Elizabeth-France Marillia

A desaturase-like protein from white spruce is a Δ9 desaturase

Gymnospermae seed lipids are characterized by a high degree of desaturation, most having a Δ9 double bond. By degenerate polymerase chain reaction (PCR) we have isolated a white spruce (Picea glauca) cDNA clone that encodes an amino acid sequence sharing a high degree of homology with other putative plant acyl‐coenzyme A (CoA) Des9 desaturases. Both in vivo and in vitro expression studies in a Δ9 desaturase‐deficient yeast strain demonstrated the desaturation functionality of the white spruce clone, and gas chromatography‐mass spectrometry (GC‐MS) analyses confirmed the regioselectivity of the encoded enzyme. This is the first report of the functional characterization of a plant membrane‐bound acyl‐CoA‐like protein Δ9 desaturase by heterologous expression in yeast.

Expression of meadowfoam Des5 and FAE1 genes in yeast and in transgenic soybean somatic embryos, and their roles in fatty acid modification ☆

Abstract Meadowfoam ( Limnanthes spp.) species are unique in that their seeds are rich in the unusual fatty acids Δ 5 -eicosenoic acid (C20:1 Δ5 ) and the diene, C22:2 Δ5, Δ13 . Previously the cloning of Δ 5 desaturase ( Des5 ) and fatty acid elongase 1 ( FAE1 ) meadowfoam genes and their expression in soybean were reported. Here, we present the first successful expression of the Limnanthes Des5 in yeast, resulting in the desaturation of C16:0, C18:0 and C20:0 to their corresponding cis Δ 5 isomers. In soybean ( Glycine max L.), Limnanthes Des5/FAE1 double transformant somatic embryos fed with radiolabeled C14:0 or C16:0 could elongate these substrates to C18:0, C20:0 and C22:0 and C24:0. However, radiolabeled C18:1 Δ9 or C20:1 Δ11 were not elongated to their respective monounsaturated very long-chain products, confirming that the cloned Limnanthes FAE1 homolog gene product was specific for elongating saturated fatty acids. To understand better the biosynthetic pathway for C22:2 Δ5, Δ13 , soybean somatic embryos transformed with the Des5 cDNA were fed in culture with 〚1- 14 C〛C 22:1 Δ13 fatty acid, which resulted in the biosynthesis of 〚1- 14 C〛-labeled C22:2 Δ5, Δ13 . Cell-free preparations enriched with detergent-solubilized Δ 5 desaturase activity extracted from both developing meadowfoam seeds and from Des5 transgenic soybean embryos, produced 14 C-22:2 Δ5, Δ13 when supplied with 〚1- 14 C〛 C22:1-CoA. Thus, both the in vivo and in vitro experiments showed that the biosynthesis of C22:2 Δ5, Δ13 can occur in somatic soybean embryos transformed with the Limnanthes Des5 cDNA, and confirmed that the pathway for C22:2 biosynthesis in meadowfoam involves further desaturation of erucoyl-CoA by a Δ 5 -regiospecific desaturase.

Cloning and Characterization of a Constitutive Lysophosphatidic Acid Acyltransferase 2 (LPAT2) Gene from Tropaeolum majus L

The cloning and characterization of a lyso-phosphatidic acid acyltransferase (LPAT2; EC 2.3.1.51) from Tropaeolum majus from a 20,000 EST collection is described. The 1358 bp TmLPAT2 gene encodes a 42.6 kD polypep- tide; the primary sequence has a membrane bound O-acyltransferase, MBOAT, Box I motif, and Boxes II, III and IV typical of LPATs from various species. Unlike many LPAT2s, the gene was constitutively expressed in all tissues. The TmLPAT2 functionality was confirmed by expression in a yeast LPAT deletion (SLC1 - ) mutant. The TmLPAT2 could use a range of acyl-CoAs as acyl donor, including 22:1-CoA and 20:1-CoA and either 18:1-LPA or 22:1-LPA as acyl acceptor. This new LPAT2 could enable the production of Brassica seed oils with enhanced levels of very long-chain fatty acids.

Production of 22:2Δ5,Δ13 and 20:1Δ5 in Brassica carinata and soybean breeding lines via introduction of Limnanthes genes

