Nitrogen deficiency system is helpful in characterizing regulation mechanisms of ectopic triacylglycerol accumulation in Arabidopsis seedlings

Triacylglycerol (TAG) is the major storage component accumulated in seed. However the regulatory mechanism of TAG synthesis and accumulation in non-seed tissues remains unknown. Recently, we found that nitrogen (N) deficiency (0.1mM N) caused an inducement of TAG biosynthesis in Arabidopsis seedlings. ABSCISIC ACID INSENSITIVE 4 (ABI4) was essential for the activation of Acyl-CoA:diacylglycerol acyltransferase1(DGAT1) expression during N deficiency in Arabidopsis seedlings. In this addendum, we further discussed the approaches to provide a net increase in total oil production in higher plants by using the low N platform. First, the N-deficient seedlings can be used to determine the key factors that regulate the ectopic expression of key genes in TAG metabolism. Second, the research on the relationship between TAG homeostasis and cell division will be helpful to find the key factors that specifically regulate TAG accumulation under the nutrient-limited condition.     

Synthesis of Novel Lipids in Saccharomyces cerevisiae by Heterologous Expression of an Unspecific Bacterial Acyltransferase

The bifunctional wax ester synthase/acyl-coenzyme A:diacylglycerol acyltransferase (WS/DGAT) is the key enzyme in storage lipid accumulation in the gram-negative bacterium Acinetobacter calcoaceticus ADP1, mediating wax ester, and to a lesser extent, triacylglycerol (TAG) biosynthesis. Saccharomyces cerevisiae accumulates TAGs and steryl esters as storage lipids. Four genes encoding a DGAT (Dga1p), a phospholipid:diacylglycerol acyltransferase (Lro1p) and two acyl-coenzyme A:sterol acyltransferases (ASATs) (Are1p and Are2p) are involved in the final esterification steps in TAG and steryl ester biosynthesis in this yeast. In the quadruple mutant strain S. cerevisiae H1246, the disruption of DGA1, LRO1, ARE1, and ARE2 leads to an inability to synthesize storage lipids. Heterologous expression of WS/DGAT from A. calcoaceticus ADP1 in S. cerevisiae H1246 restored TAG but not steryl ester biosynthesis, although high levels of ASAT activity could be demonstrated for WS/DGAT expressed in Escherichia coli XL1-Blue in radiometric in vitro assays with cholesterol and ergosterol as substrates. In addition to TAG synthesis, heterologous expression of WS/DGAT in S. cerevisiae H1246 resulted also in the accumulation of fatty acid ethyl esters as well as fatty acid isoamyl esters. In vitro studies confirmed that WS/DGAT is capable of utilizing a broad range of alcohols as substrates comprising long-chain fatty alcohols like hexadecanol as well as short-chain alcohols like ethanol or isoamyl alcohol. This study demonstrated the highly unspecific acyltransferase activity of WS/DGAT from A. calcoaceticus ADP1, indicating the broad biocatalytic potential of this enzyme for biotechnological production of a large variety of lipids in vivo in prokaryotic as well as eukaryotic expression hosts.     

ABA-insensitive (ABI) 4 and ABI5 synergistically regulate DGAT1 expression in Arabidopsis seedlings under stress

Triacylglycerol (TAG) accumulation is essential for seed maturation in plants. Diacylglycerol acyltransferase 1 (DGAT1) is the rate-limiting enzyme in TAG biosynthesis. In this study, we show that TAG accumulation in Arabidopsis seedlings is correlated with environmental stress, and both ABI4 and ABI5 play important roles in regulating DGAT1 expression. Tobacco transient assays revealed the synergistic effect of ABI4 with ABI5 in regulating DGAT1 expression. Taken together, our findings indicate ABI5 is an important accessory factor with ABI4 in the activation of DGAT1 in Arabidopsis seedlings under stress.     

