Stearoyl-acyl Carrier Protein Desaturase Research Articles

Comparative transcriptomic analysis provides insights into the genetic networks regulating oil differential production in oil crops

BackgroundPlants differ more than threefold in seed oil contents (SOCs). Soybean (Glycine max), cotton (Gossypium hirsutum), rapeseed (Brassica napus), and sesame (Sesamum indicum) are four important oil crops with markedly different SOCs and fatty acid compositions.ResultsCompared to grain crops like maize and rice, expanded acyl-lipid metabolism genes and relatively higher expression levels of genes involved in seed oil synthesis (SOS) in the oil crops contributed to the oil accumulation in seeds. Here, we conducted comparative transcriptomics on oil crops with two different SOC materials. In common, DIHYDROLIPOAMIDE DEHYDROGENASE, STEAROYL-ACYL CARRIER PROTEIN DESATURASE, PHOSPHOLIPID:DIACYLGLYCEROL ACYLTRANSFERASE, and oil-body protein genes were both differentially expressed between the high- and low-oil materials of each crop. By comparing functional components of SOS networks, we found that the strong correlations between genes in “glycolysis/gluconeogenesis” and “fatty acid synthesis” were conserved in both grain and oil crops, with PYRUVATE KINASE being the common factor affecting starch and lipid accumulation. Network alignment also found a conserved clique among oil crops affecting seed oil accumulation, which has been validated in Arabidopsis. Differently, secondary and protein metabolism affected oil synthesis to different degrees in different crops, and high SOC was due to less competition of the same precursors. The comparison of Arabidopsis mutants and wild type showed that CINNAMYL ALCOHOL DEHYDROGENASE 9, the conserved regulator we identified, was a factor resulting in different relative contents of lignins to oil in seeds. The interconnection of lipids and proteins was common but in different ways among crops, which partly led to differential oil production.ConclusionsThis study goes beyond the observations made in studies of individual species to provide new insights into which genes and networks may be fundamental to seed oil accumulation from a multispecies perspective.

A telomere-to-telomere gap-free reference genome assembly of avocado provides useful resources for identifying genes related to fatty acid biosynthesis and disease resistance

Abstract Avocado (Persea americana Mill.) is an economically valuable plant because of the high fatty acid content and unique flavor of its fruits. Its fatty acid content, especially the relatively high unsaturated fatty acid content, provides significant health benefits. We herein present a telomere-to-telomere gapless genome assembly (841.6 Mb) of West Indian avocado. The genome contains 40,629 predicted protein-coding genes. Repeat sequences account for 57.9% of the genome. Notably, all telomeres, centromeres, and a nucleolar organizing region are included in this genome. Fragments from these three regions were observed via fluorescence in situ hybridization. We identified 376 potential disease resistance-related nucleotide-binding leucine-rich repeat genes. These genes, which are typically clustered on chromosomes, may be derived from gene duplication events. Five NLR genes (Pa11g0262, Pa02g4855, Pa07g3139, Pa07g0383, and Pa02g3196) were highly expressed in leaves, stems, and fruits, indicating they may be involved in avocado disease responses in multiple tissues. We also identified 128 genes associated with fatty acid biosynthesis and analyzed their expression patterns in leaves, stems, and fruits. Pa02g0113, which encodes one of 11 stearoyl-acyl carrier protein desaturases mediating C18 unsaturated fatty acid synthesis, was more highly expressed in the leaves than in the stems and fruits. These findings provide valuable insights that enhance our understanding of fatty acid biosynthesis in avocado.

Comparative genomics points to tandem duplications of SAD gene clusters as drivers of increased α-linolenic (ω-3) content in S. hispanica seeds.

