Stearoyl-acyl Carrier Protein Desaturase Research Articles

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.

Amino Acid Change in an Orchid Desaturase Enables Mimicry of the Pollinator’s Sex Pheromone

Mimicry illustrates the power of selection to produce phenotypic convergence in biology [1]. Astriking example is the imitation of female insectsby plants that are pollinated by sexual deception of males of the same insect species [2-4]. This involves mimicry of visual, tactile, andchemical signals of females [2-7], especially their sex pheromones [8-11]. The Mediterranean orchid Ophrys exaltata employs chemical mimicry of cuticular hydrocarbons, particularly the 7-alkenes, in an insect sex pheromone to attract andelicit mating behavior in its pollinators, males of the cellophane bee Colletes cunicularius [11-13]. A difference in alkene double-bond positions is responsible for reproductive isolation between O.exaltata and closely related species, such as O.sphegodes [13-16]. We show that these7-alkenes are likely determined by the action of the stearoyl-acyl-carrier-protein desaturase (SAD) homolog SAD5. After gene duplication, changes in subcellular localization relative to the ancestral housekeeping desaturase may have allowed proto-SAD5's reaction products to undergo further biosynthesis to both 7- and 9-alkenes. Such ancestral coproduction of two alkene classes may have led to pollinator-mediated deleterious pleiotropy. Despite possible evolutionary intermediates with reduced activity, amino acid changes at the bottom of the substrate-binding cavity have conferred enzyme specificity for 7-alkene biosynthesis by preventingthe binding of longer-chained fatty acid (FA) precursors by the enzyme. This change in desaturase function enabled theorchid to perfect its chemical mimicry of pollinator sex pheromones by escape from deleterious pleiotropy, supporting a role of pleiotropy in determining the possible trajectories of adaptive evolution.

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.

Overexpression of stearoyl-ACP desaturase enhances accumulations of oleic acid in the green alga Chlamydomonas reinhardtii

FAB2, which encodes stearoyl-acyl carrier protein desaturase, catalyzes the conversion of stearic acid (18:0) to oleic acid (18:1) in fatty acid biosynthesis. In this study, we isolated FAB2 from Chlamydomonas reinhardtii, named CrFAB2, and generated CrFAB2-overexpressing transgenic lines to identify a major role of CrFAB2 in fatty acid biosynthesis of C. reinhardtii. In CrFAB2-overexpressing lines, oleic acid (18:1) content was increased by approximately 2.4-fold compared to the wild-type control plants. Interestingly, CrFAB2 overexpression resulted in the induction of CrFAD2 expression. Consistent with this result, the induction of linoleic acid (18:2) was also detected in CrFAB2-overexpressing lines, and total fatty acid content in these lines was induced by approximately 28 % by CrFAB2 overexpression compared to the wild-type control. Our results indicate that CrFAB2 overexpression enhances the synthesis of oleic acid (18:1) and that CrFAB2 may also play a key role in regulating total fatty acid content in the green alga C. reinhardtii.

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.

The Genetic Basis of Pollinator Adaptation in a Sexually Deceptive Orchid

In plants, pollinator adaptation is considered to be a major driving force for floral diversification and speciation. However, the genetic basis of pollinator adaptation is poorly understood. The orchid genus Ophrys mimics its pollinators' mating signals and is pollinated by male insects during mating attempts. In many species of this genus, chemical mimicry of the pollinators' pheromones, especially of alkenes with different double-bond positions, plays a key role for specific pollinator attraction. Thus, different alkenes produced in different species are probably a consequence of pollinator adaptation. In this study, we identify genes that are likely involved in alkene biosynthesis, encoding stearoyl-acyl carrier protein (ACP) desaturases (SAD), in three closely related Ophrys species, O. garganica, O. sphegodes, and O. exaltata. Combining floral odor and gene expression analyses, two SAD homologs (SAD1/2) showed significant association with the production of (Z)-9- and (Z)-12-alkenes that were abundant in O. garganica and O. sphegodes, supporting previous biochemical data. In contrast, two other newly identified homologs (SAD5/6) were significantly associated with (Z)-7-alkenes that were highly abundant only in O. exaltata. Both molecular evolutionary analyses and pollinator preference tests suggest that the alkenes associated with SAD1/2 and SAD5/6 are under pollinator-mediated divergent selection among species. The expression patterns of these genes in F1 hybrids indicate that species-specific expression differences in SAD1/2 are likely due to cis-regulation, while changes in SAD5/6 are likely due to trans-regulation. Taken together, we report a genetic mechanism for pollinator-mediated divergent selection that drives adaptive changes in floral alkene biosynthesis involved in reproductive isolation among Ophrys species.

