Professor Peter J Ralph

Christian Wilhelm

Johann Lavaud

Torsten Jakob

Katherina Petrou

Sven Kranz

Chapter 12: Fluorescence as an assay to understand aspects of the physiology of light regulation

Keywords: inorganic carbon DBMIB, 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone DCMU minimum fluorescence FQR, ferredoxin-plastoquinone reductase FTIR, Fourier transformed infra-red GA, glycolaldehyde LHC, light-harvesting antenna -transition quenching SL, sine light XC, xanthophyll cycle ZX, zeaxanthin

ADP: adenine diphosphate ADRY: accelerators of deactivation reactions of the water-splitting system Y ATP: adenine triphosphate AX: antheraxanthin Ci:

Introduction

The physiological state of a chloroplast is strongly influenced by both biotic and abiotic conditions. Unfavourable growth conditions lead to photosynthetic stress. Chlorophyll a fluorescence is a widely used probe of photosynthetic activity (specifically PSII), and therefore stress which specifically targets the electron transport pathway and associated alternative electron cycling pathways. By manipulating the processes which control photosynthesis, altering the chlorophyll a fluorescence can yield detailed insight into the biochemical pathways controlling these physiological responses. Light which is captured by a chlorophyll molecule can be used by three competing processes; electron transport, energy dissipation (via heat) and chlorophyll a fluorescence emission. Excess light causes a range of photoprotective biochemical responses. Electrons produced by water-splitting are not all used in carbon fixation; if the incident irradiance generates more electrons than the dark reactions can use in carbon fixation, otherwise damage will occur to the photosynthetic apparatus. If carbon fixation is inhibited by temperature or reduced inorganic carbon (Ci), ATP or NADPH availability, then the photosystem dynamically adjusts and uses alternate sinks for electrons, such as molecular oxygen (water-water cycle or Mehler ascorbate peroxidase reaction). In this chapter, we describe the processes controlling electron transport as influenced by light and lightinduced stress. The process of stress acclimation leads to a number of photoprotective pathways and we describe how inhibitors can be used to identify these particular processes.

VERSION 2 The physiological state of a chloroplast is strongly influenced by both biotic and abiotic conditions. Unfavourable growth conditions lead to photosynthetic stress. Chlorophyll a fluorescence is a widely used probe of photosynthetic activity (specifically PSII), and therefore stress which specifically targets the electron transport pathway and associated alternative electron cycling pathways. By manipulating the processes that control photosynthesis, affecting the chlorophyll a fluorescence which yields detailed insight into the biochemical pathways. Light that is captured by a chlorophyll molecule can be utilised in three competing processes; electron transport, energy dissipation (via heat) and chlorophyll a fluorescence emission. Electrons produced by water-splitting are not always used in carbon fixation; if the incident irradiance generates more electrons than the dark reactions can use in carbon fixation, otherwise damage will occur to the photosynthetic apparatus. If carbon fixation is inhibited by temperature or reduced inorganic carbon (Ci), ATP or NADPH availability, then the photosystem dynamically adjusts and uses alternate sinks for electrons, such as molecular oxygen (water-water cycle or Mehler ascorbate peroxidase reaction). The process of stress acclimation leads to a number of photoprotective pathways and we describe how inhibitors can be used to identify these particular processes. In this chapter, we describe the processes controlling electron transport as influenced by light and light-induced stress.

Electron usage in photosynthesis

Photosynthesis drives the light reactions which ultimately lead to carbon fixation; however predicting photosynthetic rates from fluorescence is a complex issue. As outlined in other chapters of this book (Laney & Oxenborough, 2008;Suggett et al., 2008) different fluorescence tools are available to measure the electron flow through Photosystem II (PSII). The quantum yield of PSII can be multiplied by the amount of absorbed quanta which can be obtained from the incident light and either the PSII absorption cross section or the spectral overlap between the light spectrum and the insitu absorption spectra. From these data, the electron transport rate per chlorophyll molecule over time can be assessed for an entire day to determine the daily primary production [START_REF] Wagner | Balancing the energy flow from captured light to biomass under fluctuating light conditions[END_REF]. However, growth and photosynthesis are rarely equivalent. Electrons transported by PSII can follow several competing pathways: the majority of the electrons are normally used to reduce CO2 to carbohydrates, allowing the synthesis of other cellular macromolecules like proteins, lipids or nucleotides, but some of them might be lost by alternate cellular processes (see alternate electron cycling) or dissipated (non-photochemical quenching). Therefore, the ratio of electron per carbon incorporated into the biomass may vary tremendously, either by losses or by the synthesis of highly reduced biomolecules like proteins or lipids.

Alternative electron cycling (AEC)

In principle, PSII electron transport rates should match the gross rates of oxygen evolution. Studies have tried to verify this assumption with divergent results [START_REF] Falkowski | Relationship of steady-state photosynthesis to fluorescence in eukaryotic algae[END_REF][START_REF] Kolber | Measurements of variable chlorophyll fluorescence using fast repetition rate techniques: defining methodology and experimental protocols[END_REF][START_REF] Suggett | Assessment of photosynthesis in a spring cyanobacterial bloom by use of a fast repetition rate fluorometer[END_REF][START_REF] Jakob | Estimation of chlorophyll content and daily primary production of the major algal groups by means of multiwavelength-excitation PAM chlorophyll fluorometry: performance and methodological limits[END_REF]. It was shown that linearity between PSII electron transport and oxygen evolution can be found, but non-linear behaviour was also observed, especially under conditions when photosynthesis was over-saturated (excess irradiance). These experiments indicated that PSII electron transport might over-estimate the primary production under some conditions, because oxygen evolution rates were found to be lower than PSII electron flow [START_REF] Gilbert | Bio-optical modelling of oxygen evolution using in-vivo fluorescence: comparison of measured and calculated P-I curves in four representative phytoplankton species[END_REF]. Several explanations for this disparity have been suggested: a. cyclic electron flow around PSII [START_REF] Prasil | Cyclic electron flow around PSII in vivo[END_REF][START_REF] Lavaud | Influence of the diadinoxanthin pool size on photoprotection in a marine planktonic diatom Phaeodactylum tricornutum[END_REF], b. water-water cycle [START_REF] Asada | The water-water cycle in chloroplasts: Scavenging of Active Oxygens and Dissipation of Excess Photons[END_REF] where oxygen uptake on the acceptor side of PSI leads to superoxide which is then dismutated to H2O2 and then detoxified to water and, c. cyclic electron flow around PSI (Bendall & Manesse, 1996).

These processes can be summarised as alternative electron cycling (AEC) which are not energetic losses (such as non-photochemical quenching: NPQ), because at least the water-water cycle and the cyclic flow around PSI generate a proton gradient which can be used for additional ATP synthesis. Therefore, it is suggested that alternative electron cycling is a normal stress response and might be of less importance under balanced growth conditions. Recently, [START_REF] Wagner | Balancing the energy flow from captured light to biomass under fluctuating light conditions[END_REF] described an experimental setup to estimate the alternative electron cycling activity by comparing the electron flow through PSII with oxygen evolution relative to the amount of absorbed quanta. The result is shown in Figure 1.

Obviously, at low light intensities in the morning and in the late afternoon, the fluorescence-based electron transport rates closely match the oxygen evolution rates as measured by a Clark-type electrode, whereas at high light intensities the "alternative electron cycling" can account for up to 40% of the fluorescence-based electron transport. This mismatch is not due to inappropriate measuring techniques, but to the physiological variability between linear and alternative electron pathways across the photosynthetic membrane. Interestingly, the ratio of linear to alternative electron cycling is not only light-dependent, but can be linked to species-specific physiological regulation, as shown in Figure 2.

