Stearoyl-acyl Carrier Protein Research Articles

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.

Multi-functional roles of TaSSI2 involved in Fusarium head blight and powdery mildew resistance and drought tolerance

The mutation of the gene encoding a stearoyl-acyl carrier protein fatty acid desaturase (ssi2) has been proved to enhance pathogen resistance in several plants, while it's potential to regulate biotic and abiotic stresses in wheat is still unclear. In this study, we cloned TaSSI2 gene in wheat and provided several evidences of its involvement in multiple biological functions. By using barley stripe mosaic virus (BSMV)-induced gene silencing (VIGS) in wheat, it was found that TaSSI2 negatively regulated both powdery mildew and Fusarium head blight (FHB) resistance, which was consistent with the phenotype observed in knock-out mutants of Kronos. The expression of TaSSI2 was down-regulated by in vitro treatments of methyl jasmonate (MeJA), but positively regulated by salicylic acid (SA) and abscisic acid (ABA), implying the cross-talk between different hormone signaling pathways involved in wheat to regulate biotic stresses is still to be elucidated. Furthermore, the up-regulated expression of PR4 and PR5 indicated that TaSSI2 probably regulated FHB resistance by depressing the SA signaling pathway in wheat. In addition, the over-expression of TaSSI2 increased the content of linolenic acid (18:3) and subsequently enhanced drought tolerance of transgenic Brachypodium. This phenomenon might be associated with its subcellular localization in the whole cytosol, partly overlapping with Golgi apparatus and the secreted vesicles. As a stearoyl-acyl carrier protein fatty acid desaturase, TaSSI2 was proposed to be involved in cell lipid metabolism and carried targets out of the cell from membrane or wax synthesis, resulting in enhanced drought tolerance in plant.

Stearoyl-Acyl Carrier Protein Desaturase Mutations Uncover an Impact of Stearic Acid in Leaf and Nodule Structure.

Stearoyl-acyl carrier protein desaturase (SACPD-C) has been reported to control the accumulation of seed stearic acid; however, no study has previously reported its involvement in leaf stearic acid content and impact on leaf structure and morphology. A subset of an ethyl methanesulfonate mutagenized population of soybean (Glycine max) 'Forrest' was screened to identify mutants within the GmSACPD-C gene. Using a forward genetics approach, one nonsense and four missense Gmsacpd-c mutants were identified to have high levels of seed, nodule, and leaf stearic acid content. Homology modeling and in silico analysis of the GmSACPD-C enzyme revealed that most of these mutations were localized near or at conserved residues essential for diiron ion coordination. Soybeans carrying Gmsacpd-c mutations at conserved residues showed the highest stearic acid content, and these mutations were found to have deleterious effects on nodule development and function. Interestingly, mutations at nonconserved residues show an increase in stearic acid content yet retain healthy nodules. Thus, random mutagenesis and mutational analysis allows for the achievement of high seed stearic acid content with no associated negative agronomic characteristics. Additionally, expression analysis demonstrates that nodule leghemoglobin transcripts were significantly more abundant in soybeans with deleterious mutations at conserved residues of GmSACPD-C. Finally, we report that Gmsacpd-c mutations cause an increase in leaf stearic acid content and an alteration of leaf structure and morphology in addition to differences in nitrogen-fixing nodule structure.

Deletion of the SACPD-C Locus Alters the Symbiotic Relationship Between Bradyrhizobium japonicum USDA110 and Soybean, Resulting in Elicitation of Plant Defense Response and Nodulation Defects.

Legumes form symbiotic associations with soil-dwelling bacteria collectively called rhizobia. This association results in the formation of nodules, unique plant-derived organs, within which the rhizobia are housed. Rhizobia-encoded nitrogenase facilitates the conversion of atmospheric nitrogen into ammonia, which is utilized by the plants for its growth and development. Fatty acids have been shown to play an important role in root nodule symbiosis. In this study, we have investigated the role of stearoyl-acyl carrier protein desaturase isoform C (SACPD-C), a soybean enzyme that catalyzes the conversion of stearic acid into oleic acid, which is expressed in developing seeds and in nitrogen-fixing nodules. In-depth cytological investigation of nodule development in sacpd-c mutant lines M25 and MM106 revealed gross anatomical alteration in the sacpd-c mutants. Transmission electron microscopy observations revealed ultrastructural alterations in the sacpd-c mutants that are typically associated with plant defense response to pathogens. In nodules of two sacpd-c mutants, the combined jasmonic acid (JA) species (JA and the isoleucine conjugate of JA) were found to be reduced and 12-oxophytodienoic acid (OPDA) levels were significantly higher relative to wild-type lines. Salicylic acid levels were not significantly different between genotypes, which is divergent from previous studies of sacpd mutant studies on vegetative tissues. Soybean nodule phytohormone profiles were very divergent from those of roots, and root profiles were found to be almost identical between mutant and wild-type genotypes. The activities of antioxidant enzymes, ascorbate peroxidase, and superoxide dismutase were also found to be higher in nodules of sacpd-c mutants. PR-1 gene expression was extremely elevated in M25 and MM106, while the expression of nitrogenase was significantly reduced in these sacpd-c mutants, compared with the parent 'Bay'. Two-dimensional gel electrophoresis and matrix-assisted laser desorption-ionization time of flight mass spectrometry analyses confirmed sacpd-c mutants also accumulated higher amounts of pathogenesis-related proteins in the nodules. Our study establishes a major role for SACPD-C activity as essential for proper maintenance of soybean nodule morphology and physiology and indicates that OPDA signaling is likely to be involved in attenuation of nodule biotic defense responses.

