Altered Lipid Composition and Enhanced Nutritional Value of <i>Arabidopsis</i> Leaves following Introduction of an Algal Diacylglycerol Acyltransferase 2

Sanjaya,; Miller, Rachel E.; Durrett, Timothy P.; Kosma, Dylan K.; Lydic, Todd A.; Muthan, Bagyalakshmi; Koo, Abraham J.; Bukhman, Yury V.; Reid, Gavin E.; Howe, Gregg A.; Ohlrogge, John B.; Benning, Christoph

doi:10.1105/tpc.112.104752

Cited by 95 publications

(84 citation statements)

References 61 publications

(89 reference statements)

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“…This second mechanism would provide fatty acids for polyunsaturated TAG synthesis by one of two pathways: (1) the head groups of membrane lipids could be cleaved off to provide diacylglycerol for acyl-CoA:diacylglycerol acyltransferase (Boyle et al, 2012;Deng et al, 2012;La Russa et al, 2012;Hung et al, 2013;Sanjaya et al, 2013) or phospholipid:diacylglycerol acyltransferase (PDAT) (Boyle et al, 2012;Yoon et al, 2012) reactions; or (2) the membrane lipids could directly serve as the polar lipid substrate for PDAT reactions.…”

Section: Lcs2 Is a Key Component Of The Poorly Characterized Pathway mentioning

confidence: 99%

An Indexed, Mapped Mutant Library Enables Reverse Genetics Studies of Biological Processes in Chlamydomonas reinhardtii

et al. 2016

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The green alga Chlamydomonas reinhardtii is a leading unicellular model for dissecting biological processes in photosynthetic eukaryotes. However, its usefulness has been limited by difficulties in obtaining mutants in specific genes of interest. To allow generation of large numbers of mapped mutants, we developed high-throughput methods that (1) enable easy maintenance of tens of thousands of Chlamydomonas strains by propagation on agar media and by cryogenic storage, (2) identify mutagenic insertion sites and physical coordinates in these collections, and (3) validate the insertion sites in pools of mutants by obtaining >500 bp of flanking genomic sequences. We used these approaches to construct a stably maintained library of 1935 mapped mutants, representing disruptions in 1562 genes. We further characterized randomly selected mutants and found that 33 out of 44 insertion sites (75%) could be confirmed by PCR, and 17 out of 23 mutants (74%) contained a single insertion. To demonstrate the power of this library for elucidating biological processes, we analyzed the lipid content of mutants disrupted in genes encoding proteins of the algal lipid droplet proteome. This study revealed a central role of the long-chain acyl-CoA synthetase LCS2 in the production of triacylglycerol from de novo-synthesized fatty acids.

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Section: Lcs2 Is a Key Component Of The Poorly Characterized Pathway mentioning

confidence: 99%

An Indexed, Mapped Mutant Library Enables Reverse Genetics Studies of Biological Processes in Chlamydomonas reinhardtii

et al. 2016

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“…In Chlamydomonas and Nannochloropsis spp., transcriptome and genetic analysis revealed contribution of acyltransferases to TAG accumulation and their role as potential targets for manipulating TAG hyperaccumulation (Boyle et al, 2012;Sanjaya et al, 2013;Li et al, 2014a). Several carotenoid biosynthesis genes exhibited similar expression patterns (Supplemental Data Set S3); one exception is that a LPAAT gene (g8553), predicted to function in the chloroplast prokaryotic pathway, was down-regulated at 72 h in dark-grown cells and then recovered by light induction.…”

Section: How Was Lipid Synthesized?mentioning

confidence: 99%

Genomic Foundation of Starch-to-Lipid Switch in Oleaginous Chlorella spp.

