Acyl-CoA elongase expression during seed development in Brassica napus

The Bn-FAE1.1 and Bn-FAE1.2 genes encode the 3-ketoacyl-CoA synthase, a component of the elongation complex responsible for the synthesis of very long chain monounsaturated fatty acids (VLCMFA) in the seeds of Brassica napus. Bn-FAE1 gene expression was studied during seed development using two different cultivars: Gaspard, a high erucic acid rapeseed (HEAR), and ISLR4, a low erucic acid rapeseed (LEAR). The mRNA developmental profiles were similar for the two cultivars, the maximal expression levels being measured at 8 weeks after pollination (WAP) in HEAR and at 9 WAP in LEAR. Differential expression of Bn-FAE1.1 and Bn-FAE1.2 genes was also studied. In each cultivar the same expression profile was observed for both genes, but Bn-FAE1.2 was expressed at a lower level than Bn-FAE1.1. Secondly, VLCMFA synthesis was measured using particulate fractions prepared from maturating seeds harvested weekly after pollination. The oleoyl-CoA and ATP-dependent elongase activities increased from the 4th WAP in HEAR and reached the maximal level at 8 WAP, whereas both activities were absent in LEAR. In contrast, the 3-hydroxy dehydratase, a subunit of the elongase complex, had a similar activity in both cultivars and reached a maximum from 7 to 9 WAP. Finally, antibodies against the 3-ketoacyl-CoA synthase revealed a protein of 57 kDa present only in HEAR. Our results show: (i) that both genes are transcribed in HEAR and LEAR cultivars; (ii) that they are coordinately regulated; (iii) that Bn-FAE1.1 is quantitatively the major isoform expressed in seeds; (iv) that the Bn-FAE1 gene encodes a protein of 57 kDa responsible for the 3-ketoacyl-CoA synthase activity.     

Acyl-CoA elongase expression during seed development in Brassica napus

The Bn-FAE1.1 and Bn-FAE1.2 genes encode the 3-ketoacyl-CoA synthase, a component of the elongation complex responsible for the synthesis of very long chain monounsaturated fatty acids (VLCMFA) in the seeds of Brassica napus. Bn-FAE1 gene expression was studied during seed development using two different cultivars: Gaspard, a high erucic acid rapeseed (HEAR), and ISLR4, a low erucic acid rapeseed (LEAR). The mRNA developmental profiles were similar for the two cultivars, the maximal expression levels being measured at 8 weeks after pollination (WAP) in HEAR and at 9 WAP in LEAR. Differential expression of Bn-FAE1.1 and Bn-FAE1.2 genes was also studied. In each cultivar the same expression profile was observed for both genes, but Bn-FAE1.2 was expressed at a lower level than Bn-FAE1.1. Secondly, VLCMFA synthesis was measured using particulate fractions prepared from maturating seeds harvested weekly after pollination. The oleoyl-CoA and ATP-dependent elongase activities increased from the 4th WAP in HEAR and reached the maximal level at 8 WAP, whereas both activities were absent in LEAR. In contrast, the 3-hydroxy dehydratase, a subunit of the elongase complex, had a similar activity in both cultivars and reached a maximum from 7 to 9 WAP. Finally, antibodies against the 3-ketoacyl-CoA synthase revealed a protein of 57 kDa present only in HEAR. Our results show: (i) that both genes are transcribed in HEAR and LEAR cultivars; (ii) that they are coordinately regulated; (iii) that Bn-FAE1.1 is quantitatively the major isoform expressed in seeds; (iv) that the Bn-FAE1 gene encodes a protein of 57 kDa responsible for the 3-ketoacyl-CoA synthase activity.     

