Elsevier

Aquatic Botany

Volume 93, Issue 3, October 2010, Pages 185-194
Aquatic Botany

Exposure times in rapid light curves affect photosynthetic parameters in algae

https://doi.org/10.1016/j.aquabot.2010.07.002Get rights and content

Abstract

Short-and long-duration light curves were applied to four macroalgae (Ulva sp., Codium fragile, Ecklonia radiata and Lessonia variegata), and two microalgal species (Chlorella emersonii and Chaetoceros muellerii). With increasing light curve duration, the maximal relative electron transport rate increased by a factor of three in E. radiata, and by factors of 1.25 and 1.23 in C. emersonii and L. variegata, respectively, but did not change in C. fragile and Ch. muellerii. The light saturation point Ek increased by 26 μmol photons m−2 s−1 in C. emersonii and 20 μmol photons m−2 s−1 in Ch. muellerii and E. radiata with elevated light curve exposure times. Oscillatory patterns of the continuous fluorescence readings reflect accumulation of QA. Continuous fluorescence values increased, or decreased, by approximately 10% within light curve increments. However, oscillations of 25% were not uncommon, which shows that cells are changing their photo-physiological response state during steady light conditions. Increasing dark acclimation times prior to light curve application lowered maximal relative electron transport rates in the C. emersonii (from 28 ± 1.7 to 25 ± 1.2 for 15 and 95 min dark acclimation in short-duration light curves respectively). This effect was counterbalanced by longer light curve application. It can therefore be concluded that manipulation of light exposure and dark incubation prior to the experiment affects the photosynthetic response, presumably due to different activation states of photosynthetic and photoprotective mechanisms. The highly species-specific photo-response patterns imply that a common rapid light curve protocol will generate artefacts in some species.

Introduction

Aquatic primary producers are subjected to fluctuations in light doses and flux. The depth of the euphotic zone, the cell's passage time within mixed layers, shading effects and light focusing events require a plastic response system to highly variable light conditions. The ability to adjust to given photon flux (PF) situations can vary with species, which may occupy very different ecological niches (e.g. MacKenzie et al., 2005, van Leeuwe et al., 2005). Cellular responses to a change in PF can occur on different timescales. The amount of accessory pigments, for instance, can follow dial patterns, with acclimation times of hours (Owens et al., 1980, Claustre et al., 2002). Short-term responses to altered PF can take place in milliseconds for activation procedures in the photosynthetic unit (Schansker et al., 2006), and in minutes for enzyme activation (Portis and Parry, 2007, Krause and Weis, 1991). Acclimation to changed PF conditions involves gene expression and processes that are carried out in hours, or days, rather than minutes. For the purpose of this paper, the sum of short-term responses, measured by changes in chlorophyll fluorescence signals, to variable incubation times in darkness and various PF will be referred to as photo-response. Estimation of in situ primary production has been of considerable interest and is an ongoing issue. An early approach was to incubate phytoplankton communities in closed containers at different water depths over the period of a day. The change in oxygen concentration, measured by Winkler titration, was used to calculate the average daily primary production. Long-term bottle incubations, however, do not allow water exchange, which entails the risk of nutrient exploitation and underestimation of photosynthetic performance (Falkowski and Howe, 1976, Falkowski and Owens, 1978). In shorter, 2–4 h, incubations photosynthetic carbon incorporation increased up to 40% (Macedo et al., 2002). More recently, measurements based on chlorophyll a fluorescence have been employed for primary productivity measurements, providing the advantage of a ‘non-intrusive’ nature of the measurement (e.g. Baker, 2008, Schreiber, 1986). However, the comparison of oxygen evolution and chlorophyll a fluorescence based electron transport rate (ETR) correlate well for many species, although deviations were observed at low and high PF (Geel et al., 1997, Kromkamp et al., 2001, Wagner et al., 2006). Additional to measurement of primary production, chlorophyll a fluorescence measurements provide information about the photo-response through non-photochemical quenching (NPQ) mechanisms (Krause and Weis, 1991, Garcia-Mendoza et al., 2002, Govindjee, 2002). NPQ mechanisms can be separated into three different categories, namely energy dependent quenching qE, state transitions qT (absent in diatoms and brown algae (Owens, 1986, Smith and Melis, 1987)), and photoinhibition qI, which operate in different timeframes (Müller et al., 2001). Generally, up-regulation of NPQ is at least biphasic with a fast component that reacts in seconds, and a slow component with full activation kinetics on the scale of minutes (Niyogi et al., 1998, Müller et al., 2001, Grouneva et al., 2009). The fast component is controlled by a trans-thylakoid pH gradient (ΔpH gradient), while de-epoxidation of xanthophyll cycle pigments is responsible for the slower phase of qE (Demming-Adams, 1990, Lavaud, 2007). Diatoms show structurally different qE control mechanisms as the Psbs protein, a protein that is essential for qE in green algae (Li et al., 2009), is absent (Armbrust et al., 2004) and xanthophyll cycle pigmentation is different compared to Chlorophyta (Lavaud, 2007). Wilhelm et al. (2006) review many of the substantial differences between diatoms and green algae for nutrient and carbon assimilation. Up-regulation of photosynthesis and full RuBisCO activation requires 5–20 min in algae (MacIntyre et al., 1997, White and Critchley, 1999), but is much more rapid in higher plants (Eichelmann et al., 2009).

