Photokinetic adaptation of sea-ice algae

Brief description

Metadata record for data from ASAC Project 2702
See the link below for public details on this project.

Sea-ice algae are the basis of the Antarctic food web and are essential for healthy functioning of the Antarctic ecosystem. These algae exploit a unique niche within this extreme environment. Using advanced photosynthetic analysis we will examine the mechanisms which influence the productivity of sea-ice algae.

The objective of this project is to understand the processes of light acclimation and photo-protection employed by sea-ice algae under extremely low temperature conditions. Several new hypotheses have been proposed in a recent review of low temperature acclimation of higher plants (Oquist and Huner, 2003). To further understand the remarkable tolerance of sea-ice algae to photoinhibition, we propose to test several of these hypotheses. Sea-ice algae fix inorganic carbon that forms the basis of the Southern Ocean food web. Sea ice covers up to 20 million km2 of the Southern Ocean each year. Global climate change will decrease the sea-ice thickness and distribution (IPCC, 2001); however subtle changes in temperature and light penetration will also have profound negative impacts on the photosynthetic efficiency of the sea-ice microalgae before any macroscale changes take place. Sea-ice algae are essentially the only food source for invertebrates and fish for up to nine months of the year. During winter and spring, krill (Euphausia sp.) have been observed feeding directly on sea-ice algae. Further, changes in sea-ice productivity will have a cascade effect further up the food web. Therefore, understanding how physical driving forces (temperature and light) affect sea-ice algae productivity will be critical to our ability to predict the effects of climate change and sustainably manage this unique and vulnerable ecosystem.

Our primary objective is: To understand the processes of light acclimation and photo-protection employed by sea-ice algae under extremely low temperature conditions, with an aim to better understanding the potential implications of global climate change on the Antarctic sea-ice ecosystem.

Lineage

Progress Code: completed

Statement: Taken from the 2005-2006 Progress Report:

This objective will be met by addressing the following questions:
1) How does light and temperature stress interact in relation with the photosynthesis of sea-ice algae?
- Do all species partition photon energy in a similar way between photochemical quenching (qP) and non-photochemical quenching (qN) when exposed to light and temperature stress?
2) Does low temperature exacerbate light stress in sea-ice algae?
- Do changes in temperature of these cryophilic cells cause the photosystems to alter their efficiency and therefore become photoinhibited?
- Does the composition and organisation of PSII apparatus change with cold acclimation?
3) How do sea-ice algae use energy dissipation pathways to protect themselves?
- Do sea-ice algae use non-photochemical quenching as a permanent energy dissipation mechanism, as opposed to a dynamic photoprotective mechanism?
- Does light and/or temperature stress correlate with particular components of non-photochemical quenching processes (qE/qT/qI)?
- Does state-transition quenching increase at lower temperatures?
4) Do sea ice algae use alternate protective mechanisms, apart from qN?
- How important is PSI cyclic electron transport (Mehler) and photorespiration in low temperature stress?

Approach:
Experiment 1: Partitioning of quenching under light/temperature manipulations
Photosynthesis of sea-ice algae is strongly influenced by light and temperature (Rochet et al 1985; Palmisano et al 1987). At low temperatures the photosynthetic apparatus (enzyme systems) functions slowly, so light can easily damage the photosystems due to the formation of free radicals. All plants attempt to achieve photostasis, where incoming photon energy is carefully balanced between maximising photosynthesis, without allowing excess energy to cause damage to the photosystems. As such, energy absorbed from irradiance can be used in photosynthesis (photochemical quenching, qP) or protectively dissipated as excess heat (non-photochemical quenching, qN). Preliminary evidence would suggest that sea-ice algae use qN to prevent excess light from damaging the photosynthetic apparatus; however the regulation of qN seems inconsistent (Ralph et al unpubl).

Photosynthetic capacity of sea-ice algae will be measured after exposure to a series of light/temperature combinations. Treatments will include light (0, 10, 50, 100, 200, 400 mmol photons m-2 s-1) and temperature (-1.8, 0 and 2 degrees C). A temperature controlled water bath will be used with 24 incubation chambers having a range of neutral density filters (0 - 400 mmol photon m-2 s-1). Four replicate samples of sea-ice algae will be exposed to the combination of light and temperature treatments (two factor random block ANOVA design). Samples will include pure cultures of Fragilariopsis sp., Polarella glacialis and Entomoneis kjellmannii (provided by McMinn).

