Soybean Aphid Infestation Induces Changes in Fatty Acid Metabolism in Soybean

The soybean aphid (Aphis glycines Matsumura) is one of the most important insect pests of soybeans in the North-central region of the US. It has been hypothesized that aphids avoid effective defenses by inhibition of jasmonate-regulated plant responses. Given the role fatty acids play in jasmonate-induced plant defenses, we analyzed the fatty acid profile of soybean leaves and seeds from aphid-infested plants. Aphid infestation reduced levels of polyunsaturated fatty acids in leaves with a concomitant increase in palmitic acid. In seeds, a reduction in polyunsaturated fatty acids was associated with an increase in stearic acid and oleic acid. Soybean plants challenged with the brown stem rot fungus or with soybean cyst nematodes did not present changes in fatty acid levels in leaves or seeds, indicating that the changes induced by aphids are not a general response to pests. One of the polyunsaturated fatty acids, linolenic acid, is the precursor of jasmonate; thus, these changes in fatty acid metabolism may be examples of “metabolic hijacking” by the aphid to avoid the induction of effective defenses. Based on the changes in fatty acid levels observed in seeds and leaves, we hypothesize that aphids potentially induce interference in the fatty acid desaturation pathway, likely reducing FAD2 and FAD6 activity that leads to a reduction in polyunsaturated fatty acids. Our data support the idea that aphids block jasmonate-dependent defenses by reduction of the hormone precursor.     

Development of marker-free transgenic Jatropha plants with increased levels of seed oleic acid

Background

Jatropha curcas is recognized as a new energy crop due to the presence of the high amount of oil in its seeds that can be converted into biodiesel. The quality and performance of the biodiesel depends on the chemical composition of the fatty acids present in the oil. The fatty acids profile of the oil has a direct impact on ignition quality, heat of combustion and oxidative stability. An ideal biodiesel composition should have more monounsaturated fatty acids and less polyunsaturated acids. Jatropha seed oil contains 30% to 50% polyunsaturated fatty acids (mainly linoleic acid) which negatively impacts the oxidative stability and causes high rate of nitrogen oxides emission.

Results

The enzyme 1-acyl-2-oleoyl-sn-glycero-3-phosphocholine delta 12-desaturase (FAD2) is the key enzyme responsible for the production of linoleic acid in plants. We identified three putative delta 12 fatty acid desaturase genes in Jatropha (JcFAD2s) through genome-wide analysis and downregulated the expression of one of these genes, JcFAD2-1, in a seed-specific manner by RNA interference technology. The resulting JcFAD2-1 RNA interference transgenic plants showed a dramatic increase of oleic acid (> 78%) and a corresponding reduction in polyunsaturated fatty acids (< 3%) in its seed oil. The control Jatropha had around 37% oleic acid and 41% polyunsaturated fatty acids. This indicates that FAD2-1 is the major enzyme responsible for converting oleic acid to linoleic acid in Jatropha. Due to the changes in the fatty acids profile, the oil of the JcFAD2-1 RNA interference seed was estimated to yield a cetane number as high as 60.2, which is similar to the required cetane number for conventional premium diesel fuels (60) in Europe. The presence of high seed oleic acid did not have a negative impact on other Jatropha agronomic traits based on our preliminary data of the original plants under greenhouse conditions. Further, we developed a marker-free system to generate the transgenic Jatropha that will help reduce public concerns for environmental issues surrounding genetically modified plants.

Conclusion

In this study we produced seed-specific JcFAD2-1 RNA interference transgenic Jatropha without a selectable marker. We successfully increased the proportion of oleic acid versus linoleic in Jatropha through genetic engineering, enhancing the quality of its oil.     

Silencing of Gm<Emphasis Type="Italic">FAD3</Emphasis> gene by siRNA leads to low α-linolenic acids (18:3) of <Emphasis Type="Italic">fad3</Emphasis>-mutant phenotype in soybean [<Emphasis Type="Italic">Glycine max</Emphasis> (Merr.)]

