Vasco Giovagnetti

Spectral radiation dependent photoprotective mechanism in the diatom Pseudo-nitzschia multistriata.

Phytoplankton, such as diatoms, experience great variations of photon flux density (PFD) and light spectrum along the marine water column. Diatoms have developed some rapidly-regulated photoprotective mechanisms, such as the xanthophyll cycle activation (XC) and the non-photochemical chlorophyll fluorescence quenching (NPQ), to protect themselves from photooxidative damages caused by excess PFD. In this study, we investigate the role of blue fluence rate in combination with red radiation in shaping photoacclimative and protective responses in the coastal diatom Pseudo-nitzschia multistriata. This diatom was acclimated to four spectral light conditions (blue, red, blue-red, blue-red-green), each of them provided with low and high PFD. Our results reveal that the increase in the XC pool size and the amplitude of NPQ is determined by the blue fluence rate experienced by cells, while cells require sensing red radiation to allow the development of these processes. Variations in the light spectrum and in the blue versus red radiation modulate either the photoprotective capacity, such as the activation of the diadinoxanthin-diatoxanthin xanthophyll cycle, the diadinoxanthin de-epoxidation rate and the capacity of non-photochemical quenching, or the pigment composition of this diatom. We propose that spectral composition of light has a key role on the ability of diatoms to finely balance light harvesting and photoprotective capacity.

The xanthophyll cycle affects reversible interactions between PsbS and light-harvesting complex II to control non-photochemical quenching.

To maintain high photosynthetic rates, plants must adapt to their light environment on a timescale of seconds to minutes. Therefore, the light-harvesting antenna system of photosystem II in thylakoid membranes, light-harvesting complex II (LHCII), has a feedback mechanism, which determines the proportion of absorbed energy dissipated as heat: non-photochemical chlorophyll fluorescence quenching (NPQ). This is crucial to prevent photo-oxidative damage to photosystem II (PSII) and is controlled by the transmembrane pH differences (ΔpH). High ΔpH activates NPQ by protonation of the protein PsbS and the enzymatic de-epoxidation of LHCII-bound violaxanthin to zeaxanthin. But the precise role of PsbS and its interactions with different LHCII complexes remain uncertain. We have investigated PsbS–LHCII interactions in native thylakoid membranes using magnetic-bead-linked antibody pull-downs. The interaction of PsbS with the antenna system is affected by both ΔpH and the level of zeaxanthin. In the presence of ΔpH alone, PsbS is found to be mainly associated with the trimeric LHCII protein polypeptides, Lhcb1, Lhcb2 and Lhcb3. However, a combination of ΔpH and zeaxanthin increases the proportion of PsbS bound to the minor LHCII antenna complex proteins Lhcb4, Lhcb5 and Lhcb6. This pattern of interaction is not influenced by the presence of PSII reactions centres. Similar to LHCII particles in the photosynthetic membrane, PsbS protein forms clusters in the NPQ state. NPQ recovery in the dark requires uncoupling of PsbS. We suggest that PsbS acts as a ‘seeding’ centre for the LHCII antenna rearrangement that is involved in NPQ.

PsbS protein modulates non-photochemical chlorophyll fluorescence quenching in membranes depleted of photosystems.

Plants with varying levels of PsbS protein were grown on lincomycin. Enhanced levels of non-photochemical fluorescence quenching (NPQ) in over-expressers of the protein have been observed. This was accompanied by increased amplitude of the irreversible NPQ component, qI, previously considered to reflect mainly photoinhibition of PSII reaction centres (RCII). However, since RCIIs were largely absent the observed qI is likely to originate from the LHCII antenna. In chloroplasts of over-expressers of PsbS grown on lincomycin an abnormally large NPQ (∼7) was characterised by a 0.34 ns average chlorophyll fluorescence lifetime. Yet the lifetime in the Fm state was similar to that of wild-type plants. 77K fluorescence emission spectra revealed a specific 700 nm peak typical of LHCII aggregates as well as quenching of the PSI fluorescence at 730 nm. The aggregated state manifested itself as a clear change in the distance between LHCII complexes detected by freeze-fracture electron microscopy. Grana thylakoids in the quenched state revealed 3 times more aggregated LHCII particles compared to the dark-adapted state. Overall, the results directly demonstrate the importance of LHCII aggregation in the NPQ mechanism and show that the PSII supercomplex structure plays no role in formation of the observed quenching.

