- Research article
- Open Access
Survey of the total fatty acid and triacylglycerol composition and content of 30 duckweed species and cloning of a Δ6-desaturase responsible for the production of γ-linolenic and stearidonic acids in Lemna gibba
https://doi.org/10.1186/1471-2229-13-201
© Yan et al.; licensee BioMed Central Ltd. 2013
- Received: 3 October 2013
- Accepted: 20 November 2013
- Published: 5 December 2013
Abstract
Background
Duckweeds, i.e., members of the Lemnoideae family, are amongst the smallest aquatic flowering plants. Their high growth rate, aquatic habit and suitability for bio-remediation make them strong candidates for biomass production. Duckweeds have been studied for their potential as feedstocks for bioethanol production; however, less is known about their ability to accumulate reduced carbon as fatty acids (FA) and oil.
Results
Total FA profiles of thirty duckweed species were analysed to assess the natural diversity within the Lemnoideae. Total FA content varied between 4.6% and 14.2% of dry weight whereas triacylglycerol (TAG) levels varied between 0.02% and 0.15% of dry weight. Three FA, 16:0 (palmitic), 18:2Δ9,12 (Linoleic acid, or LN) and 18:3Δ9,12,15 (α-linolenic acid, or ALA) comprise more than 80% of total duckweed FA. Seven Lemna and two Wolffiela species also accumulate polyunsaturated FA containing Δ6-double bonds, i.e., GLA and SDA. Relative to total FA, TAG is enriched in saturated FA and deficient in polyunsaturated FA, and only five Lemna species accumulate Δ6-FA in their TAG. A putative Δ6-desaturase designated LgDes, with homology to a family of front-end Δ6-FA and Δ8-spingolipid desaturases, was identified in the assembled DNA sequence of Lemna gibba. Expression of a synthetic LgDes gene in Nicotiana benthamiana resulted in the accumulation of GLA and SDA, confirming it specifies a Δ6-desaturase.
Conclusions
Total accumulation of FA varies three-fold across the 30 species of Lemnoideae surveyed. Nine species contain GLA and SDA which are synthesized by a Δ6 front-end desaturase, but FA composition is otherwise similar. TAG accumulates up to 0.15% of total dry weight, comparable to levels found in the leaves of terrestrial plants. Polyunsaturated FA is underrepresented in TAG, and the Δ6-FA GLA and SDA are found in the TAG of only five of the nine Lemna species that produce them. When present, GLA is enriched and SDA diminished relative to their abundance in the total FA pool.
Keywords
- Desaturase
- Fatty acid
- Triacylglycerol
- Lemnoideae
- Duckweed
- Lemna
- Wolffiela
- Renewable feedstock
- Biofuel
Background
Duckweeds are the smallest known aquatic flowering plants [1]. These monocotyledonous plants family are in the family Lemnoideae which contains five genera: Lemna, Spirodela, Wolffia, Wolffiela and Landoltia, encompassing more than 38 different species geographically distributed around the globe [2]. The morphology of different genera of duckweed differs greatly, ranging from the relatively complex structure of members of the genus Spirodela to the extremely reduced structures found in the genus Wolffia.
Duckweeds have been used for research since the 1960s [3] but genetic and molecular techniques have advanced more rapidly in other model systems. There is renewed interest in duckweed due to the high demand for renewable biomass. Many species of duckweed have rapid doubling times, as short as 48 hours in Lemna aequinoctialis and Wolffia microscopica[1], and certain duckweed species have the ability to grow on waste water [4]. Recent research has focused on the ability of duckweed to produce starch and protein, for instance, Spirodela polyrhiza has been shown to accumulate up to 20% dry weight as starch when grown on pig effluent [5]. These traits have made duckweed a desirable candidate for biomass production. Lemna gibba has also been engineered to produce monoclonal antibodies [6].
Both starch and oil are sinks for photosynthetically fixed carbon, but oil is highly reduced, having an energy density more than 2-fold that of starch. Plant oils have a wide variety of applications, including industrial feedstocks [7] biofuels and dietary supplements. Plant oil is generally harvested from seeds, but recent studies suggest that oil accumulation can be successfully engineered in vegetative tissue [8–12]. Although little data available on lipid composition and accumulation in duckweed, its short doubling time, substantial ability to store excess photosynthate as starch and ability to grow on wastewater make it a promising candidate to screen for potential vegetative oil production.
To explore the possibility of using duckweed for oil production, we conducted a survey of 30 different species spanning the overall diversity of this family with respect to FA and TAG abundance. This study also examines duckweed’s FA composition to identify strains of duckweed that accumulate specific FA that are of potential importance as industrial feedstocks or dietary supplements. The survey revealed that nine duckweed species accumulate Δ6-containing FA in the form of γ-linolenic acid (GLA) or stearidonic acid (SDA). While these fatty acids are rarely found in higher plants, they are found in borage [13], and Echium[14]. Δ6-containing FA are synthesized by desaturases that are commonly referred to as “front-end” because they introduce double bonds between the carboxyl group of the FA and an existing double bond [15] rather than between an existing double bond and the terminal methyl group as in the majority of desaturase enzymes. Front-end desaturases occur as C-terminal fusions with their electron donor, cytochrome b5 and contain a tri-partite histidine motif found in all desaturases [16], except that the first histidine in the third box is substituted for a glutamine [15]. Δ6 desaturase sequences cluster with a ubiquitous class of Δ8 sphingolipid long-chain-base (LCB) desaturases [17] preventing their functional designation to either class based on sequence alone. A detailed study of Lemna gibba, which contains both GLA and SDA, resulted in the identification of LgDes, a member of the Δ6/Δ8 desaturase family. The identity of LgDes as a Δ6 desaturase was confirmed by heterologous expression in Nicotiana benthamiana.
Methods
Plant materials and growth conditions
Duckweed lines were obtained from The Rutgers University Duckweed Stock Cooperative (http://www.ruduckweed.org), and were cultured in SH medium containing 1.6 g/L Schenk and Hildebrandt Basal Salt Mixture (Sigma) with 0.5% glucose (pH 5.7). Fronds were cultured in T-75 culture flasks containing 100 ml of the culture medium, at 22°C, under continuous fluorescent light (100 μE m-2 s-1).
