Antisense-mediated Depletion of Tomato Endoplasmic Reticulum Omega-3 Fatty Acid Desaturase Enhances Thermal Tolerance

An endoplasmic reticulum-localized tomato ω-3 fatty acid desaturase gene(LeFAD3) was isolated.The antisense tomato plants were obtained under the control of the cauliflower mosaic virus 35S promoter(35S-CaMV).Northern blot analysis confirmed that the expression of LeFAD3 was inhibited in the tomato genome.Levels of 18:3 decreased and correspondingly levels of 18:2 increased in total lipids of leaves and roots.After heat stress,the fresh weight of the aerial parts of antisense transgenic plants was higher than that of the wild type(WT) plants.The membrane system ultrastructure of chloroplasts in leaf cells and all of the subcellular organelles in the root tips of transgenic plants remained more intact than those of WT.Relative electric conductivity increased less in transgenic plants than in WT.Under heat stress,the maximal photochemical efficiency of photosystem II(Fv/Fm) and the O2 evolution rate decreased more in WT than in transgenic plants.These results suggested that the depletion of LeFAD3 increased the saturation of fatty acids and alleviated high temperature stress.     

Antisense expression of tomato chloroplast omega-3 fatty acid desaturase gene (<Emphasis Type="Italic">LeFAD7</Emphasis>) enhances the tomato high-temperature tolerance through reductions of trienoic fatty acids and alterations of physiological parameters

We studied how the reductions of trienoic fatty acids (TAs) and increases of dienoic fatty acids (DAs) enhanced high-temperature tolerance in antisense expression of tomato chloroplast omega-3 fatty acid desaturase gene (LeFAD7) transgenic tomato (Lycopersicon esculentum Mill.) plants. In transgenic plants, the content of linolenic acid (18:3) was markedly decreased, while linoleic acid (18:2) was increased correspondingly and the similar changes were observed under high-temperature stress as well. Under high-temperature stress, transgenic plants can maintain a relatively higher level of net photosynthetic rate (P _N) and chlorophyll (Chl) content than that of wild type (WT) plants. A decreased Chl/Carotenoids (xanthophylls and carotenes, Car) ratio and Chl a/b ratio were observed in transgenic plants. Transgenic plants exhibited visible decrease in the relative electrolyte conductivity, higher activities of antioxidative enzymes and lower reactive oxygen species correspondingly than WT. In addition, high-temperature stress for 24 h caused more extensive changes of chloroplast ultrastructure in WT than in transgenic plants. We therefore suggested that the enhancement of high-temperature tolerance in antisense expression of LeFAD7 transgenic plants might be raised from the reduction of TAs and increase of DAs subsequently leading to series of physiological alterations.     

Overexpression of endoplasmic reticulum omega-3 fatty acid desaturase gene improves chilling tolerance in tomato

An endoplasmic reticulum-localized tomato omega-3 fatty acid desaturase gene (LeFAD3) was isolated and characterized with regard to its sequence, response to various temperatures and function in transgenic tomato plants. Northern blot analysis showed that LeFAD3 was expressed in all organs tested and was markedly abundant in roots. Meanwhile, the expression of LeFAD3 was induced by chilling stress (4 °C), but inhibited by high temperature (40 °C). The transgenic plants were obtained under the control of the cauliflower mosaic virus 35S promoter (35S-CaMV). Northern and western blot analyses confirmed that sense LeFAD3 was transferred into tomato genome and overexpressed. Level of linolenic acids (18:3) increased and correspondingly level of linoleic acid (18:2) decreased in leaves and roots. After chilling stress, the fresh weight of the aerial parts of transgenic plants was higher than that of the wild type (WT) plants, and the membrane system ultrastructure of chloroplast in leaf cell and all the subcellular organelles in root tips of transgenic plants kept more intact than those of WT. Relative electric conductivity increased less in transgenic plants than that in WT, and the respiration rate of the transgenic plants was notably higher than that of WT. The maximal photochemical efficiency of PSII (F_v/F_m) and the O₂ evolution rate in WT decreased more than those in transgenic plants under chilling stress. Together with other data, results showed that the overexpression of LeFAD3 led to increased level of 18:3 and alleviated the injuries under chilling stress.     