Salvia hispanica L. (chia) is a source of abundant ω-3 polyunsaturated fatty acids (ω-3-PUFAs) that are highly beneficial to human health. The genomic basis for this accrued ω-3-PUFA content in this emerging crop was investigated through the assembly and comparative analysis of a chromosome-level reference genome for S. hispanica. The highly contiguous 321.5-Mbp genome assembly covering all six chromosomes enabled the identification of 32,922 protein-coding genes. Two whole-genome duplications (WGD) events were identified in the S. hispanica lineage. However, these WGD events could not be linked to the high α-linolenic acid (ALA, ω-3) accumulation in S. hispanica seeds based on phylogenomics. Instead, our analysis supports the hypothesis that evolutionary expansion through tandem duplications of specific lipid gene families, particularly the stearoyl-acyl carrier protein desaturase (ShSAD) gene family, is the main driver of the abundance of ω-3-PUFAs in S. hispanica seeds. The insights gained from the genomic analysis of S. hispanica will help establish a molecular breeding target that can be leveraged through genome editing techniques to increase ω-3 content in oil crops.

Genome-wide identification and a comparative transcriptomics approach reveal FaSAD3 as a strawberry fruit ripening regulator

Fatty acid desaturases (FADs) play a crucial role in plant growth as well as in the regulation of biotic and abiotic stresses. However, their function has not been well understood in the ripening of fruit. In this study, we identified 73 FADs in the octoploid strawberry genome and analyzed their physicochemical characteristics, chromosomal localization, phylogeny, gene structures, covariance, cis-acting elements, and transcript levels during fruit ripening stages. According to the predicted subcellular localization, FADs are mostly located in the plasma membrane, with a lower abundance in the cell wall, cytoplasm, and nucleus. Numerous cis-elements in the FADs promoter are responsive to light, hormones, and stress. Here, combining comparative transcriptomics, we identified a Δ9 stearoyl-acyl carrier protein (ACP) desaturase gene (FxaC_8 g24100, namely FaSAD3) that is negatively regulated by both key glycolysis genes, cytoplasmic glyceraldehyde-3-phosphate dehydrogenase (FaGAPC2) and pyruvate kinase (FaPKc2.2). The spatiotemporal expression analysis revealed that FaSAD3 is expressed in both vegetative and reproductive organs. Subcellular localization analysis confirmed that FaSAD3 is expressed in both the cytoplasm and nucleus. Meanwhile, the transient overexpression of FaSAD3 reduced anthocyanin, sugar, organic acid, total phenol, and flavonoid contents, while increased fruit firmness. Furthermore, the yeast one hybridization and dual-luciferase assay revealed that the ripening regulator FaGAMYB negatively regulates the transcriptional activity of FaSAD3 promoter, promoting strawberry fruit ripening. These findings indicated that FaSAD3 acts as a negative regulator in strawberry fruits ripening, providing a novel model for comprehending the mechanisms involved in strawberry fruit ripening.

Heterogeneous expression of stearoyl-acyl carrier protein desaturase genes SAD1 and SAD2 from Linum usitatissimum enhances seed oleic acid accumulation and seedling cold and drought tolerance capability in Brassica napus

Flax (Linum usitatissimum L.) as one of the versatile crops is a major source of unsaturated fatty acids in seeds. Stearoyl-acyl carrier protein desaturase (SAD) is a dehydrogenase enzyme that plays a key role in oleic acid biosynthesis as well as responses to biotic and abiotic stresses. However, the function of SAD orthologs from L. usitatissimum has not been assessed. Here, we found that two LuSAD genes, LuSAD1 and LuSAD2, are present in the genome of L. usitatissimum cultivar ‘Longya 10’. Heterogeneous expression of either LuSAD1 or LuSAD2 in Arabidopsis thaliana resulted in the higher contents of total fatty acids and oleic acid in seeds. Interestingly, ectopic expression of LuSAD2 in A. thaliana caused altered plant architecture. Consistently, the overexpression of either LuSAD1 or LuSAD2 in Brassica napus also resulted in increased contents of total fatty acids and oleic acid in seeds. Furthermore, we demonstrated that either LuSAD1 or LuSAD2 enhances seedling resistance to cold and drought stresses via improving antioxidant enzyme activity and nonenzymatic antioxidant levels, as well as reducing membrane damage. These findings not only broaden our knowledge regarding the LuSAD functions in plants, but also offer promising targets for improving oil quantity and quality, and abiotic stress tolerance of oil-producing crops through molecular manipulation.

