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Arabidopsis is wildly used as a model plant and camelina is increasingly used for oilseed research and applications. Although the Arabidopsis genome has been sequenced for two decades, the functions of many lipid‐related genes and their regulators have not been well characterized. Improvements in the efficiency and accuracy of gene investigations are key to effective discovery of gene function and downstream bioengineering of plant oil quantity and quality. In this study, a visible marker was used to quickly identify transgenic T1 seeds and a method has been developed to phenotype fatty acid compositions and oil content of single T1 seeds. A whole seed direct transmethylation method was first optimized with multiple seeds and incubation at 85°C for 2 hours in a transmethylation solvent (5% H2SO4 in methanol with 30% toluene cosolvent) is recommended. Based on this method, a single Arabidopsis seed mini‐transmethylation (SAST) method has been established in a 1.5 ml GC sample vial with 200 μl transmethylation solvent. Characteristics of the method were evaluated and it was used to phenotype transgenic T1 seeds expressing AtFAD2 or RcWRI1. Our results indicate that fatty acid composition of T1 individual seeds are consistent with those of pools of multiple seeds from higher generations. However, oil content per individual seed varied substantially and therefore pooling five seeds is recommended for phenotyping oil content of T1 seeds. Additionally, a whole camelina single‐seed direct transmethylation was evaluated and results confirm its feasibility. The suitability of partial seed analysis of camelina was investigated but variation in composition of different seed tissues limits this approach.

Cutin is an extracellular lipid polymer that contributes to protective cuticle barrier functions against biotic and abiotic stresses in land plants. Glycerol has been reported as a component of cutin, contributing up to 14% by weight of total released monomers. Previous studies using partial hydrolysis of cuticle-enriched preparations established the presence of oligomers with glycerol-aliphatic ester links. Furthermore, glycerol-3-phosphate 2-O-acyltransferases (sn-2-GPATs) are essential for cutin biosynthesis. However, precise roles of glycerol in cutin assembly and structure remain uncertain. Here, a stable isotope-dilution assay was developed for the quantitative analysis of glycerol by GC/MS of triacetin with simultaneous determination of aliphatic monomers. To provide clues about the role of glycerol in dicarboxylic acid (DCA)-rich cutins, this methodology was applied to compare wild-type (WT) Arabidopsis cutin with a series of mutants that are defective in cutin synthesis. The molar ratio of glycerol to total DCAs in WT cutins was 2:1. Even when allowing for a small additional contribution from hydroxy fatty acids, this is a substantially higher glycerol to aliphatic monomer ratio than previously reported for any cutin. Glycerol content was strongly reduced in both stem and leaf cutin from all Arabidopsis mutants analyzed (gpat4/gpat8, att1-2 and lacs2-3). In addition, the molar reduction of glycerol was proportional to the molar reduction of total DCAs. These results suggest “glycerol-DCA-glycerol” may be the dominant motif in DCA-rich cutins. The ramifications and caveats for this hypothesis are presented.

Bayberry (Myrica pensylvanica) fruits are covered with a remarkably thick layer of crystalline wax consisting of triacylglycerol (TAG) and diacylglycerol (DAG) esterified exclusively with saturated fatty acids. As the only plant known to accumulate soluble glycerolipids as a major component of surface waxes, Bayberry represents a novel system to investigate neutral lipid biosynthesis and lipid secretion by vegetative plant cells. The assembly of Bayberry wax is distinct from conventional TAG and other surface waxes, and instead proceeds through a pathway related to cutin synthesis (Simpson and Ohlrogge, 2016). In this study, microscopic examination revealed that the fruit tissue that produces and secretes wax (Bayberry knobs) is fully developed before wax accumulates and that wax is secreted to the surface without cell disruption. Comparison of transcript expression to genetically related tissues (Bayberry leaves, M. rubra fruits), cutin-rich tomato and cherry fruit epidermis, and to oil-rich mesocarp and seeds, revealed exceptionally high expression of 13 transcripts for acyl-lipid metabolism together with down-regulation of fatty acid oxidases and desaturases. The predicted protein sequences of the most highly expressed lipid-related enzyme-encoding transcripts in Bayberry knobs are 100% identical to the sequences from Bayberry leaves, which do not produce surface DAG or TAG. Together, these results indicate that TAG biosynthesis and secretion in Bayberry is achieved by both up and down-regulation of a small subset of genes related to the biosynthesis of cutin and saturated fatty acids, and also implies that modifications in gene expression, rather than evolution of new gene functions, was the major mechanism by which Bayberry evolved its specialized lipid metabolism. This article is part of a Special Issue entitled: Plant Lipid Biology edited by Kent D. Chapman and Ivo Feussner.

Plant 14-3-3 proteins are phosphopeptide-binding proteins, belonging to a large family of proteins involved in numerous physiological processes including primary metabolism, although knowledge about the function of 14-3-3s in plant lipid metabolism is sparse. WRINKLED1 (WRI1) is a key transcription factor that governs plant oil biosynthesis. At present, AtWRI1-interacting partners remain largely unknown. Here, we show that 14-3-3 proteins are able to interact with AtWRI1, both in yeast and plant cells. Transient co-expression of 14-3-3- and AtWRI1-encoding cDNAs led to increased oil biosynthesis in Nicotiana benthamiana leaves. Stable transgenic plants overproducing a 14-3-3 protein also displayed increased seed oil content. Co-production of a 14-3-3 protein with AtWRI1 enhanced the transcriptional activity of AtWRI1. The 14-3-3 protein was found to increase the stability of AtWRI1. A possible 14-3-3 binding motif was identified in one of the two AP2 domains of AtWRI1, which was also found to be critical for the interaction of AtWRI1 with an E3 ligase linker protein. Thus, we hypothesize a regulatory mechanism by which the binding of 14-3-3 to AtWRI1 interferes with the interaction of AtWRI1 and the E3 ligase, thereby protecting AtWRI1 from degradation. Taken together, our studies identified AtWRI1 as a client of 14-3-3 proteins and provide insights into a role of 14-3-3 in mediating plant oil biosynthesis.

Bayberry (Myrica pensylvanica) fruits synthesize an extremely thick and unusual layer of crystalline surface wax that accumulates to 32% of fruit dry weight, the highest reported surface lipid accumulation in plants. The composition is also striking, consisting of completely saturated triacylglycerol, diacylglycerol, and monoacylglycerol with palmitate and myristate acyl chains. To gain insight into the unique properties of Bayberry wax synthesis, we examined the chemical and morphological development of the wax layer, monitored wax biosynthesis through [14C]-radiolabeling, and sequenced the transcriptome. Radiolabeling identified sn-2 monoacylglycerol as an initial glycerolipid intermediate. The kinetics of [14C]-DAG and [14C]-TAG accumulation and the regiospecificity of their [14C]-acyl chains indicated distinct pools of acyl donors and that final TAG assembly occurs outside of cells. The most highly expressed lipid-related genes were associated with production of cutin, whereas transcripts for conventional TAG synthesis were >50-fold less abundant. The biochemical and expression data together indicate that Bayberry surface glycerolipids are synthesized by a pathway for TAG synthesis that is related to cutin biosynthesis. The combination of a unique surface wax and massive accumulation may aid understanding of how plants produce and secrete non-membrane glycerolipids and also how to engineer alternative pathways for lipid production in non-seeds.

