The giant kelp Macrocystis pyrifera presents a different nonphotochemical quenching control than higher plants

Authors

  • Ernesto García-Mendoza,

    1. Departamento de Oceanografía Biológica, Centro de Investigación Científica y de Educación Superior de Ensenada, Km 107 Carretera Tijuana-Ensenada, Ensenada, BC, México CP 22860
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  • María Florencia Colombo-Pallotta

    1. Departamento de Oceanografía Biológica, Centro de Investigación Científica y de Educación Superior de Ensenada, Km 107 Carretera Tijuana-Ensenada, Ensenada, BC, México CP 22860
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Author for correspondence: Ernesto García-Mendoza Tel. +52 646 175 0500 Fax: +52 646 175 0587 Email: ergarcia@cicese.mx

Summary

  • • Here the mechanisms involved in excitation energy dissipation of Macrocystis pyrifera were characterized to explain the high nonphotochemical quenching of chlorophyll a (Chla) fluorescence (NPQ) capacity of this alga.
  • • We performed a comparative analysis of NPQ and xanthophyll cycle (XC) activity in blades collected at different depths. The responses of the blades to dithiothreitol (DTT) and to the uncoupler NH4Cl were also assayed.
  • • The degree of NPQ induction was related to the amount of zeaxanthin synthesized in high light. The inhibition of zeaxanthin synthesis with DTT blocked NPQ induction. A slow NPQ relaxation upon the addition of NH4Cl, which disrupts the transthylakoid proton gradient, was detected. The slow NPQ relaxation took place only in the presence of de-epoxidated XC pigments and was related to the epoxidation of zeaxanthin.
  • • These results indicate that in M. pyrifera, in contrast to higher plants, the transthylakoid proton gradient alone does not induce NPQ. The role of this gradient seems to be related only to the activation of the violaxanthin de-epoxidase enzyme.

Introduction

Macrocystis pyrifera (Phaeophyceae, phylum Heterokontophyta), the giant kelp, is the dominant alga in shallow, hard-bottom coastal waters of the southern California current system (North, 1971). It has one of the highest growth rates of all macroscopic photoautotrophs (0.5 m d−1; Gerard, 1982) and can grow up to 60 m long. This alga forms aggregations known as kelp forests and the fronds form a dense canopy at the surface. Therefore, light attenuation is high inside the kelp forest. The irradiance at 20 m depth could be <1% of the incident light at the surface (Dean, 1985). Therefore, photosynthetic tissue of a single organism is exposed to a large gradient of light quantity and quality. A single organism is capable of expressing different photoacclimatization and photoprotection responses according to the position of its photosynthetic tissue in the water column. One important photoprotection response detected in M. pyrifera is that surface blades are able to have an extremely high nonphotochemical quenching of photosystem II (PSII) chlorophyll a fluorescence (NPQ) when exposed to saturating light conditions (Colombo-Pallotta et al. 2006). This is probably an efficient strategy to avoid photoinhibitory damage since the photosynthetic rate in surface blades sustains net plant production (Colombo-Pallotta et al. 2006).

Nonphotochemical fluorescence quenching is a proxy to measure the thermal dissipation of excess energy in the photosynthetic apparatus. The dissipation of excess energy as heat is one of the most important photoprotection mechanism of higher plants and algae (Pfündel & Bilger, 1994; Müller et al., 2001; Horton & Ruban, 2005). Other processes such as state transitions and photoinactivation of PSII are also involved in NPQ (Horton et al., 1991). However, a protective role has been assigned mainly to the thermal dissipation part of NPQ (Müller et al., 2001), denominated as high-energy quenching or qE (Horton et al., 1991). One important process involved in qE is the xanthophyll cycle (XC). The XC is the de-epoxidation of the carotenoid violaxanthin (Vio) into zeaxanthin (Zea) via the intermediate pigment antheraxanthin (Ant) in saturating light and the reverse reaction in low light or in darkness (Yamamoto et al., 1962). Zea synthesized via the XC is essential for thermal dissipation. However, two other elements are necessary for full expression of qE, the formation of a proton gradient across the thylakoid membrane (ΔpH; Noctor et al., 1991), and the presence of the PSII antenna subunit PsbS (Li et al., 2000). The mechanistic model of NPQ induction proposed for higher plants (reviewed in Müller et al., 2001; Govindjee, 2002; Horton & Ruban, 2005; Niyogi et al., 2005) indicates that the acidification of the lumen in high light (ΔpH formation) induces the protonation of one or more PSII antenna complexes, but especially PsbS. Protonation causes conformational changes in the antenna that promotes thermal dissipation (fast induction phase). In parallel to protonation, the low pH in the lumen activates the violaxanthin de-epoxidase enzyme (VDE), which catalyses the synthesis of Zea. This pigment binds to the protonated proteins and forms a quenching complex that enhances thermal dissipation (slow phase). There is a general agreement in the sequence of the events described above; however, what is still under debate is whether Zea is just an allosteric activator (Horton et al., 1991) or if it is directly involved in the quenching of chlorophyll a (Chla)-excited molecules (Frank et al., 1994; Ma et al., 2003).