Sequence characterization and temporal expression analysis of different SADs and FAD2-2 genes in two Iranian olive cultivars

Stearoyl-acyl carrier protein (ACP) desaturases (SADs) and fatty acid desaturases (FADs) are the main genes catalyzing the first and the second desaturation steps throughout the biosynthesis of oleic and linoleic acids, respectively, hence, the major determinants of oil quality and composition. To uncover the molecular mechanisms behind differential oil content and composition in olive fruit, the nucleotide and amino acid sequences of FAD2-2 and three SAD genes were characterized in 'Mari' and 'Shengeh' as two Iranian olive cultivars displaying high contrasting oil composition. In addition, the expression levels of these genes were screened at different time points during fruit development and ripening. Despite detection of a number of nucleotide sequence variations in four characterized genes, the results of amino acid analyses have shown most of them were synonymous and caused no differences at protein level. However, some of the single nucleotide variations caused non cognate amino acid substitution indicating possible conformational changes in the resultant peptide. In particular, regarding OeFAD2-2, a nonsynonymous SNP was detected in 'Shengeh' compared with other olive's FAD2-2 causing an amino acid lysine residue substitution for a glutamic acid, as well as a nonsynonymous substitution in 'Mari' (threonine to serine). The in silico prediction of three-dimensional structure of FAD2-2 revealed these substitutions may lead to structural changes in the final protein structure and function, hence, may contribute to the higher activity of FAD2-2 protein in mesocarp of 'Shengeh' than 'Mari' cultivars. According to results obtained from sequence analysis of oil biosynthesis genes, two Iranian olive cultivars were the most similar and had high sequence differences with international varieties. Results of temporal transcript abundance revealed that OeSAD2 had higher expression in 'Mari' (the high quality oil cultivar) than 'Shengeh' during the critical period of oil biosynthesis. Results of our data corroborates the previous records suggesting that OeSAD2 as the most pronounced isoform of stearoyl-acyl carrier protein desaturases responsible for oleic acid biosynthesis in olive mesocarp. In addition our results also suggest that OeSAD3 may have contributed to the higher oil quality of 'Mari' than 'Shengeh'. Based on our results, OeSAD2 and OeFAD2-2 are suggested as the possible targets for engineering fatty acid composition to help pyramid genes to improve quality and quantity of olive oil and creating high quality olive cultivars.

The genome of oil-Camellia and population genomics analysis provide insights into seed oil domestication

BackgroundAs a perennial crop, oil-Camellia possesses a long domestication history and produces high-quality seed oil that is beneficial to human health. Camellia oleifera Abel. is a sister species to the tea plant, which is extensively cultivated for edible oil production. However, the molecular mechanism of the domestication of oil-Camellia is still limited due to the lack of sufficient genomic information.ResultsTo elucidate the genetic and genomic basis of evolution and domestication, here we report a chromosome-scale reference genome of wild oil-Camellia (2.95 Gb), together with transcriptome sequencing data of 221 cultivars. The oil-Camellia genome, assembled by an integrative approach of multiple sequencing technologies, consists of a large proportion of repetitive elements (76.1%) and high heterozygosity (2.52%). We construct a genetic map of high-density corrected markers by sequencing the controlled-pollination hybrids. Genome-wide association studies reveal a subset of artificially selected genes that are involved in the oil biosynthesis and phytohormone pathways. Particularly, we identify the elite alleles of genes encoding sugar-dependent triacylglycerol lipase 1, β-ketoacyl-acyl carrier protein synthase III, and stearoyl-acyl carrier protein desaturases; these alleles play important roles in enhancing the yield and quality of seed oil during oil-Camellia domestication.ConclusionsWe generate a chromosome-scale reference genome for oil-Camellia plants and demonstrate that the artificial selection of elite alleles of genes involved in oil biosynthesis contributes to oil-Camellia domestication.

Critical metabolic pathways and SAD/FADs, WRI1s, and DGATs cooperate for high-oleic acid oil production in developing oil tea (Camellia oleifera) seeds.

