Fatty Acid Desaturation during Chilling Acclimation Is One of the Factors Involved in Conferring Low-Temperature Tolerance to Young Tobacco Leaves

The FAD7 gene, a gene for a chloroplast [omega]-3 fatty acid desaturase, is responsible for the trienoic fatty acid (TA) formation in leaf tissues. The TA content of the leaf tissue of the 25[deg]C-grown transgenic tobacco (Nicotiana tabacum cv SR1) plants, in which the FAD7 gene from Arabidopsis thaliana was overexpressed, increased uniformly by about 10%. Fatty acid unsaturation in all major leaf polar lipid species increased in the 25[deg]C-grown FAD7 transformants but was approximately the same between the control plants and the FAD7 transformants when grown at 15[deg]C. Therefore, the overexpression of the exogenous FAD7 gene leads to the same consequence in the tobacco plants as the low-temperature-induced TA production that may be catalyzed by an endogenous, temperature-regulated chloroplast [omega]-3 fatty acid desaturase. In the 25[deg]C-grown control plants, the chilling treatment caused symptoms of leaf chlorosis and suppression of leaf growth. The 25[deg]C-grown FAD7 transgenic plants conferred alleviation of these chilling-induced symptoms. A reductions of the chilling injury similar to that of the FAD7 transformants was also observed in the 15[deg]C-preincubated control plants. These results indicate that the increased TA production during chilling acclimation is one of the prerequisites for the normal leaf development at low, nonfreezing temperatures.     

Silencing genes encoding omega-3 fatty acid desaturase alters seed size and accumulation of Bean pod mottle virus in soybean

Omega-3 fatty acid desaturase (FAD3)-catalyzed conversion of linoleic acid to linolenic acid (18:3) is an important step for the biosynthesis of fatty acids as well as the phytohormone jasmonic acid (JA) in plants. We report that silencing three microsomal isoforms of GmFAD3 enhanced the accumulation of Bean pod mottle virus (BPMV) in soybean. The GmFAD3-silenced plants also accumulated higher levels of JA, even though they contained slightly reduced levels of 18:3. Consequently, the GmFAD3-silenced plants expressed JA-responsive pathogenesis-related genes constitutively and exhibited enhanced susceptibility to virulent Pseudomonas syringae. Increased accumulation of BPMV in GmFAD3-silenced plants was likely associated with their JA levels, because exogenous JA application also increased BPMV accumulation. The JA-derived increase in BPMV levels was likely not due to repression of salicylic acid (SA)-derived signaling because the GmFAD3-silenced plants were enhanced in SA-dependent defenses. Furthermore, neither exogenous SA application nor silencing the SA-synthesizing phenylalanine ammonia lyase gene altered BPMV levels in soybean. In addition to the altered defense responses, the GmFAD3-silenced plants also produced significantly larger and heavier seed. Our results indicate that loss of GmFAD3 enhances JA accumulation and, thereby, susceptibility to BPMV in soybean.     

Low temperature and light regulate delta 12 fatty acid desaturases (FAD2) at a transcriptional level in cotton (Gossypium hirsutum)

Lipid modifying enzymes play a key role in the development of cold stress tolerance in cold-resistant plants such as cereals. However, little is known about the role of the endogenous enzymes in cold-sensitive species such as cotton. Delta 12 fatty acid desaturases (FAD2), known to participate in adaptation to low temperatures through acyl chain modifications were used in gene expression studies in order to identify parameters of plant response to low temperatures. The induction of microsomal delta 12 fatty acid desaturases at an mRNA level under cold stress in plants is shown here for first time. Quantitative PCR showed that though both delta 12 omega 6 fatty acid desaturase genes FAD2-3 and FAD2-4 identified in cotton are induced under cold stress, FAD2-4 induction is significantly higher than FAD2-3. The induction of both isoforms was light regulated, in contrast a third isoform FAD2-2 was not affected by cold or light. Stress tolerance and light regulatory elements were identified in the predicted promoters of both FAD2-3 and FAD2-4 genes. Di-unsaturated fatty acid species rapidly increased in the microsomal fraction isolated from cotton leaves, following cold stress. Expression analysis patterns were correlated with the observed increase in both total and microsomal fatty acid unsaturation levels suggesting the direct role of the FAD2 genes in membrane adaptation to cold stress.     

