Accumulation of DNA damage in Antarctic mosses: correlations with ultraviolet-B radiation, temperature and turf water content vary among species

The susceptibility of three East Antarctic moss species to UV-B radiation was examined by measuring accumulation of cyclobutane pyrimidine dimers under natural sunlight during the austral summer season of 2002/03. The 2002/03 season was characterized by unusually low springtime ozone depletion and as such our results likely underestimate the DNA damage possible in a more typical UV-B radiation season. Despite this all three species accumulated significant DNA photoproducts. We also found a positive association between photoproduct accumulation and incident UV-B radiation in the two cosmopolitan species, Bryum pseudotriquetrum and Ceratodon purpureus , with more DNA damage in samples collected early in the season compared with later in the summer. For B. pseudotriquetrum , negative associations were also observed between photoproduct accumulation and both turf water content and the 10-day mean air temperature. Photoproduct accumulation in the endemic species Schistidium antarctici was similarly high across the season and no significant association with environmental variables was found. Our results are consistent with the two cosmopolitan species having somewhat higher UV-B-screening capabilities and possibly more efficient mechanisms for repairing DNA damage than the endemic S. antarctici .     

Moss Communities and Their Characteristics in Ice-free Areas of the Windmill Islands,Wilkes Land,Continental Antarctica

The moss vegetation of the Windmill Islands can be classified into the following seven communities: 1.Grimmia antarctici community; 2. G. antarctici-Ceratodon purpureus community; 3. Bryum pseudotriquetrum community; 4. G. antarctici-B, pseudotriquetrum community; 5. Ceratodon purpureus community; 6. C. purpureus-G, antarctici community; 7. C. purpureus B. pseudotriquetrum community. Communities 1, 3, 4 and 7 occur in wet habitats while communities 5 and 6 are found in rather dry habitats, community 2 distributes between dry and wet habitats. Microtopography governs the distribution of water supply and therefore community types. Stable moss communities usually form moss hummocks and hollows. Species composition, colour and height of the hummocks determines the vertical structure of the moss community layers with 2-3 strata being discernible and level structure forming mosaic. Other phytocoenological characteristics of the moss communities, such as the dense moss cushions, asexual reproduction (although antheridia or archegonia have been found in each of the species), abundance of epiphytic algae and lichens growing on the surface of moss hummocks, the colour change of some species in different habitats, appear related to moisture availability, light intensity, wind exposure and temperature.     

Contrasting strategies for UV-B screening in sub-Arctic dwarf shrubs

The content and distribution of UV-absorbing phenolic compounds was investigated in leaves of three species of Vaccinium co-existing at a site in north Sweden. Vaccinium myrtillus L., Vaccinium vitis-idaea L., and Vaccinium uliginosum L. exhibit markedly different strategies, in terms of localization and content of leaf phenolics and in their responses to UV-B enhancement. Plants were exposed to either ambient radiation or to enhancement of UV-B corresponding to 15% (clear sky) depletion of stratospheric ozone for approximately 10 years prior to commencement of this study. Vaccinium myrtillus contained the highest concentration of methanol-extractable UV-B-absorbing compounds, which was elevated in plants exposed to enhanced UV-B. Fluorescence and confocal laser scanning microscopy showed that these compounds were distributed throughout the leaf, and were particularly concentrated in chlorophyll-containing cells. In V. vitis-idaea, most phenolic compounds were cell wall-bound and concentrated in the walls of the epidermis; this pool increased in response to UV-B enhancement. It is suggested that these two plants represent extreme forms of two divergent strategies for UV-B screening, the different responses possibly being related to leaf longevity in the two species. The response of V. uliginosum was intermediate between the other two, with high concentrations of cell wall-bound phenolics in the epidermis but with this pool decreasing, and the methanol-soluble pool tending to increase, after exposure to enhanced UV-B. One explanation for this response is that this plant is deciduous, like V. myrtillus, but has leaves that are structurally similar to those of V. vitis-idaea.     