Biomass composition analysis
Duckweeds were harvested by filtration to remove excess media. Total dry weights, and lipid and metabolite contents were determined as previously described [18]. Briefly, duckweed tissue was homogenized in 3 mL methanol/water (4:3, v/v) with an Omni tissue grinder (Omni International, Merietta, GA) followed by the addition of 3.4 mL CHCl3 to create a biphasic solvent system (CHCl3/methanol/H2O, 8:4:3, v/v/v, [19]). The phases were separated by centrifugation at 3,000 xg at ambient temperature. The dry weight fractions of lipids (CHCl3 phase), free metabolites (methanol/water phase) and cell pellets (insoluble material) were obtained.
TAG extraction
TAG was separated from the total lipid extract by thin layer chromatography (TLC). Approximately 25% of the total lipid extract was spotted on a silica gel plate along with an Arabidopsis seed oil (TAG) standard. The plate was then developed with hexane: diethyl ether: acetic acid (80:20:1,v/v/v). After development, lipids were visualized by incubation in iodine vapour. The mobility of TAG was identified by comparison to authentic standards and the silica zone containing TAG was collected by scraping the silica from the TLC plate.
FA and TAG analysis
FA were converted to fatty acid methyl esters (FAMEs) by derivatization using boron trichloride methanol as previously reported [20, 21] after addition of 100 μg of heptadecanoic acid as an internal standard. 4,4-dimethyloxazoline (DMOX) derivatives were generated by incubation of FAMEs with 2-amino-2-methyl-1-propanol under a nitrogen atmosphere at 190°C for 16 hours [21]. Pyrrolidone adducts were generated using a standard protocol [22]. To facilitate quantitation, 100 μg of heptadecanoic acid was added to each sample as an internal standard and the abundance of FA from TAG were determined by integrating the areas of each GC-MS peak relative to the internal standard. FA and TAG profiles were obtained with the use of a Hewlett Packard 6890 gas chromatograph equipped with an Agilent J&W DB 23 capillary column (30 m × 0.25 mm × 0.25 mm) and a model 5973 mass selective detector. Each sample was analysed using an injector temperature of 250°C and a program in which the oven was heated from 80-170°C at 20°C/min, and from 170-210°C at 5°C/min. The values are presented as mean percentages ± standard deviations, n = 3 or more.
Nicotiana benthamiana transient expression system
Nicotiana benthamiana plants were grown for approximately 6 weeks in growth chambers under a 16/8 hour day/night cycle and approximately 160 μmol m-2 s-1 of light at 22°C. The transient expression protocol was based on the method of Schütze [23]. On the day before infiltration, plants were watered and, to reduce sample variability, upper and lower leaves were removed to leave 3 recently fully expanded leaves (approximately 10-12 cm in diameter) for infiltration. This procedure allows each leaf to be exposed to full light in the post-inoculation phase. A single colony of Agrobacterium containing a binary plasmid harbouring the target desaturase gene under the control of the 35S promoter [24] was cultured overnight at 30°C in 5 ml of LB medium containing appropriate antibiotics. Cells were collected by centrifugation at 4,000 ×g for 10 min, and resuspend in freshly prepared AS medium (10 mM MES-KOH, pH 5.6, 10 mM MgCl2, 150 μM acetosyringone) to an OD600 of 0.5 and incubated for 1.5 hr. at 22°C. This suspension was diluted 1:1 with an equivalent preparation of cells harbouring the p19 RNA silencing suppressor [25] and incubated for a further 1.5 hr at 22°C prior to infiltration with the use of a 1 ml needleless syringe pressed against the abaxial surface of the leaf. Approximately 8 discrete infiltrations were performed per leaf, with the position of treatment and control infiltrations assigned randomly. The perimeters of infiltrated areas and treatment codes were marked using ballpoint pen. Plants were returned to growth chambers under normal growing conditions and the leaves were harvested 4 days after infiltration for analysis.
Results
Total fatty acid content and composition
Providence of species used in this study
Species | RDSC # | ID | Continent | Country | State/City/Notes |
|---|---|---|---|---|---|
Landoltia punctata | 3 | DWC014 | South America | Venezuela | D. F., La Mariposa |
Lemna aequinoctialis | 62 | 8011 | North America | USA | Oklahoma, Kay Co., Blackwell |
Lemna disperma | 320 | 7767 | Australia | Western Australia | King River |
Lemna gibba G-3 | 9 | DWC130 | North America | not known | not known |
Lemna japonica | 196 | 8693 | Asia | Japan | Hokkaido, Setana |
Lemna minor | 376 | DWC114 | Europe | Switzerland | Ticino, Castel San Pietro |
Lemna minuta | 311 | 8430 | Europe | United Kingdom | England, Cambridge |
Lemna obscura | 281 | 7143 | North America | USA | Florida, Dade Co., Miami |
Lemna perpusilla | 274 | 8473 | North America | USA | North Carolina, Johnston Co., Gees Cross Road |
Lemna tenera | 13 | 9024 | Australia | Australia | Northern Territories, Nancar Billabong |
Lemna trisulca | 564 | 7413 | Europe | Romania | Dobrogea, Maliuc |
Lemna turionifera | 125 | 8133 | North America | USA | California, San Diego Co., Wohlford L. |
Lemna valdiviana | 198 | 8754 | South America | Bolivia | Huatajata |
Spirodela intermedia | 151 | 7178 | South America | Argentina | Buenos Aires, Buenos Aires |
Spirodela polyrhiza | 189 | 7498 | North America | USA | North Carolina, Durham Co., Durham |
Wolffia angusta | 65 | 7274 | Australia | New South Wales | Newcastle, Seaham |
Wolffia arrhiza | 52 | 7193 | Africa | Uganda | Masaka |
Wolffia australiana | 82 | DWC304 | Australia | New South Wales | Singleton, Doughboy Hollow |
Wolffia borealis | 90 | 9147 | North America | USA | Massachusetts, Franklin Co., Deerfield |
Wolffia brasiliensis | 78 | 7150 | North America | USA | Texas, Hays Co., San Marcos |
Wolffia cylindracea | 180 | 7340 | Africa | Tanzania | Iringa, Mesangati |
Wolffia globosa | 73 | 9141 | South America | Chile | Quillon, Laguna Alendano |
Wolffia neglecta | 624 | 9149 | Asia | Pakistan | Karachi, Gulshan-e-Iasbah |
Wolffiella caudata | 256 | 9139 | South America | Brazil | Amazonas, Manaus |
Wolffiella hyalina | 70 | 8640 | Africa | Tanzania | Arusha, Amboseli |
Wolffiella lingulata | 271 | 9451 | South America | Brazil | not known |
Wolffiella neotropica | 69 | 7279 | South America | Brazil | Rio de Janeiro, Cabo Frio |
Wolffiella oblonga | 46 | 9136 | South America | Brazil | Mato Grosso, Corumba |
Wolffiella repanda | 254 | 9122 | South America | Zimbabwe | Urungwe Safari Area, 12 km SE of Chirundu (from seeds) |
Wolffiella welwitschii | 85 | 7644 | Africa | Angola | Benguela, Cubal |
Ranking of total FA content as a percent of dry weight for 30 duckweed species. Means and standard deviations of three independent replicates are presented for each species.
