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Adaptation and Acclimation of Photosynthetic Microorganisms to Permanently Cold Environments

Department of Microbiology, University of Illinois, Urbana, Illinois 61801,¹ Department of Land Resources and Environmental Sciences, Montana State University, Bozeman, Montana 59717,² Department of Natural Science, Mid-Sweden University, Sundsvall, Sweden,³ ECORC, Agriculture and Agri-Food Canada, Ottawa, Ontario K1A 0C6, Canada,⁴ Department of Biology and The Biotron, University of Western Ontario, London, Ontario N6A 5B7, Canada⁵

SUMMARY

INTRODUCTION

Adaptation to Permanently Cold Environments

Goals and Scope

ADAPTATION AND ACCLIMATION TO LOW TEMPERATURE

Membrane Lipid Composition

Cold-Adapted Enzymes

Photosynthetic Electron Transport and Energy Balance

PERMANENTLY COLD ECOSYSTEMS

High-Alpine Snowfields

Polar Sea Ice

Transitory Ponds

Cryoconites

Ice-Covered Lake Systems

MCMURDO DRY VALLEY LAKE PHOTOTROPHIC COMMUNITIES

Biogeochemistry

Temperature, Light Climate, and Phytoplankton Abundance

Phytoplankton Nutrient Status

Photosynthetic Characteristics

Phototaxis

STUDY OF AN ANTARCTIC LAKE PHYTOPLANKTON

Identification and Phylogeny of the Lake Bonney Psychrophile

Cell Morphology

Growth and Photosynthetic Characteristics

Membrane Lipid and Fatty Acid Composition and Pigmentation

Biochemistry and Physiology of the Photosynthetic Apparatus

Short-Term Photoacclimation

Antenna quenching.

State transitions.

Photoinhibition and recovery.

Phototactic response.

Thermosensitivity of the photosynthetic apparatus.

Long-Term Photoacclimation

Composition and structure of light-harvesting complexes.

Photosystem stoichiometry.

Model of the Photochemical Apparatus of a Eukaryotic Psychrophile

CONCLUSIONS AND FUTURE DIRECTIONS

ACKNOWLEDGMENTS

REFERENCES

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INTRODUCTION

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In many cold ecosystems, the primary producers are the essential base of inorganic carbon fixation and autotrophic energy production, which allow food webs to develop by providing new organic carbon and nutrient sources (197, 212, 213, 270). Cold-adapted microorganisms include both psychrophilic (an organism with an optimal growth temperature at or below 15°C, and a maximum growth temperature below 20°C) (52, 170) and psychrotrophic (an organism exhibiting the ability to grow at temperatures below 15°C but exhibiting maximum growth rates at temperature optima above 18°C) organisms, and are often subjected to other extreme environmental parameters. Deep marine-dwelling species are exposed to extremely high pressures (baropsychrophiles) (280). Many microbial communities associated with the Antarctic and Arctic sea ice are subjected to salt concentrations several orders of magnitude higher than that of seawater (halopsychrophiles) (196, 266), and most of the terrestrial polar microorganisms are also exposed to osmotic stress and desiccation (53, 263). Microorganisms living in the Antarctic subglacial environment have been isolated from the atmosphere for more than 15 million years and exist at low-temperature, high-pressure, and low-nutrient levels (104, 221, 242). Phototrophic microorganisms growing on snow and ice surfaces in alpine or glacial environments are exposed to high light as well as UV radiation (72). In contrast, microalgal assemblages residing on the underside of the Arctic and Antarctic sea ice and planktonic algae growing beneath sea ice as well as under and in the permanent ice covers of the Antarctic dry valley lakes are photosynthetically active in a light environment less than 1% of incident photosynthetically active radiation (PAR) (119, 129). Importantly, all photoautotrophs residing in high-latitude polar environments must survive up to 5 months of total darkness between solar light cycles (196, 221).

To successfully colonize low-temperature environments, psychrophilic photoautotrophs have evolved a number of strategies that range from molecular to whole cell to ecosystem levels. The process of genetic change that accumulates over a time scale of many generations in response to an organism's specific environmental niche is termed adaptation. This is in contrast to acclimation, which refers to short-term physiological adjustments that occur during a lifetime in response to transitory changes in environmental conditions. Examples of adaptative mechanisms to low temperature include the evolution of cold shock and antifreeze proteins, the modulation of the kinetics of key enzymes, and the development of more fluid biological membranes through the accumulation of polyunsaturated fatty acyl chains. In contrast, acclimatory responses to transitory changes in the thermal environment are dependent upon sensor/signal pathways, and involve, for example, modulations in rates of transcription or translation of enzymes.

Photosynthetic microorganisms possess a myriad of mechanisms to acclimate to extremes in the light environment (from total darkness to extreme shade to photoinhibitory levels) to efficiently utilize photosynthetically available radiation while avoiding the detrimental effects of overexcitation of the photosynthetic apparatus. In addition, absorption of light energy (a temperature-independent process) must be tightly coordinated with the temperature-dependent processes of production of photochemically formed energy products (NADPH and ATP) and downstream catabolic consumption of these energy sources during organismal growth and metabolism. While comparative research on phototrophic organisms adapted to temperate versus low-temperature environments is in its infancy, laboratory-controlled research on pure-culture psychrophilic phototrophs (162, 163, 166) is beginning to reveal the unique adaptive mechanisms that these extremophiles employ to thrive in their environments, and how niche adaptation has impacted the capacity of low-temperature-adapted phototrophs to acclimate to environmental change (167-169).

ADAPTATION AND ACCLIMATION TO LOW TEMPERATURE

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Indeed, the role of unsaturation of lipids represents one of the most thoroughly investigated mediators of cold adaptation (30), and the production of polyunsaturated fatty acids has been used for chemotaxonomic classification of psychrophilic and psychrotolerant bacteria (81, 231) and microalgae (36), as well as a diagnostic indicator of thermal bleaching in corals (250). High polyunsaturated fatty acid concentrations have been detected in natural communities and isolated cultures of several low-temperature-adapted phototrophic microorganisms, including sea ice diatoms (85, 162, 163, 184), dinoflagellates (254), and green algae (167). In addition to temperature, the degree of unsaturation of membrane lipids in phototrophic organisms is also dependent upon factors such as high salinity (185), desiccation, light (253), and nutrient availability (227, 240).

