Aberrant light sensing and motility in the green alga Chlamydomonas priscuii from the ice-covered Antarctic Lake Bonney.

The Antarctic green alga Chlamydomonas priscuii is an obligate psychrophile and an emerging model for photosynthetic adaptation to extreme conditions. Endemic to the ice-covered Lake Bonney, this alga thrives at highly unusual light conditions characterized by very low light irradiance (<15 µmol m-2 s-1), a narrow wavelength spectrum enriched in blue light, and an extreme photoperiod. Genome sequencing of C. priscuii exposed an unusually large genome, with hundreds of highly similar gene duplicates and expanded gene families, some of which could be aiding its survival in extreme conditions. In contrast to the described expansion in the genetic repertoire in C. priscuii, here we suggest that the gene family encoding for photoreceptors is reduced when compared to related green algae. This alga also possesses a very small eyespot and exhibits an aberrant phototactic response, compared to the model Chlamydomonas reinhardtii. We also investigated the genome and behavior of the closely related psychrophilic alga Chlamydomonas sp. ICE-MDV, that is found throughout the photic zone of Lake Bonney and is naturally exposed to higher light levels. Our analyses revealed a photoreceptor gene family and a robust phototactic response similar to those in the model Chlamydomonas reinhardtii. These results suggest that the aberrant phototactic response in C. priscuii is a result of life under extreme shading rather than a common feature of all psychrophilic algae. We discuss the implications of these results on the evolution and survival of shade adapted polar algae.

Temperature stress in psychrophilic green microalgae: Minireview.

Photosynthetic algae are the main primary producers in polar regions, form the basis of polar food webs, and are responsible for a significant portion of global carbon fixation. Many cold-water algae are psychrophiles that thrive in the cold but cannot grow at moderate temperatures (≥20°C). Polar regions are at risk of rapid warming caused by climate change, and the sensitivity of psychrophilic algae to rising temperatures makes them, and the ecosystems they inhabit, particularly vulnerable. Recent research on the Antarctic psychrophile Chlamydomonas priscuii, an emerging algal model, has revealed unique adaptations to life in the permanent cold. Additionally, genome sequencing of C. priscuii and its relative Chlamydomonas sp. ICE-L has given rise to a plethora of computational tools that can help elucidate the genetic basis of psychrophily. This minireview summarizes new advances in characterizing the heat stress responses in psychrophilic algae and examines their extraordinary sensitivity to temperature increases. Further research in this field will help determine the impact of climate change on psychrophiles from threatened polar environments.

A constitutive stress response is a result of low temperature growth in the Antarctic green alga Chlamydomonas sp. UWO241.

The Antarctic green alga Chlamydomonas sp. UWO241 is an obligate psychrophile that thrives in the cold (4-6°C) but is unable to survive at temperatures ≥18°C. Little is known how exposure to heat affects its physiology or whether it mounts a heat stress response in a manner comparable to mesophiles. Here, we dissect the responses of UWO241 to temperature stress by examining its growth, primary metabolome and transcriptome under steady-state low temperature and heat stress conditions. In comparison with Chlamydomonas reinhardtii, UWO241 constitutively accumulates metabolites and proteins commonly considered as stress markers, including soluble sugars, antioxidants, polyamines, and heat shock proteins to ensure efficient protein folding at low temperatures. We propose that this results from life at extreme conditions. A shift from 4°C to a non-permissive temperature of 24°C alters the UWO241 primary metabolome and transcriptome, but growth of UWO241 at higher permissive temperatures (10 and 15°C) does not provide enhanced heat protection. UWO241 also fails to induce the accumulation of HSPs when exposed to heat, suggesting that it has lost the ability to fine-tune its heat stress response. Our work adds to the growing body of research on temperature stress in psychrophiles, many of which are threatened by climate change.

Cyclic electron flow (CEF) and ascorbate pathway activity provide constitutive photoprotection for the photopsychrophile, Chlamydomonas sp. UWO 241 (renamed Chlamydomonas priscuii).

