Production of the infant formula ingredient 1,3-olein-2-palmitin in Arabidopsis thaliana seeds.

In human milk fat, palmitic acid (16:0) is esterified to the middle (sn-2 or ß) position on the glycerol backbone and oleic acid (18:1) predominantly to the outer positions, giving the triacylglycerol (TG) a distinctive stereoisomeric structure that is believed to assist nutrient absorption in the infant gut. However, the fat used in most infant formulas is derived from plants, which preferentially esterify 16:0 to the outer positions. We have previously showed that the metabolism of the model oilseed Arabidopsis thaliana can be engineered to incorporate 16:0 into the middle position of TG. However, the fatty acyl composition of Arabidopsis seed TG does not mimic human milk, which is rich in both 16:0 and 18:1 and is defined by the high abundance of the TG molecular species 1,3-olein-2-palmitin (OPO). Here we have constructed an Arabidopsis fatty acid biosynthesis 1-1 fatty acid desaturase 2 fatty acid elongase 1 mutant with around 20% 16:0 and 70% 18:1 in its seeds and we have engineered it to esterify more than 80% of the 16:0 to the middle position of TG, using heterologous expression of the human lysophosphatidic acid acyltransferase isoform AGPAT1, combined with suppression of LYSOPHOSPHATIDIC ACID ACYLTRANSFERASE 2 and PHOSPHATIDYLCHOLINE:DIACYLGLYCEROL CHOLINEPHOSPHOTRANSFERASE. Our data show that oilseeds can be engineered to produce TG that is rich in OPO, which is a structured fat ingredient used in infant formulas.

Engineering the stereoisomeric structure of seed oil to mimic human milk fat.

Human milk fat substitute (HMFS) is a class of structured lipid that is widely used as an ingredient in infant formulas. Like human milk fat, HMFS is characterized by enrichment of palmitoyl (C16:0) groups specifically at the middle (sn-2 or ß) position on the glycerol backbone, and there is evidence that triacylglycerol (TAG) with this unusual stereoisomeric structure provides nutritional benefits. HMFS is currently made by in vitro enzyme-based catalysis because there is no appropriate biological alternative to human milk fat. Most of the fat currently used in infant formulas is obtained from plants, which exclude C16:0 from the middle position. In this study, we have modified the metabolic pathway for TAG biosynthesis in the model oilseed Arabidopsis thaliana to increase the percentage of C16:0 at the middle (vs. outer) positions by more than 20-fold (i.e., from ∼3% in wild type to >70% in our final iteration). This level of C16:0 enrichment is comparable to human milk fat. We achieved this by relocating the C16:0-specific chloroplast isoform of the enzyme lysophosphatidic acid acyltransferase (LPAT) to the endoplasmic reticulum so that it functions within the cytosolic glycerolipid biosynthetic pathway to esterify C16:0 to the middle position. We then suppressed endogenous LPAT activity to relieve competition and knocked out phosphatidylcholine:diacylglycerol cholinephosphotransferase activity to promote the flux of newly made diacylglycerol directly into TAG. Applying this technology to oilseed crops might provide a source of HMFS for infant formula.

Basic LEUCINE ZIPPER TRANSCRIPTION FACTOR67 Transactivates DELAY OF GERMINATION1 to Establish Primary Seed Dormancy in Arabidopsis.

Seed dormancy governs the timing of germination, one of the most important developmental transitions in a plant's life cycle. The DELAY OF GERMINATION1 (DOG1) gene is a key regulator of seed dormancy and a major quantitative trait locus in Arabidopsis (Arabidopsis thaliana). DOG1 expression is under tight developmental and environmental regulation, but the transcription factors involved are not known. Here we show that basic LEUCINE ZIPPER TRANSCRIPTION FACTOR67 (bZIP67) acts downstream of the central regulator of seed development, LEAFY COTYLEDON1, to transactivate DOG1 during maturation and help to establish primary dormancy. We show that bZIP67 overexpression enhances dormancy and that bZIP67 protein (but not transcript) abundance is increased in seeds matured in cool conditions, providing a mechanism to explain how temperature regulates DOG1 expression. We also show that natural allelic variation in the DOG1 promoter affects bZIP67-dependent transactivation, providing a mechanism to explain ecotypic differences in seed dormancy that are controlled by the DOG1 locus.