Fatty acid composition as a percentage of total fatty acids of thirty species of duckweed and total fatty acid as a percent of dry weight
Species | Fatty acids (% of total) | Total FA /Dry Weight | ||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|
14:0 | 16:0 | 16:1 | 18:0 | 18:1 | 18:2 | 18:3 (GLA) | 18:3 (ALA) | 18:4 | 20:0 | 22:0 | ||
Landoltia punctata | 0.3 ± 0.11 | 24.88 ± 0.3 | 1.59 ± 0.2 | 6.57 ± 5.7 | 0.9 ± 0.4 | 5.77 ± 1.4 | n.d.2 | 60 ± 8.7 | n.d. | n.d. | n.d. | 7.8 ± 0.6 |
Lemna aequinoctialis | 0.6 ± 0.1 | 26.95 ± 0.6 | 0.5 ± 0.1 | 5.51 ± 1.8 | 1.2 ± 0.2 | 18.44 ± 0.9 | n.d. | 45.99 ± 2.0 | n.d. | 0.3 ± 0.1 | 0.5 ± 0.4 | 9.3 ± 1.9 |
Lemna disperma | 0.7 ± 0.1 | 22.1 ± 0.4 | 2.1 ± 0.4 | 1.6 ± 0.3 | 2.3 ± 0.5 | 12.7 ± 0.2 | 1.6 ± 0.2 | 48.9 ± 0.9 | 7.5 ± 0.1 | 0.2 ± 0.0 | 0.3 ± 0.0 | 11.4 ± 0.9 |
Lemna gibba | 0.7 ± 0.2 | 21.51 ± 0.8 | 3.62 ± 0.3 | 3.12 ± 0.8 | 2.11 ± 0.3 | 11.06 ± 0.7 | 1.81 ± 0.2 | 47.14 ± 0.3 | 7.74 ± 0.4 | 0.7 ± 0.0 | 0.5 ± 0.2 | 11.2 ± 1.2 |
Lemna japonica | 1.1 ± 0.1 | 18.86 ± 0.2 | 2.11 ± 0.0 | 1.5 ± 0.1 | 2.21 ± 0.0 | 21.16 ± 0.1 | 4.51 ± 0.1 | 40.62 ± 0.0 | 6.92 ± 0.1 | 0.4 ± 0.0 | 0.6 ± 0.1 | 8.0 ± 0.5 |
Lemna minor | 0.55 ± 0.0 | 21.74 ± 0.5 | 2.76 ± 0.0 | 2.1 ± 0.4 | 1.77 ± 0.1 | 15.89 ± 0.3 | n.d. | 54.42 ± 0.7 | n.d. | 0.33 ± 0.0 | 0.44 ± 0.1 | 10.6 ± 0.8 |
Lemna minuta | 0.7 ± 0.1 | 19.32 ± 0.2 | 1.8 ± 0.0 | 1.5 ± 0.1 | 2.2 ± 0.1 | 15.12 ± 0.3 | n.d. | 59.06 ± 0.3 | n.d. | 0.1 ± 0.0 | 0.2 ± 0.0 | 7.7 ± 0.1 |
Lemna obscura | 0.7 ± 0.1 | 20.74 ± 0.1 | 1.4 ± 0.1 | 2.2 ± 0.2 | 2.1 ± 0.1 | 15.03 ± 0.2 | 1.8 ± 0.1 | 49.2 ± 0.3 | 5.61 ± 0.2 | 0.5 ± 0.1 | 0.7 ± 0.0 | 7.5 ± 1.3 |
Lemna perpusilla | 0.9 ± 0.1 | 22.47 ± 0.6 | 1.2.0.1 | 2.61 ± 0.5 | 3.01 ± 0.2 | 19.56 ± 0.9 | n.d. | 48.95 ± 2.5 | n.d. | 0.5 ± 0.0 | 0.8 ± 0.3 | 5.5 ± 0.7 |
Lemna tenera | 0.7 ± 0.1 | 28.06 ± 1.3 | 1 ± 0.1 | 2.1 ± 0.0 | 2.51 ± 0.1 | 17.23 ± 0.2 | n.d. | 48.4 ± 1.5 | n.d. | n.d. | n.d. | 6.7 ± 0.6 |
Lemna triscula | 1.1 ± 0.1 | 19.74 ± 0.4 | 2 ± 0.1 | 1.7 ± 0.1 | 2.2 ± 0.0 | 23.85 ± 0.7 | 2.3 ± 0.1 | 43.29 ± 0.1 | 2.81 ± 0.3 | 0.5 ± 0.1 | 0.5 ± 0.0 | 5.9 ± 0.0 |
Lemna turionefera | 1.11 ± 0.1 | 18.45 ± 0.0 | 2.22 ± 0.1 | 1.41 ± 0.1 | 1.71 ± 0.0 | 18.75 ± 0.1 | 3.02 ± 0.5 | 46.07 ± 0.7 | 6.35 ± 0.0 | 0.3 ± 0.0 | 0.6 ± 0.1 | 8.3 ± 0.1 |
Lemna valdiviana | 0.7 ± 0.0 | 18.4 ± 0.2 | 1.2 ± 0.1 | 1.8 ± 0.2 | 2.3 ± 0.1 | 15.8 ± 0.0 | 0.7 ± 0.4 | 58.8 ± 0.7 | n.d. | 0.1 ± 0.0 | 0.2 ± 0.1 | 8.0 ± 0.9 |
Spirodela intermedia | 0.1 ± 0.0 | 32.83 ± 5.1 | 2.2 ± 0.2 | 3.8 ± 0.7 | 2.9 ± 0.1 | 5.21 ± 0.3 | n.d. | 51.85 ± 5.4 | n.d. | 0.4 ± 0.1 | 0.7 ± 0.2 | 8.4 ± 0.9 |
Spirodela polyrhiza | 0.1 ± 0.1 | 24.44 ± 0.9 | 1.92 ± 0.5 | 4.04 ± 3.7 | 0.2 ± 0.0 | 4.95 ± 1.5 | n.d. | 64.04 ± 4.9 | n.d. | 0.1 ± 0.1 | 0.2 ± 0.0 | 7.5 ± 0.2 |
Wolffia angusta | 0.6 ± 0.0 | 24.82 ± 0.1 | 0.4 ± 0.1 | 3.7 ± 0.9 | 3.2 ± 0.1 | 18.62 ± 0.3 | n.d. | 47.25 ± 0.8 | n.d. | 1 ± 0.0 | 0.4 ± 0.1 | 8.0 ± 0.9 |