Commonly used photosynthesis vs. irradiance protocols differ in the PF applied, the duration of the PF increments and the dark incubation times prior to RLC application. A dark incubation period is necessary to re-oxidise the electron transport chain and relax NPQ mechanisms to allow the detection of the maximal quantum efficiency. In addition, other cell functions are down-regulated as can be seen by the significant decrease of dark respiration within minutes (Raven and Beardall, 2004, Beardall et al., 1994). Electron carriers in the photosynthetic unit, however, might still be used during chlororespiratory activity, which can affect photosynthetic and NPQ activation pattern when cells are illuminated (Wilhelm and Duval, 1990, Peltier and Cournac, 2002). Due to chlororespiration, diatoms are able to maintain a ΔpH gradient and retain some active qE in the dark (Wilhelm and Duval, 1990, Ting and Owens, 1993, Lavaud, 2007). The dark-induced de-activation of Calvin–Benson–Bassham cycle proteins can be desirable (or not) and various dark incubation durations can de-activate the Calvin–Benson–Bassham cycle to various levels. However, when employing fluorescence it has become more common to apply rapid light curves (RLC), which use light increments between 10 s (short) and 90 s (long) (White and Critchley, 1999, Kromkamp et al., 2001, López-Figueroa et al., 2003, Ralph and Gademann, 2005). The times required for full activation of photosynthesis and NPQ often exceed the duration of RLC (Perkins et al., 2006). Accordingly, application of short RLC exclude a number of photo-responses to changed PF, namely the reactions that operate on timescales longer than the RLC itself. This can be a desired effect, however short RLC bear the risk of artefacts when used inappropriately. Additionally to the duration of PF increments, experiments can include increasing, or decreasing PF increments, application of dark phases after each light increment, or using a new sample for each measurement per increment, depending on the purpose of the physiological investigation. In this study, short and long RLC have been applied to four macroalgae and two microalgae of different phylogenetic position in order to estimate the artefacts deriving from the manipulation of RLC duration.

Section snippets

Sample origins

The brown macroalga Ecklonia radiata was collected from Port Philip Bay, Australia in August 2005. Samples were kept in the dark at 18 °C until measurements were made on the same, or the following day. A representative section in the middle of the thallus was selected and RLC applied on the horizontal, thinner part of the thallus by placing the fiber optics approximately 5 mm away from the thallus surface. The temperature was held at a constant 18 °C during the measurement, and individual thalli

Results

Increasing the duration of RLC increments resulted in both, different shapes of the RLC (Fig. 1) and values of the photosynthetic parameters (Table 1) in the majority of the species tested. Prolonged exposure to low PF in the long RLC resulted in higher α values in Ulva sp. and L. variegata. In short RLC, rETR started to level off at lower PF (Fig. 1), allowing fewer data points for α calculation, leading to a less reliable α determination by the model. No significant difference in α was

Discussion

Manipulation of RLC duration and the dark incubation time prior to the RLC affected photosynthetic parameters and resulted in species-specific photo-responses in the majority of the species tested. Differences were detected in saturating, as well as sub-saturating PF and might result from a multitude of physiological response kinetics and response mechanisms. First we discuss potentially restricting factors in low PF, which can have add-on effects on the high PF phase of the RLC, when

Acknowledgments

The authors are grateful to the divers sampling macroalgae in Doubtful Sound and Port Philip bay (Christopher Hepburn, Daniel Pritchard, Derek Richards, Christopher Cornelison). Tara Higgins and Kirsten Shelly generously provided cultures of Chlorella emersonii. We thank two anonymous reviewers for their valuable comments and improvements on the manuscript. SI was funded by MGS and MIPR scholarships from Monash University.

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