Gene identification and functional characterization of a Δ12 fatty acid desaturase in Tetrahymena thermophila and its influence in homeoviscous adaptation to low temperature

Homeoviscous adaptation in poikilotherms is based in the regulation of the level of desaturation of fatty acids, variation in phospholipids head groups and sterol content in the membrane lipids, in order to maintain the membrane fluidity in response to changes in environmental temperature. Increased proportion of unsaturated fatty acids is thought to be the main response to low-temperature acclimation, which is mostly achieved by fatty acid desaturases. Genome analysis of the ciliate Tetrahymena thermophila and a gene knockout approach has allowed us to identify one Δ12 FAD and to study its activity in the original host and in a yeast heterologous expression system. The “PUFA index” -relative content of polyunsaturated fatty acids compared to the sum of saturated and monounsaturated fatty acid content- was ~57% lower at 15 °C and 35 °C in the Δ12 FAD gene knockout strain (KOΔ12) compared to WT strain. We characterized the role of T. thermophila Δ12 FAD on homeoviscous adaptation and analyzed its involvement in cellular growth, cold stress response, and membrane fluidity, as well as its expression pattern during temperature shifts. Although these alterations allowed normal growth in the KOΔ12 strain at 30 °C or higher temperatures, growth was impaired at temperatures of 20 °C or lower, where homeoviscous adaptation is impaired. These results stress the importance of Δ12 FAD in the regulation of cold adaptation processes, as well as the suitability of T. thermophila as a valuable model to investigate the regulation of membrane lipids and evolutionary conservation and divergence of the underlying mechanisms.     

Ozone-induced expression of the Arabidopsis FAD7 gene requires salicylic acid, but not NPR1 and SID2

The Arabidopsis FAD7 gene encodes a plastid omega-3 fatty acid desaturase that catalyzes the desaturation of dienoic fatty acids to trienoic fatty acids in chloroplast membrane lipids. The expression of FAD7 was rapidly and locally induced by ozone exposure, which causes oxidative responses equivalent to pathogen-induced hypersensitive responses and subsequently activates various defense-related genes. This induction was reduced in salicylic acid (SA)-deficient NahG plants expressing SA hydroxylase, but was unaffected in etr1 and jar1 mutants, which are insensitive to ethylene and jasmonic acid (JA), respectively. The SA dependence of the FAD7 induction was confirmed by the exogenous application of SA. SA-induced expression of FAD7 in the npr1 mutant which is defective in an SA signaling pathway occurred to the same extent as in the wild type. Furthermore, in the sid2 mutant which lacks an enzyme required for SA biosynthesis, the expression of FAD7 was induced by ozone exposure. These results suggest that the ozone-induced expression of FAD7 gene requires SA, but not ethylene, JA, NPR1 and SID2.     

Glycine betaine protects tomato (Solanum lycopersicum) plants at low temperature by inducing fatty acid desaturase7 and lipoxygenase gene expression

Cold stress is among the environmental stressors limiting productivity, yield and quality of agricultural plants. Tolerance to cold stress is associated with the increased unsaturated fatty acids ratio in the plant membranes which are also known to be substrates of octadecanoid pathway for jasmonate and other oxylipins biosynthesis. Accumulation of osmoprotectant, glycine betaine (GB) is well known to be effective in the protecting membranes and mitigating cold stress effects but, the mode of action is poorly understood. We studied the role of GB in cold stress responses of two tomato cultivated varieties; Gerry (cold stress sensitive) and T47657 (moderately cold stress tolerant) and compared the differences in lypoxygenase-13 (TomLOXF) and fatty acid desaturase 7 (FAD7) gene expression profiles and physiological parameters including relative growth rates, relative water content, osmotic potential, photosynthetic efficiency, membrane leakage, lipid peroxidation levels. Our results indicated that GB might have a role in inducing FAD7 and LOX expressions for providing protection against cold stress in tomato plants which could be related to the desaturation process of lipids leading to increased membrane stability and/or induction of other genes related to stress defense mechanisms via octadecanoid pathway or lipid peroxidation products.     

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.     

Wounding changes the spatial expression pattern of the arabidopsis plastid omega-3 fatty acid desaturase gene (FAD7) through different signal transduction pathways.