The importance of NPQ and XC for photoprotection in brown algae has been recognized. Species incapable of expressing efficient photoprotective mechanisms are confined to deeper waters while species possessing active xanthophyll cycling proliferate in shallow environments (Rodrigues et al., 2002). Given that XC in brown algae is similar to the one present in higher plants (Vio to Zea conversion), it is assumed that the mechanisms controlling NPQ are the same for both groups. However, other members of the phylum Heterokontophyta, such as diatoms (Bacillariophyceae), show differences in NPQ induction when compared with higher plants. In diatoms state transitions are not present (Owens & Wold, 1986). Diatoms have a XC in which the xanthophyll diadinoxanthin (DDx) is converted into diatoxanthin (DTx), however, they are capable of expressing the Vio to Zea cycle (Lohr & Wilhelm, 1999). Epoxidation rate of XC pigments is much higher in diatoms than in plants (Olaizola et al., 1994; Lavaud et al., 2004). The NPQ could reach values close to 10 in diatoms (Lavaud et al., 2002a; Ruban et al., 2004) while in plants it is lower than 4 (Demmig-Adams & Adams, 1996). Unlike in higher plants, NPQ in diatoms is very sensitive to the chemical inhibition of XC activity. Dithiothreitol (DTT), an inhibitor of the VDE, reduces significantly NPQ (Olaizola et al., 1994; Lavaud et al., 2002b).

Macrocystis pyrifera has a XC similar to higher plants but could have a super-high NPQ capacity like that of diatoms. In the present study we characterized the mechanisms involved in the excitation energy dissipation in M. pyrifera to explain the high NPQ in this alga. We measured NPQ and xanthophyll cycling in blades collected from different depths as a comparative analysis of the differential expression of photoprotection in tissue acclimatized to different light conditions. The results are discussed in the context of the NPQ model described for higher plants.

Materials and Methods

Plant material

Macrocystis pyrifera (L.) C. Agardh samples were collected by scuba diving in a kelp forest near Ensenada (31°41.96 N; 116°40.90 W), Baja California, México. Based on the holdfast diameter (North, 1971), 2-yr-old organisms were sampled in spring (March 31 and May 12, 2005) and in summer (July 12 and September 5, 2005). On each date two to three different organisms were sampled from the surface to the bottom at approx. 3-m depth intervals. Four blades for each depth (0, 3, 6, 9, 15 and 18 m) were collected. The blades were tagged, kept in darkness and transported to the laboratory in coolers with seawater. The time between collection and processing of the samples did not exceed 2 h.

Tissue discs (1.2 cm diameter) free from visible epiphytes were cut with a cork borer at 10 cm above the pneumatocyst along the central axis of each blade. The discs from the four blades collected at each depth were pooled. Discs were randomly selected from the pool for measurements of the different variables. Some discs were used immediately for determination of some variables and others were maintained in 250 ml Erlenmeyer flasks with 200 ml of filtered seawater. Tissue discs were maintained at constant temperature (17 ± 0.5°C) with vigorous air bubbling. Light (75 µmol photons m−2 s−1) was provided by cool-white fluorescent bulbs in a 12 h light/12 h dark cycle. Irradiance was measured with a LI-1000 Data Logger (Li-Cor, Lincoln, NE, USA) light sensor. Pigment content, xanthophyll cycle kinetics and NPQ induction were measured just after collecting the organisms. Inhibitors and photoinhibition experiments were performed the following day. There were no noticeable changes in pigmentation or photosynthetic characteristics between days (data not shown).

Pigment quantification

The pigment content of the tissue was measured after sample collection and during the xanthophyll cycle induction experiments. Pigments concentration was measured by high-performance liquid chromatography (HPLC) as described by Colombo-Pallotta et al. (2006).

