Cold-Adapted Enzymes

A critical adaptive feature of all cold-adapted microorganisms is the molecular adaptation of enzymes to compensate for the reduction in chemical reaction rates at low temperatures. The relationship between temperature and chemical reactions can be described by the Arrhenius equation (k = Ae^−Ea/RT), where k is the rate constant, A is the constant for a particular reaction (e.g., frequency factor), E_a is the activation energy, R is the gas constant, and T is the absolute temperature in degrees Kelvin. E_a is related to Q₁₀, the factor by which the rate changes by varying the temperature 10°C, according to the following relationship: ln Q₁₀ = (E_a × 10)/(RT₂T₁), where T₂ and T₁ are the temperature limits for which Q₁₀ is desired. In general a 10°C reduction in growth temperature causes biochemical reaction rates to decline two to three times (Q₁₀ = 2 to 3). Therefore, the activity of a mesophilic enzyme can be reduced as much as 80-fold when the growth temperature is shifted from 37°C to 0°C. Despite the severe reduction of enzymatic activity, the doubling time of a psychrophilic bacteria at 4°C can be comparable to that of mesophilic bacteria grown at 37°C (57).

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).

High-Alpine Snowfields

The presence of “colored snow” in alpine snowfields which persists through the summer is a common occurrence worldwide at high altitudes (>2,500 m) and polar regions. At this altitude, the light level is extremely high and is exacerbated by PAR reflecting off the snow, so that PAR levels as high as 5,000 μmol m⁻² s⁻¹ reach algal cells (274). In addition, algal populations can be exposed to as much as 30% more UV compared with phototrophic organisms growing at sea level. This UV effect is more pronounced for wavelengths in the UV-B region (15).

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).

Polar Sea Ice

Polar sea ice covers 5% of the Northern Hemisphere and 8% of the Southern Hemisphere, but sea ice is also found in the Baltic, Caspian, and Knots seas, making it one of the major biomes in terms of surface area (up to 13% of the Earth's surface) (124). The most prevalent sea ice ecosystem is in the high latitudes of the Southern Hemisphere, where more than 40% of the Southern Ocean is covered during winter by sea ice that often exceeds 1 m in thickness (251). Sea ice supports rich single-celled microbial ecosystems, in addition to small metazoans (195). However, annual recession of the ice as well as breakup of the pack ice by physical forcing can make this a highly variable habitat from a seasonal down to a daily and even hourly level (60). Thus, physiological and metabolic adaptation of the sea ice biota must involve the ability to acclimate to rapid fluctuations in the physical environment due to the transient nature of the sea ice.

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.

Transitory Ponds

The ice shelves of the Arctic and Antarctic support shallow pond ecosystems that are created during the summer season when pockets of the ice melt to form bodies of liquid water of various sizes. While the formation of liquid water is seasonally transitory, the ponds often melt out in the same location every year, and the microorganisms, particularly the microbial mats, exhibit several decades of seasonal growth. The microorganisms that colonize these extreme habitats must be capable of surviving daily and annual freezing-thawing cycles, persistent low temperatures, continuously high exposure to solar radiation during the summer, and long periods of dormancy. Despite these constraints, there exist diverse and productive consortia of microorganisms in the form of microbial (cyanobacterial) phototrophic mats. These biota are representative modern-day examples of how life survived and evolved during global glaciation and extended periods of extreme cold (263-265).

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 β-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.

Cryoconites

Cryoconite habitats are transitory (with respect to liquid water) environments which form during polar summers when dark wind-blown particulate matter imbedded in glacial ice is heated by solar radiation and melts, forming a cylindrical basin of liquid water despite subfreezing air temperatures. Microorganisms associated with the wind-blown material serve as the seed populations of these unusual transitory microenvironments, which have been documented in both the Arctic and Antarctic as well as alpine glaciers (25, 71, 172a).

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.

Biogeochemistry

Several important biogeochemical implications arise from the presence of the permanent ice that covers the lakes. Gas exchange between the water column and atmosphere is severely restricted (213) and vertical mixing within the liquid water column of the lakes is predominantly through molecular diffusion owing to the lack of wind- and river-induced turbulence (244, 245). One of the manifestations of these biogeochemical conditions is a highly layered distribution of chemical species in the water column of the lakes.

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. ​(Fig.5,5, 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).

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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.)