Wolffia arrhiza | 0.7 ± 0.4 | 23.94 ± 2.5 | 0.8 ± 0.3 | 2.21 ± 0.2 | 1.41 ± 0.3 | 24.45 ± 0.7 | n.d. | 45.27 ± 3.8 | n.d. | 0.7 ± 0.1 | 0.5 ± 0.1 | 8.9 ± 0.5 |
Wolffia australiana | 0.4 ± 0.2 | 24.22 ± 5.8 | 2 ± 0.6 | 2 ± 0.8 | 1.9 ± 0.5 | 17.82 ± 0.2 | 1.7 ± 0.6 | 38.24 ± 5.5 | 10.11 ± 2.8 | 0.5 ± 0.2 | 1.1 ± 0.5 | 10.0 ± 0.4 |
Wolffia borealis | 0.6 ± 0.2 | 27.67 ± 1.2 | 0.9 ± 0.4 | 7.39 ± 0.9 | 2.7 ± 1.1 | 25.47 ± 1.3 | n.d. | 34.17 ± 2.4 | n.d. | 1.1 ± 0.3 | n.d. | 14.2 ± 1.0 |
Wolffia brasiliensis | 0.4 ± 0.0 | 29.55 ± 2.2 | 1.89 ± 0.0 | 3.98 ± 0.1 | 1.99 ± 0.3 | 18.91 ± 0.1 | n.d. | 40.3 ± 1.0 | n.d. | 1.29 ± 0.2 | 1.69 ± 0.0 | 5.6 ± 0.5 |
Wolffia cylindracea | 0.3 ± 0.1 | 27.15 ± 1.1 | 1.8 ± 0.4 | 4.49 ± 1.7 | 1.1 ± 0.3 | 19.56 ± 0.2 | n.d. | 43.11 ± 2.1 | 2.1 ± 0.1 | n.d. | 0.4 ± 0.3 | 6.7 ± 0.4 |
Wolffia globosa | 0.7 ± 0.1 | 30.36 ± 0.9 | n.d. | 2.91 ± 0.3 | 0.5 ± 0.2 | 14.03 ± 0.8 | n.d. | 49.2 ± 0.8 | n.d. | 0.7 ± 0.2 | 1.6 ± 0.4 | 11.1 ± 1.0 |
Wolffia neglecta | 0.6 ± 0.1 | 24.53 ± 1.1 | 0.8 ± 0.3 | 5.38 ± 0.1 | 2.89 ± 0.7 | 24.13 ± 0.9 | n.d. | 39.98 ± 1.0 | n.d. | 0.9 ± 0.2 | 0.8 ± 0.6 | 12.5 ± 0.8 |
Wolffiella caudata | 0.6 ± 0.0 | 22.65 ± 0.7 | 0.4 ± 0.0 | 1.8 ± 0.1 | 1.9 ± 0.1 | 24.95 ± 0.4 | n.d. | 46.11 ± 1.2 | n.d. | 0.6 ± 0.1 | 1 ± 0.0 | 7.0 ± 0.4 |
Wolffiella hyalina | 0.3 ± 0.0 | 17.17 ± 0.4 | 1.5 ± 0.0 | 2.2 ± 0.3 | 3.49 ± 0.1 | 20.66 ± 0.2 | n.d. | 51.9 ± 0.9 | n.d. | 0.8 ± 0.1 | 2 ± 0.1 | 10.3 ± 1.2 |
Wolffiella lingulata | 0.8 ± 0.1 | 36.34 ± 4.3 | n.d. | 7.41 ± 1.1 | 1.3 ± 0.2 | 15.82 ± 2.2 | n.d. | 38.34 ± 2.8 | n.d. | n.d. | n.d. | 5.7 ± 0.3 |
Wolffiella neotropica | 0.6 ± 0.1 | 27.01 ± 1.6 | 0.2 ± 0.0 | 3.92 ± 0.6 | 0.6 ± 0.1 | 4.82 ± 0.6 | n.d. | 61.24 ± 1.9 | n.d. | 0.4 ± 0.0 | 1.2 ± 0.1 | 7.1 ± 0.3 |
Wolffiella oblonga | 0.8 ± 0.0 | 30.16 ± 1.0 | 0.4 ± 0.0 | 3.91 ± 0.6 | 1.3 ± 0.0 | 11.62 ± 0.8 | n.d. | 47.09 ± 1.5 | n.d. | 1.1 ± 0.1 | 3.61 ± 0.1 | 10.0 ± 1.4 |
Wolffiella repanda | 0.5 ± 0.1 | 25.45 ± 0.7 | 1 ± 0.2 | 2.51 ± 0.6 | 1.5 ± 0.8 | 16.03 ± 2.4 | n.d. | 51.3 ± 0.8 | n.d. | 0.3 ± 0.1 | 1.4 ± 0.2 | 7.6 ± 0.5 |
Wolffiella welwitschii | 0.5 ± 0.1 | 26.65 ± 0.6 | 0.5 ± 0.2 | 2 ± 0.4 | 1.2 ± 0.9 | 17.84 ± 0.5 | n.d. | 49.1 ± 1.5 | n.d. | 0.6 ± 0.1 | 1.6 ± 0.2 | 4.6 ± 0.7 |
Identification of GLA and SDA in Lemna gibba . Portions of gas chromatogram traces for FA 4,4-dimethyloxazoline (DMOX) derivatives from Spirodela polyrhiza (Panel A), and Lemna gibba (Panel B). Peak labeled 1, corresponds to ALA, 2, GLA, and 3, SDA. Mass spectra corresponding to the DMOX derivatives of GLA (Panel C), and SDA (Panel D) are shown. Ions diagnostic for the positions of the double bonds in the acyl chains and mass ions are indicated and diagrammed according to W.W. Christie (http://lipidlibrary.aocs.org/ms/masspec.html).