Colocalization of Polyphenol Oxidase and Photosystem II Proteins

Polyphenol oxidase (PPO) appears to be ubiquitous in higher plants but, as yet, no function has been ascribed to it. Herein, we report on the localization of PPO based upon biochemical fractionation of chloroplast membranes in Vicia faba (broad bean) into various complexes and immunocytochemical electron microscopic investigations. Sucrose density gradient fractionations of thylakoid membranes after detergent solubilization reveals that PPO protein (by reactivity with anti-PPO antibody) and activity (based upon ability to oxidize di-dihydroxyphenylalanine) are found only in fractions enriched in photosystem II (PSII). Furthermore, of the PSII particles isolated using three different protocols utilizing several plant species, all had PPO. Immunogold localization of PPO on thin sections reveals exclusive thylakoid labeling with a distribution pattern consistent with other PSII proteins (80% grana, 20% stroma). These data strongly indicate that PPO is at least peripherally associated with the PSII complex.     

Addition of an N-terminal epitope tag significantly increases the activity of plant fatty acid desaturases expressed in yeast cells

Saccharomyces cerevisiae shows great potential for development of bioreactor systems geared toward the production of high-value lipids such as polyunsaturated omega-3 fatty acids, the yields of which are largely dependent on the activity of ectopically expressed enzymes. Here, we show that the addition of an N-terminal epitope tag sequence (either Myc or hemagglutinin) to oleate desaturase (FAD2) or omega-3 linoleate desaturase (FAD3) enzymes from plants, which catalyze consecutive reactions in the production of long chain omega-3 fatty acids, significantly increases their activity up to fourfold when expressed in yeast cells. Quantitative protein blotting using an antibody specific for native FAD2 revealed that the steady-state amount of the epitope-tagged FAD2 protein was also approximately fourfold higher than that of its untagged counterpart, demonstrating a direct relationship between the epitope tag-induced increase in enzyme amount and fatty acid product formation. Protein half-life and RNA blotting experiments indicated that the half-lives and mRNA content of the tagged and untagged FAD2 proteins were essentially the same, suggesting that the epitope tags increased protein abundance by improving translational efficiency. Taken together, these results indicate that the addition of an epitope tag sequence to a plant fatty acid desaturase (FAD) not only provides a useful means for protein immunodetection using highly specific, commercially available antibodies, but that it also significantly increases FAD activity and the production of polyunsaturated fatty acids in yeast cells.     

Identification and evaluation of ω-3 fatty acid desaturase genes for hyperfortifying α-linolenic acid in transgenic rice seed

α-Linolenic acid (ALA) deficiency and a skewed of ω6:ω3 fatty acid ratio in the diet are a major explanation for the prevalence of cardiovascular diseases and inflammatory/autoimmune diseases. There is a need to enhance the ALA content and to reduce the ratio of linoleic acid (LA) to ALA. Six ω-3 (Δ-15) fatty acid desaturase (FAD) genes were cloned from rice and soybean. The subcellular localizations of the proteins were identified. The FAD genes were introduced into rice under the control of an endosperm-specific promoter, GluC, or a Ubi-1 promoter to evaluate their potential in increasing the ALA content in seeds. The ALA contents in the seeds of endoplasmic reticulum (ER)-localized GmFAD3-1 and OsFAD3 overexpression lines increased from 0.36 mg g?1 to 8.57 mg g?1 and 10.06 mg g?1, respectively, which was 23.8- and 27.9-fold higher than that of non-transformants. The trait of high ALA content was stably inheritable over three generations. Homologous OsFAD3 is more active than GmFAD3-1 in catalysing LA conversion to ALA in rice seeds. Overexpression of ER-localized GmFAD3-2/3 and chloroplast-localized OsFAD7/8 had less effect on increasing the ALA content in rice seeds. The GluC promoter is advantageous compared with Ubi-1 in this experimental system. The enhanced ALA was preferentially located at the sn-2 position in triacylglycerols. A meal-size portion of high ALA rice would meet >80% of the daily adult ALA requirement. The ALA-rich rice could be expected to ameliorate much of the global dietary ALA deficiency.     

