Engineering Camelina sativa (L.) Crantz for enhanced oil and seed yields by combining diacylglycerol acyltransferase1 and glycerol‐3‐phosphate dehydrogenase expression

Plant seed oil‐based liquid transportation fuels (i.e., biodiesel and green diesel) have tremendous potential as environmentally, economically and technologically feasible alternatives to petroleum‐derived fuels. Due to their nutritional and industrial importance, one of the major objectives is to increase the seed yield and oil production of oilseed crops via biotechnological approaches. Camelina sativa, an emerging oilseed crop, has been proposed as an ideal crop for biodiesel and bioproduct applications. Further increase in seed oil yield by increasing the flux of carbon from increased photosynthesis into triacylglycerol (TAG) synthesis will make this crop more profitable. To increase the oil yield, we engineered Camelina by co‐expressing the Arabidopsis thaliana (L.) Heynh. diacylglycerol acyltransferase1 (DGAT1) and a yeast cytosolic glycerol‐3‐phosphate dehydrogenase (GPD1) genes under the control of seed‐specific promoters. Plants co‐expressing DGAT1 and GPD1 exhibited up to 13% higher seed oil content and up to 52% increase in seed mass compared to wild‐type plants. Further, DGAT1‐ and GDP1‐co‐expressing lines showed significantly higher seed and oil yields on a dry weight basis than the wild‐type controls or plants expressing DGAT1 and GPD1 alone. The oil harvest index (g oil per g total dry matter) for DGTA1‐ and GPD1‐co‐expressing lines was almost twofold higher as compared to wild type and the lines expressing DGAT1 and GPD1 alone. Therefore, combining the overexpression of TAG biosynthetic genes, DGAT1 and GPD1, appears to be a positive strategy to achieve a synergistic effect on the flux through the TAG synthesis pathway, and thereby further increase the oil yield.     

Microbial production of conjugated γ‐linolenic acid from γ‐linolenic acid by Lactobacillus plantarum AKU 1009a

Aims: Optimal production conditions of conjugated γ‐linolenic acid (CGLA) from γ‐linolenic acid using washed cells of Lactobacillus plantarum AKU 1009a as catalysts were investigated. Methods and Results: Washed cells of Lact. plantarum AKU 1009a exhibiting a high level of CGLA productivity were obtained by cultivation in a nutrient medium supplemented with 0·03% (w/v) α‐linolenic acid as an inducer. Under the optimal reaction conditions with 13 mg ml^?1γ‐linolenic acid as a substrate in 5 ‐ml reaction volume, the washed cells [32% (wet cells, w/v) corresponding to 46 mg ml^?1 dry cells] as the catalysts produced 8·8 mg CGLA per millilitre reaction mixture (68% molar yield) in 27 h. The produced CGLA was a mixture of two isomers, i.e., cis‐6,cis‐9,trans‐11‐octadecatrienoic acid (CGLA1, 40% of total CGLA) and cis‐6,trans‐9,trans‐11‐octadecatrienoic acid (CGLA2, 60% of total CGLA), and accounted for 66% of total fatty acid obtained. The CGLA produced was obtained as free fatty acids adsorbed mostly on the surface of the cells of Lact. plantarum AKU1009a. Conclusion: The practical process of CGLA production from γ‐linolenic acid using washed cells of Lact. plantarum AKU 1009a was successfully established. Significance and Impact of the Study: We presented the first example of microbial production of CGLA. CGLA produced by the process is valuable for evaluating their physiological and nutritional effects, and chemical characteristics.     

Identification of a Chlamydomonas plastidial 2‐lysophosphatidic acid acyltransferase and its use to engineer microalgae with increased oil content

Despite a strong interest in microalgal oil production, our understanding of the biosynthetic pathways that produce algal lipids and the genes involved in the biosynthetic processes remains incomplete. Here, we report that Chlamydomonas reinhardtii Cre09.g398289 encodes a plastid‐targeted 2‐lysophosphatidic acid acyltransferase (CrLPAAT1) that acylates the sn‐2 position of a 2‐lysophosphatidic acid to form phosphatidic acid, the first common precursor of membrane and storage lipids. In vitro enzyme assays showed that CrLPAAT1 prefers 16:0‐CoA to 18:1‐CoA as an acyl donor. Fluorescent protein‐tagged CrLPAAT1 was localized to the plastid membrane in C. reinhardtii cells. Furthermore, expression of CrLPAAT1 in plastids led to a > 20% increase in oil content under nitrogen‐deficient conditions. Taken together, these results demonstrate that CrLPAAT1 is an authentic plastid‐targeted LPAAT in C. reinhardtii, and that it may be used as a molecular tool to genetically increase oil content in microalgae.     