Oil tea trees produce high-quality edible oils with desirably high oleic acid (18:1) and low linoleic (18:2) and linolenic (18:3) fatty acid (FA) levels, but limited understanding of tea oil biosynthesis and regulation has become a significant obstacle for the breeding of high-yield and -quality oil tea varieties. By integrating metabolite and transcriptome analyses of developing oil tea seeds, we dissected the critical metabolic pathways, including glycolysis, fatty acid, and triacylglycerol (TAG) biosynthesis, as well as genes essential for tea seed oil production. Two plastidic stearoyl-acyl carrier protein desaturases (CoSAD1 and 2) and two endoplasmic reticulum-localized FA desaturases (CoFAD2 and 3) were functionally characterized as responsible for high 18:1 and low 18:2 and 18:3 proportions in tea oils. Two diacylglycerol O-acyltransferases (CoDGAT1 and 2) that may prefer to synthesize 18:1-TAG were functionally characterized and might be also important for high 18:1-TAG production. The highly expressed CoWRI1a and b were identified and characterized as activators of glycolysis and regulators of directing source carbon flux into FA biosynthesis in developing oil tea seeds. The upregulated CoSADs with downregulated CoFAD2 and CoFAD3 at the late seed developmental stages mainly accounted for high 18:1 levels. Two CoDGATs might be responsible for assembling TAGs with oleoyl acyl chains, whilst two CoWRI1s regulated carbons from parental sources, partitioning into oil production in oil tea embryo sinks. This study provides a deep understanding of the biosynthesis of tea seed oils and information on genes that may be used as molecular markers to breed oil tea varieties with higher oil yield and quality.

Insights into triterpene synthesis and unsaturated fatty-acid accumulation provided by chromosomal-level genome analysis of Akebia trifoliata subsp. australis

Akebia trifoliata subsp. australis is a well-known medicinal and potential woody oil plant in China. The limited genetic information available for A. trifoliata subsp. australis has hindered its exploitation. Here, a high-quality chromosome-level genome sequence of A. trifoliata subsp. australis is reported. The de novo genome assembly of 682.14 Mb was generated with a scaffold N50 of 43.11 Mb. The genome includes 25,598 protein-coding genes, and 71.18% (485.55 Mb) of the assembled sequences were identified as repetitive sequences. An ongoing massive burst of long terminal repeat (LTR) insertions, which occurred ~1.0 million years ago, has contributed a large proportion of LTRs in the genome of A. trifoliata subsp. australis. Phylogenetic analysis shows that A. trifoliata subsp. australis is closely related to Aquilegia coerulea and forms a clade with Papaver somniferum and Nelumbo nucifera, which supports the well-established hypothesis of a close relationship between basal eudicot species. The expansion of UDP-glucoronosyl and UDP-glucosyl transferase gene families and β-amyrin synthase-like genes and the exclusive contraction of terpene synthase gene families may be responsible for the abundant oleanane-type triterpenoids in A. trifoliata subsp. australis. Furthermore, the acyl-ACP desaturase gene family, including 12 stearoyl-acyl-carrier protein desaturase (SAD) genes, has expanded exclusively. A combined transcriptome and fatty-acid analysis of seeds at five developmental stages revealed that homologs of SADs, acyl-lipid desaturase omega fatty acid desaturases (FADs), and oleosins were highly expressed, consistent with the rapid increase in the content of fatty acids, especially unsaturated fatty acids. The genomic sequences of A. trifoliata subsp. australis will be a valuable resource for comparative genomic analyses and molecular breeding.

Soybean TILLING-by-Sequencing+ reveals the role of novel GmSACPD members in unsaturated fatty acid biosynthesis while maintaining healthy nodules.

Developing soybean lines with high levels of stearic acid is a primary goal of the soybean industry. Most high-stearic-acid soybeans carry different GmSACPD-C mutated alleles. However, due to the dual role of GmSACPD-C in seeds and nodule development, all derived deleterious GmSACPD-C mutant alleles are of extremely poor agronomic value because of defective nodulation. The soybean stearoyl-acyl carrier protein desaturase (GmSACPD) gene family is composed of five members. Comparative genomics analysis indicated that SACPD genes were duplicated and derived from a common ancestor that is still present in chlorophytic algae. Synteny analysis showed the presence of segment duplications between GmSACPD-A/GmSACPD-B, and GmSACPD-C/GmSACPD-D. GmSACPD-E was not contained in any duplicated segment and may be the result of tandem duplication. We developed a TILLING by Target Capture Sequencing (Tilling-by-Sequencing+) technology, a versatile extension of the conventional TILLING by sequencing, and successfully identified 12, 14, and 18 ethyl methanesulfonate mutants at the GmSACPD-A, GmSACPD-B, and GmSACPD-D genes, respectively. Functional analysis of all identified mutants revealed an unprecedented role of GmSACPD-A, GmSACPD-B, and GmSACPD-D in unsaturated fatty acid biosynthesis without affecting nodule development and structure. This discovery will positively impact the development of high-stearic-acid lines to enhance soybean nutritional value without potential developmental tradeoffs.