In the paradigm for dissociative-type fatty acid biosynthesis, Escherichia coli, unsaturated fatty acids are synthesized de novo as part of the anaerobic fatty acid biosynthetic pathway: unsaturated fatty acids are produced via the isomerization of a trans-unsaturated fatty acid at the C₁₀ level of the fatty acid biosynthetic pathway. Since this pathway is active during growth, modification of membrane fluidity via the introduction of unsaturated fatty acids can only occur in actively growing cultures.

In other bacteria and eukaryotes, double bonds are introduced postsynthetically into a fatty acid via an aerobic desaturation pathway that acts on membrane lipids (174). This reaction is catalyzed by a family of enzymes called desaturases, which introduce a double bond via an energy-dependent reaction which is both fatty acyl chain and bond position specific. In a variable thermal environment, microorganisms possessing the aerobic fatty acid desaturation pathway have an advantage over those relying on the anaerobic type of unsaturated fatty acid production in that fatty acyl chain modification can occur independently of cell growth.

Acclimation to low-temperature stress via an increase in expression of desaturases has been documented in poikilothermic organisms such as bacteria, algae, plants, and animals (2, 47, 84, 135, 139, 189, 255). Cyanobacteria have been particularly exploited as models of lipid unsaturation because several of the fatty acid desaturase genes have been genetically manipulated (234, 235). In the cyanobacterium genus Synechocystis, the expression of three desaturases (encoded by desA, desB, and desD) is inducible under cold temperatures. The sensor/signal pathway for regulation of transcription of the single desaturase found in Bacillus subtillus, encoded by des, has also been thoroughly investigated (35, 142).

In contrast, while the importance of unsaturated fatty acids in low-temperature adaptation of polar microorganisms is recognized, identification of the gene products involved in unsaturated fatty acid production in polar species has been largely unexplored. One exception is the identification of several putative enzymes involved in lipid biosynthesis and the production of unsaturated diether lipids in the Antarctic archaeon Methanococcoides burtonii (isolated from Ace Lake, Antarctica) (183). Low-temperature growth of M. burtonii cultures produced a high proportion of unsaturated lipids; however, the putative pathway to produce unsaturated fatty acids in the methanogen is distinct from either the anaerobic pathway in E. coli or the desaturase-mediated pathway in other bacteria and eukaryotes.

The fluidity of the membrane is closely connected with optimal photosynthetic function at low temperatures, which relies on the correct folding of complex multisubunit membrane-associated proteins which form the photosynthetic electron transport chain. The majority of protein components of the photosynthetic apparatus are anchored in the photosynthetic membranes via specific lipid species, the galactolipids (monogalactosyldiaclylglycerol [MGDG] and digalactosyldiacylglycerol [DGDG]), which are exclusively associated with the chloroplast. Membrane fluidity is also essential for electron transport between the photosynthetic complexes via mobile carriers such as plastoquinone (230), as well as the diffusivity of gases (226). Resistance to photoinhibitory damage, particularly at low temperatures, and the photosystem II repair cycle are also dependent upon the ability to desaturate fatty acids (70, 102).

While there is clearly a strong corelationship between the fatty acyl content of the photosynthetic membranes and optimal photosynthetic function at low temperatures, the role of lipids in low-temperature photosynthesis in polar microorganisms is largely unknown. Mock and Kroon (162, 163) recently reported on the interrelationship between membrane lipid composition and photosynthetic function during acclimation to low temperatures and either nitrogen limitation (162) or low irradiance (163) in mixed cultures of three sea ice diatom species. The sea ice microalgae exhibited large chloroplasts due to adaptation to the low-light conditions of the sea ice environment, producing cells whose cellular membranes were dominated by those in the thylakoid. Thus, major lipid classes under either N-replete or N-deplete conditions were the chloroplast-related classes MGDG and DGDG. Under nitrogen deplete conditions, diatom cultures exhibited a reduction in intracellular proteins and a concomitant rise in total lipids, particularly in the storage lipid triacylglycerol. The storage lipids act as a sinks for photosynthetic electron transport, which under nitrogen-replete conditions would be utilized for the reduction of nitrate. Furthermore, N limitation also resulted in a dramatic reduction in the MGDG-to-DGDG ratio.

MGDG is a non-bilayer-forming lipid class which can form a bilayer in thylakoid proteins due to the high proportion of proteins and pigments in the photosynthetic membranes. Presumably, the increase in overall lipids and in particular DGDG compensates for the loss of membrane-bound proteins and pigments and maintain membrane stability (162). Higher levels of unsaturated fatty acids in the photosynthetic membranes may also aid in assembly of D1 into photosystem II (see Fig. 1), which has been shown to be dependent upon the degree of fatty acid unsaturation, particularly at low temperatures (174, 189). Despite these coordinated mechanism of adjusting lipid content and photosynthetic function to nitrogen stress, pools of the mobile electron acceptor plastoquinone were relatively reduced in N-limited cultures, indicative of an imbalance in absorbed light energy with utilization of stored photosynthetic energy in the form of reducing equivalents (162).

View larger version (38K): [in this window] [in a new window]   FIG. 1. Oxygenic photosynthetic electron transport chain in the thylakoids of green algae and higher plants. Three major membrane-bound protein complexes functioning in series, photosystem II (PSII), cytochrome b6f complex (Cyt b6f), and photosystem I (PSI), are required to transport electrons from water to NADP+. Light energy is absorbed by the light-harvesting complexes, and excitation energy is transferred to the reaction centers, where it is used to drive charge separation of a chlorophyll pair (P680 and P700 for PSII and PSI, respectively). Electrons are transported from PSII to cytochrome b6f across the thylakoid membrane by the mobile transporter plastoquinone (PQ/PQH2), and from cytochrome b6f to PSI in the lumenal space by the small protein plastocyanin (PC). Electrons flow from NADPH to downstream metabolic reactions such as carbon and nutrient assimilation. QA, quinone A; QB, quinone B; FNR, ferredoxin-NADP oxidoreductase; A0, A1 FX, FA, and FB, intermediate electron acceptors of Photosystem I.