Stearoyl-acyl carrier protein desaturase gene from the oleaginous microalga Chlorella zofingiensis: cloning, characterization and transcriptional analysis

The green alga Chlorella zofingiensis can accumulate high level of oleic acid (OA, C18:1△(9)) rich oils in response to stress conditions. To understand the regulation of biosynthesis of fatty acid in particular OA at the molecular level, we cloned and characterized the stearoyl acyl carrier protein (ACP) desaturase (SAD) responsible for OA formation through desaturation of stearic acid (C18:0) from C. zofingiensis. Southern blot indicated that the C. zofingiensis genome contained a single copy of SAD, from which the deduced amino acid sequence shared high identity to the corresponding homologs from other microalgae and higher plants. The desaturation activity of SAD was demonstrated in vitro using C18:0-ACP as a substrate. Stress conditions such as high light (HL), nitrogen deficiency (N(-)), or combination of HL and N(-) (HL+N(-)) drastically up-regulated the transcripts of biotin carboxylase (BC, a subunit of ACCase) and SAD, and therefore induced considerably the cellular accumulation of total fatty acids including OA. Glucose (50mM) gave rise to the similar up-regulation of the two genes and induction of fatty acid accumulation. The accumulation of intracellular reactive oxygen species was found to be associated with the up-regulation of genes. This is the first report of characterization of Chlorella-derived SAD and the results may contribute to understanding of the mechanisms involved in fatty acid/lipid biosynthesis in microalgae.

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.

Construction and Application of a Reference Plasmid Suitable for Determination of CpTI and cry1A Gene Dosages in Genetically Modified Cottons

A reference plasmid, pMD-CCS, was constructed to detect multiple target genes in the insect resistant cotton (Gossypium hirsutum L.) varieties in China. This plasmid contained cowpea trypsin inhibitor (CpTI), Bacillus thuringiensis insecticidal crystal protein (cry1A), and cotton endogenous gene Stearoyl-acyl carrier protein desaturase (Sad1) sequences targeting the key exogenous gene types. The real-time quantitative PCR methods for CpTI and cry1A were established using pMD-CCS as the reference material (RM). The dosagess of CpTI and cry1A from 9 insect resistant cotton varieties were determined. The average CpTI dosages were 0.018–0.020 copies per genome and the average cry1A dosages were 1.377–2.136 copies per genome in the 3 insect resistant cotton varieties including Kemian 3. The average cry1A dosages were 0.887–2.564 copies per genome in 6 varieties including Ezamian 1-F1. The standard deviations of the quantitative measurement ranged from 0.001 to 0.049. These results demonstrated that pMD-CCS could be used as the RM for the quantitative measurement of CpTI and cry1A genes in insect resistant cotton varieties.

An effective virus-based gene silencing method for functional genomics studies in common bean

BackgroundCommon bean (Phaseolus vulgaris L.) is a crop of economic and nutritious importance in many parts of the world. The lack of genomic resources have impeded the advancement of common bean genomics and thereby crop improvement. Although concerted efforts from the "Phaseomics" consortium have resulted in the development of several genomic resources, functional studies have continued to lag due to the recalcitrance of this crop for genetic transformation.ResultsHere we describe the use of a bean pod mottle virus (BPMV)-based vector for silencing of endogenous genes in common bean as well as for protein expression. This BPMV-based vector was originally developed for use in soybean. It has been successfully employed for both protein expression and gene silencing in this species. We tested this vector for applications in common bean by targeting common bean genes encoding nodulin 22 and stearoyl-acyl carrier protein desaturase for silencing. Our results indicate that the BPMV vector can indeed be employed for reverse genetics studies of diverse biological processes in common bean. We also used the BPMV-based vector for expressing the green fluorescent protein (GFP) in common bean and demonstrate stable GFP expression in all common bean tissues where BPMV was detected.ConclusionsThe availability of this vector is an important advance for the common bean research community not only because it provides a rapid means for functional studies in common bean, but also because it does so without generating genetically modified plants. Here we describe the detailed methodology and provide essential guidelines for the use of this vector for both gene silencing and protein expression in common bean. The entire VIGS procedure can be completed in 4-5 weeks.

Stearoyl-acyl carrier protein desaturases are associated with floral isolation in sexually deceptive orchids

The orchids Ophrys sphegodes and O. exaltata are reproductively isolated from each other by the attraction of two different, highly specific pollinator species. For pollinator attraction, flowers chemically mimic the pollinators' sex pheromones, the key components of which are alkenes with different double-bond positions. This study identifies genes likely involved in alkene biosynthesis, encoding stearoyl-acyl carrier protein (ACP) desaturase (SAD) homologs. The expression of two isoforms, SAD1 and SAD2, is flower-specific and broadly parallels alkene production during flower development. SAD2 shows a significant association with alkene production, and in vitro assays show that O. sphegodes SAD2 has activity both as an 18:0-ACP Δ(9) and a 16:0-ACP Δ(4) desaturase. Downstream metabolism of the SAD2 reaction products would give rise to alkenes with double-bonds at position 9 or position 12, matching double-bond positions observed in alkenes in the odor bouquet of O. sphegodes. SAD1 and SAD2 show evidence of purifying selection before, and positive or relaxed purifying selection after gene duplication. By contributing to the production of species-specific alkene bouquets, SAD2 is suggested to contribute to differential pollinator attraction and reproductive isolation among these species. Taken together, these data are consistent with the hypothesis that SAD2 is a florally expressed barrier gene of large phenotypic effect and, possibly, a genic target of pollinator-mediated selection.