Construction and Functional Analysis of CRISPR/Cas9 Vector of <i>FAD2</i> Gene Family in Soybean

Soybean oleic acid content is one of the important indexes to evaluate the quality of soybean oil. In the synthesis pathway of soybean fatty acids, the FAD2 gene family is the key gene that regulates the production of linoleic acid from soybean oleic acid. In this study, CRISPR/Cas9 gene editing technology was used to regulate FAD2 gene expression. Firstly, the CRISPR/Cas9 single knockout vectors GmFAD2-1B and GmFAD2-2C and double knockout vectors GmFAD2-2A-3 were constructed. Then, the three vectors were transferred into the recipient soybean variety Jinong 38 by Agrobacterium-mediated cotyledon node transformation, and the mutant plants were obtained. Functional analysis and comparison of the mutant plants of the T2 and T3 generations were carried out. The results showed that there was no significant difference in agronomic traits between the CRISPR/Cas9 single and double knockout vectors and the untransformed CRISPR/Cas9 receptor varieties. The oleic acid content of the plants that knocked out the CRISPR/Cas9 double gene vector was significantly higher than that of the single gene vector.     

PEROXIREDOXIN Q stimulates the activity of the chloroplast 16:1Δ3trans FATTY ACID DESATURASE4

Thylakoid membrane lipids, comprised of glycolipids and the phospholipid phosphatidylglycerol (PG), are essential for normal plant growth and development. Unlike other lipid classes, chloroplast PG in nearly all plants contains a substantial fraction of the unusual trans fatty acid 16:1^Δ3trans or 16:1t. We determined that, in Arabidopsis thaliana, 16:1t biosynthesis requires both FATTY ACID DESATURASE4 (FAD4) and a thylakoid‐associated redox protein, PEROXIREDOXIN Q (PRXQ), to produce wild‐type levels of 16:1t. The FAD4–PRXQ biochemical relationship appears to be very specific in planta, as other fatty acids (FA) desaturases do not require peroxiredoxins for their activity, nor does FAD4 require other chloroplast peroxiredoxins under standard growth conditions. Although most of chloroplast PG assembly occurs at the inner envelope membrane, FAD4 was primarily associated with the thylakoid membranes facing the stroma. Furthermore, co‐production of PRXQ with FAD4 was required to produce Δ3‐desaturated FAs in yeast. Alteration of the redox state of FAD4 or PRXQ through site‐directed mutagenesis of conserved cysteine residues impaired Δ3 FA production. However, these mutations did not appear to directly alter disulfide status of FAD4. These results collectively demonstrate that the production of 16:1t is linked to the redox status of the chloroplast through PRXQ associated with the thylakoids.     

Modification, processing, and subcellular localization in Escherichia coli of the pCloDF13-encoded bacteriocin release protein fused to the mature portion of beta-lactamase.

A fusion between the pCloDF13-derived bacteriocin release protein and beta-lactamase was constructed to investigate the subcellular localization and posttranslational modification of the bacteriocin release protein in Escherichia coli. The signal sequence and 25 of the 28 amino acid residues of the mature bacteriocin release protein were fused to the mature portion of beta-lactamase. The hybrid protein (Mr, 31,588) was expressed in minicells and whole cells and possessed full beta-lactamase activity. Immunoblotting of subcellular fractions revealed that the hybrid protein is present in both the cytoplasmic and outer membranes of E. coli. Radioactive labeling experiments in the presence or absence of globomycin showed that the hybrid protein is modified with a diglyceride and fatty acids and is processed by signal peptidase II, as is the murein lipoprotein. The results indicated that the pCloDF13-encoded bacteriocin release protein is a lipoprotein which is associated with both membranes of E. coli cells.     