Ultraviolet B screening potential is higher in two cosmopolitan moss species than in a co-occurring Antarctic endemic moss: implications of continuing ozone depletion

Concentrations of UVB (ultraviolet B) absorbing pigments and anthocyanins were measured in three moss species, over a summer growing season in Antarctica. Pigment concentrations were compared with a range of climatic variables to determine if there was evidence that pigments were induced by UVB radiation, or other environmental parameters, and secondly if there were differences between species in their pigment responses. Significant seasonal differences in the potential UVB screening pigments were found, with the two cosmopolitan species Bryum pseudotriquetrum and Ceratodon purpureus appearing better protected from the potentially damaging effects of ozone depletion than the Antarctic endemic Schistidium antarctici. B. pseudotriquetrum accumulated the highest concentration of UVB screening pigments and showed positive associations between UVB radiation and both UVB absorbing and anthocyanin pigments. The negative associations between water availability measures and UVB absorbing and anthocyanin pigments also suggest that B. pseudotriquetrum is well protected in the desiccated state. This could offer B. pseudotriquetrum an advantage over the other species when high UVB radiation coincides with low temperatures and low water availability, thus limiting physiological activity and consequently, active photoprotective and repair mechanisms. As these pigments could act as either direct UVB screens or antioxidants, the results suggest that B. pseudotriquetrum is best equipped to deal with the negative effects of increased exposure to UVB radiation due to ozone depletion. The most exposed species, C. purpureus, has intermediate and stable concentrations of UVB absorbing pigments suggesting it may rely on constitutive UVB screens. Anthocyanin pigments were more responsive in this species and could offer increased antioxidant protection during periods of high UVB radiation. S. antarctici appears poorly protected and showed no evidence of any UV photoprotective response, providing additional evidence that this endemic is more vulnerable to climate change.     

Ultraviolet radiation screening compounds

Amongst the diversity of methods used by organisms to reduce damage caused by ultraviolet (UV) radiation, the synthesis of UV-screening compounds is almost ubiquitous. UV-screening compounds provide a passive method for the reduction of UV-induced damage and they are widely distributed across the microbial, plant and animal kingdoms. They share some common chemical features. It is likely that on early earth strong selection pressures existed for the evolution of UV-screening compounds. Many of these compounds probably had other physiological roles, later being selected for the efficacy of UV screening. The diversity in physiological functions is one of the complications in studying UV-screening compounds and determining the true ecological importance of their UV-screening role. As well as providing protection against ambient UV radiation, species with effective screening may also be at an advantage during natural ozone depletion events. In this review the characteristics of a wide diversity of UV-screening compounds are discussed and evolutionary questions are explored. As research into the range of UV-screening compounds represented in the biosphere continues, so it is likely that the properties of many more compounds will be elucidated. These compounds, as well as providing us with insights into natural responses to UV radiation, may also have implications for the development of artificial UV-screening methods to reduce human exposure to UV radiation.     

Compositional characterization and imaging of “wall-bound” acylesters of Populus trichocarpa reveal differential accumulation of acyl molecules in normal and reactive woods

Acylesterification is one of the common modifications of cell wall non-cellulosic polysaccharides and/or lignin primarily in monocot plants. We analyzed the cell-wall acylesters of black cottonwood (Populus trichocarpa Torr. & Gray) with liquid chromatography-mass spectrometry (LC-MS), Fourier transform-infrared (FT-IR) microspectroscopy, and synchrotron infrared (IR) imaging facility. The results revealed that the cell wall of dicotyledonous poplar, as the walls of many monocot grasses, contains a considerable amount of acylesters, primarily acetyl and p-hydroxycinnamoyl molecules. The "wall-bound" acetate and phenolics display a distinct tissue specific-, bending stress responsible- and developmental-accumulation pattern. The "wall-bound" p-coumarate predominantly accumulated in young leaves and decreased in mature leaves, whereas acetate and ferulate mostly amassed in the cell wall of stems. Along the development of stem, the level of the "wall-bound" ferulate gradually increased, while the basal level of p-coumarate further decreased. Induction of tension wood decreased the accumulation of the "wall-bound" phenolics while the level of acetate remained constant. Synchrotron IR-mediated chemical compositional imaging revealed a close spatial distribution of acylesters with cell wall polysaccharides in poplar stem. These results indicate that different "wall-bound" acylesters play distinct roles in poplar cell wall structural construction and/or metabolism of cell wall matrix components.     

