Taxonomic affinities of dark septate root endophytes of Colobanthus quitensis and Deschampsia antarctica,the two native Antarctic vascular plant species

Although dark septate fungal endophytes (DSE) occur widely in association with plant roots in cold-stressed habitats, little is known of the taxonomic status of DSE in Antarctica. Here we investigate the phylogenetic affinities of DSE colonising the roots of Colobanthus quitensis and Deschampsia antarctica, the two maritime Antarctic vascular plant species. Two hundred and forty-three DSE were isolated from roots collected from 17 sites across a 1 470 km transect through maritime and sub-Antarctica. The ITS1-5.8S-ITS2 nuclear ribosomal gene cluster of representative isolates was sequenced, and the sequences were recovered in 10 sequence groups and sub-groups. Nine of the sequence groupings could be placed in the Helotiales and the remaining one showed high homology to a large number of currently unassigned anamorphic ascomycete sequences. Of the Helotiales, Leptodontidium orchidicola, Rhizoscyphus ericae and species of Tapesia and Mollisia could be confidently identified. This study demonstrates that members of the Helotiales, including several widely-recognised DSE genera, commonly colonise the roots of C. quitensis and D. antarctica in the Antarctic.     

Leishmanicidal and antitumoral activities of endophytic fungi associated with the Antarctic angiosperms Deschampsia antarctica Desv. and Colobanthus quitensis (Kunth) Bartl.

A total of 564 isolates of endophytic fungi were recovered from the plants Deschampsia antarctica and Colobanthus quitensis collected from Antarctica. The isolates were screened against parasites Leishmania amazonensis and Trypanosoma cruzi and against the human tumour cell lines. Of the 313 fungal isolates obtained from D. antarctica and 251 from C. quitensis, 25 displayed biological activity. Nineteen extracts displayed leishmanicidal activity, and six inhibited the growth of at least one tumour cell line. These fungi belong to 19 taxa of the genera Alternaria, Antarctomyces, Cadophora, Davidiella, Helgardia, Herpotrichia, Microdochium, Oculimacula, Phaeosphaeria and one unidentified fungus. Extracts of 12 fungal isolates inhibited the proliferation of L. amazonesis at a low IC₅₀ of between 0.2 and 12.5 μg ml⁻¹. The fungus Phaeosphaeria herpotrichoides displayed only leishmanicidal activity with an IC₅₀ of 0.2 μg ml⁻¹, which is equivalent to the inhibitory value of amphotericin B. The extract of Microdochium phragmitis displayed specific cytotoxic activity against the UACC-62 cell line with an IC₅₀ value of 12.5 μg ml⁻¹. Our results indicate that the unique angiosperms living in Antarctica shelter an interesting bioactive fungal community that is able to produce antiprotozoal and antitumoral molecules. These molecules may be used to develop new leishmanicidal and anticancer drugs.     

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.     

Photosynthetic and respiratory acclimation and growth response of Antarctic vascular plants to contrasting temperature regimes

