Transgenic cotton plants with increased seed oleic acid content

Abstract

Cottonseed typically contains about 15% oleic acid. Here we report the development of transgenic cotton plants with higher seed oleic acid levels. Plants were generated by Agrobacterium-mediated transformation. A binary vector was designed to suppress expression of the endogenous cottonseed ∆-12 desaturase (fad 2) by subcloning a mutant allele of a rapeseed fad 2 gene downstream from a heterologous, seedspecific promoter (phaseolin). Fatty acid profiles of total seed lipids from 43 independent transgenic lines were analyzed by gas chromatography. Increased seed oleic acid content ranged from 21 to 30% (by weight) of total fatty acid content in 22 of the primary transformants. The increase in oleic acid content was at the expense of linoleic acid, consistent with reduced activity of cottonseed FAD2. Progeny of some lines yielded oleic acid content as high as 47% (three times that of standard cottonseed oil). Molecular analyses of nuclear DNA from transgenics confirmed the integration of the canola transgene into the cotton genome. Collectively, our results extend the metabolic engineering of vegetable oils to cottonseed and should provide the basis for the development of a family of novel cottonseed oils.Paper no. J9910 in JAOCS 78, 941-947 (September 2001). KEY WORDS:Cottonseed oil, fatty acid metabolism, metabolic engineering, oilseeds.

FAD2 and FAD3 Desaturases Form Heterodimers That Facilitate Metabolic Channeling in Vivo

“…Immunological cross-linking experiments show that FAD enzymes form dimers, like their distant integral membrane desaturase homolog, yeast Ole1p. The existence of FAD dimers is also consistent with published genetic studies in which the heterologous expression of a nonfunctional Brassica oleate desaturase FAD2 mutant (Bnfad2) resulted in a dominant negative reduction in linoleic acid and a corresponding increase in oleic acid in transgenic cotton seeds (31,32). Interpretation of these results in the context of the present work suggests that the defective Bnfad2 subunit may associate with the functional WT cotton subunit, resulting in its inactivation.…”

Visualization of Lipid Droplet Composition by Direct Organelle Mass Spectrometry

“…Seeds were from cultivar Coker 312 (nontransgenic) or were from transgenic lines (T5 generation) in the Coker 312 background, expressing a nonfunctional allele of the Brassica napus ⌬ 12 -desaturase (Bnfad2) under the control of a seed-specific promoter (28,29). These seeds displayed reduced oil, elevated oleic acid phenotype relative to Coker 312 seeds (28,29). Intact embryos (mostly cotyledon tissues) were harvested from desiccated seeds following seed coat removal.…”

“…We demonstrate the capability to directly sample populations of purified LDs as well as perform single-organelle mass spectrometry and lipid characterization. To illustrate the utility of this approach, we show a compositional shift in TAG profiles in LDs purified from modified oleic cottonseed lines previously generated in our laboratory (dominant-negative expression of Bnfad2 (28,29)), the presence of cyclic fatty acids in TAGs of cotton root LDs, and the molecular comparison between Arabidopsis seed and leaf LDs. These new approaches complement existing analytical and cell biology techniques and can be extended to the analysis of LDs and organelles from other organisms.…”

Half-of-the-Sites Reactivity of the Castor Δ9-18:0-Acyl Carrier Protein Desaturase

“…For both the yeast Ole1 (Lou and Shanklin, 2010) and plant FAD2 desaturases (Chapman et al, 2001(Chapman et al, , 2008, coexpression of inactive mutant subunits results in the inactivation of the endogenous wild-type desaturases, presumably by the formation of heterodimers. Thus, for the membrane class of desaturase enzymes, two catalytically competent subunits are required for catalysis.…”