The Velocity of Light Intensity Increase Modulates the Photoprotective Response in Coastal Diatoms

In aquatic ecosystems, the superimposition of mixing events to the light diel cycle exposes phytoplankton to changes in the velocity of light intensity increase, from diurnal variations to faster mixing-related ones. This is particularly true in coastal waters, where diatoms are dominant. This study aims to investigate if coastal diatoms differently activate the photoprotective responses, xanthophyll cycle (XC) and non-photochemical fluorescence quenching (NPQ), to cope with predictable light diel cycle and unpredictable mixing-related light variations. We compared the effect of two fast light intensity increases (simulating mixing events) with that of a slower increase (corresponding to the light diel cycle) on the modulation of XC and NPQ in the planktonic coastal diatom Pseudo-nitzschia multistriata. During each light treatment, the photon flux density (PFD) progressively increased from darkness to five peaks, ranging from 100 to 650 µmol photons m−2 s−1. Our results show that the diel cycle-related PFD increase strongly activates XC through the enhancement of the carotenoid biosynthesis and induces a moderate and gradual NPQ formation over the light gradient. In contrast, during mixing-related PFD increases, XC is less activated, while higher NPQ rapidly develops at moderate PFD. We observe that together with the light intensity and its increase velocity, the saturation light for photosynthesis (Ek) is a key parameter in modulating photoprotection. We propose that the capacity to adequately regulate and actuate alternative photoprotective ‘safety valves’ in response to changing velocity of light intensity increase further enhances the photophysiological flexibility of diatoms. This might be an evolutionary outcome of diatom adaptation to turbulent marine ecosystems characterized by unpredictable mixing-related light changes over the light diel cycle.

Growth and photophysiological responses of two picoplanktonic Minutocellus species, strains RCC967 and RCC703 (Bacillariophyceae)

Reaching up to 50% of the total biomass in oligotrophic waters and armed with a set of ecological and biological properties related to their small size, picophytoplankton (<3.0 µm) are a good model to address ecophysiological questions regarding phytoplankton biodiversity. Two picoplanktonic diatoms, one isolated from an upwelling ecosystem in the Pacific Ocean (Minutocellus sp., strain RCC967), and another from oceanic waters in the Indian Ocean (Minutocellus sp., strain RCC703) were used to test hypotheses on the functional relation between ecological niche adaptation and photosynthetic regulation capacity and efficiency. Cultures were subjected to five sine light climates, each one set to peak at a different photon flux density, respectively 10, 50, 100, 250 and 500 µmol photons m−2 s−1. Growth rate, photosynthesis, non-photochemical fluorescence quenching, pigment composition, and particulate organic carbon and nitrogen content were followed daily for 5 days. Growth rate and physiological response curves were different in the two species, in agreement with their distinct habitats of origin. Such differences could be related to the diverse photoacclimative strategies displayed by the two species, revealing a clear adaptive divergence despite their close taxonomic relationship. Photoacclimative strategies of the two picoplanktonic diatoms are discussed in the light of functional diversity and ecosystem adaptation.

Assessment of the impact of photosystem I chlorophyll fluorescence on the pulse-amplitude modulated quenching analysis in leaves of Arabidopsis thaliana

In their natural environment, plants are exposed to varying light conditions, which can lead to a build-up of excitation energy in photosystem (PS) II. Non-photochemical quenching (NPQ) is the primary defence mechanism employed to dissipate this excess energy. Recently, we developed a fluorescence-quenching analysis procedure that enables the protective effectiveness of NPQ in intact Arabidopsis leaves to be determined. However, pulse-amplitude modulation measurements do not currently allow distinguishing between PSII and PSI fluorescence levels. Failure to account for PSI contribution is suggested to lead to inaccurate measurements of NPQ and, particularly, maximum PSII yield (Fv/Fm). Recently, Pfündel et al. (Photosynth Res 114:189–206, 2013) proposed a method that takes into account PSI contribution in the measurements of Fo fluorescence level. However, when PSI contribution was assumed to be constant throughout the induction of NPQ, we observed lower values of the measured minimum fluorescence level (

The causes of altered chlorophyll fluorescence quenching induction in the Arabidopsis mutant lacking all minor antenna complexes

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Detachment of the fucoxanthin chlorophyll a/c binding protein (FCP) antenna is not involved in the acclimative regulation of photoprotection in the pennate diatom Phaeodactylum tricornutum

Focalc.′) than those calculated according to the formula of Oxborough and Baker (Photosynth Res 54:135–142 1997) (