The Arabidopsis FAD7 gene encodes a plastid omega-3 fatty acid desaturase that catalyzes the desaturation of dienoic fatty acids in membrane lipids. The mRNA levels of the Arabidopsis FAD7 gene in rosette leaves rose rapidly after local wounding treatments. Wounding also induced the expression of the FAD7 gene in roots. To study wound-responsive expression of the FAD7 gene in further detail, we analyzed transgenic tobacco plants carrying the -825 Arabidopsis FAD7 promoter-beta-glucuronidase fusion gene. In unwounded transformants, FAD7 promoter activity was restricted to the tissues whose cells contained chloroplasts. Activation of the FAD7 promoter by local wounding treatments was more substantial in stems (29-fold) and roots (10-fold) of transgenic plants than it was in leaves (approximately two-fold). Significant induction by wounding was observed in the overall tissues of stems and included trichomes, the epidermis, cortex, vascular system, and the pith of the parenchyma. Strong promoter activity was found preferentially in the vascular tissues of wounded roots. These results indicate that wounding changes the spatial expression pattern of the FAD7 gene. Inhibitors of the octadecanoid pathway, salicylic acid and n-propyl gallate, strongly suppressed the wound activation of the FAD7 promoter in roots but not in leaves or stems. In unwounded plants, exogenously applied methyl jasmonate activated the FAD7 promoter in roots, whereas it repressed FAD7 promoter activity in leaves. Taken together, wound-responsive expression of the FAD7 gene in roots is thought to be mediated via the octadecanoid pathway, whereas in leaves, jasmonate-independent wound signals may induce the activation of the FAD7 gene. These observations indicate that wound-responsive expression of the FAD7 gene in aerial and subterranean parts of plants is brought about by way of different signal transduction pathways.     

细胞质膜在罗非鱼和叉尾斗鱼低温驯化过程中的功能

研究通过一系列科学的指标,比如死亡温度、累计温度处理时间和存活率曲线,检测并比较了吉富罗非鱼和叉尾斗鱼的低温耐受能力,然后系统地检测了在两种鱼中,低温对一系列与细胞膜相关的生理指标的影响,包括细胞膜流动性、内吞作用和膜蛋白Na+, K+ -ATPase的活性。实验结果显示叉尾斗鱼比吉富罗非鱼有着明显优良的耐寒力。两种鱼内与细胞膜相关的生理指标都对温度敏感,但是具体的敏感程度却在耐寒种(叉尾斗鱼)和不耐寒种(吉富罗非鱼)之间存在显著差异。在具体每个物种内部,与细胞膜相关的一系列生理功能对温度的敏感程度是一致的。所有这些种间差异性和种内一致性都使得细胞膜流动性在物种耐寒过程中的重要性凸显出来。结果揭示细胞质膜,尤其是质膜流动性可能在这两种鱼对低温适应过程中扮演重要的角色。       

Traits of fatty acid accumulation in dimorphic seeds of the euhalophyte Suaeda salsa in saline conditions

The mechanism on of how salinity affects seed fatty acids accumulation remains unclear in halophytes. The present results revealed that the content of total unsaturated fatty acids in black seeds was higher than in brown seeds in the euhalophyte Suaeda salsa under controlled saline conditions. Salinity (200?mM NaCl) significantly increased the total oil content, unsaturated acid/saturated acid ratio, and content of α-linolenic acid (C18:3) (ALA), especially in brown seeds. The most abundant fatty acid in dimorphic seeds is linoleic acid (C18:2) (>70%). It appears that more ALA accumulated in brown seeds compared to black seeds. The enzyme activity of omega-3 fatty acid desaturase (ω-3 FAD) in brown seeds was much higher than that in black seeds, but salinity had no significant effect on the activity of ω-3 FAD in both brown and black seeds. The relative expression of SsFAD7 was increased by salinity, and the value in brown seeds was much higher than that in black seeds. This means salinity can, salinity can improve the quantity of fatty acids in dimorphic seeds of S. salsa, and the enzyme of ω-3 FAD and SsFAD7 may involve in the accumulation of ALA in dimorphic seeds under salinity.     

