Kinetic complexities of triacylglycerol accumulation in developing embryos from Camelina sativa provide evidence for multiple biosynthetic systems.

Quantitative flux maps describing glycerolipid synthesis can be important tools for rational engineering of lipid content and composition in oilseeds. Lipid accumulation in cultured embryos of Camelina sativa is known to mimic that of seeds in terms of rate of lipid synthesis and composition. To assess the kinetic complexity of the glycerolipid flux network, cultured embryos were incubated with [14C/13C]glycerol, and initial and steady state rates of [14C/13Cglyceryl] lipid accumulation were measured. At steady state, the linear accumulations of labeled lipid classes matched those expected from mass compositions. The system showed an apparently simple kinetic precursor-product relationship between the intermediate pool, dominated by diacylglycerol (DAG) and phosphatidylcholine (PC), and the triacylglycerol (TAG) product. We also conducted isotopomer analyses on hydrogenated lipid class species. [13C3glyceryl] labeling of DAG and PC, together with estimates of endogenous [12C3glyceryl] dilution, showed that each biosynthetically active lipid pool is ∼30% of the total by moles. This validates the concept that lipid sub-pools can describe lipid biosynthetic networks. By tracking the kinetics of [13C3glyceryl] and [13C2acyl] labeling, we observed two distinct TAG synthesis components. The major TAG synthesis flux (∼75%) was associated with >95% of the DAG/PC intermediate pool, with little glycerol being metabolized to fatty acids, and with little dilution from endogenous glycerol; a smaller flux exhibited converse characteristics. This kinetic heterogeneity was further explored using postlabeling embryo dissection and differential lipid extractions. The minor flux was tentatively localized to surface cells across the whole embryo. Such heterogeneity must be recognized in order to construct accurate gene expression patterns and metabolic networks describing lipid biosynthesis in developing embryos.

Quantitative analysis of glycerol in dicarboxylic acid-rich cutins provides insights into Arabidopsis cutin structure.

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.

Lipid analysis of developing Camelina sativa seeds and cultured embryos.

Camelina sativa is a cultivated oilseed rich in triacylglycerols containing oleic, linoleic, α-linolenic and eicosenoic acids. As it holds promise as a model species, its lipid synthesis was characterized in vivo and in culture. Lipid accumulates at a maximum rate of about 26 µg/day/seed (11.5 mg lipid/day/g fresh seed weight), a rate comparable with other oilseeds. Noteworthy is a late stage surge in α-linolenic acid accumulation. Small amounts of unusual epoxy and hydroxy fatty acids are also present in the triacylglycerols. These include 15,16-epoxy- and 15-hydroxy-octadecadienoic acids and homologous series of ω7-hydroxy-alk-ω9-enoic and ω9/10-hydroxy-alkanoic acids. Mid-maturation embryos cultured in vitro have growth and lipid deposition rates and fatty acid compositions that closely match that of seeds, but extended culture periods allow these rates to rise and surpass those observed in planta. Optimized thin layer chromatography systems for characterization of labeled products from acetate or glycerol labeling are described. Glycerol label is only found in acylglycerols, largely as the intact glyceryl backbone, but acetate can label acyl groups and sterols, the latter to a much higher relative specific activity. This presumably occurs because mevalonic acid precursor is derived from the non-plastid pool of acetyl-CoA that is also the source for malonyl-CoA to drive FAE1-dependent chain elongation. Particular attention has been paid to the separation of sterols and diacylglycerols, and to hydrogenation of triacylglycerols to simplify their analysis. These improved methods will allow more accurate analyses of the fluxes of lipid metabolism in cultured plant embryos.

Lipid labeling from acetate or glycerol in cultured embryos of Camelina sativa seeds: A tale of two substrates.

