The coexpression of two desaturases provides an optimized reduction of saturates in camelina oil.

Reducing the saturate content of vegetable oils is key to increasing their utility and adoption as a feedstock for the production of biofuels. Expression of either the FAT5 16 : 0-CoA desaturase from Caenorhabditis elegans, or an engineered cyanobacterial 16 : 0/18 : 0-glycerolipid desaturase, DES9*, in seeds of Arabidopsis (Arabidopsis thaliana) substantially lowered oil saturates. However, because pathway fluxes and regulation of oil synthesis are known to differ across species, translating this transgene technology from the model plant to crop species requires additional investigation. In the work reported here, we found that high expression of FAT5 in seeds of camelina (Camelina sativa) provided only a moderate decrease in saturates, from 12.9% of total oil fatty acids in untransformed controls to 8.6%. Expression of DES9* reduced saturates to 4.6%, but compromised seed physiology and oil content. However, the coexpression of the two desaturases together cooperatively reduced saturates to only 4.0%, less than one-third of the level in the parental line, without compromising oil yield or seedling germination and establishment. Our successful lowering of oil saturates in camelina identifies strategies that can now be integrated with genetic engineering approaches that reduce polyunsaturates to provide optimized oil composition for biofuels in camelina and other oil seed crops.

A multigene approach secures hydroxy fatty acid production in Arabidopsis.

A central goal of green chemistry is to produce industrially useful fatty acids in oilseed crops. Although genes encoding suitable fatty acid-modifying enzymes are available from more than a dozen wild species, progress has been limited because expression of these enzymes in transgenic plants produces only low yields of the desired products. For example, fatty acid hydroxylase 12 (FAH12) from castor (Ricinus communis) produces only 17% hydroxy fatty acids (HFAs) when expressed in Arabidopsis (Arabidopsis thaliana), compared with 90% HFAs in castor seeds. The transgenic plants also have reduced oil content and seed vigor. Here, we review experiments that have provided for steady increased HFA accumulation and oil content. This research has led to exciting new discoveries of enzymes and regulatory processes in the pathways of both seed oil synthesis and lipid metabolism in other parts of the plant. Recent investigations have revealed that HFA-accumulating seeds are unable to rapidly mobilize HFA-containing triacylglycerol (TAG) storage lipid after germination to provide carbon and energy for seedling development, resulting in reduced seedling establishment. These findings present a new opportunity to investigate a different, key area of lipid metabolism-the pathways of TAG lipolysis and ß-oxidation in germinating seedlings.

Overexpression mutants reveal a role for a chloroplast MPD protein in regulation of reactive oxygen species during chilling in Arabidopsis.

Reactive oxygen species (ROS) contribute to cellular damage in several different contexts, but their role during chilling damage is poorly defined. Chilling sensitivity both limits the distribution of plant species and causes devastating crop losses worldwide. Our screen of chilling-tolerant Arabidopsis (Arabidopsis thaliana) for mutants that suffer chilling damage identified a gene (At4g03410) encoding a chloroplast Mpv17_PMP22 protein, MPD1, with no previous connection to chilling. The chilling-sensitive mpd1-1 mutant is an overexpression allele that we successfully phenocopied by creating transgenic lines with a similar level of MPD1 overexpression. In mammals and yeast, MPD1 homologs are associated with ROS management. In chilling conditions, Arabidopsis overexpressing MPD1 accumulated H2O2 to higher levels than wild-type controls and exhibited stronger induction of ROS response genes. Paraquat application exacerbated chilling damage, confirming that the phenotype occurs due to ROS dysregulation. We conclude that at low temperature increased MPD1 expression results in increased ROS production, causing chilling damage. Our discovery of the effect of MPD1 overexpression on ROS production under chilling stress implies that investigation of the nine other members of the Mpv17_PMP22 family in Arabidopsis may lead to new discoveries regarding ROS signaling and management in plants.

The biochemistry of headgroup exchange during triacylglycerol synthesis in canola.