Metabolomic profiling of wild-type and mutant soybean root nodules using laser-ablation electrospray ionization mass spectrometry reveals altered metabolism.

The establishment of the nitrogen-fixing symbiosis between soybean and Bradyrhizobium japonicum is a complex process. To document the changes in plant metabolism as a result of symbiosis, we utilized laser ablation electrospray ionization-mass spectrometry (LAESI-MS) for in situ metabolic profiling of wild-type nodules, nodules infected with a B. japonicum nifH mutant unable to fix nitrogen, nodules doubly infected by both strains, and nodules formed on plants mutated in the stearoyl-acyl carrier protein desaturase (sacpd-c) gene, which were previously shown to have an altered nodule ultrastructure. The results showed that the relative abundance of fatty acids, purines, and lipids was significantly changed in response to the symbiosis. The nifH mutant nodules had elevated levels of jasmonic acid, correlating with signs of nitrogen deprivation. Nodules resulting from the mixed inoculant displayed similar, overlapping metabolic distributions within the sectors of effective (fix+ ) and ineffective (nifH mutant, fix- ) endosymbionts. These data are inconsistent with the notion that plant sanctioning is cell autonomous. Nodules lacking sacpd-c displayed an elevation of soyasaponins and organic acids in the central necrotic regions. The present study demonstrates the utility of LAESI-MS for high-throughput screening of plant phenotypes. Overall, nodules disrupted in the symbiosis were elevated in metabolites related to plant defense.

Monitoring Adventitious Presence of Transgenes in Cotton Collections from Genebank and Experimental Plots: Ensuring GM-Free Conservation and Cultivation of Genetic Resources

Bt cotton is the commercialized genetically modified (GM) crop in India with four approved GM events, namely MON531 (Bollgard® I), MON15985 (Bollgard® II), GFM and Event 1. Several GM events of cotton with insect-resistant or herbicide-tolerant GM traits were under field trials or imported for research purposes. With the increase in adoption of Bt cotton, unintentional introgression of transgenes in the conserved diversity-rich germplasm and in the field samples needs to be monitored in a systematic manner employing efficient GM diagnostics. Adventitious presence of transgenes was checked in 100 ex situ accessions of cotton being conserved in the National Genebank. These accessions belonging to Gossypium hirsutum, G. barbadense, G. arboreum, G. hirsutum × G. barbadense, represented seven Bt cotton growing states. Pentaplex PCR assay covering screening for over 90% of globally approved GM cotton events was employed, simultaneously targeting Cauliflower Mosaic Virus 35S promoter (P-35S), Agrobacterium tumefaciens nos terminator (T-nos), neomycin phosphotransferase (nptII) and phosphinothricin-N-acetyltransferase (pat) marker genes, and endogenous stearoyl acyl carrier protein desaturase (Sad1) gene as an internal control. Real-time PCR assays targeting P-35S and T-nos, with the limit of detection up to 0.01%, were employed for further confirmation. A study was also conducted on fifty cotton samples collected from the experimental plots, which were grown at distance of 50 and 10 metres from Bt cotton event MON15985. Adventitious presence of transgenes was checked using multiplex PCR assay targeting cry1Ac, cry2Ab2 and endogenous Sad1 gene to define a strategy for screening for specific GM events. Adventitious presence of transgenes was not detected in any of the tested samples based on the screening tests conducted. This strategy targeting specific GM traits would assist in GM-free cultivation of varieties in the experimental plots. The reported approach with wide GM coverage is reliable for checking adventitious presence of transgenes in genebanks, which could be applied to test conserved germplasm on a regular basis, for ensuring GM-free conservation of genetic resources.

