stearoyl-ACP Desaturase Research Articles

Transcriptional Analysis of Stearoyl-Acyl Carrier Protein Desaturase Genes from Olive (Olea europaea) in Relation to the Oleic Acid Content of the Virgin Olive Oil.

The specific contribution of different stearoyl-ACP desaturase (SAD) genes to the oleic acid content in olive (Olea europaea) fruit has been studied. Toward that end, we isolated three distinct cDNA clones encoding three SAD isoforms from olive (cv. Picual), as revealed by sequence analysis. The expression levels of olive SAD genes were determined in different tissues from Picual and Arbequina cultivars, including developing mesocarp and seed, together with the unsaturated fatty acid content. Lipid and gene expression analyses indicate that OeSAD2 seems to be the main gene contributing to the oleic acid content of the olive fruit and, therefore, of the virgin olive oil. This conclusion was confirmed when the study was extended to Hojiblanca, Picudo, and Manzanilla cultivars. Furthermore, our data indicate that the olive microsomal oleate desaturase gene OeFAD2-2, but not OeSAD2, is responsible for the linoleic acid content in the virgin olive oil.

Natural variations in stearoyl-acp desaturase genes affect the conversion of stearic to oleic acid in maize kernel.

We identified 11 SAD genes, and mined their natural variations associated with the conservation of stearic to oleic acid, especially ZmSAD1 supported by both the QTL and an expression QTL. Maize oil is generally regarded as a healthy vegetable oil owing to its low abundance of saturated fatty acids. Stearoyl-ACP desaturase (SAD) is a key rate-limiting enzyme for the conservation of stearic (C18:0) to oleic (C18:1) acid. Here, 11 maize SAD genes were identified to have more divergent functions than Arabidopsis SAD genes. The genomic regional associations in a maize panel including 508 inbred lines identified 6 SAD genes significantly associated (P<0.01) with the C18:0/C18:1 ratio or the level of C18:0 or C18:1, one gene of which co-localized with a quantitative trait locus (QTL) and 5 of which co-localized with an expression QTL. ZmSAD1, supported by both the QTL and an expression QTL, had the largest effect on C18:0/C18:1. One nonsynonymous single-nucleotide polymorphism in exon 3 and one 5-bp insertion/deletion in the 3' untranslated region were further shown to contribute to the natural variation in C18:0/C18:1 according to ZmSAD1-based association mapping. Finally, selection tests of ZmSAD1 in teosinte, regular maize, and high-oil maize indicated that ZmSAD1 was not a selection target during the process of maize domestication and high-oil maize development. These results will guide the manipulation of the ratio between saturated and unsaturated fatty acids in maize.

Transcriptional Activation of Two Delta-9 Palmitoyl-ACP Desaturase Genes by MYB115 and MYB118 Is Critical for Biosynthesis of Omega-7 Monounsaturated Fatty Acids in the Endosperm of Arabidopsis Seeds.

In angiosperms, double fertilization of the embryo sac initiates the development of the embryo and the endosperm. In Arabidopsis thaliana, an exalbuminous species, the endosperm is reduced to one cell layer during seed maturation and reserves such as oil are massively deposited in the enlarging embryo. Here, we consider the strikingly different fatty acid (FA) compositions of the oils stored in the two zygotic tissues. Endosperm oil is enriched in ω-7 monounsaturated FAs, that represent more than 20 mol% of total FAs, whereas these molecular species are 10-fold less abundant in the embryo. Two closely related transcription factors, MYB118 and MYB115, are transcriptionally induced at the onset of the maturation phase in the endosperm and share a set of transcriptional targets. Interestingly, the endosperm oil of myb115 myb118 double mutants lacks ω-7 FAs. The identification of two Δ9 palmitoyl-ACP desaturases responsible for ω-7 FA biosynthesis, which are activated by MYB115 and MYB118 in the endosperm, allows us to propose a model for the transcriptional control of oil FA composition in this tissue. In addition, an initial characterization of the structure-function relationship for these desaturases reveals that their particular substrate specificity is conferred by amino acid residues lining their substrate pocket that distinguish them from the archetype Δ9 stearoyl-ACP desaturase.

