Quantitative profiling and pattern analysis of triacylglycerol species in Arabidopsis seeds by electrospray ionization mass spectrometry

Plant triacylglycerols (TAGs), or vegetable oils, provide approximately 25% of dietary calories to humans and are becoming an increasingly important source of renewable bioenergy and industrial feedstocks. TAGs are assembled by multiple enzymes in the endoplasmic reticulum from building blocks that include an invariable glycerol backbone and variable fatty acyl chains. It remains a challenge to elucidate the mechanism of synthesis of hundreds of different TAG species in planta. One reason is the lack of an efficient analytical approach quantifying individual molecular species. Here we report a rapid and quantitative TAG profiling approach for Arabidopsis seeds based on electrospray ionization tandem mass spectrometry with direct infusion and multiple neutral loss scans. The levels of 93 TAG molecular species, identified by their acyl components, were determined in Arabidopsis seeds. Quantitative TAG pattern analyses revealed that the TAG assembly machinery preferentially produces TAGs with one elongated fatty acid. The importance of the selectivity in oil synthesis was consistent with an observation that an Arabidopsis mutant overexpressing a patatin‐like phospholipase had enhanced seed oil content with elongated fatty acids. This quantitative TAG profiling approach should facilitate investigations aimed at understanding the biochemical mechanisms of TAG metabolism in plants.     

Enhanced seed viability and lipid compositional changes during natural ageing by suppressing phospholipase Dα in soybean seed

Changes in phospholipid composition and consequent loss of membrane integrity are correlated with loss of seed viability. Furthermore, phospholipid compositional changes affect the composition of the triacylglycerols (TAG), i.e. the storage lipids. Phospholipase D (PLD) catalyses the hydrolysis of phospholipids to phosphatidic acid, and PLDα is an abundant PLD isoform. Although wild‐type (WT) seeds stored for 33 months were non‐viable, 30%–50% of PLDα‐knockdown (PLD‐KD) soybean seeds stored for 33 months germinated. WT and PLD‐KD seeds increased in lysophospholipid levels and in TAG fatty acid unsaturation during ageing, but the levels of lysophospholipids increased more in WT than in PLD‐KD seeds. The loss of viability of WT seeds was correlated with alterations in ultrastructure, including detachment of the plasma membrane from the cell wall complex and disorganization of oil bodies. The data demonstrate that, during natural ageing, PLDα affects the soybean phospholipid profile and the TAG profile. Suppression of PLD activity in soybean seed has potential for improving seed quality during long‐term storage.     

The TAG1 locus of Arabidopsis encodes for a diacylglycerol acyltransferase

Diacylglycerol acyltransferase (DGAT, EC 2.3.1.20) is a membrane enzyme that drives the final step in the formation of oils using diacylglycerol (DAG) and acyl-CoA to yield triacylglycerol (TAG). We identified a putative plant DGAT gene (TRIACYLGLYCEROL1: TAG1) and demonstrated its function by the cloning of two mutated alleles, designated AS11 (tag1-1) and ABX45 (tag1-2). One allele, AS11, has been previously characterised at the biochemical level. Mutant seeds contained less oil with a modified fatty acid profile and have reduced germination rates compared to wild-type controls. The TAG1 cDNA encodes for a 520-aa protein that possesses multiple putative transmembrane domains and shows 70 % similarity to a human DGAT cDNA.     

Oleate from triacylglycerols is desaturated in cold-induced developing sunflower (Helianthus annuus L.) seeds

For the first time, an active fatty-acid metabolism is indicated for triacylglycerols (TAG) of developing sunflower (Helianthus annuus L.) seeds. When the developing seeds were transferred to low temperature, the total amount of oleate found in TAG decreased as that of linoleate increased, while the contents of total lipids and TAG remained unchanged. These results suggest that oleate from TAG was used for desaturation. This occurred first in microsomal TAG, but after a long cold period it was observed mainly in the oil-body fraction. Thesn-2 position of TAG was preferentially enriched in linoleate. Apparently, more linoleate than necesary for the maintenance of membrane fluidity was synthesized at the expense of TAG oleate.     

Induction of accumulation and degradation of the 18.4-kDa oleosin in a triacylglycerol-storing cell culture of anise (Pimpinella anisum L.)