Chla fluorescence measurements

Photosystem II Chla emission was measured with a pulse amplitude modulated fluorometer (Diving-PAM; Heinz Walz, Effeltrich, Germany). Nomenclature and parameters calculation were as in van Kooten & Snel (1990). Maximum PSII quantum efficiency was determined in dark-adapted discs and calculated as Fv/Fm. The variable fluorescence (Fv) is the difference between the maximum (Fm) and the minimum (F0) emission. The F0 value was the fluorescence in darkness excited only by the pulse modulated measuring beam and Fm represents the maximum fluorescence measured in darkness when all the photochemical quenching is suppressed (first electron acceptor fully reduced) by a short saturating light pulse (0.8 s).

Xanthophyll cycle and nonphotochemical quenching of Chla fluorescence (NPQ)

Both, NPQ and the change in the concentration of the xanthophyll cycle pigments violaxanthin (Vio), antheraxanthin (Ant) and zeaxanthin (Zea) upon exposure to saturating light were measured in discs extracted from the blades collected at each depth. The discs were dark adapted for 1 h before high light exposure. Each disc was exposed for a maximum of 30 min to 1500 µmol photons m−2 s−1. The discs were maintained in a home-made acrylic temperature-controlled chamber (17°C) with constant seawater flow. The NPQ was monitored by placing the PAM fiber opZtic at 60° in relation to the light exposed side of the discs; this optical geometry was maintained during all measurements. A saturating pulse was applied each minute and NPQ was calculated as:

NPQ = (Fm − F′m)/F′m(Eqn 1)

(F′m is the maximum fluorescence measured in high light). After the light treatment, the discs were frozen and preserved in liquid nitrogen for pigment analysis.

The rate constant of Vio de-epoxidation (k) was calculated by fitting the decrease of Vio concentration in time (t) to an exponential model (Olaizola et al., 1994):

[Viot − Viof] = [Vioi − Viof]ekt(Eqn 2)

(i and f are, respectively, the concentration of Vio in dark-adapted discs and after exposing the discs to 30 min of high light).

Inhibitors treatments

The effect of DTT on NPQ induction was assayed in blades collected at different depths. Discs from the blades were dark adapted for 1 h and incubated with DTT (1 mm) for 30 min before light exposure. This concentration and incubation time proved to be effective for the inhibition of the Vio de-epoxidase enzyme (VDE). After the DTT incubation period, the discs were exposed to high light as in the xanthophyll cycle experiments.

The effect of the disruption of the transthylakoid proton gradient on the fluorescence quenching and on zeaxanthin epoxidation was investigated by using NH4Cl. The discs were exposed to high light in a temperature-controlled chamber and after 20 min NH4Cl was added. A final concentration of 100 mm NH4Cl was used to disrupt the ΔpH (Ruban et al., 2004). After the uncoupler addition, the discs were maintained in light. Also, NPQ induction was monitored in discs incubated with this uncoupler for 10 min in darkness before the high light treatment.

Photoinhibition experiments

The recovery of Fv/Fm after light stress was measured in discs from blades collected at different depths. The discs were exposed to high light for 30 min as in the xanthophyll cycle experiments. Afterwards, recovery of Fv/Fm in low light (25–30 µmol photons m−2 s−1) was measured in DTT-treated and untreated discs. The rate and the t1/2 of Fv/Fm recovery were calculated by fitting the data to the model proposed by Hanelt (1998):

Y = 1 − Aexp(−at) + Bexp(−bt)(Eqn 3)

(Y is the Fv/Fm relative to the one measured in darkness before light exposure; A is the fast recovery phase with a time constant a; B with a time constant b describes the amplitude of the slow recovery phase).

Statistical and data analysis

Data of the two to three organisms collected at each sampling date (four) were averaged for the Σ XC and DPS calculation, therefore n = 4 for each depth. Two to three assays for each depth were performed for the xanthophyll cycle induction experiments. Also, for the inhibitor and photoinhibition experiments the assays were performed at least twice for each depth. The Vio de-epoxidation rate and parameters described in the Fv/Fm recovery model were calculated by fitting the data to the proposed model using the Marquardt–Levenberg algorithm (sigmaplot software; Jandel Scientific Software, San Rafael, CA, USA). The statistical tests were done using the statistica 6.0 package (StatSoft Inc., Tulsa, OK, USA). In all cases, an alpha of 0.05 significance was chosen.