Castor Stearoyl-ACP Desaturase Can Synthesize a Vicinal Diol by Dioxygenase Chemistry.

In previous work, we identified a triple mutant of the castor (Ricinus communis) stearoyl-Acyl Carrier Protein desaturase (T117R/G188L/D280K) that, in addition to introducing a double bond into stearate to produce oleate, performed an additional round of oxidation to convert oleate to a trans allylic alcohol acid. To determine the contributions of each mutation, in this work we generated individual castor desaturase mutants carrying residue changes corresponding to those in the triple mutant and investigated their catalytic activities. We observed that T117R, and to a lesser extent D280K, accumulated a novel product, namely erythro-9,10-dihydroxystearate, that we identified via its methyl ester through gas chromatography-mass spectrometry and comparison with authentic standards. The use of 18O2 labeling showed that the oxygens of both hydroxyl moieties originate from molecular oxygen rather than water. Incubation with an equimolar mixture of 18O2 and 16O2 demonstrated that both hydroxyl oxygens originate from a single molecule of O2, proving the product is the result of dioxygenase catalysis. Using prolonged incubation, we discovered that wild-type castor desaturase is also capable of forming erythro-9,10-dihydroxystearate, which presents a likely explanation for its accumulation to ∼0.7% in castor oil, the biosynthetic origin of which had remained enigmatic for decades. In summary, the findings presented here expand the documented constellation of di-iron enzyme catalysis to include a dioxygenase reactivity in which an unactivated alkene is converted to a vicinal diol.

Identification of Climate and Genetic Factors That Control Fat Content and Fatty Acid Composition of Theobroma cacao L. Beans.

The main ingredients of chocolate are usually cocoa powder, cocoa butter, and sugar. Both the powder and the butter are extracted from the beans of the cacao tree (Theobroma cacao L.). The cocoa butter represents the fat in the beans and possesses a unique fatty acid profile that results in chocolate’s characteristic texture and mouthfeel. Here, we used a linkage mapping population and phenotypic data of 3,292 samples from 420 progeny which led to the identification of 27 quantitative trait loci (QTLs) for fatty acid composition and six QTLs for fat content. Progeny showed extensive variation in fat levels and composition, with the level of palmitic acid negatively correlated to the sum of stearic acid, oleic acid, and linoleic acid. A major QTL explaining 24% of the relative level of palmitic acid was mapped to the distal end of chromosome 4, and those higher levels of palmitic acid were associated with the presence of a haplotype from the “TSH 1188” parent in the progeny. Within this region of chromosome 4 is the Thecc1EG017405 gene, an orthologue and isoform of the stearoyl-acyl carrier protein (ACP) desaturase (SAD) gene in plants, which is involved in fatty acid biosynthesis. Besides allelic differences, we also show that climate factors can change the fatty acid composition in the beans, including a significant positive correlation between higher temperatures and the higher level of palmitic acid. Moreover, we found a significant pollen donor effect from the variety “SIAL 70” which was associated with decreased palmitic acid levels.

Physiological Responses and Expression Changes of Fatty Acid Metabolism–Related Genes in Wheat (Triticum aestivum) Under Cold Stress