When light pulse frequency is manipulated, the relationship between electron transport and oxygen evolution is further altered. In the sine light (SL) climate which simulates a sunny day, the ratio PAM/oxygen is always higher than in the exponentially fluctuating light (FL) climate (Fig. 2), when the light intensity oscillates with a frequency of half an hour between the maximum value and zero. The green alga Chlorella vulgaris performs much more alternative electron transport than the diatom Phaeodactylum tricornutum under both light conditions (FL and SL). In the sine light where the cells are exposed to a photon flux which exceeds the capacity of the Calvin cycle, the alternative electron cycling is highest indicating, that it can act as a photoprotective mechanism which compliments other photoprotective processes. Given that alternative electron cycling in the green alga was higher than in the diatom, this corresponds with the observation that in diatoms the energy dissipation capacity of the diadinoxanthin/diatoxanthin xanthophyll cycle is more active than in green algae or higher plants [START_REF] Ruban | The super-excess energy dissipation in diatom algae: comparative analysis with higher plants[END_REF][START_REF] Goss | The importance of a highly active and delta-pH-regulated diatoxanthin epoxidase for the regulation of the PS II antenna function in diadinoxanthin cycle containing algae[END_REF] and will be discussed later in this chapter. It should be noted that green algae and higher plants have a different suite of xanthophyll pigments to diatoms and dinoflagellates.

Electron usage to produce new biomass

Under continuous light, the oxygen production or the uptake of inorganic carbon shows a clear linear relationship with biomass production [START_REF] Toepel | Quantitative Estimates to convert photosynthetic electron transport rates into biomass[END_REF] indicating that 55-60 µmol oxygen released is equivalent to 1 mg dry weight. However, the ratio of oxygen released to carbon incorporated is highly variable, for several reasons. Firstly, the reduction in biomass strongly depends on the species and the environmental conditions [START_REF] Kroon | From electron to biomass: a mechanistic model to describe phytoplankton photosynthesis and steady state growth rates[END_REF]. For example, under N or P limitation, the relative proportion of carbon incorporated into carbohydrates is strongly increased and therefore the reduction in biomass is relatively low. The energetic cost of converting the products of the Calvin cycle into lipid or proteins are incorporated by higher rates of mitochondrial respiration. Therefore, it can not be expected that the ratio of oxygen production in the light, per oxygen molecule consumed in the dark to be constant. This ratio is modulated, not only by the availability of nutrients and the reduction in biomass, but also by the turn-over rates of proteins and lipids. It is well documented that cells growing under high-light have significantly higher mitochondrial respiration rates, as well as under nutrient replete conditions and optimal temperature [START_REF] Wilhelm | The variability of the photosynthetic unit in Chlorella II. The effect of light intensity and cell development on photosynthesis, P-700 and cytochrome f in homocontinuous and synchronous cultures of Chlorella[END_REF]. Table 1 shows that the ratio photosynthesis/respiration varies not only in response to the light climate (sine versus fluctuating light) but also with the C/N ratio. Therefore, the ratio of photosynthetic electrons to carbon incorporated into the newly formed cells has to be variable. However, such parameters have not been measured under an adequate range of conditions or with sufficient species to make broad speculation.

The conversion of electron transport rates into actual new biomass requires accurate estimates for the ratio of electrons per carbon in the macromolecules of the new biomass. In future, the FTIR spectroscopy [START_REF] Stehfest | The application of micro-FTIR spectroscopy to analyze nutrient stress-related changes in biomass composition of phytoplankton algae[END_REF] might become a tool to measure this parameter, and is also possible with single cells. This opens the perspective to improve the robustness of estimates for primary production measure by using advanced fluorescence techniques.

Effect of light stress on fluorescence signatures and their interpretation

When captured light energy cannot be completely utilized for metabolic processes, the excess energy accumulates within the photosynthetic apparatus [START_REF] Nixon | Regulation of photosynthetic electron transport[END_REF]. This typically occurs when the light intensity is too high (over minimum saturating irradiance: Ek). However, this also occurs when the cells are suddenly switched from a dark/low light environment or to a higher irradiance (not necessarily over Ek) depending on the physiological state of the cells and their response to other environmental cues. Accumulation of excess energy within the photosynthetic apparatus can be harmful for photosynthesis, and especially for the activity of PSII, because the over-reduction of the primary electron acceptor (QA) generates free radicals which leads to oxidative stress [START_REF] Ledford | Singlet oxygen and photo-oxidative stress management in plants and algae[END_REF]; stress which will ultimately cause a decrease in the photosynthetic rate (i.e. photoinhibition). Photosynthetic organisms have developed a number of fast photoprotective (or photoacclimative) processes to minimize the level of oxidative stress, especially linked to the dissipation of the excess absorbed energy [START_REF] Niyogi | Safety valves for photosynthesis[END_REF]. Nonphotochemical quenching (NPQ) is believed to be one of the most important of these mechanisms for the fast regulation of photosynthesis in higher plants as well as in algae [START_REF] Szabo | Light and oxygenic photosynthesis: energy dissipation as a protection mechanism against photo-oxidation[END_REF][START_REF] Demmig-Adams | Photoprotection in an ecological context: the remarkable complexity of thermal energy dissipation[END_REF][START_REF] Lavaud | Fast regulation of photosynthesis in diatoms: Mechanisms, evolution and ecophysiology[END_REF]. It should be noted that NPQ is not a form of AEC, but rather it is especially efficient for organisms growing in a fluctuating light environment where it helps to balance the absorption of light energy with its use, and ultimately plays a role in the maintenance of their fitness [START_REF] Külheim | Rapid regulation of light harvesting and plant fitness in the field[END_REF][START_REF] Ralph | Operation of the xanthophyll cycle in the seagrass Zostera marina in response to variable light[END_REF][START_REF] Demmig-Adams | Photoprotection in an ecological context: the remarkable complexity of thermal energy dissipation[END_REF][START_REF] Lavaud | Fast regulation of photosynthesis in diatoms: Mechanisms, evolution and ecophysiology[END_REF]. NPQ was shown to disturb the measurement and use of in vivo chlorophyll fluorescence for ecophysiological purposes in microalgae (Chapter 7; [START_REF] Serra | Quantification of the effect of nonphotochemical quenching on the determination of in vivo chl a from phytoplankton along the water column of a freshwater reservoir[END_REF].

