Poster: Lipids

Abs # 646: The effects of a microbial 3-hydroxysteroid oxidase enzyme on steroid content and function of the chloroplast in transgenic tobacco.

Presenter:	Bedard, Mark , Contact Author
Authors	Bedard, Mark  (A)   Becker, David  (B)   Galante, Mike  (A)   Ruffino, John  (A)   MacLean, Brittany  (A)   Fordyce, Amber  (A)   Nigam, Amit  (A)   Dewitt, Matt  (A)   Grebenok, Robert  (A)
Affiliations:	(A): Canisius College, Department of Biology (B): Pomona College, Department of Biology

The transgenic tobacco lines that are the focus of the current research express a microbial 3-hydroxysteroid oxidase gene, whose actions culminate in an approximate 5-fold increase in a whole plant steroid accumulation. Furthermore, whole plant sterol analysis demonstrates transgenic tobacco line 103 maintains a 2.5-fold greater level of steryl-ester accumulation than that observed in control tobacco. Activity typical of the 3-hydroxysteroid oxidase is localized to the thylakoid membrane fraction, clearly defining the chloroplast as a sub-cellular site of the 3-hydroxysteroid oxidase activity. Approximately 70% of the entire thylakoid membrane sterol pool, including sitosterol, stigmasterol, campesterol and cholesterol is maintained as products of the 3-hydroxysteroid oxidase enzyme. Atypical thylakoid membrane steroids, including cholesta-4-en-3-one, cholestan-3-one, campestan-3-one, stigmasta-4-en-3-one, and sitostan-3-one are found exclusively as free steroid, while approximately 90% of the combined pools of stigmasterol, cholesterol, campesterol and sitosterol are found as steryl-esters in the thylakoid membrane. The modified sterol budget in the thylakoids of transgenic tobacco line 103 correlates with altered rates of in-vitro electron transport measured in transgenic thylakoid membranes at elevated and reduced temperatures when compared to controls. Furthermore, transgenic line 103 demonstrates growth rates superior to control tobacco when grown in a constant 10 or 35 degree Celsius environment. The relationships among 3-hydroxysteroid oxidase localization, atypical steroid accumulation in the thylakoid membrane and plant ontogeny are discussed.