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.