The photosynthetic characteristics of red coralline ...

DOI 10.1515/bot-2012-0135

Botanica Marina 2012; aop

Heidi L. Burdett*, Sebastian J. Hennige, Fiona T.-Y. Francis and Nicholas A. Kamenos

The photosynthetic characteristics of red coralline algae, determined using pulse amplitude modulation (PAM) fluorometry Abstract: Interest in red coralline algae is increasing due to their projected sensitivity to ocean acidification and their utility as palaeoenvironmental proxies. Thus, it is crucial to obtain a thorough understanding of their basic photosynthetic characteristics and appropriate techniques for use in both laboratory and in situ studies. This study provides fluorescence methodology and data for the ecologically important red coralline alga Lithothamnion glaciale using pulse amplitude modulation (PAM) fluorometry. Lithothamnion glaciale was sufficiently darkacclimated for in situ work following 10 s of quasi-darkness, attaining 95–98% of the maximum photochemical efficiency (Fv/Fm). Rapid light curves conducted in situ and in the laboratory determined a low light adaptation, with a saturation intensity of 4.45–54.6 μmol photons m-2 s-1. Intra-thallus heterogeneity was observed between branch tips and bases (i.e., within the thallus) using a custom-made 2 mm fibre optic probe (the heterogeneity could not be detected using the standard 5 mm probe). Branch bases were lower light acclimated than the tips, with higher maximum effective quantum yield (Fq′/Fm′max) and lower non-photochemical quenching. Samples measured in May were higher light acclimated than in March, which suggests a degree of seasonal acclimation. Light history and photon irradiance levels were thus found to significantly affect the photosynthetic characteristics of L. glaciale. Keywords: coralline algae (CCA); crustose; light; maerl; photosynthesis; rhodolith.

*Corresponding author: Heidi L. Burdett, School of Geographical and Earth Sciences, University of Glasgow, Glasgow, G12 8QQ, UK, e-mail: [email protected] Sebastian J. Hennige: School of Life Sciences, Heriot-Watt University, Edinburgh, EH14 4AS, UK Fiona T.-Y. Francis: Department of Biology, Dalhousie University, Halifax, B3H 4R2, NS, Canada Nicholas A. Kamenos: School of Geographical and Earth Sciences, University of Glasgow, Glasgow, G12 8QQ, UK; and School of Life Sciences, University of Glasgow, Glasgow, G12 8QQ, UK

Introduction Free-living, non-geniculate, red coralline algae (Rhodophyta: Corallinales) (Figure 1), commonly known as maerl or rhodoliths, are found worldwide in the coastal photic zone (Foster 2001). Dense coralline algal beds may develop under appropriate environmental circumstances, sustaining high biodiversity (Barbera et al. 2003) and providing a range of ecosystem services, such as nursery areas for commercially important gadoids and bivalves (Kamenos et al. 2004a,b). Red coralline algae may annually lay down growth bands composed of high-Mg calcite (Blake and Maggs 2003, Kamenos et al. 2008), the chemistry (Kamenos 2010, Halfar et al. 2011) and structure (Burdett et al. 2011) of which can be used in palaeoenvironmental reconstructions. The growth of these bands is dependent upon the photosynthesis of the thalli, and as such, photosynthesis is a key process to study when considering maerl bed accretion, ecosystem service provision and palaeoenvironmental reconstructions. Red coralline algae live under a wide range of photon irradiances, with active growth recorded from the deepest part of the photic zone [ > 200 m depth, 0.0015 μmol m-2 s-1 photosynthetically active radiation (PAR)] (Littler et al. 1991), to shallow tropical reefs (2000 μmol m-2 s-1 PAR) (Payri et al. 2001). Nevertheless, some studies suggest that red coralline algae are acclimated to low-light regimes (Kühl et al. 2001, Wilson et al. 2004) and are therefore particularly susceptible to light-induced stress (Payri et al. 2001). Indeed, high irradiance has been shown to cause degradation of photosynthetic pigments and subsequent bleaching of coralline algae (Irving et al. 2004, Martone et al. 2010). However, photosynthesis research on freeliving and encrusting coralline algae is currently limited, and only a few studies have used non-invasive chlorophyll-a fluorometry techniques (Irving et al. 2004, Wilson et al. 2004, Irving et al. 2005). Chlorophyll-a fluorescence induction pulse amplitude modulation (PAM) techniques provide a non-destructive way of assessing the photophysiology of a range of organisms, including phytoplankton, macroalgae and

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H.L. Burdett et al.: Photokinetics of coralline algae