Conferring high‐temperature tolerance to nontransgenic tomato scions using graft transmission of RNA silencing of the fatty acid desaturase gene

We investigated graft transmission of high‐temperature tolerance in tomato scions to nontransgenic scions from transgenic rootstocks, where the fatty acid desaturase gene (LeFAD7) was RNA‐silenced. Tomato was transformed with a plasmid carrying an inverted repeat of LeFAD7 by Agrobacterium. Several transgenic lines showed the lower amounts of LeFAD7 RNA and unsaturated fatty acids, while nontransgenic control did not, and siRNA was detected in the transgenic lines, but not in control. These lines grew under conditions of high temperature, while nontransgenic control did not. Further, the nontransgenic plants were grafted onto the silenced transgenic plants. The scions showed less of the target gene RNA, and siRNA was detected. Under high‐temperature conditions, these grafted plants grew, while control grafted plants did not. Thus, it was shown that high‐temperature tolerance was conferred in the nontransgenic scions after grafting onto the silenced rootstocks.     

Overexpression of tomato chloroplast omega-3 fatty acid desaturase gene alleviates the photoinhibition of photosystems 2 and 1 under chilling stress

In transgenic (TG) tomato (Lycopersicon esculentum Mill.) overexpressed ω-3 fatty acid desaturase gene (LeFAD7) was identified, which was controlled by the cauliflower mosaic virus 35S promoter and induced increased contents of unsaturated fatty acids in thylakoid membrane. Under chilling stress at low irradiance (4 °C, 100 μmol m⁻² s⁻¹) TG plants with higher linolenic acids (18: 3) content maintained a higher O₂ evolution rate, oxidizable P700 content, and maximal photochemical efficiency (F_v/F_m) than wild type (WT) plants. Low temperature treatment for 6 h resulted in extensive changes of chloroplast ultrastructure: in WT plants most chloroplasts became circular, the number of amyloids increased, appressed granum stacks were dissolved, grana disappeared, and the number of grana decreased, while only a few grana were found in leaves of TG plants. Hence the overexpression of LeFAD7 could increase the content of 18: 3 in thylakoid membrane, and this increase alleviated the photoinhibition of photosystem (PS) 1 and PS2 under chilling at low irradiance.     

转反义LeETR2基因番茄的表型

转反义LeETR2基因番茄植株的表型与普通番茄有所不同。用乙烯25μL／L处理,转基因番茄能够表现出正常的“三重反应”,但根的伸长和根毛形成受到显著抑制。同时,转基因番茄植株对乙烯处理的偏上生长反应敏感度不及普通番茄,叶柄和花柄的脱落被延迟。这几方面的表型特点并不完全一致,我们推测LeETR2在番茄发育的不同阶段可能发挥不同的功能。     

Enhancement of low-temperature tolerance in transgenic tomato plants overexpressing Lefad7 through regulation of trienoic fatty acids

We studied how tomato (Lycopersicon esculentum Mill.) chloroplast omega-3 fatty acid desaturase gene (Lefad7) overexpression enhanced low-temperature (LT) tolerance in transgenic tomato plants. In these plants, the content of linolenic acid (18:3) markedly increased and, correspondingly, the content of linoleic acid (18:2) decreased. Similar changes were found after 6 h under LT (4°C) treatment. Under LT stress, wild type (WT) tomato plants showed a much greater increase in relative electrolyte leakage and malondialdehyde (MDA) contents compared with transgenic plants. Transgenic plants exhibited higher activities of antioxidative enzymes and a lower content of reactive oxygen species (ROS). Transgenic plants maintained a relatively higher level of the net photosynthetic rate (P _N) and chlorophyll (Chl) content than WT plants under LT stress. Taken together, we suggested that overexpression of Lefad7 enhanced LT tolerance by changing the composition of membrane lipids in tomato plants, with the increased content of trienoic fatty acids and reduced content of dienoic fatty acids that led to series of physiological alterations.     

