Evaluating ascorbate oxidase as a plant defense against leaf-chewing insects using transgenic poplar.

Ascorbate is the major water-soluble antioxidant in plants and animals, and it is an essential nutrient for most insect herbivores. Therefore, ascorbate oxidase (AO) has been proposed to function as a plant defense that decreases the availability of ascorbate to insects. This hypothesis was tested by producing transgenic poplar (Populus tremula x Populus alba; Salicaceae) with 14- to 37-fold higher foliar AO activities than control (wild type) leaves and feeding these leaves to Lymantria dispar L. (Lepidoptera: Lymantriidae) caterpillars and Melanoplus sanguinipes (Fabricius) (Orthoptera: Acrididae) grasshoppers. To examine potential mechanisms of activity of AO in these insects, ascorbyl radical and/or ascorbate levels were measured in gut contents. No significant changes in ascorbyl radical or ascorbate levels were found in the midgut contents of L. dispar larvae that ingested the leaves of the AO-overexpressing genotypes compared to the control genotype, and no significant decreases in ascorbate levels were found in the foregut or midgut contents of M. sanguinipes. Treatment of control leaves with commercial AO also produced no changes in the midgut biochemistry of L. dispar larvae, as measured by levels of ascorbyl radicals. Likewise, no increase in oxidative stress was observed in L. dispar that consumed tannin-treated AO-overexpressing leaves compared with tannin-treated control genotype leaves. Performance experiments were carried out on first- and fourth-instar L. dispar larvae on leaf disks and on third instars feeding on intact leaves on trees. In no case was a significant difference found in the contrast between the control and three AO-overexpressing genotypes for relative consumption rate, relative growth rate, or nutritional indices. We conclude that elevated levels of AO in poplar are unlikely to serve as a defense against herbivores such as L. dispar or M. sanguinipes and that the low oxygen levels commonly found in the guts of caterpillars and grasshoppers may limit the activity of ingested AO in these leaf-chewing insects.