Johann Lavaud

Influence of the diadinoxanthin pool size on photoprotection in the marine planktonic diatom Phaeodactylum tricornutum.

The pool size of the xanthophyll cycle pigment diadinoxanthin (DD) in the diatom Phaeodactylum tricornutum depends on illumination conditions during culture. Intermittent light caused a doubling of the DD pool without significant change in other pigment contents and photosynthetic parameters, including the photosystem II (PSII) antenna size. On exposure to high-light intensity, extensive de-epoxidation of DD to diatoxanthin (DT) rapidly caused a very strong quenching of the maximum chlorophyll fluorescence yield (F m, PSII reaction centers closed), which was fully reversed in the dark. The non-photochemical quenching of the minimum fluorescence yield (F o, PSII centers open) decreased the quantum efficiency of PSII proportionally. For bothF m and F o, the non-photochemical quenching expressed asF/F′ − 1 (with F′ the quenched level) was proportional to the DT concentration. However, the quenching of F o relative to that ofF m was much stronger than random quenching in a homogeneous antenna could explain, showing that the rate of photochemical excitation trapping was limited by energy transfer to the reaction center rather than by charge separation. The cells can increase not only the amount of DT they can produce, but also its efficiency in competing with the PSII reaction center for excitation. The combined effect allowed intermittent light grown cells to down-regulate PSII by 90% and virtually eliminated photoinhibition by saturating light. The unusually rapid and effective photoprotection by the xanthophyll cycle in diatoms may help to explain their dominance in turbulent waters.

GENERAL FEATURES OF PHOTOPROTECTION BY ENERGY DISSIPATION IN PLANKTONIC DIATOMS (BACILLARIOPHYCEAE)

Planktonic diatoms (Bacillariophyceae) have to cope with large fluctuations of light intensity and periodic exposure to high light. After a shift to high light, photoprotective dissipation of excess energy characterized by the nonphotochemical quenching of fluorescence (NPQ) and the concomitant deepoxidation of diadinoxanthin to diatoxanthin (DT) were measured in four different planktonic marine diatoms (Bacillariophyceae): Skeletonema costatum (Greville) Cleve, Cylindrotheca fusiformis Reimann et Lewin, Thalassiosira weissflogii (Grunow) Fryxell et Hasle, and Ditylum brightwellii (West) Grunow in comparison to the model organism Phaeodactylum tricornutum Böhlin. Upon a sudden increase of light intensity, deepoxidation was rapid and de novo synthesis of DT also occurred. In all species, NPQ was linearly related to the amount of DT formed during high light. In this report, we focused on the role of DT in the dissipation of energy that takes place in the light‐harvesting complex. In S. costatum for the same amount of DT, less NPQ was formed than in P. tricornutum and as a consequence the photoprotection of PSII was less efficient. The general features of photoprotection by harmless dissipation of excess energy in planktonic diatoms described here partly explain why diatoms are well adapted to light intensity fluctuations.

In diatoms, a transthylakoid proton gradient alone is not sufficient to induce a non‐photochemical fluorescence quenching

Non‐photochemical fluorescence quenching (NPQ) in diatoms is associated with a xanthophyll cycle involving diadinoxanthin (DD) and its de‐epoxidized form, diatoxanthin (DT). In higher plants, an obligatory role of de‐epoxidized xanthophylls in NPQ remains controversial and the presence of a transthylakoid proton gradient (ΔpH) alone may induce NPQ. We used inhibitors to alter the amplitude of ΔpH and/or DD de‐epoxidation, and coupled NPQ. No ΔpH‐dependent quenching was detected in the absence of DT. In diatoms, both ΔpH and DT are required for NPQ. The binding of DT to protonated antenna sites could be obligatory for energy dissipation.

Photosystem II electron transfer cycle and chlororespiration in planktonic diatoms.

The dominance of diatoms in turbulent waters suggests special adaptations to the wide fluctuations in light intensity that phytoplankton must cope with in such an environment. Our recent demonstration of the unusually effective photoprotection by the xanthophyll cycle in diatoms [Lavaud et al. (2002) Plant Physiol 129 (3) (in press)] also revealed that failure of this protection led to inactivation of oxygen evolution, but not to the expected photoinhibition. Photo-oxidative damage might be prevented by an electron transfer cycle around Photosystem II (PS II). The induction of such a cycle at high light intensity was verified by measurements of the flash number dependence of oxygen production in a series of single-turnover flashes. After a few minutes of saturating illumination, the oxygen flash yields are temporarily decreased. The deficit in oxygen production amounts to at most 3 electrons per PS II, but continues to reappear with a half time of 2 min in the dark until the total pool of reducing equivalents accumulated during the illumination has been consumed by (chloro)respiration. This is attributed to an electron transfer pathway from the plastoquinone pool or the acceptor side of PS II to the donor side of PS II that is insignificant at limiting light intensity but is accelerated to milliseconds at excess light intensity. Partial filling of the 3-equivalents capacity of the cyclic electron transfer path in PS II may prevent both acceptor-side photoinhibition in oxygen-evolving PS II and donor-side photoinhibition when the oxygen-evolving complex is temporarily inactivated.