Clearly, the maintenance of appropriate reaction rates of enzyme-catalyzed reactions of essential metabolic processes must be one of the major challenges that cold-adapted microorganisms have overcome. One strategy to combat lowered reaction rates could be to increase enzyme concentrations; however, this would be energetically inefficient and there are only a few examples of this type of cold adaptation strategy (34, 185). While the underlying molecular mechanisms governing low-temperature adaptation in psychrophilic microorganisms are not well understood, there is a general consensus that a major adaptive strategy to compensate for reduced reaction rates is at the level of the catalytic efficiency (k_cat/K_m) of cold-adapted enzymes (67). This is accomplished either by higher turnover numbers (k_cat) at the expense of K_m (substrate concentration at half-maximum activity) or by optimizing both parameters (increasing k_cat and decreasing K_m). Several psychrophilic enzymes exhibit a temperature shift for maximal activity to lower temperatures and a concomitant unfolding at moderate temperatures (57, 103). These properties have been the result of amino acid substitutions that promote increased flexibility of the protein.

Recently, Napolitano and Shain (178, 179) have proposed an additional compensatory strategy for maintaining sufficient rates of biochemical reactions at low temperatures in a diverse collection of psychrophilic organisms. These authors found that in mesophilic and thermophilic organisms, levels of ATP and growth rates varied proportionally with respect to growth temperature, that is, at higher growth temperatures, an increase in energy demand (i.e., higher growth rates) coincided with an increase in energy supply (i.e., adenylate pools). Conversely, several psychrophilic organisms representing three kingdoms, Eubacteria, Fungi, and Protista (or two of the domains of life, Eucarya and Bacteria [277]) exhibited an inverse relationship between adenylate levels and growth temperature (178), despite the fact that growth rates (i.e., energy demand) varied proportionally with growth temperature in all the psychrophilic organisms. Thus, it appears that elevated ATP and total adenylate pools may represent an additional adaptive strategy to compensate for lower rates of biochemical reactions at low temperatures. It is likely that altered activity of a key enzyme(s) involved in adenylate metabolism, such as F₁ ATPase or AMP phosphatase/deaminase, governs the differences in energy metabolism between the psychrophiles and either the mesophiles or thermophiles. However, biochemical and genetic evidence is currently unavailable to address this hypothesis (178).

While the research regarding enzymes from psychrophilic microorganisms has begun to increase in recent years, little attention has been paid to cold-adapted enzymes from phototrophic microorganisms. Loppes et al. (133) investigated temperature dependence and thermolability of nitrate reductase and argininosuccinate lyase from a psychrophilic Chloromonas sp. isolated from Petrel Island in Antarctica. Both psychrophilic enzymes exhibited a shift in maximal enzyme activity to lower temperatures. In particular, argininosuccinate lyase exhibited 25% of its maximum activity at 5°C, while the enzyme isolated from the mesophilic Chloromonas reinhardtii was completely inactive at this temperature. Lastly, both psychrophilic enzymes also exhibited a lower thermal stability than the mesophilic counterparts.

In contrast with argininosuccinate lyase, the temperature maximum for carboxylase activity of ribulose-1,5-bisphosphate carboxylase (Rubisco), one of the most critical enzymes for inorganic carbon fixation in phototrophs, was not altered in two isolates of the Antarctic Chloromonas sp., and the specific activity at low temperatures was actually lower in the psychrophilic compared with the mesophilic Rubisco (42). This was one of the few exceptions described so far of a psychrophilic enzyme not exhibiting higher catalytic activity at lower temperatures. In agreement with this report, Rubisco isolated from Antarctic hairgrass also exhibited lower activity at lower temperatures, and activity increased linearly up to an incubation temperature of 50°C (236). A similar trend was observed in winter-hardened-crops species (232). Conversely, the protein was thermally sensitive to inactivation in both Antarctic algae and plants (42, 236). While sequence analysis and modeling of the large subunit (rbsL) of Rubisco in the psychrophilic Chloromonas sp. indicated no changes in amino acid residues directly involved in catalysis, several substitutions may contribute to the stability of the enzymes and interactions with the small subunit (281). In contrast, heat inactivation of Rubisco in the Antarctic grass species was interpreted as an imbalance in the rates of Rubisco inactivation and reactivation by Rubisco activase (236).

A study comparing the temperature dependence of NO₃^– and NH₄⁺ incorporation, and the activity of the assimilatory enzyme NO₃^– reductase with photosynthesis (CO₂ incorporation) in natural assemblages of psychrophilic Antarctic sea ice microalgae (218) revealed that NO₃^– and NH₄⁺ incorporation reached maximal rates between 0.5 and 2.0°C (Q₁₀ = 10.1) and 2.0 and 3.0°C (Q₁₀ = 15.7), respectively, which was close to that for CO₂ incorporation (2.5 to 3.0°C; Q₁₀ = 16.1).

These metabolic characteristics show the psychrophilic tendencies of the microalgal assemblage and, by virtue of the high Q₁₀ values, further show that their metabolic activity can change rapidly given a small change in temperature. Conversely, NO₃^– reductase showed a distinctly higher temperature maximum (10.0 to 12.0°C) and a lower Q₁₀ value (1.4) than either inorganic N or C incorporation. These results led the authors to conclude that, owing to differential temperature characteristics between N transport and N assimilation at the in situ growth temperature (–1.9°C), the incorporation of extracellular NO₃^– into cellular macromolecules may be limited by transport of NO₃^– into the cell rather than the intracellular reduction of NO₃^–. Such cases of differential temperature responses by selected biosynthetic pathways may play a role in the overall C/N acquisition ratios of organisms living in cold habitats, a contention that has been corroborated by other field studies on sea ice microalgae (216, 218).