Non-photochemical quenching (NPQ) originates in the light-harvesting antenna (LHC) of PSII. When the available excitation energy exceeds the photochemical capacity, it can then be dissipated as heat (or reallocated) before it reaches the PSII reaction center. This process arise from reactions not directly related to photochemistry, which have been defined as 'non-photochemical quenching' to be distinguished from the processes dealing with the 'photochemical quenching' (qP) which is directly related to photochemistry and the linear transport of electrons (Fig. 3a) [START_REF] Maxwell | Chlorophyll fluorescence-a practical guide[END_REF][START_REF] Baker | Chlorophyll fluorescence: A probe of photosynthesis in vivo[END_REF]. In that framework, the redox state of quinones (QA and QB) and plastoquinones can strongly influence the emission of fluorescence in parallel to NPQ under high light conditions [START_REF] Perkins | Light response curve methodology and possible implications in the application of chlorophyll fluorescence to benthic diatoms[END_REF]. NPQ reduces the lifetime of excited chlorophylls ( 1 Chl*) and thereby the quantum yield of Chl a fluorescence, which is seen by a decrease of Fm to Fm' level (see Fig. 3a). For that reason, it is calculated as (Fm-Fm')/Fm' (or Fo-Fo'/Fo'; [START_REF] Lavaud | Influence of the diadinoxanthin pool size on photoprotection in a marine planktonic diatom Phaeodactylum tricornutum[END_REF]. In higher plants, green algae and dinoflagellates, , it consists of three components (Fig. 3a) [START_REF] Stroch | Non-radiative dissipation of absorbed excitation energy within photosynthetic apparatus of higher plants[END_REF][START_REF] Hill | Impact of bleaching conditions on the components of non-photochemical quenching in the zooxanthellae of a coral[END_REF][START_REF] Szabo | Light and oxygenic photosynthesis: energy dissipation as a protection mechanism against photo-oxidation[END_REF]: the energy-dependent quenching (qE) which is regulated by the built-up of a transthylakoid proton gradient (ΔpH) and the operation of the xanthophyll cycle (XC); state-transition quenching (qT) which relies on the redistribution of excitation energy between photosystems by physical modulation of the cross-section of light-harvesting antennas [START_REF] Ruban | Dynamics of higher plant photosystem cross-section associated with state-transitions[END_REF]; and the sustained quenching which is heterogeneous [START_REF] Demmig-Adams | Photoprotection in an ecological context: the remarkable complexity of thermal energy dissipation[END_REF] which partially depends on xanthophylls [START_REF] Garcia-Mendoza | The giant kelp Macrocystis pyrifera presents a different non-photochemical quenching control than in higher plants[END_REF] as well as on photoinactivation/photoinhibition (qI) of PSII [START_REF] Stroch | Non-radiative dissipation of absorbed excitation energy within photosynthetic apparatus of higher plants[END_REF]. Quantification of these three components is either based on their relaxation kinetics in the dark [START_REF] Müller | Non-photochemical quenching. A response to excess light energy[END_REF] or requires photosynthetic inhibitors [START_REF] Horton | Studies on the induction of chlorophyll fluorescence in isolated barley protoplasts. IV. Resolution of non-photochemical quenching[END_REF]. The characteristics of their relaxation kinetics can vary according to environmental stresses and between groups of organisms. Such that, qE relaxes very rapidly (within tens of seconds after the offset of light), qT takes several minutes (shorter for cyanobacteria and rhodophytes), while qI is sustained and can last for hours even days under certain extreme environmental conditions [START_REF] Demmig-Adams | Photoprotection in an ecological context: the remarkable complexity of thermal energy dissipation[END_REF][START_REF] Garcia-Mendoza | The giant kelp Macrocystis pyrifera presents a different non-photochemical quenching control than in higher plants[END_REF]. Furthermore, in diatoms qE usually relaxes very slowly in comparison to higher plants (compare the two organisms in Fig. 3b) so that it could be confounded with qI due to overlaps with time. In general, with non-stressed leaves, qE is the major component under moderate to saturating irradiance. qI can become prominent under over-saturating irradiances and possibly in combination with other stresses (nutrient/water deficiency, temperature and salinity) [START_REF] Demmig-Adams | Photoprotection in an ecological context: the remarkable complexity of thermal energy dissipation[END_REF]. In this context, qT is not as relevant since it generally only makes a small contribution to overall relaxation of fluorescence (see Fig. 3a) [START_REF] Nixon | Regulation of photosynthetic electron transport[END_REF]. qT is usually significant only under low light levels [START_REF] Mullineaux | State-transitions: and example of acclimation to low-light stress[END_REF] while some dinoflagellates increase qT under thermal and light stress [START_REF] Hill | Impact of bleaching conditions on the components of non-photochemical quenching in the zooxanthellae of a coral[END_REF]. The amplitude and kinetics of the whole NPQ process and the importance of each component (Fig. 3b) can be extremely divergent between taxa, especially among microalgal groups [START_REF] Casper-Lindley | Fluorescence quenching in four unicellular algae with different light-harvesting and xanthophyll-cycle pigments[END_REF][START_REF] Hill | Impact of bleaching conditions on the components of non-photochemical quenching in the zooxanthellae of a coral[END_REF][START_REF] Juneau | Comparison by PAM fluorometry of photosynthetic activity of nine marine phytoplankton grown under identical conditions[END_REF], and even between species within a taxonomic group [START_REF] Lavaud | General features of photoprotection by energy dissipation in planktonic diatoms (Bacillariophyceae)[END_REF][START_REF] Lavaud | Photoprotection capacity differs among diatoms: Possible consequences on the spatial distribution of diatoms related to fluctuations in the underwater light climate[END_REF]. For example, qE shows high amplitude and fast onset in diatoms and brown macroalgae, while being of minor importance in most of the green microalgae [START_REF] Finazzi | Non-photochemical quenching of chlorophyll fluorescence in Chlamydomonas reinhardtii[END_REF] and cyanobacteria [START_REF] Kirilovsky | Photoprotection in cyanobacteria: the orange carotenoid protein (OCP)-related non-photochemical quenching mechanism[END_REF]. Nevertheless, within the diatoms (see the Chapter 7) as well as higher plants [START_REF] Johnson | The dissipation of excess excitation energy in British plant species[END_REF] there are clear differences in qE amplitude that have been highlighted. Whereas, qT is currently unknown in diatoms [START_REF] Owens | Light-harvesting function in the diatom Phaeodactylum tricornutum II. Distribution of excitation energy between the photosystems[END_REF]) and brown macroalgae [START_REF] Fork | Light energy distribution in the brown alga Macrocystis pyrifera (giant kelp)[END_REF], and of only moderate importance in higher plants and dinoflagellates [START_REF] Hill | Impact of bleaching conditions on the components of non-photochemical quenching in the zooxanthellae of a coral[END_REF], yet highly developed in some green microalgae and cyanobacteria [START_REF] Finazzi | The central role of the green alga Chlamydomonas reinhardtii in revealing the mechanism of state transitions[END_REF][START_REF] Mullineaux | State-transitions: and example of acclimation to low-light stress[END_REF].

Amongst the three components of NPQ, qE is a major influence on the Chl a fluorescence signal under normal growth conditions [START_REF] Logan | Avoiding common pitfalls of chlorophyll fluorescence analysis under field conditions[END_REF]; see also the Chapter 7). The interpretation of qE is possibly the most complex of the NPQ components, as it is linked to faster regulation of photosynthesis than qT and qI with most organisms, especially under naturally fluctuating environment [START_REF] Lavaud | Fast regulation of photosynthesis in diatoms: Mechanisms, evolution and ecophysiology[END_REF]. [START_REF] Frenkel | Hierarchy amongst photosynthetic acclimation responses for plant fitness[END_REF] demonstrated that qE is critical for maintaining the fitness of plants under natural temperate-light conditions, rather than qT. Also, in cyanobacteria and green microalgae, qT has no significant physiological importance in photoprotection towards high-light stress, yet is more relevant in low light conditions [START_REF] Mullineaux | State-transitions: and example of acclimation to low-light stress[END_REF][START_REF] Ruban | Dynamics of higher plant photosystem cross-section associated with state-transitions[END_REF] and for acclimation to different light quality [START_REF] Pfannschmidt | Acclimation to varying light qualities: toward the functional relationship of state transitions and adjustment of photosystem stochiometry[END_REF]. The qT mechanism has been documented, as well as its impact on the fluorescence signal, especially in cyanobacteria and green microalgae (see [START_REF] Campbell | Chlorophyll fluorescence analysis of cyanobacterial photosynthesis and acclimation[END_REF][START_REF] Nixon | Regulation of photosynthetic electron transport[END_REF][START_REF] Finazzi | The central role of the green alga Chlamydomonas reinhardtii in revealing the mechanism of state transitions[END_REF][START_REF] Mullineaux | State-transitions: and example of acclimation to low-light stress[END_REF]. Even though qI has been well documented in some species of higher plants growing in extreme environments [START_REF] Demmig-Adams | Photoprotection in an ecological context: the remarkable complexity of thermal energy dissipation[END_REF], its occurrence and control mechanism remains unknown in some of the algal groups. Also, the part of qI which depends on xanthophylls is also linked to the qE process (Demmig-Adams & Adams, 2006), although clear mechanistic differences have only been recently demonstrated [START_REF] Dall'osto | A mechanism of non-photochemical energy dissipation, independent from PsbS, revealed by a conformational change in the antenna protein CP26[END_REF].