WRINKLED1 (WRI1) is a key transcription factor governing plant oil biosynthesis. We characterized three intrinsically disordered regions (IDRs) in Arabidopsis WRI1, and found that one C-terminal IDR of AtWRI1 (IDR3) affects the stability of AtWRI1. Analysis by bimolecular fluorescence complementation and yeast-two-hybrid assays indicated that the IDR3 domain does not determine WRI1 stability by interacting with BTB/POZ-MATH proteins connecting AtWRI1 with CULLIN3-based E3 ligases. Analysis of the WRI1 sequence revealed that a putative PEST motif (proteolytic signal) is located at the C-terminal region of AtWRI1(IDR) (3). We also show that a 91 amino acid domain at the C-terminus of AtWRI1 without the PEST motif is sufficient for transactivation. We found that removal of the PEST motif or mutations in putative phosphorylation sites increased the stability of AtWRI1, and led to increased oil biosynthesis when these constructs were transiently expressed in tobacco leaves. Oil content was also increased in the seeds of stable transgenic wri1-1 plants expressing AtWRI1 with mutations in the IDR3-PEST motif. Taken together, our data suggest that intrinsic disorder of AtWRI1(IDR3) may facilitate exposure of the PEST motif to protein kinases. Thus, phosphorylation of the PEST motif in the AtWRI1(IDR) (3) domain may affect AtWRI1-mediated plant oil biosynthesis. The results obtained here suggest a means to increase accumulation of oils in plant tissues through WRI1 engineering.

Summary Seed oils have proved recalcitrant to modification for the production of industrially useful lipids. Here, we demonstrate the successful metabolic engineering and subsequent field production of an oilseed crop with the highest accumulation of unusual oil achieved so far in transgenic plants. Previously, expression of the Euonymus alatus diacylglycerol acetyltransferase (EaDAcT) gene in wild-type Arabidopsis seeds resulted in the accumulation of 45 mol% of unusual 3-acetyl-1,2-diacyl-sn-glycerols (acetyl-TAGs) in the seed oil (Durrett et al., 2010 PNAS 107:9464). Expression of EaDAcT in dgat1 mutants compromised in their ability to synthesize regular triacylglycerols increased acetyl-TAGs to 65 mol%. Camelina and soybean transformed with the EaDAcT gene accumulate acetyl-triacylglycerols (acetyl-TAGs) at up to 70 mol% of seed oil. A similar strategy of coexpression of EaDAcT together with RNAi suppression of DGAT1 increased acetyl-TAG levels to up to 85 mol% in field-grown transgenic Camelina. Additionally, total moles of triacylglycerol (TAG) per seed increased 20%. Analysis of the acetyl-TAG fraction revealed a twofold reduction in very long chain fatty acids (VLCFA), consistent with their displacement from the sn-3 position by acetate. Seed germination remained high, and seedlings were able to metabolize the stored acetyl-TAGs as rapidly as regular triacylglycerols. Viscosity, freezing point and caloric content of the Camelina acetyl-TAG oils were reduced, enabling use of this oil in several nonfood and food applications.

A diacylglycerol acetyltransferase, EaDAcT, from Euonymus alatus, synthesizes sn-3 acetyl triacylglycerols (acetyl-TAG) when expressed in Arabidopsis, Camelina and soybean. Compared to most vegetable oils, acetyl-TAGs have reduced viscosity and improved cold temperature properties that confer advantages in applications as biodegradable lubricants, food emulsifiers, plasticizers, and ‘drop-in’ fuels for some diesel engines. A high-oleic Camelina line was engineered to express the EaDAcT gene in order to produce acetyl-TAG oils with fatty acid compositions and physiochemical properties complementary to wild-type acetyl-TAG. The accumulation of acetyl-TAGs at 70 mol% of seed TAG in field-grown high-oleic Camelina had minor or no effect on seed weight, oil content, harvest index and seed yield. The total moles of TAG increased up to 27% reflecting the ability to synthesize more acetyl-TAG from the same supply of long-chain fatty acid. Acetyl-TAG could be separated from long-chain TAG by silica column or by reverse phase chromatography. The predominant acetyl-TAG molecular species produced in high-oleic Camelina was acetyl-dioleoyl-glycerol. The crystallization temperature of high-oleic acetyl-TAG (by differential scanning calorimetry at 1.0 °C/min) was reduced by 30 °C compared to control TAG. The viscosity of high-oleic acetyl-TAG was 27% lower than TAG from the high-oleic control and the caloric content was reduced by 5%. Field production of T4 and T5 transgenic plants yielded over 250 kg seeds for oil extraction and analysis.

Diverse fatty acid structures from different plant species are important renewable resources for industrial raw materials and as liquid fuels with high energy density. Because of its immense geographical and topographical variations, China is a country with enormous diversity of plant species, including large numbers of plants endemic to China. The richness of this resource of species provides a wide range of fatty acids in seeds or other tissues, many of which have been identified by Chinese scientists. However, in the past, most publications describing analysis of these plants were written in Chinese, making access for researchers from other countries difficult. In this study, we investigated reports on seed and fruit oil fatty acids as described in Chinese literature. Six books and more than one thousand papers were collected and the identified fatty acids and relevant plant species were summarized. In total, about 240 fatty acids from almost 1,500 plant species were identified from available Chinese literature. Only about one third of these species were retrieved in in the PhyloFAdb and SOFA online databases of plant fatty acids. By referring to a summary of plant species endemic to China, 277 Chinese endemic species from 68 families have been surveyed for seed fatty acids. These account for less than 2% of total Angiosperm species endemic to China indicating the scope of species yet to be surveyed. To discover additional new fatty acid structures that might benefit society, it is important in the future to study oilseed fatty acids of the many other Chinese endemic plants. As an example, seeds of five unsurveyed species were collected and their fatty acids were analyzed. Ricinoleic acid was detected for the first time in the Salicaceae family.

Lipid droplets in plants (also known as oil bodies, lipid bodies, or oleosomes) are well characterized in seeds, and oleosins, the major proteins associated with their surface, were shown to be important for stabilizing lipid droplets during seed desiccation and rehydration. However, lipid droplets occur in essentially all plant cell types, many of which may not require oleosin-mediated stabilization. The proteins associated with the surface of nonseed lipid droplets, which are likely to influence the formation, stability, and turnover of this compartment, remain to be elucidated. Here, we have combined lipidomic, proteomic, and transcriptomic studies of avocado (Persea americana) mesocarp to identify two new lipid droplet-associated proteins, which we named LDAP1 and LDAP2. These proteins are highly similar to each other and also to the small rubber particle proteins that accumulate in rubber-producing plants. An Arabidopsis (Arabidopsis thaliana) homolog to LDAP1 and LDAP2, At3g05500, was localized to the surface of lipid droplets after transient expression in tobacco (Nicotiana tabacum) cells that were induced to accumulate triacylglycerols. We propose that small rubber particle protein-like proteins are involved in the general process of binding and perhaps the stabilization of lipid-rich particles in the cytosol of plant cells and that the avocado and Arabidopsis protein members reveal a new aspect of the cellular machinery that is involved in the packaging of triacylglycerols in plant tissues.

Rapid turnover of stored triacylglycerol occurs after seed germination, releasing fatty acids that provide carbon and energy for seedling establishment. Glycerolipid and fatty acid turnover that occurs at other times in the plant life cycle, including senescence is less studied. Although the entire pathway of β-oxidation is induced during senescence, Arabidopsis leaf fatty acids turnover at rates 50 fold lower than in seedlings. Major unknowns in lipid turnover include the identity of lipases responsible for degradation of the wide diversity of galactolipid, phospholipid, and other lipid class structures. Also unknown is the relative flux of the acetyl-CoA product of β-oxidation into alternative metabolic pathways. We present an overview of senescence-related glycerolipid turnover and discuss its function(s) and speculate about how it might be controlled to increase the energy density and nutritional content of crops. To better understand regulation of lipid turnover, we developed a database that compiles and plots transcript expression of lipid-related genes during natural leaf senescence of Arabidopsis. The database allowed identification of coordinated patterns of down-regulation of lipid biosynthesis genes and the contrasting groups of genes that increase, including 68 putative lipases.

Engineering transgenic plants that accumulate high levels of medium-chain fatty acids (MCFA) has been least successful for shorter chain lengths (e.g., C8). We demonstrate that one limitation is the activity of acyl-ACP synthetase (AAE) that re-activates fatty acids released by acyl-ACP thioesterases. Seed expression of Cuphea pulcherrima FATB acyl-ACP thioesterase in a double mutant lacking AAE15/16 increased 8:0 accumulation almost 2-fold compared to expression in wild type. These results also provide an in planta demonstration that AAE enzymes participate not only in activation of exogenously added MCFA but also in activation of MCFA synthesized in plastids.