A very prevalent group of oxygenic phototrophs found in low-temperature environments are the chromophytes, of which diatom algae in particular dominate marine and sea ice habitats (Table 1). The diatoms possess a typical oxygenic photochemical apparatus; however, chlorophyll b is replaced by chlorophyll c, and fucoxanthin is a major carotenoid (74). Green algae play various roles in low-temperature environments, which are often more likely to be dominated by prokaryotic photosynthetic microorganisms. Notable exceptions are found in two divergent low-temperature environments, the alpine snow ecosystem, which is dominated by psychrophillic Chlamydomonas and Chloromonas spp., and the permanent ice-covered lakes of the McMurdo Dry Valleys, which are vertically stratified layers of green algae (Table 1). Both ecosystems are discussed in detail later in this review.

Cyanobacterial mats are particularly prevalent in shallow aquatic systems in both the Arctic and Antarctic. The cyanobacterial members of these systems are adapted and acclimated to a range of extremes in the thermal environment, freezing and thawing cycles, and photoprotection, as well as nutrient limitation (175). Numerous studies have shown that the microbial mats rely on adaptation of pigmentation to both maximize light-harvesting ability as well as protect against damaging light levels (16, 92, 171, 223, 262). Furthermore, Roos and Vincent (228) showed that the mat-forming cyanobacterium Phormidium murrayi isolated from the McMurdo Ice Shelf acclimated to laboratory-controlled changes in PAR, UV, and temperature by regulating ratios of light-harvesting and light-screening pigments.

Unlike the oxygenic photosynthetic process, the photochemical process of anoxygenic photosynthesis does not lead to the production of molecular oxygen. Two types of reaction centers are found in the anoxygenic phototrophic bacteria: type I, associated with green bacteria, and type II, associated with purple bacteria. Associated with the reaction center is a simple light-harvesting antenna, which can be partitioned into an inner antenna closely associated with the reaction center and a peripheral antenna. The pufM gene encodes the pigment-binding reaction center protein of all purple phototrophic bacteria, and pufM-specific primer sets have been utilized as a diagnostic tool to assess the abundance and diversity of purple sulfur and purple nonsulfur phototrophs in natural environments, including stratified lake ecosystems in the dry valleys of Antarctica (1, 106). The major light-harvesting pigments are bacteriochlorophyll and carotenoids. As in cyanobacteria, some of the photosynthetic electron transport chain components are shared with the respiratory chain.

Anoxygenic phototrophs form a diverse group that include green sulfur bacteria, green nonsulfur bacteria, heliobacteria, and purple bacteria and perform a primary role in carbon and sulfur cycling in aquatic ecosystems, particularly in anoxic zones in stratified lakes. Recently, Karr et al. (106) determined the distribution of the pufM gene to reveal the highly stratified nature of purple nonsulfur bacteria in the anoxic zone of Lake Fryxell, a perennially ice-covered lake located in the McMurdo Dry Valleys, Antarctica. While future pure-culture analyses of specific isolates are necessary to investigate adaptive and acclimative strategies specific to anaerobic photoautotrophs, the high diversity of the purple bacterial population implies successful exploitation of this extreme aquatic environment of low light intensities, near-freezing water column, and anoxic conditions by this group of anoxygenic photoautotrophs (106).

Balancing the energy flow through the process of photosynthesis is a challenge due to differential temperature sensitivities and differential rates between the photochemical reactions and the biochemical reactions. The balance of energy flow between the photophysical and photochemical processes that transform light and the metabolic sinks that consume the energy is called photostasis (93, 192). The following equation, derived by Falkowski and Chen (55), defines photostasis, _PSII x E_k = ^–1, where _PSII is the effective absorption cross section of PSII, E_k is the irradiance (I) at which the maximum photosynthetic quantum yield balances photosynthetic capacity estimated from a photosynthetic light response curve, and ^–1 is the rate at which photosynthetic electrons are consumed by a terminal electron acceptor such as CO₂ under light-saturated conditions.

An imbalance between energy absorbed versus energy utilized will occur whenever the rate at which the energy absorbed through PSII and the rate at which electrons are injected into photosynthetic electron transport exceed the metabolic electron sink capacity, that is, whenever _PSII x E_k > ^–1. Thus, photosynthetic microorganisms growing in low-temperature environments are potentially under a constant state of energy imbalance due to the decrease in ^–1. Excitation pressure (94, 147) or excessive excitation energy (105) is a relative measure of the reduction state of quinone A and reflects the redox state of the intersystem PQ pool (94). This can measured in vivo or in vitro by pulse amplitude-modulated fluorescence as either 1 – qP (in which qP is photochemical quenching) or as suggested more recently as 1 – qL ( in which qL is the fraction of open PSII centers) (113). Thus, excitation pressure is a measure of the imbalance in energy flow, that is, a measure of the extent to which I > E_k, and thus, _PSII x I > ^–1.

The inequality illustrated above also provides insights into the possible mechanisms by which phototrophic organisms may respond to the imbalance in energy budget to attain photostasis. Figure 2 illustrates the possible fates of absorbed light energy through the photosynthetic apparatus. Energy balance is attained by either reducing _PSII by reducing light-harvesting antenna size and/or reducing the effective absorption cross-sectional area of PSII by dissipating energy nonphotochemically as heat (55, 89, 114). Roos and Vincent (228) reported that cultures of the Antarctic mat-forming cyanobacterium P. murrayi exhibited a similar acclimatory response to either low temperature or high PAR in a way similar to that of the mesophilic green alga Chlorella vulgaris (147), that is, to reduce the functional size of PSII by significantly increasing the carotenoid/chlorophyll a ratio under either low temperatures or high PAR or UV levels. This provides evidence that, like mesophilic organisms, low-temperature-adapted photoautotrophs sense and respond to excitation pressure (228).

