Stearoyl-acyl Carrier Protein Desaturase Gene Research Articles

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 in Brassica napus

Flax (Linum usitatissimum L.) is a versatile crop and its seeds are a major source of unsaturated fatty acids. 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 higher contents of total fatty acids and oleic acid in the seeds. Interestingly, ectopic expression of LuSAD2 in A. thaliana caused altered plant architecture. Similarly, the overexpression of either LuSAD1 or LuSAD2 in Brassica napus also resulted in increased contents of total fatty acids and oleic acid in the seeds. Furthermore, we demonstrated that either LuSAD1 or LuSAD2 enhances seedling resistance to cold and drought stresses by improving antioxidant enzyme activity and nonenzymatic antioxidant levels, as well as reducing membrane damage. These findings not only broaden our knowledge of the LuSAD functions in plants, but also offer promising targets for improving the quantity and quality of oil, and the abiotic stress tolerance of oil-producing crops, through molecular manipulation.

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.

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.

Identification of candidate genes from the SAD gene family in cotton for determination of cottonseed oil composition.

Cotton is an economically important crop grown for natural fiber and seed oil production. Cottonseed oil ranks third after soybean oil and colza oil in terms of edible oilseed tonnage worldwide. The fatty acid composition of cottonseed oil determines its industrial application and nutritional values. However, little progress has been made in understanding cottonseed oil biogenesis. Stearoyl-acyl carrier protein desaturase (SAD), the only known enzyme to convert saturated fatty acids into unsaturated fatty acids in plants, plays key roles in determining the fatty acid composition of cottonseed oil. In this study, we identified 9, 9, 18 and 19 SAD genes in the genomes of four sequenced cotton species: diploid Gossypium raimondii (D5), G. arboreum (A2), tetraploid G. hirsutum acc. TM-1 (AD1) and G. barbadense cv. Xinhai21 (AD2), respectively. Bioinformatic and phylogenetic analyses revealed that cotton SADs can be classified into two classes. Expression patterns showed developmental and spatial regulation of SADs in cotton. GhSAD2 and GhSAD4 were preferentially expressed in developing ovules 20-35days post-anthesis, and significantly different expression patterns were found between high-oil and low-oil cotton cultivars, implying these two genes could be involved in cottonseed oil biogenesis. Association analysis further confirmed that GhSAD4-At expression was closely related to the oleic acid (O) content, linoleic acid (L) content and O/L value in cottonseed, implying GhSAD4 plays an important role in cottonseed oil composition. This study brings new perspectives for integrated genome-wide identification of SADs in cotton and provides references for the genetic improvement of cottonseed oil.

Cloning and Functional Analysis of Peanut Δ 9 -Stearoyl-Acyl Carrier Protein Desaturase Promoter

The Δ 9 -stearoyl-acyl carrier protein desaturase (SAD) plays key roles in determining the ratio of saturated to unsatu- rated fatty acids in higher plants. The 720 bp 5' flanking sequence of peanut SAD (AhSAD) gene was isolated from the genomic DNA of peanut cultivar Yuhua 9326 by nested PCR using genomic walking method. The fragment of 5' UTR was obtained using 5' RACE (Rapid Amplification of cDNA End), confirming that the 720 bp 5' flanking sequence is promoter fragment based on sequence alignments. Bioinformatics analysis indicated that AhSAD promoter contained several light, hormone responsive ele- ments and enhancer-like elements as well as CAAT box and TATA box. To study the function of this promoter, we constructed a binary expression vector pBI-PAhSAD by replacing CaMV35S promoter of pBI121 with the AhSAD promoter, which was intro- duced into Arabidopsis and transiently expressed in peanut, respectively, by Agrobacterium-mediated transformation. Histo- chemical staining analysis showed that the GUS gene mainly expressed in roots, stem, leaves, cotyledon and peanut pegs. The histochemical staining was observed in peanut whole leaves, while only in Arabidopsis veins of cauline leaves.

Characterization of a stearoyl-acyl carrier protein desaturase gene family from chocolate tree, Theobroma cacao L.

