Characterization of two chemotypes of <Emphasis Type="Italic">Pinus pinaster</Emphasis> by their terpene and acid patterns in needles

The existence of two chemotypes of Pinus pinaster, on the basis of the chemical composition of the resin acids in their needles, is known. An investigation was performed on 54 samples of needles of Spanish Pinus pinaster to study the differences between these chemotypes on the basis of monoterpene, sesquiterpene, neutral diterpene, fatty acid, and resin acid composition. One-hundred and twelve compounds were identified by GC–FID and GC–MS. Statistical analysis of the results established the existence of two groups or chemotypes, in the ratio of 5:1. In one chemotype, total acid compounds were more abundant than neutral compounds, whereas in the other the concentrations of both neutral and acid compounds were similar. Distinction of the chemotypes was based on the presence/absence of a sesquiterpene (germacrene d-4-ol acetate), neutral diterpenes (8(14),13(15)-abietadiene, anticopalol, an isomer of anticopalol, and pimarol), fatty acids (10-octadecenoic, 14-hydroxy-10-octadecenoic, and 13-hydroxy-9-octadenoic acids and an unidentified fatty acid), and resin acids (levopimaric + palustric, eperuic, and anticopalic acids, and three isomers of anticopalic acid); and on the different relative percentages of other compounds of these types. This study gives a wide view of the composition of the needles of Pinus pinaster, improving the differentiation of chemotypes on the basis of terpene and acid composition.     

Differences between Pinus pinea and Pinus pinaster as bioindicators of polycyclic aromatic hydrocarbons

The potential of Univariate and Multivariate Analysis and specifically Principal Components Analysis (PCA), has been employed to assess the performance of pine needles as bioindicators of polycyclic aromatic hydrocarbons (PAHs) and particularly emphasize the crucial importance of making a distinction between different pine species if more than one is sampled. Four sampling sessions were done in 29 sites and needles of two common pine species (Pinus pinaster Ait. in 19 sites and Pinus pinea L. in 12) were collected and analysed using gas chromatography-mass spectrometry (GC-MS) and PCA. The results obtained indicated significant differences between species, attributed to their different morphology. The mean total PAH concentration of the P. pinaster needles are over two times higher than P. pinea's. This difference is lower when the results are presented in lipid weight, but still statistically significant. Samples from the two sites with adjacent trees reinforce these conclusions, showing significant differences in terms of PAH mean concentration and aromatic ring patterns.     

Effect of light and temperature on epicuticular fatty acid and fatty alcohol of tobacco

Genetically uniform burley tobacco (Nicotiana tabacum) was grown under field and various controlled-environment conditions to determine whether environment influenced epicuticular alkane, fatty acid, and fatty-alcohol composition of the leaves. Quantity and quality of alkanes, fatty acids, and fatty alcohols were greatly influenced by environmental conditions. Highest light intensity did not result in the largest total long aliphatic carbon-chain production. Generally, long photoperiod and cool temperature were associated with highest long aliphatic carbon-chain production on a leaf area basis. Quantity of the individual alkane, fatty acid, or fatty alcohol classes present under the different growth conditions varied in relation to the leaf metabolic status and not leaf size.     

Novel conserved segments are associated with differential expression patterns for Pinaceae dehydrins

Dehydrins are thought to play an essential role in the response, acclimation and tolerance to different abiotic stresses, such as cold and drought. These proteins have been classified into five groups according to the presence of conserved and repeated motifs in their amino acid sequence. Due to their putative functions in the response to stress, dehydrins have been often used as candidate genes in studies on population variability and local adaptation to environmental conditions. However, little is still known regarding the differential role played by such groups or the mechanism underlying their function. Based on the sequences corresponding to dehydrins available in public databases we have isolated eight different dehydrins from cDNA of Pinus pinaster. We have obtained also their genomic sequences and identified their intron/exon structure. Quantitative RT-PCR analysis of their expression pattern in needles, stems and roots during a severe and prolonged drought stress, similar to the ones trees must face in nature, is also reported. Additionally, we have identified two amino acid motifs highly conserved and repeated in Pinaceae dehydrins and absent in angiosperms, presumably related to the divergent expression profiles observed.     

