A Multifunctional Lipoxygenase with Fatty Acid Hydroperoxide Cleaving Activity from the Moss Physcomitrella patens

Abstract

A complex mixture of fatty acid-derived aldehydes, ketones, and alcohols is released upon wounding of the moss Physcomitrella patens. To investigate the formation of these oxylipins at the molecular level we isolated a lipoxygenase from P. patens, which was identified in an EST library by sequence homology to lipoxygenases from plants. Sequence analysis of the cDNA showed that it exhibits a domain structure similar to that of type2 lipoxygenases from plants, harboring an N-terminal import signal for chloroplasts. The recombinant protein was identified as arachidonate 12-lipoxygenase and linoleate 13-lipoxygenase with a preference for arachidonic acid and eicosapentaenoic acid. In contrast to any other lipoxygenase cloned so far, this enzyme exhibited in addition an unusual high hydroperoxidase and also a fatty acid chain-cleaving lyase activity. Because of these unique features the pronounced formation of (2Z)-octen-1-ol, 1-octen-3-ol, the dienal (5Z,8Z,10E)-12-oxo-dodecatrienoic acid and 12-keto eicosatetraenoic acid was observed when arachidonic acid was administered as substrate. 12-Hydroperoxy eicosatetraenoic acid was found to be only a minor product. Moreover, the P. patens LOX has a relaxed substrate tolerance accepting C 18 -C 22 fatty acids giving rise to even more LOX-derived products. In contrast to other lipoxygenases a highly diverse product spectrum is formed by a single enzyme accounting for most of the observed oxylipins produced by the moss. This single enzyme might, in a fast and effective way, be involved in the formation of signal and/or defense molecules thus contributing to the broad resistance of mosses against pathogens.

Lipid metabolism in arbuscular mycorrhizal roots of Medicago truncatula

“…Indeed the analysis of two independent lines show that PpHPL is only involved in the generation of the plant like volatile (2E)-nonenal and not in the formation of octenols typically found so far mainly in mammals ( Figure 5). According to our findings described here and in recent publications (Senger et al, 2005;Wichard et al, 2005b) a so far new picture for the wound activated formation of volatile oxylipins in mosses arises that involves only one key enzyme required for the transformation of free fatty acids. This single LOX (PpLOX1) can account for high product diversity (Senger et al, 2005).…”

“…According to our findings described here and in recent publications (Senger et al, 2005;Wichard et al, 2005b) a so far new picture for the wound activated formation of volatile oxylipins in mosses arises that involves only one key enzyme required for the transformation of free fatty acids. This single LOX (PpLOX1) can account for high product diversity (Senger et al, 2005). It either produces directly octenols from AA or it provides the substrate (12-HPETE) for PpHPL described here, leading to formation of (2E)-nonenal.…”

“…As main constituents we identified oct-1-en-3-ol, oct-3-en-1-ol and (2E)-nonenal, which were exclusively derived from AA. While the production of the octenols is most likely caused by the recently cloned LOX of P. patens (Senger et al, 2005), the HPL described here might be responsible for the formation of (2E)-nonenal by a pathway found in all higher plants. To gain further insight into the involvement of PpHPL in volatile formation, we created knock-out mosses where the PpHPL-gene was disrupted.…”

“…The production of the octenols was most likely caused by the recently cloned LOX of P. patens (Senger et al, 2005). To analyze if the HPL cloned here is responsible for the formation of (3Z)-nonenal, which could be converted to (2E)-nonenal by an isomerase, we created knock-out mosses where the PpHPL-gene was disrupted by a cDNA coding for kanamycin resistance.…”

“…These volatiles are all derived from AA (Wichard et al, 2005b). While the octenols may be produced by the action of a novel LOX with a lyase activity that has been described recently (Senger et al, 2005), the enzyme responsible for C9-volatile production is still unknown. Here we present the isolation and characterization of a new HPL from the moss P. patens.…”