紫苏种子脂肪酸组成及合成代谢研究进展

紫苏是一种新型油料作物,种子含油量为35%左右,紫苏籽油脂肪酸组成丰富,含有棕榈酸(16:0)、硬脂酸(18:0)、油酸(18:1)、亚油酸(18:2)和α-亚麻酸(18:3)等,其中α-亚麻酸(ALA)含量高达60%,广泛用于功能性保健食品、药物及油脂化工业.介绍紫苏种子脂肪酸组成及合成代谢基本途径,对近年来脂肪酸合成代谢基因工程研究进行概述与展望.     

Accumulation of Phosphatidic Acid Increases Vancomycin Resistance in Escherichia coli

In Gram-negative bacteria, lipopolysaccharide (LPS) contributes to the robust permeability barrier of the outer membrane, preventing entry of toxic molecules such as antibiotics. Mutations in lptD, the beta-barrel component of the LPS transport and assembly machinery, compromise LPS assembly and result in increased antibiotic sensitivity. Here, we report rare vancomycin-resistant suppressors that improve barrier function of a subset of lptD mutations. We find that all seven suppressors analyzed mapped to the essential gene cdsA, which is responsible for the conversion of phosphatidic acid to CDP-diacylglycerol in phospholipid biosynthesis. These cdsA mutations cause a partial loss of function and, as expected, accumulate phosphatidic acid. We show that this suppression is not confined to mutations that cause defects in outer membrane biogenesis but rather that these cdsA mutations confer a general increase in vancomycin resistance, even in a wild-type cell. We use genetics and quadrupole time of flight (Q-TOF) liquid chromatography-mass spectrometry (LC-MS) to show that accumulation of phosphatidic acid by means other than cdsA mutations also increases resistance to vancomycin. We suggest that increased levels of phosphatidic acid change the physical properties of the outer membrane to impede entry of vancomycin into the periplasm, hindering access to its target, an intermediate required for the synthesis of the peptidoglycan cell wall.     

Accumulation Pattern of IgG Antibodies and Fab Fragments in Transgenic Arabidopsis thaliana Plants

For the further optimization of antibody expression in plants,it is essential to determine the final accumulation sites ofplant-made antibodies. Previously, we have shown that, uponsecretion, IgG antibodies and F_ab fragments can be detectedin the intercellular spaces of leaf mesophyil cells of transgenicArabidopsis thaliana plants. However, immunofluorescence microscopyshowed that this is probably not their final accumulation site.In leaves, IgG and F_abfragments accumulate also at the interiorside of the epidermal cell layers and in xylem vessels. Theseaccumulation sites correspond with the leaf regions where waterof the transpiration stream is entering a space impermeableto the proteins or where water is evaporating. In roots, plant-madeF_ab fragments accumulate in intercellular spaces of cortex cells,in the cytoplasm of pericycle and, to a lesser extent, endodermiscells, and in cells of the vascular cylinder. In other words,antibody accumulation occurs at the sites where water passeson its radial pathway towards and within the vascular bundle.Taken together, our results suggest that, upon secretion ofplant-made antibodies or F_ab fragments, a large proportion ofthese proteins are transported in the apoplast of A. thaliana,possibly by the water flow in the transpiration stream. ⁴Corresponding author. Fax 32-9-2645349; e-mail: anpic{at}gengenp.rug.ac.be     

七种植物籽油中脂肪酸成分的比较分析(英文)

为寻求新的食用油资源,发展了一种快速可靠的气相色谱-质谱联用方法,用于植物籽油中脂肪酸成分的定性鉴定和含量测定。所建立的方法成功用于葡萄籽、南瓜籽和猕猴桃籽等七种植物籽油中的棕榈酸、十八烷酸、油酸、亚油酸和α-亚麻酸的定性定量分析。结果表明,刺葡萄籽油、普通葡萄籽油、国外葡萄籽油、南瓜籽油、枸杞籽油和西番莲籽油均具有相似的脂肪酸谱,尽管其中它们所含上述五种脂肪酸含量不同,由于均存在人体所必需的饱和与不饱和脂肪酸,故可以用作替代食用油。猕猴桃籽油因为其存在高含量的α-亚麻酸成分,可能是更好的食用油和营养油资源。本文首次对枸杞籽油、西番莲籽油和猕猴桃籽油脂肪酸成分进行绝对含量分析,为新的食用油资源的开发提供了重要的依据。     