Identification of a Δ6-desaturase from Lemna gibba
Amino acid identities computed using the ClustalW2 multiple sequence alignment algorithm
LgDes | BoD6 | EgD6 | PfD6 | RnD6 | PvD6 | AtD8a | AtD8b | NtD8 | HaD8 | PfD8 | PvD8 | |
|---|---|---|---|---|---|---|---|---|---|---|---|---|
LgDes | ||||||||||||
BoD6 | 62 | |||||||||||
EgD6 | 65 | 85 | ||||||||||
PfD6 | 58 | 65 | 65 | |||||||||
RnD6 | 63 | 61 | 64 | 58 | ||||||||
PvD6 | 57 | 64 | 63 | 95 | 57 | |||||||
AtD8a | 61 | 58 | 62 | 58 | 65 | 57 | ||||||
AtD8b | 62 | 60 | 61 | 58 | 66 | 59 | 80 | |||||
NtD8 | 70 | 73 | 73 | 64 | 71 | 64 | 66 | 66 | ||||
HaD8 | 59 | 61 | 61 | 56 | 65 | 56 | 67 | 67 | 64 | |||
PfD8 | 61 | 61 | 63 | 80 | 61 | 80 | 62 | 63 | 71 | 59 | ||
PvD8 | 61 | 62 | 64 | 80 | 62 | 81 | 62 | 63 | 71 | 59 | 94 |
Sequence comparison of LgDes with experimentally validated homologs. Alignment was made using CLUSTALW2 [28] and BOXSHADE 3.21. (http://www.ch.embnet.org/software/BOX_form.html). Residues identical for six or more sequences in a given position are in white text on a black background, and for six or more similar residues are white with grey background. Sequences: LgDes (KF638283, this study), Δ6 desaturase sequences: Borago officinalis (BoD6, AAC49700) [13]; Echinum gentianoides (EgD6, AAL23580) [14], Primula farinosa (PfD6, AAP23034) [29], Ribes nigrum (RnD6, ADA60230) [30], Primula vialii (PvD6, AAP23036) [29]. Δ8 sequences: Arabidopsis thaliana (AtD8a, NP_191717; AtD8b, NP_182144) [26], Nicotiana tabacum (NtD8, ABO31111) [17], Helianthus annuus (HaD8, CAA60621) [31], Primula farinosa (PfD8, AAP23033, PvD8, AAP23035) [29]. Three histidine boxes common to membrane bound desaturases are underlined in red. The consensus cytochrome b5 sequence is underlined in blue and the amino acid position corresponding to L210 of LgDes, identified by CPDL, is marked with a vertical red arrow.
Phylogenetic tree of LgDes with experimentally validated homologs. Clustalw2 phylogenetic tree output was fed into the Phylodendron tree drawing tool (http://iubio.bio.indiana.edu/treeapp/treeprint-form.html) to construct the tree by neighbor-joining distance analysis. Line lengths indicate the relative distances between nodes. Sequences and gene identifiers are those described in Figure 3. Color codes: LgDes, red, Δ6-FA desaturases green and Δ8-LCB sphingolipid desaturases, blue.
Expression of LgDes results in the accumulation of GLA and SDA in N. benthamiana leaves. Portions of gas chromatogram traces for FA-pyrrolidide derivatives from Nicotiana benthamiana containing empty vector control (Panel A), LgDes (Panel B), borage Δ6-desaturase (Panel C). Peak labeled 1, corresponds to LA, 2, ALA, 3, GLA, 4, SDA, and 5, 20:0. Mass spectra corresponding to the pyrrolidide derivatives of GLA (Panel D), and SDA (Panel E) derived from the expression of LgDes shown in Panel B, peaks 3 and 4, respectively, are shown. Ions diagnostic for the positions of the double bonds in the acyl chains and the mass ions are indicated and diagrammed according to W.W. Christie (http://lipidlibrary.aocs.org/ms/masspec.html).
