Evidence of a cross-talk regulation of a GA 20-oxidase (FsGA20ox1) by gibberellins and ethylene during the breaking of dormancy in Fagus sylvatica seeds.

Gibberellin 20-oxidase (GA 20-oxidase) is an enzyme that catalyses the last three steps in the synthesis of active GAs and is a potential control point in the regulation of GA biosynthesis. Reverse transcriptase-polymerase chain reaction with degenerated oligonucleotides conserved among GA 20-oxidases was used to isolate a cDNA clone for this enzyme in Fagus sylvatica L. seeds. This clone contains all the features and exhibits homology to GA 20 oxidases from several plant species. Expression of this clone, named FsGA20ox1, as a fusion protein expressed in Escherichia coli confirmed that it was able to metabolize [(14)C]GA(12) to [(14)C]GA(9) and [(14)C]GA(53) to [(14)C]GA(20). Analysis of FsGA20ox1 transcript levels showed similar low expression during stratification at 4 degrees C and in the presence of gibberellic acid or ethephon (compound that releases ethylene in solution), treatments proved to be efficient in breaking the dormancy of beech seeds. However, there was a drastic increase of FsGA20ox1 transcript levels in the presence of paclobutrazol (PCB), a well-known GAs biosynthesis inhibitor, or of 2-aminoxyacetic acid (AOA), an inhibitor of ethylene biosynthesis. Furthermore, the effect of AOA was reversed by the addition of GA(3) and that of PCB by ethephon. This indicates that the gene product is subjected to down-regulation by GA and ethylene, and further suggests a cross-talk gene regulation by these two hormones during the transition from seed dormancy to germination.

Activation of the fatty acid alpha-dioxygenase pathway during bacterial infection of tobacco leaves. Formation of oxylipins protecting against cell death.

A pathogen-induced oxygenase showing homology to prostaglandin endoperoxide synthases-1 and -2 was recently characterized by in vitro experiments as a fatty acid alpha-dioxygenase catalyzing formation of unstable 2(R)-hydroperoxy fatty acids. To study the activity of this enzyme under in vivo conditions and to elucidate the fate of enzymatically produced 2-hydroperoxides, leaves of tobacco were analyzed for the presence of alpha-dioxygenase-generated compounds as well as for lipoxygenase (LOX) products and free fatty acids. Low basal levels of 2-hydroxylinolenic acid (0.4 nmol/g leaves fresh weight) and 8,11,14-heptadecatrienoic acid (0.1 nmol/g) could be demonstrated. These levels increased strongly upon infection with the bacterium Pseudomonas syringae pv syringae (548 and 47 nmol/g, respectively). Transgenic tobacco plants overexpressing alpha-dioxygenase were developed, and incompatible infection of such plants led to a dramatic elevation of 2-hydroxylinolenic acid (1778 nmol/g) and 8,11,14-heptadecatrienoic acid (86 nmol/g), whereas the levels of LOX products were strongly decreased. Further analysis of oxylipins in infected leaves revealed the presence of a number of 2-hydroxy fatty acids differing with respect to chain length and degree of unsaturation as well as two new doubly oxygenated oxylipins identified as 2(R),9(S)-dihydroxy-10(E),12(Z),15(Z)-octadecatrienoic acid and 2(R),9(S)-dihydroxy-10(E),12(Z)-octadecadienoic acid. alpha-Dioxygenase-generated 2-hydroxylinolenic acid, and to a lesser extent lipoxygenase-generated 9-hydroxyoctadecatrienoic acid, exerted a tissue-protective effect in bacterially infected tobacco leaves.