View larger version (19K): [in this window] [in a new window]   FIG. 2. Schematic showing the function of the photosynthetic electron transport chain as a redox sensor. The process of photosynthesis integrates the fast temperature-independent photochemical reactions of light absorption and charge separation with the "slow" processes of electron transport and downstream utilization of electron sinks through growth and metabolism. Any environmental stress that perturbs the poise between energy absorbed and energy utilized is sensed by the photoautotrophic organism at the level of the redox state of intersystem electron transport pool (excitation pressure, redox) and/or the build-up of protons across the thylakoid membranes (proton motive force). Low temperatures (Low T) cause an imbalance between energy absorbed and energy utilized by reducing rates of energy consumption by downstream metabolic processes. The energy imbalance is corrected at the level of either light absorption via modulation in the absorptive cross-section of PSII (e.g., dissipation of excess energy as heat, NPQ) or at the level of energy utilization (e.g., modulations at the level of Calvin cycle enzymes). Ox, oxidized; red, reduced.

Lomas and Gilbert (130) have proposed an alternate energy sink in diatoms under conditions when light absorption exceeds metabolic energy requirements. Under a growth environment of low-temperature-induced high excitation pressure, natural diatom-dominated populations exhibited relatively high rates of NO₃^– uptake. These authors suggested that the N uptake in excess of nutritional requirements was an adaptive mechanism to modulate the balance between photosynthetic energy production and to compensate for a reduced metabolic energy requirement at low temperatures. More recently, Parker and Armbrust (198) showed that laboratory-grown cultures of the diatom Thalassiosira pseudonana exposed to low-temperature-induced high excitation pressure balanced energy sink utilization between nitrogen metabolism and photorespiration in a complementary manner. When replete nitrate levels were available in the growth medium, excess energy was consumed via NO₃^– reduction; however, when NO₃^– levels were depleted, the photorespiration pathway became the major excess energy sink. These data indicate that diatoms exhibit an acclimatory ability to finely tune acclimatory responses to energy imbalances between photochemistry and cellular metabolism (198). This adaptation to low temperature may be an indicator of the dominance of diatoms in many permanently low-temperature marine environments, such as the polar sea ice.

PERMANENTLY COLD ECOSYSTEMS

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Phototrophic prokaryotic and eukaryotic microorganisms are widely distributed across a variety of low-temperature environments. These environments can differ widely in their physical and chemical properties, which influence the survival strategies of the organisms living within each ecotype (see Table 1). The various survival strategies often produce unique biogeochemical and consortial relationships among the organism within the habitats (194); therefore, knowledge of the physical and chemical properties of the environment is an integrative step to understanding of microbial photoadaption within each environment (61, 181, 211, 267).

UV-B and UV-A are both particularly damaging to phototrophic microorganisms, which typically exhibit suboptimal rates of growth and photosynthesis under UV stress. UV-B damages cells at the nucleotide and protein level, inhibits photosynthesis, and prolonged exposure can lead to cell death (268). UV-A exposure causes more indirect detrimental effects in the form of reactive oxygen species that attack nucleotides, proteins, and lipids (260). The potential for photooxidative stress under excessive high light is exacerbated by year-round low temperatures (around 0°C). Algal populations living below the snow surface are exposed to less damaging levels of PAR as well as UV radiation; however, during the summer months, snow melting occurs rapidly, so that cell transport in the meltwater changes the vertical profile of algal assemblages, resulting in a highly variable daily PAR exposure (75). On the other hand, snow cover in these environments can also effectively attenuate 100% of PAR, so that algal communities must also cope with variable periods of total darkness (11).

Despite this harsh environment of low temperatures and high and variable light, more than 100 species of chlorophytes (mainly Chlamydomonas and Chloromonas spp.) have been identified as the dominant organisms in the algal blooms contributing to red, yellow, gray, and green snow patches. Single cells isolated from these algal patches have been shown to be photosynthetically active (72, 274). Therefore, the algae inhabiting these environments must possess adaptive mechanisms to survive a temperature and light regimen that would cause severe photoinhibition and photooxidative damage to most plants.

One of the most-studied snow algae is Chlamydomonas nivalis, which is responsible for causing the more commonly observed red coloration of snow patches (271). The most prevalent photoinhibitory avoidance mechanism possessed by C. nivalis is the red coloration of the cells, caused by high levels of the secondary carotenoid astaxanthin. Astaxanthin accumulates around the single, centrally located chloroplast as lipid droplets. Badgered et al. (14) reported that astaxanthin was esterified to either a monounsaturated or a diunsaturated fatty acid in red vegetative cells and cysts, respectively, and the esterification of the auxiliary pigment to a fatty acid allows the chromophore to be concentrated within lipid globules to maximize photoprotection. Astaxanthin exhibits a maximal absorption wavelength of around 474 nm, and red vegetative or resting cells are almost entirely shaded from light in the shorter (blue) wavelengths. Recently, it was found that astaxanthin screens UV wavelengths in C. nivalis (72) and provides a photoprotective role by screening the chloroplast in other algae (80). C. nivalis is also known to accumulate phenolics in response to UV exposure (51) and, to a lesser extent, may also rely on mycosporine-like amino acids (strong UV-absorbing water-soluble molecules commonly synthesized by algae) (22, 72).

Since the majority of studies have focused on natural snow algal communities, evidence of acclimation to variations in environmental conditions is scant. One exception is a recent study by Baldisserotto et al. (11) regarding acclimation of thylakoid ultrastructure and photosynthetic apparatus in the filamentous snow alga Xanthonema sp. to prolonged exposure to complete darkness to mimic acclimation to the austral night. In response to prolonged darkness, Xanthonema cells exhibited a preferential disassembly of PSII while largely retaining the light-harvesting complexes of PSII for up to 35 days. Evidence of this acclimative response suggests that natural snow algal populations may preserve thylakoid ultrastructure organization via LHC-mediated stabilization of the photosynthetic membranes (11).

The food webs in the pack ice of the continent are supported primarily by photoautrophic production by phytoplankton, and many algal classes (Bacillariophyceae, Chrysophyceae, Chlorophyceae, Cryptophyceae, Dinophyceae, Prymnesiophyceae, Prasinophyceae, and Cyanobacteria) have been identified in the polar ice communities (111). The pennate diatoms (Fragilariopsis cylindrus and Fragilariopsis surta) are the most abundant organisms and are the major contributors to the brown coloration in the ice during summer algal blooms (124). During the transition from winter to spring in polar ice regions, large algal blooms are initiated and can attain very high biomass (up to 1,000 µg chlorophyll a/liter in the sea ice), much higher than typical of the diatom-dominated phytoplankton biomass of the surrounding waters of the Southern ocean (typically less than 5 µg chlorophyll a/liter). These algae are adapted to survive temperatures as low as –20°C as well as the dehydrating effects of high salinities.