High-resolution fluorescence measurements will be performed using a Water-PAM (Walz, Effeltrich Germany) to assess photosynthetic capacity of cells. We will perform two styles of analysis; short-term adaptation to light variation (rapid light curve: RLC) and quenching analysis (qP and qN). A number of photosynthetic indicators will be measured in association with Rapid Light Curves; these include a (photosynthetic efficiency below saturating light), Ek (minimum saturating irradiance), rETRmax (maximum relative electron transport rate) and Yi (initial effective quantum yield of PSII). For further details see Ralph et al (2002). To identify the relative importance of qP and qN, we will run a series of steady state light curves (a series of quenching analysis curves at increasing irradiance), where the cells are exposed to irradiance levels for 10 min before assessment of qP and qN. The irradiance levels tested will include; 0, 20, 50, 100, 200, 400, 800 mmol photon m-2 s-1 at temperatures of -1.8, 0 and 2 degrees C.

Photosynthetic capacity of sea-ice microalgae is more strongly influenced by temperature, than by light (Rochet et al (1995). This finding is confirmed by our own measurements? (Ralph et al unpubl). Usually, non-photochemical processes (qN) prevent photoinhibition at low temperature; however we have found microalgae were able to maintain photosynthetic activity of a wide range of temperatures without excessive use of qN. High levels of qN could result in inactivated PSII reaction centres functioning as heat sinks, which could theoretically use light energy to provide heat which improves metabolic activity. It is anticipated that sensitive species (Nitzchia stelleta) will have limited capacity for modulating the qN, whereas robust species (such as Fragilariopsis sp and Polarella glacialis) will use qN to tolerate a wide range of light and temperature conditions.

Experiment 2: Does low temperature exacerbate light stress
Huner et al (1993) suggested that microalgae which acclimate to low temperature (non-freezing) and moderate irradiance could acquire resistance to irradiance up to 5 to 6 times growth irradiance. This response appears to be linked to the photosynthetic apparatus having the ability to maintain the primary electron acceptor (QA) in an oxidised form. However, once Photosystem II is overwhelmed by either thermal stress or excess light, qN is unable to re-oxidise QA resulting in accumulation of reduced QA, thus decreasing photochemical efficiency (FPSII). The redox state of QA appears to be a factor that could differentiate species sensitivity to low-temperature photoinhibition. Successful species may keep QA oxidised (or prevent over-reduction of QA) under low-temperature and high-light conditions. Tolerance to photoinhibition can also occur by reducing the proportion of active PSII reaction centres (PSIIA), while inactive centres (PSIIA) become energy sinks to protect the remaining photosynthetic apparatus (Oquist and Huner, 2003).

We will use fast induction kinetics to measure the activity of early responses (millisecond) of photosynthesis, which can infer the redox state of QA and PQ. The QA redox state will be examined under both light-limited and saturated light conditions (20 and 200 mmol photon m-2 s-1, respectively). Four replicate samples of cultured sea-ice algae for each treatment will be measured. We postulate that the primary electron acceptor (QA) and oxygen-evolving complex (OEC) are the most sensitive components of PSII to low-temperature photoinhibition; however the pool size of plastoquinone (PQ) could also be sensitive to freezing stress. Preliminary data (Ryan et al unpubl.) indicated that oxygen production was reduced by thermal/light stress; however this has not been linked to biophysical changes in OEC.

Experiment 3: Energy dissipation protection at low temperature
Once we have identified the overall importance of qN (objective 1), we will subdivide the qN response into 3 distinct components (linked to specific biochemical processes); (1) photoinhibitory quenching (qI); (2) state transition quenching (qT); and (3) energy-dependent quenching (qE). Photoinhibitory quenching usually occurs once the photosystems are damaged, whereas state transition quenching is very important in fluctuating light conditions to optimise the distribution of light between PSII and PSI, while energy-dependent quenching is usually linked to xanthophylls pigment protection of photosystems. It is anticipated that qE will increase with lower temperatures, as effective quantum yield decreases (Horton and Hague, 1988; Hill et al. in review). We also anticipate that qT will be strongly modulated under environmental light fluctuations. To identify each component of qN, it will be necessary to block electron transport chain using a range of photosynthetic inhibitors. The complete qN process will be blocked using dithiotreitol (DTT ~1 mM), while DCMU (~10 mM) will inhibit only energy-dependent quenching and state transition quenching is blocked with sodium fluoride (NaF, 50 mM). We are not sure of the concentrations and time of exposure required for these microalgae, however based on temperate species; this should be in the range of 10 mM - 10 mM (Neidhardt et al, 1998).