Cold stress changes the molecular and physiological responses in wheat genotypes according to their genetic potential and duration of stress. Cold stress (6 °C) has been applied in time series of 9 days (1, 3, 6, and 9 days) and evaluated the morpho-physiological responses and genes involved in fatty acid metabolisms such as desaturases and lipid transfer protein by real-time PCR technique in two cultivars of wheat, including MV-17 (cold-tolerant) and Kuhdasht (cold-sensitive). Results indicated that cold stress had a significant effect on morpho-physiological traits in MV-17 when compared with Kuhdasht. Moreover, cold stress for a long period of time could induce more bioactive compounds like hydrogen peroxide and malondialdehyde in MV-17 cultivar than in Kuhdasht. The contents of proline and glycine betaine as osmoregulators increased during times after cold exposure in both cultivars, but ascorbic acids as an antioxidant significantly increased in MV-17 cultivar during prolonged cold treatments. Meanwhile, fatty acid desaturase and lipid transfer genes are involved in fatty acid biosynthesis and lipid changes and differently expressed in cold stress. The fatty acid desaturase 2 (FAD2) leads to lipid unsaturation and its transcript significantly increased in MV-17 cultivar, but it was non-significant in Kuhdasht cultivar. With prolonged cold exposure, fatty acid desaturase 3 (FAD3) and lipid transfer protein 1 (LTP1) gene expression increased and showed the similar trend in both cultivars, while stearoyl-acyl carrier protein desaturases (SAD1) reached to maximum level after 6 days of cold exposure. We found that MV-17 cultivar could be resistant to cold stress during prolonged exposure to 6 °C by the increase in the contents of osmolytes, antioxidant, and fatty acid desaturases especially FAD2 in comparison with Kuhdasht cultivar.

Transcriptional Regulation of Stearoyl-Acyl Carrier Protein Desaturase Genes in Response to Abiotic Stresses Leads to Changes in the Unsaturated Fatty Acids Composition of Olive Mesocarp.

In higher plants, the stearoyl-acyl carrier protein desaturase (SAD) catalyzes the first desaturation step leading to oleic acid, which can be further desaturated to linoleic and α-linolenic acids. Therefore, SAD plays an essential role in determining the overall content of unsaturated fatty acids (UFA). We have investigated how SAD genes expression and UFA composition are regulated in olive (Olea europaea) mesocarp tissue from Picual and Arbequina cultivars in response to different abiotic stresses. The results showed that olive SAD genes are transcriptionally regulated by temperature, darkness and wounding. The increase in SAD genes expression levels observed in Picual mesocarp exposed to low temperature brought about a modification in the UFA content of microsomal membrane lipids. In addition, darkness caused the down-regulation of SAD genes transcripts, together with a decrease in the UFA content of chloroplast lipids. The differential role of olive SAD genes in the wounding response was also demonstrated. These data point out that different environmental stresses can modify the UFA composition of olive mesocarp through the transcriptional regulation of SAD genes, affecting olive oil quality.

Loop-Mediated Isothermal Amplification for Detection of Endogenous Sad1 Gene in Cotton: An Internal Control for Rapid Onsite GMO Testing.

Confirming the integrity of seed samples in powdered form is important prior to conducting a genetically modified organism (GMO) test. Rapid onsite methods may provide a technological solution to check for genetically modified (GM) events at ports of entry. In India, Bt cotton is the commercialized GM crop with four approved GM events; however, 59 GM events have been approved globally. GMO screening is required to test for authorized GM events. The identity and amplifiability of test samples could be ensured first by employing endogenous genes as an internal control. A rapid onsite detection method was developed for an endogenous reference gene, stearoyl acyl carrier protein desaturase (Sad1) of cotton, employing visual and real-time loop-mediated isothermal amplification (LAMP). The assays were performed at a constant temperature of 63°C for 30min for visual LAMP and 62ºC for 40min for real-time LAMP. Positive amplification was visualized as a change in color from orange to green on addition of SYBR® Green or detected as real-time amplification curves. Specificity of LAMP assays was confirmed using a set of 10 samples. LOD for visual LAMP was up to 0.1%, detecting 40 target copies, and for real-time LAMP up to 0.05%, detecting 20 target copies. The developed methods could be utilized to confirm the integrity of seed powder prior to conducting a GMO test for specific GM events of cotton. LAMP assays for the endogenous Sad1 gene of cotton have been developed to be used as an internal control for onsite GMO testing in cotton.

Constitutive BiP protein accumulation in Arabidopsis mutants defective in a gene encoding chloroplast-resident stearoyl-acyl carrier protein desaturase.