Many pathways of primary metabolism are substantially conserved within and across plant families. However, significant differences in organization and fluxes through a reaction network may occur, even between plants in closely related genera. Assessing and understanding these differences is key to appreciating metabolic diversity, and to attempts to engineer plant metabolism for higher crop yields and desired product profiles. To better understand lipid metabolism and seed oil synthesis in canola (Brassica napus), we have characterized four canola homologues of the Arabidopsis (Arabidopsis thaliana) ROD1 gene. AtROD1 encodes phosphatidylcholine:diacylglycerol cholinephosphotransferase (PDCT), the enzyme that catalyzes a major flux of polyunsaturated fatty acids (PUFAs) in oil synthesis. Assays in yeast indicated that only two of the canola genes, BnROD1.A3 and BnROD1.C3, encode active isozymes of PDCT, and these genes are strongly expressed during the period of seed oil synthesis. Loss of expression of BnROD1.A3 and BnROD1.C3 in a double mutant, or by RNA interference, reduced the PUFA content of the oil to 26.6% compared with 32.5% in the wild type. These results indicate that ROD1 isozymes in canola are responsible for less than 20% of the PUFAs that accumulate in the seed oil compared with 40% in Arabidopsis. Our results demonstrate the care needed when translating results from a model species to crop plants.

Phosphatidylglycerol Composition Is Central to Chilling Damage in the Arabidopsis fab1 Mutant.

The Arabidopsis (Arabidopsis thaliana) fatty acid biosynthesis1 (fab1) mutant has increased levels of the saturated fatty acid 16:0, resulting from decreased activity of 3-ketoacyl-ACP synthase II. In fab1 leaves, phosphatidylglycerol, the major chloroplast phospholipid, contains >40% high-melting-point molecular species (HMP-PG; molecules that contain only 16:0, 16:1-trans, and 18:0 fatty acids)-a trait associated with chilling-sensitive plants-compared with <10% in wild-type Arabidopsis. Although they do not exhibit short-term chilling sensitivity when exposed to low temperatures (2°C to 6°C) for long periods, fab1 plants do suffer collapse of photosynthesis, degradation of chloroplasts, and eventually death. To test the relevance of HMP-PG to the fab1 phenotype, we used transgenic 16:0 desaturases targeted to the endoplasmic reticulum and the chloroplast to lower 16:0 in leaf lipids of fab1 plants. We produced two lines that had very similar lipid compositions except that one, ER-FAT5, contained high HMP-PG, similar to the fab1 parent, while the second, TP-DES9*, contained <10% HMP-PG, similar to the wild type. TP-DES9* plants, but not ER-FAT5 plants, showed strong recovery and growth following 75 d at 2°C, demonstrating the role of HMP-PG in low-temperature damage and death in fab1, and in chilling-sensitive plants more broadly.

Castor LPCAT and PDAT1A Act in Concert to Promote Transacylation of Hydroxy-Fatty Acid onto Triacylglycerol.

Oilseeds produce abundant triacylglycerol (TAG) during seed maturation to fuel the establishment of photoautotrophism in the subsequent generation. Commonly, TAG contains 18-carbon polyunsaturated fatty acids (FA), but plants also produce oils with unique chemical properties highly desirable for industrial processes. Unfortunately, plants that produce such oils are poorly suited to agronomic exploitation, leading to a desire to reconstitute novel oil biosynthesis in crop plants. Here, we studied the production and incorporation of hydroxy-fatty acids (HFA) onto TAG in Arabidopsis (Arabidopsis thaliana) plants expressing the castor (Ricinus communis) FAH12 hydroxylase. One factor limiting HFA accumulation in these plants is the inefficient removal of HFA from the site of synthesis on phosphatidylcholine (PC). In Arabidopsis, lysophosphatidic acid acyltransferase (LPCAT) cycles FA to and from PC for modification. We reasoned that the castor LPCAT (RcLPCAT) would preferentially remove HFA from PC, resulting in greater incorporation onto TAG. However, expressing RcLPCAT in Arabidopsis expressing FAH12 alone (line CL37) or together with castor acyl:coenzyme A:diacylglycerol acyltransferase2 reduced HFA and total oil yield. Detailed analysis indicated that RcLPCAT reduced the removal of HFA from PC, possibly by competing with the endogenous LPCAT isozymes. Significantly, coexpressing RcLPCAT with castor phospholipid:diacylglycerol acyltransferase increased novel FA and total oil contents by transferring HFA from PC to diacylglycerol. Our results demonstrate that a detailed understanding is required to engineer modified FA production in oilseeds and suggest that phospholipase A2 enzymes rather than LPCAT mediate the highly efficient removal of HFA from PC in castor seeds.

