CONFAB: Controlling fatty acid breakdown in order to produce viable oilseeds with increased yields of novel oils

• Research unit: EU RFP
• Project number: 99.0692

Key words (English)

Fatty acids; oils; polyhydroxyalkanoate; B-oxidation; peroxisome

Alternative project number (English)

EU project number: GLRT-1999-00213

Research programs (English)

EU-programme: 5. Frame Research Programme - 1.1.3 The 'cell factory'

Short description (English)

See abstract

Further information (English)

Full name of research-institution/enterprise:
Université de Lausanne
Institut de Biologie et de Physiologie végétale

Partners and International Organizations (English)

Coordinator: University of York (UK)

Abstract (English)

Part A. Analysis of futile cycling in plants producing novel oils.
This second year was devoted to the analysis of transgenic plants. Well over 100 lines have been analyzed for expression (under napin and CaMV35S promoter) of the peroxisomal PHA synthase along with the fatty acid hydroxylase, acetylenase and epoxigenase. Plants co-expressing these corresponding proteins at a high and stable level over several generations have been selected. PHA expression was confirmed by Western analysis while expression of the fatty acid modifying enzymes were confirmed by analysis of fatty acids in mature seeds. For the lines expressing the hydroxylase, accumulation of ricinoleic acid and densipolic acid up to 5.7% in seeds was measured. For lines expressing the acetylenase, accumulation of crepenynic acid in seeds up to 1.6% was measured, while for lines expressing the epoxigenase, accumulation of vernolic acid in seeds up to 3.4% was measured. With a number of selected lines, the quantity and quality of the PHA produced is presently being analyzed in order to uncover the presence of a futile cycle of fatty acids towards the peroxisome in transgenic plants accumulating unusual fatty acids.
B. Expression profile of genes involved in fatty acid biosynthesis and ß-oxidation in transgenic plants expressing fatty acid modifying enzymes
After having selected a set of approximately 300 genes involved in fatty acid metabolism (synthesis and degradation), peroxisome metabolism and development, as well as a number of controls, we have printed a series of cDNA microarrays on glass slides. A number of technical problems needed to be resolved. Chief among them was the sensitivity of the detection since the majority of the genes present on the chip are expressed at very low level. A number of protocols have therefore been tested for the labeling of probes, isolation of mRNA as well as for printing the DNA on the slides. Preliminary analysis of plants expressing the fatty acid modifying enzymes (hydroxylase, epoxigenase, acetylenase) under the control of the CaMV35S promoter have been obtained. Remarkably, synthesis of unusual fatty acids in whole plants affected the expression of very few genes, despite the fact that for some transgenes (e.g. hydroxylase) the growth of the plant was affected. We are presently re-analyzing these plants with a new, more sensitive protocol. We will also analyze the expression profile of plants expressing the fatty acid modifying genes under the control of a seed specific promoter.

References in databases (English)

Swiss Database: Euro-DB of the
State Secretariat for Education and Research
Hallwylstrasse 4
CH-3003 Berne, Switzerland
Tel. [phone]
Swiss Project-Number: 99.0692