Alteration of low-temperature susceptibility of the cyanobacterium Synechococcus sp. PCC 7002 by genetic manipulation of membrane lipid unsaturation

Cyanobacteria acclimate to low temperature by desaturating their membrane lipids. Mutant strains of Synechococcus sp. PCC 7002 containing insertionally inactivated desA (Δ12 acyl-lipid desaturase) and desB (ω3 acyl-lipid desaturase) genes were produced, and their low-temperature susceptibility was characterized. The desA mutant synthesized no linoleic acid or α-linolenic acid, and the desB mutant did not produce α-linolenic acid. The desA mutant grew more slowly than the wild-type at 22° C and could not grow at 15° C. The desB mutant could not continuously grow at 15° C, although no observable phenotype appeared at higher temperatures. It has been shown that expression of the desA gene occurs at 38° C and is up-regulated at 22° C, and that the desB gene is only expressed at 22° C. These results indicate that the expression of the desA and desB genes occurs at higher temperatures than those at which a significant decline in physiological activities is caused by the absence of their products. The temperature dependency of photosynthesis was not affected by these mutations. Since chlorosis and inability to grow at 15° C with nitrate was suppressed by the substitution of urea as a nitrogen source, it is very likely that the chilling susceptibility of the desaturase mutants is attributable to nutrient limitation. Received: 24 April 1997 / Accepted: 5 August 1997     

Improvement of polyunsaturated fatty acids synthesis by the coexpression of CYB5 with desaturase genes in Saccharomyces cerevisiae

Since Saccharomyces cerevisiae contains Δ9 fatty acid desaturase (OLE1) as a sole fatty acid desaturase, it produces saturated and monounsaturated fatty acids of 16- and 18-carbon compounds. We showed earlier that Kluyveromyces lactis Δ12 (KlFAD2) and ω3 (KlFAD3) fatty acid desaturase genes enabled S. cerevisiae to make also polyunsaturated fatty acids (PUFAs), linoleic (18:2n-6), and α-linolenic (18:3n-3) acids. Unlike Δ9 fatty acid desaturase Ole1p, the two added fatty acid desaturases (KlFAD2and KlFAD3) do not contain a cytochrome b5 domain, and we now report on effects of the overexpression of K. lactis and S. cerevisiae cytochrome b5 (CYB5) genes as well as temperature effects on PUFA synthesis. Without extra cytochrome b5, while PUFA synthesis is significant at low temperature (20 °C), it was marginal at 30 °C. Overexpression of cytochrome b5 at 20 °C did not affect the fatty acid synthesis so much, but it significantly enhanced the synthesis of PUFA at 30 °C.     

Effect of weak constant magnetic field on the composition and content of lipids in radish seedlings at various temperatures

We studied the effects of weak permanent homogenous hirizontal magnetic field (PMF) (400 A/m) on the composition and content of lipids and composition of their fatty acids (FAs) in radish (Raphanus sativus L. var. radicula D.C., cv. Rosovo-krashyi s belym konchikom) seedlings at temperatures of 20 and 10°C. We compared lipid composition and content in seedlings at the phase of developed cotyledons (20°C, 5-day-old, and 10°C, 8-day-old seedlings) under low light and in darkness with the lipid composition and content in dry seeds. The seedlings grown in geomagnetic field (GMF) served as a control. In dry seeds, about 99% of total lipids comprised neutral lipids (NL) and only 1% were polar lipids (PL). Triacylglycerols predominated among NL comprising 93% of total seed lipids. During seed germination, NLs were consumed and PL were produced: the amount of glycolipids increased in control by 3.5–5 times and the amount of phospholipis, by 1.5–2 times.In the light at 20°C, PMF suppressed the formation of PL (by 18%), whereas in darkness, it stimulated it approximately by 80% as compared with control. In the light at 10°C, PMF slightly stimulated PL formation; in darkness, it did not almost affect their synthesis. In all treatments, PMF increased the ratio of phospholipids to sterols by 30–100%. Among FA, PMF exerted the strongest effect on the content of erucic acid: it increased in the light and in darkness at 20°C approximately by 25% and decreased at 10°C in the light by 13%. PMF behaved as a correction factor affecting lipid metabolism on the background of light and temperature action.     