Natural variation in acyl editing is a determinant of seed storage oil composition.

Seeds exhibit wide variation in the fatty acid composition of their storage oil. However, the genetic basis of this variation is only partially understood. Here we have used a multi-parent advanced generation inter-cross (MAGIC) population to study the genetic control of fatty acid chain length in Arabidopsis thaliana seed oil. We mapped four quantitative trait loci (QTL) for the quantity of the major very long chain fatty acid species 11-eicosenoic acid (20:1), using multiple QTL modelling. Surprisingly, the main-effect QTL does not coincide with FATTY ACID ELONGASE 1 and a parallel genome wide association study suggested that LYSOPHOSPHATIDYLCHOLINE ACYLTRANSFERASE 2 (LPCAT2) is a candidate for this QTL. Regression analysis also suggested that LPCAT2 expression and 20:1 content in seeds of the 19 MAGIC founder accessions are related. LPCAT is a key component of the Lands cycle; an acyl editing pathway that enables acyl-exchange between the acyl-Coenzyme A and phosphatidylcholine precursor pools used for microsomal fatty acid elongation and desaturation, respectively. We Mendelianised the main-effect QTL using biparental chromosome segment substitution lines and carried out complementation tests to show that a single cis-acting polymorphism in the LPCAT2 promoter causes the variation in seed 20:1 content, by altering the LPCAT2 expression level and total LPCAT activity in developing siliques. Our work establishes that oilseed species exhibit natural variation in the enzymic capacity for acyl editing and this contributes to the genetic control of storage oil composition.

Genome Wide Analysis of Fatty Acid Desaturation and Its Response to Temperature.

Plants modify the polyunsaturated fatty acid content of their membrane and storage lipids in order to adapt to changes in temperature. In developing seeds, this response is largely controlled by the activities of the microsomal ω-6 and ω-3 fatty acid desaturases, FAD2 and FAD3. Although temperature regulation of desaturation has been studied at the molecular and biochemical levels, the genetic control of this trait is poorly understood. Here, we have characterized the response of Arabidopsis (Arabidopsis thaliana) seed lipids to variation in ambient temperature and found that heat inhibits both ω-6 and ω-3 desaturation in phosphatidylcholine, leading to a proportional change in triacylglycerol composition. Analysis of the 19 parental accessions of the multiparent advanced generation intercross (MAGIC) population showed that significant natural variation exists in the temperature responsiveness of ω-6 desaturation. A combination of quantitative trait locus (QTL) analysis and genome-wide association studies (GWAS) using the MAGIC population suggests that ω-6 desaturation is largely controlled by cis-acting sequence variants in the FAD2 5' untranslated region intron that determine the expression level of the gene. However, the temperature responsiveness of ω-6 desaturation is controlled by a separate QTL on chromosome 2. The identity of this locus is unknown, but genome-wide association studies identified potentially causal sequence variants within ∼40 genes in an ∼450-kb region of the QTL.

Step changes in leaf oil accumulation via iterative metabolic engineering.

Synthesis and accumulation of plant oils in the entire vegetative biomass offers the potential to deliver yields surpassing those of oilseed crops. However, current levels still fall well short of those typically found in oilseeds. Here we show how transcriptome and biochemical analyses pointed to a futile cycle in a previously established Nicotiana tabacum line, accumulating up to 15% (dry weight) of the storage lipid triacylglycerol in leaf tissue. To overcome this metabolic bottleneck, we either silenced the SDP1 lipase or overexpressed the Arabidopsis thaliana LEC2 transcription factor in this transgenic background. Both strategies independently resulted in the accumulation of 30-33% triacylglycerol in leaf tissues. Our results demonstrate that the combined optimization of de novo fatty acid biosynthesis, storage lipid assembly and lipid turnover in leaf tissue results in a major overhaul of the plant central carbon allocation and lipid metabolism. The resulting further step changes in oil accumulation in the entire plant biomass offers the possibility of delivering yields that outperform current oilseed crops.