Summary Triacylglycerol (TAG) is the main storage lipid in plant seeds and the major form of plant oil used for food and, increasingly, for industrial and biofuel applications. Several transcription factors, including FUSCA3 (At3 g26790, FUS3), are associated with embryo maturation and oil biosynthesis in seeds. However, the ability of FUS3 to increase TAG biosynthesis in other tissues has not been quantitatively examined. Here, we evaluated the ability of FUS3 to activate TAG accumulation in non-seed tissues. Overexpression of FUS3 driven by an estradiol-inducible promoter increased oil contents in Arabidopsis seedlings up to 6% of dry weight; more than 50-fold over controls. Eicosenoic acid, a characteristic fatty acid of Arabidopsis seed oil, accumulated to over 20% of fatty acids in cotyledons and leaves. These large increases depended on added sucrose, although without sucrose TAG increased three- to four-fold. Inducing the expression of FUS3 in tobacco BY2 cells also increased TAG accumulation, and co-expression of FUS3 and diacylglycerol acyltransferase 1 (DGAT1) further increased TAG levels to 4% of dry weight. BY2 cell growth was not altered by FUS3 expression, although Arabidopsis seedling development was impaired, consistent with the ability of FUS3 to induce embryo characteristics in non-seed tissues. Microarrays of Arabidopsis seedlings revealed that FUS3 overexpression increased the expression of a higher proportion of genes involved in TAG biosynthesis than genes involved in fatty acid biosynthesis or other lipid pathways. Together these results provide additional insights into FUS3 functions in TAG metabolism and suggest complementary strategies for engineering vegetative oil accumulation.

Plant epidermal cells have evolved specialist functions associated with adaptation to stress. These include the synthesis and deposition of specialized metabolites such as waxes and cutin together with flavonoids and anthocyanins, which have important roles in providing a barrier to water loss and protection against UV radiation, respectively. Characterization of the sticky peel (pe) mutant of tomato (Solanum lycopersicum) revealed several phenotypes indicative of a defect in epidermal cell function, including reduced anthocyanin accumulation, a lower density of glandular trichomes, and an associated reduction in trichome-derived terpenes. In addition, pe mutant fruit are glossy and peels have increased elasticity due to a severe reduction in cutin biosynthesis and altered wax deposition. Leaves of the pe mutant are also cutin deficient and the epicuticular waxes contain a lower proportion of long-chain alkanes. Direct measurements of transpiration, together with chlorophyll-leaching assays, indicate increased cuticular permeability of pe leaves. Genetic mapping revealed that the pe locus represents a new allele of CUTIN DEFICIENT2 (CD2), a member of the class IV homeodomain-leucine zipper gene family, previously only associated with cutin deficiency in tomato fruit. CD2 is preferentially expressed in epidermal cells of tomato stems and is a homolog of Arabidopsis (Arabidopsis thaliana) ANTHOCYANINLESS2 (ANL2). Analysis of cuticle composition in leaves of anl2 revealed that cutin accumulates to approximately 60% of the levels observed in wild-type Arabidopsis. Together, these data provide new insight into the role of CD2 and ANL2 in regulating diverse metabolic pathways and in particular, those associated with epidermal cells.

Cell cultures allow rapid kinetic labeling experiments that can provide information on precursor-product relationships and intermediate pools. T-87 suspension cells are increasingly used in Arabidopsis (Arabidopsis thaliana) research, but there are no reports describing their lipid composition or biosynthesis. To facilitate application of T-87 cells for analysis of glycerolipid metabolism, including tests of gene functions, we determined composition and accumulation of lipids of light- and dark-grown cultures. Fatty acid synthesis in T-87 cells was 7- to 8-fold higher than in leaves. Similar to other plant tissues, phosphatidylcholine (PC) and phosphatidylethanolamine were major phospholipids, but galactolipid levels were 3- to 4-fold lower than Arabidopsis leaves. Triacylglycerol represented 10% of total acyl chains, a greater percentage than in most nonseed tissues. The initial steps in T-87 cell lipid assembly were evaluated by pulse labeling cultures with [14C]acetate and [14C]glycerol. [14C]acetate was very rapidly incorporated into PC, preferentially at sn-2 and without an apparent precursor-product relationship to diacylglycerol (DAG). By contrast, [14C]glycerol most rapidly labeled DAG. These results indicate that acyl editing of PC is the major pathway for initial incorporation of fatty acids into glycerolipids of cells derived from a 16:3 plant. A very short lag time (5.4 s) for [14C]acetate labeling of PC implied channeled incorporation of acyl chains without mixing with the bulk acyl-CoA pool. Subcellular fractionation of pea (Pisum sativum) leaf protoplasts indicated that 30% of lysophosphatidylcholine acyltransferase activity colocalized with chloroplasts. Together, these data support a model in which PC participates in trafficking of newly synthesized acyl chains from plastids to the endoplasmic reticulum.

Endosperm and embryo tissues from the seeds of Euonymus alatus (Burning Bush) accumulate high levels of 3-acetyl-1,2-diacyl- sn -glycerols (acTAGs) as their major storage lipids. In contrast, the aril tissue surrounding the seed produces long-chain triacylglycerols (lcTAGs) typical of most other organisms. The presence of the sn -3 acetyl group imparts acTAGs with different physical and chemical properties, such as a 30% reduction in viscosity, compared to lcTAGs. Comparative transcriptome analysis of developing endosperm and aril tissues using pyrosequencing technology was performed to isolate the enzyme necessary for the synthesis of acTAGs. An uncharacterized membrane-bound O -acyltransferase (MBOAT) family member was the most abundant acyltransferase in the endosperm but was absent from the aril. Expression of this MBOAT in yeast resulted in the accumulation of acTAGs but not lcTAG; hence, the enzyme was named EaDAcT ( Euonymus alatus d iacylglycerol ac etyl t ransferase). Yeast microsomes expressing EaDAcT possessed acetyl-CoA diacylglycerol acetyltransferase activity but lacked long-chain acyl-CoA diacylglycerol acyltransferase activity. Expression of EaDAcT under the control of a strong, seed-specific promoter in Arabidopsis resulted in the accumulation of acTAGs, up to 40 mol % of total TAG in the seed oil. These results demonstrate the utility of deep transcriptional profiling with multiple tissues as a gene discovery strategy for low-abundance proteins. They also show that EaDAcT is the acetyltransferase necessary and sufficient for the production of acTAGs in Euonymus seeds, and that this activity can be introduced into the seeds of other plants, allowing the evaluation of these unusual TAGs for biofuel and other applications.

All plants produce suberin, a lipophilic barrier of the cell wall that controls water and solute fluxes and restricts pathogen infection. It is often described as a heteropolymer comprised of polyaliphatic and polyaromatic domains. Major monomers include ω-hydroxy and α,ω-dicarboxylic fatty acids, glycerol, and ferulate. No genes have yet been identified for the aromatic suberin pathway. Here we demonstrate that Arabidopsis (Arabidopsis thaliana) gene AT5G41040, a member of the BAHD family of acyltransferases, is essential for incorporation of ferulate into suberin. In Arabidopsis plants transformed with the AT5G41040 promoter:YFP fusion, reporter expression is localized to cell layers undergoing suberization. Knockout mutants of AT5G41040 show almost complete elimination of suberin-associated ester-linked ferulate. However, the classic lamellar structure of suberin in root periderm of at5g41040 is not disrupted. The reduction in ferulate in at5g41040-knockout seeds is associated with an approximate stoichiometric decrease in aliphatic monomers containing ω-hydroxyl groups. Recombinant AT5G41040p catalyzed acyl transfer from feruloyl-coenzyme A to ω-hydroxyfatty acids and fatty alcohols, demonstrating that the gene encodes a feruloyl transferase. CYP86B1, a cytochrome P450 monooxygenase gene whose transcript levels correlate with AT5G41040 expression, was also investigated. Knockouts and overexpression confirmed CYP86B1 as an oxidase required for the biosynthesis of very-long-chain saturated α,ω-bifunctional aliphatic monomers in suberin. The seed suberin composition of cyp86b1 knockout was surprisingly dominated by unsubstituted fatty acids that are incapable of polymeric linkages. Together, these results challenge our current view of suberin structure by questioning both the function of ester-linked ferulate as an essential component and the existence of an extended aliphatic polyester.