Results

Chla fluorescence quenching analysis

Nonphotochemical quenching of Chla fluorescence in M. pyrifera surface blades collected in spring was unusually high, reaching values of 10 (Colombo-Pallotta et al., 2006). To characterize the mechanisms that control NPQ in M. pyrifera we performed fluorescence quenching analysis in blades obtained from sampled depths. Figure 1 shows the fluorescence traces of the quenching analysis experiments for blades collected from three depths during the summer period. The blades from different depths presented clear differences in the induction of fluorescence quenching in high light and its relaxation in darkness (Fig. 1). Reduction of Fm in high light was rapid and was the highest in surface blades (Fig. 1a). The F′m value decreased below F0 in these blades and maximum NPQ values were around 5–6. The NPQ capacity decreased with depth and in samples collected below 6 m, the Fm reduction in high light was the lowest (Fig. 1c). Although surface blades showed a high NPQ, this was lower than NPQ of blades collected in spring.

Figure 1.

Quenching analysis in Macrocystis pyrifera blades collected at different depths. Fluorescence was measured in dark-adapted discs exposed to actinic light (AL; 1500 µmol photons m−2 s−1) for approx. 30 min (up and down arrows). Recovery of fluorescence yields (F, Fm) was measured afterwards in darkness. Maximum fluorescence (Fm, F′m) was determined by a short saturating pulse of light (0.8 s) applied every minute. (a) Surface blades; (b) blades from 6 m depth; (c) blades from 18 m depth.

In relation to the quenching relaxation in darkness, Fm recovery was slower than the induction of this process in high light (Fig. 1a,b) and the highest recovery of Fm in darkness was present in surface blades (Fig. 1a). By contrast, in the 18-m depth blades Fm recovery after light stress was minimal (Fig. 1c). Parallel to Fm quenching relaxation, variable fluorescence also recovered in darkness after light stress. In surface blades, the recovery of Fv was the highest and decreased with depth. The 18-m depth blades showed minimal recovery of Fv (Fig. 1c).

NPQ and XC activity

The amount of xanthophyll cycle pigments (Σ XC) and their interconversion upon high light exposure were measured to compare the xanthophyll cycle activity and fluorescence quenching of blades collected at sampled depths. Table 1 shows the average of these variables measured in organisms collected on the four sampling dates. As expected, surface blades that were exposed to higher irradiances presented the largest XC pigment pool, and the maximum XC de-epoxidation state (DPS; Table 1) after collection of the samples from the field.

Table 1.  Xanthophyll cycle (XC) characteristics of Macrocystis pyrifera blades collected at different depths
Depth (m)Σ XC (mol mol1 Chla) k (min1)DPS (relative units)DPS (HL) (relative units)DPS (HL) +DTT
  1. The XC pigment pool (Σ XC) is the sum of the concentration of violaxanthin (Vio), anthexanthin (Ant), and zeaxanthin (Zea). The de-epoxidation state of Σ XC (DPS) was calculated as [Zea] + [Ant]/Σ XC and was measured before and after 30 min of high light exposure (HL) in dithiothreitol (+DTT) and untreated samples. The calculation of the de-epoxidation rate constant (k) is described in the Material and Methods section. The standard deviation is presented in parenthesis; n = 4. Statistical differences between depths are denoted by different superscript letters (Student's paired t-test, P > 0.05).

00.25 (0.07)a0.480.08 (0.02)0.36 (0.06)a0.09
30.22 (0.04)a0.320.08 (0.01)0.20 (0.06)a 
60.12 (0.05)b0.280.04 (0.01)0.180.03
90.11 (0.01)b0.100.06 (0.02)0.12 (0.04)b 
150.10 (0.01)b0.150.07 (0.01)0.10 (0.05)b 
180.10 (0.01)b0.080.05 (0.03)0.11 (0.03)b0.04

Upon exposure to saturating light Vio was converted into Ant and Zea. The amount and rate of Ant and Zea synthesis in 30 min of high light were related to the position of the blades in the water column (Table 1). The de-epoxidation rate constant (k) and maximum DPS in high light were the highest in surface blades and decreased significantly from surface to 6-m depth (Table 1). These parameters were the lowest and did not present significant differences (Student's paired t-test, P > 0.05) in blades collected from depths below 6 m (Table 1).

Synthesis of Zea was highly correlated with the induction of NPQ in blades from different depths (Pearson's correlation coefficient; r = 0.9). The relationship between the Zea concentration measured in high light and the associated NPQ of blades collected from all depths and from the different sampling dates are presented in Fig. 2. There was a strong relationship between Zea synthesis and NPQ formation (Fig. 2; linear regression model, r2 = 0.84). The slope of the NPQ and Zea concentration relationship (Fig. 2) was not different from 1 and the intercept to the origin was not different from 0 (Student's t-test, P < 0.05). The variability of this relationship (Fig. 2) could be related mainly to the heterogeneity among the different blades collected at each depth. In Fig. 2, data obtained from samples maintained in darkness after high light exposure were also included. Reversibility of XC (epoxidation of Zea) took place under these conditions. Data from these measurements followed the NPQ and Zea relationship. However, some NPQ remained with low concentrations of Zea (Fig. 2), probably related to PSII damage (see later).