A novel mutant allele of SSI2 confers a better balance between disease resistance and plant growth inhibition on Arabidopsis thaliana.

BackgroundResistance and growth are opposing characteristics in plants. SA INSENSITIVITY OF npr1-5 (SSI2) encodes a stearoyl-ACP desaturase (S-ACP DES) that has previously been reported to simultaneously enhance resistance and repress growth.ResultsHere, we characterize ssi2-2, a novel mutant allele of SSI2 that has two amino acid substitutions. Compared with wild-type and two other mutants of SSI2, ssi2-2 showed intermediate phenotypes in growth size, punctate necrosis, resistance to the bacterial pathogen Pst DC3000, salicylic acid (SA) content, pathogenesis-related (PR) gene levels and 18:1 content. These results indicate that ssi2-2 is a weak mutant of SSI2. Additionally, by using ssi2-2 as an intermediate control, a number of differentially expressed genes were identified in transcriptome profiling analysis. These results suggest that constitutive expression of defense-related genes and repression of IAA signaling-associated genes is present in all SSI2 mutants.ConclusionsTaken together, our results suggest that the weak ssi2-2 mutant maintains a better balance between plant immunity and vegetative growth than other mutants, consequently providing a basis to genetically engineer disease resistance in crop plants.Electronic supplementary materialThe online version of this article (doi:10.1186/s12870-016-0898-x) contains supplementary material, which is available to authorized users.

Crop-specific GMO matrix-multiplex PCR: A cost-efficient screening strategy for genetically modified maize and cotton events approved globally

Crop-specific GMO matrices of 199 genetically modified (GM) events, comprising 143 GM maize events with 75 genetic elements and 56 cotton events with 45 genetic elements, were developed to screen globally approved GM maize and cotton events. As per the compiled information in the matrix, frequently present genetic elements were identified using GMOSeek algorithm: eight genetic elements ([P-35S] [r-act] [T-35S] [T-nos] [pinII] [pat] [aad-1] [cp4 epsps]) in maize and four ([P-35S] [T-nos] [pat] [nptII]) in cotton. Based on the cost-efficiency, feasibility of plexing and coverage of GM events, maize-specific tetraplex PCR, targeting Cauliflower Mosaic Virus 35S promoter (P-35S), Agrobacterium tumefaciens nos terminator (T-nos), Cauliflower Mosaic Virus 35S terminator (T-35S) and endogenous alcohol dehydrogenase (Adh1) gene, with limit of detection (LOD) up to 0.5% was developed. For screening of GM cotton events, pentaplex PCR, targeting P-35S, T-nos, neomycin phosphotransferase II (nptII), phosphinothricin acetyltransferase (pat) and endogenous stearoyl-ACP desaturase (Sad1) gene, with LOD up to 0.25% was developed. Practical applicability of multiplex PCR assays was confirmed with six maize samples of proficiency testing and eight spiked cotton samples. The reported tetraplex and pentaplex PCR assays could efficiently screen 94% of maize and 93% of cotton events approved globally. The developed GMO matrices in combination with multiplex PCR could facilitate checking the GM status of seed samples or food and feed products, and monitoring for presence of authorized GMOs in food and supply chain. This approach can be easily employed by low resource GM testing laboratories in the developing countries, as the multiplex PCR assays are easy to operate with less time and cost inputs. The GMO matrices being presented herein are based on the current information of approved GM events of maize and cotton, which can be further upgraded by including new approved GM events. As most of the newly approved GM events are stacked versions of already commercialized GM events, the developed multiplex PCR assays could also be employed to screen for their presence.

Oil biosynthesis in a basal angiosperm: transcriptome analysis of Persea Americana mesocarp.