Elicitor-induced changes of phenylalanine ammonia-lyase activity in barley cell suspension cultures

Suspension-cultured barley cells responded to treatments with crude yeast extract and purified glucan preparation by rapidly and transiently (4 h postelicitation) inducing L-phenylalanine ammonia-lyase activity. Similarly, treatment of cell cultures with chitosan resulted in increased phenylalanine ammonia-lyase activity 2–4 h after elicitation, whereas a mycelium preparation of a fungal pathogen, Bipolaris sorokiniana, and purified chitin caused a more delayed induction of phenylalanine ammonia-lyase (8 h postelicitation). The most abundant of the plant cell wall degrading enzymes produced by Bipolaris sorokiniana, β-1,4-xylanase, had only a weak elicitor activity in barley cells suggesting that fungal cell wall components rather than the hydrolytic enzymes secreted by the fungus function as recognizable components that cause barley cells to induce defences. Treatment of the elicited cells with a phenylalanine ammonia-lyase inhibitor, α-aminooxy-β-phenylpropionic acid, resulted in the superinduction of the enzyme indicating the blocking of the feedback regulation mechanisms, whereas in the presence of 1 mM trans-cinnamic acid the elicitor-induction of phenylalanine ammonia-lyase was completely inhibited. Elicitor treatments increased the accumulation of wall-bound phenolics as evidenced by phloroglucinol-HCl staining and thioglycolic acid methods. However, α-aminooxy-β-phenylpropionic acid applied in combination with the elicitor did not prevent the accumulation of phenolics in barley cell walls. This suggested that phenylalanine ammonia-lyase might not play an important role in the synthesis wall-bound phenolic compounds in barley. However, cinnamic acid, whether applied alone or together with the elicitor, increased the amount of wall-bound phenolics in suspension-cultured barley cells. This revised version was published online in June 2006 with corrections to the Cover Date.     

Accumulation of tyrosol glucoside in transgenic potato plants expressing a parsley tyrosine decarboxylase

As part of the response to pathogen infection, potato plants accumulate soluble and cell wall-bound phenolics such as hydroxycinnamic acid tyramine amides. Since incorporation of these compounds into the cell wall leads to a fortified barrier against pathogens, raising the amounts of hydroxycinnamic acid tyramine amides might positively affect the resistance response. To this end, we set out to increase the amount of tyramine, one of the substrates of the hydroxycinnamoyl-CoA:tyramine N-(hydroxycinnamoyl)-transferase reaction, by placing a cDNA encoding a pathogen-induced tyrosine decarboxylase from parsley under the control of the 35S promoter and introducing the construct into potato plants via Agrobacterium tumefaciens-mediated transformation. While no alterations were observed in the pattern and quantity of cell wall-bound phenolic compounds in transgenic plants, the soluble fraction contained several new compounds. The major one was isolated and identified as tyrosol glucoside by liquid chromatography-electrospray ionization-high resolution mass spectrometry and NMR analyses. Our results indicate that expression of a tyrosine decarboxylase in potato does not channel tyramine into the hydroxycinnamoyl-CoA:tyramine N-(hydroxycinnamoyl)-transferase reaction but rather unexpectedly, into a different pathway leading to the formation of a potential storage compound.     

Metabolic diversion of the phenylpropanoid pathway causes cell wall and morphological changes in transgenic tobacco stems

Studies involving transgenic plants with modifications in the lignin pathway reported to date, have received a relatively preliminary characterisation in relation to the impact on vascular integrity, biomechanical properties of tissues and carbon allocation to phenolic pools. Therefore, in this study transgenic tobacco plants (Nicotiana tabacum cv XHFD 8) expressing various levels of a bacterial 4-hydroxycinnamoyl-CoA hydratase/lyase (HCHL) gene have been characterised for cell wall and related morphological changes. The HCHL enzyme converts p-coumaroyl-CoA to 4-hydroxybenzaldehyde thereby rerouting the phenylpropanoid pathway. Plants expressing high levels of HCHL activity exhibited reduced lignin deposition, impaired monolignol biosynthesis and vascular integrity. The plants also exhibited reduction in stem toughness concomitant with a massive reduction in both the cell wall esterified and soluble phenolics. A notable result of redirecting the carbon flux was the wall-bound accretion of vanillin and vanillic acid, probably due to the shunt pathway. Intracellular accumulation of novel metabolites such as hydroxybenzoic and vanillic acid derivatives also occurred in the transgenic plants. A line with intermediate levels of HCHL expression conferred correspondingly reduced lignin deposition, toughness and phenolics. This line displayed a normal morphology but distorted vasculature. Coloration of the xylem has been previously attributed to incorporation of alternative phenolics, whereas results from this study indicate that the coloration is likely to be due to the association of low molecular weight phenolics. There was no evidence of increased growth or enhanced cellulose biosynthesis as a result of HCHL expression. Hence, rerouting the phenylpropanoid biosynthetic pathway quantitatively and qualitatively modifies cell wall-bound phenolics and vascular structure.     