Responses of pigmentation and δ13C in Qilian juniper to cold and drought stresses under natural conditions in the Qilian Mountains,China

The seasonal changes of pigments and stable carbon isotope composition (δ¹³C values) were investigated in needles of Qilian juniper (Sabina przewalskii Kom.) from two typical sites, one drier and the other wetter, in the Qilian Mountains, China. The anthocyanins and rhodoxanthin content from both sites were much higher in winter than in summer. Plant needles contained more carotenoids and xanthophylls in winter at the wetter site, while no seasonal difference appeared at the drier site. However, lower chlorophyll content and higher proline and δ¹³C were observed at the drier site. Average tree height was shorter at the drier site trees than at the wetter site. Our results suggested that under natural conditions, pigmentation in S. przewalskii may serve to improve the energy balance of the photosynthetic apparatus under cold and drought stresses. Proline and δ¹³C could be used as drought indicators for S. przewalskii.     

Effects of aqueous sulphur dioxide on pine needle glycolipids

The major glycolipids in the fully developed and young needle tissues of lodgepole pine (Pinus contorta Dougl. var. latifolia Engelm.) were monogalactosyl diglyceride (MGDG) digalactosyl diglyceride (DGDG), and sulphoquinovosyl diglyceride (SQDG). The concentration of these glycolipids was considerably higher in the fully developed needles than in the young needles. The major fatty acid in the MGDG fraction (from both tissues) and DGDG fraction (from fully developed tissues) was linolenic acid. However, palmitic acid was the major fatty acid in the DGDG fraction from the young tissues and the SQDG fraction from both tissues. Treatment of needles with aq. SO₂ solutions produced marked changes in the concentration and composition of these glycolipid fractions. At 100 ppm, SO₂ produced a considerable drop in the linolenic acid content of all glycolipid fractions, more pronounced in the young needles than in the fully developed ones. SO₂ also had an effect on the release of soluble sugars from the needle tissues of both ages.     

Conversion of Digalactosyl Diglyceride (Extra Long Carbon Chain Conjugates) into Monogalactosyl Diglyceride of Pine Needle Chloroplasts upon Dehardening

Changes in lipid and fatty acid composition of pine needle chloroplasts were determined upon transfer of the trees from outside (-5°C) to 32°C. Within 7 1/2 hours after transfer, conversion of a portion of digalactosyldiglyceride into monogalactosyl diglyceride was observed. This portion consisted of a conjugate with extra long chain fatty acids (cerotic acid, 26: 0, and C₂₆-cyclo-propane fatty acid). Only minor further changes in lipid and fatty acid composition were observed. Dehardening of the needles occurred within the same period. The data support the hypothesis that the degalactosidation reaction specifically depends on the fatty acid composition. The extra long chain fatty acids may link different sub-units of the chloroplasts and may contribute to an additional layer of H-bonded water on the membrane surface because of the increased exposure of the galactose groups.     

Quality evaluation of Semen persicae (seed of Prunus persica L. Batsch) by gas chromatography–mass spectrometry

Seeds of Prunus persica L. Batsch were collected at various times from different regions of China. The fatty acid composition of the seeds was analysed using gas chromatography-mass spectrometry (GC-MS). The aim was to evaluate whether genetic and environmental factors influenced the chemical profile of fatty acids in Semen persicae. Saturated and unsaturated fatty acids were present at 4.8-8.7% and 90.7-94.8% of total fatty acids, respectively. Oleic and linoleic acids were dominant in all samples and ranged from 59.3 to 81.4% and from 11.6 to 31.0%, respectively. All samples had high levels of unsaturated fatty acids. Hierarchical clustering analysis and principal components analysis were performed to differentiate and classify the samples based on the contents of the characteristic fatty acid constituents. This study provides evidence that metabolites may reflect genetic and environmental similarities, such as management practices used in cultivars (irrigation, fertilization and sanitary treatments), and evidence of the variability of genetic make up and local environmental conditions in samples grown in the wild.     

Influence of environmental and genetic factors on the stability of essential oil composition of Thymus pulegioides

Thymus pulegioides plants were collected from various natural habitats of Lithuania and transferred into a new uniform environment. The plants were cloned annually at controlled conditions and their essential oil composition was monitored by capillary GC and GC/MS. The geraniol/geranial/neral (G/G/N), thymol (T), linalool (L), carvacrol/γ-terpinene/p-cymene (C/γT/pC) and thymol/carvacrol/γ-terpinene/p-cymene (T/C/γT/pC) chemotypes of T. pulegioides were studied. It was found that according to the stability of essential oil composition on a sudden change of environmental conditions two types of T. pulegioides can be distinguished: (1) plants which preserve their chemical composition of the essential oils; (2) plants, which considerably change their chemical composition of the essential oils.     