Figure 2.

Relationship of nonphotochemical quenching of chlorophyll a (Chla) fluorescence (NPQ) and zeaxanthin concentration in Macrocystis pyrifera. The data of several experiments performed with samples collected in spring and summer are included (open symbols). The results obtained from samples maintained in darkness after high light exposure are also included (closed symbols). The fit (r2 = 0.84) to a linear regression model (solid line) and the 95% confidence intervals (dashed lines) are presented.

The comparative analysis of Zea synthesis and NPQ in blades acclimatized to different light conditions (different depths) showed the need of this pigment for thermal dissipation. Given that surface blades presented the highest DPS and Σ XC (Table 1), these blades accumulated high levels of Zea under light stress conditions, which is in agreement with their high NPQ capacity. By contrast, basal blades with a minimal NPQ capacity synthesized low amounts of Zea through the XC (Table 1). The importance of Zea synthesis through the xanthophyll cycle for NPQ formation was further elucidated by inhibiting the VDE with DTT. The inhibition of Zea synthesis (Table 1) suppressed 80% of NPQ in surface blades (Fig. 3a). By contrast, in basal blades (18 m) DTT did not produce any noticeable effect on NPQ induction (Fig. 3b). Dithiothreitol also affected the NPQ relaxation in darkness after light stress. The NPQ relaxation in surface blades was fast in darkness after the high light treatment (Fig. 3a), and DTT completely suppressed this relaxation (Fig. 3b). The effect of DTT in samples from all depths was to set the NPQ to a minimum value. All DTT-treated samples presented similar induction and relaxation kinetics and these characteristics were similar to the 18-m depth untreated blades, which presented the lowest NPQ (Fig. 3) and the lowest XC activity (Table 1).

Figure 3.

Dithiothreitol (DTT) effect on nonphotochemical quenching of chlorophyll a fluorescence (NPQ) in blades of Macrocystis pyrifera collected from different depths. (a) NPQ in untreated samples (control); (b) NPQ in discs incubated with DTT before high light exposure.

Effect of NH4Cl on NPQ

Because the formation of a transthylakoid proton gradient is essential for NPQ, the disruption of this gradient relaxes immediately the fluorescence quenching formed in high light. A dissimilar response of NPQ upon the addition of the uncoupler NH4Cl was detected in blades collected from different depths. Fluorescence quenching formed in 20 min of high light exposure relaxed slowly after the addition of NH4Cl in surface blades (Fig. 4a). The effect of NH4Cl on fluorescence quenching relaxation diminished with depth and this uncoupler did not present any effect on basal blades (Fig. 4b,c).

Figure 4.

Effect of NH4Cl on fluorescence quenching in Macrocystis pyrifera blades collected from different depths. Discs obtained from blades collected from (a) surface, (b) 6-m and (c) 18-m depths were exposed to actinic light (up arrow) and NH4Cl was added after 20 min.

Fluorescence quenching relaxation after the addition of NH4Cl seemed to be correlated with the degree of NPQ expression associated with XC activity. In fact, surface blades incubated with DTT before light exposure did not present the slow NPQ relaxation after the addition of NH4Cl (Fig. 5a). These results indicate that the slow reversibility of NPQ took place only in the presence of de-epoxidated XC pigments. The effect of incubating the samples with NH4Cl before light exposure was also assayed. Samples incubated with NH4Cl did not synthesize Zea in high light ([Zea] = 0.007 mol Zea mol−1 Chla) and therefore fluorescence quenching was minimal (Fig. 5b). The NPQ after 15 min of high light exposure for surface samples incubated with NH4Cl (NPQ = 0.48) was not different from the one measured in samples incubated with DTT (NPQ = 0.63; Fig. 5a). In blades treated with the inhibitors that blocked Zea synthesis the NPQ was comparable to that measured in basal blades (NPQ = 0.55, Fig. 3b). These results indicate that NPQ in M. pyrifera was dependent principally on Zea formation and that the residual NPQ detected in basal blades and DTT-treated samples could be associated with a reduction of Fm independent of XC activity, like PSII inactivation. The effect of NH4Cl on NPQ relaxation seemed to be related to the back conversion of Zea into Vio since most likely the VDE activity was lost after the addition of the uncoupler. To support this idea, the rate of Zea epoxidation in darkness after 20 min of high light was measured and compared with the effect of NH4Cl on Zea concentration in blades maintained in high light. The Zea concentration formed in high light (20 min) decreased rapidly in darkness (Fig. 6). In approx. 30 min the epoxidation reaction restored Zea concentration close to values measured before light stress (Fig. 6). Addition of NH4Cl to blades exposed to high light induced the epoxidation of Zea (Fig. 6). The amount of Zea epoxidated in high light after the addition of NH4Cl was not different from the one measured in darkness; the rate of Zea epoxidation was also similar between both treatments (Student's t-test, P < 0.05).