Functional complementation of a periila ω3 fatty acid desaturase under the seed-specific SeFAD2 promoter

Functional characterization of the fatty acid desaturase genes and seed-specific promoters is prerequisite for altering the unsaturated fatty acid content of oilseeds by genetic manipulation. The ω-6 fatty acid desaturase (FAD2) and ω-3 fatty acid desaturase (FAD3) catalyze extra-plastidial desaturation of oleic acid to linoleic acid and linoleic acid to linolenic acid, respectively. These are major constituents in seed storage oils. Here, we report the complementation of a perilla linoleic acid desaturase (PrFAD3) cDNA under the seed-specific sesame FAD2 (SeFAD2) promoter in the Arabidopsis fad3 mutant. PrFAD3 is functionally active and the SeFAD2 promoter is applicable for modifying fatty acid composition in developing seeds. Transient expression of the GUS gene under that promoter in the developing seeds and leaves of sesame, soybean, and corn via microprojectile bombardment indicated that the SeFAD2 promoter likely will be useful for altering the seed phenotypes of dicot and monocot crops.     

Contribution of omega-3 fatty acid desaturase and 3-ketoacyl-ACP synthase II (KASII) genes in the modulation of glycerolipid fatty acid composition during cold acclimation in birch leaves

Temperate and boreal tree species respond to low positive temperatures (LT) or a shortening of the photoperiod (SD) by inducing cold acclimation. One of the metabolic consequences of cold acclimation is an increase in fatty acid (FA) desaturation in membrane lipids, which allows functional membrane fluidity to be maintained at LT. The molecular mechanisms of FA desaturation were investigated in leaves of birch seedlings (Betula pendula) during cold acclimation. Four genes involved in FA biosynthesis were isolated: a 3-ketoacyl-ACP synthase II gene (BpKASII) involved in the elongation of palmitoyl-ACP to stearoyl-ACP, and three omega-3 FA desaturase genes (BpFAD3, BpFAD7, and BpFAD8) involved in the desaturation of linoleic acid (18:2) to alpha-linolenic acid (18:3). BpFAD7 was the main omega-3 FAD gene expressed in birch leaves, and it was down-regulated by LT under SD conditions. LT induced the expression of BpFAD3 and BpFAD8 and a synchronous increase in 18:3 occurred in glycerolipids. Changes in the photoperiod did not affect the LT-induced increase in 18:3 in chloroplast lipids (MGDG, DGDG, PG), but it modulated the LT response detected in extra-chloroplastic lipids (PC, PE, PI, PS). A decrease in the proportion of the 16-carbon FAs in lipids occurred at LT, possibly in relation to the regulation of BpKASII expression at LT. These results suggest that LT affects the whole FA biosynthesis pathway. They support a co-ordinated action of microsomal (BpFAD3) and chloroplast enzymes (BpFAD7, BpFAD8) in determining the level of 18:3 in extra-chloroplastic membranes, and they highlight the importance of dynamic lipid trafficking.     

生菜种子低温处理后耐冻性和脂肪酸合成代谢的关系研究

以生菜(Lactuca sativa)种子为研究对象,通过不同时间的吸水处理分析其含水量变化,再通过程序降温处理,分析不同含水量种子发芽率的差异,以及脂肪酸合成有关基因(FAD2、FAD3、PPT、ELOVL)和冷调节基因ICE1的表达。结果表明,种子含水量随吸水时间增加而升高。程序降温至同样的低温冷冻条件下(-20℃、-22℃),吸水时间小于6 h的种子发芽率较高,而吸水8 h以上的种子发芽率显著降低。种子吸水8 h含水量处于饱和状态,在此状态下种子对低温较为敏感,说明含水量对种子耐冻性有影响。冷冻处理后生菜种子基因表达检测结果表明,脂肪酸去饱和酶基因(FAD2、FAD3)、蛋白质棕榈酰基硫脂酶相关基因(PPT）、长链脂肪酸延伸酶相关基因(ELOVL)的表达水平均随着种子含水量增加呈上升趋势,吸胀10 h的种子表达量最高,此时种子由于高含水量所受冷冻伤害最大。基因ICE1在冷冻处理种子中的表达也随着吸水时间增加而升高,在吸水10 h时种子中表达量到最高水平。综上,种子含水量越高,所受冷冻伤害越大。但种子在低温条件下具有一定的抗冷反应,可通过相关基因的过表达调控合成更多不饱和脂肪酸、抗冻蛋白等提高含水种子耐冻性。     

A report of progressive provisioning in a stink bug,Parastrachia japonensis (Hemiptera: Cydnidae)

The majority (75%) of femaleParastrachia japonensis (Hemiptera: Cydnidae), while caring for 1st and 2nd instar nymphs, foraged for and provisioned nests with drupes of the host plant,Schoepfia jasminodora (Olacacae), which the young then fed on. The maximum number of drupes found in a given nest was 27. Females could travel as fast as 61.4 cm/min while carrying drupes that were nearly double their weight, and were directly observed to travel as far as 10 m when encumbered with a drupe. It was found that nests which were far from the food source were provisioned with at least as many drupes as those nests that were within the drupe range. While females could not distinguish their own eggs from others, apparently they could distinguish their own nests containing their nymphs. Eggs failed to survive in the absence of a female, however, a small number of nymphs reached adulthood even in the absence of a female when drupes were provided. This report represents the first direct observation of progressive provisioning by a phytophagous hemipteran under field conditions.     