Identification, characterization and field testing of Brassica napus mutants producing high-oleic oils.

Producing healthy, high-oleic oils and eliminating trans-fatty acids from foods are two goals that can be addressed by reducing activity of the oleate desaturase, FAD2, in oilseeds. However, it is essential to understand the consequences of reducing FAD2 activity on the metabolism, cell biology and physiology of oilseed crop plants. Here, we translate knowledge from studies of fad2 mutants in Arabidopsis (Arabidopsis thaliana) to investigate the limits of non-GMO approaches to maximize oleic acid in the seed oil of canola (Brassica napus), a species that expresses three active FAD2 isozymes. A series of hypomorphic and null mutations in the FAD2.A5 isoform were characterized in yeast (Saccharomyes cerevisiae). Then, four of these were combined with null mutations in the other two isozymes, FAD2.C5 and FAD2.C1. The resulting mutant lines contained 71-87% oleic acid in their seed oil, compared with 62% in wild-type controls. All the mutant lines grew well in a greenhouse, but in field experiments we observed a clear demarcation in plant performance. Mutant lines containing less than 80% oleate in the seed oil were indistinguishable from wild-type controls in growth parameters and seed oil content. By contrast, lines with more than 80% oleate in the seed oil had significantly lower seedling establishment and vigor, delayed flowering and reduced plant height at maturity. These lines also had 7-11% reductions in seed oil content. Our results extend understanding of the B. napusFAD2 isozymes and define the practical limit to increasing oil oleate content in this crop species.

Tri-Hydroxy-Triacylglycerol Is Efficiently Produced by Position-Specific Castor Acyltransferases.

Understanding the biochemistry of triacylglycerol (TAG) assembly is critical in tailoring seed oils to produce high-value products. Hydroxy-fatty acid (HFA) is one such valuable modified fatty acid, which can be produced at low levels in Arabidopsis (Arabidopsis thaliana) seed through transgenic expression of the castor (Ricinus communis) hydroxylase. The resulting plants have low seed oil content and poor seedling establishment, indicating that Arabidopsis lacks efficient metabolic networks for biosynthesis and catabolism of hydroxy-containing TAG. To improve utilization of such substrates, we expressed three castor acyltransferase enzymes that incorporate HFA at each stereochemical position during TAG synthesis. This produced abundant tri-HFA TAG and concentrated 44% of seed HFA moieties into this one TAG species. Ricinoleic acid was more abundant than any other fatty acid in these seeds, which had 3-fold more HFA by weight than that in seeds following simple hydroxylase expression, the highest yet measured in a nonnative plant. Efficient utilization of hydroxy-containing lipid substrates increased the rate of TAG synthesis 2-fold, leading to complete relief of the low-oil phenotype. Partition of HFA into specific TAG molecules increased the storage lipid available for mobilization during seedling development, resulting in a 1.9-fold increase in seedling establishment. Expression of a complete acyltransferase pathway to efficiently process HFA establishes a benchmark in the quest to successfully produce modified oils in plants.

Development Defects of Hydroxy-Fatty Acid-Accumulating Seeds Are Reduced by Castor Acyltransferases.

Researchers have long endeavored to produce modified fatty acids in easily managed crop plants where they are not natively found. An important step toward this goal has been the biosynthesis of these valuable products in model oilseeds. The successful production of such fatty acids has revealed barriers to the broad application of this technology, including low seed oil and low proportion of the introduced fatty acid and reduced seed vigor. Here, we analyze the impact of producing hydroxy-fatty acids on seedling development. We show that germinating seeds of a hydroxy-fatty acid-accumulating Arabidopsis (Arabidopsis thaliana) line produce chlorotic cotyledons and suffer reduced photosynthetic capacity. These seedlings retain hydroxy-fatty acids in polar lipids, including chloroplast lipids, and exhibit decreased fatty acid synthesis. Triacylglycerol mobilization in seedling development also is reduced, especially for lipids that include hydroxy-fatty acid moieties. These developmental defects are ameliorated by increased flux of hydroxy-fatty acids into seed triacylglycerol created through the expression of either castor (Ricinus communis) acyltransferase enzyme ACYL-COA:DIACYLGLYCEROL ACYLTRANSFERASE2 or PHOSPHOLIPID:DIACYLGLYCEROL ACYLTRANSFERASE1A. Such expression increases both the level of total stored triacylglycerol and the rate at which it is mobilized, fueling fatty acid synthesis and restoring photosynthetic capacity. Our results suggest that further improvements in seedling development may require the specific mobilization of triacylglycerol-containing hydroxy-fatty acids. Understanding the defects in early development caused by the accumulation of modified fatty acids and providing mechanisms to circumvent these defects are vital steps in the development of tailored oil crops.