Can dual chlorophyll fluorescence excitation be used to assess the variation in the content of UV-absorbing phenolic compounds in leaves of temperate tree species along a light gradient?

The present study assesses light-induced variations in phenolic compounds in leaves of saplings of two co-occurring temperate species (Acer platanoides L., and Fraxinus excelsior L.) along a light gradient using a new non-invasive optical method (Dualex). The Dualex-derived UV absorbance of leaf epidermis (the sum of the adaxial and abaxial faces, AUV) increased significantly with increasing light in both species. AUV values were correlated with absorbance of the leaf extract at 305 nm and 375 nm (A305 and A375) in both species with similar slopes for both species. However, a large difference in intercept was observed between the two species when A305 was regressed against AUV. Similarly, AUV values were well correlated with the amount of phenolics in the leaf extracts assessed by the Folin-Ciocalteu method, but slopes were significantly different for the two species. Thus, the UV-A epidermal transmittance, despite being a reliable indicator of the UV-screening capacity of the leaf epidermis, cannot be used for any quantitative estimate of UV-B screening capacity or of energetic requirement for leaf construction without a species-specific calibration.     

Elongation of teichuronic acid chains by a wall-membrane preparation from Micrococcus luteus.

A wall-plus-membrane preparation from Micrococcus luteus catalyzes the incorporation of [14C]glucose from UDP-[14C]glucose, into two fractions of teichuronic acid, which is the cell wall polysaccharide consisting of alternating residues of glucose and N-acetylmannosaminuronic acid (ManNAcUA). Membrane-associated teichuronic acid was extracted from the wall-membrane fraction of reaction mixtures by sodium dodecyl sulfate. The synthesis of membrane-associated teichuronic acid required UDP-glucose, UDP-ManNAcUA, and UDP-N-acetylglucosamine and was inhibited by tunicamycin. Glucose incorporated into wall-bound teichuronic acid remained in wall fragments after extraction with sodium dodecyl sulfate, and its incorporation required UDP-glucose and UDP-ManNAcUA (but not UDP-N-acetylglucosamine) and was insensitive to tunicamycin. Radioactive material incorporated into wall-bound teichuronic acid could be released by treatment with mild acid or by digestion with lysozyme, indicating that the wall-bound teichuronic acid was covalently linked to peptidoglycan. There were about 600 pmol of wall-bound teichuronic acid acceptor sites for glucose per mg of protein as measured in incorporation reaction mixtures lacking UDP-ManNAcUA. In the presence of both UDP-glucose and UDP-ManNAcUA, elongation of teichuronic acid acceptor sites occurred, with the addition of six to eight disaccharide units to each acceptor site.     

Profiling C6-C3 and C6-C1 phenolic metabolites in Cocos nucifera

This paper reports the detection and identification of phenolic metabolites (C6-C3 and C6-C1 compounds) in Cocos nucifera. An HPLC/UV system was used to analyze the soluble and wall-associated phenolics in mesocarp and leaf tissues of C. nucifera. Alkaline hydrolysis of the cell wall material of the mesocarpic and leaf tissues yielded 4-hydroxybenzoic acid as the major phenolic compound. Other phenolic acids identified were ferulic acid, 4-coumaric acid, 4-hydroxybenzaldehyde and vanillic acid. No significant qualitative differences in composition were observed between leaf and mesocarp, but there were quantitative variations in the metabolite levels.     