Figure 5.

Fluorescence quenching in Macrocystis pyrifera surface blades incubated with (a) dithiothreitol (DTT) and (b) NH4Cl before high light exposure (up arrow). The NH4Cl was added after 15 min of high light exposure to the DTT-treated samples.

Figure 6.

Epoxidation of zeaxanthin in darkness after exposing Macrocystis pyrifera surface blades to 20 min of high light (closed circles, solid line). The reduction of zeaxanthin concentration in light after the addition of NH4Cl is represented by the open circles and the dashed line. The lines represent the best fit to an exponential model and the epoxidation rate constants are presented for both treatments. The dotted line represents the amount of Zea present in darkness before light exposure.

Photoprotective role of XC

To tests the importance of Zea formation through the XC as a photoprotective mechanism, the recovery of PSII maximum quantum efficiency (Fv/Fm) after light stress was measured in the samples from different depths. The highest recovery rate was measured in surface blades. In these blades, the fast phase (A) represented > 90% of the PSII maximum efficiency recovery. Therefore, Fv/Fm recovered rapidly after the light stress with a t1/2 of 5 min (Fig. 7a). The relative importance of the Fv/Fm fast recovery phase decreased in blades from 6-m and 18-m depths, and they presented a t1/2 of 25 min and 132 min, respectively (Fig. 7b,c). The impairment of XC activity with DTT caused a negative effect on Fv/Fm recovery in the surface and 6-m depth samples but not in the 18-m depth samples. In the DTT-treated surface and 6-m depth blades the fast recovery phase of Fv/Fm disappeared. The t1/2 of Fv/Fm recovery increased 12-fold in DTT-treated surface blades and 3.5-fold in DTT-treated 6-m depth samples. By contrast, the t1/2 of Fv/Fm recovery was similar in DTT-treated and untreated 18-m depth samples.

Figure 7.

Photosystem II maximum quantum efficiency (Fv/Fm) recovery after light stress in Macrocystis pyrifera blades collected from (a) surface, (b) 6-m and (c) 18-m depths. The Fv/Fm recovery was measured in dithiothreitol (DTT)-treated (closed circles) and untreated (open symbols) samples. The fit to the model proposed by Hanelt (1998, see the Materials and Methods section) is represented by the continuous lines.

Discussion

Relationship between NPQ and XC

The analysis of Chla fluorescence quenching and XC activity in M. pyrifera photosynthetic tissue acclimatized to different light conditions (blades collected from different depths) showed that, unlike in higher plants, NPQ in M. pyrifera could be explained mainly by the synthesis of Zea. Therefore, the high NPQ capacity of this alga is associated with a high accumulation of Zea in light saturating conditions.

The fluorescence quenching analysis and the inhibitor treatments were performed in blades collected in summer. These blades showed a lower NPQ than blades collected in spring (Colombo-Pallotta et al., 2006). During spring, M. pyrifera shows the highest frond elongation rate (González-Fregoso et al., 1991) while in summer, photosynthetic performance and growth decrease owing to elevated temperatures and low concentration of nutrients at the surface (Gerard, 1984). Therefore, particular physiological characteristics are represented in this study. Nevertheless, the relationship of NPQ and Zea constructed with all the data acquired for the different sampling dates indicates that the mechanisms of thermal dissipation are similar between different physiological conditions of the blades.