RNA interference (RNAi) has been recently employed as an effective experimental tool for both basic and applied biological studies in various organisms including plants. RNAi deploys small RNAs, mainly small interfering RNAs (siRNAs), to mediate the degradation of mRNA for regulating gene expression in plants. Here we report an efficient siRNA-mediated gene silencing of the omega-3 fatty acid desaturase (FAD3) gene family in a complex genome, the soybean (Glycine max). The FAD3 enzyme is responsible for the synthesis of alpha-linolenic acids (18:3) in the polyunsaturated fatty acid pathway. It is this fatty acid that contributes mostly to the instability of soybean and other seed oils. Therefore, a significant reduction of this fatty acid will increase the stability of the seed oil, enhancing the seed agronomical value. A conserved nucleotide sequence, 318-nt in length, common to the three gene family members was used as an inverted repeat for RNA interference. The RNAi expression cassette was driven by a seed-specific promoter. We show that the transgene-produced siRNA caused silencing of FAD3 that was comparable to the fad3 mutant phenotype and, furthermore, that such a silencing is stably inherited in engineered soybean lines. Since the pool size of the alpha-linolenic acids is small relative to the other polyunsaturated fatty acids in soybean, the significant reduction of this fatty acid suggests a role and great potential for the siRNA strategy in silencing gene families in a complex genome.     

Omega-3 fatty acid desaturase (FAD3, FAD7, FAD8) gene expression and linolenic acid content in cowpea leaves submitted to drought and after rehydration

Membrane polyunsaturated fatty acids (PUFA) and particularly linolenic acid (18:3, LA) are known to be implicated in plant tolerance to low temperature. Their role in resistance to drought is much less investigated. In this work, three full-length cDNAs corresponding to omega-3 fatty acid desaturases: fad3 (endoplasmic reticulum), fad7 and fad8 (chloroplastic) were isolated from Vigna unguiculata leaves. Two cowpea cultivars, one drought-tolerant, EPACE-1, and one drought-susceptible, 1183, were compared in terms of fad isoform gene expression and leaf LA contents in plants submitted to water stress followed by rehydration. In EPACE-1, LA content in the main leaf polar lipids increased in response to mild water deficit. Severe water deficits induced a decrease in MGDG LA content while those of PC and DGDG continued to increase. Variations in FAD gene expression, matched those in LA contents. In 1183, LA contents decreased in all lipid classes in response to water stress, as did FAD3 and FAD8 gene expression levels. Rehydration after a moderate water stress induced stimulation mostly in FAD3 gene expression in both cvs. LA contents were equivalent to control levels in EPACE-1. In 1183, they were back to control levels in PC shortly after rehydration but remained low in galactolipids. These results suggested that omega-3 FAD activities were involved in the increase in leaf membrane unsaturation, in the drought tolerant plants whereas the sensitive plants lost PUFAs in response to the treatment. The significance of this discrepancy between the two cvs. in terms of adaptation to drought is discussed.     

Changes in the fatty acid composition of steelhead trout, Salmo gairdnerii Richardson, associated with parr-smolt transformation

Fatty acids from the several lipid classes of selected steelhead trout (Salmo gairdnerii) parr and smolt tissues, previously separated by thin-layer chromatography, were analyzed by gas-liquid chromatography. The fatty acid composition of the parr was markedly different from that of the smolt; the former being characterized by relatively low amounts of polyunsaturated fatty acids and relatively high amounts of linoleic acid, much like the typical freshwater lipid pattern. The fatty acid composition of the smolt was characterized by large proportions of long-chain polyunsaturated fatty acids. Generally, the fatty acid composition of the smolt resembled the typical seawater lipid pattern. The change in fatty acid composition of the smolt is anticipatory to seawater entry and is independent of diet and water temperature. These alterations suggest that the assumption of a polyunsaturated lipid pattern during parr-smolt transformation (smoltification) is preadaptive to seawater entry.     

Analysis of interspecific variation in relative fatty acid composition: use of flow cytometry to estimate unsaturation index and relative polyunsaturated fatty acid content in microalgae

Relative polyunsaturated fatty acid content and unsaturation index are very important composition variables in the use of microalgae both for animal and human nutrition and biofuel production. A readily available technique to rapidly and inexpensively estimate relative fatty acid composition is very important for mass screening of new strains for the production of different types of oil. This study demonstrates the validity of Nile Red staining and flow cytometry for quick estimation of unsaturation index and relative fatty acid content in microalgae. Nile Red staining allows polar and neutral lipid contents to be estimated, and in this study a significant correlation was observed between polar/neutral ratio and fatty acid content in the species studied, corresponding to higher polyunsaturated fatty acid content in the polar lipid fraction of microalgae. This technique enables quick estimation of relative polyunsaturated fatty acid content and interspecific variation, as well as variations caused by culture conditions. In the species studied, most variability in fatty acid composition was due to variation in monounsaturated and polyunsaturated fatty acids, with less variation observed in saturated fatty acid content.     