The atf2 gene is involved in triacylglycerol biosynthesis and accumulation in the oleaginous Rhodococcus opacus PD630

Rhodococcus opacus PD630 is an oleaginous bacterium able to accumulate large amounts of triacylglycerols (TAG) in different carbon sources. The last reaction for TAG biosynthesis is catalyzed by the bifunctional wax ester synthase/acyl-CoA:diacylglycerol acyltransferase (WS/DGAT) enzymes encoded by atf genes. R. opacus PD630 possesses at least 17 putative atf homologous genes in its genome, but only atf1 and atf2 exhibited a significant DGAT activity when expressed in E. coli, as revealed in a previous study. The contribution of atf1 gene to TAG accumulation by strain PD630 has been demonstrated previously, although additional Atfs may also contribute to lipid accumulation, since the atf1-disrupted mutant is still able to produce significant amounts of TAG (Alvarez et al., Microbiology 154:2327–2335, 2008). In this study, we investigated the in vivo role of atf2 gene in TAG accumulation by R. opacus PD630 by using different genetic strategies. The atf2-disrupted mutant exhibited a decrease in TAG accumulation (up to 25–30 %, w/w) and an approximately tenfold increase in glycogen formation in comparison with the wild-type strain. Surprisingly, in contrast to single mutants, a double mutant generated by the disruption of atf1 and atf2 genes only showed a very low effect in TAG and in glycogen accumulation under lipid storage conditions. Overexpression of atf1 and atf2 genes in strain PD630 promoted an increase of approximately 10 % (w/w) in TAG accumulation, while heterologous expression of atf2 gene in Mycobacterium smegmatis caused an increase in TAG accumulation during cultivation in nitrogen-rich media. This study demonstrated that, in addition to atf1 gene, atf2 is actively involved in TAG accumulation by the oleaginous R. opacus PD630.     

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.     

Arabidopsis mutants deficient in diacylglycerol acyltransferase display increased sensitivity to abscisic acid,sugars, and osmotic stress during germination and seedling development

Arabidopsis seeds store triacylglycerol (TAG) as the major carbon reserve, which is used to support postgerminative seedling growth. Diacylglycerol acyltransferase (DGAT) catalyzes the final step in TAG synthesis, and two isoforms of DGAT have previously been identified in Arabidopsis. It has been shown that DGAT1 plays an important role in seed development because Arabidopsis with mutations at the TAG1 locus accumulate less seed oil. There is also evidence showing that DGAT1 is active after seed germination. The aim of this study is to investigate the effect of mutations of DGAT1 on postembryonic development in Arabidopsis. We carried out detailed analyses of two tag1 mutants in different ecotypic backgrounds of Arabidopsis. Results show that during germination and seedling growth, seed storage TAG degradation was not affected in the tag1 mutants. However, sugar content of the mutant seedlings is altered, and activities of the hexokinases are significantly increased in the tag1 mutant seedlings. The tag1 mutants are also more sensitive to abscisic acid, glucose, and osmotic strength of the medium in germination and seedling growth.     

Synthesis of novel lipids in Saccharomyces cerevisiae by heterologous expression of an unspecific bacterial acyltransferase

The bifunctional wax ester synthase/acyl-coenzyme A:diacylglycerol acyltransferase (WS/DGAT) is the key enzyme in storage lipid accumulation in the gram-negative bacterium Acinetobacter calcoaceticus ADP1, mediating wax ester, and to a lesser extent, triacylglycerol (TAG) biosynthesis. Saccharomyces cerevisiae accumulates TAGs and steryl esters as storage lipids. Four genes encoding a DGAT (Dga1p), a phospholipid:diacylglycerol acyltransferase (Lro1p) and two acyl-coenzyme A:sterol acyltransferases (ASATs) (Are1p and Are2p) are involved in the final esterification steps in TAG and steryl ester biosynthesis in this yeast. In the quadruple mutant strain S. cerevisiae H1246, the disruption of DGA1, LRO1, ARE1, and ARE2 leads to an inability to synthesize storage lipids. Heterologous expression of WS/DGAT from A. calcoaceticus ADP1 in S. cerevisiae H1246 restored TAG but not steryl ester biosynthesis, although high levels of ASAT activity could be demonstrated for WS/DGAT expressed in Escherichia coli XL1-Blue in radiometric in vitro assays with cholesterol and ergosterol as substrates. In addition to TAG synthesis, heterologous expression of WS/DGAT in S. cerevisiae H1246 resulted also in the accumulation of fatty acid ethyl esters as well as fatty acid isoamyl esters. In vitro studies confirmed that WS/DGAT is capable of utilizing a broad range of alcohols as substrates comprising long-chain fatty alcohols like hexadecanol as well as short-chain alcohols like ethanol or isoamyl alcohol. This study demonstrated the highly unspecific acyltransferase activity of WS/DGAT from A. calcoaceticus ADP1, indicating the broad biocatalytic potential of this enzyme for biotechnological production of a large variety of lipids in vivo in prokaryotic as well as eukaryotic expression hosts.     