Effect of sodium nitroprusside on heat resistance of wheat coleoptiles: Dependence on the formation and scavenging of reactive oxygen species

The effect of nitric oxide donor sodium nitroprusside (SNP) on resistance of coleoptiles of 4-day-old etiolated seedlings of wheat (Triticum aestivum L., cv. Elegiya) to damaging heating (10 min at 43°C) and possible dependence of this effect on changes in the activities of enzymes producing and scavenging reactive oxygen species (ROS) were studied. Treatment of coleoptiles with 500 μM SNP considerably boosted generation of superoxide anion radical therein. This effect was substantially suppressed by blocker of calcium channels (lanthanum chloride), calmodulin antagonist (chlorpromazine), and inhibitor of NADPH-oxidase (imidazole) but not by peroxidase inhibitor (salicylhydroxamic acid). NO donor activated antioxidant enzymes (superoxide dismutase, catalase, and soluble peroxidase) and elevated heat resistance of wheat coleoptiles. NO scavenger methylene blue, antioxidant agent ionol, calcium antagonists, and NADPH-oxidase inhibitor imidazole substantially reduced the elevation of heat resistance of wheat coleoptiles induced by NO donor. It was concluded that SNP-induced heat resistance of coleoptiles depended on calcium and ROS, whose production is probably boosted by activation of NADPH-oxidase.     

Presence of an alpha-amylase isozyme with high temperature optima in the wheat variety tolerant to high temperature at juvenile plant stage

In the present study α-amylase was partially purified from detached grains of five day old seedlings of two wheat (Triticum aestivum L.) varieties, showing differential responses to high temperature stress at seedling stage. A three step purification via ammonium sulphate precipitation, DEAE-cellulose column chromatography and gel filtration on Sephadex G-150 was employed. A single α-amylase was detected in the high temperature sensitive PBW-175 variety, while two isozymes namely, α-amylase-1 and α-amylase-2 were obtained in the relatively tolerant WL-711 variety. The pH optima of the three α-amylases were in 5.0–5.5 range and comparable to the cereal amylases. The temperature optima of PBW-175 α-amylase and α-amylase-1 of WL-711, which appeared to be the major isozyme of the variety, were same at 45 °C and also comparable to cereal amylases. On the other hand the optimum temperature for α-amylase-2 was high at 70 °C, which is unusual and not reported earlier for cereal amylases. The Km of PBW-175 α-amylase was lower than the Km values of WL-711 isozymes, this was well co-related with an overall high α-amylase activity detected in the detached grains of five day old seedlings of PBW-175 compared to WL-711. However WL-711 variety showed a better inherent seedling growth, vigour and EUE than PBW-175, may be because it had two α-amylase isozymes which could compensate for the higher enzyme activity detected in PBW-175. Moreover, the presence of α-amylase-2 in the grain of WL-711 having temperature optima of 70 °C, possibly rendered its seedlings tolerant to HS of 50 °C, while the seedlings of PBW-175 succumbed to this temperature shock.     

Synergistic effect of high-light and low temperature on cell growth of the Δ12 fatty acid desaturase mutant in Synechococcus sp. PCC 7002

The effect of polyunsaturated fatty acids on photosynthesis and the growth of the marine cyanobacterium Synechococcus sp. PCC 7002 was examined using wild-type and Δ12 fatty acid desaturase mutant strains. Under a light intensity of 250 μmol m⁻² s⁻¹, wild-type cells could grow exponentially in a temperature range of 20–38 °C, but growth was non-exponential below 20 °C and ceased at 12 °C. The Δ12 desaturase mutant cells lacking polyunsaturated fatty acids had the same growth rate as wild-type cells in a temperature range of 25–38 °C but grew slowly at 22 °C, and no cell growth took place below 18 °C. Under a very high-light intensity of 2.5 mmol m⁻² s⁻¹, wild-type cells could grow exponentially in a temperature range of 30–38 °C, although the high-light grown cells became chlorotic because of nitrogen limitation. The temperature sensitive phenotype in the Δ12 desaturase mutant was enhanced in cells grown under high-light illumination; the mutant cells could grow at 38 °C, but were killed at 30 °C. The decrease of oxygen evolution and nitrate consumption by whole cells as a function of temperature was similar in both wild type and the Δ12 desaturase mutant. No differences were observed in either light-induced damage of oxygen evolution or recovery from this damage. No inactivation of oxygen evolution took place at 22 °C under the normal light intensity of 250 μmol m⁻² s⁻¹. These results suggest that growth of the Δ12 desaturase mutant at low temperature is not directly limited by the inactivation of photosynthesis, and raise new questions about the functions of polyunsaturated membrane lipids on low temperature acclimation in cyanobacteria. This revised version was published online in June 2006 with corrections to the Cover Date.     