Development of a model system to identify differences in spring and winter oat

Our long-term goal is to develop a Swedish winter oat (Avena sativa). To identify molecular differences that correlate with winter hardiness, a winter oat model comprising of both non-hardy spring lines and winter hardy lines is needed. To achieve this, we selected 294 oat breeding lines, originating from various Russian, German, and American winter oat breeding programs and tested them in the field in south- and western Sweden. By assaying for winter survival and agricultural properties during four consecutive seasons, we identified 14 breeding lines of different origins that not only survived the winter but also were agronomically better than the rest. Laboratory tests including electrolytic leakage, controlled crown freezing assay, expression analysis of the AsVrn1 gene and monitoring of flowering time suggested that the American lines had the highest freezing tolerance, although the German lines performed better in the field. Finally, six lines constituting the two most freezing tolerant lines, two intermediate lines and two spring cultivars were chosen to build a winter oat model system. Metabolic profiling of non-acclimated and cold acclimated leaf tissue samples isolated from the six selected lines revealed differential expression patterns of 245 metabolites including several sugars, amino acids, organic acids and 181 hitherto unknown metabolites. The expression patterns of 107 metabolites showed significant interactions with either a cultivar or a time-point. Further identification, characterisation and validation of these metabolites will lead to an increased understanding of the cold acclimation process in oats. Furthermore, by using the winter oat model system, differential sequencing of crown mRNA populations would lead to identification of various biomarkers to facilitate winter oat breeding.     

Characterization of 19 Genes Encoding Membrane-Bound Fatty Acid Desaturases and their Expression Profiles in Gossypium raimondii Under Low Temperature

To produce unsaturated fatty acids, membrane-bound fatty acid desaturases (FADs) can be exploited to introduce double bonds into the acyl chains of fatty acids. In this study, 19 membrane-bound FAD genes were identified in Gossypium raimondii through database searches and were classified into four different subfamilies based on phylogenetic analysis. All 19 membrane-bound FAD proteins shared three highly conserved histidine boxes, except for GrFAD2.1, which lost the third histidine box in the C-terminal region. In the G. raimondii genome, tandem duplication might have led to the increasing size of the FAD2 cluster in the Omega Desaturase subfamily, whereas segmental duplication appeared to be the dominant mechanism for the expansion of the Sphingolipid and Front-end Desaturase subfamilies. Gene expression analysis showed that seven membrane-bound FAD genes were significantly up-regulated and that five genes were greatly suppressed in G. raimondii leaves exposed to low temperature conditions.     

Role of salicylic acid and fatty acid desaturation pathways in ssi2-mediated signaling

