No abstract

A mutant line of Pisum fulvum was identified that lacked seed lipoxygenase-2 (LOX-2). The mutant phenotype was introgressed into a standard Pisum sativum cv. Birte to provide near-isogenic lines with or without seed LOX-2. Genetic analyses showed the mutation to behave as a single, recessive Mendelian gene. Northern and dot-blot analyses showed a large reduction in LOX-2 mRNA from developing seeds of the LOX-2-null mutant. A restriction fragment length polymorphism associated with the 5' end of the LOX-2 gene(s) co-segregated with the null phenotype, indicating that the reduction of LOX-2 mRNA was neither a consequence of deletion of the LOX genes nor a consequence of the action of a genetically distant regulatory gene. Analysis of the 5'-flanking sequences of LOX-2 genes from Birte and the near-isogenic LOX-2-null mutant revealed a number of insertions, deletions and substitutions within the promoter from the LOX-2-null mutant that could be responsible for the null phenotype. Incubation of crude seed LOX preparations from Birte and the LOX-2-null mutant showed that the latter generated relatively less 13-hydroperoxides and also produced relatively more hydroxy- and ketoacid compounds that have implications for the fresh-frozen pea industry.

Part of the 5'-flanking sequence of a pea (Pisum sativum) lipoxygenase (LOX) gene was cloned, after amplification from genomic DNA by inverse polymerase chain reaction. Translational and transcriptional fusions of 818 bp of the 5'-flanking region and its deletion derivatives (-513 and -356) were made to a beta-glucuronidase (GUS)-coding sequence and introduced into tobacco. Analysis of T1 transformants showed that the 818 bp 5'-flanking sequence drove GUS expression in seeds that was temporally regulated in a fashion similar to the accumulation of LOX mRNA in developing pea seeds. Contrary to expectations, however, expression of the 818 bp promoter-GUS fusion was not seed-specific; GUS activity was highest in leaves and also present in stems and, to a lesser extent, roots. Deletion analyses identified the region between -818 and -513 as essential for high-level, temporally regulated expression in seeds and also indicated that the sequence between -513 and -356 plays a negative role in leaf/stem, but not seed, expression. Comparison of translational and transcriptional fusions indicated that the LOX initiation codon was used more efficiently than the GUS initiation codon by the tobacco leaf translational apparatus.