Fibre optic probe size 5 mm 2 mm

Figure 1 Lithothamnion glaciale: free living thallus (scale bar = 2 cm) and diameters of the two fibre optic probes used in this study for comparison to thallus branch size.

higher plants (e.g., Edwards and Kim 2010, Ritchie and Bunthawin 2010, White et al. 2011). By supplying saturating pulses of actinic light at pre-set intensities and frequencies, PAM fluorometry provides detailed information on the photosynthetic response of organism under light limitation and saturation. This provides information on the current photosynthetic state of the organism along with photoacclimation (short term, e.g., hours to days) and adaptation (longer term, e.g., seasonality) potential in response to PAR (Falkowski and Laroche 1991). The characterisation of acclimation and adaptation of an organism is key to understanding its productivity and its eventual growth, and for this, comprehensive baseline data and techniques need to be established. A number of studies have used chlorophyll-a fluorescence techniques to assess the photosynthetic characteristics of macroalgae, both in the field and in the laboratory. Tropical, temperate and polar species typically harbour short-term photoacclimation mechanisms to minimise photodamage throughout a diurnal light cycle, with species-specific adaptations in response to tidal exposure (Hanelt et al. 1993, Figueroa et al. 2003b), low water temperatures (Hanelt and Nultsch 1995) and depth (Häder et al. 1998). Similarly, seasonal patterns in photoacclimation may occur, with enhanced photoacclimation during the summer months (Kutser et al. 2006). Light quality can affect the photosynthetic capacity of red macroalgae, increasing pigment and protein production under blue light and stimulating growth under red light (Figueroa et al. 1995). Exposure to high irradiance and or UV radiation may cause damage to DNA, the photosynthetic apparatus and light harvesting pigments (Rundquist et al. 1995, Kutser et al. 2006).

The use of rapid light curves (RLCs) has become well established in PAM fluorometry, whereby photosynthetic organisms are exposed to increasing levels of irradiance, interrupted with bursts of saturating actinic light (Ralph and Gademann 2005, Perkins et al. 2006). This technique provides information on the dissipation of energy from limiting levels of irradiance through to saturating levels and can act as a proxy to the electron transport rate (ETR) through photosystem II. Photosynthesis-irradiance (P-E) curves allow calculation of the maximum (darkadapted) and effective (light-adapted) quantum yield of fluorescence, the light saturation coefficient (Ek) and the maximum theoretical ETR. RLCs also provide information on the quenching coefficients qP, qN and NPQ, where qP describes the photochemical quenching (energy used for photosynthesis), and qN and NPQ both describe non-photochemical quenching, where absorbed photon energy is dissipated as heat and/or re-emitted as fluorescence when photosynthesis is saturated (White and Critchley 1999). However, in contrast to traditional light curves, a steady state is not reached during each light step of a RLC (Ralph and Gademann 2005). Thus, results from RLCs yield information on the actual, rather than optimal, photosynthetic state as suggested by traditional light curves (Ralph and Gademann 2005). In addition, comparison of RLCs from different species or under different environmental conditions should be conducted with care, as the irradiance absorption of a photosynthetic organism may change, affecting ETR (Saroussi and Beer 2007). With interest in red coralline algae increasing due to their predicted sensitivity to ocean acidification (e.g., Burdett et al. 2012) and use as palaeoenvironmental proxies (e.g., Kamenos 2010), it is important to obtain a thorough understanding of the baseline photosynthetic characteristics of these key algae. This understanding is needed before more complex ecological and photosynthesis studies can be considered. This study therefore sought to clarify the fluorescence methodology and techniques required to study the ecologically important temperate red coralline alga Lithothamnion glaciale Kjellman and to provide the first comparison of PAM fluorescence RLCs between laboratory and in situ studies.

Materials and methods Algal material Free-living thalli of Lithothamnion glaciale were collected by hand from Loch Sween on the west coast of Scotland



(56°01.99′N, 05°36.13′W) using SCUBA from 6 m below chart datum. Thalli were transported to the University of Glasgow in seawater (ambient temperature, in the dark) and transferred to 120 l re-circulating seawater tanks at ambient conditions.