Under environmental stress, plants and algae employ a variety of strategies to protect the photosynthetic apparatus and maintain photostasis. To date, most studies on stress acclimation have focused on model organisms which possess limited to no tolerance to stressful extremes. We studied the ability of the Antarctic alga Chlamydomonas sp. UWO 241 (UWO 241) to acclimate to low temperature, high salinity or high light. UWO 241 maintained robust growth and photosynthetic activity at levels of temperature (2 °C) and salinity (700 mM NaCl) which were nonpermissive for a mesophilic sister species, Chlamydomonas raudensis SAG 49.72 (SAG 49.72). Acclimation in the mesophile involved classic mechanisms, including downregulation of light harvesting and shifts in excitation energy between photosystem I and II. In contrast, UWO 241 exhibited high rates of PSI-driven cyclic electron flow (CEF) and a larger capacity for nonphotochemical quenching (NPQ). Furthermore, UWO 241 exhibited constitutively high activity of two key ascorbate cycle enzymes, ascorbate peroxidase and glutathione reductase and maintained a large ascorbate pool. These results matched the ability of the psychrophile to maintain low ROS under short-term photoinhibition conditions. We conclude that tight control over photostasis and ROS levels are essential for photosynthetic life to flourish in a native habitat of permanent photooxidative stress. We propose to rename this organism Chlamydomonas priscuii.

Photosynthetic adaptation to polar life: Energy balance, photoprotection and genetic redundancy.

The persistent low temperature that characterize polar habitats combined with the requirement for light for all photoautotrophs creates a conundrum. The absorption of too much light at low temperature can cause an energy imbalance that decreases photosynthetic performance that has a negative impact on growth and can affect long-term survival. The goal of this review is to survey the mechanism(s) by which polar photoautotrophs maintain cellular energy balance, that is, photostasis to overcome the potential for cellular energy imbalance in their low temperature environments. Photopsychrophiles are photosynthetic organisms that are obligately adapted to low temperature (0°- 15 °C) but usually die at higher temperatures (≥20 °C). In contrast, photopsychrotolerant species can usually tolerate and survive a broad range of temperatures (5°- 40 °C). First, we summarize the basic concepts of excess excitation energy, energy balance, photoprotection and photostasis and their importance to survival in polar habitats. Second, we compare the photoprotective mechanisms that underlie photostasis and survival in aquatic cyanobacteria and green algae as well as terrestrial Antarctic and Arctic plants. We show that polar photopsychrophilic and photopsychrotolerant organisms attain energy balance at low temperature either through a regulated reduction in the efficiency of light absorption or through enhanced capacity to consume photosynthetic electrons by the induction of O2 as an alternative electron acceptor. Finally, we compare the published genomes of three photopsychrophilic and one photopsychrotolerant alga with five mesophilic green algae including the model green alga, Chlamydomonas reinhardtii. We relate our genomic analyses to photoprotective mechanisms that contribute to the potential attainment of photostasis. Finally, we discuss how the observed genomic redundancy in photopsychrophilic genomes may confer energy balance, photoprotection and resilience to their harsh polar environment. Primary production in aquatic, Antarctic and Arctic environments is dependent on diverse algal and cyanobacterial communities. Although mosses and lichens dominate the Antarctic terrestrial landscape, only two extant angiosperms exist in the Antarctic. The identification of a single 'molecular key' to unravel adaptation of photopsychrophily and photopsychrotolerance remains elusive. Since these photoautotrophs represent excellent biomarkers to assess the impact of global warming on polar ecosystems, increased study of these polar photoautotrophs remains essential.

Draft genome sequence of the Antarctic green alga Chlamydomonas sp. UWO241.

Antarctica is home to an assortment of psychrophilic algae, which have evolved various survival strategies for coping with their frigid environments. Here, we explore Antarctic psychrophily by examining the ∼212 Mb draft nuclear genome of the green alga Chlamydomonas sp. UWO241, which resides within the water column of a perennially ice-covered, hypersaline lake. Like certain other Antarctic algae, UWO241 encodes a large number (≥37) of ice-binding proteins, putatively originating from horizontal gene transfer. Even more striking, UWO241 harbors hundreds of highly similar duplicated genes involved in diverse cellular processes, some of which we argue are aiding its survival in the Antarctic via gene dosage. Gene and partial gene duplication appear to be an ongoing phenomenon within UWO241, one which might be mediated by retrotransposons. Ultimately, we consider how such a process could be associated with adaptation to extreme environments but explore potential non-adaptive hypotheses as well.

Chlamydomonas sp. UWO 241 Exhibits High Cyclic Electron Flow and Rewired Metabolism under High Salinity.