The qE mechanism has been described in a molecular context for higher plants and green microalgae [START_REF] Standfuss | Mechanisms of photoprotection and non-photochemical quenching in pea light-harvesting complex at 2.5 Å resolution[END_REF][START_REF] Cogdell | The structural basis of non-photochemical quenching revealed?[END_REF][START_REF] Ruban | Identification of a mechanism of photoprotective energy dissipation in higher plants[END_REF]. The machinery triggering and controlling qE amplitude and kinetics is now quite well known for groups of algae like the diatoms and brown macroalgae [START_REF] Goss | The importance of a highly active and delta-pH-regulated diatoxanthin epoxidase for the regulation of the PS II antenna function in diadinoxanthin cycle containing algae[END_REF][START_REF] Lavaud | Fast regulation of photosynthesis in diatoms: Mechanisms, evolution and ecophysiology[END_REF], as well as in the cyanobacteria and prochlorophytes [START_REF] Bailey | The occurrence of rapidly reversible non-photochemical quenching of chlorophyll a fluorescence in cyanobacteria[END_REF][START_REF] Kirilovsky | Photoprotection in cyanobacteria: the orange carotenoid protein (OCP)-related non-photochemical quenching mechanism[END_REF]. The NPQ process is based on a feed-back reaction from the linear electron transport through the build-up of a trans-thylakoid ΔpH and subsequent acidification of the thylakoid lumen (see [START_REF] Nixon | Regulation of photosynthetic electron transport[END_REF]. Consequently, the activity of the ATP synthase [START_REF] Dal Bosso | Inactivation of the chloroplast ATP synthase g subunit results in high nonphotochemical fluorescence quenching and altered nuclear gene expression in Arabidopsis thaliana[END_REF] the cytochrome b6f [START_REF] Munekage | Cytochrome b6f mutation specifically affects thermal dissipation of absorbed light energy in Arabidopsis[END_REF], or the cyclic electron flow around PSI [START_REF] Miyake | Effects of light intensity on cyclic electron flow around PSI and its relationship to non-photochemical quenching of Chl fluorescence in tobacco leaves[END_REF] can indirectly influence qE. Hence, in a simple direct relationship, the higher the irradiance, the higher the electron transport rate, the higher the accumulation of protons in the lumen, the higher qE. In some organisms such as diatoms, it appears there is a relative independence of the PSII redox-state from the proton-motive electron transfer and subsequent NPQ [START_REF] Ruban | The super-excess energy dissipation in diatom algae: comparative analysis with higher plants[END_REF][START_REF] Lavaud | Photoprotection capacity differs among diatoms: Possible consequences on the spatial distribution of diatoms related to fluctuations in the underwater light climate[END_REF]. To summarize NPQ responses, the lumen acidification triggers two events (Fig. 4): 1) the protonation of specific sites of the LHC antenna, and 2) the activation of an enzyme, a de-epoxidase, which drives the conversion of epoxized xanthophyll to a de-epoxidized form. This conversion is reversible as the backward reaction is driven by an epoxidase which also depends on the trans-thylakoid ΔpH. The accumulation of de-epoxidized xanthophylls thus depends on the balance between the activity of both enzymes within the xanthophyll cycle (XC) (see [START_REF] Lavaud | Fast regulation of photosynthesis in diatoms: Mechanisms, evolution and ecophysiology[END_REF] for a detailed description). In higher plants, green microalgae and brown macroalgae, the XC involves the conversion of violaxanthin to zeaxanthin (ZX) via antheraxanthin (AX) (Fig. 4) while the diatoms and dinoflagellates use diadinoxanthin (DD) which is converted to diatoxanthin (DT) under elevated light (see Chapter 7). Both protonated LHC protein(s) and the presence of DT or ZX/AX in the LHC antenna of PSII are thought to act together as the trigger of the qE. The whole LHC antenna switches into a dissipative mode when excess excitation energy should be converted into heat while Chl a fluorescence is quenched (Fig. 4) [START_REF] Stroch | Non-radiative dissipation of absorbed excitation energy within photosynthetic apparatus of higher plants[END_REF][START_REF] Szabo | Light and oxygenic photosynthesis: energy dissipation as a protection mechanism against photo-oxidation[END_REF]. More precisely, protonation would promote and transduce conformational changes ('aggregations') which bring pigments closer together and especially chlorophyll/xanthophyll molecules. In higher plants the 'special' polypeptide which undergoes protonation is PsbS [START_REF] Niyogi | Is PsbS the site of nonphotochemical quenching in photosynthesis?[END_REF]. PsbS function is essentially to sense the lumen pH thanks to several H + -binding amino acids residues present on the luminal loops of the protein [START_REF] Dominici | Biochemical properties of the PsbS subunit of photosystem II either purified from chloroplast or recombinant[END_REF][START_REF] Li | Structure-function analysis of photosystem II subunit S (PsbS) in vivo[END_REF]. Its ability to bind pigments in vivo remains unclear [START_REF] Niyogi | Is PsbS the site of nonphotochemical quenching in photosynthesis?[END_REF] with contradictory reports from in vitro works [START_REF] Aspinall-O'dea | In vitro reconstitution of the activated zeaxanthin state associated with energy dissipation in plants[END_REF][START_REF] Dominici | Biochemical properties of the PsbS subunit of photosystem II either purified from chloroplast or recombinant[END_REF]. In green microalgae (Graham & Niyogi, pers. com.) and diatoms [START_REF] Zhu | Light-harvesting and photoprotection in diatoms: Identification and expression of L818-like proteins[END_REF], the Li-818 proteins which are up-regulated under high light, could play a similar role as PsbS in qE. Simultaneously as PsbS protonation, de-epoxidized xanthophylls would also act as 'allosteric regulators' by amplifying the conformational changes within the whole LHC antenna. The physical process by which excitation energy is effectively converted into heat has only recently been understood [START_REF] Holt | Carotenoid cation formation and the regulation of photosynthetic light harvesting[END_REF][START_REF] Pascal | Molecular basis of photoprotection and control of photosynthetic light-harvesting[END_REF][START_REF] Ruban | Identification of a mechanism of photoprotective energy dissipation in higher plants[END_REF]. The qE mechanism is rather similar in other organisms like the diatoms and the brown macroalgae given some peculiarities (see Chapter 7). In other groups like the red algae, cyanobacteria and prochlorophytes, the process is quite different even though it involves xanthophylls and special proteins of the antenna system [START_REF] Lavaud | Fast regulation of photosynthesis in diatoms: Mechanisms, evolution and ecophysiology[END_REF]. Therefore, qE in these taxa is not as controlled as in higher plants or diatoms since these organisms do not display a finely regulated xanthophyll cycle, also cyanobacteria and prochlorophytes show no involvement of a trans-thylakoid ΔpH [START_REF] Kirilovsky | Photoprotection in cyanobacteria: the orange carotenoid protein (OCP)-related non-photochemical quenching mechanism[END_REF].