Fluorescence measurements All discrete chlorophyll-a fluorescence measurements were made using a Diving-PAM fluorometer and WinControl software (Walz GmbH, Effeltrich, Germany). To ensure a sufficient fluorescence signal, fibre optic probes were placed approximatey 1 mm away from the sample at an angle to minimise shading (Wilson et al. 2004). Measurements were conducted in situ at Loch Sween and in the laboratory using the standard PAM fibre optic probe (5 mm) and a custom 2 mm fibre optic probe (Figure 1). In a fully relaxed, dark-acclimated state, the minimum and maximum fluorescence yields are termed Fo and Fm, respectively (see Table 1 for a full list of parameters). These parameters are termed Fo′ and Fm′, respectively, under actinic light. The maximum quantum yield Fv/Fm (dimensionless) is defined as (Fm-Fo)/Fm. This describes the maximum photochemical efficiency of energy transfer to the PSII reaction centres. Under actinic light, the effective quantum yield, Fq′/Fm′, is defined as (Fm′-F′)/Fm′.

Dark acclimation To assess the dark-acclimation time needed for discrete fluorescence measurements, thalli (n = 5) kept in the

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laboratory under actinic light were exposed to saturating light pulses at 5-min intervals (t = 0, 5, 10 min). Tank lights were switched off after 14 min 50 s, and a further eight saturation fluorescence measurements were taken at t+15, 20, 25, 30, 35, 40, 60 and 100 min. Measurements were made at the thallus branch tips using 5 mm and 2 mm probes and the branch base using a 2 mm probe only (as the 5 mm probe would not pass between the red coralline algal branches). Quantum efficiency was stable in the light and in the dark at the thallus tips (5 mm and 2 mm probes) and base (2 mm probe only). These experiments suggested that a 5 min dark period was sufficient to fully relax PSII (as indicated by increased quantum efficiency, Figure 2) and that 10 s of ‘quasi’ dark acclimation was sufficient in situ if there were time constraints on sample collection. Quantum yield increased by an average of 12.5% (SE ± 0.7%) after 10 s of darkness and increased to a maximum after 5 min of darkness for the 5 mm and 2 mm branch tip measurements. Ten second dark-acclimation yield values represented approximately 95–98% of the maximum yield measurements achieved after 5 min of dark adaptation and as such were deemed suitable for in situ dark-acclimation.

Rapid light curves RLCs were measured using the light curve function of the Diving-PAM set to the lowest intensity (LC-INT = 1). Actinic light illumination was increased in nine incremental 10 s intensities of PAR, from 2 to 997 μmol photons m-2 s-1, each followed by a saturating light pulse. RLCs were conducted at the tips of branches using a 5 mm and 2 mm fibre optic

Parameter

Definition

Fo Fm F′ Fv Fv′ Fq′ Fv/Fm Fq′/Fm′ Fq′/Fm′max Fv′/Fm′ Fq′/Fv′ NPQ α rETRmax Ek

Minimum fluorescence (dark-acclimated) Maximum fluorescence (dark-acclimated) Fluorescence under actinic light Variable fluorescence (dark-acclimated); (Fm-Fo) Variable fluorescence yield under actinic light; (Fm′-Fo′) Fluorescence quenched; (Fm-F ′) Maximum photochemical efficiency or quantum yield of PSII (dark-acclimated) Effective photochemical efficiency or quantum yield of PSII under actinic light; (Fm-F ′)/Fm′ Calculated maximum photochemical efficiency of PSII Non photochemical quenching, qN, under actinic light; presented as 1-qN Photochemical quenching, qP, under actinic light Non-photochemical quenching; (Fm-Fm′)/Fm′ Initial slope of the light dependent part of a rapid light curve Maximum relative electron transport rate (μmol electrons m-2 s-1) Minimum saturating intensity (μmol photons m-2 s-1)

Table 1 Photosynthetic parameters and definitions used in this study. Fluorescence yields have instrument-specific units, and ratios are dimensionless.


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0.65

Photochemical efficiency

account that 15% of chlorophyll-a in red algae is associated with PSII (Goldstein et al. 1992, Figueroa et al. 2003a). The light-response of electron transport was then fitted to the following model using least squares non-linear regression (Hennige et al. 2008) modified from Jassby and Platt (1976) to derive α and rETRmax:

Dark

Light 0.60

0.55

0.50

rETR = rETRmax×[1-exp(-α×E/rETRmax)]

0.45

5 mm 2 mm Tip 2 mm Base

0.40 0

5

10

15

20

25

30

35

40

60

100

Time (min)

Figure 2 Lithothamnion glaciale: yield measurements of thalli in the light (0–10 min) and dark (15–100 min). Lights were turned off after 14 min 50 s. Measurements were taken at thallus branch tips using a 5 mm fibre optic probe (black circle) and a 2 mm probe (open circles) and from the branch base using a 2 mm fibre optic probe (black triangles). Values are mean ± SE, n = 5.