Studies on the metabolism of lipids in seeds frequently use radiolabeled acetate and glycerol supplied to excised developing seeds to track the biosynthesis of acyl and lipid head groups, respectively. Such experiments are generally restricted to shorter time periods and the results may not quantitatively reflect in planta rates. These limitations can be removed by using cultured embryos, provided they mimic growth and lipid deposition observed for embryos in planta. Mid-maturation embryos from Camelina sativa were cultured in vitro to assess the use of sufficient acetate or glycerol concentrations and labeling periods for stable isotope labeling and mass spectrometric detection. Maximum incorporation of exogenous acetate into fatty acids occurred at 1mM and above. This provides about 5% of the total carbon flux entering fatty acids, enough for (13)C isotopomer analysis while maintaining normal biosynthetic rates for over 24h. Labeling analysis indicates that acetate reports lipid metabolism uniformly across the embryo. At higher acetate concentrations with longer incubations, the rate of fatty acid synthesis is reduced and the composition of newly synthesized fatty acids changes. While the mole fractions of oleate that undergo Δ12-desaturation or elongation are independent of biosynthetic flux, Δ15-desaturation shows a bimodal dependence. These observations are consistent with changes occurring in planta over seed development. Incorporation rates of the glyceryl moiety into lipids saturates at about 0.5mM exogenous glycerol. At saturation, the exogenous glycerol almost completely replaces the endogenous supply of glycerol-3-phosphate without affecting net lipid accumulation or fatty acid composition. It is concluded that acetate and glycerol labeling of cultured C. sativa embryos can provide an accurate representation of lipid metabolism in embryos in vivo, and that in Camelina embryos glycerol-3-phosphate levels do not co-limit triacylglycerol synthesis.

Analysis of aliphatic waxes associated with root periderm or exodermis from eleven plant species.

Aliphatic waxes can be found in association with suberized tissues, including roots. Non-polar lipids were isolated by rapid solvent extraction of mature regions of intact roots from eleven angiosperms, including both monocots and dicots. The majority of roots analyzed were taproots or tuberous taproots that had undergone secondary growth and thus were covered by a suberized periderm. The exceptions therein were maize (Zea mays L.) and rice (Oryza sativa L.), which present a suberized exodermis. The analysis herein focused on aliphatic waxes, with particular emphasis on alkyl hydroxycinnamates (AHCs). AHCs were widely distributed, absent from only one species, were found in both aerial and subterranean portions of tuberous taproots, and were associated with the fibrous roots of both maize and rice. Most species also contained monoacylglycerols, fatty alcohols and/or free fatty acids. Carrot (Daucus carrota L.) was the outlier, containing only free fatty acids, sterols, and polyacetylenes as identified components. Sterols were the only ubiquitous component across all roots analyzed. Monoacylglycerols of ω-hydroxy fatty acids were present in maize and rice root waxes. For species within the Brassiceae, wax compositions varied between subspecies or varieties and between aerial and subterranean portions of taproots. In addition, reduced forms of photo-oxidation products of ω-hydroxy oleate and its corresponding dicarboxylic acid (10,18-dihydroxy-octadec-8-enoate, 9,18-dihydroxy-octadec-10-enoate and 9-hydroxyoctadec-10-ene-1,18-dioate) were identified as naturally occurring suberin monomers in rutabaga (Brassica napus subsp. rapifera Metzg.) periderm tissues.

Metabolic engineering of oilseed crops to produce high levels of novel acetyl glyceride oils with reduced viscosity, freezing point and calorific value.

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.

The impact of automation on pharmacy staff experience of workplace stressors.

OBJECTIVE: Determine the effect of installing an original pack automated dispensing system (ADS) on staff experience of occupational stressors. METHODS: Pharmacy staff in a National Health Service hospital in Wales, UK, were administered an anonymous occupational stressor questionnaire pre- (n = 45) and post-automation (n = 32). Survey responses pre- and post-automation were compared using Mann-Whitney U test. Statistical significance was P ≤ 0.05. Four focus groups were conducted (two groups of accredited checking technicians (ACTs) (group 1: n = 4; group 2: n = 6), one group of pharmacists (n = 17), and one group of technicians (n = 4) post-automation to explore staff experiences of occupational stressors. Focus group transcripts were analysed according to framework analysis. KEY FINDINGS: Survey response rate pre-automation was 78% (n = 35) and 49% (n = 16) post-automation. Automation had a positive impact on staff experience of stress (P = 0.023), illogical workload allocation (P = 0.004) and work-life balance (P = 0.05). All focus-group participants reported that automation had created a spacious working environment. Pharmacists and ACTs reported that automation had enabled the expansion of their roles. Technicians felt like 'production-line workers.' Robot malfunction was a source of stress. CONCLUSION: The findings suggest that automation had a positive impact on staff experience of stressors, improving working conditions and workload. Technicians reported that ADS devalued their skills. When installing ADS, pharmacy managers must consider the impact of automation on staff. Strategies to reduce stressors associated with automation include rotating staff activities and role expansions.

The impact of automation on workload and dispensing errors in a hospital pharmacy.