Overexpression of Seipin1 Increases Oil in Hydroxy Fatty Acid-Accumulating Seeds.

While plant oils are an important source of food, plants also produce oils containing specialized fatty acids with chemical and physical properties valued in industry. Ricinoleic acid, a hydroxy fatty acid (HFA) produced in the seed of castor (Ricinus communis), is of particular value, with a wide range of applications. Since castor cultivation is currently successful only in tropical climates, and because castor seed contain the toxin ricin, there are ongoing efforts to develop a temperate crop capable of HFA biosynthesis. In castor, ricinoleic acid is incorporated into triacylglycerol (TAG) which accumulates in the seed lipid droplets. Research in the model plant Arabidopsis (Arabidopsis thaliana) has successfully produced HFA constituting 30% of the total seed oil, but this is far short of the level required to engineer commercially viable crops. Strategies to increase HFA have centered on co-expression of castor TAG biosynthesis enzymes. However, since lipid droplets are the location of neutral lipid storage, manipulating droplets offers an alternative method to increase oil that contains specialized fatty acids. The Arabidopsis Seipin1 protein modulates TAG accumulation by affecting lipid droplet size. Here, we overexpress Seipin1 in a hydroxylase-expressing Arabidopsis line, increasing seed HFA by 62% and proportionally increasing total oil. Increased seed oil was concomitant with a 22% increase in single seed weight and a 69% increase in harvest weight, while seed germination rose by 45%. Because Seipin1 function is unaffected by the structure of the HFA, these results demonstrate a novel strategy that may increase accumulation of many specialized seed oils.

Trimethylguanosine Synthase1 (TGS1) Is Essential for Chilling Tolerance.

Chilling stress is a major factor limiting plant development and crop productivity. Because the plant response to chilling is so complex, we are far from understanding the genes important in the response to chilling. To identify new genes important in chilling tolerance, we conducted a novel mutant screen, combining a confirmed SALK T-DNA insertion collection with traditional forward genetics. We screened a pool of more than 3700 confirmed homozygous SALK T-DNA insertion lines for visible defects under prolonged growth at 5°C. Of the chilling-sensitive mutants we observed, mutations at one locus were characterized in detail. This gene, At1g45231, encodes an Arabidopsis (Arabidopsis thaliana) trimethylguanosine synthase (TGS1), previously uncharacterized in the plant kingdom. We confirmed that Arabidopsis TGS1 is a functional ortholog of other trimethylguanosine synthases based both on its in vitro methyltransferase activity and on its ability to rescue the cold-growth inhibition of a Saccharomyces cerevisiae tgs1Δ mutant in vivo. While tgs1 mutant plants grew normally at 22°C, their vegetative and reproductive growth was severely compromised under chilling conditions. When we transgenically expressed TGS1 in the mutant plants, the chilling-sensitive phenotype was relieved, demonstrating that TGS1 is required for chilling tolerance.

Epidermal jasmonate perception is sufficient for all aspects of jasmonate-mediated male fertility in Arabidopsis.