Stearoyl-acyl carrier protein desaturase-mediated conversion of stearic acid to oleic acid (18:1) is the key step that regulates the levels of unsaturated fatty acids (FAs) in cells. Our previous work with the Arabidopsis (Arabidopsis thaliana) ssi2/fab2 mutant and its suppressors demonstrated that a balance between glycerol-3-phosphate (G3P) and 18:1 levels is critical for the regulation of salicylic acid (SA)- and jasmonic acid-mediated defense signaling in the plant. In this study, we have evaluated the role of various genes that have an impact on SA, resistance gene-mediated, or FA desaturation (FAD) pathways on ssi2-mediated signaling. We show that ssi2-triggered resistance is dependent on EDS1, PAD4, EDS5, SID2, and FAD7 FAD8 genes. However, ssi2-triggered defects in the jasmonic acid pathway, morphology, and cell death phenotypes are independent of the EDS1, EDS5, PAD4, NDR1, SID2, FAD3, FAD4, FAD5, DGD1, FAD7, and FAD7 FAD8 genes. Furthermore, the act1-mediated rescue of ssi2 phenotypes is also independent of the FAD2, FAD3, FAD4, FAD5, FAD7, and DGD1 genes. Since exogenous application of glycerol converts wild-type plants into ssi2 mimics, we also studied the effect of exogenous application of glycerol on mutants impaired in resistance-gene signaling, SA, or fad pathways. Glycerol increased SA levels and induced pathogenesis-related gene expression in all but sid2, nahG, fad7, and fad7 fad8 plants. Furthermore, glycerol-induced phenotypes in various mutant lines correlate with a concomitant reduction in 18:1 levels. Inability to convert glycerol into G3P due to a mutation in the nho1-encoded glycerol kinase renders plants tolerant to glycerol and unable to induce the SA-dependent pathway. A reduction in the NHO1-derived G3P pool also results in a partial age-dependent rescue of the ssi2 morphological and cell death phenotypes in the ssi2 nho1 plants. The glycerol-mediated induction of defense was not associated with any major changes in the lipid profile and/or levels of phosphatidic acid. Taken together, our results suggest that glycerol application and the ssi2 mutation in various mutant backgrounds produce similar effects and that restoration of ssi2 phenotypes is not associated with the further desaturation of 18:1 to linoleic or linolenic acids in plastidal or extraplastidal lipids.     

Selective Foraging Behavior in Nest-Provisioning Females of Parastrachia japonensis (Hemiptera: Cydnidae): Cues for Preferred Food

Females of Parastrachia japonensis (Hemiptera: Cydnidae) provision nymph-containing nests with drupes of the single host tree, Schoepfia jasminodora (Rosidae: Santales). P. japonensis feeds on the endosperm of these drupes. Foraging females approached and accepted only a few selected drupes for transport to their nests from among the hundreds of drupes scattered on the ground. The characteristics of the drupes that elicited an acceptance response under field conditions were examined. Females estimated the degree of endosperm development using volatile and nonvolatile chemicals in the outer skin as cues and selected only those drupes which had a well-developed endosperm. Although the cues that prompted approach and inspection of a drupe are in the fleshy outer skin, females presented with various substitute drupes coated with outer skin juices of acceptable drupes selected only those they could recognize as a solid mass, suggesting that they used volatile chemicals in the outer skin as cues for approaching and inspecting a drupe and then taste and tactile stimuli as cues for acceptance. Thus, this bug possesses a complex, ecologically sensitive cognitive system that enables it to recognize valuable food items by using reliable cues. The importance of selective foraging by P. japonensis females and the evolutionary significance of this behavior are discussed.     

龙眼叶片膜脂脂肪酸组分与龙眼耐寒性的关系

龙眼叶片膜脂不饱和脂肪酸含量和脂肪酸不饱和度与龙眼不同品种的耐寒性呈正相关;在年周期中,不饱和脂肪酸含量的变化与龙眼耐寒性的变化呈现平行关系;龙眼叶片膜脂脂肪酸组分含量反映着龙眼品种间耐寒性遗传上的差异,可作为耐寒性的鉴定指标。     

木薯MeMYC2.2基因耐低温功能研究

MYC2（MYeloCytomatosis）转录因子是植物应对逆境胁迫过程中茉莉酸信号传导相关的核心转录因子。本研究旨在初步分析木薯MeMYC2.2基因在低温胁迫响应中的功能。利用生物信息学分析木薯MeMYC2.1和MeMYC2.2基因的结构及其编码蛋白的理化性质;通过定量PCR分析了上述2个基因在木薯组培苗叶片中对低温胁迫的响应;通过转基因拟南芥研究MeMYC2.2的抗冻功能。木薯组培苗叶片中2个MeMYC2基因的表达均在低温胁迫早期被诱导,其中,与MeMYC2.1相比,MeMYC2.2差异表达更显著。MeMYC2.2蛋白主要定位于细胞核中,且在酵母中具有明显转录自激活功能,表明该蛋白具有转录因子特性。与野生型相比,过表达MeMYC2.2的转基因拟南芥抗冻能力显著提高。在低温处理下,CBF3基因在转基因拟南芥中的表达量要明显高于其在野生型的表达量,但另外3个CBF基因在转基因拟南芥中的表达量明显下降。木薯MeMYC2.2的表达受低温和茉莉酸调控,可以提高植物的抗冻性,且可能影响CBF基因对低温的响应。本研究为进一步利用MeMYC2基因改良木薯的低温耐受性奠定了理论基础。