Conversion efficiencies of desaturases expressed in Nicotiana benthamiana
Desaturase | Conversion | Conversion % |
|---|---|---|
LgDes | 18:2 to 18:3 | 3.76 ± 0.04 1 |
18:3 to 18:4 | 3.28 ± 1.27 | |
BoD6 | 18:2 to 18:3 | 6.89 ± 1.17 |
18:3 to 18:4 | 6.07 ± 2.73 |
Triacyl Glycerol content and composition
Fatty acid composition of TAG as a percentage of total TAG, and TAG as a percentage of total lipid or dry weight
Fatty acids (% of total) | % TAG per mg of lipid | % TAG per mg dry weight | |||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
14:0 | 16:0 | 16:1 | 18:0 | 18:1 | 18:2 | 18:3 (GLA) | 18:3 (ALA) | 18:4 | 20:0 | 22:0 | |||
Landoltia punctata | 0.6 ± 0.21 | 39.6 ± 9.4 | n.d. 2 | 5.4 ± 0.6 | 6.7 ± 1.4 | 12.2 ± 1.2 | n.d. | 35.5 ± 7.9 | n.d. | n.d. | n.d. | 0.7 ± 0.1 | 0.06 ± 0.01 |
Lemna aequinoctialis | 2 ± 0.5 | 36.1 ± 1.9 | n.d. | 28.4 ± 1.8 | 3.8 ± 1.2 | 11.9 ± 1.6 | n.d. | 17.8 ± 3 | n.d. | n.d. | n.d. | 0.6 ± 0.1 | 0.06 ± 0.01 |
Lemna disperma | 2.7 ± 0.4 | 44 ± 12.6 | n.d. | 22.1 ± 10.2 | 7.3 ± 7.3 | 10.4 ± 1.7 | n.d. | 13.4 ± 3.1 | n.d. | n.d. | n.d. | 0.5 ± 0.1 | 0.05 ± 0.02 |
Lemna gibba | 0.3 ± 0.1 | 30.5 ± 2.3 | n.d. | 6.2 ± 0.6 | 7.1 ± 1.8 | 17 ± 1.3 | n.d. | 33 ± 2.3 | 5.9 ± 1.1 | n.d. | n.d. | 0.5 ± 0.1 | 0.06 ± 0.00 |
Lemna japonica | 1.9 ± 0.7 | 36.2 ± 1.7 | n.d. | 6.6 ± 0.6 | 3.4 ± 0.1 | 22.9 ± 0.9 | 5.5 ± 0.5 | 18.5 ± 0.5 | 5.1 ± 0.1 | n.d. | n.d. | 0.9 ± 0.0 | 0.08 ± 0.01 |
Lemna minor | 2.5 ± 0.4 | 44 ± 1.9 | n.d. | 15 ± 0.5 | 4.1 ± 1.0 | 12.5 ± 1.9 | n.d. | 22 ± 0.9 | n.d. | n.d. | n.d. | 0.3 ± 0.0 | 0.03 ± 0.01 |
Lemna minuta | 1.1 ± 0.1 | 38.4 ± 0.5 | n.d. | 11.1 ± 0.5 | 6.9 ± 0.1 | 16.3 ± 0.7 | n.d. | 26.2 ± 1.1 | n.d. | n.d. | n.d. | 0.8 ± 0.1 | 0.06 ± 0.01 |
Lemna obscura | 1.7 ± 0.2 | 29.2 ± 4.2 | n.d. | 16.1 ± 1.6 | 3.8 ± 1.2 | 13.7 ± 1.6 | n.d. | 31.5 ± 6.1 | 3.9 ± 0.9 | n.d. | n.d. | 0.7 ± 0.2 | 0.05 ± 0.00 |
Lemna perpusilla | 0.8 ± 0.0 | 35.2 ± 0.2 | n.d. | 8.4 ± 1.3 | 5.2 ± 0.5 | 23.3 ± 0.0 | n.d. | 23.8 ± 3.2 | n.d. | 1.6 ± 0.2 | 1.6 ± 0.9 | 1.4 ± 0.2 | 0.08 ± 0.01 |
Lemna tenera | 1.2 ± 0.1 | 46.7 ± 1.1 | n.d. | 17 ± 3.5 | 5.9 ± 0.1 | 12.3 ± 1.1 | n.d. | 17 ± 2.9 | n.d. | n.d. | n.d. | 0.9 ± 0.2 | 0.06 ± 0.01 |
Lemna triscula | 1.1 ± 0.1 | 20.7 ± 1.3 | n.d. | 7 ± 1.3 | 1.8 ± 0.1 | 32.7 ± 0.5 | 3.3 ± 0.7 | 33.5 ± 0.6 | n.d. | n.d. | n.d. | 0.6 ± 0.1 | 0.04 ± 0.01 |
Lemna turionefera | 1.6 ± 0.3 | 30.5 ± 2.2 | n.d. | 9.4 ± 0.2 | 2.5 ± 0.7 | 20.3 ± 2.7 | 5.8 ± 0.1 | 25.9 ± 1.4 | 4.1 ± 1.1 | n.d. | n.d. | 0.6 ± 0.1 | 0.05 ± 0.01 |
Lemna valdiviana | 1.3 ± 0.0 | 36.3 ± 2.2 | n.d. | 13 ± 2.0 | 5.4 ± 0.4 | 15.7 ± 0.9 | n.d. | 28.4 ± 2.3 | n.d. | n.d. | n.d. | 0.6 ± 0.1 | 0.05 ± 0.01 |
Spirodela intermedia | 2 ± 0.6 | 41.8 ± 2.1 | n.d. | 22.7 ± 3.1 | 3.3 ± 1.3 | 4.7 ± 1.3 | n.d. | 25.6 ± 1.7 | n.d. | n.d. | n.d. | 0.4 ± 0.1 | 0.03 ± 0.00 |
Spirodela polyrhiza | 1.6 ± 0.3 | 42.4 ± 3.9 | n.d. | 12.2 ± 4.1 | 3.7 ± 0.9 | 10.1 ± 1.0 | n.d. | 29.9 ± 0.1 | n.d. | n.d. | n.d. | 0.2 ± 0.0 | 0.02 ± 0.00 |
Wolffia angusta | 1.9 ± 0.1 | 34.2 ± 1.2 | n.d. | 14.2 ± 0.4 | 9.80.02 | 29.6 ± 0.7 | n.d. | 10.3 ± 0.8 | n.d. | n.d. | n.d. | 1.3 ± 0.2 | 0.1 ± 0.01 |
Wolffia arrhiza | 5.3 ± 0.3 | 33.9 ± 2.9 | n.d. | 17.4 ± 0.8 | 6.8 ± 2.0 | 23.7 ± 1.0 | n.d. | 12.9 ± 1.7 | n.d. | n.d. | n.d. | 0.6 ± 0.1 | 0.05 ± 0.00 |