In plants, the conversion of stearoyl-ACP to oleoyol-ACP is catalyzed by a plastid-localized soluble stearoyl-acyl carrier protein (ACP) desaturase (SAD). The activity of SAD significantly impacts the ratio of saturated and unsaturated fatty acids, and is thus a major determinant of fatty acid composition. The cacao genome contains eight putative SAD isoforms with high amino acid sequence similarities and functional domain conservation with SAD genes from other species. Sequence variation in known functional domains between different SAD family members suggested that these eight SAD isoforms might have distinct functions in plant development, a hypothesis supported by their diverse expression patterns in various cacao tissues. Notably, TcSAD1 is universally expressed across all the tissues, and its expression pattern in seeds is highly correlated with the dramatic change in fatty acid composition during seed maturation. Interestingly, TcSAD3 and TcSAD4 appear to be exclusively and highly expressed in flowers, functions of which remain unknown. To test the function of TcSAD1 in vivo, transgenic complementation of the Arabidopsis ssi2 mutant was performed, demonstrating that TcSAD1 successfully rescued all AtSSI2 related phenotypes further supporting the functional orthology between these two genes. The identification of the major SAD gene responsible for cocoa butter biosynthesis provides new strategies for screening for novel genotypes with desirable fatty acid compositions, and for use in breeding programs to help pyramid genes for quality and other traits such as disease resistance.

Cloning and functional characterization of SAD genes in potato.

Stearoyl-acyl carrier protein desaturase (SAD), locating in the plastid stroma, is an important fatty acid biosynthetic enzyme in higher plants. SAD catalyzes desaturation of stearoyl-ACP to oleyl-ACP and plays a key role in determining the homeostasis between saturated fatty acids and unsaturated fatty acids, which is an important player in cold acclimation in plants. Here, four new full-length cDNA of SADs (ScoSAD, SaSAD, ScaSAD and StSAD) were cloned from four Solanum species, Solanum commersonii, S. acaule, S. cardiophyllum and S. tuberosum, respectively. The ORF of the four SADs were 1182 bp in length, encoding 393 amino acids. A sequence alignment indicated 13 amino acids varied among the SADs of three wild species. Further analysis showed that the freezing tolerance and cold acclimation capacity of S. commersonii are similar to S. acaule and their SAD amino acid sequences were identical but differed from that of S. cardiophyllum, which is sensitive to freezing. Furthermore, the sequence alignments between StSAD and ScoSAD indicated that only 7 different amino acids at residues were found in SAD of S. tuberosum (Zhongshu8) against the protein sequence of ScoSAD. A phylogenetic analysis showed the three wild potato species had the closest genetic relationship with the SAD of S. lycopersicum and Nicotiana tomentosiformis but not S. tuberosum. The SAD gene from S. commersonii (ScoSAD) was cloned into multiple sites of the pBI121 plant binary vector and transformed into the cultivated potato variety Zhongshu 8. A freeze tolerance analysis showed overexpression of the ScoSAD gene in transgenic plants significantly enhanced freeze tolerance in cv. Zhongshu 8 and increased their linoleic acid content, suggesting that linoleic acid likely plays a key role in improving freeze tolerance in potato plants. This study provided some new insights into how SAD regulates in the freezing tolerance and cold acclimation in potato.

A stearoyl-acyl carrier protein desaturase, NbSACPD-C, is critical for ovule development in Nicotiana benthamiana.

Stearoyl-acyl carrier protein desaturase (SACPD) activity is essential for production of the major unsaturated fatty acids (UFAs) in plant lipids. We report here the characterization of three SACPD genes from Nicotiana benthamiana, NbSACPD-A, -B, and -C. All three genes share high similarity to AtSSI2/FAB2 (Suppressor of Salicylic acid-Insensitivity2/Fatty Acid Biosynthesis2), the primary SACPD isoform in Arabidopsis. Knocking down the expression of individual or combinations of NbSACPDs by an artificial microRNA approach resulted in significantly reduced accumulation of 18C UFAs and elevated levels of 18:0-FA (Fatty acids) in leaves, indicating that all three genes participated in fatty acid desaturation. The triple knockdown (KD) plants displayed severe growth phenotypes, including spontaneous cell death and dwarfing. While no vegetative morphologic abnormality was observed in NbSACPD-A, -B, or -C KD plants, strikingly, NbSACPD-C KD plants produced small fruits with aborted ovules. Reciprocal crosses with wild-type and NbSACPD-C KD plants revealed that knocking down NbSACPD-C expression caused female, but not male, sterility. Furthermore, arrested ovule development and significantly altered lipid composition in ovaries were observed in NbSACPD-C KD plants, consistent with the predominant NbSACPD-C expression in ovules. The ovule development defect was fully complemented by coexpressing an amiRNA-resistant NbSACPD-C variant in the NbSACPD-C KD background, further supporting a specific requirement for NbSACPD-C in female fertility. Our results thus indicated that NbSACPD-C plays a critical role maintaining membrane lipid composition in ovule development for female fertility in N.benthamiana, complementing and extending prior understanding on the well-demonstrated roles of SACPDs in biotic and abiotic stresses.

