Purification and characterization of lipoxygenase isozymes from potato tubers: Mechanistic aspects of lipoxygenase in disease resistance.

摘要

The potential role of the enzyme lipoxygenase (LOX) in potato tuber resistance to the oomycete Phytophthora infestans was investigated. In order to test the role of tuber LOX in disease resistance, the LOX isozymes in tuber tissue were purified to electrophoretic homogeneity. These isozymes were then characterized as to their primary products, kinetic parameters, and sequence similarities. Finally, the primary products from LOX activity using the P. infestans elicitor arachidonic acid (AA) as a substrate were tested for their ability to differentially regulate the isozymes of HMG-CoA reductase (HMGR) known to be differentially regulated in potato tuber during the oomycete defense response.; Using a combination of column chromatography and HPLC, three purified isozymes of potato tuber LOX were obtained. The analysis of these isozymes revealed that they were all 5-LOXs with respect to the primary products from AA. However, there were differences in kinetic parameters, percentages of primary products and their chiral enantiomers, amino acid compositions, and tryptic digest peptide fragments. These differences indicate that there may be more than one gene encoding the three isozymes.; Products from LOX activity on AA were used to elicit tuber tissue and to test whether these products could induce differential regulation of HMGR isozyme transcript levels in a manner similar to AA. The elicitor AA is thought to decrease steroid and increase sesquiterpene production through the differential regulation of the rate-limiting HMGR isozymes hmg 1 and hmg 2 or hmg 3, respectively. It was found that the main product from LOX activity using AA as a substrate, as well as a minor product, were able to effect the decrease in hmg 1 transcript levels similar to that of AA. However, the products were unable to up-regulate hmg 3 transcript levels. This suggests that LOX may only be involved in decreasing hmg 1 transcript levels leading to decreases in steroid production, and that there is more than one route by which AA elicits tuber defense responses.