Rhizoremediation of Diesel-Contaminated Soil with Two Rapeseed Varieties and Petroleum degraders Reveals Different Responses of the Plant Defense Mechanisms

Plant-assisted bioremediation (rhizoremediation) stands out as a potential tool to inactivate or completely remove xenobiotics from the polluted environment. Therefore, it is of key importance to find an adequate combination of plant species and microorganisms that together enhance the clean-up process. To understand the response of plants upon bioaugmentation, the antioxidative and detoxification system was analyzed in high and low erucic acid rapeseed varieties (HEAR and LEAR, respectively), after 8 weeks of their treatment with petroleum degraders and 6000 mg diesel oil/kg dry soil. The oxidative stress was enhanced in LEAR being exposed to sole diesel oil, in comparison with HEAR. However, when LEAR plants were additionally inoculated with bacteria, suppression of total catalase (CAT) and ascorbate peroxidase (APX) activity were observed. Interestingly, glutathione transferase (GST) activity was found in these plants at a much higher level than in HEAR, which correlated with a more efficient diesel removal performed by LEAR in the polluted soil and upon bioaugmentation. A distinct profile of polycyclic aromatic hydrocarbons (PAH) was detected in leaves of these plants. Neither LEAR nor HEAR experienced any changes in the photosynthetic capacity upon diesel pollution and presence of petroleum degraders, which supports the usefulness of rhizoremediation with rapeseed.     

Increasing erucic acid content through combination of endogenous low polyunsaturated fatty acids alleles with Ld-LPAAT + Bn-fae1 transgenes in rapeseed (Brassica napus L.)

High erucic acid rapeseed (HEAR) oil is of interest for industrial purposes because erucic acid (22:1) and its derivatives are important renewable raw materials for the oleochemical industry. Currently available cultivars contain only about 50% erucic acid in the seed oil. A substantial increase in erucic acid content would significantly reduce processing costs and could increase market prospects of HEAR oil. It has been proposed that erucic acid content in rapeseed is limited because of insufficient fatty acid elongation, lack of insertion of erucic acid into the central sn-2 position of the triaclyglycerol backbone and due to competitive desaturation of the precursor oleic acid (18:1) to linoleic acid (18:2). The objective of the present study was to increase erucic content of HEAR winter rapeseed through over expression of the rapeseed fatty acid elongase gene (fae1) in combination with expression of the lysophosphatidic acid acyltransferase gene from Limnanthes douglasii (Ld-LPAAT), which enables insertion of erucic acid into the sn-2 glycerol position. Furthermore, mutant alleles for low contents of polyunsaturated fatty acids (18:2 + 18:3) were combined with the transgenic material. Selected transgenic lines showed up to 63% erucic acid in the seed oil in comparison to a mean of 54% erucic acid of segregating non-transgenic HEAR plants. Amongst 220 F₂ plants derived from the cross between a transgenic HEAR line and a non-transgenic HEAR line with a low content of polyunsaturated fatty acids, recombinant F₂ plants were identified with an erucic acid content of up to 72% and a polyunsaturated fatty acid content as low as 6%. Regression analysis revealed that a reduction of 10% in polyunsaturated fatty acids content led to a 6.5% increase in erucic acid content. Results from selected F₂ plants were confirmed in the next generation by analysing F₄ seeds harvested from five F₃ plants per selected F₂ plant. F₃ lines contained up to 72% erucic acid and as little as 4% polyunsaturated fatty acids content in the seed oil. The 72% erucic acid content of rapeseed oil achieved in the present study represents a major breakthrough in breeding high erucic acid rapeseed.     

Breeding high-stearic oilseed rape (Brassica napus) with high- and low-erucic background using optimised promoter-gene constructs

Seed lipids of oilseed rape (Brassica napus) usually contain small proportions (<3%) of stearic acid. The objective of this study was to increase the content of stearic fatty␣acid in rapeseed oil. An antisense down-regulation of the endogenous stearoyl-ACP desaturase (SAD) catalysing the reaction step from stearic to oleic acid in two different genetic backgrounds was studied. The result of down-regulation of the SAD yielded an about 10-fold increase of stearic acid from 3.7% up to 32% in single seeds of transgenic low-erucic acid rapeseed (LEAR), while high-erucic acid rapeseed (HEAR) showed a 4-fold increase of C18:0 from 1% up to 4%. It could be shown in pooled T2 seed material of LEAR rapeseed, that the stearic acid content is highly correlated with the down-regulation of SAD as indicated by the␣stearate desaturation proportion (SDP). The importance of the promoter strength for the alteration of a trait was confirmed in this study as no change in the fatty acid composition of transgenic plants was achieved with gene constructs controlled by the weak FatB4 seed-specific promoter from Cuphea lanceolata.Karim Zarhloul and Christof Stoll have contributed in equal parts to the present work     