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.

Analysis of codon use features of stearoyl-acyl carrier protein desaturase gene in Camellia sinensis

The stearoyl-acyl carrier protein desaturase (SAD) gene widely exists in all kinds of plants. In this paper, the Camellia sinensis SAD gene (CsSAD) sequence was firstly analyzed by Codon W, CHIPS, and CUSP programs online, and then compared with genomes of the tea plant, other species and SAD genes from 11 plant species. The results show that the CsSAD gene and the selected 73 of C. sinensis genes have similar codon usage bias. The CsSAD gene has a bias toward the synonymous codons with A and T at the third codon position, the same as the 73 of C. sinensis genes. Compared with monocotyledons such as Triticum aestivum and Zea mays, the differences in codon usage frequency between the CsSAD gene and dicotyledons such as Arabidopsis thaliana and Nicotiana tobacum are less. Therefore, A. thaliana and N. tobacum expression systems may be more suitable for the expression of the CsSAD gene. The analysis result of SAD genes from 12 plant species also shows that most of the SAD genes are biased toward the synonymous codons with G and C at the third codon position. We believe that the codon usage bias analysis presented in this study will be essential for providing a theoretical basis for discussing the structure and function of the CsSAD gene.

Fatty acid elongase 1 (FAE1) promoter as a candidate for genetic engineering of fatty acids to improve seed oil composition

As an important cis-regulatory element, a promoter plays a key role in plant gene expression and regulation, and has been widely used in plant genetic engineering. The fatty acid elongase 1 (FAE1) promoter was isolated from Arabidopsis thaliana . Sequence analysis showed that the FAE1 promoter contains two Skn-1 motifs, one O 2 -site, and one G-box, which play important roles in controlling seed-preferred expression. Stearoyl-acyl carrier protein desaturase (SAD) catalyzes the conversion of stearic to oleic acid. 23 FAE1pro:GUS and 31 FAE1pro:SAD transgenic lines were obtained and confirmed by polymerase chain reaction (PCR). In the FAE1pro:GUS transgenic lines, β-glucuronidase (GUS) staining was observed in immature leaves, young stems, flowers, and pods, indicating that FAE1 promoter is highly active in these tissues. Real-time PCR revealed that the FAE1 promoter-driven maize SAD gene was highly expressed in the seeds, followed by the pods and flowers; however, its expression level was low in the roots, stems, and leaves. Stearic acid and long-chain saturated fatty acid contents of seeds were significantly decreased; further, the ratio of total saturated fatty acids to unsaturated fatty acids was also reduced. These results indicate that FAE1 promoter is ideal for genetic engineering to improve seed oil composition. Key words : Arabidopsis thaliana, FAE1 promoter, stearoyl-acyl carrier protein desaturase (SAD), seed fatty acid composition.

Isolation and Analysis of DNA Fragment of Genes Related to Kopyor Trait in Coconut Plant

Kopyor coconut is a natural mutant that has abnormal endosperm development. For the first time several genes that were suspected to be related to kopyor trait were identified based on the chemical compounds of the endosperm that different from that of normal coconut. Sucrose synthase (SUS), Stearoyl acyl carrier protein desaturase (SACPD), and Absicid acid insensitive (ABI) genes were isolated and analyzed. Four DNA fragments with length of 746, 738, 780, and 687 bp (CnSus1A, CnSus1B, CnSus2A, and CnSus2B) were obtained from SUS gene. Sequence analysis at DNA and amino acid level showed that CnSus1A, CnSus1B, CnSus2A, and CnSus2B were classified into monocot SUS group with nongrass SUS type. Isolation of SACPD gene resulted in one DNA fragment with DNA length of 716 bp. CnSacpd shared a high homology with SACPD gene of oil palm and soybean. Isolation of ABI gene resulted in two DNA fragments, CnAbi3A and CnAbi3B, with DNA length of 760 and 728 bp, respectively. CnAbi3A and CnAbi3B showed a high homology with ABI3 gene of several plants. All DNA fragment obtained from SUS, SACPD, ABI genes were used as templates to design spesific markers for each corresponding gene. There were 7 specific primer sets designed, i.e., CnSUS1A, CnSUS1B, CnSUS2A, CnSUS2B, CnSACPD, CnABI3A, and CnABI3B.<

Cloning and Characterization of a Stearoyl–Acyl Carrier Protein Desaturase Gene from Cinnamomum longepaniculatum