油菜BnrbcS基因超表达提高拟南芥种子重量和含油量

为深入分析光合作用关键酶二磷酸核酮糖羧化酶（Rubisco）小亚基在油菜等＂绿色种子＂发育过程中的作用,采用RT-PCR技术从油菜种胚中克隆了一个含Rubisco小亚基全长编码区的cDNA序列,命名为BnrbcS（GenBank登录号DQ242646）。BnrbcS编码181个氨基酸残基,其推导的氨基酸序列中包含典型的Rubisco小亚基功能域并与其他高等植物Rubisco小亚基具有很高的同源性,暗示BnrbcS基因产物与拟南芥等植物的Rubisco小亚基在结构和功能上可能十分相似。除了在叶、子叶、角果皮等光合器官中有很高表达外,BnrbcS在贮藏物质高速积累时期的未成熟油菜种子中亦有中等水平的表达,且其＂钟型＂表达模式与一些脂肪酸合成酶系基因的表达模式相类似。将NAPIN启动子驱动的BnrbcS种子特异超表达结构转入拟南芥,转化株系的种子含油量和种子重量有一定程度提高,显示BnrbcS在调控种子油脂等贮藏物质积累过程中具有作用。     

In Vivo Inhibition of Seed Development and Reserve Protein Accumulation in Recombinants of Abscisic Acid Biosynthesis and Responsiveness Mutants in Arabidopsis thaliana

In Arabidopsis thaliana, seed development in recombinants of the ABA-deficient aba mutant with the ABA response mutants abi1 or abi3 is compared to wild type and the monogenic parents. Aberrant seed development occurred in the aba,abi3 recombinant and was normal in aba,abi1, abi3 and aba,abi1 seeds. Embryos of the recombinant aba,abi3 seeds maintained the green color until maturity, the seeds kept a high water content, did not form the late abundant 2S and 12S storage proteins, were desiccation intolerant, and often showed viviparous germination. Application of ABA, and particularly of an ABA analog, to the roots of plants during seed development partially alleviated the aberrant phenotype. Seeds of aba,abi3 were normal when they developed on a mother plant heterozygous for Aba. In contrast to seed development, the induction of dormancy was blocked in all monogenic mutants and recombinants. Dormancy was only induced by embryonic ABA; it could not be increased by maternal ABA or ABA applied to the mother plant. It is concluded that endogenous ABA has at least two different effects in developing seeds. The nature of these responses and of the ABA response system is discussed.     

Microsomal Lyso-Phosphatidic Acid Acyltransferase from a Brassica oleracea Cultivar Incorporates Erucic Acid into the sn-2 Position of Seed Triacylglycerols

Developing seeds from Brassica oleracea (L.) var botrytis cv Sesam were examined for the ability to biosynthesize and incorporate erucic acid into triacylglycerols (TAGs). Seed embryos at mid-development contained a high concentration of erucic acid in diacylglycerols and TAGs, and substantial levels were also detected in free fatty acids, acyl-coenzyme A (CoA), phosphatidic acid, and phosphatidylcholine. Homogenates and microsomal fractions from seeds at mid-development produced [14C]eicosenoyl- and [14C]erucoyl-CoAs from [14C]oleoyl-CoA in the presence of malonyl-CoA and reducing equivalents in vitro. These fatty acids were incorporated into TAGs via the Kennedy pathway. However, unlike most Brassicaceae, the B. oleracea was able to insert significant erucic acid into the sn-2 position of TAGs. It was shown that the lyso-phosphatidic acid acyltransferase (LPAT) incorporated erucic acid into the sn-2 position of lyso-phosphatidic acid. The erucoyl-CoA:LPAT activity during seed development and the sn-2 erucic acid content of the TAG fraction in mature seed were compared to those in B. napus, Tropaeolum majus, and Limnanthes douglasii. There was a correlation between the in vitro erucoyl-CoA:LPAT activity and the sn-2 erucic acid content in seed TAGs. To our knowledge, this is the first member of the Brassicaceae reported to have an LPAT able to use erucoyl-CoA. This observation has important implications for efforts being made to increase the erucic acid content in B. napus, to supply strategic industrial feedstocks.     

n-3 Fatty Acid Deficiency Increases Brain Protein Synthesis in the Free-Moving Adult Rat

Abstract: The autoradiographic method with l -[³⁵S]methionine was used to determine the effects of an n-3 fatty acid deficiency on brain protein synthesis. Brain protein synthesis was significantly increased (from 50 to 150%) in 45 of the 52 brain structures studied in n-3 fatty acid-deficient rats as compared with control animals. Biochemical analysis confirmed the increase in overall rate of protein synthesis in brain as a whole.     