Temperature, Light Climate, and Phytoplankton Abundance

Temperatures in Lake Bonney range from near 0°C just beneath the ice cover to a maximum of 6.1°C at 14 m and to a minimum of −1°C in the deep saline water at 36 m (Fig. ​(Fig.5,5, lower panels). Despite the unusual temperature profile, the water column remains highly stable (i.e., it is not mixed by buoyant forces) because of the salt gradient (Fig. ​(Fig.5,5, upper panels). Spigel and Priscu (244, 245) have shown that the salinity profile is extraordinarily constant from year to year and that the curvature of the profiles is consistent with the effects of diffusion.

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. ​(Fig.6).6). Measurable PAR is present only from late September through mid-March for most seasons.

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FIG. 6.

Underwater PAR plots taken from the east lobe of Lake Bonney over the time period of 1999-2003.

Measurements made within each field season show that phytoplankton productivity and biomass increase when solar radiation first penetrates the ice cover of the lake, generally reaching their highest levels in January, when field studies typically end owing to logistic constraints (221). The depth of transmission also varies considerably with the 2000 to 2001 season, showing much deeper penetration than other seasons. This long-term variability in PAR reflects changes in environmental factors, such as irradiance (through cloud cover, changes in ice transparency, or turbidity associated with stream flow), UV radiation, and variation in stream-derived nutrients related to glacial melt over this period. In addition, temperatures were warmer during the late 1980s, resulting in higher ice cover porosity (62) and higher stream flow (27, 154), the latter yielding greater nutrient loading to the lake (see also reference 58). Lastly, Vincent et al. (267) also showed that sufficient UV radiation penetrates the ice cover of certain dry valley lakes to significantly inhibit algal growth. These facts, together with the region's sensitivity to ozone depletion (141) and climate warming and cooling (49, 150), make measurements of phytoplankton photosynthesis in these lakes an important gauge of environmental change.

Figure ​Figure77 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).

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FIG. 7.

Vertical profiles of the major phytoplankton species in the east lobe of Lake Bonney at selected time intervals over the past decade.

Phytoplankton Nutrient Status

Early reports on nutrient deficiency in the lakes of the McMurdo Dry Valleys were based primarily on indirect evidence such as nitrogen-to-phosphorus ratios in the water column (86), nutrient ratios in streams entering the lakes (20), photobiological responses of phytoplankton (126, 181), and direct measurement of nitrogen uptake using ¹⁵N-labeled compounds (210, 220). With the inception of studies focusing on photosynthesis (126, 128, 180, 181) and nitrogen transformations (120, 209, 213, 220), it became clear that detailed information on nutrient regulation of phytoplankton photosynthesis was necessary to understand the microbial dynamics of the lake ecosystems.

Priscu (211) conducted the first detailed long-term phytoplankton nutrient bioassays in these lakes and showed that phytoplankton photosynthesis in Lake Bonney just beneath the ice cover (5 m; Chroomonas sp. dominated) and at 13 m (Ochromonas spp. dominated), was extremely phosphorus deficient. However, photosynthesis in a sample from 18 m (dominated by a Chlamydomonas species later identified as C. raudensis UWO 241; see below) did not respond to either N or P addition. Priscu (211) showed that it was the upward diffusion of deep nutrient pools that were formed many thousands of years ago (138) that supports much of the phytoplankton photosynthesis now observed in Lake Bonney, particularly in the deep waters just above the chemocline (i.e., between 17 and 18 m). Phosphorus deficiency in both phytoplankton and bacterioplankton has been confirmed in Lake Bonney by other investigators (50, 58).

Photosynthetic Characteristics

Detailed photosynthesis-irradiance curves conducted on phytoplankton populations of the east lobe of Lake Bonney from 5 m (just beneath the ice; 0°C), 6 m (3°C), 10 m (5.5°C), and 17 m (6°C) by Lizotte and Priscu (127, 128) showed evidence of extreme shade adaptation, including low saturation points for photosynthesis (E_k = 15 to 45 μmol photons m⁻² s⁻¹), and extremely low maximal photosynthetic rates, P^B_m < 0.3 μg C (μg chlorophyll a)⁻¹ h⁻¹. Deeper phytoplankton (10 and 17 m) were shown to have P^B_m and photosynthetic efficiencies (α, slope of initial portion of P versus E curve) three to five times higher than those at the ice-water interface, despite Q₁₀ values of only ~2 for P^B_m, implying that a simple temperature response cannot explain all of the differences between phytoplankton populations. Lizotte and Priscu (128) concluded that the deep chlorophyll layers in Lake Bonney may be caused by factors such as in situ growth of phytoplankton enhanced by higher nutrient availability at a nutricline, physiological adaptation to decreased irradiance, decrease in sinking rate of phytoplankton with depth, and/or behavioral aggregation of phytoplankton.