OBJECTIVES: To determine the effect of installing an original-pack automated dispensing system (ADS) on dispensary workload and prevented dispensing incidents in a hospital pharmacy. METHODS: Data on dispensary workload and prevented dispensing incidents, defined as dispensing errors detected and reported before medication had left the pharmacy, were collected over 6 weeks at a National Health Service hospital in Wales before and after the installation of an ADS. Workload was measured by non-participant observation using the event recording technique. Prevented dispensing incidents were self-reported by pharmacy staff on standardised forms. Median workloads (measured as items dispensed/person/hour) were compared using Mann-Whitney U tests and rate of prevented dispensing incidents were compared using Chi-square test. Spearman's rank correlation was used to examine the association between workload and prevented dispensing incidents. A P value of ≤0.05 was considered statistically significant. KEY FINDINGS: Median dispensary workload was significantly lower pre-automation (9.20 items/person/h) compared to post-automation (13.17 items/person/h, P < 0.001). Rate of prevented dispensing incidents was significantly lower post-automation (0.28%) than pre-automation (0.64%, P < 0.0001) but there was no difference (P = 0.277) between the types of dispensing incidents. A positive association existed between workload and prevented dispensing incidents both pre- (ρ = 0.13, P = 0.015) and post-automation (ρ = 0.23, P < 0.001). Dispensing incidents were found to occur during prolonged periods of moderate workload or after a busy period. CONCLUSION: Study findings suggest that automation improves dispensing efficiency and reduces the rate of prevented dispensing incidents. It is proposed that prevented dispensing incidents frequently occurred during periods of high workload due to involuntary automaticity. Prevented dispensing incidents occurring after a busy period were attributed to staff experiencing fatigue after-effects.

A land-plant-specific glycerol-3-phosphate acyltransferase family in Arabidopsis: substrate specificity, sn-2 preference, and evolution.

Arabidopsis (Arabidopsis thaliana) has eight glycerol-3-phosphate acyltransferase (GPAT) genes that are members of a plant-specific family with three distinct clades. Several of these GPATs are required for the synthesis of cutin or suberin. Unlike GPATs with sn-1 regiospecificity involved in membrane or storage lipid synthesis, GPAT4 and -6 are unique bifunctional enzymes with both sn-2 acyltransferase and phosphatase activity resulting in 2-monoacylglycerol products. We present enzymology, pathway organization, and evolutionary analysis of this GPAT family. Within the cutin-associated clade, GPAT8 is demonstrated as a bifunctional sn-2 acyltransferase/phosphatase. GPAT4, -6, and -8 strongly prefer C16:0 and C18:1 ω-oxidized acyl-coenzyme As (CoAs) over unmodified or longer acyl chain substrates. In contrast, suberin-associated GPAT5 can accommodate a broad chain length range of ω-oxidized and unsubstituted acyl-CoAs. These substrate specificities (1) strongly support polyester biosynthetic pathways in which acyl transfer to glycerol occurs after oxidation of the acyl group, (2) implicate GPAT specificities as one major determinant of cutin and suberin composition, and (3) argue against a role of sn-2-GPATs (Enzyme Commission 2.3.1.198) in membrane/storage lipid synthesis. Evidence is presented that GPAT7 is induced by wounding, produces suberin-like monomers when overexpressed, and likely functions in suberin biosynthesis. Within the third clade, we demonstrate that GPAT1 possesses sn-2 acyltransferase but not phosphatase activity and can utilize dicarboxylic acyl-CoA substrates. Thus, sn-2 acyltransferase activity extends to all subbranches of the Arabidopsis GPAT family. Phylogenetic analyses of this family indicate that GPAT4/6/8 arose early in land-plant evolution (bryophytes), whereas the phosphatase-minus GPAT1 to -3 and GPAT5/7 clades diverged later with the appearance of tracheophytes.

Identification of an Arabidopsis fatty alcohol:caffeoyl-Coenzyme A acyltransferase required for the synthesis of alkyl hydroxycinnamates in root waxes.

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 P(At5g63560)::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.

Pleiotropic phenotypes of the sticky peel mutant provide new insight into the role of CUTIN DEFICIENT2 in epidermal cell function in tomato.

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.

Solving the puzzles of cutin and suberin polymer biosynthesis.

Cutin and suberin are insoluble lipid polymers that provide critical barrier functions to the cell wall of certain plant tissues, including the epidermis, endodermis and periderm. Genes that are specific to the biosynthesis of cutins and/or aliphatic suberins have been identified, mainly in Arabidopsis thaliana. They notably encode acyltransferases, oxidases and transporters, which may have either well-defined or more debatable biochemical functions. However, despite these advances, important aspects of cutin and suberin synthesis remain obscure. Central questions include whether fatty acyl monomers or oligomers are exported, and the extent of extracellular assembly and attachment to the cell wall. These issues are reviewed. Greater emphasis on chemistry and biochemistry will be required to solve these unknowns and link structure with function.