As discussed above, adaptation of the lipid membranes via enrichment in polyunsaturated fatty acids is a common low-temperature adaptation mechanism in psychrophilic bacteria (157) isolated from sea ice as well as the sea ice diatoms isolated from the Antarctic (162, 163) and Arctic ice shelves (85). A higher degree of unsaturated fatty acids in the membrane lipids is also an adaptive advantage under high-salinity stress. Sea ice algae produce high levels of osmoregulators and cryoprotectants such as proline, polyols, betaines, and dimethylsulfonioproprionate (43, 46). Dimethylsulfonioproprionate production in the sea ice communities has received heightened attention, as it is a significant biological source of the volatile product dimethyl sulfide, which contributes to the sulfur load in the atmosphere (23, 45, 110).

The major environmental factor affecting the growth of sea ice communities is light. The light environment of sea ice is extremely low due to the reflection of the majority of PAR (85 to 99% surface irradiance) via the snow cover and sea ice and relatively high attenuation by the snow and ice itself (117, 149, 225). UV stress on the sea ice algae was initially assumed to be low owing to attenuation by the overlying ice; however, with the widening of the ozone hole over the Southern Hemisphere, sea ice communities are now being exposed to higher levels of UV-B radiation (258). A number of studies on natural and isolated cultures have indicated that the sea ice diatoms exhibit extreme shade adaptation (33, 96). Very low light compensation points (0.2 to 1 µmol m^–2 s^–1) (31) have been reported for natural populations residing on the underside of the ice sheet, with light saturation of growth reported for some ice algae to be below 20 µmol m^–2 s^–1 (73, 225, 273). Sea ice algae are adapted to low light levels by an augmented light-harvesting apparatus as well as by heterotrophic acquisition of carbon and energy sources, and can actively take up amino acids, sugars, and organic acids. However, heterotrophic growth appears to play a minor role in ice algal blooms. Palmisano et al. (196) estimated the heterotrophic capacity of ice algae to be about 0.3% relative to rates of photosynthesis.

Natural populations possess the ability to adjust pigmentation in response to changes in the light environment. In sea ice microalgal assemblages dominated by diatom algae, the ratio of the xanthophyll diatoxanthin relative to diadinoxanthin increased proportionally with an increase in irradiance levels from sunrise to afternoon (107). Variability in photosynthetic capability as well as pigmentation has also been observed at the level of changes in vertical positions in the sea ice (32, 108). Furthermore, ice algae were shown to possess a functional xanthophylls cycle, the diadinoxanthin cycle, when isolated cultures were exposed to irradiance levels that were four times higher than the natural light environment (116), and microalgae populations in Antarctic sea ice exhibit the ability to avoid short-term photoinhibitory conditions by dissipating excess light energy nonphotochemically (NPQ) (224). Thus, despite extreme shade adaptation, isolated diatom cultures have retained the xanthophyll cycle-mediated acclimatory mechanisms of dissipation of excess light energy via heat.

Photoacclimation of psychrophilic diatoms to variations in temperature and irradiance has also been reported in laboratory-controlled conditions (161, 164). Mock and Hock (161) reported that while initial exposure of laboratory-controlled cultures of the polar diatom Fragilariopsis cylindrus to a downshift in temperature induced a cold shock response at the level of both PSII photochemical efficiency and photosynthetic capacity, recovery from cold shock was observed after a few days of exposure; F. cylindrus possesses the ability to photoacclimate to changes in temperature environment. Furthermore, cultures acclimated to lower temperatures exhibited a higher capacity for NPQ in a manner that was comparable to high-light acclimation.

Mock and Valentin (164) recently constructed one of the first expressed sequence tag libraries in F. cylindrus. By monitoring gene expression during a downshift in temperature from 5°C to –1.8°C, these authors showed that when exposed to moderate irradiance levels, cells acclimated to lower temperatures by downregulating expression of PSII and carbon fixation genes while upregulating genes encoding chaperons and those involved in plastid protein synthesis and turnover. In contrast, cultures grown under low light did not respond to the temperature downshift by upregulating genes involved in chaperone or protein turnover function (164). These studies are some of the first to provide evidence that polar diatoms photoacclimate to low temperatures in a light-dependent manner and are probably sensing changes in excitation pressure.

According to the snowball Earth hypotheses, around 600 million years ago, the Earth was completely covered by ice exceeding 1 km in thickness. It has been suggested that photosynthetic cyanobacteria and bacteria may have survived global glaciation by residing in bacterial mats similar to present-day cyanobacterial mats found in Arctic and Antarctic ponds (87, 264, 265). The close association of microorganisms in these microrefugia would favor the development of symbiotic relationships, and even perhaps influence the development of the eukaryotic cell in a manner similar to the theory for eukaryotic cell development in thermal microbial mat ecosystems (263).

In contrast with the sea ice assemblages, which are dominated by diatom algae, the pond microbial mats are dominated by filamentous cyanobacteria. These organisms are tolerant to high UV, desiccation, and freezing-thawing cycles. They form mucilaginous mats which act as microrefugia, or small-scale refuges, for a plethora of other less-tolerant photosynthetic and heterotrophic forms of microbial life, including eukaryotic microalgae and microinvertebrates. Until recently, it was believed that ice shelf mat communities were restricted to Antarctica. However, Vincent and coworkers have documented widespread communities at the Ward Hunt Ice Shelf as well as the Markham Ice Shelf, both located on Northern Ellesmere Island, Nunavut, in the Canadian high Arctic (263, 266). The Arctic and Antarctic ice shelves are one of the environments most vulnerable to climate warming (88, 95, 122, 151, 261); increasing global temperatures has already had a significant negative impact on accelerating the fragmentation and loss of the Ward Hunt Ice Shelf and the draining of a 3,000-year-old Arctic lake that had been dammed behind it (172).