A mutant of Arabidopsis deficient in desaturation of palmitic Acid in leaf lipids

The overall fatty acid composition of leaf lipids in a mutant of Arabidopsis thaliana was characterized by elevated amounts of palmitic acid and a decreased amount of unsaturated 16-carbon fatty acids as a consequence of a single nuclear mutation. Quantitative analysis of the fatty acid composition of individual lipids suggested that the mutant is deficient in the activity of a chloroplast ω9 fatty acid desaturase which normally introduces a double bond in 16-carbon acyl chains esterified to monogalactosyldiacylglycerol (MGD). The mutant exhibited an increased ratio of 18- to 16-carbon fatty acids in MGD due to a change in the relative contribution of the prokaryotic and eukaryotic pathways of lipid biosynthesis. This appears to be a regulated response to the loss of chloroplast ω9 desaturase and presumably reflects a requirement for polyunsaturated fatty acids for the normal assembly of chloroplast membranes. The reduction in mass of prokaryotic MGD species involved both a reduction in synthesis of MGD by the prokaryotic pathway and increased turnover of MGD molecular species which contain 16:0.     

Ultrastructure and lipid alterations induced by cadmium in tomato (Lycopersicon esculentum) chloroplast membranes

The effects of cadmium (Cd) uptake on ultrastructure and lipid composition of chloroplasts were investigated in 28-day-old tomato plants (Lycopersicon esculentum var. Ibiza F1) grown for 10 days in the presence of various concentrations of CdCl2. Different growth parameters, lipid and fatty acid composition, lipid peroxidation, and lipoxygenase activity were measured in the leaves in order to assess the involvement of this metal in the generation of oxidative stress. We first observed that the accumulation of Cd increased with external metal concentration, and was considerably higher in roots than in leaves. Cadmium induced a significant inhibition of growth in both plant organs, as well as a reduction in the chlorophyll and carotenoid contents in the leaves. Ultrastructural investigations revealed that cadmium induced disorganization in leaf structure, essentially marked by a lowered mesophyll cell size, reduced intercellular spaces, as well as severe alterations in chloroplast fine structure, which exhibits disturbed shape and dilation of thylakoid membranes. High cadmium concentrations also affect the main lipid classes, leading to strong changes in their composition and fatty acid content. Thus, the exposure of tomato plants to cadmium caused a concentration-related decrease in the fatty acid content and a shift in the composition of fatty acids, resulting in a lower degree of fatty acid unsaturation in chloroplast membranes. The level of lipid peroxides and the activity of lipoxygenase were also significantly enhanced at high Cd concentrations. These biochemical and ultrastructural changes suggest that cadmium, through its effects on membrane structure and composition, induces premature senescence of leaves.     

Effect of cadmium on lipid composition of pepper

Seedlings (2 weeks old) of pepper (Capsicum annum) were grown in nutrient solution with added CdCl(2) (10 or 50 microM) for 7 days. In Cd-treated plants, changes in acyl lipids and fatty acid composition were investigated. Cd particularly lowered the amount of monogalactosyldiacylglycerol (MGDG) and enhanced accumulation of phospholipids [phosphatidylcholine (PC), phosphatidylethanolamine and phosphatidylglycerol] in leaves. In contrast, content of PC and galactolipids (MGDG and digalactosyldiacylglycerol) decreased in roots. Fatty acid composition of leaves was also changed by Cd addition to external medium, but no important changes occurred in roots. Levels of leaf polyunsaturated fatty acids, especially 18:3 and 16:3, were reduced. Lipid and fatty acid composition changes in roots are discussed in relation to Cd tolerance in pepper.     

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.     

Arabidopsis mutants deficient in polyunsaturated fatty acid synthesis. Biochemical and genetic characterization of a plant oleoyl-phosphatidylcholine desaturase.