Two closely related anise cell-culture lines, Pa15 and Pa19, differ considerably in growth rate, potential to form somatic embryoids, triacylglycerol (TAG) storage and pattern of lipid-body proteins. Line Pa15 grows very fast (doubling rate: 3 d), mainly as single cells, exhibits a low potential for somatic embryogenesis and its TAG content is relatively low (5–20 mg TAG per g dry weight). In contrast, the line Pa19 shows lower growth rates (doubling rate: 8 d), tends to form clusters of somatic cells, has a higher TAG content (100–150 mg TAG per g dry weight), and somatic embryoids are easily induced. Under defined culture conditions, the TAG content of the line Pa19 can be increased to approximately 70% of that of ripe anise seeds (150 and 220 mg TAG per g dry weight, respectively). Polyclonal antibodies prepared against the most abundant protein (relative molecular mass 18.4 kDa) from the lipid-body fraction of anise seeds (Radetzky et al. 1993, Planta 191, 166–172) react also with a 18.4-kDa protein from the lipid-body fraction of cells of the Pa19 culture. In contrast, only fairly low levels of the 18.4-kDa oleosin were detected in Pal5. Limited sucrose supply in the medium resulted in TAG degradation and the concomitant decrease in the amount of immunodetectible 18.4-kDa protein in the Pa19 cell culture. Treatment with sorbitol, or abscisic acid and sorbitol in combination, enhanced TAG contents and also the amount of immunostained 18.4-kDa protein in the cell culture Pa19, whereas no effect was found on either TAG content or 18.4-kDa protein in the cell-culture line Pa15. The 18.4-kDa protein can be classified as an oleosin, a proposal which is supported by the similarity in molecular mass compared with other known oleosins, its occurrence in the lipid-body fraction and the fact that its amount correlates with the TAG content. The results of this study indicate that the Pa19 cell culture provides a valid model system for investigations of lipid storage and mobilization in higher-plant cells.Abbreviations ABA cis-abscisic acid - TAG triacylglycerol(s) - 2,4-D 2,4-dichlorophenoxyacetic acid The authors thank Christiane Bernshausen for kind technical assistance.     

Reactive extraction of acylglycerides using a column bioreactor containing Rhizopus oryzae resting-cells

The work evaluated the use of a bioreactor for the enzymatic transesterification of oilseed. Rhizopus oryzae resting-cells, isolated from Foeniculum vulgare Fennel, were used as a biocatalyst. The bioreactor consisted of two glass-jacketed flasks, interconnected by a circuit using isooctane as a solvent, which was recirculated with the help of a peristaltic pump. The system enabled the extraction and hydrolysis of triacylglycerides (TAG), and the subsequent esterification of the free fatty acids formed (FFA). Esterification was carried out by the addition of alcohol using a peristaltic pump. This reactive extraction of vegetable oil was optimized for sunflower seeds using 4-hydroxymethyl-2,2-dimethyl-1,3-dioxolane (solketal) as alcohol. Subsequently, a variety of oil seeds and alcohols were tested. Final ester conversions ranged from 84 to 98% depending on the alcohol and the oilseed used.     

The phosphatidylcholine diacylglycerol cholinephosphotransferase is required for efficient hydroxy fatty acid accumulation in transgenic Arabidopsis

We previously identified an enzyme, phosphatidylcholine diacylglycerol cholinephosphotransferase (PDCT), that plays an important role in directing fatty acyl fluxes during triacylglycerol (TAG) biosynthesis. The PDCT mediates a symmetrical interconversion between phosphatidylcholine (PC) and diacylglycerol (DAG), thus enriching PC-modified fatty acids in the DAG pool prior to forming TAG. We show here that PDCT is required for the efficient metabolism of engineered hydroxy fatty acids in Arabidopsis (Arabidopsis thaliana) seeds. When a fatty acid hydroxylase (FAH12) from castor (Ricinus communis) was expressed in Arabidopsis seeds, the PDCT-deficient mutant accumulated only about half the amount of hydroxy fatty acids compared with that in the wild-type seeds. We also isolated a PDCT from castor encoded by the RcROD1 (Reduced Oleate Desaturation1) gene. Seed-specific coexpression of this enzyme significantly increased hydroxy fatty acid accumulation in wild type-FAH12 and in a previously produced transgenic Arabidopsis line coexpressing a castor diacylglycerol acyltransferase 2. Analyzing the TAG molecular species and regiochemistry, along with analysis of fatty acid composition in TAG and PC during seed development, indicate that PDCT acts in planta to enhance the fluxes of fatty acids through PC and enrich the hydroxy fatty acids in DAG, and thus in TAG. In addition, PDCT partially restores the oil content that is decreased in FAH12-expressing seeds. Our results add a new gene in the genetic toolbox for efficiently engineering unusual fatty acids in transgenic oilseeds.     