Quantitative Proteomic Analysis of Low Linolenic Acid Transgenic Soybean Reveals Perturbations of Fatty Acid Metabolic Pathways

To understand the effect of fatty acid desaturase gene (GmFAD3) silencing on perturbation of fatty acid (FA) metabolic pathways, the changes are compared in protein profiling in control and low linolenic acid transgenic soybeans using tandem mass tag based mass spectrometry. Protein profiling of the transgenic line unveiled changes in several key enzymes of FA metabolism. This includes enzymes of lower abundance; fabH, fabF, and thioestrase associated with FA initiation, elongation, and desaturation processes and LOX1_5, ACOX, ACAA1, MFP2 associated with β‐oxidation of α‐linolenic acids pathways. In addition, the GmFAD3 silencing results in a significant reduction in one of the major allergens, Gly m 4 (C6T3L5). These results are important for exploring how plants adjust in their biological processes when certain changes are induced in the genetic makeup. A complete understanding of these processes will aid researchers to alter genes for developing value‐added soybeans.     

Bioinformatics study of delta-12 fatty acid desaturase 2 (FAD2) gene in oilseeds

Fatty acid desaturases constitute a group of enzymes that introduce double bonds into the hydrocarbon chains of fatty acids to produce unsaturated fatty acids. In plants, seed-specific delta-12 fatty acid desaturase 2 (FAD2) is responsible for the high content of linoleic acid by inserting a double bond at the delta-12 (omega-6) position of oleic acid. In this study, sixteen FAD2 and FAD2-2 protein sequences from oilseeds were analyzed by computational tools including two databases of the NCBI and EXPASY and data management tools such as SignalP, TMHMM, Psort, ProtParam, TargetP, PLACE and PlantCARE. These services were used to predict the protein properties such as molecular mass, pI, signal peptide, transmembrane and conserved domains, secondary and spatial structures. The polypeptide sequences were aligned and a neighbour-joining tree was constructed using MEGA5.1 to elucidate phylogenetic relationships among FAD2 genes. Based on the phylogenetic analysis species with high similarity in FAD2 sequence grouped together. FAD2 proteins include highly conserved histidine-rich motifs (HECGHH, HRRHH and HV[A/C/T]HH) that are located by three to five transmembrane anchors. For further investigations Sesamum indicum FAD2 was selected and analyzed by bioinformatics tools. Analysis showed no N-terminal signal peptide for probable localization of FAD2 protein in cytoplasmic organelles such as chloroplast, mitochondria and Golgi. Instead the C-terminal signaling motif YNNKL, Y(K/N)NKF or YRNKI allows FAD2 protein to selectively bind to and embed in the endoplasmic reticulum. FAD2 promoter contains different cis-regulatory elements involve in the biotic and abiotic stresses response or control of gene expression specifically in seeds.     

A Proline-, Threonine-, and Glycine-Rich Protein Down-Regulated by Drought Is Localized in the Cell Wall of Xylem Elements

A cDNA clone encoding a proline-, threonine-, and glycine-rich protein (PTGRP) was isolated from a wild tomato species (Lycopersicon chilense) (L.X. Yu, H. Chamberland, J.G. Lafontain, Z. Tabaeizadeh [1996] Genome 39: 1185-1193). Northern-blot analysis and in situ hybridization studies revealed that PTGRP is down-regulated by drought stress. The level of the mRNA in leaves and stems of 8-d drought-stressed plants decreased 5- to 10-fold compared with that in regularly watered plants. The mRNA re-accumulated when drought-stressed plants were rewatered. Antibodies raised against a glutathione S-transferase/PTGRP fusion protein were used to elucidate the subcellular localization of the protein by immunogold labeling. In regularly watered L. chilense plants, PTGRP protein was found to be localized in xylem pit membranes and disintegrated primary walls. Examination of sections from drought-stressed plants revealed a significant decrease in the levels of labeling. In these samples, only a few scattered gold particles were detected in the same areas. In the leaf tissues of plants that had been rewatered for 3 d following an 8-d drought stress, the labeling pattern was similar to that of the regularly watered plants. To our knowledge, PTGRP is the first drought-regulated protein that has been precisely localized in the cell wall.     