Loss of function of FATTY ACID DESATURASE7 in tomato enhances basal aphid resistance in a salicylate-dependent manner

We report here that disruption of function of the ω-3 FATTY ACID DESATURASE7 (FAD7) enhances plant defenses against aphids. The suppressor of prosystemin-mediated responses2 (spr2) mutation in tomato (Solanum lycopersicum), which eliminates the function of FAD7, reduces the settling behavior, survival, and fecundity of the potato aphid (Macrosiphum euphorbiae). Likewise, the antisense suppression of LeFAD7 expression in wild-type tomato plants reduces aphid infestations. Aphid resistance in the spr2 mutant is associated with enhanced levels of salicylic acid (SA) and mRNA encoding the pathogenesis-related protein P4. Introduction of the Naphthalene/salicylate hydroxylase transgene, which suppresses SA accumulation, restores wild-type levels of aphid susceptibility to spr2. Resistance in spr2 is also lost when we utilize virus-induced gene silencing to suppress the expression of NONEXPRESSOR OF PATHOGENESIS-RELATED PROTEINS1 (NPR1), a positive regulator of many SA-dependent defenses. These results indicate that FAD7 suppresses defenses against aphids that are mediated through SA and NPR1. Although loss of function of FAD7 also inhibits the synthesis of jasmonate (JA), the effects of this desaturase on aphid resistance are not dependent on JA; other mutants impaired in JA synthesis (acx1) or perception (jai1-1) show wild-type levels of aphid susceptibility, and spr2 retains aphid resistance when treated with methyl jasmonate. Thus, FAD7 may influence JA-dependent defenses against chewing insects and SA-dependent defenses against aphids through independent effects on JA synthesis and SA signaling. The Arabidopsis (Arabidopsis thaliana) mutants Atfad7-2 and Atfad7-1fad8 also show enhanced resistance to the green peach aphid (Myzus persicae) compared with wild-type controls, indicating that FAD7 influences plant-aphid interactions in at least two plant families.     

Cold-induced accumulation of ω-3 polyunsaturated fatty acid in a liverwort, Marchantia polymorpha L

The liverwort Marchantia polymorpha L. synthesizes various long-chain polyunsaturated fatty acids including arachidonic acid and eicosapentaenoic acid, neither of which is produced by higher plants. Here we report the effects of temperature on long-chain polyunsaturated fatty acid accumulation in the liverwort. The accumulation of ω-3 polyunsaturated fatty acids increased significantly as the growth temperature decreased. Specifically, the relative content of eicosapentaenoic acid to total fatty acids at 5 °C was approximately 3-fold higher than at 25 °C. On the other hand, the accumulation of ω-6 polyunsaturated fatty acids decreased at low temperatures. An analysis of gene expression indicated that the mRNA of the MpFAD3 gene for ER ω-3 desaturase increased significantly at 5 °C. These results indicate that in the liverwort the n-3 pathway was enhanced at low temperature, mainly via expression of the cold-induced ω-3 desaturase gene, leading to increased accumulation of eicosapentaenoic acid.     

多聚半乳糖醛酸酶反义基因在转基因番茄中的表达

番茄的多聚半乳糖醛酸是一种在果实成熟阶段特异性表达的酶。为了研究它在果实成熟中的作用,将其ｃＤＮＡ与花椰菜花叶病毒３５Ｓ启动子嵌合后,以反义基因的形式经农杆菌介导导入番茄植株,进一步分析了反义基因的整合与表达。结果表明,在转基因番茄中,反义基因的表达能明显抑制果实内源多聚半乳糖醛酸酶的活性。     

Antisense-mediated suppression of tomato zeaxanthin epoxidase alleviates photoinhibition of PSII and PSI during chilling stress under low irradiance