probe, and at the base of branches using the 2 mm probe only. The quantum yield measurements from each RLC were fitted against the following model to describe the light-response of quantum efficiency using non-linear least squares regression (Suggett et al. 2007, Hennige et al. 2008): Fq′/Fm′ = [(Fq′/Fm′ max×Ek)(1-exp(-E/Ek))]/E

[1]

where Ek (μmol photons m-2 s-1) is the minimum saturation intensity (Hill et al. 2004), the light level at which photochemical efficiency shifts from light limitation to light saturation. E is equivalent to PAR (μmol photons m-2 s-1). PAR was determined throughout using an Apogee (Logan, UT, USA) QSO-E quantum sensor. For the first data point, measured on dark-acclimated thalli, Fv/Fm was used. An estimate of the maximum effective quantum yield under actinic light is also derived from Eq. [1] (Moore et al. 2005, Suggett et al. 2007, Hennige et al. 2008), termed here as Fq′/Fm′ max. As Fq′/Fm′ max is derived from RLC illumination, differences between samples may be accounted for by differences in light acclimation rather than environmental light availability. Using values of measured Fq′/Fm′, the relative electron transport rate (rETR μmol electrons m-2 s-1) was calculated for each actinic light intensity (E) of the RLC: rETR = Effective quantum yield×PAR×0.15

[2]

where PAR is the actinic irradiance in μmol photons m-2 s-1, and 0.15 is a multiplication factor to take into

[3]

where α (dimensionless) and rETRmax (μmol electrons m-2 s-1) describe the light-dependent and light-saturated rETR, respectively. Non-photochemical quenching (NPQ) was calculated as Stern-Volmer quenching [(Fm-Fm′)/Fm′], which is usually the preferred method (Ralph and Gademann 2005, Hennige et al. 2008), and also as qN (Fv′/Fm′) as effective photochemical efficiency (Fq′/Fm′) is a product of photochemical quenching, qP (Fq′/Fv′), and non-photochemical quenching under actinic light, qN (Fv′/Fm′) (Hennige et al. 2008). Fo′ used for the determination of qP and qN was derived according to Suggett et al. (2003). qP and qN describe the decrease in fluorescence due to photochemical and non-photochemical quenching, and thus decreasing qN values represent increased non-photochemical quenching. For ease of interpretation, data are presented as 1-qN.

Experimental design Field RLCs were conducted in situ in Loch Sween in May 2011 on undisturbed thalli at 6 m depth, following 10 s quasi-dark acclimation. Measurements were conducted on thalli tips (n = 5) using both 5 mm and 2 mm probes. Logistical constraints prevented 2 mm probe measurements in the field.

Laboratory Thalli were collected in March and May 2011 for laboratory measurements. Thalli were maintained under ambient collection conditions, viz., March: 7°C ± 1, salinity 32 and 10:14 h light:dark cycle; May: 12°C ± 1, salinity 32 and 17:7 h light:dark cycle. Irradiance was maintained at 90 μmol photons m-2 s-1 during daylight hours, corresponding to average light intensities in Loch Sween (Rix et al. 2012). RLCs were conducted after 5 min dark acclimation as follows: (1) 5 mm fibre optic: the tips of five independent thalli, (2) 2 mm fibre optic: tips and bases each of five independent thalli.



Comparison In situ – 2 mm vs. 5 mm May Ek May Fq′/Fm′ Laboratory – tip vs. base March Ek May Ek March Fq′/Fm′ May Fq′/Fm′ Laboratory – March vs. May Seasonal Ek Seasonal Fq′/Fm′ Seasonal α Seasonal rETRmax Laboratory – 2 mm vs. 5 mm, March vs. May Probe size Ek Probe size Fq′/Fm′ Laboratory vs. in situ May Ek May Fq′/Fm′ May α May rETRmax

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Statistic

Grouping

F1,8 = 17.07, p = 0.003 F1,8 = 0.27, p = 0.617

(5TF) (2TF) (5TF, 2TF)

F1,18 = 1.56, p = 0.227 F1,8 = 0.35, p = 0.568 F1,18 = 5.26, p = 0.034 F1,8 = 5.77, p = 0.043

(2TL, 2BL) (2TL, 2BL) (2TL) (2BL) (2BL) (2TL)

F1,13 = 13.20, p = 0.003 F1,13 = 4.98, p = 0.044 F1,13 = 2.97, p = 0.109 F1,13 = 1.03, p = 0.328

(5TL March) (5TL May) (5TL March) (5TL May) (5TL March, 5TL May) (5TL March, 5TL May)

F1,52 = 61.97, p