Very long chain and hydroxylated fatty acids in offspring of somatic hybrids between Brassica napus and Lesquerella fendleri

 Offspring of somatic hybrids between the zero-erucic acid rapeseed cv Hanna and Lesquerella fendleri were analysed regarding their fatty acid profiles. In the first back-cross generation one plant was found that produced a seed containing up to 16.5% erucic acid and 15% eicosaenoic acid (Line 1), as well as a seed having 4.3% ricinoleic acid (Line 2). This was interpreted as due to a contribution of elongase and hydroxylase genes from the L. fendleri genome since these two fatty acids are not produced in the recipient rapeseed cultivar Hanna. Crosses between Line 1 and cv Hanna resulted in the production of seeds with 35% erucic acid (F₂). Furthermore, crosses between the F₂ plants and the rapeseed cultivar Gulle, producing 35% erucic acid in the seeds, resulted in F₃ seeds with 48% erucic acid. The highest amount of erucic acid, 61.5%, was found in the F₆ generation after crossing Line 1 with a high erucic acid rapeseed line, HEAR, followed by self-fertilisation for two generations. When performing Southern-blot analysis on the F₆ plants, seven of the nine analysed plants hybridised with the L. fendleri species-specific repetitive probe. The presence of the hydroxylase gene was also observed in the F₆ generation of Line 1 according to Southern-blot analysis. Hybridisation with a hydroxylase probe was seen although no hydroxy fatty acids could be detected in any of the F₆ plants. In parallel, Line 2 was crossed with HEAR cv Gulle and self fertilised. No hydroxy fatty acids were detected in the F₂ generation of Line 2 and no specific hybridisation patterns could be found in the Southern-blot analysis. Received: 12 December 1998 / Accepted: 4 January 1999     

油菜品质育种现状及展望

国际油菜品质改良始于20世纪60年代,以降低油菜籽中芥酸和硫代葡萄糖苷为主要目标.随着历史的不断发展,油菜的品质改良已不再局限于这两个指标.在食用油方面,已将提高油酸、亚油酸含量,降低饱和脂肪酸和亚麻酸含量作为今后的主攻方向,使之成为最健康的食用油;在工业用油方面,高芥酸和中等长度的脂肪酸改良已逐渐展开.今后常规育种、杂种优势利用和生物技术的有机结合,将使油菜品种的改良进入到一个新的阶段.     

Production of human surfactant protein C in milk of transgenic mice

Respiratory distress syndrome (RDS), caused by lack of pulmonary surfactant, affects 65 000 infants annually in the USA. Surfactant replacement therapy reduces the morbidity and mortality associated with RDS. Human surfactant protein C (SP-C) is an important component of pulmonary surfactant. To produce human SP-C, a construct using the rat whey acidic protein (WAP) promoter and 3 untranslated regions to target expression of the human SP-C gene to the mammary gland of transgenic mice was created. WAP/SP-C mRNA expression was detected in all transgenic lines analysed. SP-C was expressed in a copy-number-dependent and integration-site-independent fashion, with levels of expression ranging from 0.01% to 36.0% of the endogenous mouse WAP mRNA, and WAP/SP-C mRNA expresison levels were greater than those of the endogenous mouse lung SP-C mRNA. Expression at the RNA level was specific to the mammary gland and paralleled the endogenous WAP expression pattern during mammary gland development. Expression and secretion of the SP-C protein in the lactating mammary gland was demonstrated by western blots performed on whole milk using an anti-SP-C polyclonal antibody. Immunoreactive proteins of MW 22 and 12–14 kDa appeared only in transgenic milk. The 22 kDa protein represents the proprotein, and the 12–14 kDa is a processed form of SP-C.     