A tomato (Lycopersicon esculentum Mill.) zeaxanthin epoxidase gene (LeZE) was isolated and antisense transgenic tomato plants were produced. Northern, southern, and western blot analyses demonstrated that antisense LeZE was transferred into the tomato genome and the expression of LeZE was inhibited. The ratio of (A+Z)/(V+A+Z) in antisense transgenic plants was maintained at a higher level than in the wild type (WT) plants under high light and chilling stress with low irradiance. The value of non-photochemical quenching (NPQ) in WT and transgenic plants was not affected during the stresses. The oxidizable P700 and the maximal photochemical efficiency of PSII (F_v/F_m) in transgenic plants decreased more slowly at chilling temperature under low irradiance. These results suggested that suppression of LeZE caused zeaxanthin accumulation, which was helpful in alleviating photoinhibition of PSI and PSII in tomato plants under chilling stress.     

利用番茄为受体表达轮状病毒外壳蛋白VP7的研究

将轮状病毒外壳蛋白VP7基因克隆到植物表达载体pBll21,并转化到根癌农杆菌(Agrobacterium tumefaciens)菌株EHAl05中,采用叶盘转化法转化番茄(1ycopersicon esculentumMill．)栽培品种TX0014,获得了转基因植株．经PCR、PCR—Southernblot和Southernblot分析表明：VP7基因已整合到转基因番茄植株的核基因组中．RT—PCR、Westernblot分析表明：VP7蛋白在叶片和果实中均获得了表达．     

ω3 fatty acid desaturases from microorganisms: structure, function, evolution, and biotechnological use

The biosynthesis of very-long-chain polyunsaturated fatty acids involves an alternating process of fatty acid desaturation and elongation catalyzed by complex series of enzymes. ω3 desaturase plays an important role in converting ω6 fatty acids into ω3 fatty acids. Genes for this desaturase have been identified and characterized in a wide range of microorganisms, including cyanobacteria, yeasts, molds, and microalgae. Like all fatty acid desaturases, ω3 desaturase is structurally characterized by the presence of three highly conserved histidine-rich motifs; however, unlike some desaturases, it lacks a cytochrome b5-like domain. Understanding the structure, function, and evolution of ω3 desaturases, particularly their substrate specificities in the biosynthesis of very-long-chain polyunsaturated fatty acids, lays the foundation for potential production of various ω3 fatty acids in transgenic microorganisms.     

Characterization of Transgenic Rice Plants Expressing an Arabidopsis FAD7

Fatty acid -3 desaturase (FAD) is the key enzyme catalyzing the formation of trienoic fatty acids. We utilized an Arabidopsis FAD7 gene and the seven independent transgenic rice plants harbouring 1 to 3 copies of this gene were generated. The expression of FAD7 mRNA was different among independent transgenic lines regardless of the copy number. The total linolenic acid (18:3) contents reduced by about 7 – 32 % in transgenic rice plants but the linoleic acid (18:2) content increased accordingly. With or without wounding treatments, the jasmonate content was higher in transgenic lines than in wild-type rice plant. The transgenic lines overproducing jasmonate also showed increased expression of PR1b mRNA and allene oxide synthase inresponse to wounding.     

深黄被孢霉△^6—脂肪酸脱氢酶基因在转基因烟草中的表达

γ—亚麻酸(GLA)是人体和动物饮食中具有营养作用的重要的多烯不饱和脂肪酸,在大多数油料作物种子中不含有GLA,而只含有其前体物亚油酸,只有少数油料植物种子中含有GLA,如夜来香(Oenothera spp),琉璃苣(Borago officinalis)等。△^6—脂肪酸脱氢酶可将亚油酸转化为γ—亚麻酸,为了能够在传统的油料作物种子中产生GLA,我们将从深黄被孢霉中克隆的△^6—脂肪酸脱氢酶基因,与植物表达载体pGA643连接,构建了重组质粒pGAM—ICL6,将其通过农杆菌介导法,导入模式植物烟草中。经PCR和Southern杂交分析表明该基因已导入并整合到烟草的基因组中,Northern杂交结果表明该基因在转基因烟草的mRNA水平上获得表达。对转基因植株进行脂肪酸分析,结果显示,GLA和十八碳四烯酸(OTA)分别占总脂肪酸含量的19．7％和3．5％。