BackgroundThe mechanism by which plants synthesize and store high amounts of triacylglycerols (TAG) in tissues other than seeds is not well understood. The comprehension of controls for carbon partitioning and oil accumulation in nonseed tissues is essential to generate oil-rich biomass in perennial bioenergy crops. Persea americana (avocado), a basal angiosperm with unique features that are ancestral to most flowering plants, stores ~ 70 % TAG per dry weight in its mesocarp, a nonseed tissue. Transcriptome analyses of select pathways, from generation of pyruvate and leading up to TAG accumulation, in mesocarp tissues of avocado was conducted and compared with that of oil-rich monocot (oil palm) and dicot (rapeseed and castor) tissues to identify tissue- and species-specific regulation and biosynthesis of TAG in plants.ResultsRNA-Seq analyses of select lipid metabolic pathways of avocado mesocarp revealed patterns similar to that of other oil-rich species. However, only some predominant orthologs of the fatty acid biosynthetic pathway genes in this basal angiosperm were similar to those of monocots and dicots. The accumulation of TAG, rich in oleic acid, was associated with higher transcript levels for a putative stearoyl-ACP desaturase and endoplasmic reticulum (ER)-associated acyl-CoA synthetases, during fruit development. Gene expression levels for enzymes involved in terminal steps to TAG biosynthesis in the ER further indicated that both acyl-CoA-dependent and -independent mechanisms might play a role in TAG assembly, depending on the developmental stage of the fruit. Furthermore, in addition to the expression of an ortholog of WRINKLED1 (WRI1), a regulator of fatty acid biosynthesis, high transcript levels for WRI2-like and WRI3-like suggest a role for additional transcription factors in nonseed oil accumulation. Plastid pyruvate necessary for fatty acid synthesis is likely driven by the upregulation of genes involved in glycolysis and transport of its intermediates. Together, a comparative transcriptome analyses for storage oil biosynthesis in diverse plants and tissues suggested that several distinct and conserved features in this basal angiosperm species might contribute towards its rich TAG content.ConclusionsOur work represents a comprehensive transcriptome resource for a basal angiosperm species and provides insight into their lipid metabolism in mesocarp tissues. Furthermore, comparison of the transcriptome of oil-rich mesocarp of avocado, with oil-rich seed and nonseed tissues of monocot and dicot species, revealed lipid gene orthologs that are highly conserved during evolution. The orthologs that are distinctively expressed in oil-rich mesocarp tissues of this basal angiosperm, such as WRI2, ER-associated acyl-CoA synthetases, and lipid-droplet associated proteins were also identified. This study provides a foundation for future investigations to increase oil-content and has implications for metabolic engineering to enhance storage oil content in nonseed tissues of diverse species.Electronic supplementary materialThe online version of this article (doi:10.1186/s12870-015-0586-2) contains supplementary material, which is available to authorized users.

Indole-3-acetic acid (IAA) induced changes in oil content, fatty acid profiles and expression of four fatty acid biosynthetic genes in Chlorella vulgaris at early stationary growth phase

Microalgae lipids and oils are potential candidates for renewable biodiesel. Many microalgae species accumulate a substantial amount of lipids and oils under environmental stresses. However, low growth rate under these adverse conditions account for the decrease in overall biomass productivity which directly influence the oil yield. This study was undertaken to investigate the effect of exogenously added auxin (indole-3-acetic acid; IAA) on the oil content, fatty acid compositions, and the expression of fatty acid biosynthetic genes in Chlorella vulgaris (UMT-M1). Auxin has been shown to regulate growth and metabolite production of several microalgae. Results showed that oil accumulation was highest on days after treatment (DAT)-2 with enriched levels of palmitic (C16:0) and stearic (C18:0) acids, while the linoleic (C18:2) and α-linolenic (C18:3n3) acids levels were markedly reduced by IAA. The elevated levels of saturated fatty acids (C16:0 and C18:0) were consistent with high expression of the β-ketoacyl ACP synthase I (KAS I) gene, while low expression of omega-6 fatty acid desaturase (ω-6 FAD) gene was consistent with low production of C18:2. However, the increment of stearoyl-ACP desaturase (SAD) gene expression upon IAA induction did not coincide with oleic acid (C18:1) production. The expression of omega-3 fatty acid desaturase (ω-3 FAD) gene showed a positive correlation with the synthesis of PUFA and C18:3n3.

