What limits production of unusual monoenoic fatty acids in transgenic plants?

Abstract

Unusual monounsaturated fatty acids are major constituents (greater than 80%) in seeds of Coriandrum sativum L. (coriander) and Thunbergia alata Bojer, as well as in glandular trichomes (greater than 80% derived products) of Pelargonium x hortorum (geranium). These diverged fatty acid structures are produced via distinct plastidial acyl-acyl carrier protein (ACP) desaturases. When expressed in Arabidopsis thaliana (L.) Heynh. under strong seed-specific promoters the unusual acyl-ACP desaturases resulted in accumulation of unusual monoene fatty acids at 1-15% of seed fatty acid mass. In this study, we have examined several factors that potentially limit higher production of unusual monoenes in transgenic oilseeds. (i) Immunoblots indicated that the introduced desaturases were expressed at levels equivalent to or higher than the endogenous delta9 18:0-ACP desaturase. However, the level of unusual fatty acid produced in transgenic plants was not correlated with the level of desaturase expression. (ii) The unusual desaturases were expressed in several backgrounds, including antisense 18:0-ACP desaturase plants, in fab1 mutants, and co-expressed with specialized ACP or ferredoxin isoforms. None of these experiments led to high production of expected products. (iii) No evidence was found for degradation of the unusual fatty acids during seed development. (iv) Petroselinic acid added to developing seeds was incorporated into triacylglycerol as readily as oleic acid, suggesting no major barriers to its metabolism by enzymes of glycerolipid assembly. (v) In vitro and in situ assay of acyl-ACP desaturases revealed a large discrepancy of activity when comparing unusual acyl-ACP desaturases with the endogenous delta9 18:0-ACP desaturase. The combined results, coupled with the sensitivity of acyl-ACP desaturase activity to centrifugation and low salt or detergent suggests low production of unusual monoenes in transgenic plants may be due to the lack of, or incorrect assemble of, a necessary multi-component enzyme association.

Efficiency for increasing seed oil content using WRINKLED1 and DGAT1 under the control of two seed-specific promoters, FAE1 and Napin

“…Tissue-specific promoters, such as leaf-, phloem-, root-, fruit-, pollen-, flowerand green tissue-specific promoters, have also been used for such purposes (van Tunen et al 1988;Pear et al 1989;Yamamoto et al 1991;Rogers et al 2001;Husebye et al 2002;Gowik et al 2004;Bakhsh et al 2011). In particular, several seed-specific promoters, including Napin, USP, FAE1, oleosin, and SeFAD2 have been widely used to modify seed oil composition and content (Josefsson et al 1987;Rossak et al 2001;Suh et al 2002;Schmid et al 2005;Kim et al 2006;. In this study, the activities of the two seed-specific promoters, AtFAE1 from Arabidopsis Fatty Acid Elongase 1 gene and the BnNapin from Brassica napus seed storage protein gene, were compared by the expression of AtWRI1 and AtDGAT1 genes under the control of those promoters in Arabidopsis.…”

Identification and characterization of genes involved in metabolism of n5 monoene precursors to n5 anacardic acids in the trichomes of Pelargonium x hortorum.

“…UFAs are hypothesized to be produced through a distinct "metabolic channel" of the primary metabolic pathways that results in production of common fatty acids found in all plants 11,12 . In plants, fatty acid biosynthesis occurs in plastids through the activity of fatty acid synthase 13 .…”

“…Very long chain fatty acids or glycerolipids are produced via substrate channeling of acyl-ACPs in the cytosol 3, 11 whereas unusual monoenes that are produced in the plastid that can either get incoporated into triacylglycerol or undergo enzymatic modifications that lead to production of specialized metabolites in specific tissues 3,11 . Most of the UFAs become part of storage lipids whereas very few readily enter plastidial or cytosolic membrane lipids 3 .…”

“…Some UFAs can exist in cytosolic or epidermal membranes (specifically long chain UFAs) 7 that are used as substrates by plants for the production of plant specialized/secondary metabolites and other lipids that prevent water loss on plant surfaces by forming a hydrophobic barrier and additionally provide protection against plant pathogens and environmental stress 6 . Some of these metabolites (like anacardic acids) not only protect plants from biotic and abiotic challenges but also have clinical applications 8 . If the production of UFAs can be enhanced in seeds and other UFA specific tissues, then they can serve as a petroleum substitute and have various industrial, chemical and nutraceuticals appliacations 3,[8][9][10] .…”

“…Some of these metabolites (like anacardic acids) not only protect plants from biotic and abiotic challenges but also have clinical applications 8 . If the production of UFAs can be enhanced in seeds and other UFA specific tissues, then they can serve as a petroleum substitute and have various industrial, chemical and nutraceuticals appliacations 3,[8][9][10] . This fundamental and applied application of UFAs in plant biology has led to considerable interest in studying the biosynthesis of UFAs and identifying specific enzymes involved in their production.…”