Small Changes in Environmental Parameters Lead to Alterations in Antibiotic Resistance,Cell Morphology and Membrane Fatty Acid Composition in Staphylococcus lugdunensis

Staphylococcus lugdunensis has emerged as a major cause of community-acquired and nosocomial infections. This bacterium can rapidly adapt to changing environmental conditions to survive and capitalize on opportunities to colonize and infect through wound surfaces. It was proposed that S. lugdunensis would have underlying alterations in metabolic homeostasis to provide the necessary levels of adaptive protection. The aims of this project were to examine the impacts of subtle variations in environmental conditions on growth characteristics, cell size and membrane fatty acid composition in S. lugdunensis. Liquid broth cultures of S. lugdunensis were grown under varying combinations of pH (6–8), temperature (35–39°C) and osmotic pressure (0–5% sodium chloride w/w) to reflect potential ranges of conditions encountered during transition from skin surfaces to invasion of wound sites. The cells were harvested at the mid-exponential phase of growth and assessed for antibiotic minimal inhibitory concentration (MIC), generation time, formation of small colony variants, cell size (by scanning electron microscopy) and membrane fatty acid composition. Stress regimes with elevated NaCl concentrations resulted in significantly higher antibiotic resistance (MIC) and three of the combinations with 5% NaCl had increased generation times (P<0.05). It was found that all ten experimental growth regimes, including the control and centroid cultures, yielded significantly different profiles of plasma membrane fatty acid composition (P<0.0001). Alterations in cell size (P<0.01) were also observed under the range of conditions with the most substantial reduction occurring when cells were grown at 39°C, pH 8 (514±52 nm, mean ± Standard Deviation) compared with cells grown under control conditions at 37°C with pH 7 (702±76 nm, P<0.01). It was concluded that S. lugdunensis responded to slight changes in environmental conditions by altering plasma membrane fatty acid composition, growth rates and morphology to achieve optimal adaptations for survival in changing environments.     

Light and Nutrient Dependent Responses in Secondary Metabolites of Plantago lanceolata Offspring Are Due to Phenotypic Plasticity in Experimental Grasslands

A few studies in the past have shown that plant diversity in terms of species richness and functional composition can modify plant defense chemistry. However, it is not yet clear to what extent genetic differentiation of plant chemotypes or phenotypic plasticity in response to diversity-induced variation in growth conditions or a combination of both is responsible for this pattern. We collected seed families of ribwort plantain (Plantago lanceolata) from six-year old experimental grasslands of varying plant diversity (Jena Experiment). The offspring of these seed families was grown under standardized conditions with two levels of light and nutrients. The iridoid glycosides, catalpol and aucubin, and verbascoside, a caffeoyl phenylethanoid glycoside, were measured in roots and shoots. Although offspring of different seed families differed in the tissue concentrations of defensive metabolites, plant diversity in the mothers'' environment did not explain the variation in the measured defensive metabolites of P. lanceolata offspring. However secondary metabolite levels in roots and shoots were strongly affected by light and nutrient availability. Highest concentrations of iridoid glycosides and verbascoside were found under high light conditions, and nutrient availability had positive effects on iridoid glycoside concentrations in plants grown under high light conditions. However, verbascoside concentrations decreased under high levels of nutrients irrespective of light. The data from our greenhouse study show that phenotypic plasticity in response to environmental variation rather than genetic differentiation in response to plant community diversity is responsible for variation in secondary metabolite concentrations of P. lanceolata in the six-year old communities of the grassland biodiversity experiment. Due to its large phenotypic plasticity P. lanceolata has the potential for a fast and efficient adjustment to varying environmental conditions in plant communities of different species richness and functional composition.     

Intraspecific variation in Pinus pinaster PSII photochemical efficiency in response to winter stress and freezing temperatures

As part of a program to select maritime pine (Pinus pinaster Ait.) genotypes for resistance to low winter temperatures, we examined variation in photosystem II activity by chlorophyll fluorescence. Populations and families within populations from contrasting climates were tested during two consecutive winters through two progeny trials, one located at a continental and xeric site and one at a mesic site with Atlantic influence. We also obtained the LT₅₀, or the temperature that causes 50% damage, by controlled freezing and the subsequent analysis of chlorophyll fluorescence in needles and stems that were collected from populations at the continental trial site. P. pinaster showed sensitivity to winter stress at the continental site, during the colder winter. The combination of low temperatures, high solar irradiation and low precipitation caused sustained decreases in maximal photochemical efficiency (F_v/F_m), quantum yield of non-cyclic electron transport (Φ_PSII) and photochemical quenching (qP). The variation in photochemical parameters was larger among families than among populations, and population differences appeared only under the harshest conditions at the continental site. As expected, the environmental effects (winter and site) on the photochemical parameters were much larger than the genotypic effects (population or family). LT₅₀ was closely related to the minimum winter temperatures of the population''s range. The dark-adapted F_v/F_m ratio discriminated clearly between interior and coastal populations.In conclusion, variations in F_v/F_m, Φ_PSII, qP and non-photochemical quenching (NPQ) in response to winter stress were primarily due to the differences between the winter conditions and the sites and secondarily due to the differences among families and their interactions with the environment. Populations from continental climates showed higher frost tolerance (LT₅₀) than coastal populations that typically experience mild winters. Therefore, LT₅₀, as estimated by F_v/F_m, is a reliable indicator of frost tolerance among P. pinaster populations.     