Seasonal dynamics of fatty acids in the lipids of water moss <Emphasis Type="Italic">Fontinalis antipyretica</Emphasis> from the Yenisei River

Identification of lipid fatty acids (FA) and studying of their seasonal dynamics in water moss Fontinalis antipyretica from the Yenisei River were carried out by means of gas chromatography-mass spectrometry. FA composition of water moss was notable for a relatively low level of saturated acids and predominance of polyunsaturated acids (PUFA) with double bonds (accounting for more than 30% of total FA) and polyunsaturated acids with double and triple bonds (acetylenic acids, accounting for more than 40% of total FA). Among PUFA, α- and γ-linolenic (18:3ω3 and 18:3ω6), arachidonic (20:4ω6), and eicosapentaenoic (20:5ω3) acids prevailed. Relative content of PUFA from ω3-family was the greatest in spring, and the level of PUFA from ω6-group was essentially the same throughout all the seasons. In the biomass of water moss, we identified seven acetylenic acids; among them octadeca-9,12-dien-6-ynoic (6a,9,12-18:3), octadeca-9,12,15-trien-6-ynoic (6a,9,12,15-18:4), and eicosa-11,14-dien-8-ynoic (8a,11,14-20:3) acids were predominant. For the first time, in the lipids of water moss we identified an acetylenic eicosa-11,14,17-trien-8-ynoic acid (8a,11,14,15-20:4). Relative content of acetylenic acids in the total FA was great throughout the entire period of investigation with the peak accumulation in summer. Owing to a steadily high level in the biomass of water moss and the lack of other producers of these acids in the ecosystem, acetylenic FA are highly specific biochemical markers useful for the investigation of trophic interactions between higher aquatic plants and zoobenthos.     

Occurrence and synthesis of lectin in coleoptiles of germinating wheat,rye and rice seedlings

Occurrence, synthesis and localization of lectins in coleoptiles of 3-day old seedlings of wheat, rye, barley and rice were studied by a combination of high resolution ion-exchange chromatography, in vivo labelling with ³⁵S-cysteine and immunocytochemistry. Whereas no lectin can be isolated or localized in barley coleoptile, 1.9 and 40 ng of lectin per coleoptile was obtained from wheat and rye respectively. Wheat germ agglutinin was localized in the outer layer of the wheat coleoptile and both inner and outer layers of rye coleoptile displayed a specific reaction. In rice, 250 ng of lectin is present in the coleoptile and is distributed throughout this organ. Wheat coleoptiles synthesize no lectin and rye coleoptiles synthesize minute amounts while those from developing rice seedlings incorporate reasonable amounts of ³⁵S-cysteine into lectin.Abbreviations FPLC Fast Protein Liquid Chromatography - GlcNAc N-acetylglucosamine - PBS phosphate buffered saline - SDS-PAGE sodium dodecyl sulphate polyacrylamide gel electrophoresis     

Isolation and Characterisation of a High-Efficiency Desaturase and Elongases from Microalgae for Transgenic LC-PUFA Production

The production of long-chain polyunsaturated fatty acids from precursor molecules linoleic acid (LA; 18:2ω6) and α-linolenic acid (ALA; 18:3ω3) is catalysed by sequential desaturase and elongase reactions. We report the isolation of a front-end Δ6-desaturase gene from the microalgae Ostreococcus lucimarinus and two elongase genes, a Δ6-elongase and a Δ5-elongase, from the microalga Pyramimonas cordata. These enzymes efficiently convert their respective substrates when transformed in yeast (39–75% conversion for ω3 substrate fatty acids), and the Δ5-elongase in particular displays higher elongation efficiency (75% for conversion of eicosapentaenoic acid (20:5ω3) to docosapentaenoic acid (22:5ω3)) than previously reported genes. In addition, the Δ6-desaturase is homologous with acyl-CoA desaturases and shows a strong preference for the ω3 substrate ALA.     