The extreme shade adaptation shown by Lizotte and Priscu (128) for the in situ phytoplankton populations indicates that these organisms are highly efficient at converting light to photosynthetic energy and may have a large number of chlorophyll pigments associated with each of the photosynthetic reaction centers. Neale and Priscu (180-182) used in vivo fluorescence yield to define the depth profile of relative changes in quantum yield of photosynthesis. The dark-adapted in vivo fluorescence per unit chlorophyll was higher and the photochemical yield, measured as the fluorescence parameter F_v/F_m (where F_v is variable chlorophyll a fluorescence and F_m is maximal chlorophyll a fluorescence) (19) were lower in the shallow populations dominated by the cryptophyte Chroomonas sp. compared to the deep populations dominated by Chlamydomonas sp. The F_v/F_m data in particular imply a low quantum yield in shallow populations, increasing quantum yield in the region of 10 to 15 m, and nealy maximal values in the Chlamydomonas sp.-dominated deep populations between 16 and 18 m (181, 182). This is in agreement with the trend of increasingly higher initial slopes of the photosynthesis-irradiance curves measured at 5, 10, and 17 m by Lizotte and Priscu (127, 128) and increasing quantum yields of photosynthesis and fluorescence with depth computed by Lizotte and Priscu (126).

Neale and Priscu (180) obtained further information on the structure and function of the photosynthetic apparatus in phytoplankton populations from several dry valley lakes through analysis of the slow (minute time scale) fluorescence transients. The steady-state fluorescence yield (F_s) in samples from Lake Bonney after 5 min of illuminations was lower (quenched) for irradiances greater than 10 μmol photons m⁻² s⁻¹, indicating induction of protective mechanisms to dissipate excess excitation energy (nonphotochemical quenching) at an unprecedented low irradiance. These results are consistent with adaptation to a constant-shade environment and maintenance of low excitation pressure.

Cell Morphology

A recent morphological study (205) showed that C. raudensis is a biflagellate single cell of approximately 10 to 15 μm in length (Fig. ​(Fig.8A).8A). C. raudensis also exists as nonmotile colonies of approximately 30 μm that contain 16 to 32 flagellated daughter cells (Fig. ​(Fig.8B).8B). A single cup-shaped chloroplast is present, as well as a basally located pyrenoid body which is surrounded by starch platelets (Fig. ​(Fig.8A).8A). The eye spots of C. raudensis were typically very small (<1.1 μm) and difficult to locate, in comparison with the very long eyespots of C. subcaudata and the multirowed eyespots of C. reinhardtii (205). Morphological investigation supports the conclusions from the phylogenetic analysis that the Lake Bonney psychrophile is distinct from its former namesake, C. subcaudata.

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FIG. 8.

Electron micrographs of C. raudensis grown under laboratory-controlled conditions (8°C/20 μmol photons m⁻² s⁻¹) showing a single cell (A) and a membrane-bound colony (B). Note starch accumulation within the pyrenoids (P). C, chloroplast; N, nucleus; G, Golgi apparatus; M, mitochondrion; F, flagellum. Scale bars, 1.0 μm. (Reprinted and modified from reference 205 with permission of Blackwell Publishing.)

Growth and Photosynthetic Characteristics

Photosynthetic function and acclimation to environmental change in Chlamydomonas raudensis UWO241 have been studied for over a decade, primarily by Huner and coworkers (166-169, 205). Early studies characterized environmental limitations for growth of axenic cultures under laboratory conditions (Fig. ​(Fig.9).9). C. raudensis exhibited an optimal growth temperature below 10°C, although cultures exhibited exponential growth rates up to a temperature of 16°C. However, cultures failed to grow and cells were nonviable at growth temperatures above 18°C, which classifies C. raudensis as a true psychrophilic phytoplankton (Fig. ​(Fig.9A9A).

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FIG. 9.

Dependence of rates of growth in C. raudensis on temperature (A) and irradiance (B). A. Cultures were grown under low irradiance (20 μmol m⁻² s⁻¹) and variable growth temperatures. B. Cultures were grown under optimal growth temperature (8°C) and variable growth irradiances.