Analysis of 16S rRNA genes from oscillatorians isolated from Antarctic and Arctic ice-shelf microbial mat communities indicates that filamentous cyanobacteria in both polar environments originated from temperate species (176). However, 16S rRNA analysis of psychrophilic filamentous cyanobacteria of the order Oscillatoria isolated from certain Antarctic algal mats shows they are more distantly related to temperate strains of the same genera, implying that these organisms may have been introduced to Antarctica as the continent cooled (20 million years before the present) and evolved in their present-day habitat, compared to psychrotrophic strains that may have been introduced more recently (176). Tang et al. (249) found that many of the Arctic and Antarctic mat-forming cyanobacteria are not psychrophilic and exhibit growth temperature optima far above the temperatures found in their natural environments. Therefore it appears that adaptation to growth at low temperatures is not a requirement for successful colonization of these habitats, and other characteristics, such as UV screening and protection against photoinhibition, may have a greater selective advantage in the Arctic ice shelf environment (249).

The phototrophic microbial mats are dominated by the filamentous cyanobacteria of the order Oscillatoriales. Atmospheric nitrogen-fixing cyanobacteria, such as Nostoc and Anabaena spp., were found to be codominant, suggesting that bound inorganic nitrogen may be depleted in these environments and that N₂ fixation may be an important source of nitrogen to these organisms as well as the entire mat community (223). An Antarctic algal mat from a pond near Ross Island, Antarctica, was also the sampling site for the isolation of the first psychrophilic anoxygenic phototrophic bacterium (Rhodoferax antarcticus sp. nov.) (140).

N₂ fixation has also been shown to be important in the survival of filamentous cyanobacteria inhabiting the permanent ice covers of certain Antarctic lakes (194, 215). The UV-screening and N₂-fixing cyanobacteria aid in the formation of a rich microhabitat that supports a highly concentrated assortment of other organisms, including bacteria, eukaryotic algae, ciliates, flagellates, nematodes, rotifers, and platyhelminthes. Their success in surviving and proliferating in these environments is likely due to a complexity of adaptive characteristics, such as maintenance of overwintering populations. One such strategy is the production of the mucopolysaccharide matrix, which not only traps sediment particles and aids in mat cohesion but also probably provides protection against freezing-thawing cycles and desiccation, thus allowing the mat communities to survive the winters and form seed populations for the resumption of growth in the summer (263).

In conjunction with habitat-specific stresses such as low temperatures and desiccation, the microbial mats during the summer are exposed to continuous high levels of UV radiation. Cyanobacteria possess a wide variety of avoidance and repair mechanisms to combat the negative effects of UV exposure, including the synthesis of intracellular (97) and extracellular (66) UV-screening compounds. Screening of UV and excessive PAR levels is a major adaptive strategy in the upper layers of the ice shelf mats, as was evident by the high carotenoid-to-chlorophyll a ratios.

Photoprotective carotenoids such as lutein, echinenone, and ß-carotene have also been detected, but by far the major pigment present in both the upper and bottom layers of the mats is the UV-screening sheath pigment scytonemin as well as its degradation product, scytonemin-red (266). Scytonemin is a common UV-screening pigment found in many extreme environments and is effective at screening maximally in the UV-A and UV-C regions, as well as the UV-B region (222). De novo synthesis of this screening pigment occurs in response to exposure to UV-A, but can be induced by increases in temperature and photooxidative conditions in isolates from epilithic desert crust communities (44). The high levels of UV-screening pigments appear to screen out close to 100% of the short-wavelength radiation, so that the biota residing in the lower layers of the mats were exposed to wavelengths restricted to low intensity (<2% PAR) in the yellow-red waveband. Therefore, phototrophic microorganisms residing in the lower layers of the mat are likely adapted to shade conditions.

Photosynthetically active microalgal and cyanobacterial populations that fix inorganic carbon and nitrogen provide the nutrient foundation for a surprisingly complex microbial assemblage which includes bacteria, algae, and diatoms, as well as simple metazoans such as rotifers, tardigrades, and nematodes. During the polar winter, the holes refreeze and the organisms survive the cold, dark winter through dormancy. Each cryoconite hole potentially forms a unique ecosystem with relatively complex biogeochemical processes (256, 257).

Molecular analysis of the small-subunit rRNA gene of cryoconite holes that form in the McMurdo Dry Valley region have shown several sequences to be similar to rRNA gene species isolated from either microbial aggregates or microbial mats associated with the adjacent dry valley lake communities (25, 71). Cryoconite holes serve as a repository for terrestrial populations and may serve as glacial refuges for microorganisms in cold polar deserts. While there has been minimal work on adaptative mechanisms of organisms residing in cryoconite holes, there are presumably similarities with the ponds that form on the ice shelves.

View larger version (118K): [in this window] [in a new window]   FIG. 3. Map of the McMurdo Dry Valleys, Antarctica. Unusual meteorological conditions in the dry valleys produce the only permanently ice-covered lakes on Earth and the liquid water beneath the ice allows one of the few refugia for microbial communities in continental Antarctica. Each lake exhibits a unique biogeochemistry and therefore a unique microbial consortium. A Chlamydomonas sp. has been identified in many of the lakes. C. raudensis UWO 241 was isolated from the east lobe of Lake Bonney, Taylor Valley.

Photosynthetic production of organic carbon drives biogeochemical reactions (that is, the partitioning and cycling of chemical elements and/or compounds between living and nonliving parts of an ecosystem) and influences species abundance and diversity in all ecosystems. Polar lake systems have exceptional stresses imposed on photosynthesis by amplified seasonal patterns in sunlight and the permanent ice covers that greatly reduce the amount of light that reaches the water column. Despite the apparent lack of ecological complexity, we know now that these polar desert lakes harbor a complex assemblage of interacting autotrophic and heterotrophic microorganisms (123, 137, 210, 221) (Fig. 4).