Wolffia australiana | 2.5 ± 0.5 | 38.1 ± 5.5 | n.d. | 24.3 ± 2.7 | 7.4 ± 2.7 | 17 ± 3.7 | n.d. | 10.8 ± 1.9 | n.d. | n.d. | n.d. | 0.2 ± 0.1 | 0.02 ± 0.01 |
Wolffia borealis | 6.4 ± 0.4 | 38.5 ± 3.4 | n.d. | 34.5 ± 3.0 | n.d. | 9 ± 4.3 | n.d. | 11.7 ± 2.9 | n.d. | n.d. | n.d. | 0.3 ± 0.1 | 0.04 ± 0.02 |
Wolffia brasiliensis | 1.3 ± 0.0 | 46.1 ± 0.3 | n.d. | 32 ± 1.5 | 3.7 ± 0.0 | 8 ± 0.1 | n.d. | 8.9 ± 0.2 | n.d. | n.d. | n.d. | 0.8 ± 0.1 | 0.04 ± 0.00 |
Wolffia cylindracea | 1.1 ± 0.2 | 54.7 ± 1.0 | n.d. | 7 ± 0.7 | 4.5 ± 0.9 | 24 ± 0.8 | n.d. | 8.7 ± 1.7 | n.d. | n.d. | n.d. | 1.0 ± 0.3 | 0.07 ± 0.02 |
Wolffia globosa | 2.5 ± 0.7 | 43.8 ± 2.4 | n.d. | 25.2 ± 4.8 | 6.5 ± 6.1 | 9.3 ± 2.2 | n.d. | 12.6 ± 6.2 | n.d. | n.d. | n.d. | 0.6 ± 0.3 | 0.06 ± 0.03 |
Wolffia neglecta | 4.5 ± 0.9 | 48.5 ± 1.5 | n.d. | 29.1 ± 0.3 | 4.9 ± 1.2 | 6.6 ± 1.5 | n.d. | 6.5 ± 1.3 | n.d. | n.d. | n.d. | 0.3 ± 0.0 | 0.04 ± 0.00 |
Wolffiella caudata | 1.6 ± 0.1 | 51.2 ± 1.3 | n.d. | 9.8 ± 1.5 | 3 ± 0.3 | 17.1 ± 0.1 | n.d. | 17.3 ± 1.6 | n.d. | n.d. | n.d. | 0.7 ± 0.1 | 0.05 ± 0.01 |
Wolffiella hyalina | 2.1 ± 0.6 | 47.1 ± 3.2 | n.d. | 15.7 ± 1.4 | 9.8 ± 1.6 | 14.5 ± 2.0 | n.d. | 10.9 ± 1.4 | n.d. | n.d. | n.d. | 0.6 ± 0.1 | 0.06 ± 0.01 |
Wolffiella lingulata | 1.8 ± 0.2 | 47 ± 2.1 | n.d. | 32.3 ± 3.7 | 3.8 ± 0.7 | 5.9 ± 0.7 | n.d. | 9.2 ± 2.7 | n.d. | n.d. | n.d. | 2.7 ± 0.3 | 0.15 ± 0.02 |
Wolffiella neotropica | 1.9 ± 0.4 | 47.2 ± 2.7 | n.d. | 23.8 ± 5.0 | 5.1 ± 4.6 | 7.1 ± 3.7 | n.d. | 14.9 ± 3.7 | n.d. | n.d. | n.d. | 0.9 ± 0.1 | 0.06 ± 0.01 |
Wolffiella oblonga | 2.4 ± 0.1 | 39.3 ± 2.5 | n.d. | 24.8 ± 2.6 | 4.9 ± 3.5 | 12.7 ± 5.5 | n.d. | 15.9 ± 4.1 | n.d. | n.d. | n.d. | 1.1 ± 0.2 | 0.11 ± 0.00 |
Wolffiella repanda | 1.7 ± 0.5 | 48.8 ± 4.9 | n.d. | 11.5 ± 1.3 | 3.5 ± 1.1 | 17.1 ± 1.5 | n.d. | 17.4 ± 2.9 | n.d. | n.d. | n.d. | 1.0 ± 0.1 | 0.07 ± 0.00 |
Wolffiella welwitschii | 0.7 ± 0.3 | 52.9 ± 1.2 | n.d. | 19.3 ± 9.6 | 2.5 ± 0.6 | 14.3 ± 5.3 | n.d. | 10.3 ± 4.2 | n.d. | n.d. | n.d. | 0.9 ± 0.4 | 0.04 ± 0.01 |
Whereas nine duckweed species accumulate Δ6-containing FA, only five Lemna species (L. gibba, L. japonica, L. obscura, L. triscula, L. turionefera) accumulated them in TAG. In the three species (L. japonica, L. triscula, L. turionefera) that accumulated GLA in TAG, levels were significantly higher (student T-test, p < 0.05) by approximately 50% in TAG than in the total FA whereas SDA was significantly decreased (student T-test, p < 0.05) in TAG relative to total FA by approximately 50%. Only two of the nine species of duckweed (L. japonica, L. turionefera) contain both GLA and SDA in both their total FA and TAG (Table 5).
Discussion
This study represents the most comprehensive survey of both FA and TAG composition and content of duckweed undertaken to date with the 30 species chosen to represent the range of diversity of Lemnoideae family with respect to both morphology and geographic origins.
The FA content shows a 3-fold range; Wolffia borealis having the highest at 14.2% of dry weight with Wolffiella welwitschii at 4.6%. Total FA profiles show little variation with ALA and palmitic acid together accounting for >60%. All the surveyed species have similar FA compositions within their TAG in which > 50% of the FA is composed of palmitic acid plus ALA. Stearic acid represents >10% of FA in the TAG in 22 of the species surveyed.