The Antarctic green alga Chlamydomonas sp. UWO 241 (UWO 241) is adapted to permanent low temperatures, hypersalinity, and extreme shade. One of the most striking phenotypes of UWO 241 is an altered PSI organization and constitutive PSI cyclic electron flow (CEF). To date, little attention has been paid to CEF during long-term stress acclimation, and the consequences of sustained CEF in UWO 241 are not known. In this study, we combined photobiology, proteomics, and metabolomics to understand the underlying role of sustained CEF in high-salinity stress acclimation. High salt-grown UWO 241 exhibited increased thylakoid proton motive flux and an increased capacity for nonphotochemical quenching. Under high salt, a significant proportion of the up-regulated enzymes were associated with the Calvin-Benson-Bassham cycle, carbon storage metabolism, and protein translation. Two key enzymes of the shikimate pathway, 3-deoxy-d-arabinoheptulosonate 7-phosphate synthase and chorismate synthase, were also up-regulated, as well as indole-3-glycerol phosphate synthase, an enzyme involved in the biosynthesis of l-Trp and indole acetic acid. In addition, several compatible solutes (glycerol, Pro, and Suc) accumulated to high levels in high salt-grown UWO 241 cultures. We suggest that UWO 241 maintains constitutively high CEF through the associated PSI-cytochrome b 6 f supercomplex to support robust growth and strong photosynthetic capacity under a constant growth regime of low temperatures and high salinity.

Presence and absence of light-independent chlorophyll biosynthesis among Chlamydomonas green algae in an ice-covered Antarctic lake.

The cold, permanently ice-covered waters of Lake Bonney, Antarctica, may seem like an uninviting place for an alga, but they are home to a diversity of photosynthetic life, including Chlamydomonas sp. UWO241, a psychrophile residing in the deep photic zone. Recently, we found that UWO241 has lost the genes responsible for light-independent chlorophyll biosynthesis, which is surprising given that this green alga comes from a light-limited environment and experiences extended periods of darkness during the Antarctic winter. Why discard such a process? We argued that it might be linked to the very high dissolved oxygen concentration of Lake Bonney at the depth at which UWO241 is found. Oxygen is the Achilles' heel of the key enzyme involved in light-independent chlorophyll biosynthesis: DPOR. If this hypothesis is true, then other algae in Lake Bonney should also be susceptible to losing DPOR, such as Chlamydomonas sp. ICE-MDV, which predominantly resides in the chemocline, a depth with an even higher oxygen concentration than that where UWO241 exists. Here, we report that, contrary to our earlier prediction, ICE-MDV has maintained the genes encoding DPOR. We briefly discuss the implications of this finding in relation to the loss of light-independent chlorophyll synthesis in UWO241.

Cold-Adapted Protein Kinases and Thylakoid Remodeling Impact Energy Distribution in an Antarctic Psychrophile.

The Antarctic psychrophile Chlamydomonas sp. UWO241 evolved in a permanently ice-covered lake whose aquatic environment is characterized not only by constant low temperature and high salt but also by low light during the austral summer coupled with 6 months of complete darkness during the austral winter. Since the UWO241 genome indicated the presence of Stt7 and Stl1 protein kinases, we examined protein phosphorylation and the state transition phenomenon in this psychrophile. Light-dependent [γ-33P]ATP labeling of thylakoid membranes from Chlamydomonas sp. UWO241 exhibited a distinct low temperature-dependent phosphorylation pattern compared to Chlamydomonas reinhardtii despite comparable levels of the Stt7 protein kinase. The sequence and structure of the UWO241 Stt7 kinase domain exhibits substantial alterations, which we suggest predisposes it to be more active at low temperature. Comparative purification of PSII and PSI combined with digitonin fractionation of thylakoid membranes indicated that UWO241 altered its thylakoid membrane architecture and reorganized the distribution of PSI and PSII units between granal and stromal lamellae. Although UWO241 grown at low salt and low temperature exhibited comparable thylakoid membrane appression to that of C. reinhardtii at its optimal growth condition, UWO241 grown under its natural condition of high salt resulted in swelling of the thylakoid lumen. This was associated with an upregulation of PSI cyclic electron flow by 50% compared to growth at low salt. Due to the unique 77K fluorescence emission spectra of intact UWO241 cells, deconvolution was necessary to detect enhancement in energy distribution between PSII and PSI, which was sensitive to the redox state of the plastoquinone pool and to the NaCl concentrations of the growth medium. We conclude that a reorganization of PSII and PSI in UWO241 results in a unique state transition phenomenon that is associated with altered protein phosphorylation and enhanced PSI cyclic electron flow. These data are discussed with respect to a possible PSII-PSI energy spillover mechanism that regulates photosystem energy partitioning and quenching.

Characterization of photosynthetic ferredoxin from the Antarctic alga Chlamydomonas sp. UWO241 reveals novel features of cold adaptation.