A dominant‐negative form of Arabidopsis AP‐3 β‐adaptin improves intracellular pH homeostasis

Intracellular pH (pH_i) is a crucial parameter in cellular physiology but its mechanisms of homeostasis are only partially understood. To uncover novel roles and participants of the pH_i regulatory system, we have screened an Arabidopsis mutant collection for resistance of seed germination to intracellular acidification induced by weak organic acids (acetic, propionic, sorbic). The phenotypes of one identified mutant, weak acid‐tolerant 1‐1D (wat1‐1D) are due to the expression of a truncated form of AP‐3 β‐adaptin (encoded by the PAT2 gene) that behaves as a as dominant‐negative. During acetic acid treatment the root epidermal cells of the mutant maintain a higher pH_i and a more depolarized plasma membrane electrical potential than wild‐type cells. Additional phenotypes of wat1‐1D roots include increased rates of acetate efflux, K⁺ uptake and H⁺ efflux, the latter reflecting the in vivo activity of the plasma membrane H⁺‐ATPase. The in vitro activity of the enzyme was not increased but, as the H⁺‐ATPase is electrogenic, the increased ion permeability would allow a higher rate of H⁺ efflux. The AP‐3 adaptor complex is involved in traffic from Golgi to vacuoles but its function in plants is not much known. The phenotypes of the wat1‐1D mutant can be explained if loss of function of the AP‐3 β‐adaptin causes activation of channels or transporters for organic anions (acetate) and for K⁺ at the plasma membrane, perhaps through miss‐localization of tonoplast proteins. This suggests a role of this adaptin in trafficking of ion channels or transporters to the tonoplast.     

The hybrid Four‐CBS‐Domain KINβγ subunit functions as the canonical γ subunit of the plant energy sensor SnRK1

The AMPK/SNF1/SnRK1 protein kinases are a family of ancient and highly conserved eukaryotic energy sensors that function as heterotrimeric complexes. These typically comprise catalytic α subunits and regulatory β and γ subunits, the latter function as the energy‐sensing modules of animal AMPK through adenosine nucleotide binding. The ability to monitor accurately and adapt to changing environmental conditions and energy supply is essential for optimal plant growth and survival, but mechanistic insight in the plant SnRK1 function is still limited. In addition to a family of γ‐like proteins, plants also encode a hybrid βγ protein that combines the Four‐Cystathionine β‐synthase (CBS)‐domain (FCD) structure in γ subunits with a glycogen‐binding domain (GBD), typically found in β subunits. We used integrated functional analyses by ectopic SnRK1 complex reconstitution, yeast mutant complementation, in‐depth phylogenetic reconstruction, and a seedling starvation assay to show that only the hybrid KINβγ protein that recruited the GBD around the emergence of the green chloroplast‐containing plants, acts as the canonical γ subunit required for heterotrimeric complex formation. Mutagenesis and truncation analysis further show that complex interaction in plant cells and γ subunit function in yeast depend on both a highly conserved FCD and a pre‐CBS domain, but not the GBD. In addition to novel insight into canonical AMPK/SNF/SnRK1 γ subunit function, regulation and evolution, we provide a new classification of plant FCD genes as a convenient and reliable tool to predict regulatory partners for the SnRK1 energy sensor and novel FCD gene functions.     