Air temperatures have risen over the past 50 yr along the Antarctic Peninsula, and it is unclear what impact this is having on Antarctic plants. We examined the growth response of the Antarctic vascular plants Colobanthus quitensis (Caryophyllaceae) and Deschampsia antarctica (Poaceae) to temperature and also assessed their ability for thermal acclimation, in terms of whole-canopy net photosynthesis (P(n)) and dark respiration (R(d)), by growing plants for 90 d under three contrasting temperature regimes: 7°C day/7°C night, 12°C day/7°C night, and 20°C day/7°C night (18 h/6 h). These daytime temperatures represent suboptimal (7°C), near-optimal (12°C), and supraoptimal (20°C) temperatures for P(n) based on field measurements at the collection site near Palmer Station along the west coast of the Antarctic Peninsula. Plants of both species grown at a daytime temperature of 20°C had greater RGR (relative growth rate) and produced 2.2-3.3 times as much total biomass as plants grown at daytime temperatures of 12° or 7°C. Plants grown at 20°C also produced 2.0-4.1 times as many leaves, 3.4-5.5 times as much total leaf area, and had 1.5-1.6 times the LAR (leaf area ratio; leaf area:total biomass) and 1.1-1.4 times the LMR (leaf mass ratio; leaf mass:total biomass) of plants grown at 12° or 7°C. Greater RGR and biomass production at 20°C appeared primarily due to greater biomass allocation to leaf production in these plants. Rates of P(n) (leaf-area basis), when measured at their respective daytime growth temperatures, were highest in plants grown at 12°C, and rates of plants grown at 20°C were only 58 (C. quitensis) or 64% (D. antarctica) of the rates in plants grown at 12°C. Thus, lower P(n) per leaf area in plants grown at 20°C was more than offset by much greater leaf-area production. Rates of whole-canopy P(n) (per plant), when measured at their respective daytime growth temperatures, were highest in plants grown at 20°C, and appeared well correlated with differences in RGR and total biomass among treatments. Colobanthus quitensis exhibited only a slight ability for relative acclimation of P(n) (leaf-area basis) as the optimal temperature for P(n) increased from 8.4° to 10.3° to 11.5°C as daytime growth temperatures increased from 7° to 12° to 20°C. There was no evidence for relative acclimation of P(n) in D. antarctica, as plants grown at all three temperature regimes had a similar optimal temperature (10°C) for P(n). There was no evidence for absolute acclimation of P(n) in either species, as rates of P(n) in plants grown at a daytime temperature of 12°C were higher than those of plants grown at daytime temperatures of 7° or 20°C, when measured at their respective growth temperatures. The poor ability for photosynthetic acclimation in these species may be associated with the relatively stable maritime temperature regime during the growing season along the Peninsula. In contrast to P(n), both species exhibited full acclimation of R(d), and rates of R(d) on a leaf-area basis were similar among treatments when measured at their respective daytime growth temperature. Our results suggest that in the absence of interspecific competition, continued warming along the Peninsula will lead to improved vegetative growth of these species due to (1) greater biomass allocation to leaf-area production (as opposed to improved rates of P(n) per leaf area) and (2) their ability to acclimate R(d), such that respiratory losses per leaf area do not increase under higher temperature regimes.     

Leaf micromorphology of Antarctic pearlwort Colobanthus quitensis (Kunth) Bartl.

The leaf micromorphology of Antarctic pearlwort, Colobanthus quitensis (Kunth) Bartl. was analyzed. Plants were collected at King George Island (62°5′S, 58°23′W). Leaves were analyzed by optical and scanning electron microscopy, and quantitative analyses performed on leaf tissues and their internal geometry. C. quitensis leaves are ca. 588 μm thick, and composed of palisade and spongy parenchyma, respectively ca. 171 and 312 μm thick. Cuticles are thin and cover short epidermal cells. The central vein is surrounded by two bundles of achlorophyllous cells. Sclerenchymatic tissues are poorly developed. SEM analysis reveals faint striations over leaf surfaces. Stomata are present on both surfaces, but restricted to the leaf margins on the abaxial side. The ratio of mesophyll cell surface area per unit leaf area (Ames/A) is 34.2. The number of cells per cross-sectional area occupied was 24% higher for the palisade than for the spongy tissue, which determines a higher cell surface area per cross-sectional area for the former tissue. The authors correlate these results to plant ecological distribution in Antarctica and to water and carbon economy. Accepted: 14 January 2000     