Quenching based in the PSII reaction center (as opposed to LHC antenna) has also been observed in higher plants [START_REF] Bukhov | Energy dissipation in photosyntesis: Does the quenching of chlorophyll fluorescence originate from antenna complexes of photosystem II or from the reaction center?[END_REF][START_REF] Stroch | Non-radiative dissipation of absorbed excitation energy within photosynthetic apparatus of higher plants[END_REF]) and green microalgae [START_REF] Finazzi | A zeaxanthin-independent nonphotochemical quenching mechanism localized in the photosystem II core complex[END_REF], and possibly in diatoms [START_REF] Eisenstadt | Changes in the photosynthetic reaction centre II in the diatom Phaeodactylum tricornutum result in nonphotochemical fluorescence quenching[END_REF]. Appearance of reaction centre quenching depends on the balance between light and carbon fixation fluxes [START_REF] Finazzi | A zeaxanthin-independent nonphotochemical quenching mechanism localized in the photosystem II core complex[END_REF] along with a clear temperature influence [START_REF] Kornyeyev | Minimization of the photon energy absorbed by 'closed' reaction centers of photosystem 2 as a photoprotective strategy in higher plants[END_REF]. This quenching appears to drive both qE and qI components of NPQ with both fast and slow relaxation kinetics, respectively. In contrast to the antenna-based quenching, it cannot cause changes in the Fo level [START_REF] Maxwell | Chlorophyll fluorescence-a practical guide[END_REF]. Nevertheless, as well as the antenna-based quenching it requires thylakoid acidification, but it does not require de-epoxidized xanthophylls [START_REF] Bukhov | Energy dissipation in photosyntesis: Does the quenching of chlorophyll fluorescence originate from antenna complexes of photosystem II or from the reaction center?[END_REF][START_REF] Finazzi | A zeaxanthin-independent nonphotochemical quenching mechanism localized in the photosystem II core complex[END_REF]. The qI part of this reaction center based quenching is associated with a reversible inactivation of a sub-population of the PS II [START_REF] Finazzi | A zeaxanthin-independent nonphotochemical quenching mechanism localized in the photosystem II core complex[END_REF] as well as with PSII photodamage [START_REF] Kornyeyev | Minimization of the photon energy absorbed by 'closed' reaction centers of photosystem 2 as a photoprotective strategy in higher plants[END_REF].

Use of chemicals for the differentiation of photosynthetic processes

Photosynthesis is a complex interaction of complementary processes such as alternate electron cycling (AEC) and non-photochemical quenching (NPQ). A common method of isolating specific processes is using biochemical inhibitors such as herbicides. Electron transport inhibitors, uncouplers, artificial electron acceptors and donors have all proved to be essential tools in elucidating the function of various components of the photosynthetic electron transport chain, metabolic pathways and photosynthetic regulatory processes. Using herbicides to understand the regulation of photosynthesis and related biochemical pathways requires the basic understanding of how these herbicides interact with the photosynthetic apparatus.

Determining the appropriate concentration of herbicide is very important and often problematic, because depending on the organism, cells can have different cell wall composition, membrane transporters and a variation in the number of reaction centres per cell, thus requiring different concentrations of herbicide [START_REF] Durnford | Assessing the potential for chloroplast redox regulation of nuclear gene expression[END_REF]. Therefore, any concentrations specified herein are only an indication of what has been used based the range of concentrations found in the literature. The most effective and correct way to determine the concentration at which a cellular response occurs is by titration of the herbicide against a known cell density or chlorophyll a concentration while measuring the physiological impact (oxygen evolution or chl a fluorescence). -1',1'-dimethylurea), also known as Diuron, is the most extensively used inhibitor of photosynthetic electron transport. DCMU inhibits electron transport between PSII and PSI, impacting on the acceptor side of PSII by supplanting a bound plastoquinone from the QB -binding site of PSII (Fig 5). Binding of this herbicide to the QB -site of PSII, results in the effective blocking of electron flow and leads to the subsequent inhibition of photosynthesis. Blocking of electron flow is a consequence of the herbicide being incapable of receiving electrons, and therefore electrons remain trapped in QA, the first quinone acceptor [START_REF] Kleczkowski | Glucose activation and metabolism through UDP-glucose pyrophosphorylase in plants[END_REF]. This trapping of electrons prevents the reduction of plastoquinone, by holding the electrons in the D1 dimer, thus affecting the redox state of the PQ pool, which becomes completely oxidised [START_REF] Durnford | Assessing the potential for chloroplast redox regulation of nuclear gene expression[END_REF].

Inhibitors of linear electron transport

DCMU causes a rapid increase to maximum fluorescence (Fm), where all PSII reaction centres are closed and the plastoquinone pool fully oxidised [START_REF] Trebst | Inhibitors in the functional dissection of the photosynthetic electron transport system[END_REF]. DCMU has no impact on the membrane potential of the thylakoid in darkness, yet completely inhibits light-induced membrane pH gradient. The amount of DCMU required for the inhibition of 50% of PSII reaction centres will vary depending on cell concentration and species. Published concentrations range from 1-20 µM [START_REF] Falkowski | Aquatic Photosynthesis 2 nd edition[END_REF]. However, incremental increases in the amount of DCMU added to cells will result in changes in variable fluorescence and the rate of QA re-oxidation [START_REF] Durnford | Assessing the potential for chloroplast redox regulation of nuclear gene expression[END_REF], which will invariably allow for the determination of the appropriate concentration of DCMU needed to elicit the desired effect. In addition, the light acclimation state of the cells needs to taken into account, as cells grown at low PFD will have a plastiquinone (PQ) pool that is predominantly oxidized and therefore the addition of DCMU will have very little effect on the redox state of the PQ pool [START_REF] Durnford | Assessing the potential for chloroplast redox regulation of nuclear gene expression[END_REF] similarly, chlororespiration can alter the PQ redox state, allowing fluorescence yield to occur with saturating DCMU concentrations [START_REF] Wilhelm | Fluorescence induction kinetics as a tool to detect chlororespiratorian activity in the prasinophycean alga Mantoniella squamata[END_REF]). In the presence of saturating DCMU concentrations, fluorescence yield becomes maximum (Fo⇒Fm so Fv/Fm⇒0), as QA can no longer pass electrons to PQ, so electron transport stops and the maximum amount of captured energy is dissipated as fluorescence.

Like DCMU, DBMIB (2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone) is also an inhibitor of electron transport, however it blocks further along the electron transport chain near the Cytochrome b6f complex [START_REF] Trebst | Inhibitors in the functional dissection of the photosynthetic electron transport system[END_REF]. DBMIB is thought to interfere at the Reiske iron-sulfur centre [START_REF] Trebst | Inhibitors in the functional dissection of the photosynthetic electron transport system[END_REF], thus blocking photosynthetic electron flow through the Cytochrome b6f complex. DBMIB binds close to the Qo pocket [START_REF] Cramer | Transmembrane Traffic in the Cytochrome b 6 f Complex[END_REF], the plastoquinol binding site of the Cytochrome b6f complex (Fig. 5), inhibiting the reoxidation of PQH2 thus keeping the PQ pool completely reduced [START_REF] Trebst | Inhibitors in electron flow: Tools for the functional and structural localization of carriers and energy conservation sites[END_REF]. Some precaution should be taken when using DBMIB, because site of action is concentration dependent, as well as redox sensitive, where DBMIB becomes reduced under light and oxidised in the dark [START_REF] Bukhov | Interaction of exogenous quinones with membranes of higher plant chloroplasts: modulation of quinone capacities as photochemical and non-photochemical quenchers of energy in Photosystem II during light-dark transitions[END_REF]. At low concentrations, DBMIB inhibits electron transport on the reducing side of the PQ, but at higher concentrations excess DBMIB will inhibit the QB site of PSII, located on the oxidising side of the PQ [START_REF] Moreland | Mechanisms of Action of Herbicides[END_REF][START_REF] Rich | The interaction of duroquinol, DBMIB and NQNO with the chloroplast cytochrome bf complex[END_REF]. To prevent fluorescence quenching from oxidised DBMIB, it can be used in conjunction with an excess of sodium ascorbate [START_REF] Kufryk | Sll1717 Affects the Redox State of the Plastoquinone Pool by Modulating Quinol Oxidase Activity in Thylakoids[END_REF]. For every Cytochrome b6f complex, one molecule of DBMIBred is needed for complete inhibition of electron transfer through the Cytochrome b6f complex [START_REF] Rich | The interaction of duroquinol, DBMIB and NQNO with the chloroplast cytochrome bf complex[END_REF]. DBMIB can also inhibit mitochondrial electron transport [START_REF] Durnford | Assessing the potential for chloroplast redox regulation of nuclear gene expression[END_REF]. This is a good example of co-inhibition, where some inhibitors have more than one impact site, and therefore interpretation of results must carefully consider the possibility of an alternate component of the cell machinery being affected by the inhibitor. DBMIB reduces minimum fluorescence (Fo) as well as the rise of variable fluorescence (Fv). While DBMIBred quenches chlorophyll a fluorescence, it does so less efficiently than the oxidised form (DBMIBox) and both forms alter NPQ estimates [START_REF] Tyystjarvi | The Krautsky curve is a built-in barcode[END_REF].