Genetic enhancement of palmitic acid accumulation in cotton seed oil through RNAi down‐regulation of ghKAS2 encoding β‐ketoacyl‐ACP synthase II (KASII)

Palmitic acid (C16:0) already makes up approximately 25% of the total fatty acids in the conventional cotton seed oil. However, further enhancements in palmitic acid content at the expense of the predominant unsaturated fatty acids would provide increased oxidative stability of cotton seed oil and also impart the high melting point required for making margarine, shortening and confectionary products free of trans fatty acids. Seed‐specific RNAi‐mediated down‐regulation of β‐ketoacyl‐ACP synthase II (KASII) catalysing the elongation of palmitoyl‐ACP to stearoyl‐ACP has succeeded in dramatically increasing the C16 fatty acid content of cotton seed oil to well beyond its natural limits, reaching up to 65% of total fatty acids. The elevated C16 levels were comprised of predominantly palmitic acid (C16:0, 51%) and to a lesser extent palmitoleic acid (C16:1, 11%) and hexadecadienoic acid (C16:2, 3%), and were stably inherited. Despite of the dramatic alteration of fatty acid composition and a slight yet significant reduction in oil content in these high‐palmitic (HP) lines, seed germination remained unaffected. Regiochemical analysis of triacylglycerols (TAG) showed that the increased levels of palmitic acid mainly occurred at the outer positions, while C16:1 and C16:2 were predominantly found in the sn‐2 position in both TAG and phosphatidylcholine. Crossing the HP line with previously created high‐oleic (HO) and high‐stearic (HS) genotypes demonstrated that HP and HO traits could be achieved simultaneously; however, elevation of stearic acid was hindered in the presence of high level of palmitic acid.     

Arabidopsis plants expressing only the redox‐regulated Rca‐α isoform have constrained photosynthesis and plant growth

Rubisco activase (Rca) facilitates the release of sugar‐phosphate inhibitors from the active sites of Rubisco and thereby plays a central role in initiating and sustaining Rubisco activation. In Arabidopsis, alternative splicing of a single Rca gene results in two Rca isoforms, Rca‐α and Rca‐β. Redox modulation of Rca‐α regulates the function of Rca‐α and Rca‐β acting together to control Rubisco activation. Although Arabidopsis Rca‐α alone less effectively activates Rubisco in vitro, it is not known how CO₂ assimilation and plant growth are impacted. Here, we show that two independent transgenic Arabidopsis lines expressing Rca‐α in the absence of Rca‐β (‘Rca‐α only’ lines) grew more slowly in various light conditions, especially under low light or fluctuating light intensity, and in a short day photoperiod compared to wildtype. Photosynthetic induction was slower in the Rca‐α only lines, and they maintained a lower rate of CO₂ assimilation during both photoperiod types. Our findings suggest Rca oligomers composed of Rca‐α only are less effective in initiating and sustaining the activation of Rubisco than when Rca‐β is also present. Currently there are no examples of any plant species that naturally express Rca‐α only but numerous examples of species expressing Rca‐β only. That Rca‐α exists in most plant species, including many C3 and C4 food and bioenergy crops, implies its presence is adaptive under some circumstances.     

Constitutive or seed‐specific overexpression of Arabidopsis G‐protein γ subunit 3 (AGG3) results in increased seed and oil production and improved stress tolerance in Camelina sativa

Heterotrimeric G‐proteins consisting of Gα, Gβ and Gγ subunits play an integral role in mediating multiple signalling pathways in plants. A novel, recently identified plant‐specific Gγ protein, AGG3, has been proposed to be an important regulator of organ size and mediator of stress responses in Arabidopsis, whereas its potential homologs in rice are major quantitative trait loci for seed size and panicle branching. To evaluate the role of AGG3 towards seed and oil yield improvement, the gene was overexpressed in Camelina sativa, an oilseed crop of the Brassicaceae family. Analysis of multiple homozygous T4 transgenic Camelina lines showed that constitutive overexpression of AGG3 resulted in faster vegetative as well as reproductive growth accompanied by an increase in photosynthetic efficiency. Moreover, when expressed constitutively or specifically in seed tissue, AGG3 was found to increase seed size, seed mass and seed number per plant by 15%–40%, effectively resulting in significantly higher oil yield per plant. AGG3 overexpressing Camelina plants also exhibited improved stress tolerance. These observations draw a strong link between the roles of AGG3 in regulating two critical yield parameters, seed traits and plant stress responses, and reveal an effective biotechnological tool to dramatically increase yield in agricultural crops.     