During leaf senescence, macromolecule breakdown occurs and nutrients are translocated to support growth of new vegetative tissues, seeds, or other storage organs. In this study, we determined the fatty acid levels and profiles in Arabidopsis (Arabidopsis thaliana), Brachypodium distachyon, and switchgrass (Panicum virgatum) leaves during natural senescence. In young leaves, fatty acids represent 4% to 5% of dry weight and approximately 10% of the chemical energy content of the leaf tissues. In all three species, fatty acid levels in leaves began to decline at the onset of leaf senescence and progressively decreased as senescence advanced, resulting in a greater than 80% decline in fatty acids on a dry weight basis. During senescence, Arabidopsis leaves lost 1.6% of fatty acids per day at a rate of 2.1 μg per leaf (0.6 μg mg−1 dry weight). Triacylglycerol levels remained less than 1% of total lipids at all stages. In contrast to glycerolipids, aliphatic surface waxes of Arabidopsis leaves were much more stable, showing only minor reduction during senescence. We also examined three Arabidopsis mutants, acx1acx2, lacs6lacs7, and kat2, which are blocked in enzyme activities of β-oxidation and are defective in lipid mobilization during seed germination. In each case, no major differences in the fatty acid contents of leaves were observed between these mutants and the wild type, indicating that several mutations in β-oxidation that cause reduced breakdown of reserve oil in seeds do not substantially reduce the degradation of fatty acids during leaf senescence.

In traditional mutant screening approaches, genetic variants are tested for one or a small number of phenotypes. Once bona fide variants are identified, they are typically subjected to a limited number of secondary phenotypic screens. Although this approach is excellent at finding genes involved in specific biological processes, the lack of wide and systematic interrogation of phenotype limits the ability to detect broader syndromes and connections between genes and phenotypes. It could also prevent detection of the primary phenotype of a mutant. As part of a systems biology approach to understand plastid function, large numbers of Arabidopsis thaliana homozygous T-DNA lines are being screened with parallel morphological, physiological, and chemical phenotypic assays (www.plastid.msu.edu). To refine our approaches and validate the use of this high-throughput screening approach for understanding gene function and functional networks, approximately 100 wild-type plants and 13 known mutants representing a variety of phenotypes were analyzed by a broad range of assays including metabolite profiling, morphological analysis, and chlorophyll fluorescence kinetics. Data analysis using a variety of statistical approaches showed that such industrial approaches can reliably identify plant mutant phenotypes. More significantly, the study uncovered previously unreported phenotypes for these well-characterized mutants and unexpected associations between different physiological processes, demonstrating that this approach has strong advantages over traditional mutant screening approaches. Analysis of wild-type plants revealed hundreds of statistically robust phenotypic correlations, including metabolites that are not known to share direct biosynthetic origins, raising the possibility that these metabolic pathways have closer relationships than is commonly suspected.

Mature seeds of Arabidopsis thaliana and Brassica napus contain complex mixtures of aliphatic monomers derived from non-extractable lipid polyesters. Most of the monomers are deposited in the seed coat, and their compositions suggest the presence of both cutin and suberin layers. The location of these polyesters within the seed coat, and their contributions to permeability of the seed coat and other functional properties are unknown. Polyester deposition was followed over Brassica seed development and distinct temporal patterns of monomer accumulation were observed. Octadecadiene-1,18-dioate, the major leaf cutin monomer, was transiently deposited. In contrast, the saturated dicarboxylates maintained a constant level during seed desiccation, whereas the fatty alcohols and saturated omega-hydroxy fatty acids continually increased. Dissection and analysis of Brassica seed coats showed that suberization is not specific to the chalaza. Analysis of the Arabidopsis ap2-7 mutant suggested that suberin monomers are preferentially associated with the outer integument. Several Arabidopsis knockout mutant lines for genes involved in polyester biosynthesis (att1, fatB and gpat5) were examined for seed monomer load and composition. The variance in polyester monomers of these mutants is correlated with dye penetration assays. Furthermore, stable transgenic plants expressing promoter::YFP fusions showed ATT1 promoter activity in the inner integument, whereas GPAT5 promoter is active in the outer integument. Together, the Arabidopsis data indicated that there is a suberized layer associated with the outer integument and a cutin-like polyester layer associated with the inner seed coat.

Two Brassicaceae species, Physaria fendleri and Camelina sativa, are genetically very closely related to each other and to Arabidopsis thaliana. Physaria fendleri seeds contain over 50% hydroxy fatty acids (HFAs), while Camelina sativa and Arabidopsis do not accumulate HFAs. To better understand how plants evolved new biochemical pathways with the capacity to accumulate high levels of unusual fatty acids, transcript expression and protein sequences of developing seeds of Physaria fendleri, wild-type Camelina sativa, and Camelina sativa expressing a castor bean (Ricinus communis) hydroxylase were analyzed. A number of potential evolutionary adaptations within lipid metabolism that probably enhance HFA production and accumulation in Physaria fendleri, and, in their absence, limit accumulation in transgenic tissues were revealed. These adaptations occurred in at least 20 genes within several lipid pathways from the onset of fatty acid synthesis and its regulation to the assembly of triacylglycerols. Lipid genes of Physaria fendleri appear to have co-evolved through modulation of transcriptional abundances and alterations within protein sequences. Only a handful of genes showed evidence for sequence adaptation through gene duplication. Collectively, these evolutionary changes probably occurred to minimize deleterious effects of high HFA amounts and/or to enhance accumulation for physiological advantage. These results shed light on the evolution of pathways for novel fatty acid production in seeds, help explain some of the current limitations to accumulation of HFAs in transgenic plants, and may provide improved strategies for future engineering of their production.

All vascular plants are protected from the environment by a cuticle, a lipophilic layer synthesized by epidermal cells and composed of a cutin polymer matrix and waxes. The mechanism by which epidermal cells accumulate and assemble cuticle components in rapidly expanding organs is largely unknown. We have begun to address this question by analyzing the lipid compositional variance, the surface micromorphology, and the transcriptome of epidermal cells in elongating Arabidopsis (Arabidopsis thaliana) stems. The rate of cell elongation is maximal near the apical meristem and decreases steeply toward the middle of the stem, where it is 10 times slower. During and after this elongation, the cuticular wax load and composition remain remarkably constant (32 μg/cm2), indicating that the biosynthetic flux into waxes is closely matched to surface area expansion. By contrast, the load of polyester monomers per unit surface area decreases more than 2-fold from the upper (8 μg/cm2) to the lower (3 μg/cm2) portion of the stem, although the compositional variance is minor. To aid identification of proteins involved in the biosynthesis of waxes and cutin, we have isolated epidermal peels from Arabidopsis stems and determined transcript profiles in both rapidly expanding and nonexpanding cells. This transcriptome analysis was validated by the correct classification of known epidermis-specific genes. The 15% transcripts preferentially expressed in the epidermis were enriched in genes encoding proteins predicted to be membrane associated and involved in lipid metabolism. An analysis of the lipid-related subset is presented.