While the total FA compositions and the TAG FA compositions are similar across the Lemnoideae, the total FA composition differs from that of TAG. The level of saturated FA in TAG is approximately twice that of the total FA pool, with ALA decreasing to compensate; specifically stearic acid comprises <7% of the total FA, but represents 10-34% of TAG in 22 of the duckweed species. The Δ6-double bond–containing FA SDA follows the same pattern, its levels being 51% lower in TAG relative to the total FA pool, whereas GLA, like saturated FAs, is a preferred substrate for TAG incorporation in the three Lemna species, japonica, triscula and turionefera, with its levels increasing by 50% in TAG relative to the total FA pool. The increase in saturates and GLA in TAG relative to the total FA pool implies that these three duckweed species contain acyltransferases with preference for saturated FA and GLA. An alternate explanation is that each duckweed species has several acyl transferases that show differential selectivity with respect to saturated (and GLA), and polyunsaturated FA substrates which are expressed at different levels in different duckweed species. These results provide information that may be helpful to identify duckweed species that contain TAG biased towards desired FA compositions.
The fronds of duckweeds have much lower oil content than the seeds of soybean, rapeseed, cottonseed and peanut, the world’s major oil crops. Wolffiella lingulata exhibits the highest oil content at 0.14% of total dry mass, which is 200-fold lower than that of soybean seed [32] and 400 times lower than that of sunflower seed [33]. This large difference is mainly due to the fact that duckweed harvested for this study comprises vegetative tissue, and a more appropriate comparison would be to that of other vegetative tissues. For instance, Arabidopsis leaves contain approximately 0.06% of dry mass as TAG [8], crabapple leaves contains approximately 0.15% TAG and L. serriola leaves contain around 0.5% TAG [34]. Thus, Wolffiella lingulata’s TAG content of 0.15% falls within the range reported for terrestrial vegetative tissues.
Scatter plot of TAG per DW as a function of total FA per DW.
Lipid composition is relatively uniform across all the surveyed duckweeds, with the exception of the presence of Δ6-FA in nine duckweed species. GLA and SDA are uncommon, having being found only in a small number of higher plants [38], including borage [13] and Echium[14]. Our finding that the Lemna gibba Δ6 desaturase LgDes showed stronger homology to a tobacco Δ8-shingolipid desaturase [17] than to the borage Δ6 desaturase, which was used as a homology probe to identify LgDes, supports the observation that Δ6-FA desaturases and Δ8-LCB sphingolipid desaturases are closely related and likely have evolved from each other a number of times during evolution [15]. It is interesting in this regard that expression experiments using closely related Δ6-FA and Δ8-LCB desaturases in yeast showed them to have non-overlapping functions [39]. The availability of both substrates in yeast suggests that the context of the acyl chain as a FA or LCB does not influence regioselectivity for these enzymes as it does for a family of ADS enzymes in which the nature of the head group determines whether a Δ7 or Δ9 double bond is introduced to the 16:0 FA substrate [40]. It would be interesting to determine whether duckweed species that lack detectable Δ6-FA also lack the Δ6-desaturase gene, or whether it is present but not expressed under our growth conditions. The traditional approach of southern or northern blotting is not useful in this case because of the generally high sequence similarity between Δ6-FA and Δ8-LCB desaturase gene sequences (Figures 3 and 4 and [15]). We therefore searched the unpublished genomes of a Δ6-FA-containing species Lemna gibba, and the Δ6-FA-lacking species, Spirodela polyrhiza for sequences homologous to LgDes. One putative desaturase was identified in each genome, which showed higher homology to each other than to LgDes, suggesting they encode Δ8-LCB desaturases. Our search therefore failed to provide evidence for a Spirodela polyrhiza Δ6-FA desaturase with close homology to LgDes.
In an attempt to identify locations that might help explain the difference in enzyme specificity between Δ6-FA and six Δ8-LCB desaturases we performed conserved property difference locator (CPDL) analysis [41] using six functionally enzymes from each class (Additional file 1: CPDL analysis of six D6 and six D8 desaturases). Many amino acid locations were identified as potential specificity determining residues however, only one location (at amino acid 210), showed a consistent property difference between the Δ6-FA and Δ8-LCB class of desaturases (see Figure 3, vertical red arrow). The position occupied by the neutral amino acids Leu210, in LgDes, and Tyr in the other five Δ6 desaturases is occupied by a charged His residue in all six Δ8 desaturase sequences. The hypothesis that the nature of the amino acid at position 210 along with other factor(s) that lie between amino acids 80 and 350 specify Δ6 FA or Δ8 LCB activity is consistent with the available borage chimera data [39].
Conclusions
The survey of 30 species duckweeds showed total FA content varied approximately 3-fold between 4.6% and 14.2% of dry weight; however, there is surprisingly low variability in FA composition between the 30 duckweed species surveyed, with three FA, palmitic, LN and ALA comprising more than 80% of total duckweed FA. However, the Δ6-FA GLA and/or SDA were identified in seven Lemna and two Wolffiela species. LgDes, a desaturase, with homology to a family of front-end Δ6-FA and Δ8-spingolipid desaturases, comprising a cytochrome b5-desaturase fusion, was isolated from Lemna gibba and functionally confirmed as a Δ6-FA desaturase by expression in Nicotiana benthamiana. In duckweed, TAG enriched in saturated FA at the expense of polyunsaturated FA accumulates at up to 0.15% of total FA, i.e., at levels comparable to those found in the leaves of terrestrial plants. Δ6-FA occur in the TAG of only five Lemna species, within which GLA is enriched, and SDA diminished, relative to their abundance in the total FA pool.
Declarations
Acknowledgements
This work was supported by the Office of Basic Energy Sciences of the U.S. Department of Energy (JS and JS), and DOE EERE to JS, JS, YY, EE, RM and HS. We thank Dr. F. W. Studier and Dr. Xiao-Hong Yu for critical reading of the manuscript and Dr. Sean McCorkle for technical assistance.
Authors’ Affiliations
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