Jasmonate (JA) signaling is essential for several environmental responses and reproductive development in many plant species. In Arabidopsis thaliana, the most obvious phenotype of JA biosynthetic and perception mutants is profound sporophytic male sterility characterized by failure of stamen filament elongation, severe delay of anther dehiscence and pollen inviability. The site of action of JA in the context of reproductive development has been discussed, but the ideas have not been tested experimentally. To this end we used targeted expression of a COI1-YFP transgene in the coi1-1 mutant background. As COI1 is an essential component of the JA co-receptor complex, the null coi1-1 mutant is male sterile due to lack of JA perception. We show that expression of COI1-YFP in the epidermis of the stamen filament and anther in coi1 mutant plants is sufficient to rescue filament elongation, anther dehiscence and pollen viability. In contrast, filament expression alone or expression in the tapetum do not restore dehiscence and pollen viability. These results demonstrate that epidermal JA perception is sufficient for anther function and pollen viability, and suggest the presence of a JA-dependent non-autonomous signal produced in the anther epidermis to synchronize both anther dehiscence and pollen maturation.

WRINKLED1 Rescues Feedback Inhibition of Fatty Acid Synthesis in Hydroxylase-Expressing Seeds.

Previous attempts at engineering Arabidopsis (Arabidopsis thaliana) to produce seed oils containing hydroxy fatty acids (HFA) have resulted in low yields of HFA compared with the native castor (Ricinus communis) plant and caused undesirable effects, including reduced total oil content. Recent studies have led to an understanding of problems involved in the accumulation of HFA in oils of transgenic plants, which include metabolic bottlenecks and a decrease in the rate of fatty acid synthesis. Focusing on engineering the triacylglycerol assembly mechanisms led to modest increases in the HFA content of seed oil, but much room for improvement still remains. We hypothesized that engineering fatty acid synthesis in the plastids to increase flux would facilitate enhanced total incorporation of fatty acids, including HFA, into seed oil. The transcription factor WRINKLED1 (WRI1) positively regulates the expression of genes involved in fatty acid synthesis and controls seed oil levels. We overexpressed Arabidopsis WRI1 in seeds of a transgenic line expressing the castor fatty acid hydroxylase. The proportion of HFA in the oil, the total HFA per seed, and the total oil content of seeds increased to an average of 20.9%, 1.26 µg, and 32.2%, respectively, across five independent lines, compared with 17.6%, 0.83 µg, and 27.9%, respectively, for isogenic segregants. WRI1 and WRI1-regulated genes involved in fatty acid synthesis were up-regulated, providing for a corresponding increase in the rate of fatty acid synthesis.

Identification of Arabidopsis GPAT9 (At5g60620) as an Essential Gene Involved in Triacylglycerol Biosynthesis.

The first step in the biosynthesis of nearly all plant membrane phospholipids and storage triacylglycerols is catalyzed by a glycerol-3-phosphate acyltransferase (GPAT). The requirement for an endoplasmic reticulum (ER)-localized GPAT for both of these critical metabolic pathways was recognized more than 60 years ago. However, identification of the gene(s) encoding this GPAT activity has remained elusive. Here, we present the results of a series of in vivo, in vitro, and in silico experiments in Arabidopsis (Arabidopsis thaliana) designed to assign this essential function to AtGPAT9. This gene has been highly conserved throughout evolution and is largely present as a single copy in most plants, features consistent with essential housekeeping functions. A knockout mutant of AtGPAT9 demonstrates both male and female gametophytic lethality phenotypes, consistent with the role in essential membrane lipid synthesis. Significant expression of developing seed AtGPAT9 is required for wild-type levels of triacylglycerol accumulation, and the transcript level is directly correlated to the level of microsomal GPAT enzymatic activity in seeds. Finally, the AtGPAT9 protein interacts with other enzymes involved in ER glycerolipid biosynthesis, suggesting the possibility of ER-localized lipid biosynthetic complexes. Together, these results suggest that GPAT9 is the ER-localized GPAT enzyme responsible for plant membrane lipid and oil biosynthesis.

Fatty acid synthesis is inhibited by inefficient utilization of unusual fatty acids for glycerolipid assembly.