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.

Application of Glycerol for Induced Powdery Mildew Resistance in Triticum aestivum L.

Previous work has demonstrated that glycerol-3-phosphate (G3P) and oleic acid (18:1) are two important signal molecules associated with plant resistance to fungi. In this article, we provide evidence that a 3% glycerol spray application 1–2 days before powdery mildew infection and subsequent applications once every 4 days was sufficient to stimulate the plant defense responses without causing any significant damage to wheat leaves. We found that G3P and oleic acid levels were markedly induced by powdery mildew infection. In addition, TaGLI1 (encoding a glycerol kinase) and TaSSI2 (encoding a stearoylacyl carrier protein fatty acid desaturase), two genes associated with the glycerol and fatty acid (FA) pathways, respectively, were induced by powdery mildew infection, and their promoter regions contain some fungal response elements. Moreover, exogenous application of glycerol increased the G3P level and decreased the level of oleic acid (18:1). Glycerol application induced the expression of pathogenesis-related (PR) genes (TaPR-1, TaPR-2, TaPR-3, TaPR-4, and TaPR-5), induced the generation of reactive oxygen species (ROS) before powdery mildew infection, and induced salicylic acid (SA) accumulation in wheat leaves. Further, we sprayed glycerol in a wheat field and found that it significantly (p < 0.05) reduced the severity of powdery mildew disease and lessened disease-associated kernel weight loss, all without causing any noticeable degradation in wheat seed quality.

Modification of the fatty acid composition in Arabidopsis and maize seeds using a stearoyl-acyl carrier protein desaturase-1 (ZmSAD1) gene.

BackgroundStearoyl-acyl carrier protein desaturase (SAD) is a key enzyme that catalyses the conversion of stearoyl-acyl carrier protein (ACP) to oleoyl-ACP, a precursor for the biosynthesis of polyunsaturated fatty acids. ZmSAD1 (GenBank: KU949326) is a major QTL for stearic acid content in maize seeds. To investigate the biological function and the application potential of maize ZmSAD1 in oil biosynthesis, we isolated the full-length ZmSAD1 cDNA from maize B73 and overexpressed it in Arabidopsis and maize.ResultsUnder seed-specific overexpression of ZmSAD1 in Arabidopsis, the stearic acid content and the ratio of saturated to unsaturated fatty acids in the seeds were significantly decreased relative to those in the control. Conversely, in transgenic ZmSAD1 RNAi Arabidopsis seeds, the contents of stearic acid and long-chain saturated acids and the ratio of saturated to unsaturated fatty acids were significantly increased; in addition, the oleic acid content was significantly decreased. More importantly, transgenic ZmSAD1 maize that expressed high levels of ZmSAD1 in its mature seeds showed reduced stearic acid content (1.57 %) and a lower saturated to unsaturated fatty acid ratio (20.40 %) relative to those (1.64 % and 20.61 %, respectively) of the control. Conversely, down-regulation of ZmSAD1 in maize resulted in increased levels of stearic acid (1.78 %), long-chain saturated acids (0.85 %) and the ratio of saturated to unsaturated fatty acids (21.54 %) relative to those (1.64 %, 0.74 %, and 20.61 %, respectively) of the control, whereas the oleic acid (32.01 %) level was significantly decreased relative to that (32.68 %) of the control.ConclusionsOur work demonstrates that the contents of stearic acid, oleic acid, and long-chain saturated acids, and the ratio of saturated to unsaturated fatty acids, are modified in maize seeds by seed-specific overexpression or down-regulation of ZmSAD1. Therefore, the ZmSAD1 gene is a useful tool for engineering the seed oil composition in maize and other crops.Electronic supplementary materialThe online version of this article (doi:10.1186/s12870-016-0827-z) contains supplementary material, which is available to authorized users.

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.

Modeling the two-locus architecture of divergent pollinator adaptation: how variation in SAD paralogs affects fitness and evolutionary divergence in sexually deceptive orchids.