Cyclin-dependent kinase activity enhances phosphatidylcholine biosynthesis in Arabidopsis by repressing phosphatidic acid phosphohydrolase activity.

Coordination of endomembrane biogenesis with cell cycle progression is considered to be important in maintaining cell function during growth and development. We previously showed that the disruption of PHOSPHATIDIC ACID PHOSPHOHYDROLASE (PAH) activity in Arabidopsis thaliana stimulates biosynthesis of the major phospholipid phosphatidylcholine (PC) and causes expansion of the endoplasmic reticulum. Here we show that PC biosynthesis is repressed by disruption of the core cell cycle regulator CYCLIN-DEPENDENT KINASE A;1 (CDKA;1) and that this repression is reliant on PAH. Furthermore, we show that cyclin-dependent kinases (CDKs) phosphorylate PAH1 at serine 162, which reduces both its activity and membrane association. Expression of a CDK-insensitive version of PAH1 with a serine 162 to alanine substitution represses PC biosynthesis and also reduces the rate of cell division in early leaf development. Together our findings reveal a physiologically important mechanism that couples the rate of phospholipid biosynthesis and endomembrane biogenesis to cell cycle progression in Arabidopsis.

ACYL-ACYL CARRIER PROTEIN DESATURASE2 and 3 Are Responsible for Making Omega-7 Fatty Acids in the Arabidopsis Aleurone.

Omega-7 monounsaturated fatty acids (ω-7s) are specifically enriched in the aleurone of Arabidopsis (Arabidopsis thaliana) seeds. We found significant natural variation in seed ω-7 content and used a Multiparent Advanced Generation Inter-Cross population to fine-map a major quantitative trait loci to a region containing ACYL-ACYL CARRIER PROTEIN DESATURASE1 (AAD1) and AAD3 We found that AAD3 expression is localized to the aleurone where mutants show an approximately 50% reduction in ω-7 content. By contrast, AAD1 is localized to the embryo where mutants show a small reduction in ω-9 content. Enzymatic analysis has previously shown that AAD family members possess both stearoyl- and palmitoyl-ACP Δ(9) desaturase activity, including the predominant isoform SUPPRESSOR OF SALICYLIC ACID INSENSITIVE2. However, aad3 ssi2 aleurone contained the same amount of ω-7s as aad3 Within the AAD family, AAD3 shares the highest degree of sequence similarity with AAD2 and AAD4. Mutant analysis showed that AAD2 also contributes to ω-7 production in the aleurone, and aad3 aad2 exhibits an approximately 85% reduction in ω-7s Mutant analysis also showed that FATTY ACID ELONGASE1 is required for the production of very long chain ω-7s in the aleurone. Together, these data provide genetic evidence that the ω-7 pathway proceeds via Δ(9) desaturation of palmitoyl-ACP followed by elongation of the product. Interestingly, significant variation was also identified in the ω-7 content of Brassica napus aleurone, with the highest level detected being approximately 47% of total fatty acids.

Regulation of endomembrane biogenesis in arabidopsis by phospatidic acid hydrolase.

