Investigation of substrate binding and product stereochemistry issues in two linoleate 9-lipoxygenases

Herein we characterize the Arabidopsis thaliana AtLOX1 and tomato (Solanum lycopersicum) LOXA proteins as linoleate 9S-lipoxygenases (9-LOX), and use the enzymes to test a model that predicts a relationship between substrate binding orientation and product stereochemistry. The cDNAs were heterologously expressed in E. coli and the proteins partially purified by nickel affinity chromatography using a N-terminal (His)(6)-tag. Both enzymes oxygenated linoleic acid almost exclusively to the 9S-hydroperoxide with turnover numbers of 300-400/s. AtLOX1 showed a broad range of activity over the range pH 5-9 (optimal at pH 6); tomato LOXA also showed optimal activity around pH 5-7 dropping off more sharply at pH 9. Site-directed mutagenesis of a conserved active site Ala (Ala562 in AtLOX1, Ala 564 in tomato LOXA, and typically conserved as Ala in S-LOX and Gly in R-LOX), revealed that substitution with Gly led to the production of a mixture of 9S- and 13R-hydroperoxyoctadecadienoic acids from linoleic acid. To follow up on earlier reports of 9-LOX metabolism of anandamide (van Zadelhoff et al. Biochem. Biophys. Res. Commun. 248:33-38, 1998), we also tested this substrate with the mutants, which produced predictable shifts in product profile, including a shift from the prominent 11S-hydroperoxy derivative of wild-type to include the 15R-hydroperoxide. These results conform to a model that predicts a head-first substrate binding orientation for 9S-LOX. We also found that linoleoyl-phosphatidylcholine is not a 9S-LOX substrate, which is consistent with this conclusion.

Source: Lipids (2008) vol. 43, p. 979-987

Agrobacterium-mediated transformation of an elite indica rice for insect resistance

In India, rice occupies the largest area among all crops and accounts for as much as 21 % of the total cropped area. Rice plants are prone to attack from many pests and pathogens. Among them coleopteran pests are well known for causing extensive damage. Sitophilus oryzae causes severe damage to rice seeds during storage. Alpha amylase inhibitor gene isolated from Phaseolus vulgaris seeds was introduced into Basmati rice (PB1) through Agrobacterium -mediated transformation. A total of 174 hygromycin-resistant plants were regenerated. Most of these plants were GUS-positive. PCR analysis and Southern hybridization confirmed the presence of 4.9 kb alpha amylase inhibitor gene in transformed plants. Western blot confirmed presence of alpha amylase inhibitor protein. Results of the bioassay study revealed significant reduction in survival rate of rice weevil, S. oryzae reared on transgenic rice seeds.

Source: Current Science, Vol 90 (2006) Page 829-835

Efficient transformation of rice (Oryza sativa L.) mediated by Agrobacterium and sequence analysis of the boundaries of the T-DNA

A large number of morphologically normal, fertile, transgenic rice plants were obtained by co-cultivation of rice tissues with Agrobacterium tumefaciens. The efficiency of transformation was similar to that obtained by the methods used routinely for transformation of dicotyledons with the bacterium. Stable integration, expression and inheritance of transgenes were demonstrated by molecular and genetic analysis of transformants in the R0, R1 and R2 generations. Sequence analysis revealed that the boundaries of the T-DNA in transgenic rice plants were essentially identical to those in transgenic dicotyledons. Calli induced from scutella were very good starting materials. A strain of A. tumefaciens that carried a so-called 'super-binary' vector gave especially high frequencies of transformation of various cultivars of japonica rice that included Koshihikari, which normally shows poor responses in tissue culture.

Source: The Plant Journal (1994) vol. 6, p. 271-282