Water relations and osmotic adjustment in Apium graveolens during long‐term NaCl stress and subsequent relief

Pea plants ( Pisum sativum L. cv. Feltham First) exposed to a heat stress of 37°C for 6 h accumulated two low molecular weight (LMW) heat shock proteins (HSPs) of molecular mass 22 kDa. The two LMW HSPs were associated with purified mitochondria. N‐terminal amino acid sequencing analysis indicates that the more basic of these proteins is a novel protein. The response of other cultivars of P. sativum to heat shock revealed that up to three 22‐kDa HSPs were expressed in a cultivar‐specific manner. Evidence presented suggests that the different 22‐kDa HSPs arise as a result of there being multiple 22‐kDa HSP genes. The expression of the most basic novel HSP was studied in the Feltham First cultivar using two dimensional SDS‐PAGE. Treatment of intact plants with chloramphenicol and cycloheximide prior to heat stress treatment indicated that the LMW HSPs were nuclear encoded and de novo synthesised. The response to heat shock was rapid with protein expression detected within 45 min and the protein remained in excess of 6 days following removal of the stress. The protein accumulated to very high levels with maximal expression being 2% of the total mitochondrial protein. The results are discussed in relation to the likely role of LMW HSPs in thermotolerance.     

Erythrocyte membrane in rats fed high erucic acid-containing mustard oil: osmotic fragility, lipid composition, and (Na+, K+)- and (Ca2+, Mg2+)-ATPases

Erythrocyte hemolytic properties, cholesterol/phospholipid ratios, fatty acid composition, and activities of the membrane-bound enzymes (Na+, K+)- and (Ca2+, Mg2+)-ATPase were studied in male and female rats fed low erucic acid rapeseed (LEAR) and high erucic acid mustard oils (HEAM) for a period of 16 months. Rats receiving groundnut oil (GNO) served as controls. Erythrocytes from HEAM-receiving male and female rats showed increased resistance to hypotonic hemolysis. In male rats this was associated with an 85% increase (P less than 0.07) in the cholesterol/phospholipid molar ratio. The fatty acid double-bond index showed an increase in male rats receiving HEAM as well as LEAR oils. In the erythrocytes from female rats, the cholesterol/phospholipid molar ratio and double bond index remained unaffected. Specific activity of ouabain-sensitive (Na+, K+)-ATPase showed a small (+20%) but significant (P less than 0.05) increase in male but not female rats of HEAM group. Total (Na+, K+)-ATPase, ouabain-insensitive component, and (Ca2+, Mg2+)-ATPase were not altered in rats receiving LEAR or HEAM.     

A rapeseed FAE1 gene is linked to the E1 locus associated with variation in the content of erucic acid

 The synthesis of very long chain fatty acids occurs in the cytoplasm via an elongase complex. A key component of this complex is the β-ketoacyl-CoA synthase, a condensing enzyme which in Arabidopsis is encoded by the FAE1 gene. Two sequences homologous to the FAE1 gene were isolated from a Brassica napus immature embryo cDNA library. The two clones, CE7 and CE8, contain inserts of 1647 bp and 1654 bp, respectively. The CE7 gene encodes a protein of 506 amino acids and the CE8 clone, a protein of 505 amino acids, each having an approximate molecular mass of 56 kDa. The sequences of the two cDNA clones are highly homologous yet distinct, sharing 97% nucleotide identity and 98% identity at the amino acid level. Southern hybridisation showed the rapeseed β-ketoacyl-CoA synthase to be encoded by a small multigene family. Northern hybridisation showed the expression of the rapeseed FAE1 gene(s) to be restricted to the immature embryo. One of the FAE1 genes is tightly linked to the E1 locus, one of two loci controlling erucic acid content in rapeseed. The identity of the second locus, E2, is discussed. Received: 4 April 1997 / Accepted: 30 July 1997     