Photosynthetic light response curves were performed under a range of measuring temperatures on C. raudensis cultures grown at 8°C and 20 μmol O₂ m⁻² s⁻¹ (204). Rates of apparent O₂ evolution (ΦO₂) were temperature dependent in C. raudensis in a laboratory setting, exhibiting the highest ΦO₂ at 8°C and then a steady decline at higher measuring temperatures. The ΦO₂ was exceptionally high at 8°C in C. raudensis, indicating maximal efficiency in converting light to photosynthetic energy at low temperature.

A lower ΦO₂ at higher temperatures can be explained in part by the temperature-dependent induction of the energy-dissipative xanthophyll cycle. The maximum photosynthetic capacity (P_max) in C. raudensis measured under light-saturating conditions was highest at 8°C and, unlike ΦO₂, was little affected by temperature up to temperatures of 25°C. Relative to the optimum temperature of 8°C, P_max in C. raudensis was 82% lower at 35°C and 98% lower at 45°C. This indicates that the limitation for growth of C. raudensis at temperatures above 20°C is not immediately due to high-temperature damage to the photosynthetic apparatus or primary carbon metabolism.

Earlier ecophysiological studies by Priscu and coworkers (180, 181) of Lake Bonney phytoplankton communities indicated that photoprotective mechanisms have been sacrificed in favor of augmentation of light-harvesting capacity. However, isolated cultures of C. raudensis grown at the optimal growth temperature (8°C) and variable irradiance can tolerate growth irradiance levels that were significantly higher than the natural light environment (about 10-fold), and exhibited a linear increase in growth rate with respect to growth irradiance up to 150 μmol m⁻² s⁻¹ (Fig. ​(Fig.9B).9B). This ability to modulate growth rate at low temperatures under variable irradiance differs from that of low-temperature-grown cultures of mesophilic algae (such as the model organism Chlorella vulgaris), which are unable to adjust growth rate in response to variable growth irradiance (147, 275).

The psychrophilic C. raudensis also exhibits unusual growth response to light of specific qualities. While cultures of the Antarctic alga grew exponentially under either white light or blue light, cultures failed to grow under red light (although cells were still viable and resumed exponential growth when transferred back to white light) (169). Presumably, the inability to grow under red light is a consequence of natural populations of C. raudensis being exposed to virtually no light in the longer-wavelength spectrum. Therefore, they are adapted to a stable light environment of low intensity enriched in blue-green wavelengths.

Membrane Lipid and Fatty Acid Composition and Pigmentation

C. raudensis exhibits a typical distribution of lipid species that has been characterized in other Chlamydomonas sp., including the mesophile C. reinhardtii. The galactolipids (MGDG, DGDG, and sulfoquinovosyldiacyglycerol), lipids specifically associated with the chloroplast, made up more than 75% of the total lipid content, a common feature in organisms where more than 80% of the total membranes are associated with the single large plastid. In contrast, fatty acid content differed significantly between the mesophilic and the psychrophilic membranes. C. raudensis exhibited a significantly higher unsaturated fatty acyl bond index in comparison with C. reinhardtii (167). Most notably, the all chloroplast galactolipids (MGDG, DGDG, and sulfoquinovosyldiacyglycerol) of C. raudensis possessed high levels of polyunsaturated fatty acids (167), suggesting low-temperature adaptation specifically associated with the photosynthetic membranes.

In contrast, characteristic of Chlamydomonas sp., the majority of polyunsaturated fatty acids in photosynthetic membranes of the mesophile C. reinhardtii were restricted to the galactolipid MGDG, while the other major plastid lipids possessed saturated or monounsaturated fatty acyl species. Lastly, C. raudensis exhibited several novel polyunsaturated fatty acids with positional shifts in the unsaturated bond closer to the lipid head group. It is hypothesized that a shift in the double bond to positions closer to the head group increases the cross-sectional area of the fatty acyl tails, causing higher degrees of disruption in the lipid membrane and therefore higher degrees of fluidity. Since the desaturation of the fatty acyl chains is catalyzed by position-specific desaturases, presumably C. raudensis must also possess enzymes with novel positional specificity.