DGAT1 from the arachidonic-acid-producing microalga <Emphasis Type="Italic">Lobosphaera incisa</Emphasis> shows late gene expression under nitrogen starvation and substrate promiscuity in a heterologous system

We report the identification and characterization of an acyl-CoA:diacylglycerol acyltransferase 1 (DGAT1)-encoding gene from the green oleaginous microalga Lobosphaera incisa (SAG 2468), a prolific photosynthetic producer of the n-6 very long chain polyunsaturated fatty acid (VLC-PUFA), arachidonic acid. The gene expression pattern of LiDGAT1 in L. incisa cells showed a weak increase in mRNA abundance in the course of nitrogen starvation under low light; however, LiDGAT1 expression was significantly upregulated with the progression of N-starvation under high light. Heterologous expression of LiDGAT1 in the neutral lipid-deficient mutant H1246 of Saccharomyces cerevisiae complemented the mutant phenotype and demonstrated an excelling TAG production compared to the yeast endogenous DGAT gene (DGA1). The TAG that formed in the LiDGAT1-expressing H1246 cells contained higher proportions of C16:0 and C18:0 fatty acids, suggesting that at least in a heterologous system, lacking PUFA biosynthesis, the enzyme seems to favor saturated over monounsaturated fatty acids. LiDGAT1 expression prompted an incorporation of several tested exogenous C18 PUFA and C20 VLC-PUFA into TAG. LiDGAT1-driven activity mediated the incorporation of either n-3 or n-6 VLC-PUFA, supplied as substrates for the TAG assembly; however, somewhat of a preference for 18:3n-3 over 20:4n-6 was demonstrated by lipidomics analysis. A structure-functional analysis of LiDGAT1 revealed that the N-terminal Pleckstrin homology (PH) domain is important but not essential for TAG generation in the yeast expression system. Deletion of the PH domain led to decreased TAG formation and ARA incorporation into TAG in yeast. Remarkably, we found the PH domain to be present in the DGAT1 of a number of chlorophytes, in a charophyceaen multicellular alga, in two diatoms and in the liverwort Marchantia polymorpha, but absent from those of red algae, higher plants and animals. Our findings indicate the promiscuity of LiDGAT1 for VLC-PUFA and suggest a specific role for this enzyme in the neutral lipid metabolism of L. incisa that needs to be further investigated by molecular engineering approaches.     

Dictyostelium discoideum Dgat2 Can Substitute for the Essential Function of Dgat1 in Triglyceride Production but Not in Ether Lipid Synthesis

Triacylglycerol (TAG), the common energy storage molecule, is formed from diacylglycerol and a coenzyme A-activated fatty acid by the action of an acyl coenzyme A:diacylglycerol acyltransferase (DGAT). In order to conduct this step, most organisms rely on more than one enzyme. The two main candidates in Dictyostelium discoideum are Dgat1 and Dgat2. We show, by creating single and double knockout mutants, that the endoplasmic reticulum (ER)-localized Dgat1 enzyme provides the predominant activity, whereas the lipid droplet constituent Dgat2 contributes less activity. This situation may be opposite from what is seen in mammalian cells. Dictyostelium Dgat2 is specialized for the synthesis of TAG, as is the mammalian enzyme. In contrast, mammalian DGAT1 is more promiscuous regarding its substrates, producing diacylglycerol, retinyl esters, and waxes in addition to TAG. The Dictyostelium Dgat1, however, produces TAG, wax esters, and, most interestingly, also neutral ether lipids, which represent a significant constituent of lipid droplets. Ether lipids had also been found in mammalian lipid droplets, but the role of DGAT1 in their synthesis was unknown. The ability to form TAG through either Dgat1 or Dgat2 activity is essential for Dictyostelium to grow on bacteria, its natural food substrate.     