Comparison of rapeseed cultivars and resynthesized lines based on allozyme and RFLP markers

It has frequently been suggested to use the resynthesis of rapeseed (Brassica napus) from B. campestris and B. oleracea to broaden its genetic base. The objective of the present study is twofold: (1) to compare the genetic variation within resynthesized rapeseed with a world-wide collection of oilseed rape cultivars, and (2) to compare genetic distances estimated from RFLP markers with distances estimated from a relatively small number of allozyme markers. We investigated 17 resynthesized lines and 24 rapeseed cultivars. Genetic distances were estimated either based on the electrophoresis of seven allozymes, with a total of 38 different bands, or based on RFLP data of 51 probe/enzyme combinations, with a total of 355 different bands. The results of allozyme and RFLP analyses agreed reasonably well. Genetic distances, estimated from two independent sets of RFLP data with 25 and 26 probe/enzyme combinations respectively, were highly correlated; hence about 50 RFLP markers are sufficient to characterize rapeseed material with a large genetic diversity. The cultivars were clustered into three groups: (1) spring rapeseed of European and Northern American origin, (2) winter rapeseed of European and Northern American origin, and (3) rapeseed of Asian origin. Several of the resynthesized rapeseed lines were similar to European winter rapeseed cultivars, whereas others had quite unique patterns. It is concluded, that resynthesized rapeseed is a valuable source for broadening the genetic variation in present breeding material of Brassica napus. However, different lines differ widely in their suitability for this purpose.     

Partial purification of the acyl-CoA elongase of Allium porrum leaves

Acyl-CoA elongase has been partially purified from leek (Allium porrum L.) epidermal cells. The microsomal elongase is first solubilized by Triton X-100. The solubilized proteins are then submitted to anion exchange chromatography on DEAE-cellulose and, finally, to gel filtration on Ultrogel 34 AcA. The purification of the elongase activity is accompanied by the enrichment in three major protein bands of 59, 61, and 65 kDa. The partially purified elongase is highly delipidated (about 10 mol lipid/mol of 60- to 65-kDa protein) and phosphatidylserine and phosphatidylethanolamine account respectively for 60 and 40% of the remaining phospholipids. The partially purified elongase retains some activities associated with fatty acid biosynthesis. The overall activity is strongly stimulated by the addition of exogenous lipids. In the presence of a mixture of PS, PE, and PC the C18-CoA elongase activity is increased more than sixfold. The Km value of stearoyl-CoA, in the presence of lipid vesicles, was determined to be 1.7 microM.     

Accumulation of pathogenesis-related (PR) 10/Bet v 1 protein homologues in mulberry (Morus bombycis Koidz.) tree during winter

Seasonal evaluation of total soluble protein fractions extracted from cortical parenchyma cells of mulberry (Morus bombycis Koidz.) tree identified a predominant 18 kDa protein that was directly correlated to periods of cold acclimation. The 18 kDa protein, designated as WAP18 (winter accumulating 18 kDa proteins) increased from September to December and then gradually decreased until June. The maximum levels of WAP18 were detected in mid‐winter, which corresponds to the maximum freeze tolerance in cortical parenchyma cells of mulberry tree. Two‐dimensional gel electrophoresis confirmed that WAP18 consists of at least three proteins that range between an isoelectric point of 5.0 and 6.0. All three proteins reacted with anti‐WAP18 antibodies, thereby suggesting that they represent individual isoforms. Furthermore, N‐terminal amino acid sequence analysis demonstrated that all three proteins contain high sequence similarity to each other and high homology to pathogenesis‐related (PR) ?10/Bet v 1 protein families. The purified WAP18 exhibited in vitro cryoprotective activity for the freeze labile l ‐lactate dehydrogenase (LDH) enzyme. These results suggest that WAP18 may function in the freezing tolerance mechanism of cortical parenchyma cells of mulberry tree during winter.     