C. raudensis grown under a regimen of low temperature and low light exhibited a pigment complement typical of a Chlamydomonas species, with lutein and neoxanthin being the most abundant light-harvesting carotenoids (166, 205). One of the most striking characteristics of the pigment composition of C. raudensis was an unusually low chlorophyll a to b ratio (1.6 to 1.8) (166, 205), consistent with the notion that C. raudensis exhibits an enhanced light-harvesting capacity as a consequence of adaptation to a constant low-light environment in its natural habitat (181). Under controlled growth conditions, C. raudensis also exhibited significantly lower levels of both antheraxanthin and zeaxanthin (166), products of the xanthophyll cycle which are involved in energy dissipation during conditions of excessive light absorption (40).

Biochemistry and Physiology of the Photosynthetic Apparatus

Given the complex set of extreme natural environmental parameters to which the psychrophile is adapted, it was not surprising to find that the organization and composition of the photosynthetic apparatus of this enigmatic green alga exhibit a number of unusual features (166, 168, 181). At the level of the photosynthetic reaction center complexes, C. raudensis possesses comparable levels of D1, a major reaction center protein of photosystem II, but reduced levels of PSI reaction center proteins (PsaA/B) in comparison with the model species, C. reinhardtii (Fig. 10A). The low PSI-PSII reaction center stoichiometry was accompanied by the absence or reduction of all light-harvesting I proteins (for, e.g., LHCI proteins p18.1 and p22.1; Fig. 10A) determined by immunoblot analysis, and was confirmed by a low ratio of chlorophyll-bound LHCI to LHCII on nondenaturing polyacrylamide gel electrophoresis (166). Furthermore, C. raudensis possesses significantly higher levels of the oligomeric form of light-harvesting II complexes, the major chlorophyll b-binding multiprotein complexes of the photosynthetic apparatus (166, 168, 169). Therefore, the organization of the photosynthetic apparatus in the Antarctic alga favors photosystem II under growth conditions simulating its natural habitat, resulting in an unusually high PSII-to-PSI ratio. This strategy would be an adaptive advantage under constant exposure to blue light of low fluence levels, since the light-harvesting apparatus of PSII utilizes chlorophyll b and short-wavelength-absorbing chlorophyll a to absorb light predominantly in the blue region (155).

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FIG. 10.

A. Abundance of major thylakoid proteins of photosystems I and II in the mesophilic species C. reinhardtii (M) and the psychrophillic C. raudensis (P) grown at the optimal growth temperature (29 and 8°C, respectively) and low irradiance levels. PsaA/B, photosystem I core reaction center proteins; D1, major photosystem II reaction protein; p18.1 and p22.1, representative light-harvesting I polypeptides. B. Low-temperature 77 K fluorescence emission spectra measured in vivo in C. reinhardtii (dotted line) and C. raudensis (solid line) cultures grown under same conditions as in A. (Modified from reference 166 with kind permission of Springer Science and Business Media.)

C. raudensis exhibits an apparent molecular mass of cytochrome f, a major polypeptide of the cytochrome b₆f complex, which is about 7 kDa smaller than the 41-kDa cytochrome f from C. reinhardtii (168). Despite this mass difference, cytochrome f from either Chlamydomonas species exhibited the ability to bind the heme cofactor, and no differences in the apparent molecular mass of either cytochrome b₆ or the Rieske Fe-S centers was observed. The petA gene, encoding the cytochrome f protein from UWO 241, was isolated and sequenced (AY039799) (79). The amino acid sequence of cytochrome f from C. raudensis was 79% identical to that of C reinhardtii (Fig. ​(Fig.1111).

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FIG. 11.

Sequence alignment of cytochrome f proteins from C. raudensis and C. reinhardtii. The protein sequence was predicted from the DNA sequence and aligned using the DNAman program. Black boxes indicate identical amino acids. Green shading, similar amino acids. Black arrow, N terminus of mature cytochrome f protein; red box, heme binding motif; brown box, transmembrane helix; gray underline, C terminus localized in stromal side of thylakoid membrane; blue arrows, lysine residues involved in plastocyanin docking; yellow arrows, amino acid residues involved in water chain formation.

The major difference in sequence was the presence of three cysteine residues (C21, C24, and C261) in the psychrophile rather than the two Cys residues (C21 and C24) typically found in other cyanobacterial, algal, and plant species and involved in covalent heme binding. The C261 residue is present in the transmembrane helix of cytochrome f of C. raudensis, is not involved in heme binding, and does not appear to be redox sensitive (78). However, the heme in cytochrome f of C. raudensis is significantly less stable to high temperature than that of C. reinhardtii, which has been interpreted to reflect an adaptation to a psychrophilic environment at the protein sequence level. This interpretation is consistent with the observation that components of the thylakoid membrane of C. raudensis denature at a temperature that is about 10°C lower than that of the mesophile C. reinhardtii (78, 167).