Conjugated fatty acids accumulate to high levels in phospholipids of metabolically engineered soybean and Arabidopsis seeds

Expression of Delta(12)-oleic acid desaturase-related fatty acid conjugases from Calendula officinalis, Momordica charantia, and Vernicia fordii in seeds of soybean (Glycine max) or an Arabidopsis thaliana fad3/fae1 mutant was accompanied by the accumulation of the conjugated fatty acids calendic acid or alpha-eleostearic acid to amounts as high as 20% of the total fatty acids. Conjugated fatty acids, which are synthesized from phosphatidylcholine (PC)-linked substrates, accumulated in PC and phosphatidylethanolamine, and relative amounts of these fatty acids were higher in PC than in triacylglycerol (TAG) in the transgenic seeds. The highest relative amounts of conjugated fatty acids were detected in PC from seeds of soybean and A. thaliana that expressed the C. officinalis and M. charantia conjugases, where they accounted for nearly 25% of the fatty acids of this lipid class. In these seeds, >85% of the conjugated fatty acids in PC were detected in the sn-2 position, and these fatty acids were also enriched in the sn-2 position of TAG. In marked contrast to the transgenic seeds, conjugated fatty acids composed <1.5% of the fatty acids in PC from seeds of five unrelated species that naturally synthesize a variety of conjugated fatty acid isomers, including seeds that accumulate conjugated fatty acids to >80% of the total fatty acids. These results suggest that soybean and A. thaliana seeds are deficient in their metabolic capacity to selectively catalyze the flux of conjugated fatty acids from their site of synthesis on PC to storage in TAG.     

Characterization of desnutrin functional domains: critical residues for triacylglycerol hydrolysis in cultured cells

Murine desnutrin/human ATGL is a triacylglycerol (TAG) hydrolase with a predicted catalytic dyad within an α-β hydrolase fold in the N-terminal region. In humans, mutations resulting in C-terminal truncation cause neutral lipid storage disease with myopathy. To identify critical functional domains, we measured TAG breakdown in cultured cells by mutated or truncated desnutrin. In vitro, C-terminally truncated desnutrin displayed an even higher apparent V_max than the full-length form without changes in K_m, which may be explained by our finding of an interaction between the C- and N-terminal domains. In live cells, however, C-terminally truncated adenoviral desnutrin had lower TAG hydrolase activity. We investigated a role for the phosphorylation of C-terminal S406 and S430 residues but found that these were not necessary for TAG breakdown or lipid droplet localization in cells. The predicted N-terminal active sites, S47 and D166, were both critical for TAG hydrolysis in live cells and in vitro. We also identified two overlapping N-terminal motifs that predict lipid substrate binding domains, a glycine-rich motif (underlined) and an amphipathic α-helix (bold) within amino acid residues 10–24 (ISFAGCGFLGVYHIG). G14, F17, L18, and V20, but not G16 and G19, were important for TAG hydrolysis, suggesting a potential role for the amphipathic α-helix in TAG binding. This study identifies for the first time critical sites in the N-terminal region of desnutrin and reveals the requirement of the C-terminal region for TAG hydrolysis in cultured cells.     

Developmental Regulation of Diacylglycerol Acyltransferase Family Gene Expression in Tung Tree Tissues

Diacylglycerol acyltransferases (DGAT) catalyze the final and rate-limiting step of triacylglycerol (TAG) biosynthesis in eukaryotic organisms. DGAT genes have been identified in numerous organisms. Multiple isoforms of DGAT are present in eukaryotes. We previously cloned DGAT1 and DGAT2 genes of tung tree (Vernicia fordii), whose novel seed TAGs are useful in a wide range of industrial applications. The objective of this study was to understand the developmental regulation of DGAT family gene expression in tung tree. To this end, we first cloned a tung tree gene encoding DGAT3, a putatively soluble form of DGAT that possesses 11 completely conserved amino acid residues shared among 27 DGAT3s from 19 plant species. Unlike DGAT1 and DGAT2 subfamilies, DGAT3 is absent from animals. We then used TaqMan and SYBR Green quantitative real-time PCR, along with northern and western blotting, to study the expression patterns of the three DGAT genes in tung tree tissues. Expression results demonstrate that 1) all three isoforms of DGAT genes are expressed in developing seeds, leaves and flowers; 2) DGAT2 is the major DGAT mRNA in tung seeds, whose expression profile is well-coordinated with the oil profile in developing tung seeds; and 3) DGAT3 is the major form of DGAT mRNA in tung leaves, flowers and immature seeds prior to active tung oil biosynthesis. These results suggest that DGAT2 is probably the major TAG biosynthetic isoform in tung seeds and that DGAT3 gene likely plays a significant role in TAG metabolism in other tissues. Therefore, DGAT2 should be a primary target for tung oil engineering in transgenic organisms.     