杜仲籽油与紫苏籽油脂肪酸组成的比较研究

利用气相色谱法,对杜仲籽油和紫苏籽油的脂肪酸组成、α-亚麻酸含量等进行了比较研究。结果发现,两者不仅脂肪酸GC指纹图谱较为相似(脂肪酸组成、含量基本相同),而且外观、气味、折光率等质量指标也非常相近。说明了杜仲籽油具有与紫苏籽油同样的开发价值。     

A Novel Polyamine Acyltransferase Responsible for the Accumulation of Spermidine Conjugates in Arabidopsis Seed

Hydroxycinnamic acid amides are a class of secondary metabolites distributed widely in plants. We have identified two sinapoyl spermidine derivatives, N-((4′-O-glycosyl)-sinapoyl),N′-sinapoylspermidine and N,N′-disinapoylspermidine, which comprise the two major polyamine conjugates that accumulate in Arabidopsis thaliana seed. Using metabolic profiling of knockout mutants to elucidate the functions of members of the BAHD acyltransferase family in Arabidopsis, we have also identified two genes encoding spermidine disinapoyl transferase (SDT) and spermidine dicoumaroyl transferase (SCT) activities. At2g23510, which is expressed mainly in seeds, encodes a spermidine sinapoyl CoA acyltransferase (SDT) that is required for the production of disinapoyl spermidine and its glucoside in Arabidopsis seed. The structurally related BAHD enzyme encoded by At2g25150 is expressed specifically in roots and has spermidine coumaroyl CoA acyltransferase (SCT) activity both in vitro and in vivo.     

Quantitative Proteomic Analysis of Low Linolenic Acid Transgenic Soybean Reveals Perturbations of Fatty Acid Metabolic Pathways

To understand the effect of fatty acid desaturase gene (GmFAD3) silencing on perturbation of fatty acid (FA) metabolic pathways, the changes are compared in protein profiling in control and low linolenic acid transgenic soybeans using tandem mass tag based mass spectrometry. Protein profiling of the transgenic line unveiled changes in several key enzymes of FA metabolism. This includes enzymes of lower abundance; fabH, fabF, and thioestrase associated with FA initiation, elongation, and desaturation processes and LOX1_5, ACOX, ACAA1, MFP2 associated with β‐oxidation of α‐linolenic acids pathways. In addition, the GmFAD3 silencing results in a significant reduction in one of the major allergens, Gly m 4 (C6T3L5). These results are important for exploring how plants adjust in their biological processes when certain changes are induced in the genetic makeup. A complete understanding of these processes will aid researchers to alter genes for developing value‐added soybeans.     

Arabidopsis ECERIFERUM2 Is a Component of the Fatty Acid Elongation Machinery Required for Fatty Acid Extension to Exceptional Lengths