Effects of salinity and temperature on Deschampsia antarctica

Deschampsia antarctica is one of two species of vascular plants native to Antarctica. Populations of D. antarctica have become established on recently exposed glacial forelands on the Antarctic Peninsula and these plants may rely upon nutrient inputs from hauled out mammals, seabirds and sea spray. However, not much is known about the ability of these plants to tolerate salinity stress. We examined the effects of salinity and temperature on growth, reproduction, chlorophyll fluorescence and water relations of D. antarctica. In addition, we analysed concentrations of free proline in leaves and roots as previous studies have found large increases in the concentration of this amino acid in response to environmental stress. The growth chamber experiment was a 3 × 3 (temperature × salinity) complete factorial. Plants were grown at three temperature regimes: 7°/7°C, 12°/7°C, and 20°/7°C day/night and three salinity levels: <0.02 decismen per metre (dS m⁻¹; “low salinity”), 2.5 dS m⁻¹ (“medium salinity”), and 5.0 dS m⁻¹ (“high salinity”) for 66 days. Warmer temperatures improved leaf and tiller production as well as leaf and root length, which is consistent with previous findings on this species. Salinity reduced final root length by 6 and 13% in the medium and high-salinity treatments, respectively. Plants growing in medium and high-salinity treatments had xylem pressures that were more negative and higher free-proline concentrations, suggesting that proline may act as an osmoregulant in D. antarctica.     

Low-temperature tolerance of the Antarctic species Deschampsia antarctica: A complex metabolic response associated with nutrient remobilization

The species Deschampsia antarctica (DA) is one of the only two native vascular species that live in Antarctica. We performed ecophysiological, biochemical, and metabolomic studies to investigate the responses of DA to low temperature. In parallel, we assessed the responses in a non-Antarctic reference species (Triticum aestivum [TA]) from the same family (Poaceae). At low temperature (4°C), both species showed lower photosynthetic rates (reductions were 70% and 80% for DA and TA, respectively) and symptoms of oxidative stress but opposite responses of antioxidant enzymes (peroxidases and catalase). We employed fused least absolute shrinkage and selection operator statistical modelling to associate the species-dependent physiological and antioxidant responses to primary metabolism. Model results for DA indicated associations with osmoprotection, cell wall remodelling, membrane stabilization, and antioxidant secondary metabolism (synthesis of flavonols and phenylpropanoids), coordinated with nutrient mobilization from source to sink tissues (confirmed by elemental analysis), which were not observed in TA. The metabolic behaviour of DA, with significant changes in particular metabolites, was compared with a newly compiled multispecies dataset showing a general accumulation of metabolites in response to low temperatures. Altogether, the responses displayed by DA suggest a compromise between catabolism and maintenance of leaf functionality.     

Leaf functional and micro-morphological photoprotective attributes in two ecotypes of Colobanthus quitensis from the Andes and Maritime Antarctic

Colobanthus quitensis (Kunth) Bartl. (Cariophyllaceae) is distributed from Mexico to the Maritime Antarctic. It grows forming inconspicuous populations in humid and cold sites along high elevations in the Andes Mountains. Mediterranean Andes is characterized by a wider oscillation of diurnal and seasonal temperature, while the Maritime Antarctic is characterized by permanent low temperatures. Both places may experience high irradiance during sunny days (reaching up to 2,000 μmol photons m⁻² s⁻¹); however, the frequency of sunny days in the Maritime Antarctica is significantly lower (less than 20% of the whole growing season). We study whether acclimation to each environment relies on different photoprotective mechanisms. The Andean ecotype that has a longer growing season and a higher light integral reduces light absorption by the development of smaller chloroplasts with lower stacking granum area and down-regulation of Lhcb2. It also enhances the dissipation of the excess of absorbed energy by higher level of de-epoxidation of xanthophylls pool. On the other hand, the Antarctic ecotype which has developed under a shorter growing season, with lower total irradiance and continuous low temperatures, maximizes photochemical process even at low temperatures and it has a lower light-harvesting/core complex ratio and higher level of photoprotection supplied by an unusually high β-carotene and xanthophylls cycle pool. It resembles a well full light acclimated plant, probably due to higher excitation pressure imposed by lower temperature even at moderate irradiance. It is suggested that the biochemical plasticity of this species, highlighted by the development of these different strategies, is essential to cope successfully with these particular environments.     