Genetic and biochemical evidence for involvement of HOTHEAD in the biosynthesis of long-chain α-,ω-dicarboxylic fatty acids and formation of extracellular matrix

In plants, extracellular matrix polymers built from polysaccharides and cuticular lipids have structural and protective functions. The cuticle is found to be ten times thinner in Arabidopsis thaliana (L.) Heynh than in many other plants, and there is evidence that it is unusual in having a high content of α-,ω-dicarboxylic fatty acids (FAs) in its polyesters. We designated the new organ fusion mutant hth-12 after it appeared to be allelic to adhesion of calyx edges (ace) and hothead (hth), upon molecular cloning of the gene by transposon tagging. This mutant is deficient in its ability to oxidize long-chain ω-hydroxy FAs to ω-oxo FAs, which results in leaf polyesters in decreased α-,ω-dicarboxylic FAs and increased ω-hydroxy FAs. These chemical phenotypes lead to disorder of the cuticle membrane structure in hth-12. ACE/HTH is a single-domain protein showing sequence similarity to long-chain FA ω-alcohol dehydrogenases from Candida species, and we hypothesize that it may catalyze the next step after cytochrome P450 FA ω-hydroxylases in the ω-oxidation pathway. We show that ACE/HTH is specifically expressed in epidermal cells. It appears very likely therefore that the changes in the amount of α-,ω-dicarboxylic FAs in hth-12 reflect the different composition of cuticular polyesters. The ACE/HTH gene is also expressed in root epidermal cells which do not form a polyester membrane on the exterior surface, thereby making it possible that the end products of the pathway, α-,ω-dicarboxylic FAs, are generally required for the cross-linking that ensures the integrity of the outer epidermal cell wall.     

Influence of soil temperature on niacin and thiamine in honey mesquite

Summary The influence of soil temperature was examined on niacin and thiamine concentration in honey mesquite (Prosopis glandulosa var.glandulosa) seedlings. The seedlings were grown in soil temperature regimes of 21, 27, and 32°C in a controlled environment growth room. Nodulation randomly occurred on the roots of the seedlings, necessitating separate analysis according to the occurrence of nodulation. Roots of nodulated seedlings from the 21°C soil temperature regime contained greater quantities of niacin and thiamine compared to root samples from seedlings grown in either 27 or 32°C regimes. Niacin concentration of non-nodulated seedlings was highest in samples from seedlings grown in the 27°C soil temperature regime and lowest in samples from seedlings grown in the 21°C regime. Thiamine concentration was the greatest from non-nodulated seedlings grown in the 27°C soil temperature regime, while the thiamine concentration of non-nodulated samples from the 32°C regime was the least. Optimal soil temperature for honey mesquite root growth appears to be about 27°C. At sub-optimal soil temperatures niacin might have limited ‘growth’ while at supra-optimal soil temperatures, thiamine might be a limiting factor. College of Agricultural Sciences Contribution No. T-9-164.     

Cold hardening reduces photoinhibition of Eucalypts nitens and E. pauciflora at frost temperatures

Photoinhibition of photosynthesis at low temperatures was investigated in two species of subalpine eucalypt, Eucalypts nitens (Deane and Maiden) Maiden and E. pauciflora Sieb. ex Spreng. Imposition of an artificial cold-hardening treatment increased the frost tolerance of leaf tissue and increased tolerance to excess light. Cold-hardened seedlings of both species had a higher photosynthetic capacity than non-hardened seedlings at 6 and 16°C and lower levels of non-photochemical quenching (NPQ) at 20 and 5°C. Furthermore, hardened seedlings had faster rates of NPQ development at 5 and −3.5°C. An increase in minimal fluorescence, which indicates slowly reversible photoinhibition, was evident in all seedlings at −1.5 and −3.5°C but was less pronounced in hardened seedlings, with a threefold faster rate of development of NPQ, at −3.5°C than non-hardened seedlings. Hardened seedlings also recovered faster from photoinhibition at −3.5°C. Thus cold hardening increased tolerance to high light in these species. Differences between E. nitens and E. pauciflora in their response to excess light were small and significant only at −3.5°C. Faster recovery from photoinhibition of E. pauciflora was consistent with its occurrence in colder habitats than E. nitens. Received: 27 April 1997 / Accepted: 9 September 1997     