Although the surface waxes from Arabidopsis thaliana leaves and stems have been thoroughly characterized, the monomer composition of the polyesters of the cuticular membrane has not been analyzed. Delipidated Arabidopsis leaves or stems, when depolymerized under conditions to cleave polyesters, produced typical omega-hydroxy fatty acid cutin monomers such as 16-hydroxy-palmitate, 10,16-dihydroxy-palmitate and 18-hydroxy-9,10-epoxy-stearate. However, the major monomer was octadeca-cis-6, cis-9-diene-1,18-dioate, with lesser amounts of octadec-cis-9-ene-1,18-dioate and hexadeca-1,16-dioate. These dicarboxylates were found predominantly in epidermal peels from Arabidopsis stems and are therefore likely to be associated with the cuticular membrane. They were also found in analyses of canola leaves but were absent in tomato and apple fruit cutins. In the fad2-1 mutant line of Arabidopsis, which has reduced levels of linoleate and linolenate and elevated oleate in cytosolic phospholipids, the amount of octadeca-cis-6, cis-9-diene-1,18-dioate was 50% reduced, with a concomitant increase in octadec-cis-9-ene-1,18-dioate. In a fatb-ko line of Arabidopsis, where the availability of cytosolic palmitate is impaired, there was an 80% loss of C16 monomers and a compensating increase in C18 monomers. The presence of substantial amounts of dicarboxylates in cuticular membranes is unexpected. High amounts of aliphatic dicarboxylates are usually considered as an indicator of suberin, and are reported only as very minor components of cutin. The high level of polyunsaturation is also unusual in cuticles; saturated fatty acid monomers usually predominate, with lesser amounts of monounsaturates. These novel findings for Arabidopsis demonstrate that a broad range of monomer compositions are possible for polyesters of the epidermis.

A new method has been developed to rapidly determine the total inorganic carbon concentration (gaseous [CO2] + aqueous [CO(2)] + [HCO3-] + [CO3(2)-]) in developing seeds. Seeds are rapidly dissected and homogenized in 1 N HCl in gas-tight vials. The headspace gas is then analyzed by infrared gas analysis. Developing rapeseed (Brassica napus L.) and soybean [Glycine max (L.) Merr.] seeds were analyzed and found to have up to 40 and 12 mM total inorganic carbon, respectively. These concentrations are ca. 600-2000-fold higher than in ambient air or values reported for leaves. Carbon dioxide concentrations in rapeseed peaked during the stage of maximum oil synthesis and declined as seeds matured. The consequences for seed metabolism, physiology and carbon economy are discussed.

Plastid envelope proteins from the Arabidopsis nuclear genome were predicted using computational methods. Selection criteria were: first, to find proteins with NH2-terminal plastid-targeting peptides from all annotated open reading frames from Arabidopsis; second, to search for proteins with membrane-spanning domains among the predicted plastidial-targeted proteins; and third, to subtract known thylakoid membrane proteins. Five hundred forty-one proteins were selected as potential candidates of the Arabidopsis plastid inner envelope membrane proteins (AtPEM candidates). Only 34% (183) of the AtPEM candidates could be assigned to putative functions based on sequence similarity to proteins of known function (compared with the 69% function assignment of the total predicted proteins in the genome). Of the 183 candidates with assigned functions, 40% were classified in the category of “transport facilitation,” indicating that this collection is highly enriched in membrane transporters. Information on the predicted proteins, tissue expression data from expressed sequence tags and microarrays, and publicly available T-DNA insertion lines were collected. The data set complements proteomic-based efforts in the increased detection of integral membrane proteins, low-abundance proteins, or those not expressed in tissues selected for proteomic analysis. Digital northern analysis of expressed sequence tags suggested that the transcript levels of most AtPEM candidates were relatively constant among different tissues in contrast to stroma and the thylakoid proteins. However, both digital northern and microarray analyses identified a number of AtPEM candidates with tissue-specific expression patterns.

We have used cDNA microarrays to examine changes in gene expression during Arabidopsis seed development and to compare wild-type and mutant wrinkled1 (wri1) seeds that have an 80% reduction in oil. Between 5 and 13 days after flowering, a period preceding and including the major accumulation of storage oils and proteins, ∼35% of the genes represented on the array changed at least twofold, but a larger fraction (65%) showed little or no change in expression. Genes whose expression changed most tended to be expressed more in seeds than in other tissues. Genes related to the biosynthesis of storage components showed several distinct temporal expression patterns. For example, a number of genes encoding core fatty acid synthesis enzymes displayed a bell-shaped pattern of expression between 5 and 13 days after flowering. By contrast, the expression of storage proteins, oleosins, and other known abscisic acid–regulated genes increased later and remained high. Genes for photosynthetic proteins followed a pattern very similar to that of fatty acid synthesis proteins, implicating a role in CO2 refixation and the supply of cofactors for oil synthesis. Expression profiles of key carbon transporters and glycolytic enzymes reflected shifts in flux from cytosolic to plastid metabolism. Despite major changes in metabolism between wri1 and wild-type seeds

All higher plants express several different acyl carrier protein (ACP) isoforms in a tissue-specific manner. We provide evidence that expression of mRNA for the most abundant ACP isoform in Arabidopsis leaves (ACP4) is increased severalfold by light, whereas mRNA levels for ACP isoforms 2 and 3 are independent of light. The presence of GATA-like motifs in the upstream region of theAcl1.4 gene (encoding for ACP4) and the similarity in light-mediated induction to ferredoxin-A mRNA suggests a direct role of light in Acl1.4 gene activation. Polyribosomal analysis indicated that light also affects the association of ACP transcripts with polysomes, similarly to mRNAs encoding ferredoxin-A. ACP2, ACP3, and ACP4 mRNA levels were also examined in Arabidopsis cell suspension culture and were found to be differentially controlled by metabolic and/or growth derived signals. Comparison of 5′-untranslated regions (UTRs) of ACP mRNAs of diverse plant species revealed two motifs that have been conserved during evolution, a CTCCGCC box and C-T-rich sequences. Fusions of the 5′-UTR sequences of ACP1 and ACP2 to luciferase and expression in transgenic plants indicated that the ACP1 leader contributes to preferential expression in seeds, whereas the ACP2 5′-UTR favored expression in roots. The deletion of 58 bp containing the conserved motifs of the ACP1 5′-UTR resulted in 10- to 20-fold lower gene expression in leaf and seed tissues of transgenic Arabidopsis plants.

[This corrects the article DOI: 10.1371/journal.pgen.1003064.].

Key message We have constructed and annotated a web-based database of over 280 Arabidopsis genes that have characterized mutants associated with Arabidopsis acyl lipid metabolism. Abstract Mutants have played a fundamental role in gene discovery and in understanding the function of genes involved in plant acyl lipid metabolism. The first mutant in Arabidopsis lipid metabolism (fad4) was described in 1985. Since that time, characterization of mutants in more than 280 genes associated with acyl lipid metabolism has been reported. This review provides a brief background and history on identification of mutants in acyl lipid metabolism, an analysis of the distribution of mutants in different areas of acyl lipid metabolism and presents an annotated database (ARALIPmutantDB) of these mutants. The database provides information on the phenotypes of mutants, pathways and enzymes/proteins associated with the mutants, and allows rapid access via hyperlinks to summaries of information about each mutant and to literature that provides information on the lipid composition of the mutants. In addition, the database of mutants is integrated within the ARALIP plant acyl lipid metabolism website (http://aralip.plantbiology.msu.edu) so that information on mutants is displayed on and can be accessed from metabolic pathway maps. Mutants for at least 30 % of the genes in the database have multiple names, which have been compiled here to reduce ambiguities in searches for information. The database should also provide a tool for exploring the relationships between mutants in acyl lipid-related genes and their lipid phenotypes and point to opportunities for further research. Electronic supplementary material The online version of this article (doi:10.1007/s00299-014-1710-8) contains supplementary material, which is available to authorized users.