Heterologous expression of two <Emphasis Type="Italic">Glycine max</Emphasis> ω-3 fatty acid desaturases in <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

α-Linolenic acid (ALA, C 18:3^Δ9,12,15) has many important biological functions. ω-3 Fatty acid desaturase (FAD3), existing in cytosolic and plastidic compartments of higher plants, catalyzes linoleic acid (LA) desaturation to produce ALA. GmFAD3A-2 and GmFAD3C genes encoding cytosolic FAD3 from Qihuang 29 soybean were cloned and inserted into p416 vector and expressed in K601 yeast strain. Gas chromatography showed that the transformed yeast strains could produce ALA. The ALA accumulation levels for the strains transformed with GmFAD3A-2 or GmFAD3C genes were 0.77 ± 0.1 and 4.13 ± 0.4% of total fatty acids, respectively, while, as compared with that of the control, the contents of LA decreased from 14.34 ± 0.8 to 10.93 ± 0.0 and 7.85 ± 0.1%, respectively, implying that the GmFAD3C enzyme is more vigorous or stable, than GmFAD3A-2. Published in Russian in Fiziologiya Rastenii, 2009, Vol. 56, No. 4, pp. 629–634. This text was submitted by the authors in English.     

FAD assembly and thylakoid membrane binding of ferredoxin:NADP+ oxidoreductase in chloroplasts

We investigated the process of flavin adenine dinucleotide (FAD) incorporation into the ferredoxin (Fd):NADP(+) oxidoreductase (FNR) polypeptide during FNR biosynthesis, using pull-down assay with resin-immobilized Fd which bound strongly to FAD-assembled holo-FNR, but hardly to FAD-deficient apo-FNR. After FNR precursor was imported into isolated chloroplasts and processed to the mature size, the molecular form pulled down by Fd-resin increasingly appeared. The mature-sized FNR (mFNR) accumulated transiently in the stroma as the apo-form, and subsequently bound on the thylakoid membranes as the holo-form. Thus, FAD is incorporated into the mFNR inside chloroplasts, and this assembly process is followed by the thylakoid membrane localization of FNR.     

Cutting edge of chloroplast proteolysis

Chloroplasts have a dynamic protein environment and, although proteases are presumably major contributors, the identities of these crucial regulatory proteins have only recently been revealed. There are defined proteases within each of the major chloroplast compartments: the ATP-dependent Clp and FtsH proteases in the stroma and stroma-exposed thylakoid membranes, respectively, the ATP-independent DegP proteases within the thylakoid lumen and on both sides of thylakoid membranes, and the SppA protease on the stromal side of the thylakoid. All four types are homologous to proteases characterized in bacteria, but most have many isomers in higher plants. With such diversity, the challenge is to link the mode of action of each protease to the chloroplast enzymes and regulatory proteins that it targets.     

大豆ω-3脂肪酸脱氢酶基因GmFAD3C在酿酒酵母中的表达

α亚麻酸(ALA)被称为必需脂肪酸,对人体有一系列的保健作用。ω-3脂肪酸脱氢酶(FAD)催化亚油酸(LA)生成ALA。大豆种子油中ALA含量较高,为了研究大豆ω3FAD的功能,用RTPCR方法从大豆未成熟种子中扩增出GmFAD3C的cDNA,克隆到酵母表达载体p416中,并用醋酸锂法转化酿酒酵母营养缺陷型K601,经筛选鉴定,得到阳性克隆。气相色谱分析脂肪酸成分,发现工程菌产生了新的脂肪成分ALA,含量占总脂肪酸的3.1%,LA含量与对照相比相应地下降,证明该基因编码的蛋白具有催化18碳多不饱和脂肪酸(PUFA)底物LA在Δ15位脱氢生成ALA的ω3FAD功能,首次实现大豆ω-3脂肪酸脱氢酶基因在酿酒酵母K601p416系统中的表达,建立了一种新的高效低成本的FAD酵母表达系统。