Analysis of Nb-9-LOX gene expression and LOX activity in N. benthamiana plants treated with agrobacterial suspensions containing different mixtures of viral provectors: 1) 3'-provector; 2) 3'-provector and integrase provector; 3) 5'-provector and integrase provector; 4) 3'-provector, 5'-provector and integrase provector; C) untreated N. benthamiana; B) leaf infiltrated with buffer. Leaves were harvested 12 days after infiltration. (A) Quantitative real-time RT-PCR analysis was performed using Nb-9-LOX and 18S-26S interspacer gene specific primers, the latter used as an internal control for normalization. Values of Nb-9-LOX gene expression are means ± SEM of three different evaluations carried out with two sets of cDNAs. (B) LOX activities. LOX activity was measured at pH 7.0 using linoleic acid as a substrate. The reaction products were analyzed by LC-MS. Concentrations of product (9-HPOD) were determined using a standard curve calculated from various known concentrations of 9-HPOD against the UV peak areas which were recorded at 234 nm by LC-MS. Each bar represents the mean and standard error of two replicates. (C) LC-MS analysis of product formed from the reaction containing crude protein extracts of infiltrated tobacco leaf and linoleic acid. 9-HPOD (m/z 335→195, positive mode) was found to be the main product.