Suppression of late-flowering and semi-dwarf phenotypes in the arabidopsis clock mutant lhy-12;cca1-101 by phyB under continuous light

Photoperiodic flowering in Arabidopsis is controlled not only by floral activators such as GI, CO and FT, but also by repressors such as SVP and FLC. Double mutations in LHY and CCA1 (lhy;cca1) accelerated flowering under short days, mainly by the GI-CO dependent pathway. In contrast, lhy;cca1 showed delayed flowering under continuous light (LL), probably due to the GI-CO independent pathway. This late-flowering phenotype was suppressed by svp, flc and elf3. However, how SVP, FLC and ELF3 mediate LHY/CCA1 and flowering time is not fully understood. We found that lhy;cca1 exhibited short hypocotyls and petioles under LL, but the molecular mechanism for these effects has not been elucidated.To address these questions, we performed a screen for mutations that suppress either or both of the lhy;cca1 phenotypes under LL, using two different approaches. We identified two novel mutations, a dominant (del1) and a recessive (phyB-2511) allele of phyB. The flowering times of single mutants of three phyB alleles, hy3-1, del1 and phyB-2511, are almost the same and earlier than those of wild-type plants. A similar level of acceleration of flowering time was observed in all three phyB mutants tested when combined with the late-flowering mutations co-2 and SVPox. However, the effect of phyB-2511 on lhy;cca1 was different from those by hy3-1 or del1. svp-3 did not strongly enhance the early-flowering phenotypes of phyB-2511 or del1. These results suggest that light signaling via PhyB may affect factors downstream of the clock proteins, controlling flowering time and organ elongation. phyB mutations with different levels of effects on lhy;cca1-dependent late flowering would be useful to determine a specific role for PHYB in the flowering pathway controlled by lhy;cca1 under LL.Key words: Arabidopsis thaliana, CCA1, circadian clock, CO, FT, LHY, organ elongation, photoperiodic flowering, PHYB, SVP     

Directed evolution of acyl-CoA:diacylglycerol acyltransferase: Development and characterization of Brassica napus DGAT1 mutagenized libraries

Metabolic flux to triacylglycerol (TAG) may be limited by the level of acyl-CoA:diacylglycerol acyltransferase (DGAT, EC 2.3.1.20) activity. In some species, this enzyme also appears to play a role in the channeling of specific fatty acyl moieties into TAG. The objective of this work is to implement a directed evolution approach to enhance the catalytic efficiency of type-1 DGAT from Brassica napus (BnDGAT1). We generated randomly mutagenized libraries of BnDGAT1 in a yeast expression vector using error-prone PCR. The mutagenized libraries were used to transform a Saccharomyces cerevisiae strain devoid of neutral lipid biosynthesis and analyzed using a high-throughput screening (HTS) system. The HTS, recently developed for this purpose, consisted of a positive selection of clones expressing active DGAT mutants followed by quantification of DGAT activity by fluorescence detection of TAG in yeast cells. The initial results indicated that the positive selection system efficiently eliminated DGAT mutants lacking enzyme activity. Screening of 1528 selected mutants revealed that some DGAT clones had enhanced ability to synthesize TAG in yeast. This was confirmed by analysis of individual clones that could carry mutations resulting in an increased catalytic efficiency. The directed evolution approach could lead to the development of an improved plant DGAT1 for increasing seed oil content in oleaginous crops.     

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.     

DGAT1 and PDAT1 Acyltransferases Have Overlapping Functions in Arabidopsis Triacylglycerol Biosynthesis and Are Essential for Normal Pollen and Seed Development

Triacylglycerol (TAG) biosynthesis is a principal metabolic pathway in most organisms, and TAG is the major form of carbon storage in many plant seeds. Acyl-CoA:diacylglycerol acyltransferase 1 (DGAT1) is the only acyltransferase enzyme that has been confirmed to contribute to TAG biosynthesis in Arabidopsis thaliana seeds. However, dgat1 null mutants display only a 20 to 40% decrease in seed oil content. To determine whether other enzymes contribute to TAG synthesis, candidate genes were expressed in TAG-deficient yeast, candidate mutants were crossed with the dgat1-1 mutant, and target genes were suppressed by RNA interference (RNAi). An in vivo role for phospholipid:diacylglycerol acyltransferase 1 (PDAT1; At5g13640) in TAG synthesis was revealed in this study. After failing to obtain double homozygous plants from crossing dgat1-1 and pdat1-2, further investigation showed that the dgat1-1 pdat1-2 double mutation resulted in sterile pollen that lacked visible oil bodies. RNAi silencing of PDAT1 in a dgat1-1 background or DGAT1 in pdat1-1 background resulted in 70 to 80% decreases in oil content per seed and in disruptions of embryo development. These results establish in vivo involvement of PDAT1 in TAG biosynthesis, rule out major contributions by other candidate enzymes, and indicate that PDAT1 and DGAT1 have overlapping functions that are essential for normal pollen and seed development of Arabidopsis.