The effects of spacer sequences on silencing efficiency of plant RNAi vectors

RNA interference (RNAi) has been used to suppress gene expression in various eukaryotic organisms. In plants, RNAi can be induced by introduction of an RNAi vector that transcribes a self-complementary hairpin RNA. Most basic RNAi constructs have an inverted repeat interrupted with a spacer sequence. To test silencing capability of RNAi constructs, we developed an in vivo assay that is based on the RNAi-mediated changes of the α-linolenic acid content in hairy roots. A tobacco endoplasmic reticulum ω-3 fatty acid desaturase (NtFAD3) is the main enzyme for production of α-linolenic acid of root membrane lipids. Tobacco hairy roots transformed with the RNAi vectors against the NtFAD3 gene showed a decrease in α-linolenic acid content. The frequency of RNA silencing was more affected by spacer sequence than by spacer length, at least between 100 and 1800 bp. Since significant amounts of hairpin RNA against the NtFAD3 gene remained in the transgenic plants displaying a weak silencing phenotype, low degree of silencing was attributed to low efficiency of hairpin RNA processing mediated by Dicer-like proteins. Our results show the possibility of producing a broad range of the RNAi-induced silencing phenotypes by replacing the spacer sequence of RNAi construct.     

Impact of feeding and short-term temperature stress on the content and isotopic signature of fatty acids,sterols, and alcohols in the scleractinian coral <Emphasis Type="Italic">Turbinaria reniformis</Emphasis>

This study assesses the combined effect of feeding and short-term thermal stress on various physiological parameters and on the fatty acid, sterol, and alcohol composition of the scleractinian coral Turbinaria reniformis. The compound-specific carbon isotope composition of the lipids was also measured. Under control conditions (26°C), feeding with Artemia salina significantly increased the symbiont density and chlorophyll content and the growth rates of the corals. It also doubled the concentrations of almost all fatty acid (FA) compounds and increased the n-alcohol and sterol contents. δ¹³C results showed that the feeding enhancement of FA concentrations occurred either via a direct pathway, for one of the major polyunsaturated fatty acid (PUFA) compounds of the food (18:3n-3 FA), or via an enhancement of photosynthate transfer (indirect pathway), for the other coral FAs. Cholesterol (C₂₇Δ⁵) was also directly acquired from the food. Thermal stress (31°C) affected corals, but differently according to their feeding status. Chlorophyll, protein content, and maximal photosynthetic efficiency of photosystem II (PSII) decreased to a greater extent in starved corals. In such corals, FA concentrations were reduced by 33%, (especially C16, C18 FAs, and n-3 PUFA) and the sterol content by 27% (especially the C₂₈∆^5,22 and C₂₈∆⁵). The enrichment in the δ¹³C signature of the storage and structural FAs suggests that they were the main compounds respired during the stress to maintain the coral metabolism. Thermal stress had less effect on the lipid concentrations of fed corals, as only FA levels were reduced by 13%, with no major changes in their isotope carbon signatures. In conclusion, feeding plays an essential role in sustaining T. reniformis metabolism during the thermal stress.     

Acclimation of chlorophyll biosynthetic reactions to temperature stress in cucumber (Cucumis sativus L.)