The overall fatty acid composition of leaf lipids in a mutant of Arabidopsis thaliana was characterized by reduced levels of polyunsaturated 18-carbon fatty acids and an increased proportion of oleate as a consequence of a single recessive nuclear mutation. Quantitative analysis of the fatty acid composition of individual lipids demonstrated that all the major phospholipids of the extrachloroplast membranes are affected by the mutation, whereas the chlorplast lipids show fatty acid compositions only slightly different from those of wild type plants. These results are consistent with the parallel operation of two pathways of lipid synthesis in plant leaf cells (the prokaryotic pathway in the chloroplast and the eukaryotic pathway in the endoplasmic reticulum) and with genetic evidence (Browse, J., Kunst, L., Anderson, S., Hugly, S., and Somerville, C.R. (1989) Plant Physiol 90, 522-529) that an independent 18:1/16:1 desaturase operates on chloroplast membrane lipids. Direct enzyme assays confirmed that the mutant plants are deficient in the activity of a microsomal oleoyl-phosphatidycholine desaturase and demonstrated that this desaturase is the major enzyme responsible for the synthesis of polyunsaturated phospholipids. Despite this deficiency in 18:1-desaturase activity, mutant plants contained relatively high levels of 18:3 in their leaf phospholipids. This finding is interpreted as additional evidence that considerable two-way exchange of lipid occurs between the chloroplast and endoplasmic reticulum and that this exchange allows the chloroplast desaturases to provide lipids containing 18:3 to the extrachloroplast compartment, thus partially alleviating the deficiency in 18:1 desaturase activity.     

Membrane lipid polyunsaturation mediated by FATTY ACID DESATURASE 2 (FAD2) is involved in endoplasmic reticulum stress tolerance in Arabidopsis thaliana

Unsaturation of membrane glycerolipid classes at their hydrophobic fatty acid tails critically affects the physical nature of the lipid molecule. In Arabidopsis thaliana, 7 fatty acid desaturases (FADs) differently desaturate each glycerolipid class in plastids and the endoplasmic reticulum (ER). Here, we showed that polyunsaturation of ER glycerolipids is required for the ER stress response. Through systematic screening of FAD mutants, we found that a mutant of FAD2 resulted in a hypersensitive response to tunicamycin, a chemical inducer of ER stress. FAD2 converts oleic acid to linoleic acid of the fatty acyl groups of ER‐synthesized phospholipids. Our functional in vivo reporter assay revealed the ER localization and distinct tissue‐specific expression patterns of FAD2. Moreover, glycerolipid profiling of both mutants and overexpressors of FAD2 under tunicamycin‐induced ER stress conditions, along with phenotypic screening of the mutants of the FAD family, suggested that the ratio of monounsaturated fatty acids to polyunsaturated fatty acids, particularly 18:1 to 18:2 species, may be an important factor in allowing the ER membrane to cope with ER stress. Therefore, our results suggest that membrane lipid polyunsaturation mediated by FAD2 is involved in ER stress tolerance in Arabidopsis.     

Changes in fatty acids composition,hydrogen peroxide generation and lipid peroxidation of salt-stressed corn (<Emphasis Type="Italic">Zea mays</Emphasis> L.) roots

Comparative study about the salt-induced oxidative stress and lipid composition has been realised in primary root tissues for two varieties of maize (Zea mays L.) in order to evaluate their responses to salt stress. The root growth, root water content (WC), hydrogen peroxide (H₂O₂) generation, lipid peroxidation, membrane stability index and the changes in the profile of fatty acids composition were investigated. Salinity impacts in term of root growth, water content, H₂O₂ generation, lipid peroxidation and membrane destabilisation were more pronounced in primary roots of Aristo than in those of Arper indicating more sensitivity of the first variety. It was confirmed by gas chromatography that the composition of fatty acids in roots of both varieties was constituted mainly by 16:0 and 18:0 as major saturated fatty acids and 18:1ω9, 18:2ω6 and 18:3ω3 as major unsaturated fatty acids. Total lipid extracts from the roots of both varieties showed that the lipid saturation level increased under salt stress, notwithstanding the increased proportion of polyunsaturated fatty acids. The changes in lipid saturation being predominantly due to decreases in oleic acid (18:1ω9) and increases in palmitic acid (16:0). However, Arper root extracts contained a lower proportion of saturated lipids than Aristo. The enhanced proportion of highly polyunsaturated fatty acids especially linolenic and eicosapentaenoic acids was considered to be the characteristic of the relatively salt tolerance in Arper roots.