Petroselinic acid (18:1 delta6) is the major component of the seed oil of Umbelliferae species such as coriander (Coriandrum sativum) as well as Araliaceae and Garryaceae species. This unusual fatty acid is synthesized in plastids by the delta4 desaturation of palmitoyl-acyl carrier protein (16:0-ACP) and subsequent elongation of delta4-hexadecenoyl (16:1 delta4)-ACP. To characterize the enzymatic nature of the elongation reaction, an in vitro assay was developed with 16:1 delta4-ACP and 16:0-ACP as substrates. Extracts from developing coriander seeds elongated 16:1 delta4-ACP in a competitive assay at rates ten-fold greater than that with 16:0-ACP. In contrast, extracts from castor seeds, which do not synthesize petroselinic acid, displayed a strong preference for the elongation of 16:0-ACP rather than 16:1 A4-ACP. In addition, the elongation of 16:1 A4-ACP and 16:0-ACP by coriander seed extracts was strongly inhibited by cerulenin at concentrations as low as 10 microM. This finding suggested that the elongation of 16:1 A4-ACP and 16:0-ACP in coriander seed is catalyzed by a 3-ketoacyl-ACP synthase (KAS) 1-type enzyme(s), rather than a KAS II-type activity that is typically associated with 16:0-ACP elongation. Consistent with this, a cDNA for a diverged form of KAS I was isolated from a cDNA library prepared from developing coriander seed. Using a variety of heterologous probing techniques, no KAS II-type cDNAs could be identified in this library. Multiple alignment of KAS amino acid sequences indicated that, although the polypeptide corresponding to the coriander cDNA is more closely related to KAS I. its active site motif deviates from those found in both KAS I and KAS II enzymes. Also suggestive of a possible role in petroselinic acid synthesis, antibodies raised to the recombinant protein recognize an abundant 45 kDa polypeptide in coriander endosperm that is not detected in coriander leaves. These antibodies also recognize a major band of similar size in developing seeds of English ivy (Hedera helix), an Araliaceae species that also accumulates petroselinic acid in a seed-specific manner.

Now that we know the complete sequence of the Arabidopsis genome, how can plant biologists most effectively use this 130 million-bp database to move toward fuller understanding of plants? Microarray technology is one of a collection of tools that can accelerate our transition from raw data toward

Large-scale single-pass sequencing of cDNAs from different plants has provided an extensive reservoir for the cloning of genes, the evaluation of tissue-specific gene expression, markers for map-based cloning, and the annotation of genomic sequences. Although as of January 2000 GenBank contained over 220,000 entries of expressed sequence tags (ESTs) from plants, most publicly available plant ESTs are derived from vegetative tissues and relatively few ESTs are specifically derived from developing seeds. However, important morphogenetic processes are exclusively associated with seed and embryo development and the metabolism of seeds is tailored toward the accumulation of economically valuable storage compounds such as oil. Here we describe a new set of ESTs from Arabidopsis, which has been derived from 5- to 13-d-old immature seeds. Close to 28,000 cDNAs have been screened by DNA/DNA hybridization and approximately 10,500 new Arabidopsis ESTs have been generated and analyzed using different bioinformatics tools. Approximately 40% of the ESTs currently have no match in dbEST, suggesting many represent mRNAs derived from genes that are specifically expressed in seeds. Although these data can be mined with many different biological questions in mind, this study emphasizes the import of photosynthate into developing embryos, its conversion into seed oil, and the regulation of this pathway.

De novo fatty acid biosynthesis occurs predominantly in plastids. The committed step for this pathway is the production of malonyl-CoA catalysed by acetyl-CoA carboxylase (ACCase). In most plants, plastidial ACCase is a multisubunit complex minimally comprised of four polypeptides, which catalyse two reactions. In the simple oilseed plant, Arabidopsis thaliana two cDNAs encoding biotin carboxyl carrier protein (BCCP) isoforms have been identified. The remaining three subunits of ACCase appear to be single gene members in A. thaliana [Mekhedov, Martinez de Ilarduya and Ohlrogge (2000) Plant Physiol. 122, 389–401]. Transcript and protein analyses indicate that BCCP isoform 1 is constitutively expressed while isoform 2 is predominantly expressed in developing seeds. The apparent masses of constitutive and seed-enriched BCCP isoforms agree with the apparent masses of recombinantly expressed isoforms 1 and 2, respectively. In a related oilseed, Brassica napus multiple putative BCCP polypeptides were also observed in developing seeds. The presence of a divergent class of BCCP genes in A. thaliana and B. napus coincident with appropriately sized biotin-containing proteins expressed specifically in developing seeds, suggests that these BCCPs play an evolutionarily conserved role in oil deposition.

The acyl-acyl carrier protein thioesterase B1 from Arabidopsis (AtFATB1) was previously shown to exhibit in vitro hydrolytic activity for long chain acyl-acyl carrier proteins (P. Dörmann, T.A. Voelker, J.B. Ohlrogge [1995] Arch Biochem Biophys 316: 612–618). In this study, we address the question of which role in fatty acid biosynthesis this enzyme plays within the plant. Over-expression of the AtFATB1 cDNA under a seed-specific promoter resulted in accumulation of high amounts of palmitate (16:0) in seeds. RNA and protein-blot analysis in Arabidopsis and rapeseed (Brassica napus) showed that the endogenous AtFATB1 expression was highest in flowers and lower in leaves. All floral tissues of wild-type plants contained elevated amounts of 16:0, and in the polar lipid fraction of flowers close to 50 mol % of the fatty acids were 16:0. Therefore, flowers contain polar lipids with an unusually high amount of saturated fatty acids as compared to all other plant tissues. Antisense expression of theAtFATB1 cDNA under the cauliflower mosaic virus 35S promoter resulted in a reduction of seed and flower 16:0 content, but no changes in leaf fatty acids. We conclude that the AtFATB1 thioesterase contributes to 16:0 production particularly in flowers, but that additional factors are involved in leaves.

The seed oil of Thunbergia alata has an unusual fatty acid composition which consists of more than 80 % 16:1 Δ6 . This fatty acid is produced in the plastid by the action of a Δ6 palmitoyl (16:0)-ACP desaturase. To examine the biosynthesis of triacylglycerol (TAG) containing high concentrations of this unusual monoenoic fatty acid, endosperm dissected from developing T. alata seeds was labeled with [1- 14 C]-acetate. At early time points (5–15 min), the predominant labeled lipid was PC whereas at later time points (greater than 30 min) TAG became the major labeled lipid. Analysis of the acyl group composition of each lipid revealed that radiolabeled 16:1 Δ6 was highest at early time points in PC while at later time points, it was found to be highest in TAG. Further analysis of the distribution of labeled acyl groups within PC indicated that 16:1 Δ6 at the sn -2 position comprised the majority (55–78 %) of total labeled acyl groups whereas 16:1 Δ6 at the sn -1 position constituted only a small fraction (12–15 %) of the total labeled acyl groups. In contrast, unlabeled PC contained lower amounts of 16:1 Δ6 (16 %) at the sn -2 position. These results are consistent with previous studies suggesting a flux of novel monoenoic acids through PC during TAG biosynthesis, and furthermore imply a stereospecific flux through the sn- 2 position of PC.

Public databases now include vast amounts of recently acquired DNA sequences that are only partially annotated and, furthermore, are often annotated by automated methods that are subject to errors. Maximum information value of these databases can be derived only by further detailed analyses that frequently require careful examination of records in the context of biological functions. In this study we present an example of such an analysis focused on plant glycerolipid synthesis. Public databases were searched for sequences corresponding to 65 plant polypeptides involved in lipid metabolism. Comprehensive search results and analysis of genes, cDNAs and expressed sequence tags (ESTs) are available online (http://www.canr.msu.edu/lgc). Multiple alignments provided a method to estimate the number of genes in gene families. Further analysis of sequences allowed us to tentatively identify several previously undescribed genes in Arabidopsis. For example, two genomic sequences were identified as candidates for the palmitate-specific monogalactosyldiacylglycerol desaturase (FAD5). A candidate genomic sequence for 3-ketoacyl-acyl-carrier protein (ACP) synthase involved in mitochondrial fatty acid biosynthesis was also identified. Biotin carboxyl carrier protein (BCCP) in Arabidopsis is encoded by at least two genes, but the most abundant BCCP transcript so far has not been characterized. The large number (>165,000) of plant ESTs also provides an opportunity to perform “digital northern” comparisons of gene expression levels across many genes. EST abundance in general correlated with biochemical and flux characteristics of the enzymes in Arabidopsis leaf tissue. In a few cases, statistically significant differences in EST abundance levels were observed for enzymes that catalyze similar reactions in fatty acid metabolism. For example, ESTs for the FatB acyl-ACP thioesterase occur 21 times compared with 7 times for FatA acyl-ACP thioesterase, although flux through the FatA reaction is several times higher than through FatB. Such comparisons may provide initial clues toward previously undescribed regulatory phenomena. The abundance of ESTs for ACP compared with that of stearoyl-ACP desaturase and FatB acyl-ACP thioesterase suggests that concentrations of some enzymes of fatty acid synthesis may be higher than their acyl-ACP substrates.