Coordination of membrane lipid biosynthesis is important for cell function during plant growth and development. Here we summarize our recent work on PHOSPHATIDIC ACID PHOSPHOHYDROLASE (PAH) which suggests that this enzyme is a key regulator of phosphaticylcholine (PC) biosynthesis in Arabidopsis thaliana. Disruption of PAH activity elevates phosphatidic acid (PA) levels and stimulates PC biosynthesis and biogenesis of the endoplasmic reticulum (ER). Furthermore, the activity of PHOSPHOCHOLINE CYTIDYLYLTRANSFERASE (CCT), which is the key enzyme controlling the rate of PC biosynthesis, is directly stimulated by PA and expression of a constitutively active version of CCT replicates the effects of PAH disruption. Hence PAH activity can control the abundance of PA, which in turn can modulate CCT activity to govern the rate of PC biosynthesis. Crucially it is not yet clear how PAH activity is regulated in Arabidopsis but there is evidence that PAH1 and PAH2 are both phosphorylated and further work will be required to investigate whether this is functionally significant.

PHOSPHATIDIC ACID PHOSPHOHYDROLASE Regulates Phosphatidylcholine Biosynthesis in Arabidopsis by Phosphatidic Acid-Mediated Activation of CTP:PHOSPHOCHOLINE CYTIDYLYLTRANSFERASE Activity.

Regulation of membrane lipid biosynthesis is critical for cell function. We previously reported that disruption of PHOSPHATIDIC ACID PHOSPHOHYDROLASE1 (PAH1) and PAH2 stimulates net phosphatidylcholine (PC) biosynthesis and proliferation of the endoplasmic reticulum (ER) in Arabidopsis thaliana. Here, we show that this response is caused specifically by a reduction in the catalytic activity of the protein and positively correlates with an accumulation of its substrate, phosphatidic acid (PA). The accumulation of PC in pah1 pah2 is suppressed by disruption of CTP:PHOSPHOCHOLINE CYTIDYLYLTRANSFERASE1 (CCT1), which encodes a key enzyme in the nucleotide pathway for PC biosynthesis. The activity of recombinant CCT1 is stimulated by lipid vesicles containing PA. Truncation of CCT1, to remove the predicted C-terminal amphipathic lipid binding domain, produced a constitutively active enzyme. Overexpression of native CCT1 in Arabidopsis has no significant effect on PC biosynthesis or ER morphology, but overexpression of the truncated constitutively active version largely replicates the pah1 pah2 phenotype. Our data establish that membrane homeostasis is regulated by lipid composition in Arabidopsis and reveal a mechanism through which the abundance of PA, mediated by PAH activity, modulates CCT activity to govern PC content.

The metabolic flux phenotype of heterotrophic Arabidopsis cells reveals a flexible balance between the cytosolic and plastidic contributions to carbohydrate oxidation in response to phosphate limitation.

Understanding the mechanisms that allow plants to respond to variable and reduced availability of inorganic phosphate is of increasing agricultural importance because of the continuing depletion of the rock phosphate reserves that are used to combat inadequate phosphate levels in the soil. Changes in gene expression, protein levels, enzyme activities and metabolite levels all point to a reconfiguration of the central metabolic network in response to reduced availability of inorganic phosphate, but the metabolic significance of these changes can only be assessed in terms of the fluxes supported by the network. Steady-state metabolic flux analysis was used to define the metabolic phenotype of a heterotrophic Arabidopsis thaliana cell culture grown on a Murashige and Skoog medium containing 0, 1.25 or 5 mm inorganic phosphate. Fluxes through the central metabolic network were deduced from the redistribution of (13) C into metabolic intermediates and end products when cells were labelled with [1-(13) C], [2-(13) C], or [(13) C6 ]glucose, in combination with (14) C measurements of the rates of biomass accumulation. Analysis of the flux maps showed that reduced levels of phosphate in the growth medium stimulated flux through phosphoenolpyruvate carboxylase and malic enzyme, altered the balance between cytosolic and plastidic carbohydrate oxidation in favour of the plastid, and increased cell maintenance costs. We argue that plant cells respond to phosphate deprivation by reconfiguring the flux distribution through the pathways of carbohydrate oxidation to take advantage of better phosphate homeostasis in the plastid.