View larger version (80K): [in this window] [in a new window]   FIG. 4. Pictorial representation of the major food web components and their linkages within Lake Bonney (Taylor Valley, Antarctica). The rotifer is a Philodina sp., the large ciliate is a Euplotes sp., and the phytoplankton is a Chlamydomonas sp. Note the bacteria attached to the surface of the Chlamydomonas cells which appear as threadlike structures on the upper portion of the cell; flagella are evident on the right apex of the cell. Note the absence of higher trophic levels. (Reprinted from reference 221 with permission of the publisher.)

MCMURDO DRY VALLEY LAKE PHOTOTROPHIC COMMUNITIES

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The McMurdo Dry Valleys are the site of numerous permanently covered lake systems. Lakes Bonney, Hoare, and Fryxell located within Taylor Valley (Fig. 3) have been the focus of the U.S. National Science Foundation's McMurdo Dry Valleys Long-Term Ecological Research (LTER) program, which has been studying these lakes systematically over the past 13 years (http://huey.colorado.edu/LTER/). Phytoplankton play an essential role in the functioning of the food web of the dry valley lake ecosystems (Fig. 4), and understanding the diversity and variability in the phytoplankton numbers is paramount to the overall goal of the McMurdo LTER.

Lake Bonney, which lies at the snout of the Taylor Glacier (Fig. 3), consists of two basins, the east lobe (3.5 km²) and the east lobe (1.3 km²) (207). A narrow sill separates the two lobes, isolating the saline, nutrient-rich deep waters of each basin while allowing the fresher surface waters to exchange (207). As discussed below, the presence of a year-round ice cover strongly influences the aquatic biology, chemistry, and physical properties of the lake. The ice cap itself is a habitat for microorganisms due to the high porosity of the lake ice (214).

Each lobe of Lake Bonney has a distinct geochemistry and associated biology related to climate evolution and input from subglacial outflows (58, 158). For example, the west lobe contains the highest levels of dimethyl sulfide ever sampled in a natural system, whereas the east lobe has the highest dimethyl sulfoxide levels encountered in natural waters (121). These biogenetic sulfur compounds are thought to be produced by cryptophyte algae, which occupy certain layers in the water column in Lake Bonney (120). The east lobe also contains nitrous oxide concentrations that exceed 700,000% of air saturation (209, 213). The sources and sinks of these compounds are not completely understood and are often not supported by the thermodynamics of the system (120). This thermodynamic paradox has led several authors to suggest that the gradients now observed in Lake Bonney may have formed many thousands of years ago (121, 209, 211).

The dissolved oxygen concentration from just beneath the ice to 15 m exceeds 1,000 µM, which is between 250 and 350% higher than what would occur if the water was saturated with air above the lake. Oxygen at depths below 20 m shows less than 10% air saturation. The dissolved oxygen levels beneath the chemocline in the west lobe of Lake Bonney support anaerobic processes such as denitrification (58, 209, 213), whereas no significant bulk anaerobic metabolism occurs beneath the chemocline in the east lobe. Vertical dissolved inorganic nitrogen and phosphorus profiles are similar to the salinity profiles, with relatively low values above 15 m followed by large increases below 15 m (Fig. 5, upper panels). The average molar ratio of dissolved inorganic nitrogen to soluble reactive phosphorus ranges from 64 to 616 between 5 and 17 m, reaches a maximum of 1,620 at 20 m, and then averages about 600 from 21 m to the bottom. These ratios are well above that required for balanced phytoplankton growth, indicating phosphorus limitation, a contention that has been supported by experimental work on both phototrophic and heterotrophic production (50, 58, 211).

View larger version (49K): [in this window] [in a new window]   FIG. 5. Physical, chemical, and biological parameters from the west and east lobes of Lake Bonney. SRP, soluble reactive phosphorus; PPR, phytoplankton primary productivity; Chl-a, chlorophyll a; TdR, bacterial productivity measured as [3H]thymidine incorporation into DNA; PSU, practical salinity units. (Reprinted from reference 221 with permission of the publisher.)

Lizotte and Priscu (129) conducted a detailed study of spectral irradiance in Lake Bonney and other dry valley lakes and showed that irradiance was always less than 50 µmol photons m^–2 s^–1 beneath the permanent ice covers. The wavelength of maximum transmission through the water column of Lake Bonney and other lakes is in the range from 480 to 520 nm (126), with longer wavelengths (>600 nm) being greatly diminished. Lizotte and Priscu (129) showed that the water itself was the dominant absorber of available light under the ice (38 to 75% of the total absorption coefficient), but that phytoplankton played a major role in attenuating shorter wavelengths (<520 nm) (11 to 47%).

Unlike many pelagic systems, where spring growth of phytoplankton is triggered by a combination of decreasing mixed layer depth and increasing incident PAR (243), the initiation of spring phytoplankton growth in the nonturbulent waters of Lake Bonney is solely a function of the seasonal increase in incident PAR. The annual underwater light climate from 1999 through 2003 shows the large seasonal difference in underwater PAR both within and between seasons (Fig. 6). Measurable PAR is present only from late September through mid-March for most seasons.

View larger version (32K): [in this window] [in a new window]   FIG. 6. Underwater PAR plots taken from the east lobe of Lake Bonney over the time period of 1999-2003.

Figure 7 shows the distinct vertical stratification of phytoplankton species, determined by inverted light microscopy of gravity-settled samples, in the east lobe of Lake Bonney at selected intervals between 1989 and 2000. The taxonomic scheme of Seaburg et al. was utilized (239a). The layer immediately beneath the ice is consistently dominated by the cryptomonad Chroomonas sp. over the entire period. Chlamydomonas intermedia also inhabits the upper part of the water column, but at much lower biomass levels than Chroomonas organisms. The chrysophyte Ochromonas sp. displays the greatest variation in vertical distribution but is often confined to the upper and middle depths of the water column. This species reaches some of the highest biomass levels within Lake Bonney. A Chlamydomonas sp. (later identified as C. raudensis UWO241 [205]; see below) is confined to the deep saline and low-irradiance portion of the photic zone (15 to 18 m). These vertical profiles are supported by chemotaxonomic pigment analysis (125, 128).

View larger version (44K): [in this window] [in a new window]   FIG. 7. Vertical profiles of the major phytoplankton species in the east lobe of Lake Bonney at selected time intervals over the past decade.