The metabolic factors that determine oil yield in seeds are still not well understood. To begin to examine the limits on triacylglycerol (TAG) production, developingCuphea lanceolata, Ulmus carpinifolia, and Ulmus parvifolia embryos were incubated with factors whose availability might limit oil accumulation. The addition of glycerol or sucrose did not significantly influence the rate of TAG synthesis. However, the rate of 14C-TAG synthesis upon addition of 2.1 mm14C-decanoic acid (10:0) was approximately four times higher than the in vivo rate of TAG accumulation in C. lanceolata and two times higher than the in vivo rate in U. carpinifolia andU. parvifolia. In C. lanceolata embryos, the highest rate of 14C-TAG synthesis (14.3 nmol h−1 embryo−1) was achieved with the addition of 3.6 mm decanoic acid. 14C-Decanoic acid was incorporated equally well in all three acyl positions of TAG. The results suggest that C. lanceolata, U. carpinifolia, and U. parvifolia embryos have sufficient acyltransferase activities and glycerol-3-phosphate levels to support rates of TAG synthesis in excess of those found in vivo. Consequently, the amount of TAG synthesized in these oilseeds may be in part determined by the amount of fatty acid produced in plastids.

The plastid acetyl-coenzyme A carboxylase (ACCase) catalyzes the first committed step of fatty acid synthesis and in most plants is present as a heteromeric complex of at least four different protein subunits: the biotin carboxylase (BC), the biotin carboxyl carrier protein, and the α- and [beta] subunits of the carboxyltransferase. To gain insight into the subunit organization of this heteromeric enzyme complex and to further evaluate the role of ACCase in regulating fatty acid synthesis, BC expression was altered in transgenic plants. Tobacco (Nicotiana tabacum) was transformed with antisense-expression and overexpression tobacco BC constructs, which resulted in the generation of plants with BC levels ranging from 20 to 500% of wild-type levels. Tobacco plants containing elevated or moderate decreases in leaf BC were phenotypically indistinguishable from wild-type plants. However, plants with less than 25% of wild-type BC levels showed severely retarded growth when grown under low-light conditions and a 26% lower leaf fatty acid content than wild-type plants. A comparison of leaf BC and biotin carboxyl carrier protein levels in plants with elevated and decreased BC expression revealed that these two subunits of the plastid ACCase are not maintained in a strict stoichiometric ratio.

Acyl carrier proteins (ACPs) from spinach and from Escherichia coli have been used to demonstrate the utility of proton NMR for comparison of homologous structures. The structure of E. coli ACP had been previously determined and modeled as a rapid equilibrium among multiple conformational forms (Kim and Prestegard, Biochemistry 28:8792–8797, 1989). Spinach ACP showed two slowly exchanging forms and could be manipulated into one form for structural study. Here we compare this single form to postulated multiple forms of E. coli ACP using the limited amount of NOE data available for the spinach protein. A number of long-range NOE contacts were present between homologous residues in both spinach and E. coli ACP, suggesting tertiary structural homology. To allow a more definitive structural comparison, a method was developed to use spinach ACP NOE constraints to search for regions of structural divergence from two postulated forms of E. coli ACP. The homologous regions of the two protein sequences were aligned, additional distance constraints were extracted from the E. coli structure, and these were mapped onto the spinach sequence. These distance constraints were combined with experimental NOE constraints and a distance geometry simulated annealing protocol was used to test for compatibility of the constraints. All of the experimental spinach NOE constraints could be successfully combined with the E. coli data, confirming the general hypothesis of structural homology. A better fit was obtained with one form, suggesting a preferential stabilization of that form in the spinach case. Proteins 27:131–143 © 1997 Wiley-Liss, Inc.

In the plastids of most plants, acetyl-CoA carboxylase (ACCase; EC 6.4.1.2) is a multisubunit complex consisting of biotin carboxylase (BC), biotin-carboxyl carrier protien (BCCP), and carboxytransferase (alpha-CT, beta-CT) subunits. To better understand the regulation of this enzyme, we have isolated and sequenced a BC genomic clone from Arabidopsis and partially characterized its promoter. Fifteen introns were identified. The deduced amino acid sequence of the mature BC protein is highly conserved between Arabidopsis and tobacco (92.6% identity). BC expression was evaluated using northern blots and BC/GUS fusion constructs in transgenic Arabidopsis. GUS activity in the BC/GUS transgenics as well as transcript level of the native gene were both found to be higher in silique and flower than in root and leaf. Analysis of tobacco suspension cells transformed with truncated BC promoter/GUS gene fusions indicated the region from -140 to +147 contained necessary promoter elements which supported basal gene expression. A positive regulatory region was found to be located between -2100 and -140, whereas a negative element was possibly located in the first intron. In addition, several conserved regulatory elements were identified in the BC promoter. Surprisingly, although BC is a low-abundance protein, the expression of BC/GUS fusion constructs was similar to 35S/GUS constructs.

Progress is reported in these areas: acyl-editing in initial eukaryotic lipid assembly in soybean seeds; identification and characterization of two Arabidopsis thaliana lysophosphatidyl acyltransferases with preference for lysophosphatidylethanolamine; and characterization and subcellular distribution of lysolipid acyl transferase activity of pea leaves.

Enhancement of acyl-CoA–dependent triacylglycerol (TAG) synthesis in vegetative tissues is widely discussed as a potential avenue to increase the energy density of crops. Here, we report the identification and characterization of Chlamydomonas reinhardtii diacylglycerol acyltransferase type two (DGTT) enzymes and use DGTT2 to alter acyl carbon partitioning in plant vegetative tissues. This enzyme can accept a broad range of acyl-CoA substrates, allowing us to interrogate different acyl pools in transgenic plants. Expression of DGTT2 in Arabidopsis thaliana increased leaf TAG content, with some molecular species containing very-long-chain fatty acids. The acyl compositions of sphingolipids and surface waxes were altered, and cutin was decreased. The increased carbon partitioning into TAGs in the leaves of DGTT2-expressing lines had little effect on transcripts of the sphingolipid/wax/cutin pathway, suggesting that the supply of acyl groups for the assembly of these lipids is not transcriptionally adjusted. Caterpillars of the generalist herbivore Spodoptera exigua reared on transgenic plants gained more weight. Thus, the nutritional value and/or energy density of the transgenic lines was increased by ectopic expression of DGTT2 and acyl groups were diverted from different pools into TAGs, demonstrating the interconnectivity of acyl metabolism in leaves.