Macrocystis pyrifera exhibits a similar XC to higher plants; however, NPQ and associated controlling factors are very similar to diatoms. One remarkable similarity is that the relationship between Zea and NPQ in M. pyrifera is comparable to the DTx and NPQ relationship reported for diatoms (Lavaud et al., 2002a, 2004). A similar concentration of Zea or DTx relative to Chla induces comparable NPQ in both algae groups. Therefore, they express an extremely high NPQ capacity by accumulating a large amount of photoprotective pigments. The super-high NPQ in M. pyrifera was measured in blades acclimatized to high light (surface) while in the diatom Phaeodactylum tricornutum it was present in cells acclimatized to an intermittent light regime (Lavaud et al., 2002a; Ruban et al., 2004). These two conditions induce a considerable enlargement of the Σ XC. The size of XC pigment pool relative to Chla in M. pyrifera surface blades was three times larger than in basal blades, and it is approx. five times larger compared with higher plants (Jahns, 1995). Diatoms also have a much larger Σ XC than higher plants (Wilhelm, 1990; Lavaud et al., 2004) and the intermittent light condition induces a 100% increase of this pool (Lavaud et al., 2002a). These results indicate that the regulation of the Σ XC is an important acclimatization strategy in brown algae and diatoms to cope with light stress.

The amount of the interconvertible XC pigments was related to acclimatization state of the blades. Surface blades showed the fastest de-epoxidation rate and the highest accumulation of zeaxanthin upon high light exposure. The increase in the amount of Vio that can be de-epoxidated is a well characterized response of sun-acclimatized plants (Demmig-Adams & Adams, 1996). The main difference between M. pyrifera and higher plant de-epoxidation characteristics is that the maximum DPS was much lower in the alga. Maximum DPS in M. pyrifera was c. 35% whereas DPS values of sun-acclimatized plants (Pfündel & Bilger, 1994) and green algae (Casper-Lindley & Björkman, 1998; García-Mendoza et al., 2002) could be as high as 80%. In higher plants, the amount of Vio converted into Zea is related to the size of PSII peripheral light-harvesting complex (LHCII). Maximum DPS is higher when LHCII is smaller, as in high-light acclimatized plants (Jahns, 1995; Härtel et al., 1996; Färber & Jahns, 1998). The regulation of the size of the peripheral light-harvesting complex in brown algae in relation to XC activity has not been investigated, but probably the arrangement of this antenna affects the maximum amount of violaxanthin that can be de-epoxidated.

Mechanisms involved in NPQ control

Macrocystis pyrifera does not have state transitions (Fork et al., 1991) and since no ΔpH-related NPQ is present (see later), then the reduction of Fm according to the Stern–Volmer relationship was caused mainly by the accumulation of the quencher Zea. Therefore, DTT blocked NPQ induction considerably. Surface and subsurface DTT-treated blades showed a NPQ similar to the 18-m depth samples. The NPQ in the 18-m depth samples presented null sensitivity to DTT since XC activity was minimal in these blades. The residual NPQ in the 18-m depth and DTT samples could be explained by the presence of preformed Zea (or Ant) before the high light treatment but most likely by the reduction of Fm related to PSII damage.

Inhibition of Zea synthesis with DTT in diatoms also blocks NPQ induction (Olaizola et al., 1994; Casper-Lindley & Björkman, 1998; Lavaud et al., 2002b). By contrast, higher plants and green algae have a large DTT-insensitive NPQ (Pfündel & Bilger, 1994). This NPQ is associated with the protonation of LHC subunits upon formation of the ΔpH (Müller et al., 2001; Horton & Ruban, 2005). In higher plants (Horton et al., 1991) and green algae (García-Mendoza et al., 2002) high light–dark transition is accompanied with a fast decay of NPQ associated with the disappearance of the ΔpH and it is independent of the presence of Zea since it is also observed in DTT-treated samples (García-Mendoza et al., 2002). Macrocystis pyrifera does not show a fast Fm quenching induction in high light and more importantly, NPQ relaxation in darkness is slow. The NPQ induction and relaxation follow closely the synthesis of Zea and the epoxidation of this pigment, respectively. Therefore, M. pyrifera does not have a direct ΔpH-dependent NPQ. The role of the ΔpH seems to be related only to the activation of the VDE. A key observation that indicates the lack of direct ΔpH-dependent NPQ is the response of the blades to NH4Cl. A slow relaxation of NPQ after the disruption of ΔpH with NH4Cl was observed only in the presence of Zea. By contrast, in higher plants (Noctor et al., 1991; Ruban et al., 2004) and green algae (García-Mendoza et al., 2002) NPQ relaxes in seconds after addition of NH4Cl in DTT-treated and untreated samples. Thus, M. pyrifera does not have the fast NPQ induction phase that has been attributed to protonation of certain PSII antenna proteins in higher plants. Alternatively, protonation does take place and in the presence of Zea there is a slow release of protons from the quenching locus. This was suggested by Ruban et al. (2004) and Horton & Ruban (2005) to explain the slow NPQ relaxation observed in P. tricornutum upon the addition of an uncoupler. However, in M. pyrifera the slow NPQ relaxation after addition of NH4Cl was related to the epoxidation of Zea since the VDE became inactive upon disruption of the ΔpH. In any case, if protonation of some PSII antenna proteins exists, it does not induce any NPQ. Lavaud et al. (2002b) reported also that ΔpH alone does not induce NPQ in the diatom P. tricornutum.