Degradation of unusual fatty acids through ß-oxidation within transgenic plants has long been hypothesized as a major factor limiting the production of industrially useful unusual fatty acids in seed oils. Arabidopsis seeds expressing the castor fatty acid hydroxylase accumulate hydroxylated fatty acids up to 17% of total fatty acids in seed triacylglycerols; however, total seed oil is also reduced up to 50%. Investigations into the cause of the reduced oil phenotype through in vivo [(14)C]acetate and [(3)H]2O metabolic labeling of developing seeds surprisingly revealed that the rate of de novo fatty acid synthesis within the transgenic seeds was approximately half that of control seeds. RNAseq analysis indicated no changes in expression of fatty acid synthesis genes in hydroxylase-expressing plants. However, differential [(14)C]acetate and [(14)C]malonate metabolic labeling of hydroxylase-expressing seeds indicated the in vivo acetyl-CoA carboxylase activity was reduced to approximately half that of control seeds. Therefore, the reduction of oil content in the transgenic seeds is consistent with reduced de novo fatty acid synthesis in the plastid rather than fatty acid degradation. Intriguingly, the coexpression of triacylglycerol synthesis isozymes from castor along with the fatty acid hydroxylase alleviated the reduced acetyl-CoA carboxylase activity, restored the rate of fatty acid synthesis, and the accumulation of seed oil was substantially recovered. Together these results suggest a previously unidentified mechanism that detects inefficient utilization of unusual fatty acids within the endoplasmic reticulum and activates an endogenous pathway for posttranslational reduction of fatty acid synthesis within the plastid.

A Caenorhabditis elegans model for ether lipid biosynthesis and function.

Ether lipids are widespread in nature, and they are structurally and functionally important components of membranes. The roundworm, Caenorhabditis elegans, synthesizes numerous lipid species containing alkyl and alkenyl ether bonds. We isolated C. elegans strains carrying loss-of-function mutations in three genes encoding the proteins required for the initial three steps in the ether lipid biosynthetic pathway, FARD-1/FAR1, ACL-7/GNPAT, and ADS-1/AGPS. Analysis of the mutant strains show that they lack ether lipids, but possess the ability to alter their lipid composition in response to lack of ether lipids. We found that increases in de novo fatty acid synthesis and reduction of stearoyl- and palmitoyl-CoA desaturase activity, processes that are at least partially regulated transcriptionally, mediate the altered lipid composition in ether lipid-deficient mutants. Phenotypic analysis demonstrated the importance of ether lipids for optimal fertility, lifespan, survival at cold temperatures, and resistance to oxidative stress.Caenorhabditis.

Male sterility in Arabidopsis induced by overexpression of a MYC5-SRDX chimeric repressor.

Jasmonate hormone (JA) plays critical roles in both plant defense and reproductive development. Arabidopsis thaliana plants deficient in JA-biosynthesis or -signaling are male-sterile, with defects in stamen and pollen development. MYC2, MYC3 and MYC4 are JAZ-interacting bHLH transcription factors that play a major role in controlling JA responses in vegetative tissue, but are not likely to play a role in reproductive tissue. We found that a closely related transcription factor, MYC5 (bHLH28), was able to induce JAZ promoters that control some of the early JA-responsive genes in a Daucus carota (carrot) protoplast expression system. A G-box sequence in the JAZ2 promoter was necessary and sufficient for induction by MYC5 (as it is for MYC2, MYC3 and MYC4), and induction of JAZ genes was repressed by co-expression of a stabilized, JAZ1ΔJas repressor. Two allelic myc5 mutants exhibited no overt phenotype; however, transgenic lines expressing MYC5 fused to an SRDX (SUPERMAN repressive domain X) motif phenocopied mutants defective in JA signaling. In particular, MYC5-SRDX plants were male-sterile, with defects in stamen filament elongation, anther dehiscence and pollen viability. Importantly, expression of MYB21 and other transcription factors required for stamen and pollen maturation was strongly reduced in stamens of MYC5-SRDX plants relative to the wild type. Taken together, these results indicate that MYC5, probably together with other, redundant transcription factors, may be activated by JA signaling to induce the expression of MYB21 and components required for male fertility.

50 years of Arabidopsis research: highlights and future directions.