An additional structural alteration in the stoichiometry of the thylakoid protein complexes was observed at the level chloroplast ATP synthase. C. raudensis exhibited higher levels of both the α and β subunits of CF₁ relative to the mesophilic control, C. reinhardtii (166). This may be an adaptive strategy to maintain sufficient levels of plastid-derived energy and avoid low-temperature-induced reduction in ATPase reaction rates. Alternatively, the higher levels of plastid ATP synthase may indicate greater pool sizes of ATP in the psychrophile. As discussed above, one adaptive explanation for higher adenylate pools in the psychrophiles may be to compensate for reduced rates of molecular diffusion at low temperatures. Thus, higher levels of adenylates could represent an adaptive mechanism to provide adequate energy for ATP-dependent biochemical reactions at low temperatures (178, 179).

The unusual structural characteristics of the thylakoid apparatus of the psychrophilic alga had unique consequences on the functional photochemistry at the level of low-temperature (77 K) fluorescence emission. The model species C. reinhardtii possesses a classic 77 K fluorescence emission spectra, with a major emission maxima at 685 and 717 nm (Fig. 10B) (166), corresponding to chlorophyll a fluorescence originating from LHCII and LHCI-PSI complexes, respectively (114). In striking contrast, cells of C. raudensis possessed virtually no long-wavelength (>700 nm) fluorescence emission, indicating that the psychrophile possessed little to no fluorescence emission originating from PSI-LHCI complexes (166-169).

The absence of the PSI-associated 77 K fluorescence in the psychrophile is clearly associated with the reduced levels of PSI reaction center and, in particular, LHCI polypeptides (Fig. 10A). This phenomenon has been reported in a number of mutants harboring deficiencies in PSI content (38, 136, 278), as well as under specific stress conditions that target PSI function, such as iron deficiency in cyanobacteria (98), hyperosmotic stress, and low temperature (233). However, C. raudensis represents the first naturally occurring organism to exhibit this characteristic under controlled laboratory growth conditions.

Functional differences in photosynthetic electron transport between the psychrophile and the model mesophilic species C. reinhardtii have revealed striking differences in intersystem electron transport and the redox status of the intersystem pool between the two algal species. In one study, PSI activity was monitored in vivo by measuring the change in absorbance at 820 nm as an estimate of the oxidation-reduction of P₇₀₀+ (168). The mesophilic species exhibited a 1.5-fold higher steady-state change in A₈₂₀ (ΔA₈₂₀/A₈₂₀) in comparison with the psychrophilic alga, in support of earlier findings that C. raudensis possesses a structurally and functionally down-regulated PSI. However C. raudensis exhibited 1.5- to 2-fold higher levels of dark reduction of P₇₀₀ (168), indicative of higher rates of PSI-driven cyclic electron transport (9).

Neale and Priscu (181, 182) studied the light-harvesting characteristics of C. raudensis isolated from the east lobe of Lake Bonney through measurement of the PS I reaction center (P₇₀₀) content and spectral variation in PSII fluorescence kinetics. Based on their experiments, Neale and Priscu (181) concluded that the stable light environment experienced by Lake Bonney phytoplankton enables a high degree of acclimation to low-intensity blue-green light. This acclimation is accomplished partially by augmentation of the antenna size of chloroplast PSII with light-harvesting carotenoids and reducing the content of photoprotective carotenoids.

Short-Term Photoacclimation

The ability of the organism to adjust quickly and efficiently to maximize photosynthetic efficiency while avoiding the damaging effects of overexcitation of the photochemical apparatus involves a variety of regulatory processes. These photoacclimatory mechanisms can be broadly separated into two distinct groups, based on differential time scales: short-term acclimation allows the organism to adjust within minutes, while long-term acclimation involves response mechanisms that respond on time scales that vary from hours to days to months.

State transitions.