High-level expression of the rat whey acidic protein gene is mediated by elements in the promoter and 3'' untranslated region.

The high-level expression of the rat whey acidic protein (WAP) gene in transgenic mice depends on the interaction of 5'-flanking promoter sequences and intragenic sequences. Constructs containing 949 bp of promoter sequences and only 70 bp of 3'-flanking DNA were expressed at uniformly high levels, comparable to or higher than that of the endogenous gene. Although this WAP transgene was developmentally regulated, it was expressed earlier during pregnancy than was the endogenous WAP gene. Replacement of 3' sequences, including the WAP poly(A) addition site, with simian virus 40 late poly(A) sequences resulted in an approximately 20-fold reduction in the expression of WAP mRNA in the mammary gland during lactation. Nevertheless, position-independent expression of the transgene was still observed. Further deletion of 91 bp of conserved WAP 3' untranslated region (UTR) led to integration site-dependent expression. Position independence was restored following reinsertion of the WAP 3' UTR into the deleted construct at the same location, but only when the insertion was in the sense orientation. The marked differences observed between the expression levels of the 3'-end deletion constructs in transgenic mice were not seen in transfected CID 9 mammary epithelial cells. In these cells, expression of the endogenous WAP gene was dependent on the interaction of these cells with a complex extracellular matrix. In contrast, the transfected WAP constructs were not dependent on extracellular matrix for expression. Thus, both the abnormal expression of WAP in cells cultured on plastic and the precocious developmental expression of WAP in transgenic mice may reflect the absence of a negative control element(s) within these recombinant constructs.     

Expression and subcellular targeting of a soybean oleosin in transgenic rapeseed. Implications for the mechanism of oil-body formation in seeds

Two genomic clones, encoding isoforms A and B of the 24 kDa soybean oleosin and containing 5 kbp and 1 kbp, respectively, of promoter sequence, were inserted separately into rapeseed plants. T₂ seeds from five independent transgenic lines, three expressing isoform A and two expressing isoform B, each containing one or two copies of the transgene, were analysed in detail. In all five lines, the soybean transgenes exhibited the same patterns of mRNA and protein accumulation as the resident rapeseed oleosins, i.e. their expression was absolutely seed-specific and peaked at the mid-late stages of cotyledon development. The 24 kDa soybean oleosin was targeted to and stably integrated into oil bodies, despite the absence of a soybean partner isoform. The soybean protein accumulated in young embryos mainly as a 23 kDa polypeptide, whereas a 24 kDa protein predominated later in development. The ratio of rapeseed:soybean oleosin in the transgenic plants was about 5:1 to 6:1, as determined by SDS-PAGE and densitometry. Accumulation of these relatively high levels of soybean oleosin protein did not affect the amount of endogenous rapeseed oleosin. Immunoblotting studies showed that about 95% of the recombinant soybean 24 kDa oleosin (and the endogenous 19 kDa rapeseed oleosin) was targeted to oil bodies, with the remainder associated with the microsomal fraction. Sucrose density-gradient centrifugation showed that the oleosins were associated with a membrane fraction of buoyant density 1.10–1.14 g ml^?1, which partially overlapped with several endoplasmic reticulum (ER) markers. Unlike oleosins associated with oil bodies, none of the membrane-associated oleosins could be immunoprecipitated in the presence of protein A-Sepharose, indicating a possible conformational difference between the two pools of oleosin. Complementary electron microscopy-immunocytochemical studies of transgenic rapeseed revealed that all oil bodies examined could be labelled with both the soybean or rapeseed anti-oleosin antibodies, indicating that each oil body contained a mixed population of soybean and rapeseed oleosins. A small but significant proportion of both soybean and rapeseed oleosins was located on ER membranes in the vicinity of oil bodies, but none were detected on the bulk ER cisternae. This is the first report of apparent targeting of oleosins via ER to oil bodies in vivo and of possible associated conformational/ processing changes in the protein. Although oil-body formation per se can occur independently of oleosins, it is proposed that the relative net amounts of oleosin and oil accumulated during the course of seed development are a major determinant of oil-body size in desiccation-tolerant seeds.     