Aridity promotes differences in proline and phytohormone levels in <Emphasis Type="Italic">Pinus pinaster</Emphasis> populations from contrasting environments

We analysed proline, abscisic acid, (ABA), jasmonic acid (JA), indole acetic acid (IAA) and salicylic acid (SA) accumulation after summer drought at two Pinus pinaster provenance-progeny trial sites. The aim of the study was to evaluate P. pinaster phenotypic plasticity and intraspecific variation in the endogenous concentrations of these metabolites and to determine the best stress indicators for family and population discrimination. The environmental effect was remarkable, as striking differences between the sites were obtained for all indicators except for SA, which was unaffected by the environmental conditions. The levels of proline, ABA and IAA were higher in the xeric than in the mesic site. In contrast, JA was higher in the mesic site. The higher variation displayed at the family level led to family differences for all parameters and sites. Differences in proline and ABA between populations were exclusively found in the xeric site, where the population from the wet climate showed higher accumulation. This study provides evidence for differentiation among P. pinaster populations and families in their plastic responses to drought and highlights the importance of considering intraspecific variability when evaluating biochemical stress indicators in environmental studies.     

Ontogenetic differentiation between Mediterranean and Eurasian pines (sect. Pinus) at the seedling stage

Heteroblastic development in pine seedlings includes extreme morphological changes with still unclear adaptive and evolutionary significance. In particular, Mediterranean and Eurasian pines (section Pinus) living in the Mediterranean basin seem to follow quite distinct developmental trajectories at the seedling stage. Aiming to confirm this ontogenetic differentiation we performed a nursery experiment with seedlings of five Mediterranean pines (Pinus pinaster, P. brutia, P. halepensis, P. pinea and P. canariensis) and three Eurasian pines (P. sylvestris, P. uncinata, and P. nigra subsp. salzmannii), also including P. radiata as an outgroup. After destructive analyses at two harvest times (9 and 32 weeks), we found sharp differentiation between Mediterranean and Eurasian pines in a combination of traits linked to shoot heteroblasty. In particular, Mediterranean pines showed a marked delay in the proportion of adult needles to total needles in the shoot compared to Eurasian species, especially at the second harvest. However, two Mediterranean pines, P. pinaster and P. brutia showed a slightly higher proportion of secondary needles, and a higher rate of budset at a more advanced stage (68 weeks) compared to the other three Mediterranean species. Meaningfully, the outgroup P. radiata was the only species combining a high proportion of adult foliage since the first harvest with a delayed formation of the first terminal bud. We discussed the adaptive significance of these findings at the light of species’ climatic niches and life histories.     

Metabolic control of the membrane fluidity in Bacillus subtilis during cold adaptation

Membrane fluidity adaptation to the low growth temperature in Bacillus subtilis involves two distinct mechanisms: (1) long-term adaptation accomplished by increasing the ratio of anteiso- to iso-branched fatty acids and (2) rapid desaturation of fatty acid chains in existing phospholipids by induction of fatty acid desaturase after cold shock. In this work we studied the effect of medium composition on cold adaptation of membrane fluidity. Bacillus subtilis was cultivated at optimum (40 °C) and low (20 °C) temperatures in complex medium with glucose or in mineral medium with either glucose or glycerol. Cold adaptation was characterized by fatty acid analysis and by measuring the midpoint of phospholipid phase transition T_m (differential scanning calorimetry) and membrane fluidity (DPH fluorescence polarization). Cells cultured and measured at 40 °C displayed the same membrane fluidity in all three media despite a markedly different fatty acid composition. The T_m was surprisingly the highest in the case of a culture grown in complex medium. On the contrary, cultivation at 20 °C in the complex medium gave rise to the highest membrane fluidity with concomitant decrease of T_m by 10.5 °C. In mineral media at 20 °C the corresponding changes of T_m were almost negligible. After a temperature shift from 40 to 20 °C, the cultures from all three media displayed the same adaptive induction of fatty acid desaturase despite their different membrane fluidity values immediately after cold shock.     

