High-Level Expression of Cold-Tolerant Pyruvate, Orthophosphate Dikinase from a Genomic Clone with Site-Directed Mutations in Transgenic Maize

Abstract

Pyruvate, orthophosphate dikinase (PPDK) is a key enzyme in the C4 photosynthetic pathway of maize. To improve the cold tolerance of the enzyme in maize, we designed two genomic sequence-based constructs in which the carboxy-terminal region of the enzyme was modified to mimic the amino acid sequence of the cold-tolerant PPDK of Flaveria brownii (Asteraceae). A large amount of PPDK was found to have accumulated in the leaves of many of the maize plants transformed with one of these constructs – that which introduced 17 amino acid substitutions without any alteration of the exon-intron structure – although there was a wide range of variation in the amount of PPDK among the separate plants. In contrast, the production was much less in maize transformed with the second construct in which a cDNA fragment for the same carboxy-terminal region was inserted. The specific activity of PPDK in the plants transformed with the gene with the amino acid substitutions was inversely correlated with the amount of enzyme in the leaves. In addition, the activity of the cold-tolerant recombinant enzyme was judged to be regulated by the PPDK regulatory protein, similar to that of the native PPDK. The cold tolerance of PPDK in crude leaf extracts was greatly improved in plants that produced a large amount of the engineered PPDK. The photosynthetic rate at 8°C increased significantly (by 23%, p<0.05), but there was no obvious effect at higher temperatures. These results support the hypothesis that PPDK is one of the limiting factors in the C4 photosynthesis of maize under cold conditions.