potato varies with both gene and cultivar.

Patatin is a family of glycoproteins that contributes about 40% of the total soluble protein in tubers of potato (Solanum tuberosum L.). The protein is encoded by a multigene family of 50–70 genes which have been divided into classes I and II on the basis of sequence homology. The promoters of two class I genes, PS20 and PS3/27, were transcriptionally fused to β-glucuronidase and transformed into the potato cultivars Désirée and Maris Bard. Examination of the expression levels in large populations of microtubers indicated that the PS20 promoter produced β-glucuronidase activities 5-fold lower in Désirée than Maris Bard whereas the PS3/27 promoter showed similar levels in both cultivars. Furthermore, the relative expression levels from the two promoters were reversed in the two cultivars. The β-glucuronidase enzyme activity was correlated with the mRNA level but not the copy number of the introduced gene. The implications for the use of patatin promoters in the genetic modification of tubers is discussed.

Differential expression of members of the napin storage protein gene family during embryogenesis inBrassica napus

S1 nuclease analysis and sub-family-specific oligonucleotide probes were used to characterize the expression during embryogenesis of the napin storage protein gene family ofBrassica napus (oilseed rape). The expression of one sub-class represented by the napin gene gNa peaks and declines earlier than the other members of the family. This sub-class was highly expressed representing ca. 20% of napin mRNA at 26 days after anthesis.