Endoplasmic reticulum-located PDAT1-2 from castor bean enhances hydroxy fatty acid accumulation in transgenic plants

Ricinoleic acid (12-hydroxy-octadeca-9-enoic acid) is a major unusual fatty acid in castor oil. This hydroxy fatty acid is useful in industrial materials. This unusual fatty acid accumulates in triacylglycerol (TAG) in the seeds of the castor bean (Ricinus communis L.), even though it is synthesized in phospholipids, which indicates that the castor plant has an editing enzyme, which functions as a phospholipid:diacylglycerol acyltransferase (PDAT) that is specific to ricinoleic acid. Transgenic plants containing fatty acid Δ12-hydroxylase encoded by the castor bean FAH12 gene produce a limited amount of hydroxy fatty acid, a maximum of around 17% of TAGs present in Arabidopsis seeds, and this unusual fatty acid remains in phospholipids of cell membranes in seeds. Identification of ricinoleate-specific PDAT from castor bean and manipulation of the phospholipid editing system in transgenic plants will enhance accumulation of the hydroxy fatty acid in transgenic seeds. The castor plant has three PDAT genes; PDAT1-1 and PDAT2 are homologs of PDAT, which are commonly found in plants; however, PDAT1-2 is newly grouped as a castor bean-specific gene. PDAT1-2 is expressed in developing seeds and localized in the endoplasmic reticulum, similar to FAH12, indicating its involvement in conversion of ricinoleic acid into TAG. PDAT1-2 significantly enhances accumulation of total hydroxy fatty acid up to 25%, with a significant increase in castor-like oil, 2-OH TAG, in seeds of transgenic Arabidopsis, which is an identification of the key gene for oilseed engineering in production of unusual fatty acids.     

Insertional mutant analysis reveals that long-chain acyl-CoA synthetase 1 (LACS1), but not LACS8, functionally overlaps with LACS9 in Arabidopsis seed oil biosynthesis

Triacylglycerols (TAGs) are major storage materials that accumulate in developing seeds and serve as carbon and energy reserves for germination and growth of the seedling. One of the critical reactions in TAG biosynthesis is activation of fatty acyl chains to fatty acyl CoAs, catalyzed by long-chain acyl CoA synthetases (LACSs). Of the nine LACSs identified in Arabidopsis, only LACS9 is known to reside in the plastid, the site of de novo fatty acid synthesis, and is considered the major LACS isoform involved in plastidial fatty acid export for TAG formation. Because the lacs9 null mutant did not show any detectable phenotype, it was hypothesized that at least one additional LACS enzyme must be active in the plastid. Expression analyses to identify potential plastid-localized LACSs involved in TAG biosynthesis revealed that, in addition to LACS9, isoforms LACS1, LACS2, LACS4 and LACS8 are transcribed in the seed. LACS8 showed the highest expression level in the embryo and a high sequence similarity with LACS9, and was therefore characterized further and shown to be associated with the ER, not the plastid. Furthermore, disruption of LACS8 in the lacs8 mutant and lacs8 lacs9 double mutant, and over-expression of LACS8, did not affect the seed fatty acid content. In contrast, 11 and 12% decreases in fatty acid content were detected in lacs1 lacs9 and lacs1 lacs8 lacs9 seeds, respectively, indicating that LACS1 and LACS9 have overlapping functions in TAG biosynthesis. This result is surprising because, unlike LACS9, LACS1 is localized in the ER and has been shown to be involved in cuticular lipid synthesis.     

Seed storage oil mobilization is important but not essential for germination or seedling establishment in Arabidopsis