The objective of this work was to characterize photosynthetic ferredoxin from the Antarctic green alga Chlamydomonas sp. UWO241, a key enzyme involved in distributing photosynthetic reducing power. We hypothesize that ferredoxin possesses characteristics typical of cold-adapted enzymes, namely increased structural flexibility and high activity at low temperatures, accompanied by low stability at moderate temperatures. To address this objective, we purified ferredoxin from UWO241 and characterized the temperature dependence of its enzymatic activity and protein conformation. The UWO241 ferredoxin protein, RNA, and DNA sequences were compared with homologous sequences from related organisms. We provide evidence for the duplication of the main ferredoxin gene in the UWO241 nuclear genome and the presence of two highly similar proteins. Ferredoxin from UWO241 has both high activity at low temperatures and high stability at moderate temperatures, representing a novel class of cold-adapted enzymes. Our study reveals novel insights into how photosynthesis functions in the cold. The presence of two distinct ferredoxin proteins in UWO241 could provide an adaptive advantage for survival at cold temperatures. The primary amino acid sequence of ferredoxin is highly conserved among photosynthetic species, and we suggest that subtle differences in sequence can lead to significant changes in activity at low temperatures.

In planta analysis of leaf mitochondrial superoxide and nitric oxide.

Superoxide (O2(-)) and nitric oxide (NO) are produced within plant mitochondria and may have signaling functions within the cell. Here we describe semiquantitative fluorescence imaging-based approaches to estimate the mitochondrial amount of these reactive and short-lived species within intact leaf tissue. We also outline a biochemical method using oxyhemoglobin to measure NO within a whole leaf tissue extract. This quantitative method, while not specifically evaluating mitochondrial localized NO, does nonetheless provide another independent measure of NO that can be useful.

Knockdown of mitochondrial alternative oxidase induces the 'stress state' of signaling molecule pools in Nicotiana tabacum, with implications for stomatal function.

The mitochondrial electron transport chain (ETC) includes an alternative oxidase (AOX) that may control the generation of reactive oxygen species (ROS) and reactive nitrogen species (RNS). ROS and RNS act as signaling intermediates in numerous plant processes, including stomatal movement. The role of AOX in controlling ROS and RNS concentrations under both steady-state and different stress conditions was evaluated using Nicotiana tabacum plants lacking AOX as a result of RNA interference. A potential functional implication of changes in ROS and RNS homeostasis was also evaluated by examining stomatal function. The leaves of nonstressed AOX knockdowns maintained concentrations of H2O2 and nitric oxide (NO) normally seen in wildtype plants only under stress conditions. Further, guard cell NO amounts were much higher in knockdowns. These guard cells were altered in size and were less responsive to NO as a signal for stomatal closure. This, in turn, compromised the stomatal response to changing irradiance. The results reveal a role for AOX in stomata. A working model is that guard cell AOX respiration maintains NO homeostasis by preventing over-reduction of the ETC, particularly during periods when high concentrations of NO acting as a signal for stomatal closure may also be inhibiting cyt oxidase respiration.

Alternative oxidase impacts the plant response to biotic stress by influencing the mitochondrial generation of reactive oxygen species.

Previously, we showed that inoculation of tobacco with Pseudomonas syringae incompatible pv. maculicola results in a rapid and persistent burst of superoxide (O(2) (-) ) from mitochondria, no change in amount of mitochondrial alternative oxidase (AOX) and induction of the hypersensitive response (HR). However, inoculation with incompatible pv. phaseolicola resulted in increased AOX, no O(2) (-) burst and no HR. Here, we show that in transgenic plants unable to induce AOX in response to pv. phaseolicola, there is now a strong mitochondrial O(2) (-) burst, similar to that normally seen only with pv. maculicola. This interaction did not however result in a HR. This indicates that AOX amount is a key determinant of the mitochondrial O(2) (-) burst but also that the burst itself is not sufficient to induce the HR. Surprisingly, the O(2) (-) burst normally seen towards pv. maculicola is delayed in plants lacking AOX. This delay is associated with a delayed HR, suggesting that the burst does promote the HR. A O(2) (-) burst can also be induced by the complex III inhibitor antimycin A (AA), but is again delayed in plants lacking AOX. The similar mitochondrial response induced by pv. maculicola and AA suggests that electron transport is a target during HR-inducing biotic interactions.

The signaling role of a mitochondrial superoxide burst during stress.