The TOC159 mutant of Arabidopsis thaliana accumulates altered levels of saturated and polyunsaturated fatty acids

We evaluated whether the TOC159 mutant of Arabidopsis called plastid protein import 2-2 (ppi2-2) accumulates normal levels of fatty acids, and transcripts of fatty acid desaturases and galactolipid synthesis enzymes. The ppi2-2 mutant accumulates decreased pigments and total fatty acid content. The MGD1 gene was downregulated and the mutant accumulates decreased levels of monogalactosyldiacylglycerol (MGDG) and 16:3, which suggests that the prokaryotic pathway was impaired in the mutant. The HY5 gene, which encodes long hypocotyl5 transcription factor, was upregulated in the mutant. The DGD1 gene, an HY5 target was marginally increased and the mutant accumulates digalactosyldiacylglycerol at the control level. The mutant had increased expression of 3-ketoacyl-ACP synthase II gene, which encodes a plastid enzyme that elongates 16:0 to 18:0. Interestingly, glycerolipids in the mutant accumulate increased levels of 18:0. A gene that encodes stearoyl-ACP desaturase (SAD) was expressed at the control level and 18:1 was increased, which suggest that SAD may be strongly regulated at the posttranscriptional level. The molar ratio of MGDG to bilayer forming plastid lipids was decreased in the cold-acclimated wild type but not in the ppi2-2 mutant. This indicates that the mutant was unresponsive to cold-stress, and is consistent with increased levels of 18:0, and decreased 16:3 and 18:3 in the ppi2-2 mutant. Overall, these data indicate that a defective Toc159 receptor impaired the synthesis of MGDG, and affected desaturation of 16 and 18-carbon fatty acids. We conclude that expression of the MGD1 gene and synthesis of MGDG are tightly linked to plastid biogenesis.

Identification and expression of a stearoyl-ACP desaturase gene responsible for oleic acid accumulation in Xanthoceras sorbifolia seeds

Xanthoceras sorbifolia Bunge is an oilseed tree that grows well on barren lands in dry climate. Its seeds contain a large amount of oil rich in oleic acid (18:1Δ9) and linoleic acid (18:2Δ9, 12). However, the molecular regulation of oil biosynthesis in X. sorbifolia seeds is poorly understood. Stearoyl-ACP desaturase (SAD, EC 1.14.99.6) is a plastid-localized soluble desaturase that catalyzes the conversion of stearic acid (18:0) to oleic acid, which plays a key role in determining the ratio of saturated to unsaturated fatty acids. In this study, a full-length cDNA of XsSAD was isolated from developing X. sorbifolia embryos. The XsSAD open reading frame had 1194-bp, encoding a polypeptide of 397 amino acids. XsSAD expression in Escherichia coli cells resulted in increased 18:1Δ9 level, confirming the biological activity of the enzyme encoded by XsSAD. XsSAD expression in Arabidopsis ssi2 mutants partially restored the morphological phenotype and effectively increased the 18:1Δ9 level. The levels of other unsaturated fatty acids synthesized with 18:1Δ9 as the substrate also increased to some degree. XsSAD in X. sorbifolia had a much higher expression in embryos than in leaves and petals. XsSAD expression also correlated well with the oleic acid, unsaturated fatty acid, and total fatty acid levels in developing embryos. These data suggested that XsSAD determined the synthesis of oleic acid and contributed to the accumulation of unsaturated fatty acid and total oil in X. sorbifolia seeds. A preliminary tobacco rattle virus-based virus-induced gene silencing system established in X. sorbifolia can also be helpful for further analyzing the functions of XsSAD and other oil synthesis-related genes in woody plants.

Association mapping of seed oil and protein content in Sesamum indicum L. using SSR markers.