The unfolded protein response (UPR) occurs when protein folding and maturation are disturbed in the endoplasmic reticulum (ER). During the UPR, a number of genes including those encoding ER-resident molecular chaperones are induced. In Arabidopsis, BiP3 has been used as a UPR marker gene whose expression is strongly induced in response to ER stress. In this study, we mutagenized Arabidopsis plants expressing β-glucuronidase (GUS) gene under the control of BiP3 promoter and isolated a mutant that exhibits strong GUS activity without treatment with ER stress inducers. By whole genome sequencing, we identified a causal gene in the mutant as SUPPRESSOR OF SALICYLIC ACID INSENSITIVITY2 (SSI2), which encodes stearoyl-acyl carrier protein desaturase that converts stearic acids to oleic acids in the chloroplasts. In addition to GUS proteins, the ssi2 mutant accumulates endogenous BiP3 proteins without treatment by any stress reagents. Interestingly, although the degree of endogenous BiP3 protein accumulation in the ssi2 mutant was comparable to that in wild-type plants treated with the ER stress inducer tunicamycin, much less BiP3 transcripts were detected in the ssi2 mutant compared to tunicamycin-treated wild-type plants. Our finding suggests a genetic link between fatty acid metabolism in the chloroplasts and ER functions.

Enhancing freezing tolerance of Brassica napus L. by overexpression of a stearoyl-acyl carrier protein desaturase gene (SAD) from Sapium sebiferum (L.) Roxb.

Sapium sebiferum (L.) Roxb. is an important woody oil tree and traditional herbal medicine in China. Stearoyl-acyl carrier protein desaturase (SAD) is a dehydrogenase enzyme that plays a key role in the transformation of saturated fatty acids into unsaturated fatty acids in oil; these fatty acids greatly influence the freezing tolerance of plants. However, it remains unclear whether freezing tolerance can be regulated by the expression level of SsSAD in S. sebiferum L. Our research indicated that SsSAD expression in S. sebiferum L. increased under freezing stress. To further confirm this result, we constructed a pEGAD-SsSAD vector and transformed it into B. napus L. W10 by Agrobacterium tumefaciens-mediated transformation. Transgenic plants that overexpressed the SsSAD gene exhibited significantly higher linoleic (18:2) and linolenic acid (18:3) content and advanced freezing tolerance. These results suggest that SsSAD overexpression in B. napus L. can increase the content of polyunsaturated fatty acids (PUFAs) such as linoleic (18:2) and linolenic acid (18:3), which are likely pivotal in improving freezing tolerance in B. napus L. plants. Thus, SsSAD overexpression could be useful in the production of freeze-tolerant varieties of B. napus L.

First study of correlation between oleic acid content and SAD gene polymorphism in olive oil samples through statistical and bayesian modeling analyses

BackgroundVirgin olive oil is appreciated for its particular aroma and taste and is recognized worldwide for its nutritional value and health benefits. The olive oil contains a vast range of healthy compounds such as monounsaturated free fatty acids, especially, oleic acid. The SAD.1 polymorphism localized in the Stearoyl-acyl carrier protein desaturase gene (SAD) was genotyped and showed that it is associated with the oleic acid composition of olive oil samples. However, the effect of polymorphisms in fatty acid-related genes on olive oil monounsaturated and saturated fatty acids distribution in the Tunisian olive oil varieties is not understood.MethodsSeventeen Tunisian olive-tree varieties were selected for fatty acid content analysis by gas chromatography. The association of SAD.1 genotypes with the fatty acids composition was studied by statistical and Bayesian modeling analyses.ResultsFatty acid content analysis showed interestingly that some Tunisian virgin olive oil varieties could be classified as a functional food and nutraceuticals due to their particular richness in oleic acid. In fact, the TT-SAD.1 genotype was found to be associated with a higher proportion of mono-unsaturated fatty acids (MUFA), mainly oleic acid (C18:1) (r = − 0.79, p < 0.000) as well as lower proportion of palmitic acid (C16:0) (r = 0.51, p = 0.037), making varieties with this genotype (i.e. Zarrazi and Tounsi) producing more monounsaturated oleic acid (C18: 1) than saturated acid. These varieties could be thus used as nutraceuticals and functional food.ConclusionThe SAD.1 association with the oleic acid composition of olive oil was identified among the studied varieties. This correlation fluctuated between studied varieties, which might elucidate variability in lipidic composition among them and therefore reflecting genetic diversity through differences in gene expression and biochemical pathways. SAD locus would represent an excellent marker for identifying interesting amongst virgin olive oil lipidic composition.