Importance of 3-Hydroxy Fatty Acid Composition of Lipopeptides for Biosurfactant Activity

Biosurfactant production may be an economic approach to improving oil recovery. To obtain candidates most suitable for oil recovery, 207 strains, mostly belonging to the genus Bacillus, were tested for growth and biosurfactant production in medium with 5% NaCl under aerobic and anaerobic conditions. All strains grew aerobically with 5% NaCl, and 147 strains produced a biosurfactant. Thirty-five strains grew anaerobically with 5% NaCl, and two produced a biosurfactant. In order to relate structural differences to activity, eight lipopeptide biosurfactants with different specific activities produced by various Bacillus species were purified by a new protocol. The amino acid compositions of the eight lipopeptides were the same (Glu/Gln:Asp/Asn:Val:Leu, 1:1:1:4), but the fatty acid compositions differed. Multiple regression analysis showed that the specific biosurfactant activity depended on the ratios of both iso to normal even-numbered fatty acids and anteiso to iso odd-numbered fatty acids. A multiple regression model accurately predicted the specific biosurfactant activities of four newly purified biosurfactants (r² = 0.91). The fatty acid composition of the biosurfactant produced by Bacillus subtilis subsp. subtilis strain T89-42 was altered by the addition of branched-chain amino acids to the growth medium. The specific activities of biosurfactants produced in cultures with different amino acid additions were accurately predicted by the multiple regression model derived from the fatty acid compositions (r² = 0.95). Our work shows that many strains of Bacillus mojavensis and Bacillus subtilis produce biosurfactants and that the fatty acid composition is important for biosurfactant activity.     

Seasonal and within-plant gradients in the intramolecular carbon isotopic composition of amino acids of Spartina alterniflora

Autotrophy and heterotrophy create different patterns of carbon flux through the central metabolic pathway. One consequence of these different fluxes is that the α-carboxyl carbon of amino acids is derived from different carbon sources and has a different isotopic composition under autotrophic and heterotrophic conditions. In Spartina alterniflora, a C4 grass and a common and ecologically important component of coastal ecosystems, the isotopic composition of bulk acid hydrolyzable carbon and total amino acid carboxyl carbon were compared over a seasonal cycle. The isotopic composition of plants varied significantly between aboveground and below ground tissues, and the δ¹³C of both hydrolyzable organic carbon and total amino acid carboxyl carbon showed significant variation among seasons. The isotopic heterogeneity within amino acids was used to infer seasonal changes in source/sink relationships for amino acid carbon among plant organs. Comparison of the intramolecular isotope data for Spartina and C3 freshwater marsh plants indicates that the patterns and processes inferred for Spartina are not unique to this taxon.     

Seasonally Induced Changes in Acyl Lipids and Fatty Acids of Chloroplast Thylakoids of Pinus silvestris: A CORRELATION BETWEEN THE LEVEL OF UNSATURATION OF MONOGALACTOSYLDIGLYCERIDE AND THE RATE OF ELECTRON TRANSPORT

Current-year needles were sampled regularly from an approximately 20-year-old natural stand of Pinus silvestris. Chloroplast thylakoids were isolated. The electron transport capacities of photosystem II + photosystem I, as well as of the partial photoreactions, were measured. The amounts and the fatty acid compositions of monogalactosyldiglyceride, digalactosyldiglyceride, and sulfolipid of the thylakoids were analyzed. The fatty acid composition of the phospholipids (total) was also determined.     

Effect of frost hardening on lipid and Fatty Acid composition of chloroplast thylakoid membranes in two wheat varieties of contrasting hardiness

Lipid and fatty acid composition of chloroplast thylakoid membranes was determined in two varieties of wheat (Triticum aestivum L.), the hardy Miranovskaja and the sensitive Penjamo. Plants were grown at room temperature or under frost hardening conditions (1.5°C). Changes in lipid and fatty acid composition of the isolated thylakoids could be related to the temperature dependence of light-stimulated proton uptake. Changes in the thylakoid phospholipids upon hardening of the two varieties did not show any direct relation with low temperature tolerance of light-dependent H⁺ uptake; neither did changes in phospholipid fatty acid chain lengthening to 20 and 22 C-atoms in combination with increased desaturation up to 6 double bonds. Increased low temperature tolerance of light-induced H⁺ uptake by hardening was correlated with the following glycolipid changes: maintained glycolipid level, a proportionally increased digalactosyl diglyceride fraction, a decrease in thylakoid monogalactosyl diglyceride, increased sulfolipid fatty acid chain lengthening (20 and 22 C-atoms), and increased sulfolipid desaturation (4-6 double bonds). We suggest that the above mentioned changes in glycolipids have adaptive value for low temperature tolerance of light-dependent proton uptake.