Isolation of lipoxygenase cDNA clones from pea nodule mRNA

Abstract

The pattern of lipoxygenase (LOX) gene expression was investigated in pea nodule tissues using the technique of in situ hybridization. Five lipoxygenase cDNAs were cloned from nodule mRNA by the RT-PCR and 3' RACE procedures. These clones (loxN1 to loxN5) show a high degree of sequence homology, except in the 3'-untranslated region. Gene-specific riboprobes were therefore generated from subclones carrying the 3'-untranslated regions in order to investigate tissue-specific gene expression. Northern blotting analysis revealed that loxN1 corresponded to a transcript that was expressed exclusively in roots and nodules but not in the aerial parts of the plant. However, none of the LOX genes appeared to be up-regulated in nodule tissue relative to uninfected roots. Starting with the incomplete cDNA clone for loxN1, the full coding sequence termed lox1:P.s:1 was obtained by further rounds of RT-PCR and 5' RACE procedures. In situ hybridization with nodule tissues revealed several different patterns of expression for the various LOX probes. However, none of the corresponding transcripts was expressed exclusively in the invasion zone, as might have been expected if one LOX gene product had been uniquely associated with the invasion process. In conclusion, this study provides no evidence for a direct role for any LOX gene product in plant-microbe interaction or host defence, but the fact that all the transcripts were expressed at the nodule apex suggests that LOX could be involved in the development of this organ.