Sesame is an important oil crop for the high oil content and quality. The seed oil and protein contents are two important traits in sesame. To identify the molecular markers associated with the seed oil and protein contents in sesame, we systematically performed the association mapping among 369 worldwide germplasm accessions under 5 environments using 112 polymorphic SSR markers. The general linear model (GLM) was applied with the criteria of logP≥3.0 and high stability under all 5 environments. Among the 369 sesame accessions, the oil content ranged from 27.89%–58.73% and the protein content ranged from 16.72%–27.79%. A significant negative correlation of the oil content with the protein content was found in the population. A total of 19 markers for oil content were detected with a R2 value range from 4% to 29%; 24 markers for protein content were detected with a R2 value range from 3% to 29%, of which 19 markers were associated with both traits. Moreover, partial markers were confirmed using mixed linear model (MLM) method, which suggested that the oil and protein contents are controlled mostly by major genes. Allele effect analysis showed that the allele associated with high oil content was always associated with low protein content, and vice versa. Of the 19 markers associated with oil content, 17 presented near the locations of the plant lipid pathway genes and 2 were located just next to a fatty acid elongation gene and a gene encoding Stearoyl-ACP Desaturase, respectively. The findings provided a valuable foundation for oil synthesis gene identification and molecular marker assistant selection (MAS) breeding in sesame.

Characterization of a stearoyl-acyl carrier protein desaturase gene from potential biofuel plant, Pongamia pinnata L.

A new full length cDNA clone encoding stearoyl-ACP desaturase (SAD) was isolated from seeds of Pongamia pinnata, an oil yielding legume plant. The cDNA clone (PpSAD) contained a single open reading frame of 1182-bp coding for 393 amino acids with a predicted molecular mass of 45.04 kDa, and shares similarity with SAD from other plants. Characteristics of the deduced protein were predicted and analyzed using molecular homology modeling; its three dimensional structure strongly resembled the crystal structure of Ricinus communis (RcSAD). Southern blot analysis indicated that 'sad' is a multiple copy gene and was a member of a small gene family. Expression analysis using quantitative real-time PCR revealed that the gene showed marked distinct expression during different stages of seed developments. The results of the expression analysis in this study, combined with existing research, suggest that 'sad' gene may be involved in the regulation of plant seed growth and development.

A mutant of the Arabidopsis thaliana TOC159 gene accumulates reduced levels of linolenic acid and monogalactosyldiacylglycerol

Previous studies have shown that a mutant of Arabidopsis that lacks the Toc159 receptor is impaired in chloroplast biogenesis. The mutant is referred as plastid protein import 2 or ppi2 and has an albino phenotype due to its inability to import the photosynthetic proteins. In this study, we measured fatty acid composition and transcript levels of plastid-localized fatty acid desaturases in the wild type and ppi2 mutant. The objective was to evaluate whether the Toc159 receptor was critical in the import of lipid-synthesizing enzymes. The ppi2 mutant accumulated decreased levels of oleic acid (18:1) and α-linolenic acid (18:3). The mutant accumulated drastically reduced amounts of the chloroplast lipid monogalactosyldiacylglycerol (MGDG), which contains more than 80% of 18:3. The expression of genes that encode stearoyl-ACP desaturase and MGD1 synthase were down-regulated in the ppi2 mutant, and this corresponded to decreased levels of 18:1 and MGDG, respectively. We conclude that in the ppi2 mutant the impaired synthesis of MGDG resulted in decreased amounts of 18:3. The mutant however, had a 30-fold increase in fad5 transcript levels; this increase was mirrored by a 16- to 50-fold accumulation of hexadecatrienoic acid (16:3), a fatty acid found exclusively in MGDG. Taken together, these data suggest that the Toc159 receptor is required in the import of stearoyl-ACP desaturase and MGD1 synthase into the chloroplasts. Since the expression of fad5 gene was up-regulated in the ppi2 mutant, we propose that fad5 desaturase is imported into plastids through the atToc132/atToc120 protein import pathway.