In terrestrial plants as well as green algae, light absorbed preferentially by PSII relative to PSI (state 2) leads to an overreduction of the PQ pool, whereas preferential excitation of PSI relative to PSII (state 1) results in oxidation of the PQ pool. The redox state of the PQ pool regulates a thylakoid protein kinase which controls the phosphorylation state of the peripheral Lhcb antenna polypeptides and energy transfer between PSII and PSI (4). The regulation of energy transfer by the redox state of the thylakoid PQ pool reflects a short-term photoprotective mechanism to counteract the potential for uneven absorption of light by PSI and PSII and to maintain maximum photosynthetic efficiency. Thus, the state transition is a dynamic mechanism that enables photoautotrophs as varied as cyanobacteria, green algae, and terrestrial plants to respond rapidly to changes in illumination.

By monitoring the increase in the 77 K chlorophyll a fluorescence emission at 720 nm associated with PSI relative to the decrease in PSII chlorophyll a fluorescence emission at 688 nm, Morgan-Kiss et al. (168) detected state I to state II transition in C. reinhardtii which was sensitive to the redox state of the PQ pool and was associated with the phosphorylation of the major LHCII polypeptides. Furthermore, the state I-state II transition in C. reinhardtii was inhibited by the protein kinase inhibitor staurosporine. In contrast, state I-state II transitions could not be detected in the psychrophilic C. raudensis UWO 241 (Fig. 12A), although reversible phosphorylation of one major LHCII polypeptide was observed (168) (Fig. 12B). It was concluded that the psychrophile is locked in state 1 and as a consequence is considered a “natural state transition mutant” (168). Thus, adaptation of C. raudensis to the unique environment of Lake Bonney has resulted in loss of the fundamental short-term photoprotective mechanism of state transitions.

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FIG. 12.

State transitions in the mesophilic C reinhardtii (left panel) versus the psychrophilic C. raudensis (right panel). A. The 77 K fluorescence emission spectra of whole cells exposed to state I (solid line) or state II (dotted line) conditions. B. Immunoblot of isolated thylakoid membranes with antiphosphothreonine antibodies to detect phosphorylation patterns in proteins of thylakoids exposed to state I (I) or state II (II) conditions. A state I response was induced by exposure of freshly harvested cells from an exponentially growing culture to far-red light for 15 min. A state II response was induced by incubation of samples in the dark under anaerobic conditions. (Modified from reference 168 with kind permission of Springer Science and Business Media.)

The cytochrome b_6f complex has been shown to play a critical role in the regulation of LHCII phosphorylation, and mutants of this complex are unable to undergo state transitions (5, 282). To test whether the altered structure and stability of cytochrome f of C. raudensis is a determining factor in the absence of state transitions in this psychrophile, a petA deletion mutant of C. reinhardtii WT.11 (FIBE ΔpetA) was complemented with the petA gene from C. raudensis (78). The C. reinhardtii mutant expressed a cytochrome f that exhibited an apparent molecular mass that was 7 kDa smaller rather than the molecular mass expected for the WT.11 cytochrome f. Despite this complementation, the transformant was still able to undergo state transitions comparable to that observed for WT.11.

Clearly, the alteration in the structure and stability of cytochrome f from C. raudensis is not a limiting factor regulating state transitions (79). As an alternate hypothesis, Morgan-Kiss et al. (168) proposed that adaptation of C. raudensis to the unique conditions of Lake Bonney has altered the structure and function of its photosynthetic apparatus such that the PSII and stromal flux of electrons through the intersystem electron transport chain are lowered relative to the rate of cyclic PSI electron transport compared to that of C. reinhardtii, which predisposes C. raudensis to remain in state 1.

Long-Term Photoacclimation

Composition and structure of light-harvesting complexes.

A major long-term photoacclimatory mechanism in green algae (55, 118, 155, 246) and the phycobilisomes of cyanobacteria (55, 77) involves the modulation of the physical size and composition of LHCII. Cyanobacteria have proven to be an excellent model system to elucidate the molecular mechanism regulating light-harvesting in response to nutrient limitations. Seminal research by Grossman and coworkers (76, 77) provided important insights into the global regulation of phycobilisome turnover in response to N and S limitations in Synechococcus spp. In addition, Huner and coworkers (159, 160) have observed in the filamentous cyanobacterium Plectonema boyanum that there is an inverse relationship between growth irradiance and light-harvesting antenna size regardless of growth temperature. Exposure to increased irradiance in cultures grown under either low (15°C) or moderate (29°C) temperatures results in a reduction in the rod lengths of the phycobilisomes. In addition, in response to higher irradiance levels, decreases in chlorophyll a content are associated with concomitant increases in the major carotenoid myxoxanthophyll.