Expression of a whey acidic protein transgene during mammary development. Evidence for different mechanisms of regulation during pregnancy and lactation

Expression of the mouse whey acidic protein (WAP) gene is specific to the mammary gland, is induced several thousand-fold during pregnancy, and is under the control of steroid and peptide hormones. To study developmental regulation of the mouse WAP gene, a 7.2-kilobase (kb) WAP transgene, including 2.6 kb of 5'- and 1.6 kb of 3'-flanking sequences, was introduced into mice. Of the 13 lines of mice examined, 6 expressed the transgenes during lactation at levels between 3 and 54% of the endogenous gene. Although expression was dependent on the site of integration, the transgenes within a given locus were expressed in a copy number-dependent manner and were coordinately regulated. The WAP transgenes were expressed specifically in the mammary gland, but showed a deregulated pattern of expression during mammary development. In all six lines of mice, induction of the WAP transgenes during pregnancy preceded that of the endogenous gene. During lactation, expression in two lines increased coordinately with the endogenous gene, and in three other lines of mice, transgene expression decreased to a basal level. These data indicate that the 7.2-kb gene contains some but not all of the elements necessary for correct developmental regulation. At a functional level it appears as if a repressor element, which inactivates the endogenous gene until late pregnancy, and an element necessary for induction during lactation are absent from the transgene. Complementary results from developmental and hormone induction studies suggest that WAP gene expression during pregnancy and lactation is mediated by different mechanisms.     

Detection of lactobacillic acid in low erucic rapeseed oil—A note of caution when quantifying cyclic fatty acid monomers in vegetable oils

The purpose of this work was to identify an unknown component which has been detected during the analysis of cyclic fatty acid monomers (CFAMs) in low erucic acid rapeseed oils (LEAR). A sample of crude LEAR was transformed into fatty acid methyl esters (FAMEs) and hydrogenated using PtO₂. The hydrogenated sample was fractionated by reversed-phase high-performance liquid chromatography (RP-HPLC) and the fraction containing the CFAMs transformed into picolinyl esters. Analysing these picolinyl derivatives by gas–liquid chromatography coupled to mass spectrometry (GC–MS) showed that the unknown product observed in LEAR is the 11,12-methylene-octadecanoic acid. This cyclic fatty acid was also found in crude LEAR and in the corresponding seeds but was not detected in crude soya and sunflower oils. As this acid is present in the same fraction as CFAMs, known to be formed during heat treatment, great care must therefore be taken for not including it when quantifying CFAMs. It is thus necessary to verify by mass spectrometry the structures of the CFAMs in the isolated cyclic fatty acid fraction prior to quantification.     

The alteration of mRNA expression of SOD and GPX genes,and proteins in tomato (Lycopersicon esculentum Mill) under stress of NaCl and/or ZnO nanoparticles

Five cultivars of tomato having different levels of salt stress tolerance were exposed to different treatments of NaCl (0, 3 and 6 g L⁻¹) and ZnO-NPs (0, 15 and 30 mg L⁻¹). Treatments with NaCl at both 3 and 6 g L⁻¹ suppressed the mRNA levels of superoxide dismutase (SOD) and glutathione peroxidase (GPX) genes in all cultivars while plants treated with ZnO-NPs in the presence of NaCl, showed increments in the mRNA expression levels. This indicated that ZnO-NPs had a positive response on plant metabolism under salt stress. Superior expression levels of mRNA were observed in the salt tolerant cultivars, Sandpoint and Edkawy while the lowest level was detected in the salt sensitive cultivar, Anna Aasa. SDS–PAGE showed clear differences in patterns of protein expression among the cultivars. A negative protein marker for salt sensitivity and ZnO-NPs was detected in cv. Anna Aasa at a molecular weight of 19.162 kDa, while the tolerant cultivar Edkawy had two positive markers at molecular weights of 74.991 and 79.735 kDa.