922 I. 922 II. 922 III. 925 IV. 925 V. 926 VI. 927 VII. 928 VIII. 929 IX. 930 X. 931 XI. 932 XII. 933 XIII. Natural variation and genome-wide association studies 934 XIV. 934 XV. 935 XVI. 936 XVII. 937 937 References 937 SUMMARY: The year 2014 marked the 25(th) International Conference on Arabidopsis Research. In the 50 yr since the first International Conference on Arabidopsis Research, held in 1965 in Göttingen, Germany, > 54 000 papers that mention Arabidopsis thaliana in the title, abstract or keywords have been published. We present herein a citational network analysis of these papers, and touch on some of the important discoveries in plant biology that have been made in this powerful model system, and highlight how these discoveries have then had an impact in crop species. We also look to the future, highlighting some outstanding questions that can be readily addressed in Arabidopsis. Topics that are discussed include Arabidopsis reverse genetic resources, stock centers, databases and online tools, cell biology, development, hormones, plant immunity, signaling in response to abiotic stress, transporters, biosynthesis of cells walls and macromolecules such as starch and lipids, epigenetics and epigenomics, genome-wide association studies and natural variation, gene regulatory networks, modeling and systems biology, and synthetic biology.

Mutations in the Prokaryotic Pathway Rescue the fatty acid biosynthesis1 Mutant in the Cold.

The Arabidopsis (Arabidopsis thaliana) fatty acid biosynthesis1 (fab1) mutant has increased levels of the saturated fatty acid 16:0 due to decreased activity of 3-ketoacyl-acyl carrier protein (ACP) synthase II. In fab1 leaves, phosphatidylglycerol, the major chloroplast phospholipid, contains up to 45% high-melting-point molecular species (molecules that contain only 16:0, 16:1-trans, and 18:0), a trait associated with chilling-sensitive plants, compared with less than 10% in wild-type Arabidopsis. Although they do not exhibit typical chilling sensitivity, when exposed to low temperatures (2°C-6°C) for long periods, fab1 plants do suffer collapse of photosynthesis, degradation of chloroplasts, and eventually death. A screen for suppressors of this low-temperature phenotype has identified 11 lines, some of which contain additional alterations in leaf-lipid composition relative to fab1. Here, we report the identification of two suppressor mutations, one in act1, which encodes the chloroplast acyl-ACP:glycerol-3-phosphate acyltransferase, and one in lpat1, which encodes the chloroplast acyl-ACP:lysophosphatidic acid acyltransferase. These enzymes catalyze the first two steps of the prokaryotic pathway for glycerolipid synthesis, so we investigated whether other mutations in this pathway would rescue the fab1 phenotype. Both the gly1 mutation, which reduces glycerol-3-phosphate supply to the prokaryotic pathway, and fad6, which is deficient in the chloroplast 16:1/18:1 fatty acyl desaturase, were discovered to be suppressors. Analyses of leaf-lipid compositions revealed that mutations at all four of the suppressor loci result in reductions in the proportion of high-melting-point molecular species of phosphatidylglycerol relative to fab1. We conclude that these reductions are likely the basis for the suppressor phenotypes.

A small phospholipase A2-α from castor catalyzes the removal of hydroxy fatty acids from phosphatidylcholine in transgenic Arabidopsis seeds.

Ricinoleic acid, an industrially useful hydroxy fatty acid (HFA), only accumulates to high levels in the triacylglycerol fraction of castor (Ricinus communis) endosperm, even though it is synthesized on the membrane lipid phosphatidylcholine (PC) from an oleoyl ester. The acyl chains of PC undergo intense remodeling through the process of acyl editing. The identities of the proteins involved in this process, however, are unknown. A phospholipase A2 (PLA2) is thought to be involved in the acyl-editing process. We show here a role for RcsPLA2α in the acyl editing of HFA esterified to PC. RcsPLA2α was identified by its high relative expression in the castor endosperm transcriptome. Coexpression in Arabidopsis (Arabidopsis thaliana) seeds of RcsPLA2α with the castor fatty acid hydroxylase RcFAH12 led to a dramatic decrease in seed HFA content when compared with RcFAH12 expression alone in both PC and the neutral lipid fraction. The low-HFA trait was heritable and gene dosage dependent, with hemizygous lines showing intermediate HFA levels. The low seed HFA levels suggested that RcsPLA2α functions in vivo as a PLA2 with HFA specificity. Activity assays with yeast (Saccharomyces cerevisiae) microsomes showed a high specificity of RcsPLA2α for ricinoleic acid, superior to that of the endogenous Arabidopsis PLA2α. These results point to RcsPLA2α as a phospholipase involved in acyl editing, adapted to specifically removing HFA from membrane lipids in seeds.