Similar trends have been shown for photoacclimation in model mesophilic green algae such as Chlorella vulgaris (146, 275), Dunaliella tertiolecta (54, 246), and Dunaliella salina (82, 145, 248). Recently, it was shown that both the redox state of the PQ pool and the transthylakoid ΔpH act as signals in the retrograde regulation of nuclear gene expression of the Lhcb gene family and the subsequent polypeptide and pigment composition of LHCII in green algae (24, 54, 147, 148, 275).

The regulation of LHCII size in green algae and phycobilisomes in cyanobacteria is not a simple response to light intensity, since low growth temperature (5°C) mimics the effects of high light on the composition and function of LHCII (146, 148, 275, 276) and phycobilisomes (159, 160). Thus, photoacclimation reflects a response to excitation pressure rather than a response to either temperature or light per se. Thus, photostasis is attained upon exposure of green algae and cyanobacteria to high excitation pressure generated either by high light or low temperature through a decrease in the efficiency of light harvesting.

In contrast to the green algae C. vulgaris and D. salina and the cyanobacterium P. boryanum, C raudensis exhibits minimal changes in pigmentation LHCII abundance when grown at either low temperature or increasing irradiance (see Fig. 14A to C). Furthermore, unlike the mesophilic species Chlorella vulgaris, C. raudensis exhibited the ability to modulate growth rate in response to the higher growth irradiance (Fig. ​(Fig.9B,9B, Fig. 13D). This was a remarkable characteristic of the psychrophile, considering the light conditions were more than 10-fold higher than the natural light environment to which C. raudensis is adapted. This ability to increase growth rates correlates with an increase in photosynthetic energy utilization, and accounts for the lower excitation pressure exhibited in cultures of C. raudensis in comparison with cultures of the mesophilic species. Thus, on the basis of pigment composition and structural alteration of LHCII, C. raudensis may to respond to increased excitation pressure in a manner similar to wheat, rye, and Arabidopsis thaliana as well as Antarctic vascular plants (17, 279).

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FIG. 13.

Effect of a low-temperature growth regimen on pigmentation (A), abundance of thylakoid polypeptides (B), LHCII abundance (C), and growth and chlorophyll fluorescence parameters (D) in cultures of mesophilic (Chlorella vulgaris) and psychrophilic (Chlamydomonas raudensis UWO 241) green algae. A. Mid-log-phase cultures grown under regimens indicated above each culture(°C/μmol m⁻² s⁻¹) B. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of thylakoid polypeptides isolated from cultures grown under the above regimens C. Immunoblots of major light-harvesting polypeptides and Lhcb. D. Growth and fluorescence parameters of cultures. Growth rate is expressed as h⁻¹. Room-temperature chlorophyll a fluorescence parameter 1 − qP measured as growth temperature/light regimen.

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FIG. 14.

Putative model illustrating the functional changes in the organization of PSII as a survival strategy in populations of C. raudensis during the transition between winter and short Antarctic summer. Left panel, microbial populations exhibit cessation in growth during the transition from summer to winter. Cells exhibit minimal photosynthesis and high respiration rates. PSII is functionally down-regulated by the disconnection of major LHCII from the PSII core, and light is dissipated nonphotochemically as heat via an energy-dependent fluorescence quenching (q_E)-type mechanism. The PQ pool is largely reduced due to metabolically derived (e.g., starch breakdown) electron donors. In summer, excysting populations provide the seed cultures for active growth. Reassociation of oligomeric LHCII antenna with the PSII core allows rapid transition from energy-dissipative processes to efficient light energy capture and utilization during the short growing season. Respiration is low and starch reserves are replenished for the upcoming winter season.

Despite the ability of the psychrophile to maintain lower excitation pressure under lower temperature and higher irradiance conditions by increasing utilization of photosynthetic energy products (by increasing growth rates), functional changes in the light energy distribution between LHCII and PSII were observed at the level of 77 K fluorescence emission. Cultures grown under high light exhibited a rise in LHCII-associated fluorescence in comparison with cultures grown at the low-irradiance regimen (R. Morgan-Kiss, unpublished data). It is likely that the increase in LHCII fluorescence is related to a functional switch in LHCII from efficient energy transfer to PSII to nonradiative dissipation of excessive absorbed light energy.

We thank Alexander Ivanov and Marianna Król for their contributions to the research described as well as valuable discussion over the years.

This work was supported by the National Science and Engineering Research Council of Canada (NSERC) and the National Science Foundation (NSF).