Antarctic moss carpets facilitate growth of Deschampsia antarctica but not its survival

The vegetation of the Antarctic tundra is dominated by mosses and lichens. Deschampsia antarctica, the Antarctic hairgrass, is one of two vascular plant species which grow along the west coast of the Antarctic Peninsula. However, little is known about its recruitment and interaction with non-vascular tundra plants. Although several authors propose that tolerance and/or competition should be the main forms of interaction between moss carpets and D. antarctica, no relevant studies exist so far. We investigated whether positive interactions are predominant at the Shetland Islands and the west coast of the Antarctic Peninsula and focussed on the role that moss carpets play in the recruitment of D. antarctica. Across the studied zone, D. antarctica showed a significant association with moss carpets, with higher frequencies as well as more and larger individuals than on bare ground. At one site, we conducted moss removal and seedlings transplant experiments to assess the relevance of the moss carpets for different life stages of hairgrass. All experimental individuals survived until the following summer whether the moss carpet was removed or not, but growth rate was significantly lower in tussocks with moss carpets removed. Likewise, tiller size was higher in plants growing in moss carpets than on bare ground. The detected positive interactions with mosses seem to be important for the expansion of D. antarctica, raising the question about their importance under future climate change scenarios.     

Regional genetic diversity patterns in Antarctic hairgrass (Deschampsia antarctica Desv.)

Aim  To determine patterns in diversity of a major Antarctic plant species, including relationships of Antarctic populations with those outside the Antarctic zone.
Location  Antarctic Peninsula, Maritime Antarctica, sub-Antarctic islands, Falkland Islands and South America.
Methods  Amplified fragment length polymorphisms (AFLPs) and chloroplast sequences were used to study patterns of genetic diversity in Antarctic hairgrass ( Deschampsia antarctica Desv.) and the genetic relationships between populations over its distribution range. Thirty-eight populations were sampled from a large part of the distribution of D. antarctica , and additionally, herbarium specimens were included for areas from which we could not obtain fresh samples.
Results  A gradient in AFLP diversity was observed going from the Falklands southwards into the Antarctic. This gradient in diversity was also observed within the Antarctic Peninsula: diversity was lower further south. Diversity in the chloroplast genome of D. antarctica was low. Only three chloroplast haplotypes were found, each with a strong regional distribution.
Main conclusions  The phylogenetic construction of AFLP marker frequencies in meta-populations of D. antarctica supports a stepping-stone model of colonization, whereby gene flow mainly occurs between neighbouring populations. It is concluded that long-distance gene flow is very limited in D. antarctica . A very low diversity was found in the sub-Antarctic islands in the Indian Ocean, indicating that these populations have experienced a recent evolutionary bottleneck.     

Expressed sequence tag analysis of Antarctic hairgrass Deschampsia antarctica from King George Island, Antarctica

Deschampsia antarctica is the only monocot that thrives in the tough conditions of the Antarctic region. It is an invaluable resource for the identification of genes associated with tolerance to various environmental pressures. In order to identify genes that are differentially regulated between greenhouse-grown and Antarctic field-grown plants, we initiated a detailed gene expression analysis. Antarctic plants were collected and greenhouse plants served as controls. Two different cDNA libraries were constructed with these plants. A total of 2,112 cDNA clones was sequenced and grouped into 1,199 unigene clusters consisting of 243 consensus and 956 singleton sequences. Using similarity searches against several public databases, we constructed a functional classification of the ESTs into categories such as genes related to responses to stimuli, as well as photosynthesis and metabolism. Real-time PCR analysis of various stress responsive genes revealed different patterns of regulation in the different environments, suggesting that these genes are involved in responses to specific environmental factors.     