Measuring dispensary workload: a comparison of the event recording and direct time techniques.

OBJECTIVES: To compare dispensary workload, determined using the Welsh benchmarking event recording technique and the direct time technique, at two district general UK National Health Service hospitals within different university local health Boards (hospital A--manual dispensing system; hospital B--automated dispensing system). METHODS: Data on dispensary workload were collected, over a period of 6 weeks (hospital A: 8 May-18 June 2007; hospital B: 1 October-11 November 2007), by a non-participant observer using two simultaneous methods of workload measurement: direct time and event recording. Direct time technique involved timing each task involved in dispensing a sample of prescriptions from receipt to issue of dispensed medicines to patients. Welsh benchmarking event recording involved continuously logging staff activities that deviated from the dispensary rota on a data collection form to enable calculation of total staff time involved in dispensing activities. Data on number of items dispensed were obtained from the pharmacy computer system and also by manual counting of prescription items. The mean dispensary workloads were calculated as the number of items dispensed per person per hour. Two-sample t-tests were used to compare dispensary workload measurements determined using direct time and event recording technique reported by each individual hospital. Mean workloads for hospitals A and B were compared using a two-sample t-test. Statistical significance was taken as P≤0.05. KEY FINDINGS: Hospital A was associated with a lower workload (direct time: 7.27±7.16 items per person per hour; event recording: 9.57±10.6 items per person per hour). In contrast, hospital B gave a higher workload (direct time: 11.93±8.3 items per person per hour; event recording: 12.6±8.80 items per person per hour). There was a significant difference between workload (direct time: P<0.01; event recording: P<0.01) reported for both hospitals. The direct time and event recording techniques produced consistent results at each hospital (hospital A: t=0.02, P=0.99; hospital B: t=0.004, P=0.1). CONCLUSION: The direct time and Welsh benchmarking event recording techniques produced consistent results at both hospitals. Thus the Welsh benchmarking event recording technique is a valid and reproducible method of measuring dispensary workload. Hospital B (automated) had a higher workload than hospital A (manual). Further work is required to investigate the impact of automation on dispensary workload.

A distinct type of glycerol-3-phosphate acyltransferase with sn-2 preference and phosphatase activity producing 2-monoacylglycerol.

The first step in assembly of membrane and storage glycerolipids is acylation of glycerol-3-phosphate (G3P). All previously characterized membrane-bound, eukaryotic G3P acyltransferases (GPATs) acylate the sn-1 position to produce lysophosphatidic acid (1-acyl-LPA). Cutin is a glycerolipid with omega-oxidized fatty acids and glycerol as integral components. It occurs as an extracellular polyester on the aerial surface of all plants, provides a barrier to pathogens and resistance to stress, and maintains organ identity. We have determined that Arabidopsis acyltransferases GPAT4 and GPAT6 required for cutin biosynthesis esterify acyl groups predominantly to the sn-2 position of G3P. In addition, these acyltransferases possess a phosphatase domain that results in sn-2 monoacylglycerol (2-MAG) rather than LPA as the major product. Such bifunctional activity has not been previously described in any organism. The possible roles of 2-MAGs as intermediates in cutin synthesis are discussed. GPAT5, which is essential for the accumulation of suberin aliphatics, also exhibits a strong preference for sn-2 acylation. However, phosphatase activity is absent and 2-acyl-LPA is the major product. Clearly, plant GPATs can catalyze more reactions than the sn-1 acylation by which they are currently categorized. Close homologs of GPAT4-6 are present in all land plants, but not in animals, fungi or microorganisms (including algae). Thus, these distinctive acyltransferases may have been important for evolution of extracellular glycerolipid polymers and adaptation of plants to a terrestrial environment. These results provide insight into the biosynthetic assembly of cutin and suberin, the two most abundant glycerolipid polymers in nature.

A distinct DGAT with sn-3 acetyltransferase activity that synthesizes unusual, reduced-viscosity oils in Euonymus and transgenic seeds.

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 diacylglycerol acetyltransferase). 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.

Nanoridges that characterize the surface morphology of flowers require the synthesis of cutin polyester.