Comparative analysis of NPQ in different organisms is important for the acquisition of fundamental knowledge of NPQ (Horton & Ruban, 2005). Our results show that the control of one of the most important responses to light stress of phototropic eukaryotes seems to differ among evolutionary lineages. Macrocystis pyrifera (red algae lineage) presents a XC similar to higher plants (green algae lineage); however, NPQ characteristics and associated controlling factors are different. The NPQ characteristics in M. pyrifera are hard to explain using the higher plant model. The main inconsistency is that the modulator of thermal dissipation that enables a fast induction and relaxation of NPQ does not seem to be present in M. pyrifera. The on–off switch (modulator) in higher plants is the protonation of some LHC subunits, specifically PsbS (Horton & Ruban, 2005). In diatoms and brown algae, no PsbS has been found (Müller et al., 2001). Since PSII light harvesting antenna architecture is different between higher plants and brown algae (De Martino et al., 2000) it is feasible to consider differences in NPQ control between these groups of organisms. In higher plants, qE is the key element in the regulation of thermal dissipation. However, zeaxanthin-dependent and pH- and PsbS-independent light-inducible quenching is also present (Dall’Osto et al., 2005). This quenching, which is insensitive to uncouplers, corresponds in part to the slowly relaxing component of NPQ referred as qI (Dall’Osto et al., 2005). It has been shown that qI, overexpressed in plants acclimatized to low temperatures, efficiently protects the photosynthetic apparatus (Verhoeven et al., 1999; Gilmore & Ball, 2000). This condition induces sustained high values of zeaxanthin and probably the aggregation of LHCII is the molecular mechanism that promotes high qI (Gilmore & Ball, 2000; Horton et al., 2005). More recently, Dall’Osto et al. (2005) identified that the origin of the pH-independent quenching could be associated with conformational changes of LHC proteins upon the binding of zeaxanthin. It is feasible to consider that the slowly relaxing, uncoupler-insensitive quenching in brown algae is similar to the qI quenching observed in higher plants. Light harvesting architecture of brown algae probably promotes this type of quenching and might be responsible for the high NPQ measured in M. pyrifera.

The importance of XC for photoprotection has been recognized for higher plants (Havaux & Niyogi, 1999) and diatoms (Olaizola et al., 1994; Lavaud et al., 2002a). The recovery of Fv/Fm after light stress in surface blades indicates that a high NPQ represents an effective photoprotection strategy in M. pyrifera. Two processes with different kinetics contribute to the recovery of Fv/Fm after light stress (Hanelt, 1998). Slow recovery of Fv/Fm is associated with the repair of damaged PSII centres when there is a strong degradation of the D1 protein (chronic photoinhibition). The fast recovery phase is related to a rapid reversibility of PSII down-regulation activated in high light. Our data showed that this phase is related to xanthophyll cycling. Fast recovery phase is minimal in the absence of XC activity as seen in basal blades. Also, inhibition of XC activity with DTT blocked the fast recovery of Fv/Fm. Dithiothreitol is a nonspecific inhibitor of the VDE; however, no secondary effects were detected. Dithiothreitol did not increase photoinhibition in basal blades and no noticeable reduction of Fv/Fm in darkness before light stress or a reduction of oxygen evolving capacity (data not show) were detected. Therefore, the slow Fv/Fm recovery in DTT-treated surface samples and basal blades with a minimal capacity of Zea accumulation indicates the need of this pigment for photoprotection.

In conclusion, in the brown alga M. pyrifera with a XC similar to higher plants, NPQ and controlling factors resemble that present in diatoms. In both groups, NPQ depends mainly on the amount of photoprotective pigments synthesized in high light. Diatoms present a faster induction of NPQ since synthesis of diatoxanthin is achieved through a one-step de-epoxidation reaction. Diatoms proliferate in highly variable light environments such as unstratified waters; therefore, they need a faster response to light stress than macroalgae. In the latter, according to their position in the water column, the expression of a highly efficient photoprotective response (large Σ XC, high NPQ) is probably more important than a fast response.

Acknowledgements

Doctoral scholarship to M.F.C.P. was provided by project AMELIS (J37689-V), CONACYT.

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