Inhibitors of cyclic electron transport

The antibiotic, antimycin A, is an effective inhibitor of one of the alternate electron cycles (AEC), cyclic electron transport around PSI [START_REF] Tagawa | Role of chloroplast ferredoxin in the energy conversion process of photosynthesis[END_REF]. It has been proposed that inhibition of photosynthetic electron transport by antimycin A is associated with the ferredoxin-plastoquinone reductase (FQR) activity in cyclic electron transport [START_REF] Simonis | Photophosphorylation in Vivo[END_REF][START_REF] Moss | Cyclic electron transport in chloroplasts. The Q-cycle and the site of action of antimycin[END_REF][START_REF] Cleland | Photosystem I cyclic electron transport: Measurement of ferredoxin-plastoquinone reductase activity[END_REF]. In addition to inhibiting cyclic electron transport, antimycin A is also known to inhibit excess light energy dissipation measured through NPQ [START_REF] Oxborough | Characterization of the effects of antimycin A upon high energy state quenching of chlorophyll fluorescence (qE) in spinach and pea chloroplasts[END_REF]. The decline in qE (energy-dependent quenching) formation in the presence of this antibiotic is due to a change in the redox state of the electron transport chain. However, since antimycin A has no direct impact on linear electron transport rate, the redox change is most likely the result of a change in the redox state of a component located in the cytochrome complex [START_REF] Oxborough | Characterization of the effects of antimycin A upon high energy state quenching of chlorophyll fluorescence (qE) in spinach and pea chloroplasts[END_REF]. Before inhibiting cyclic electron transport, it is important to understand that there are two potential transport pathways that cycle around PSI [START_REF] Joët | Increased sensitivity of photosynthesis to antimycin A induced by inactivation of the chloroplast ndhB gene. Evidence for a participation of the NADH-dehydrogenase complex to cyclic electron flow around photosystem I[END_REF][START_REF] Munekage | Cyclic electron flow around photosystem I is essential for photosynthesis[END_REF]. The first, cycles electrons from ferrodoxin to the PQ pool and is sensitive to antimycin A, while the second, involves the NDH complex which is insensitive to the antibiotic [START_REF] Joët | Increased sensitivity of photosynthesis to antimycin A induced by inactivation of the chloroplast ndhB gene. Evidence for a participation of the NADH-dehydrogenase complex to cyclic electron flow around photosystem I[END_REF]. In the case of the NDH cycle, it is not yet fully understood and no known inhibitor has been identified. Published concentrations of antimycin A range from 0.1-50 µM [START_REF] Falkowski | Aquatic Photosynthesis 2 nd edition[END_REF].

Inhibitors of alternative electron cycling (AEC)

Distinguishing between different electron pathways is important to describe the discrepancies often seen between oxygen evolution and chlorophyll a fluorescence under stressful conditions. Molecular oxygen can be reduced downstream of PSII at various sites, using different forms of AEC. In the case of the Mehler reaction, oxygen is reduced at the acceptor side of PSI [START_REF] Mehler | Studies on reactions of illuminated chloroplasts I. Mechanism of the reduction of oxygen and other Hill reagents[END_REF] where it competes for electrons with both linear and cyclic electron transport pathways [START_REF] Heber | Irrungen, Wirrungen? The Mehler reaction in relation to cyclic electron transport in C3 plants[END_REF]. The Mehler reaction itself can not be inhibited; however, the addition of potassium cyanide (KCN) can be used to inhibit the formation of H2O and monodehydroascorbate (MDA) during the ascorbate peroxidase reaction, which is part of the Mehler cycle [START_REF] Neubauer | Mehler-peroxidase reaction mediates zeaxanthin formation and zeaxanthin-related fluorescence quenching in intact chloroplasts[END_REF]. The inhibition of H2O formation as a result of altered peroxide turnover, impacts on the zeaxanthin-dependent light energy dissipation, by suppressing zeaxanthing formation and consequently NPQ [START_REF] Neubauer | Mehler-peroxidase reaction mediates zeaxanthin formation and zeaxanthin-related fluorescence quenching in intact chloroplasts[END_REF]. In fluorescence, the addition of KCN results in a decline in NPQ as well as a decrease in linear electron flow, shown as a suppression of qP [START_REF] Neubauer | Mehler-peroxidase reaction mediates zeaxanthin formation and zeaxanthin-related fluorescence quenching in intact chloroplasts[END_REF]. Published KCN concentrations vary from 0.1mM to 3mM (Neubauer and Yamamota 1992, Hormann et al 1994[START_REF] Singh | Reactivation of photosynthesis in the photoinhibited green alga Chlamydomonas reinhardtii: Role of dark respiration and of light[END_REF].

Another more recently discovered pseudo-cyclic electron transport pathway which cycles around PSII via the plastoquinol terminal oxidase (PTOX), reduces molecular oxygen by utilising electrons from the PQ pool to generate H2O [START_REF] Cournac | Electron flow between photosystem II and oxygen in chloroplasts of photosystem I-deficient algae is mediated by a quinol oxidase involved in chlororespiration[END_REF][START_REF] Peltier | Chlororespiration[END_REF]Josse et al., 2003). This alternative electron flow around PSII (upstream of PSI & Cytochrome b6f) is believed to be advantageous in both a high-light environment and under iron limitation [START_REF] Bailey | Alternative photosynthetic electron flow to oxygen in marine Synechococcus[END_REF], as it alleviates PSII excitation pressure by transporting electrons directly to oxygen while simultaneously ensuring that the electrons bypass the iron-demanding cytochrome b6f and PSI complexes [START_REF] Mackey | A photosynthetic strategy for coping in a high-light, low nutrient environment[END_REF]. Propyl-gallate (PGal) is an oxidase inhibitor specific to PTOX [START_REF] Cournac | Electron flow between photosystem II and oxygen in chloroplasts of photosystem I-deficient algae is mediated by a quinol oxidase involved in chlororespiration[END_REF][START_REF] Bailey | Alternative photosynthetic electron flow to oxygen in marine Synechococcus[END_REF]. PGal helps determine the role PTOX plays in alternative electron flow, and establish whether or not electrons are being used to reduce oxygen through PTOX activity [START_REF] Mackey | A photosynthetic strategy for coping in a high-light, low nutrient environment[END_REF]. The addition of 1 mM PGal results in a decrease in electron flow through PSII [START_REF] Bailey | Alternative photosynthetic electron flow to oxygen in marine Synechococcus[END_REF][START_REF] Mackey | A photosynthetic strategy for coping in a high-light, low nutrient environment[END_REF], highlighting the role the oxidase plays in keeping the PSII reaction centres oxidised in cells where Cytochrome b6f and PSI activity are limiting. As in the case of DBMIB, PGal has more than one impact site in eukaryotes, as it can also lead to the inhibition of mitochondrial electron transport [START_REF] Bailey | Alternative photosynthetic electron flow to oxygen in marine Synechococcus[END_REF].