We will examine the relationship between the epoxidation state of the xanthophyll pigments and relative importance of qI quenching. Samples will be collected at the end of each incubation, and held in liquid nitrogen (-196 degrees C) until pigment analysis (HPLC) is performed at AAD (in collaboration with Dr S. Wright). We will measure total xanthophyll pool size, as well as the specific xanthophyll pigments such as zeaxanthin, violaxanthin, diadinoxanthin, diatoxanthin and fucoxanthin. The relative changes in these pigments will indicate whether sea-ice algae are able to modulate their xanthophyll cycle, or whether they remain in a semi-permanent state of epoxidation, as suggested from preliminary qN data (Ralph unpubl).

Experiment 4: Alternate photoprotective mechanisms
Preliminary data from the 2002 voyage 1 suggests that the oxygen-evolving complex (OEC) of photosystem II of sea-ice algae is sensitive to thermal stress (Ralph et al submitted). We will use both fluorescence and oxygen measurements to examine low temperature energy dissipation. Schroeter et al (1995) found poor correlation between fluorescence and oxygen production in Antarctic moss and lichen, and attributed it to increased symbiont respiration. This implies that electron transport and the oxygen evolution process become uncoupled under low temperature stress. A number of alternate explanations for these observations could include energy dissipation processes such as Mehler Ascorbate Peroxidase reaction (MAP; PSI cyclic electron transport) and photorespiration (occurs under low CO2, high O2 conditions), which both consume oxygen whilst electron transport continues (Huner et al 1993). MAP can be used as the terminal electron acceptor for PSI.

To identify MAP activity we will perform quenching analysis under moderate light (200 mmol photon m-2 s-1) to generate a qN response, then re-run the same analysis in the absence of oxygen (5 min under N2 gas bubbling). If qN and PSII collapse in the absence of O2, then MAP was responsible for the oxygen-dependent electron flow (Jones et al 1998). We will use gas manipulations (N2 and O2) to identify the activity of Mehler reaction and inhibitors (salicyclhydroxamic acid, 1.5 mM) to examine the impact of photorespiration. Once again the concentration and exposure period needed for these inhibitors to impact these polar microalgae is unknown.

Project overview:
This project will provide new insight into the influence of physical drivers on sea-ice productivity. Given global climate change, temperature and light conditions will change the rates of photosynthesis of all autotrophs. We are specifically examining whether the occurrence of low temperature photoinhibition when environmental conditions become unfavourable for photosynthesis, such that normal levels of absorbed light can become excessive and result in damage to the photosynthetic apparatus. Cells respond in a number of ways; (1) down-regulation where by photosystem II (PSII) reaction centres are closed and excess energy is dissipated as heat (non-photochemical quenching; qN), and (2) cyclic electron transport, or (3) if (1) and (2) fail to protect the cell, then photooxidative damage can occur. Non-photochemical quenching is mediated by an accessory pigment system called the xanthophyll cycle. Low temperature photoinhibition occurs as cells balance the energy flow between the temperature-insensitive photochemical processes and the temperature-sensitive biochemical processes. Ideally, a cell will maintain photostasis; however under unfavourable conditions an imbalance will occur in the reduction state of the primary plastoquinone (QA) as it is the slowest step in the PSII electron transport chain. An increase in QA reduction could be linked to either high-light stress or low temperature stress resulting in lower metabolic activity.

Public summary of the season progress:
We had a partially successful season. Two projects (Objective 1 and 3) were attempted during V1 on Aurora Australis. it took several days ship time before we had sorted out technical problems with the light and temperature experiments (Objective 1). We eventually modified the fluid dynamics within the experimental cuvette to measure the response of sea-ice algae to pharmacological manipulations to measure photoprotective responses (qI/qT/qE; Objective 3). Experiments at UTS have not been able to progress due to the delays in delivery of the 5C incubator (arrived Feb 06). We have commenced work on experimental manipulations.

Variations to work plan or objectives:
Objective 2 was attempted in conjunction with objective 1; however we generally found insufficient algae to be concentrated to allow the high-resolution fluorometer to detect the fluorescence in the samples. We focussed our attention on successfully completing objective 1.
Objective 4 was not attempted after substantial time was committed to objective 1-3. We have only recently been able to install a 5C incubator at UTS. Once sea-ice cultures are able to be regularly maintained within this facility, I will attempt to examine the Mehler pathway under controlled conditions at UTS, in preparation for 2006/7 field season.