TRANSPARENT TESTA2 regulates embryonic fatty acid biosynthesis by targeting FUSCA3 during the early developmental stage of Arabidopsis seeds

TRANSPARENT TESTA2 (TT2) regulates the biosynthesis of proanthocyanidins in the seed coat of Arabidopsis. We recently found that TT2 also participates in inhibition of fatty acid (FA) biosynthesis in the seed embryo. However, the mechanism by which TT2 suppresses the accumulation of seed FA remains unclear. In this study, we show that TT2 is expressed in embryos at an early developmental stage. TT2 is directly bound to the regulatory region of FUSCA3 (FUS3), and mediates the expression of numerous genes in the FA biosynthesis pathway. These genes include BCCP2, CAC2, MOD1 and KASII, which encode proteins involved in the initial steps of FA chain formation, FAD2 and FAD3, which are responsible for FA desaturation, and FAE1, which catalyzes very‐long‐chain FA elongation. Loss of function of TT2 results in reduced expression of GLABRA2 but does not cause a significant reduction in the mucilage attached to the seed coats, which competes with FA for photosynthates. TT2 is expressed in both maternal seed coats and embryonic tissues, but proanthocyanidins are only found in wild‐type seed coats and not in embryonic tissues. The amount of proanthocyanidins in the seed coat is negatively correlated with the amount of FAs in the embryo.     

Inhibition of Arabidopsis growth by the allelopathic compound azetidine‐2‐carboxylate is due to the low amino acid specificity of cytosolic prolyl‐tRNA synthetase

The toxicity of azetidine‐2‐carboxylic acid (A2C), a structural analogue of L‐proline, results from its incorporation into proteins due to misrecognition by prolyl‐tRNA synthetase (ProRS). The growth of Arabidopsis thaliana seedling roots is more sensitive to inhibition by A2C than is cotyledon growth. Arabidopsis contains two ProRS isozymes. AtProRS‐Org (At5g52520) is localized in chloroplasts/mitochondria, and AtProRS‐Cyt (At3g62120) is cytosolic. AtProRS‐Cyt mRNA is more highly expressed in roots than in cotyledons. Arabidopsis ProRS isoforms were expressed as His‐tagged recombinant proteins in Escherichia coli. Both enzymes were functionally active in ATP‐PPi exchange and aminoacylation assays, and showed similar K_m for L‐proline. A major difference was observed in the substrate specificity of the two enzymes. AtProRS‐Cyt showed nearly identical substrate specificity for L‐proline and A2C, but for AtProRS‐Org the specificity constant was 77.6 times higher for L‐proline than A2C, suggesting that A2C‐sensitivity may result from lower amino acid specificity of AtProRS‐Cyt. Molecular modelling and simulation results indicate that this specificity difference between the AtProRS isoforms may result from altered modes of substrate binding. Similar kinetic results were obtained with the ProRSs from Zea mays, suggesting that the difference in substrate specificity is a conserved feature of ProRS isoforms from plants that do not accumulate A2C and are sensitive to A2C toxicity. The discovery of the mode of action of A2C toxicity could lead to development of biorational weed management strategies.     

Serum fatty acid analysis and digestibility study in the Vancouver Island marmot (Marmota vancouverensis) fed a captive diet supplemented with α‐linolenic acid

Mammals that hibernate must rely on endogenous lipid reserves to survive over winter. This study was conducted to compare the difference in serum fatty acid composition, dietary intake, and apparent digestibility in the Vancouver Island marmot (N = 6) fed the Metro Zoo lagomorph diet supplemented with α‐linolenic acid [C(18:3) n‐3 (α‐LA)]. The experiment was designed as a 3 × 3 Latin square with three 17‐day collection periods. The test diets contained 12.16, 14.85, and 17.05% α‐LA as a percentage of fatty acids in the diet supplied through the addition of flaxseed oil (～53% α‐LA). Across treatments, dry matter intake (g/d), dry matter digestibility, apparent fat digestibility, and apparent neutral detergent fiber digestibility did not differ significantly (P > 0.05). There were no significant differences in serum α‐LA concentration between the three levels of α‐LA supplementation. However, this supplementation did elevate serum α‐LA, eicosapentaenoic acid [C20:5 (n‐3)], and docosahexaenoic acid [C22:6 (n‐3)] levels compared with feeding the basal zoo lagomorph diet (P < 0.05). Thus, supplementation of the basal zoo lagomorph diet with α‐LA elevated the serum levels of essential fatty acids in the Vancouver Island marmot. Zoo Biol 20:251–259, 2001. © 2001 Wiley‐Liss, Inc.