Inhibitors of CO2 fixation

Iodoacetamide has been used as an inhibitor of carbon fixation [START_REF] Miller | Active transport of CO2 by the cyanobacterium Synechococcus UTEX 625[END_REF][START_REF] Miller | Glycoaldehyde inhibits CO2 fixation in the cyanobacterium Synechococcus UTEX 625 without inhibiting the accumulation of inorganic carbon or the associated quenching of chlorophyll a fluorescence[END_REF], however when added during steady state photosynthesis, it inhibits CO2 very slowly and may induce O2 uptake in the light [START_REF] Miller | Glycoaldehyde inhibits CO2 fixation in the cyanobacterium Synechococcus UTEX 625 without inhibiting the accumulation of inorganic carbon or the associated quenching of chlorophyll a fluorescence[END_REF]). An alternative inhibitor of CO2 fixation is D, L-glyceraldehyde [START_REF] Stokes | Photosynthesis by isolated chloroplasts. Inhibition by DL-glyceraldehyde of carbon dioxide fixation[END_REF]), which at very high concentrations (>25 mM) completely inhibits CO2 fixation (Shelp & Canvin, 1989) and blocks the conversion of triose-P to ribulose-1,5bisphosphate [START_REF] Stokes | Photosynthesis by isolated chloroplasts. Inhibition by DL-glyceraldehyde of carbon dioxide fixation[END_REF]. However, more recently, glycolaldehyde (GA) has become the preferred inhibitor of CO2 fixation, as it uses concentrations an order of magnitude lower than those of D, L-glyceraldehyde, while rapidly and effectively inhibiting CO2 fixation [START_REF] Sicher | Glycolaldehyde inhibition of photosynthetic carbon assimilation by isolated chloroplasts and protoplasts[END_REF] without inhibiting CO2 or HCO3 - transport [START_REF] Miller | Glycoaldehyde inhibits CO2 fixation in the cyanobacterium Synechococcus UTEX 625 without inhibiting the accumulation of inorganic carbon or the associated quenching of chlorophyll a fluorescence[END_REF][START_REF] Rotatore | Active uptake of CO2 during photosynthesis in the green alga Eremosphaera viridis is mediated by a CO2-ATPase[END_REF]. The addition of GA to cells eliminates the chlorophyll a fluorescence quenching that is seen with the addition of inorganic carbon; however, oxygen evolution is greatly impacted by the presence of GA [START_REF] Miller | Glycoaldehyde inhibits CO2 fixation in the cyanobacterium Synechococcus UTEX 625 without inhibiting the accumulation of inorganic carbon or the associated quenching of chlorophyll a fluorescence[END_REF].

Electron transport uncouplers

Uncouplers function by dissociating electron transport from ATP synthesis during photosynthetic phosphorylation [START_REF] Moreland | Mechanisms of Action of Herbicides[END_REF][START_REF] Mccauley | Protonophores Induce Plastoquinol Oxidation and Quench Chloroplast Fluorescence: Evidence for a Cyclic, Proton-Conducting Pathway in Oxygenic Photosynthesis[END_REF]. This is accomplished by dissipating the energised state (H + ) of the membrane (ΔpH) before the energy can be utilised in ADP phosphorylation [START_REF] Moreland | Mechanisms of Action of Herbicides[END_REF] and thus prevent the formation of the trans-thylakoid ∆pH gradient. In addition to this major effect on the energy budget of the cell, the electron flow continues but the collapsed proton gradient no longer regulates electron transport rate. This type of inhibitor can be useful when examining processes triggered by ∆pH, such as NPQ and in particular qE. Common uncouplers of photophosphorylation include ammonia chloride (HN4Cl), carbonyl cyanide 4-trifluoromethyloxyphenylhydazone (FCCP) and nigericin.

Ammonium chloride (NH4Cl) is a potent uncoupler of electron transport. As described above, it works in the classical way by relaxing the pH gradient across the thylakoid membrane, inhibiting ATP synthesis. The addition of NH4Cl before the application of saturating light will prevent all quenching of Fm'. In contrast, if the uncoupler is added after fluorescence quenching has already formed (following a series of saturating pulses), it will result in a complete reversal of all Fm' quenching [START_REF] Delphin | Delta pH-dependent photosystem II fluorescence quenching induced by saturating, multiturnover pulses in red algae[END_REF].

Carbonyl cyanide p-trifluoromethoxy phenylhydrazone (FCCP) is a powerful uncoupler of photophosphorylation. It acts as an ionophore completely dissipating the pH gradient, while leaving the electron transport system uninhibited [START_REF] Canaani | Evidence for a Biological Role in Photosynthesis for Cytochrome b-559-A Component of Photosystem II Reaction Center[END_REF]. FCCP prevents the long-term fluorescence induction, meaning that the inhibition of the induction is likely the result of an increase in the dark decay processes [START_REF] Canaani | Evidence for a Biological Role in Photosynthesis for Cytochrome b-559-A Component of Photosystem II Reaction Center[END_REF]. Typical concentrations of FCCP are 1-10 µM [START_REF] Shyam | Role of dark respiration in photoinhibition of photosynthesis and its reactivation in the cyanobacterium Anacystis nidulans[END_REF][START_REF] Sigalat | Force-flow relationship in lettuce thylakoids. 1. Strict control of election flow by internal pH[END_REF][START_REF] Singh | Reactivation of photosynthesis in the photoinhibited green alga Chlamydomonas reinhardtii: Role of dark respiration and of light[END_REF]. At low concentrations FCCP quenches PSII fluorescence, indicative of the reoxidation of QA - [START_REF] Mccauley | Protonophores Induce Plastoquinol Oxidation and Quench Chloroplast Fluorescence: Evidence for a Cyclic, Proton-Conducting Pathway in Oxygenic Photosynthesis[END_REF], while it requires much higher concentrations to perform in its function as an uncoupler of oxidative phosphorylation [START_REF] Canaani | Evidence for a Biological Role in Photosynthesis for Cytochrome b-559-A Component of Photosystem II Reaction Center[END_REF]. When incubated with cells under photoinhibitory light, FCCP accelerates photoinhibition and rapidly quenches fluorescence yield [START_REF] Mccauley | Protonophores Induce Plastoquinol Oxidation and Quench Chloroplast Fluorescence: Evidence for a Cyclic, Proton-Conducting Pathway in Oxygenic Photosynthesis[END_REF][START_REF] Shyam | Role of dark respiration in photoinhibition of photosynthesis and its reactivation in the cyanobacterium Anacystis nidulans[END_REF][START_REF] Singh | Reactivation of photosynthesis in the photoinhibited green alga Chlamydomonas reinhardtii: Role of dark respiration and of light[END_REF].

Another type of uncoupler is the protonophore, such as nigericin which dissipates the proton gradient across the thylakoid membrane. Nigericin relaxes the ∆pH gradient by antiporting H + at the expense of K + across membranes, resulting in the collapse qE [START_REF] Pressman | Antibiotic-mediated transport of alkali ions across lipid bilayers[END_REF]. As a result of a breakdown in the pH gradient, the addition of nigericin to illuminated samples, results in an increase in Fm' and strong inhibition of NPQ with a concomitant large increase in steady state fluorescence Ft. The typical concentration range for nigericin is 1-5 mM [START_REF] Falkowski | Aquatic Photosynthesis 2 nd edition[END_REF].