Genetic variation of six desaturase genes in flax and their impact on fatty acid composition.

Flax (Linum usitatissimum L.) is one of the richest plant sources of omega-3 fatty acids praised for their health benefits. In this study, the extent of the genetic variability of genes encoding stearoyl-ACP desaturase (SAD), and fatty acid desaturase 2 (FAD2) and 3 (FAD3) was determined by sequencing the six paralogous genes from 120 flax accessions representing a broad range of germplasm including some EMS mutant lines. A total of 6 alleles for sad1 and sad2, 21 for fad2a, 5 for fad2b, 15 for fad3a and 18 for fad3b were identified. Deduced amino acid sequences of the alleles predicted 4, 2, 3, 4, 6 and 7 isoforms, respectively. Allele frequencies varied greatly across genes. Fad3a, with 110 SNPs and 19 indels, and fad3b, with 50 SNPs and 5 indels, showed the highest levels of genetic variations. While most of the SNPs and all the indels were silent mutations, both genes carried nonsense SNP mutations resulting in premature stop codons, a feature not observed in sad and fad2 genes. Some alleles and isoforms discovered in induced mutant lines were absent in the natural germplasm. Correlation of these genotypic data with fatty acid composition data of 120 flax accessions phenotyped in six field experiments revealed statistically significant effects of some of the SAD and FAD isoforms on fatty acid composition, oil content and iodine value. The novel allelic variants and isoforms identified for the six desaturases will be a resource for the development of oilseed flax with unique and useful fatty acid profiles.Electronic supplementary materialThe online version of this article (doi:10.1007/s00122-013-2161-2) contains supplementary material, which is available to authorized users.

Overexpression of a wheat stearoyl-ACP desaturase (SACPD) gene TaSSI2 in Arabidopsis ssi2 mutant compromise its resistance to powdery mildew

Fatty acids and their derivatives play important roles in plant defense responses. It has been shown that a mutation in a gene encoding one of stearoyl acyl carrier protein fatty acid desaturase isoforms (ssi2 mutant) enhances the resistance of Arabidopsis to multiple pathogens, and similar results were obtained in rice and soybean. However, it is unknown whether the ssi2 mutant is also resistant to powdery mildew (Golovinomyces cichoracearum). In this study, the ssi2 mutant showed enhanced resistance to powdery mildew. Furthermore, we described the cloning and characterization of the TaSSI2 gene (ortholog of AtSSI2) from wheat. Functional analysis of TaSSI2 was performed by overexpressing TaSSI2 in ssi2 mutant of Arabidopsis. The result indicated that ectopic expression of TaSSI2 restored the WT like morphology in the ssi2 background, the 35S:TaSSI2/ssi2 plants accumulated WT-like levels of oleic acid (18:1) and the transcript levels of R genes were significantly lower than that in ssi2 plants. In contrast to the constitutive PR gene expression in ssi2 plants, the transcript accumulation of PR1 and PR2 was similar in the 35S:TaSSI2/ssi2 and wild type both before and after inoculation. Trypan blue staining showed that extensive fungal hyphae and conidiophores were produced in wild-type and 35S:TaSSI2/ssi2 leaves while no visible powdery mildew growth was observed, but dramatic lesions developed at the infection sites in the ssi2 mutant leaves. Our results demonstrated that TaSSI2 is involved in the negative regulation of defense responses in powdery mildew infection, similar to its counterparts in Arabidopsis, indicating a highly conserved function of SSI2 gene in diverse plants.