Primary aerial surfaces of land plants are coated by a lipidic cuticle, which forms a barrier against transpirational water loss and protects the plant from diverse stresses. Four enzymes of a fatty acid elongase complex are required for the synthesis of very-long-chain fatty acid (VLCFA) precursors of cuticular waxes. Fatty acid elongase substrate specificity is determined by a condensing enzyme that catalyzes the first reaction carried out by the complex. In Arabidopsis (Arabidopsis thaliana), characterized condensing enzymes involved in wax synthesis can only elongate VLCFAs up to 28 carbons (C28) in length, despite the predominance of C29 to C31 monomers in Arabidopsis stem wax. This suggests additional proteins are required for elongation beyond C28. The wax-deficient mutant eceriferum2 (cer2) lacks waxes longer than C28, implying that CER2, a putative BAHD acyltransferase, is required for C28 elongation. Here, we characterize the cer2 mutant and demonstrate that green fluorescent protein-tagged CER2 localizes to the endoplasmic reticulum, the site of VLCFA biosynthesis. We use site-directed mutagenesis to show that the classification of CER2 as a BAHD acyltransferase based on sequence homology does not fit with CER2 catalytic activity. Finally, we provide evidence for the function of CER2 in C28 elongation by an assay in yeast (Saccharomyces cerevisiae).Land plants have a lipidic cuticle that seals the outer surface of all of their primary aerial organs. Structurally, the cuticle consists of two components, cutin and cuticular waxes. Together these form a hydrophobic barrier that plays a critical role in plant survival by restricting nonstomatal water loss (Riederer and Schreiber, 2001). Cuticles also protect the plant from biotic and abiotic stresses, profoundly affect plant-insect interactions (Müller, 2006), prevent epidermal fusions (Sieber et al., 2000), and are involved in drought stress signaling (Wang et al., 2011).Cutin is a polymer of mainly midchain- and ω-hydroxy and -epoxy 16 carbon (C16) and C18 fatty acids, which are cross-linked in ester bonds directly or through a glycerol backbone (Pollard et al., 2008). Cuticular waxes are aliphatic monomers that are deposited within the cutin matrix as intracuticular wax, and on top of it as epicuticular wax film and crystals. Wax is a heterogeneous mixture of very-long-chain fatty acids (VLCFAs) and their alkane, aldehyde, alcohol, ketone, and ester derivatives, which typically range from C24 to C32 in length (Samuels et al., 2008). The composition of cuticular wax varies greatly among species and tissues, often providing physical and chemical properties to the plant surface that are advantageous in specific environments.Genetic analyses have revealed that a fatty acid elongase (FAE) complex is responsible for the synthesis of VLCFA wax precursors (Millar et al., 1999; Fiebig et al., 2000; Kunst and Samuels, 2009). FAE complexes are heterotetramers of independently transcribed, monofunctional proteins localized to the endoplasmic reticulum (ER). Together, they catalyze a series of four reactions to elongate long-chain acyl-CoAs or very-long-chain acyl-CoAs by sequential addition of two carbon units. The condensing enzyme, or β-ketoacyl-CoA synthase (KCS), catalyzes the first reaction in this sequence and is both rate limiting and specific for the chain length of acyl-CoA synthesized (Millar and Kunst, 1997). Two very dissimilar families of KCSs have been identified in Arabidopsis (Arabidopsis thaliana): a FAE1-type family homologous to the first such KCS enzyme discovered in association with seed oil biosynthesis (Kunst et al., 1992; James et al., 1995; Lassner et al., 1996), and an ELONGATION DEFECTIVE (ELO)-like family homologous to the yeast (Saccharomyces cerevisiae) ELO family responsible for sphingolipid synthesis (Dunn et al., 2004). To date, no function has been ascribed to Arabidopsis ELOs. Of the 21 FAE1-type KCS enzymes in Arabidopsis (Joubès et al., 2008), 11 have been shown by microarray analysis to be up-regulated in the stem epidermis (Suh et al., 2005). Only one of these, ECERIFERUM6 (CER6/KCS6/CUT1; Millar et al., 1999; Fiebig et al., 2000; Joubès et al., 2008), has a dominant role in the elongation of VLCFAs for cuticular wax synthesis, as CER6 suppression results in a dramatic reduction of all wax monomers longer than C24 (Millar et al., 1999). Heterologous expression of CER6 in yeast has demonstrated that the CER6 condensing enzyme can produce C28 VLCFAs (O. Rowland and L. Kunst, unpublished data). However, CER6 appears to be unable to produce VLCFAs longer than C28 in yeast; this presents a problem as the bulk of Arabidopsis stem wax is made up of C29 alkanes, secondary alcohols, and ketones derived from C30 VLCFAs. Mutant screens have not revealed any other KCS enzymes necessary for VLCFA elongation past C28 in Arabidopsis. Therefore, there may be other proteins unrelated to condensing enzymes that are required for acyl chain extension beyond C28 that remain unknown.The wax-deficient mutant cer2 shows a dramatic reduction in all stem waxes longer than C28 and increased accumulation of waxes C28 or shorter, suggesting that CER2 has a role in the final steps of VLCFA elongation. Surprisingly, the cer2 mutation has been mapped to At4g24510 (Negruk et al., 1996; Xia et al., 1996), a gene homologous to plant BAHD acyltransferases. However, the CER2 protein was reported to localize exclusively to the nucleus (Xia et al., 1997). This does not fit with CER2 annotation as a BAHD acyltransferase, as all characterized BAHD acyltransferases are soluble cytosolic enzymes (D’Auria, 2006).The objective of this work was to more precisely evaluate the role of CER2 in fatty acid elongation using a new CER2 allele, cer2-5 (Columbia-0 [Col-0] ecotype). We provide evidence that CER2 has a metabolic function specific to wax synthesis, and that the CER2 homolog CER2-LIKE1 has an analogous role in leaf wax synthesis. Despite the classification of CER2 as a BAHD acyltransferase based on sequence homology, we demonstrate that CER2 cannot share the catalytic mechanism that has been confirmed for other members of the BAHD family, and provide biochemical support for a function of CER2 in VLCFA elongation by an assay in yeast.     

Lipid Accumulation during Canola Seed Germination in Response to Cinnamic Acid Derivatives

The objective of this research was to investigate how ferulic and p-coumaric acids affect lipid and fatty acid composition during canola (Brassica napus L.) seed germination. Data showed that both compounds increased total lipid and fatty acid contents in the cotyledons during germination. The largest accumulation in lipids occurred at 1.0 mM p-coumaric acid with an increase in all unsaturated fatty acids. The results suggest that allelochemicals interfere in canola seed germination by reducing lipid mobilization.