Cell wall alterations in the leaves of fusariosis-resistant and susceptible pineapple cultivars

Fusariosis, caused by the fungus Fusarium subglutinans f. sp. ananas (Syn. F. guttiforme), is one of the main phytosanitary threats to pineapple (Ananas comosus var. comosus). Identification of plant cell responses to pathogens is important in understanding the plant–pathogen relationship and establishing strategies to improve and select resistant cultivars. Studies of the structural properties and phenolic content of cell walls in resistant (Vitoria) and susceptible (Perola) pineapple cultivars, related to resistance to the fungus, were performed. The non-chlorophyll base of physiologically mature leaves was inoculated with a conidia suspension. Analyses were performed post-inoculation by light, atomic force, scanning and transmission electron microscopy, and measurement of cell wall-bound phenolic compounds. Non-inoculated leaves were used as controls to define the constitutive tissue characteristics. Analyses indicated that morphological differences, such as cell wall thickness, cicatrization process and lignification, were related to resistance to the pathogen. Atomic force microscopy indicated a considerable difference in the mechanical properties of the resistant and susceptible cultivars, with more structural integrity, associated with higher levels of cell wall-bound phenolics, found in the resistant cultivar. p-Coumaric and ferulic acids were shown to be the major phenolics bound to the cell walls and were found in higher amounts in the resistant cultivar. Leaves of the resistant cultivar had reduced fungal penetration and a faster and more effective cicatrization response compared to the susceptible cultivar.     

全日光强下5种植物叶片的UV-B防护

通过测定马尾松(Pinus massoniana)和荷木(Schima superba),及黄果厚壳桂(Cryptocarw concinna)、藜蒴(Castanopsis fissa)和锥树(Castartopsis chinensis)叶片中甲醇提取物和细胞壁碱性酚类提取物中UV-B吸收物质的含量,以及叶片叶绿素含量和比叶面积,研究全日光强下植物对UV-B的防护策略。结果表明,在全日光强下植物叶片甲醇提取物的UV-B吸收能力较遮阴下的高,如藜蒴和锥树分别高出42．6％和32．6％,而马尾松仅高出4．2％。全日光强下的黄果厚壳桂和荷木叶片细胞壁碱性酚类提取物的UV-B吸收能力亦分别比遮阴下的高3596和11．7％,而马尾松、锥树和藜蒴则较遮阴下的低,可能这些树种在全日光下细胞壁UV-B吸收物质有部分转移到细胞质,以增强栅栏组织细胞的保护。全目光强下这几种植物叶片的叶绿素含量较遮阴下的低,但有较高的比叶面积,这可能有利于减少对光的吸收和对深层组织细胞器的保护。可见不同植物是采取不同的策略来适应增高的UV-B辐射。     

Soluble and wall-bound phenolics and phenolic polymers in Musa acuminata roots exposed to elicitors from Fusarium oxysporum f.sp. cubense

The accumulation of soluble and wall-bound phenolics and phenolic polymers in Musa acuminata roots exposed to cell wall-derived elicitor from the pathogen, Fusarium oxysporum, f.sp. cubense, race four, was investigated. The root tissue from the banana cultivar "Goldfinger" was found to respond strongly and rapidly towards the elicitor through the increased synthesis of phenolic compounds. Following elicitation, the conjugated and non-conjugated phenolic metabolites in the induced root tissue were extracted and quantified. Induced phenolic synthesis was rapid and reached near maximum values after 16 h. High-performance liquid chromatography revealed both compositional and quantitative differences between induced phenolics (p-coumaric, ferulic, and sinapic acids) and those constitutively present (p-coumaric- and ferulic acid). In addition, vanillic acid was found in the ester-bound fraction and protocatechuic acid in the cell-wall bound fraction of elicited tissue. The deposition and accumulation kinetics of polymerized phenolic monomers as lignin and lignin-like polymers was quantified over a time period of 0-36 h and found to reach maximum values after 24 h. Ionization difference UV spectra of lignin indicated compositional differences in the newly synthesized lignin fraction and correlated with increased concentrations of ferulic acid and sinapic acids esters. The results show that the increased flux through the phenylpropanoid pathway resulted in the synthesis of cinnamic acid and benzoic acid derivatives that were esterified and incorporated into the cell wall fraction as part of the anti-microbial defenses activated in the root tissue.     