The stearoyl–acyl carrier protein desaturase (SAD) is a key enzyme that determines the ratio of saturated to unsaturated fatty acids in higher plants. Using reverse transcriptase polymerase chain reaction and rapid amplification of cDNA ends, a full-length cDNA of SAD was obtained from developing leaves of Cinnamomum longepaniculatum. Sequence analysis showed that the deduced amino acid sequence had high similarity to other reported SADs. The CSAD gene was functionally expressed in Escherichia coli, and the desaturating activity of the recombinant protein was markedly detected when assayed in vitro by adding spinach ferredoxin. Southern blot analysis indicated that the gene was a member of a small gene family. Northern blotting revealed that the CSAD gene was highly expressed in developing leaves of C. longepaniculatum. These results would provide opportunities for modifying fatty acid composition in C. longepaniculatum.

Cloning and characterization of a stearoyl-ACP desaturase gene from Jatropha curcas

Using degenerate primers and RT-PCR, RACE techniques, a 1491 bp cDNA segment of stearoyl-acyl carrier protein desaturase (SAD) is cloned from developing seeds of Jatropha curcas L. The segment contains a 1191 bp of complete open reading frame (ORF). Analysis in the BLAST on NCBl shows that Jatropha curcas SAD (JSAD) gene encodes a protein precursor composed of a signal peptide of 33 amino acids and a mature peptide of 363 amino acids. The homological analysis shows that JSAD has high level of homology both in nucleotide sequence and in amino acid sequence to other plants SADs. The nucleotide and peptide identity of JSAD to Ricinus communis SAD (RSAD) is up to 89% and 96.2% respectively. Molecular modeling of JSAD indicates that its three-dimensional structure strongly resembled the crystal structure of RSAD.

Two Genes from Soybean Encoding Soluble Δ9 Stearoyl‐ACP Desaturases

The Δ9 stearoyl acyl‐carrier protein desaturase (SACPD) gene of soybean [Glycine max (L.) Merrill] encodes a soluble enzyme that converts stearic to oleic acid. Understanding the regulation of SACPD expression and enzyme activity are thus important steps toward developing soybean lines with altered stearic or oleic acid content. Using primers designed to a G. max SACPD cDNA sequence, a 3648‐bp product was cloned and sequenced from the genome of cultivar Dare. Comparison of the third SACPD exon protein sequence with other available Glycine SACPD sequences revealed unique amino acid variability at positions 310 and 313. Sequence‐specific primers were designed for Real‐time RT‐PCR (reverse transcriptase‐polymerase chain reaction) for this region of exon 3. Diagnostic and specific products were recovered with these primers using Dare cDNA template and Dare genomic DNA. Sequencing of a second genomic clone from Dare confirmed that there were two SACPD genes, designated A and B, in this cultivar. Survey of the genomes of 51 soybean lines and cultivars with PCR and the gene‐specific primers indicated that all 51 had both A and B Differences between SACPD‐A and ‐B transcript abundance in soybean tissues, while quantifiable, were not dramatic. SACPD‐A and ‐B transcript accumulation for three seed developmental stages between R5 and R6 was essentially equal. Biochemical analysis of the proteins encoded by these two SACPD genes may reveal whether the amino acid variability uncovered in this study has any relation to enzyme activity.

The Primary Structure of a cDNA Clone of the Stearoyl-Acyl Carrier Protein Desaturase Gene from Potato (Solanum tuberosum L.)

The Primary Structure of a cDNA Clone of the Stearoyl-Acyl Carrier Protein Desaturase Gene from Potato (<i>Solanum tuberosum</i> L.)

Modification of Brassica seed oil by antisense expression of a stearoyl-acyl carrier protein desaturase gene.

Molecular gene transfer techniques have been used to engineer the fatty acid composition of Brassica rapa and Brassica napus (canola) oil. Stearoyl-acyl carrier protein (stearoyl-ACP) desaturase (EC 1.14.99.6) catalyzes the first desaturation step in seed oil biosynthesis, converting stearoyl-ACP to oleoyl-ACP. Seed-specific antisense gene constructs of B. rapa stearoyl-ACP desaturase were used to reduce the protein concentration and enzyme activity of stearoyl-ACP desaturase in developing rapeseed embryos during storage lipid biosynthesis. The resulting transgenic plants showed dramatically increased stearate levels in the seeds. A continuous distribution of stearate levels from 2% to 40% was observed in seeds of a transgenic B. napus plant, illustrating the potential to engineer specialized seed oil compositions.