Plant mitochondria are proposed to act as signaling organelles in the orchestration of defense responses to biotic stress and acclimation responses to abiotic stress. However, the primary signal(s) being generated by mitochondria and then interpreted by the cell are largely unknown. Recently, we showed that mitochondria generate a sustained burst of superoxide (O 2(-)) during particular plant-pathogen interactions. This O 2(-) burst appears to be controlled by mitochondrial components that influence rates of O 2(-) generation and scavenging within the organelle. The O 2(-) burst appears to influence downstream processes such as the hypersensitive response, indicating that it could represent an important mitochondrial signal in support of plant stress responses. The findings generate many interesting questions regarding the upstream factors required to generate the O 2(-) burst, the mitochondrial events that occur in support of and in parallel with this burst and the downstream events that respond to this burst.

Alternative oxidase modulates leaf mitochondrial concentrations of superoxide and nitric oxide.

• The nonenergy-conserving alternative oxidase (AOX) has been hypothesized to modulate the amount of reactive oxygen species (ROS) and reactive nitrogen species (RNS) in plant mitochondria but there is sparse direct in planta evidence to support this. • Laser scanning fluorescent confocal microscopy and biochemical methods were used to directly estimate in planta leaf concentrations of superoxide (O2(-)), nitric oxide (NO), peroxynitrite (ONOO(-)) and hydrogen peroxide (H(2)O(2)) in wildtype (Wt) tobacco (Nicotiana tabacum) and transgenic tobacco with altered amounts of AOX. • We found that plants lacking AOX have increased concentrations of leaf mitochondrial-localized O2(-) and leaf NO in comparison to the Wt, while leaf concentrations of H(2)O(2) were similar or lower in the AOX-suppressed plants. • Based on our results, we suggest that AOX respiration acts to reduce the generation of ROS and RNS in plant mitochondria by dampening the leak of single electrons from the electron transport chain to O(2) or nitrite. This may represent a universal role for AOX in plants. More work is now needed to establish the functional implications of this role, such as during abiotic and biotic stress.

Coordination of a mitochondrial superoxide burst during the hypersensitive response to bacterial pathogen in Nicotiana tabacum.

We characterized responses of Nicotiana tabacum to pathovars of the bacterial pathogen Pseudomonas syringae. These included a compatible response associated with necrotic cell death (pv. tabaci), an incompatible response that included hypersensitive response (HR) cell death (pv. maculicola) and an incompatible response that induced defences but lacked the HR (pv. phaseolicola). Signalling molecules (salicylic acid, nitric oxide, H(2)O(2)) known to induce the stress responsive tobacco Aox1a gene [that encodes the mitochondrial electron transport chain (ETC) component alternative oxidase (AOX)] accumulated preferentially during the HR, but this did not elevate Aox1a transcript or AOX protein, while the transcript and protein were strongly elevated during the defence response to pv. phaseolicola. In addition, matrix manganese superoxide dismutase (MnSOD) activity declined during the HR, unlike its response to the other pathovars, and unlike the response of other superoxide dismutase (SOD) enzymes. Finally, the HR (but not the response to pv. phaseolicola or pv. tabaci) was accompanied by an early and persistent mitochondrial superoxide (O(2)(-)) burst prior to cell death. We propose that a coordinated response of the major ETC mechanism to avoid O(2)(-) generation (AOX) and the sole enzymatic means to scavenge mitochondrial O(2)(-) (MnSOD) is important in the determination of cell fate during responses to pathogen.

Is the maintenance of homeostatic mitochondrial signaling during stress a physiological role for alternative oxidase?

All plants maintain a non-energy-conserving pathway of mitochondrial electron transport referred to as alternative oxidase (AOX) respiration. Here, we briefly review some of the most prevailing themes for the metabolic and physiological roles of this respiratory pathway. Many of these themes relate to the potential of AOX to provide metabolic homeostasis in response to fluctuating cellular conditions, such as is often seen during stress. We then review reverse genetic experiments that have been used to test these hypotheses. To date, such experiments have been limited to just two dicot species and have only targeted one member (a stress-induced member) of the AOX multigene family. Nonetheless, the experiments to date strongly reinforce the idea that AOX respiration is of particular importance during abiotic and biotic stress. Finally, we propose that another core role of AOX may be to modulate the strength of a stress-signaling pathway from the mitochondrion that controls cellular responses to stress. In this way, AOX could be acting to provide a degree of signaling homeostasis from the mitochondrion. This hypothesis may provide explanation for some of the disparate results seen in reverse genetic experiments regarding the impact of AOX on the reactive oxygen network and oxidative damage.