Divergent selection by pollinators can bring about strong reproductive isolation via changes at few genes of large effect. This has recently been demonstrated in sexually deceptive orchids, where studies (1) quantified the strength of reproductive isolation in the field; (2) identified genes that appear to be causal for reproductive isolation; and (3) demonstrated selection by analysis of natural variation in gene sequence and expression. In a group of closely related Ophrys orchids, specific floral scent components, namely n-alkenes, are the key floral traits that control specific pollinator attraction by chemical mimicry of insect sex pheromones. The genetic basis of species-specific differences in alkene production mainly lies in two biosynthetic genes encoding stearoyl–acyl carrier protein desaturases (SAD) that are associated with floral scent variation and reproductive isolation between closely related species, and evolve under pollinator-mediated selection. However, the implications of this genetic architecture of key floral traits on the evolutionary processes of pollinator adaptation and speciation in this plant group remain unclear. Here, we expand on these recent findings to model scenarios of adaptive evolutionary change at SAD2 and SAD5, their effects on plant fitness (i.e., offspring number), and the dynamics of speciation. Our model suggests that the two-locus architecture of reproductive isolation allows for rapid sympatric speciation by pollinator shift; however, the likelihood of such pollinator-mediated speciation is asymmetric between the two orchid species O. sphegodes and O. exaltata due to different fitness effects of their predominant SAD2 and SAD5 alleles. Our study not only provides insight into pollinator adaptation and speciation mechanisms of sexually deceptive orchids but also demonstrates the power of applying a modeling approach to the study of pollinator-driven ecological speciation.

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.

Cloning and functional expression of a cDNA encoding stearoyl-ACP Δ9-desaturase from the endosperm of coconut (Cocos nucifera L.)

Coconut (Cocos nucifera L.) is an economically tropical fruit tree with special fatty acid compositions. The stearoyl-acyl carrier protein (ACP) desaturase (SAD) plays a key role in the properties of the majority of cellular glycerolipids. In this paper, a full-length cDNA of a stearoyl-acyl carrier protein desaturase, designated CocoFAD, was isolated from cDNA library prepared from the endosperm of coconut (C. nucifera L.). An 1176bp cDNA from overlapped PCR products containing ORF encoding a 391-amino acid (aa) protein was obtained. The coded protein was virtually identical and shared the homology to other Δ9-desaturase plant sequences (greater than 80% as similarity to that of Elaeis guineensis Jacq). The real-time fluorescent quantitative PCR result indicated that the yield of CocoFAD was the highest in the endosperm of 8-month-old coconut and leaf, and the yield was reduced to 50% of the highest level in the endosperm of 15-month-old coconut. The coding region showed heterologous expression in strain INVSc1 of yeast (Saccharomyces cerevisiae). GC–MS analysis showed that the levels of palmitoleic acid (16:1) and oleic acid (18:1) were improved significantly; meanwhile stearic acid (18:0) was reduced. These results indicated that the plastidial Δ9 desaturase from the endosperm of coconut was involved in the biosynthesis of hexadecenoic acid and octadecenoic acid, which was similar with other plants. These results may be valuable for understanding the mechanism of fatty acid metabolism and the genetic improvement of CocoFAD gene in palm plants in the future.

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.

Identification of the molecular genetic basis of the low palmitic acid seed oil trait in soybean mutant line RG3 and association analysis of molecular markers with elevated seed stearic acid and reduced seed palmitic acid

The fatty acid composition of vegetable oil is becoming increasingly critical for its ultimate functionality and utilization in foods and industrial products. Partial chemical hydrogenation of soybean [Glycine max (L.) Merr.] oil increases oxidative stability and shelf life but also results in the introduction of trans fats as an unavoidable byproduct. Due to mandatory labeling of consumer products containing trans fats, conventional soybean oil has lost the ability to deliver the most appropriate economical functionality and oxidative stability, particularly for baking applications. Genetic improvement of the fatty acid profile of soybean oil is one method of meeting these new requirements for oil feedstocks. In this report, we characterized three mutant genetic loci controlling the saturated fatty acid content of soybean oil: two genes additively reduce palmitic acid content (fap1 and fap3-ug), and one gene independently elevates stearic acid content (fas). We identified a new null allele of fap3-ug/GmFATB1A (derived from line ELLP2) present in line RG3. The splicing defect mutation in a beta-ketoacyl-[acyl-carrier-protein] synthase III candidate gene located in the region mapped to fap1, derived originally from ethyl methane sulphonate mutant line C1726 (Cardinal et al. in Theor Appl Genet 127:97–111, 2014), was also present in line RG3. We also utilized the elevated stearic acid line RG7, which has previously been shown to contain novel mutant fas/SACPD-C alleles encoding stearoyl-acyl carrier protein desaturase (Boersma et al. in Crop Sci 52:1736–1742, 2012). Molecular marker assays have been developed to track these causative mutations and understand their contributions to seed oil fatty acid profiles in a recombinant inbred line population segregating for fap1, fap3-ug, and fas alleles.