Effects of ultraviolet-B radiation on leaf elongation, production and phenylpropanoid concentrations of Deschampsia antarctica and Colobanthus quitensis in Antarctica

Stratospheric ozone depletion by anthropogenic chlorofluorocarbons has lead to increases in ultraviolet‐B radiation (UV‐B; 280–320 nm) along the Antarctic Peninsula during the austral spring. We manipulated UV‐B levels around plants of Antarctic hair grass (Deschampsia antarctica; Poaceae) and Antarctic pearlwort (Colobanthus quitensis; Caryophyllaceae) for one field season near Palmer Station along the west coast of the Antarctic Peninsula. Treatments involved placing frames over naturally growing plants that either (1) held filters that absorbed most biologically effective radiation (UV‐B_BE; ‘reduced UV‐B’, 22% of ambient UV‐B_BE levels), (2) held filters that transmitted most UV‐B_BE (‘near‐ambient UV‐B’, 87% of ambient UV‐B_BE levels), or (3) lacked filters (‘ambient UV‐B’). Leaves on D. antarctica exposed to near‐ambient and ambient UV‐B were 16–17% shorter than those exposed to reduced UV‐B, and this was associated with shorter epidermal cells at the leaf base and tip. Leaves on C. quitensis exposed to near‐ambient and ambient UV‐B tended to be shorter (P=0.18) and epidermal cells at the leaf base tended to be smaller than those under reduced UV‐B (P<0.10). In order to further explain reductions in leaf length, we examined leaf concentrations of insoluble (cell‐wall bound) phenylpropanoids, since it has been proposed that wall‐bound phenylpropanoids such as ferulic acid may constrain cell expansion and leaf elongation. In both species, HPLC analysis revealed that ferulic and p‐coumaric acid were major components of both insoluble and soluble phenylpropanoids. Although there were no significant differences in concentrations between UV‐B treatments, concentrations of insoluble ferulic acid in D. antarctica tended to be higher under ambient and near‐ambient UV‐B than under reduced UV‐B (P=0.17). We also examined bulk‐leaf concentrations of soluble (methanol extractable) UV‐B‐absorbing compounds and found that concentrations were higher in plants exposed to near‐ambient and ambient UV‐B than in plants exposed to reduced UV‐B. We also assessed the UV‐B‐screening effectiveness of leaves that had developed on plants at the field site with a fiber‐optic microprobe. Leaf epidermal transmittance of 300‐nm UV‐B was 4.0 and 0.6% for D. antarctica and C. quitensis, respectively, which is low compared to grasses and herbaceous dicotyledonous plants found in more temperate climates. While the leaves of Antarctic vascular plants are relatively effective at screening UV‐B, levels of UV‐B in Antarctica are sufficient to reduce leaf epidermal cell size and leaf elongation in these species, although the mechanisms for these reductions remain unclear.     

Creation of a Metabolic Sink for Tryptophan Alters the Phenylpropanoid Pathway and the Susceptibility of Potato to Phytophthora infestans

The creation of artificial metabolic sinks in plants by genetic engineering of key branch points may have serious consequences for the metabolic pathways being modified. The introduction into potato of a gene encoding tryptophan decarboxylase (TDC) isolated from Catharanthus roseus drastically altered the balance of key substrate and product pools involved in the shikimate and phenylpropanoid pathways. Transgenic potato tubers expressing the TDC gene accumulated tryptamine, the immediate decarboxylation product of the TDC reaction. The redirection of tryptophan into tryptamine also resulted in a dramatic decrease in the levels of tryptophan, phenylalanine, and phenylalanine-derived phenolic compounds in transgenic tubers compared with nontransformed controls. In particular, wound-induced accumulation of chlorogenic acid, the major soluble phenolic ester in potato tubers, was found to be two- to threefold lower in transgenic tubers. Thus, the synthesis of polyphenolic compounds, such as lignin, was reduced due to the limited availability of phenolic monomers. Treatment of tuber discs with arachidonic acid, an elicitor of the defense response, led to a dramatic accumulation of soluble and cell wall-bound phenolics in tubers of untransformed potato plants but not in transgenic tubers. The transgenic tubers were also more susceptible to infection after inoculation with zoospores of Phytophthora infestans, which could be attributed to the modified cell wall of these plants. This study provides strong evidence that the synthesis and accumulation of phenolic compounds, including lignin, could be regulated by altering substrate availability through the introduction of a single gene outside the pathway involved in substrate supply. This study also indicates that phenolics, such as chlorogenic acid, play a critical role in defense responses of plants to fungal attack.     