Distinctive nanoridges on the surface of flowers have puzzled plant biologists ever since their discovery over 75 years ago. Although postulated to help attract insect pollinators, the function, chemical nature, and ontogeny of these surface nanostructures remain uncertain. Studies have been hampered by the fact that no ridgeless mutants have been identified. Here, we describe two mutants lacking nanoridges and define the biosynthetic pathway for 10,16-dihydroxypalmitate, a major cutin monomer in nature. Using gene expression profiling, two candidates for the formation of floral cutin were identified in the model plant Arabidopsis thaliana: the glycerol-3-phosphate acyltransferase 6 (GPAT6) and a member of a cytochrome P450 family with unknown biological function (CYP77A6). Plants carrying null mutations in either gene produced petals with no nanoridges and no cuticle could be observed by either scanning or transmission electron microscopy. A strong reduction in cutin content was found in flowers of both mutants. In planta overexpression suggested GPAT6 preferentially uses palmitate derivatives in cutin synthesis. Comparison of cutin monomer profiles in knockouts for CYP77A6 and the fatty acid omega-hydroxylase CYP86A4 provided genetic evidence that CYP77A6 is an in-chain hydroxylase acting subsequently to CYP86A4 in the synthesis of 10,16-dihydroxypalmitate. Biochemical activity of CYP77A6 was demonstrated by production of dihydroxypalmitates from 16-hydroxypalmitate, using CYP77A6-expressing yeast microsomes. These results define the biosynthetic pathway for an abundant and widespread monomer of the cutin polyester, show that the morphology of floral surfaces depends on the synthesis of cutin, and identify target genes to investigate the function of nanoridges in flower biology.

Identification of an Arabidopsis feruloyl-coenzyme A transferase required for suberin synthesis.

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 omega-hydroxy and alpha,omega-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 omega-hydroxyl groups. Recombinant AT5G41040p catalyzed acyl transfer from feruloyl-coenzyme A to omega-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 alpha,omega-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.

Analysis of acyl fluxes through multiple pathways of triacylglycerol synthesis in developing soybean embryos.

The reactions leading to triacylglycerol (TAG) synthesis in oilseeds have been well characterized. However, quantitative analyses of acyl group and glycerol backbone fluxes that comprise extraplastidic phospholipid and TAG synthesis, including acyl editing and phosphatidylcholine-diacylglycerol interconversion, are lacking. To investigate these fluxes, we rapidly labeled developing soybean (Glycine max) embryos with [(14)C]acetate and [(14)C]glycerol. Cultured intact embryos that mimic in planta growth were used. The initial kinetics of newly synthesized acyl chain and glycerol backbone incorporation into phosphatidylcholine (PC), 1,2-sn-diacylglycerol (DAG), and TAG were analyzed along with their initial labeled molecular species and positional distributions. Almost 60% of the newly synthesized fatty acids first enter glycerolipids through PC acyl editing, largely at the sn-2 position. This flux, mostly of oleate, was over three times the flux of nascent [(14)C]fatty acids incorporated into the sn-1 and sn-2 positions of DAG through glycerol-3-phosphate acylation. Furthermore, the total flux for PC acyl editing, which includes both nascent and preexisting fatty acids, was estimated to be 1.5 to 5 times the flux of fatty acid synthesis. Thus, recycled acyl groups (16:0, 18:1, 18:2, and 18:3) in the acyl-coenzyme A pool provide most of the acyl chains for de novo glycerol-3-phosphate acylation. Our results also show kinetically distinct DAG pools. DAG used for TAG synthesis is mostly derived from PC, whereas de novo synthesized DAG is mostly used for PC synthesis. In addition, two kinetically distinct sn-3 acylations of DAG were observed, providing TAG molecular species enriched in saturated or polyunsaturated fatty acids.

Building lipid barriers: biosynthesis of cutin and suberin.

Cutin and suberin are the polymer matrices for lipophilic cell wall barriers. These barriers control the fluxes of gases, water and solutes, and also play roles in protecting plants from biotic and abiotic stresses and in controlling plant morphology. Although they are ubiquitous, cutin and suberin are the least understood of the major plant extracellular polymers. The use of forward and reverse genetic approaches in Arabidopsis has led to the identification of oxidoreductase and acyltransferase genes involved in the biosynthesis of these polymers. However, major questions about the underlying polymer structure, biochemistry, and intracellular versus extracellular assembly remain to be resolved. The analysis of plant lines with modified cutins and suberins has begun to reveal the inter-relationships between the composition and function of these polymers.

Deposition and localization of lipid polyester in developing seeds of Brassica napus and Arabidopsis thaliana.

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.