Nitrogen form influences the response of Deschampsia antarctica to dark septate root endophytes

Fungi with dematiaceous septate hyphae, termed dark septate endophytes (DSE), are common in plant roots, particularly in cold-stressed habitats, but their effects on their host plants remain obscure. Here, we report a study that assessed the effects of six DSE on the growth and nutrient balance of Deschampsia antarctica when plants were supplied with the same amount of nitrogen in organic (casein hydrolysate) or inorganic (ammonium sulphate) form under controlled conditions. After 60 days, the DSE, that had each been isolated from D. antarctica and which analyses of internal transcribed spacer and large subunit regions indicated were similar to members of the Helotiales (Oculimacula yallundae, Mollisia and Tapesia spp.) and unassigned anamorphic ascomycetes, typically had no effect on, or reduced by 33–71%, shoot and root dry weights relative to uninoculated controls when plants had been supplied with nitrogen in inorganic form. In contrast, the DSE usually enhanced shoot and root dry weights by 51–247% when plants had been supplied with organic nitrogen. In the presence of inorganic nitrogen, only sporadic effects of DSE were recorded on shoot and root nitrogen or phosphorus concentrations, whereas in the presence of organic nitrogen, three to six of the DSE isolates increased shoot and root nitrogen and phosphorus contents. Most of the isolates decreased the phosphorus concentrations of shoots and roots when plants had been supplied with nitrogen in organic form. Our data suggest that DSE are able to mineralise peptides and amino acids in the rhizosphere, making nitrogen more freely available to roots.     

Optimized protoplast isolation and establishment of transient gene expression system for the Antarctic flowering plant Colobanthus quitensis (Kunth) Bartl.

Plant Cell, Tissue and Organ Culture (PCTOC) - Colobanthus quitensis is one of two terrestrial plants that grow in the maritime Antarctic. Despite its important ecological niche in extreme...     

Deschampsia antarctica Desv. primary photochemistry performs differently in plants grown in the field and laboratory

Primary photochemistry of photosystem II (F _v/F _m) of the Antarctic hair grass Deschampsia antarctica growing in the field (Robert Island, Maritime Antarctic) and in the laboratory was studied. Laboratory plants were grown at a photosynthetic photon flux density (PPFD) of 180 μmol m⁻² s⁻¹ and an optimal temperature (13 ± 1.5°C) for net photosynthesis. Subsequently, two groups of plants were exposed to low temperature (4 ± 1.5°C day/night) under two levels of PPFD (180 and 800 μmol m⁻² s⁻¹) and a control group was kept at 13 ± 1.5°C and PPFD of 800 μmol m⁻² s⁻¹. Chlorophyll fluorescence was measured during several days in field plants and weekly in the laboratory plants. Statistically significant differences were found in F _v/F _m (=0.75–0.83), F ₀ and F _m values of field plants over the measurement period between days with contrasting irradiances and temperature levels, suggesting that plants in the field show high photosynthetic efficiency. Laboratory plants under controlled conditions and exposed to low temperature under two light conditions showed significantly lower F _v/F _m and F _m. Moreover, they presented significantly less chlorophyll and carotenoid content than field plants. The differences in the performance of the photosynthetic apparatus between field- and laboratory-grown plants indicate that measurements performed in ex situ plants should be interpreted with caution.     

Ecophysiology of Antarctic vascular plants

Most of the ice and snow-free land in the Antarctic summer is found along the Antarctic Peninsula and adjacent islands and coastal areas of the continent. This is the area where most of the Antarctic vegetation is found. Mean air temperature tends to be above zero during the summer in parts of the Maritime Antarctic. The most commonly found photosynthetic organisms in the Maritime Antarctic and continental edge are lichens (around 380 species) and bryophytes (130 species). Only two vascular plants, Deschampsia antarctica Desv. and Colobanthus quitensis (Kunth) Bartl., have been able to colonize some of the coastal areas. This low species diversity, compared with the Arctic, may be due to permanent low temperature and isolation from continental sources of propagules. The existence of these plants in such a permanent harsh environment makes them of particular interest for the study of adaptations to cold environments and mechanisms of cold resistance in plants. Among these adaptations are high freezing resistance, high resistance to light stress and high photosynthetic capacity at low temperature. In this paper, the ecophysiology of the two vascular plants is reviewed, including habitat characteristics, photosynthetic properties, cold resistance, and biochemical adaptations to cold.