Phenolic acids and peroxidase activity in alfalfa (Medicago sativa) embryogenic cultures after ethephon treatment

Treatment with ethephon increased the concentration of exogenous ethylene in Medicago sativa L. embryogenic cell suspension cultures (consisting of single cells, small cellular clumps and globular somatic embryos) and induced changes in the metabolism of phenolic substances, activities of peroxidase (EC 1.11.1.7) and caused significant suppression of suspension culture growth. Treatment with the ethylene-releasing substance, ethephon, resulted in a several-fold increase in phenylalanine ammonia-lyase (PAL; EC 4.3.1.5) activity above the basal level and was accompanied by an elevated accumulation of phenolic acids (significant increase of methoxy-substituted acids). The majority of newly synthesised phenolic acids was incorporated into the fractions of glycosides and esters bound to the cell wall. Phenolic glycosides seemed to serve as a metabolic pool from which the phenolics were utilised during further culture. The increased activity of wall-bound ionic peroxidase after ethephon application correlated with the pronounced incorporation of ferulic acid in the cell walls. In contrast, the increased level of exogenous ethylene did not influence the growth of culture of more advanced embryos nor did it significantly alter phenylpropanoid metabolism.     

Dynamic superheated liquid extraction of anthocyanins and other phenolics from red grape skins of winemaking residues

Grape skins from a grape pomace were subject to extraction with superheated ethanol-water mixtures for quantitative extraction of anthocyans and other phenolic compounds. The variables affecting dynamic extraction of these compounds were studied and identification and quantification of the extracted compounds were performed by both direct spectrophotometry or after HPLC separation using UV or MS detectors. The optimal working conditions for total extraction of anthocyans were: 1:1 (v/v) ethanol-water acidified with 0.8% (v/v) HCl, 120 degrees C, 30 min, 1.2 ml/min and 80 bar. The yields of anthocyanins, total phenolics and flavanols thus obtained were much higher (3 times for anthocyanins, 7 times for total phenolics and 11 times for flavanols) than those provided by dynamic conventional solid-liquid extraction. Several sample preparation procedures for skins as alternatives to free-drying were also investigated and drying at 40 degrees C for 24h provided the best results. Extraction with acidified water provides similar composition and poorer efficiency than 1:1 ethanol-water; also similar to two commercial grape skin extracts used as natural colorants.     

Changes in Phenolic Compounds and Cellular Ultrastructure of Arctic and Antarctic Strains of Zygnema (Zygnematophyceae, Streptophyta) after Exposure to Experimentally Enhanced UV to PAR Ratio

Ultraviolet (UV) radiation has become an important stress factor in polar regions due to anthropogenically induced ozone depletion. Although extensive research has been conducted on adaptations of polar organisms to this stress factor, few studies have focused on semi-terrestrial algae so far, in spite of their apparent vulnerability. This study investigates the effect of UV on two semi-terrestrial arctic strains (B, G) and one Antarctic strain (E) of the green alga Zygnema, isolated from Arctic and Antarctic habitats. Isolates of Zygnema were exposed to experimentally enhanced UV A and B (predominant UV A) to photosynthetic active radiation (PAR) ratio. The pigment content, photosynthetic performance and ultrastructure were studied by means of high-performance liquid chromatography (HPLC), chlorophyll a fluorescence and transmission electron microscopy (TEM). In addition, phylogenetic relationships of the investigated strains were characterised using rbcL sequences, which determined that the Antarctic isolate (E) and one of the Arctic isolates (B) were closely related, while G is a distinct lineage. The production of protective phenolic compounds was confirmed in all of the tested strains by HPLC analysis for both controls and UV-exposed samples. Moreover, in strain E, the content of phenolics increased significantly (p?=?0.001) after UV treatment. Simultaneously, the maximum quantum yield of photosystem II photochemistry significantly decreased in UV-exposed strains E and G (p?<?0.001), showing a clear stress response. The phenolics were most probably stored at the cell periphery in vacuoles and cytoplasmic bodies that appear as electron-dense particles when observed by TEM after high-pressure freeze fixation. While two strains reacted moderately on UV exposure in their ultrastructure, in strain G, damage was found in chloroplasts and mitochondria. Plastidal pigments and xanthophyll cycle pigments were investigated by HPLC analysis; UV A- and UV B-exposed samples had a higher deepoxidation state as controls, particularly evident in strain B. The results indicate that phenolics are involved in UV protection of Zygnema and also revealed different responses to UV stress across the three strains, suggesting that other protection mechanisms may be involved in these organisms.