et al. 2015

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The ability to rapidly switch the intracellular energy storage form from starch to lipids is an advantageous trait for microalgae feedstock. To probe this mechanism, we sequenced the 56.8-Mbp genome of Chlorella pyrenoidosa FACHB-9, an industrial production strain for protein, starch, and lipids. The genome exhibits positive selection and gene family expansion in lipid and carbohydrate metabolism and genes related to cell cycle and stress response. Moreover, 10 lipid metabolism genes might be originated from bacteria via horizontal gene transfer. Transcriptomic dynamics tracked via messenger RNA sequencing over six time points during metabolic switch from starch-rich heterotrophy to lipid-rich photoautotrophy revealed that under heterotrophy, genes most strongly expressed were from the tricarboxylic acid cycle, respiratory chain, oxidative phosphorylation, gluconeogenesis, glyoxylate cycle, and amino acid metabolisms, whereas those most down-regulated were from fatty acid and oxidative pentose phosphate metabolism. The shift from heterotrophy into photoautotrophy highlights up-regulation of genes from carbon fixation, photosynthesis, fatty acid biosynthesis, the oxidative pentose phosphate pathway, and starch catabolism, which resulted in a marked redirection of metabolism, where the primary carbon source of glycine is no longer supplied to cell building blocks by the tricarboxylic acid cycle and gluconeogenesis, whereas carbon skeletons from photosynthesis and starch degradation may be directly channeled into fatty acid and protein biosynthesis. By establishing the first genetic transformation in industrial oleaginous C. pyrenoidosa, we further showed that overexpression of an NAD(H) kinase from Arabidopsis (Arabidopsis thaliana) increased cellular lipid content by 110.4%, yet without reducing growth rate. These findings provide a foundation for exploiting the metabolic switch in microalgae for improved photosynthetic production of food and fuels.

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“…By comparison, the FAs recovered from wild-type plants by analysis of the TAG band from thin layer chromatography (TLC) plates were largely saturated acyl chains ( Figure 2B). In contrast with TAGs isolated from Arabidopsis seedlings ectopically overproducing a Chlamydomonas reinhardtii DGAT type-two enzyme (Sanjaya et al, 2013) or transcription factors involved in storage product accumulation, such as LEAF COTYLEDON2 (LEC2) (Santos Mendoza et al, 2005), TAGs derived from leaves of PDAT1 overexpressors contained very limited amounts of very-long-chain fatty acids ( Figure 2B). …”

Section: Overexpression Of Pdat1 Enhances Tag Accumulation Leading Tmentioning

confidence: 99%

Dual Role for Phospholipid:Diacylglycerol Acyltransferase: Enhancing Fatty Acid Synthesis and Diverting Fatty Acids from Membrane Lipids to Triacylglycerol in Arabidopsis Leaves

et al. 2013

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There is growing interest in engineering green biomass to expand the production of plant oils as feed and biofuels. Here, we show that PHOSPHOLIPID:DIACYLGLYCEROL ACYLTRANSFERASE1 (PDAT1) is a critical enzyme involved in triacylglycerol (TAG) synthesis in leaves. Overexpression of PDAT1 increases leaf TAG accumulation, leading to oil droplet overexpansion through fusion. Ectopic expression of oleosin promotes the clustering of small oil droplets. Coexpression of PDAT1 with oleosin boosts leaf TAG content by up to 6.4% of the dry weight without affecting membrane lipid composition and plant growth. PDAT1 overexpression stimulates fatty acid synthesis (FAS) and increases fatty acid flux toward the prokaryotic glycerolipid pathway. In the trigalactosyldiacylglycerol1-1 mutant, which is defective in eukaryotic thylakoid lipid synthesis, the combined overexpression of PDAT1 with oleosin increases leaf TAG content to 8.6% of the dry weight and total leaf lipid by fourfold. In the plastidic glycerol-3-phosphate acyltransferase1 mutant, which is defective in the prokaryotic glycerolipid pathway, PDAT1 overexpression enhances TAG content at the expense of thylakoid membrane lipids, leading to defects in chloroplast division and thylakoid biogenesis. Collectively, these results reveal a dual role for PDAT1 in enhancing fatty acid and TAG synthesis in leaves and suggest that increasing FAS is the key to engineering high levels of TAG accumulation in green biomass.

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Altered Lipid Composition and Enhanced Nutritional Value of Arabidopsis Leaves following Introduction of an Algal Diacylglycerol Acyltransferase 2

Cited by 95 publications

References 61 publications

An Indexed, Mapped Mutant Library Enables Reverse Genetics Studies of Biological Processes in Chlamydomonas reinhardtii

An Indexed, Mapped Mutant Library Enables Reverse Genetics Studies of Biological Processes in Chlamydomonas reinhardtii

Genomic Foundation of Starch-to-Lipid Switch in Oleaginous Chlorella spp.

Dual Role for Phospholipid:Diacylglycerol Acyltransferase: Enhancing Fatty Acid Synthesis and Diverting Fatty Acids from Membrane Lipids to Triacylglycerol in Arabidopsis Leaves

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