Triacylglycerol (TAG) is a major storage reserve in many plant seeds. We previously identified a TAG lipase mutant called sugar-dependent1 (sdp1) that is impaired in TAG hydrolysis following Arabidopsis (Arabidopsis thaliana) seed germination (Eastmond, 2006). The aim of this study was to identify additional lipases that account for the residual TAG hydrolysis observed in sdp1. Mutants were isolated in three candidate genes (SDP1-LIKE [SDP1L], ADIPOSE TRIGLYCERIDE LIPASE-LIKE, and COMPARATIVE GENE IDENTIFIER-58-LIKE). Analysis of double, triple, and quadruple mutants showed that SDP1L is responsible for virtually all of the residual TAG hydrolysis present in sdp1 seedlings. Oil body membranes purified from sdp1 sdp1L seedlings were deficient in TAG lipase activity but could still hydrolyze di- and monoacylglycerol. SDP1L is expressed less strongly than SDP1 in seedlings. However, SDP1L could partially rescue TAG breakdown in sdp1 seedlings when expressed under the control of the SDP1 or 35S promoters and in vitro assays showed that both SDP1 and SDP1L can hydrolyze TAG, in preference to diacylglycerol or monoacylglycerol. Seed germination was slowed in sdp1 sdp1L and postgerminative seedling growth was severely retarded. The frequency of seedling establishment was also reduced, but sdp1 sdp1L was not seedling lethal under normal laboratory growth conditions. Our data show that together SDP1 and SDP1L account for at least 95% of the rate of TAG hydrolysis in Arabidopsis seeds, and that this hydrolysis is important but not essential for seed germination or seedling establishment.     

Crystallization and preliminary X-ray diffraction study of a chimaeric Fab' fragment of antibody binding tumour cells

Crystals have been obtained of a chimaeric Fab' fragment that binds to a tumour-associated mucin-like glycoprotein TAG72. The Fab' fragment comprises the variable heavy and light-chain domains of a murine monoclonal antibody, B72.3, coupled to human gamma 4 and kappa constant regions. The crystals are orthorhombic and belong to the space group P2(1)2(1)2(1), with unit cell dimensions a = 67.9 A, b = 94.2 A and c = 208.8 A. Diffraction to 2.6 A resolution was observed using synchrotron radiation. Despite the acute radiation sensitivity of the crystals a full native data set has been collected using the Weissenberg camera at the Photon Factory synchrotron. These data will be used for molecular replacement calculations in an attempt to elucidate the structure of this chimaeric Fab' fragment.     

Lipid profiling of developing Jatropha curcas L. seeds using (1)H NMR spectroscopy

Seed development in Jatropha curcas L. was studied with respect to phenology, oil content, lipid profile and concentration of sterols. Seeds were collected at various stages of development starting from one week after fertilization and in an interval of five days thereafter till maturity. These were classified as stage I to stage VII. Moisture content of the seeds ranged from 8.8 to 90.3%; the lowest in mature seeds in stage VII and highest in stage I. The seed area increased as the seed grew from stage I to stage VI (0.2-10.2mm(2) per seed), however, the seed area shrunk at stage VII. Increase in seed area corresponded to increase in fresh weight of the seeds. (1)H NMR spectroscopy of hexane extracts made at different stages of seed development revealed the presence of free fatty acids (FFA), methyl esters of fatty acids (FAME) and triglycerol esters (TAG), along with small quantity of sterols. The young seeds synthesized predominantly polar lipids. Lipid synthesis was noticed nearly three weeks after fertilization. From the fourth week the seeds actively synthesized TAG. Stage III is a turning point in seed development since at this stage, the concentration of sterols decreased to negligible, there was very little FAME formation, accumulation of TAG increased substantially, and there was a sudden decrease in FFA concentration. The findings can be helpful in understanding the biosynthesis and in efforts to improve biosynthesis of TAG and reduce FFA content in the mature seeds.     

Expression pattern of diacylglycerol acyltransferase-1, an enzyme involved in triacylglycerol biosynthesis,in Arabidopsis thaliana

Triacylglycerol (TAG) is the major carbon storage reserve in oilseeds such as Arabidopsis. Acyl-CoA:diacylglycerol acyltransferase (DGAT) catalyses the final step of the TAG synthesis pathway. Although TAG is mainly accumulated during seed development, and DGAT has presumably the highest activity in developing seeds, we show here that TAG synthesis is also actively taking place during germination and seedling development in Arabidopsis. The expression pattern of the DGAT1 gene was studied in transgenic plants containing the reporter gene -glucuronidase (GUS) fused with DNA sequences flanking the DGAT1coding region. GUS activity was not only detected in developing seeds and pollen, which normally accumulate storage TAG, but also in germinating seeds and seedlings. Western blots showed that DGAT1 protein is present in several tissues, though is most abundant in developing seeds. In seedlings, DGAT1 is expressed in shoot and root apical regions, correlating with rapid cell division and growth. The expression of GUS in seedlings was consistent with the results of RNA gel blot analyses, precursor feeding and DGAT assay. In addition, DGAT1gene expression is up-regulated by glucose and associated with glucose-induced changes in seedling development.