Seed oils of meadowfoam (Limnanthes douglasii, L. alba) contain very long-chain fatty acids of strategic importance for a number of industrial applications. These include the monoene 20 1Δ5 and the diene 22:2Δ5,Δ13. Engineering of meadowfoam-type oils in other oilseed crops is desirable for the production of these fatty acids as industrial feedstocks. Accordingly, we have targeted Brassica carinata and soybean (Glycine max) to trangenically engineer the biosynthesis of these unusual fatty acids. An L. douglasii seed-specific cDNA (designated Lim Des5) encoding a homolog of acyl-coenzyme A desaturases found in animals, fungi and cyanobacteria was expressed in B. carinata, which resulted in the accumulation of up to 10% 22:2Δ5,Δ13 in the seed oil. In soybean, co-expression of Lim Des5 with a cDNA (Lim FAE1) encoding an FAEl (elongase complex condensing enzyme) homolog from L. douglasii resulted in the accumulation of 20:1Δ5 to approximately 10% of the total fatty acids of seeds. The content of C20 and C22 fatty acids was also increased from <0.5% in non-transformed soybean seeds to >25% in seeds co-expressing the Lim. douglasii Des5 and FAE1 cDNAs. In contrast, expression of the Lim Des5 in Arabidopsis did not produce the expected 20:2Δ5,Δ11 in the seed oil. Cumulatively, these results demonstrate the utility of soybean and B. carinata for the production of vegetable oils containing novel C20 and C22 fatty acids, and confirm that the preferred substrates of the Lim Des5 are 20:0 and 22:1Δ3, respectively.

Enhancing biomass production and yield by maintaining enhanced capacity for CO2 uptake in response to elevated CO2

Dahal, K., Weraduwage, S. M., Kane, K., Rauf, S. A., Leonardos, E. D., Gadapati, W., Savitch, L., Singh, J., Marillia, E.-F., Taylor, D. C., Micallef, M. C., Knowles, V., Plaxton, W., Barron, J., Sarhan, F., Hüner, N., Grodzinski, B. and Micallef, B. J. 2014. Enhancing biomass production and yield by maintaining enhanced capacity for CO2 uptake in response to elevated CO2. Can. J. Plant Sci. 94: 1075-1083. Using four model plants, two members of the Gramineae, rye and wheat, and two Brassicaceae, Brassica napus and Arabidopsis thaliana, two fundamental approaches were exploited to determine how regulating source-sink development would alter photosynthesis, productivity and yield during long-term acclimation to elevated CO2. In one approach we exploited the cold acclimation response of winter wheat, rye and B. napus. In the other approach we modified the dark respiration in A. thaliana to alter availability of respiratory substrates required for anabolic processes, such as fatty acid metabolism, thus reducing sink limitations on canopy photosynthesis at elevated CO2. Taken together, the data show the importance of maintaining strong demand from active sinks when the above-ground canopy is being exposed to elevated levels of the primary substrate of photosynthesis, CO2.

Trichostatin A increases embryo and green plant regeneration in wheat

Key messageChemical agents such as trichostatin A (TSA) can assist in optimization of doubled haploidy for rapid improvements in wheat germplasm and addressing recalcitrance issues in cell culture responses.AbstractIn wheat, plant regeneration through microspore culture is an integral part of doubled haploid (DH) production. However, low response to tissue culture and genotype specificity are two major constraints in the broad deployment of this breeding tool. Recently, the structure of chromatin was shown to be linked with cell transitions during tissue culture. Specifically, repression of genes that are required for cell morphogenesis, through acetylation of histones, may play an important role in this process. Reduction of histone acetylation by chemical inhibition may increase tissue culture efficiency. Here, the role of trichostatin A (TSA) in inducing microspore-derived embryos was investigated in wheat. The optimal dose of TSA was determined for wheat cultivars and subsequently validated in F1 hybrids. A significant increase in the efficiency of DH production was observed in both cultivated varieties and F1 hybrids. Thus, the inclusion of TSA in DH protocols for wheat breeding programs is advocated.

Increased mtPDH Activity Through Antisense Inhibition of Mitochondrial Pyruvate Dehydrogenase Kinase Enhances Inflorescence Initiation, and Inflorescence Growth and Harvest Index at Elevated CO2 in Arabidopsis thaliana.

Mitochondrial pyruvate dehydrogenase (mtPDH) is a key respiratory enzyme that links glycolysis and the tricarboxylic acid cycle, and it is negatively regulated by mtPDH kinase (mtPDHK). Arabidopsis lines carrying either a constitutive or seed-specific antisense construct for mtPDHK were used to test the hypothesis that alteration of mtPDH activity in a tissue- and dosage-dependent manner will enhance reproductive growth particularly at elevated CO2 (EC) through a combined enhancement of source and sink activities. Constitutive transgenic lines showed increased mtPDH activity in rosette leaves at ambient CO2 (AC) and EC, and in immature seeds at EC. Seed-specific transgenic lines showed enhanced mtPDH activity in immature seeds. A strong relationship existed between seed mtPDH activity and inflorescence initiation at AC, and at EC inflorescence stem growth, silique number and seed harvest index were strongly related to seed mtPDH activity. Leaf photosynthetic rates showed an increase in rosette leaves of transgenic lines at AC and EC that correlated with enhanced inflorescence initiation. Collectively, the data show that mtPDHK plays a key role in regulating sink and source activities in Arabidopsis particularly during the reproductive phase.