The Two Alpha-dox Genes of Nicotiana Attenuata: Overlapping but Distinct Functions in Development and Stress Responses

Background: Plant fatty acid alpha-dioxygenases (alpha-DOX) are oxylipin-forming enzymes induced by biotic and abiotic stresses, which also participate in developmental processes. In Nicotiana attenuata, herbivory strongly induces the expression of an alpha-dox1 gene. To determine its role, we silenced its expression using Agrobacterium-mediated plant transformation with an inverted repeat construct. More than half of the transformed lines showed a severe dwarf growth phenotype that was very similar to the phenotype of tomato plants mutated at a second alpha-dox isoform. This led us to identify the corresponding alpha-dox2 gene in N. attenuata and examine the regulation of both alpha-dox genes as well as the consequences of their silencing in plant development and anti-herbivore defense.

Results: The transformed lines exhibiting a dwarf growth phenotype are co-silenced for both alpha-dox genes resulting in a nearly complete suppression of alpha-DOX activity, which is associated with increases in ABA, JA and anthocyanin levels, all metabolic signatures of oxidative stress. The other lines, only silenced for alpha-dox1, developed similarly to wild-type plants, exhibited a 40% reduction of alpha-DOX activity resulting in a 50% reduction of its main product in planta (2-HOT) and showed no signs of oxidative stress. In contrast to alpha-dox1, the expression of alpha-dox2 gene is not induced by wounding or elicitors in the oral secretions of Manduca sexta. Instead, alpha-dox2 is expressed in roots and flowers which lack alpha-dox1 expression, but both genes are equally regulated during leaf maturation. We transiently silenced alpha-dox gene copies with gene-specific constructs using virus induced gene silencing and determined the consequences for plant development and phytohormone and 2-HOT levels. While individual silencing of alpha-dox1 or alpha-dox2 had no effects on plant growth, the co-suppression of both alpha-dox genes decreased plant growth. Plants transiently silenced for both alpha-dox genes had increased constitutive levels of JA and ABA but silencing alpha-dox1 alone resulted in lower M. sexta-induced levels of JA, 2-HOT and ABA.

Conclusions: Thus, both alpha-dox isoforms function in the development of N. attenuata. In leaf maturation, the two alpha-dox genes have overlapping functions, but only alpha-dox2 is involved in root and flower development and only alpha-dox1 functions in anti-herbivore defense.