SMART – Sunflower Mutant population And Reverse genetic Tool for crop improvement

BackgroundSunflower (Helianthus annuus L.) is an important oilseed crop grown widely in various areas of the world. Classical genetic studies have been extensively undertaken for the improvement of this particular oilseed crop. Pertaining to this endeavor, we developed a “chemically induced mutated genetic resource for detecting SNP by TILLING” in sunflower to create new traits.ResultsTo optimize the EMS mutagenesis, we first conducted a “kill curve” analysis with a range of EMS dose from 0.5% to 3%. Based on the observed germination rate, a 50% survival rate i.e. LD50, treatment with 0.6% EMS for 8 hours was chosen to generate 5,000 M2 populations, out of which, 4,763 M3 plants with fertile seed set. Phenotypic characterization of the 5,000 M2 mutagenised lines were undertaken to assess the mutagenesis quality and to identify traits of interest. In the M2 population, about 1.1% of the plants showed phenotypic variations. The sunflower TILLING platform was setup using Endo-1-nuclease as mismatch detection system coupled with an eight fold DNA pooling strategy. As proof-of-concept, we screened the M2 population for induced mutations in two genes related to fatty acid biosynthesis, FatA an acyl-ACP thioesterase and SAD the stearoyl-ACP desaturase and identified a total of 26 mutations.ConclusionBased on the TILLING of FatA and SAD genes, we calculated the overall mutation rate to one mutation every 480 kb, similar to other report for this crop so far. As sunflower is a plant model for seed oil biosynthesis, we anticipate that the developed genetic resource will be a useful tool to identify novel traits for sunflower crop improvement.

Molecular characterization of a novel isolate of Candida tropicalis for enhanced lipid production

To characterize a new isolate of Candida tropicalis for its enhanced storage lipid accumulation with respect to lipid composition, fatty acid profile and transcriptional regulation of four key genes involved in lipid productivity using different carbon sources. Upon growing C. tropicalis on various carbon substrates, glucose was found to be the best followed by xylose for the production of both biomass and storage lipid. On glucose (100 g l(-1)) medium having specific nitrogen stress (C:N 150 : 1), the yeast was capable of yielding about 58% lipid content of its dry biomass, and neutral lipid accounted for about 75% of the total lipid. Fatty acid profiles revealed that the glucose contributed to the highest yield of total fatty acids, maximum proportion of saturated fatty acids and a significant amount of oleic acid. The enhanced lipid production with specific fatty acid profile correlated with the strong upregulation of acetyl Co-A carboxylase, stearoyl-ACP desaturase and diacylglycerol acyltransferase genes, but not the malic enzyme gene. Together, the results documented the differential regulation of four genes of lipid biosynthesis in the newly isolated C. tropicalis oleaginous strain by various carbon sources. We report here on optimization of the carbon substrate for improved lipid yield and transcriptional regulation of lipid biosynthetic genes in C. tropicalis. Our study paves the way for further enhancement of lipid production by metabolic engineering in this organism, which has potential to be the lipid feedstock as a cocoa-butter substitute.

Cloning and characterization of stearoyl–ACP desaturase gene (SAD) in oil palm (Elaeis guineensis Jacq.)

The Oil palm (Elaeis guineensis Jacq.) is the highest producing oil seed crop on a per hectare basis in the world. Current estimates are that within the next decade, palm oil will become the largest single oil traded globally. In this study, the stearyl–ACP desaturase gene (SAD) was cloned and thoroughly characterized in oil palm. Mesocarp tissue of oil palm fruit at different developmental stages including 0, 30, 60, 90, 120, 150, and 180 days after pollination (DAP) was used for expression pattern analysis. The results showed that SAD gene was highly expressed at 90 DAP. SAD gene is 1,179 base pairs in length, comprised of 3 exons and 2 introns, and translated into 393 amino acids. The phylogenetic analysis was constructed using nucleotide sequences, showing that it is closely related to SAD gene from Elaeis oleifera.

Cloning and Expression of Stearoyl-ACP Desaturase and Two Oleate Desaturases Genes from Ginkgo biloba L.