Unicellular marine algae have promise for providing sustainable and scalable biofuel feedstocks, although no single species has emerged as a preferred organism. Moreover, adequate molecular and genetic resources prerequisite for the rational engineering of marine algal feedstocks are lacking for most candidate species. Heterokonts of the genus Nannochloropsis naturally have high cellular oil content and are already in use for industrial production of high-value lipid products. First success in applying reverse genetics by targeted gene replacement makes Nannochloropsis oceanica an attractive model to investigate the cell and molecular biology and biochemistry of this fascinating organism group. Here we present the assembly of the 28.7 Mb genome of N. oceanica CCMP1779. RNA sequencing data from nitrogen-replete and nitrogen-depleted growth conditions support a total of 11,973 genes, of which in addition to automatic annotation some were manually inspected to predict the biochemical repertoire for this organism. Among others, more than 100 genes putatively related to lipid metabolism, 114 predicted transcription factors, and 109 transcriptional regulators were annotated. Comparison of the N. oceanica CCMP1779 gene repertoire with the recently published N. gaditana genome identified 2,649 genes likely specific to N. oceanica CCMP1779. Many of these N. oceanica–specific genes have putative orthologs in other species or are supported by transcriptional evidence. However, because similarity-based annotations are limited, functions of most of these species-specific genes remain unknown. Aside from the genome sequence and its analysis, protocols for the transformation of N. oceanica CCMP1779 are provided. The availability of genomic and transcriptomic data for Nannochloropsis oceanica CCMP1779, along with efficient transformation protocols, provides a blueprint for future detailed gene functional analysis and genetic engineering of Nannochloropsis species by a growing academic community focused on this genus., Author Summary Algae are a highly diverse group of organisms that have become the focus of renewed interest due to their potential for producing biofuel feedstocks, nutraceuticals, and biomaterials. Their high photosynthetic yields and ability to grow in areas unsuitable for agriculture provide a potential sustainable alternative to using traditional agricultural crops for biofuels. Because none of the algae currently in use have a history of domestication, and bioengineering of algae is still in its infancy, there is a need to develop algal strains adapted to cultivation for industrial large-scale production of desired compounds. Model organisms ranging from mice to baker's yeast have been instrumental in providing insights into fundamental biological structures and functions. The algal field needs versatile models to develop a fundamental understanding of photosynthetic production of biomass and valuable compounds in unicellular, marine, oleaginous algal species. To contribute to the development of such an algal model system for basic discovery, we sequenced the genome and two sets of transcriptomes of N. oceanica CCMP1779, assembled the genomic sequence, identified putative genes, and began to interpret the function of selected genes. This species was chosen because it is readily transformable with foreign DNA and grows well in culture.

While suberin is an insoluble heteropolymer, a number of soluble lipids can be extracted by rapid chloroform dipping of roots. These extracts include esters of saturated long-chain primary alcohols and hydroxycinnamic acids. Such fatty alcohols and hydroxycinnamic acids are also present in suberin. We demonstrate that alkyl coumarates and caffeates, which are the major components of Arabidopsis (Arabidopsis thaliana) root waxes, are present primarily in taproots. Previously we identified ALIPHATIC SUBERIN FERULOYL TRANSFERASE (At5g41040), a HXXXD-type acyltransferase (BAHD family), responsible for incorporation of ferulate into aliphatic suberin of Arabidopsis. However, aliphatic suberin feruloyl transferase mutants were unaffected in alkyl hydroxycinnamate ester root wax composition. Here we identify a closely related gene, At5g63560, responsible for the synthesis of a subset of alkyl hydroxycinnamate esters, the alkyl caffeates. Transgenic plants harboring PAt5g63560::YFP fusions showed transcriptional activity in suberized tissues. Knockout mutants of At5g63560 were severely reduced in their alkyl caffeate but not alkyl coumarate content. Recombinant At5g63560p had greater acyltransferase activity when presented with caffeoyl-Coenzyme A (CoA) substrate, thus we have named this acyltransferase FATTY ALCOHOL:CAFFEOYL-CoA CAFFEOYL TRANSFERASE. Stress experiments revealed elevated alkyl coumarate content in root waxes of NaCl-treated wild-type and fatty alcohol:caffeoyl-CoA caffeoyl transferase plants. We further demonstrate that FATTY ACYL-CoA REDUCTASEs (FARs) FAR5 (At3g44550), FAR4 (At3g44540), and FAR1 (At5g22500) are required for the synthesis of C18, C20, and C22 alkyl hydroxycinnamates, respectively. Collectively, these results suggest that multiple acyltransferases are utilized for the synthesis of alkyl hydroxycinnamate esters of Arabidopsis root waxes and that FAR1/4/5 provide the fatty alcohols required for alkyl hydroxycinnamate synthesis.

Oil palm can accumulate up to 90% oil in its mesocarp, the highest level observed in the plant kingdom. In contrast, the closely related date palm accumulates almost exclusively sugars. To gain insight into the mechanisms that lead to such an extreme difference in carbon partitioning, the transcriptome and metabolite content of oil palm and date palm were compared during mesocarp development. Compared with date palm, the high oil content in oil palm was associated with much higher transcript levels for all fatty acid synthesis enzymes, specific plastid transporters, and key enzymes of plastidial carbon metabolism, including phosphofructokinase, pyruvate kinase, and pyruvate dehydrogenase. Transcripts representing an ortholog of the WRI1 transcription factor were 57-fold higher in oil palm relative to date palm and displayed a temporal pattern similar to its target genes. Unexpectedly, despite more than a 100-fold difference in flux to lipids, most enzymes of triacylglycerol assembly were expressed at similar levels in oil palm and date palm. Similarly, transcript levels for all but one cytosolic enzyme of glycolysis were comparable in both species. Together, these data point to synthesis of fatty acids and supply of pyruvate in the plastid, rather than acyl assembly into triacylglycerol, as a major control over the storage of oil in the mesocarp of oil palm. In addition to greatly increasing molecular resources devoted to oil palm and date palm, the combination of temporal and comparative studies illustrates how deep sequencing can provide insights into gene expression patterns of two species that lack genome sequence information.

The introduction of dwarfing traits into crops was a major factor in increased grain yields during the "Green Revolution." In most cases those traits were the consequence of altered synthesis or response to the gibberellin (GA) plant hormones. Our current understanding of GA synthesis and physiology has been facilitated by the characterization of mutants. To introduce concepts about GA hormone physiology and plant transformation in an undergraduate laboratory course we have used ga5, a semi-dwarf Arabidopsis mutant with reduced activity of GA 20-oxidase. In this laboratory exercise, Arabidopsis ga5 mutant plants are transformed by the floral-dip method using Agrobacterium tumefaciens carrying plasmid constructs conferring kanamycin resistance and containing the GA5 gene. Within 4 weeks, seeds of transformed plants can be easily screened by antibiotic resistance on plates. After transfer to soil the dwarf mutant plants transformed with a wild-type version of the gene show normal size. In addition to offering a visual understanding of the effect of GA on stem elongation, students learn additional techniques in this experiment, including PCR and agarose gel electrophoresis. This experiment is cost effective and can be completed within a 4-month term.

Date palm is one of the most economically important woody crops cultivated in the Middle East and North Africa and is a good candidate for improving agricultural yields in arid environments. Nonetheless, long generation times (5-8 years) and dioecy (separate male and female trees) have complicated its cultivation and genetic analysis. To address these issues, we assembled a draft genome for a Khalas variety female date palm, the first publicly available resource of its type for a member of the order Arecales. The ∼380 Mb sequence, spanning mainly gene-rich regions, includes >25,000 gene models and is predicted to cover ∼90% of genes and ∼60% of the genome. Sequencing of eight other cultivars, including females of the Deglet Noor and Medjool varieties and their backcrossed males, identified >3.5 million polymorphic sites, including >10,000 genic copy number variations. A small subset of these polymorphisms can distinguish multiple varieties. We identified a region of the genome linked to gender and found evidence that date palm employs an XY system of gender inheritance.

The extent to which acetyl-acyl carrier protein (acetyl-ACP) is an intermediate in fatty acid biosynthesis was examined. Acetyl-ACP was the least effective primer of fatty acid synthesis by spinach extracts when compared to acetyl-CoA, butyryl-ACP or hexanoyl-ACP. Furthermore, the rate of acetyl-ACP-primed fatty acid synthesis was inhibited significantly by cerulenin, indicating that the slow utilization of acetyl-ACP was predominantly by 3-oxoacyl-ACP synthase I. In light-incubated isolated chloroplasts with high rates of fatty acid synthesis (greater than 800 nmol.h-1.mg chlorophyll-1), the rate of acetyl-ACP metabolism was at least 10-30-fold slower than the rate of butyryl-ACP metabolism. The relatively slow metabolism of acetyl-ACP provided in situ evidence that (a) butyryl-ACP was formed principally from condensation of malonyl-ACP with acetyl-CoA and (b) acetyl-ACP was a minor participant in fatty acid biosynthesis.