The adaptive responses of the greening process of plants to temperature stress were studied in cucumber (Cucumis sativus L. cv. Poinsette) seedlings grown at ambient (25 °C), low (7 °C) and high (42 °C) temperatures. Plastids isolated from these seedlings were incubated at different temperatures and the net syntheses of various tetrapyrroles were monitored. In plastids isolated from control seedlings grown at 25 °C, the optimum temperature for synthesis of Mg-protoporphyrin IX monoester or protochlorophyllide was 35 °C. Temperature maxima for Mg-protoporphyrin IX monoester and protochlorophyllide syntheses were shifted to 30 °C in chill-stressed seedlings. The net synthesis of total tetrapyrroles was severely reduced in heat-stressed seedlings and the optimum temperature for Mg-protoporphyrin IX monoester or protochlorophyllide synthesis shifted slightly towards higher temperatures, i.e. a broader peak was observed. To further study the temperature acclimation of seedlings with respect to the greening process, tetrapyrrole biosynthesis was monitored at 25 °C after pre-heating the plastids (28–70 °C) isolated from control, chill- and heat-stressed seedlings. In comparison to 28 °C-pre-heated plastids the percent inhibition of protochlorophyllide synthesis in 40 °C-pre-heated plastids was higher than for the control (25 °C-grown) in chill-stressed seedlings and lower than for the control in heat-stressed seedlings. Maximum synthesis of total tetrapyrroles and protoporphyrin IX was observed when chloroplasts were heated at 50 °C, which was probably due to heat-induced activation of the enzymes involved in protoporphyrin IX synthesis. Prominent shoulders towards lower or higher temperatures were seen in chill-stressed or heat-stressed seedlings, respectively. The shift in optimum temperature for tetrapyrrole biosynthesis in chill- and heat-stressed seedlings was probably due to acclimation of membranes possibly undergoing desaturation or saturation of membrane lipids. Proteins synthesized in response to temperature-stress may also play an important role in conferring stress-tolerance in plants. Received: 8 October 1998 / Accepted: 19 November 1998     

Evolution-related amino acids play important role in determining regioselectivity of fatty acid desaturase from <Emphasis Type="Italic">Pichia pastoris</Emphasis>

ω³-fatty acid desaturase and Δ¹²-fatty acid desaturase of Pichia pastoris with distinguishable regioselectivity and high degree of sequence similarity were chosen for regioselectivity research. Chimeras were constructed in which Histidine-rich boxes 1, 2 and the carboxyl terminal region of ω³-fatty acid desaturase were replaced with corresponding region of Δ¹²-fatty acid desaturase. The replacement was found to result in a change of regioselectivity from ωy to x + 3 by functionally characterizing these chimeric enzymes in Saccharomyces cerevisae strain INVScI. Using site-directed mutagenesis, we further demonstrated that seven conserved amino acids of ω³-fatty acid desaturase within the first two Histidine-rich regions are responsible for the regioselectivity switch. Therefore, the regioselectivity of fatty acid desaturases may be better understood by investigating the evolutionary relationships of different fatty acid desaturases. Dongsheng Wei is the partake of first-author’s profits.     

Tyrosine 39 of GH13 <Emphasis Type="Italic">α</Emphasis>-amylase from <Emphasis Type="Italic">Thermococcus hydrothermalis</Emphasis> contributes to its thermostability

The presented work is focused on the naturally thermostable α-amylase from the archaebacterium Thermococcus hydrothermalis. From the evolutionary point of view, the archaeal α-amylases are most closely related to plant α-amylases. In a wider sense, especially when the evolutionary trees are based on the less conserved part of their amino acid sequences (e.g. domain C succeeding the catalytic TIM-barrel), also the representatives of bacterial liquefying (Bacillus licheniformis) and saccharifying (Bacillus subtilis) α-amylases as well as the one from Thermotoga maritima should be included into the relatedness with the archaeal and plant α-amylases. Based on the bioinformatics analysis of the α-amylase from T. hydrothermalis, the position of tyrosine 39 (Y16 if the putative 23-residue long signal peptide is considered) was mutated to isoleucine (present in the α-amylase from T. maritima) by the in vitro mutagenesis. The biochemical characterization of the wild-type α-amylase and its Y39I mutant revealed that: (i) the specific activity of both enzymes was approximately equivalent (0.55 ± 0.13 U/mg for the wild-type and 0.52 ± 0.15 U/mg for the Y39I); (ii) the mutant exhibited decreased temperature optimum (from 85°C for the wild-type to 80°C for the Y39I); and (iii) the pH optimum remained the same (pH 5.5 for both enzymes). The remaining activity of the α-amylases was also tested by one-hour incubation at 80°C, 85°C, 90°C and 100°C. Since the wild-type α-amylase lost only 13% of its activity after one-hour incubation at the highest tested temperature (100°C), whereas 27% decrease was seen for the mutant Y39I under the same conditions, it is possible to conclude that the position of tyrosine 39 could contribute to the thermostability of the α-amylase from T. hydrothermalis.