Ginkgo (Ginkgo biloba L.) exhibits high level of resistance to various abiotic stresses, yet has undergone only minor morphological changes through millions of years. The conversion of membrane lipid components may play an important role in the temperature acclimation of plants. Stearoyl-ACP desaturase, microsomal ω-6 (Δ12) desaturase, and plastidial ω-6 (Δ12) desaturase are important enzymes in the unsaturated fatty acid biosynthetic pathway. In this study, full-length cDNA sequences of GbSAD, GbFAD2, and GbFAD6 were isolated from fresh leaves of ginkgo by rapid amplification of cDNA ends and polymerase chain reaction (PCR). Sequence analysis demonstrated that the cDNAs and deduced proteins had high similarity to other reported corresponding cDNAs and proteins. Real-time PCR revealed that transcripts of GbSAD and GbFAD2 in leaves were induced by cold stress (4 °C, 15 °C), but inhibited by high temperature (35 °C, 45 °C), whereas GbFAD6 is expressed constitutively at different temperatures.

Identification of genes possibly related to loss of quality in late-season ‘Hass’ avocados in Chile

The ‘Hass’ avocado (Persea americana Mill.) is the most important avocado variety cultivated worldwide. To reach destination markets, long-term cold storage and transit is usually needed to reduce fruit deterioration. However, low temperatures can affect fruit quality during ripening by increasing the incidence of several physiological disorders. In this work, ‘Hass’ avocados harvested in Chile in the early summer showed more incidence of physiological disorders than fruit harvested in the spring after storage for 40d at 5°C. As an initial approach to understand the types of metabolism affected during ripening after low temperature exposure, a forward suppression subtractive hybridization (SSH) cDNA library was constructed. A total of 117 differentially expressed genes encoding proteins involved in different biochemical pathways were identified. Genes related to stress response, protein synthesis and signal transduction were the most abundant. To corroborate our results with some of the previously found key differentially expressed genes, full length cDNA was obtained by rapid amplification of cDNA ends (RACE-PCR). Analysis of gene expression using real-time quantitative PCR was performed. Six avocado genes relating to cell wall, sugar and lipid metabolism were analyzed: polygalacturonase (PamPG), putative NAD+-dependent sorbitol dehydrogenase (PamSDH), stearoyl-ACP desaturase (SAD) and three putative acyl-CoA synthetase (PamACoAS1, 2, 3). The expression of all genes was up-regulated after ripening, especially in avocados harvested in the spring, except for PamSAD. These results imply that these genes could be related to the loss in quality observed in the fruit from the second harvest. Further studies should be performed to identify markers for quality loss in ‘Hass’ avocados.

The stearoyl-acyl-carrier-protein desaturase promoter (Des) from oil palm confers fruit-specific GUS expression in transgenic tomato

The stearoyl-acyl-carrier-protein (ACP) desaturase is a plastid-localized enzyme that catalyzes the conversion of stearoyl-ACP to oleoyl-ACP and plays an important role in the determination of the properties of the majority of cellular glycerolipids. Functional characterization of the fatty acid desaturase genes and their specific promoters is a prerequisite for altering the composition of unsaturated fatty acids of palm oil by genetic engineering. In this paper, the specificity and strength of the oil palm stearoyl-ACP desaturase gene promoter (Des) was evaluated in transgenic tomato plants. Transcriptional fusions between 5′ deletions of the Des promoter (Des1–4) and the β-glucuronidase (GUS) reporter gene were generated and their expression analyzed in different tissues of stably transformed tomato plants. Histochemical analysis of the Des promoter deletion series revealed that GUS gene expression was confined to the tomato fruits. No expression was detected in vegetative tissues of the transgenic plants. The highest levels of GUS activity was observed in different tissues of ripe red fruits (vascular tissue, septa, endocarp, mesocarp and columella) and in seeds, which harbored the promoter region located between −590 and +10. A comparison of the promoter-deletion constructs showed that the Des4 promoter deletion (314bp) produced a markedly low level of GUS expression in fruits and seeds. Fluorometric analysis of the GUS activity revealed a 4-fold increase in the activity of the full-length Des promoter compared to the CaMV35S promoter. RNA-hybridization analyses provided additional evidence of increased GUS expression in fruits driven by a Des fragment. Taken together, these results demonstrate the potential of the Des promoter as a tool for the genetic engineering of oil palms and other species, including dicots, in improving the quality and nutritional value of the fruits.