Electron acceptors

Electron acceptors are compounds with very strong reducing capacity, such as methyl viologen (N,N'-Dimethyl-4,4'-bipyridinium dichloride; MV 2+ ) also known as Paraquat. Methyl viologen is an artificial electron acceptor, intercepting electron flow between PSI and the Calvin cycle by competing with ferrodoxin for the binding site at PSI (Fig. 5) [START_REF] Hess | Review: light-dependent herbicides: an overview[END_REF]. MV 2+ is an extremely powerful electron acceptor, due to the nature of the bipyridinium salts, which temporarily become a stable radical with the addition of an electron, neutralising the positive charge of the cation [START_REF] Moreland | Mechanisms of Action of Herbicides[END_REF][START_REF] Peon | Excited State Dynamics of Methyl Viologen. Ultrafast Photoreduction in Methanol and Fluorescence in Acetonitrile[END_REF]. MV 2+ oxidizes the primary acceptor (ferrodoxin) of linear electron transport, allowing a ∆pH to become established. However, this temporary neutral radical rapidly reverts back to its ion form, a process that results in the production of superoxide radicals [START_REF] Hormann | Intact chloroplasts display pH 5 optimum of O2-reduction in the absence of methyl viologen: Indirect evidence for a regulatory role of superoxide protonation[END_REF][START_REF] Hess | Review: light-dependent herbicides: an overview[END_REF]. MV 2+ can be used to demonstrate damage to the electron transport chain beyond PSI (typically Calvin cycle), where incubation with MV 2+ will oxidise the electron transport chain and increase ΦPSII by supplementing the slow carbon fixation rate. In the presence of MV 2+ , non-photochemical quenching (NPQ) is reduced, because the excess electrons that are usually held up by the Calvin cycle, are being accepted by the MV 2+ allowing for continual rapid electron transport and a reduced need for excess light energy dissipation in the form of NPQ. Published concentrations of MV 2+ range from 0.05 to 1 mM [START_REF] Falkowski | Aquatic Photosynthesis 2 nd edition[END_REF].

In conclusion, we have illustrated how photosynthetic electron transport is strongly influenced by a range of internal feedback processes (AEC and NPQ) to ensure maximum efficiency, whilst preventing potential damage from excess excitation energy. Light stimulated processes such as non-photochemical quenching are closely linked with pigments, however the control mechanisms are species-specific and show wide variability. Chemical inhibitors can be used to isolate specific components of the electron transport chain allowing a mechanistic understanding of the control of these photosynthetic pathways.

Tables

Species

Growth where qP is the photosynthetic quantum efficiency at Photosystem II measured by PAM fluorescence. The amount of quanta lost by alternative electron sinks was calculated from the difference of fluorescence and oxygen-based photosynthesis rates and the assumption of a quantum efficiency of 0.125 [mol O2 (mol quanta) -1 ]. The amount of quanta used for biomass formation was derived from Φµ according to [START_REF] Jakob | Uncoupling of growth rates and biomass production under nitrate limitation in combination with dynamic light conditions in the diatom Phaeodactylum tricornutum[END_REF]. Energy losses which are not directly quantifiable as absorbed quanta, like mitochondrial respiration, have been depicted as 'losses'.

Figure captions Figure 1 :

 captions1 Figure captions

Figure 2 :

 2 Figure 2: The fate of absorbed photons in the comparison of Phaeodactylum tricornutum and Chlorella vulgaris grown in a turbidostat under dynamic light conditions (fluctuating light, FL; sine light, SL). Data are given as percentage of absorbed quanta (Qphar). Energy losses by dissipation include the conversion of absorbed light into heat and fluorescence and were derived from [(1-qP) ɬ Qphar],where qP is the photosynthetic quantum efficiency at Photosystem II measured by PAM fluorescence. The amount of quanta lost by alternative electron sinks was calculated from the difference of fluorescence and oxygen-based photosynthesis rates and the assumption of a quantum efficiency of 0.125 [mol O2 (mol quanta) -1 ]. The amount of quanta used for biomass formation was derived from Φµ according to[START_REF] Jakob | Uncoupling of growth rates and biomass production under nitrate limitation in combination with dynamic light conditions in the diatom Phaeodactylum tricornutum[END_REF]. Energy losses which are not directly quantifiable as absorbed quanta, like mitochondrial respiration, have been depicted as 'losses'.

Figure 3 .

 3 Figure 3. (A) Chlorophyll a (Chl a) fluorescence signal as measured with a PAM fluorometer on an Arabidopsis thalania leaf. After dark-adaptation, in the presence of the detector beam (dashed bottom bar), the minimal fluorescence level (Fo) is

Figure 4 .

 4 Figure 4. Simplified model of the qE mechanism in higher plants (see the text for a full description). The numbering refers to the sequence of the qE process steps. AX, antheraxanthin; H + , protons; PS II, photosystem II; VDE, violaxanthin de-epoxidase; VX, violaxanthin; ZX, zeaxanthin; ∆pH, transthylakoid proton gradient. Adapted from[START_REF] Lavaud | Fast regulation of photosynthesis in diatoms: Mechanisms, evolution and ecophysiology[END_REF].

Figure 5 .

 5 Figure 5. Electron transport flow through PSII, cytochrome b6f complex and PSI. The D1, D2 and Cyt b559 proteins are shown. The thin arrows indicate the electron flow pathway through PSII, while the thick arrow indicates the site of inhibition by DCMU. The figure shows the DCMU molecule binding to the QB site of PSII, thereby effectively blocking the continuation of electron flow from PSII to the plastoquinone, cytochrome b6f complex and onto PSI. After QB the thin arrows indicate electron flow pathways and proton (H + ) pathways. The thick arrow in the lower middle of the diagram indicates the site of impact by the inhibitors DBMIB and antimycin A.One DBMIB molecule competes with the PQH2 resulting in the blocking of the release of electrons from the plastoquinone to PSI at the Fe-S complex. Antimycin A (upper middle) inhibits the reduction of ferrodoxin in PSI, intercepting electron transport at the ferrodoxin-plastoquinone reductase, resulting in the blocking of cyclic electron transport. Electrons move through PSI indicated by thin arrows, while the thick arrow (RHS) indicates the impact site of methyl viologen. Methyl viologen interacts at the binding site of ferredoxin, accepting the terminal electron, thus preventing the reduction of ferredoxin and the continued pathway of the electron to carbon fixation or cyclic electron transport.

  Figure 1

Figure

  Figure 3 a and b

Table 1 :

 1 Comparison of Phaeodactylum tricornutum and Chlorella vulgaris with respect to the activity of alternative electron transport (expressed as the ratio of fluorescence-based to oxygen-based photosynthesis rates; PF/PO), to C/N ratios (given as mol mol -1 ), and the activity of mitochondrial respiration (expressed as the ratio of respiration rate to net photosynthesis rate; R/Pnet). Algal cultures were grown in a turbidostat under dynamic light conditions (10h and 12h light periods) which have been applied either as a non-fluctuating sine light climate or as oscillating light (osc. Light). In addition, P. tricornutum was exposed to nitrate-limited conditions (N-limited). Data are adapted from[START_REF] Wagner | Light emission originating from photosystem II radical pair recombination is sensitive to zeaxanthin related nonphotochemical quenching (NPQ)[END_REF] and[START_REF] Jakob | Uncoupling of growth rates and biomass production under nitrate limitation in combination with dynamic light conditions in the diatom Phaeodactylum tricornutum[END_REF].

		condition	PF/PO	C/N	R/Pnet
	P.tricornutum. Replete -sine light (10 h) 1.4	7.7	0.8
	P.tricornutum. Replete -osc. light (10 h) 1.3	7.9	1.0
	P.tricornutum Replete -sine light (12 h) 1.6	6.6	0.4
	P.tricornutum Replete -osc. light (12 h) 1.1	6.7	1.4
	P.tricornutum N-limited -sine light (10 h) 1.6	14.5	0.7
	P.tricornutum N-limited -osc. light (10 h) 1.2	10.8	1.5
	C.vulgaris.	Replete -sine light (12 h) 2.1	6.8	0.4
	C.vulgaris	Replete -osc. light (12 h) 2.0	6.8	0.9

Acknowledgement.

PR thanks the Australian Research Council for financial support. CW and TJ thank the deutsche Forschungsgemeinschaft for financial support Wi 764/12. JL thanks the Centre National de la Recherche Scientifique (CNRS), the Agence Nationale de la Recherche (ANR program VasiRéMi) and the deutsche Forschungsgemeinschaft (DFG, grant LA2368/2-1) for their financial support. Ross Hill is thanked for editorial comments.