High Light Acclimation in the Secondary Plastids Containing Diatom Phaeodactylum tricornutum is Triggered by the Redox State of the Plastoquinone Pool

Summary: The redox state of the plastidic plastoquinone pool triggers a retrograde signal in diatoms, organisms with plastids evolved by secondary endosymbiosis. In diatoms, the process of energy-dependent chlorophyll fluorescence quenching (qE) has an important role in photoprotection. Three components are essential for qE: (1) the light-dependent generation of a transthylakoidal proton gradient; (2) the deepoxidation of the xanthophyll diadinoxanthin (Dd) into diatoxanthin (Dt); and (3) specific nucleus-encoded antenna proteins, called Light Harvesting Complex Protein X (LHCX). We used the model diatom Phaeodactylum tricornutum to investigate the concerted light acclimation response of the qE key components LHCX, proton gradient, and xanthophyll cycle pigments (Dd+Dt) and to identify the intracellular light-responsive trigger. At high-light exposure, the up-regulation of three of the LHCX genes and the de novo synthesis of Dd+Dt led to a pronounced rise of qE. By inhibiting either the conversion of Dd to Dt or the translation of LHCX genes, qE amplification was abolished and the diatom cells suffered from stronger photoinhibition. Artificial modification of the redox state of the plastoquinone (PQ) pool via 3-(3,4-dichlorophenyl)-1,1-dimethylurea and 5-dibromo-6-isopropyl-3-methyl-1,4-benzoquinone resulted in a disturbance of Dd+Dt synthesis in an opposite way. Moreover, we could increase the transcription of two of the four LHCX genes under low-light conditions by reducing the PQ pool using 5-dibromo-6-isopropyl-3-methyl-1,4-benzoquinone. Altogether, our results underline the central role of the redox state of the PQ pool in the light acclimation of diatoms. Additionally, they emphasize strong evidence for the existence of a plastid-to-nucleus retrograde signaling mechanism in an organism with plastids that derived from secondary endosymbiosis.

An explanation for the inter-species variability of the photoprotective non-photochemical chlorophyll fluorescence quenching in diatoms.

Diatoms are a major group of microalgae whose photosynthetic productivity supports a substantial part of the aquatic primary production. In their natural environment they have to cope with strong fluctuations of the light climate which can be harmful for photosynthesis. In order to prevent the damage of their photosynthetic machinery, diatoms use fast regulatory processes among which the non-photochemical quenching of chlorophyll a fluorescence (NPQ) is one of the most important. In a previous work, we highlighted differences in the kinetics and extent of NPQ between diatom species/strains originating from different aquatic habitats. We proposed that the NPQ differences observed between strains/species could potentially participate to their ecophysiological adaptation to the light environment of their respective natural habitat. In order to better understand the molecular bases of such differences, we compared the NPQ features of four strains/species of diatoms known for their NPQ discrepancy. We could identify new spectroscopic fingerprints concomitant to NPQ and the related xanthophyll cycle. These fingerprints helped us propose a molecular explanation for the NPQ differences observed between the diatom species/strains examined. The present work further strengthens the potential role of NPQ in the ecophysiology of diatoms.

The light-harvesting antenna of the diatom Phaeodactylum tricornutum : Evidence for a diadinoxanthin-binding subcomplex

Diatoms differ from higher plants by their antenna system, in terms of both polypeptide and pigment contents. A rapid isolation procedure was designed for the membrane‐intrinsic light harvesting complexes (LHC) of the diatom Phaeodactylum tricornutum to establish whether different LHC subcomplexes exist, as well to determine an uneven distribution between them of pigments and polypeptides. Two distinct fractions were separated that contain functional oligomeric complexes. The major and more stable complex (≈ 75% of total polypeptides) carries most of the chlorophyll a, and almost only one type of carotenoid, fucoxanthin. The minor complex, carrying ≈ 10–15% of the total antenna chlorophyll and only a little chlorophyll c, is highly enriched in diadinoxanthin, the main xanthophyll cycle carotenoid. The two complexes also differ in their polypeptide composition, suggesting specialized functions within the antenna. The diadinoxanthin‐enriched complex could be where the de‐epoxidation of diadinoxanthin into diatoxanthin mostly occurs.

A model for describing the light response of the nonphotochemical quenching of chlorophyll fluorescence.

The operation of photosynthetic energy-dissipating processes is commonly characterized by measuring the light response of the nonphotochemical quenching (NPQ) of chlorophyll fluorescence, or NPQ versus E curves. This study proposes a mathematical model for the quantitative description of the generic NPQ versus E curve. The model is an adaptation of the Hill equation and is based on the close dependence of NPQ on the xanthophyll cycle (XC). The model was tested on NPQ versus E curves measured in the plant Arabidopsis thaliana and the diatom Nitzschia palea, representing the two main types of XC, the violaxanthin–antheraxanthin–zeaxanthin (VAZ) type and the diadinoxanthin–diatoxanthin (DD–DT) type, respectively. The model was also fitted to a large number of published light curves, covering the widest possible range of XC types, taxa, growth conditions, and experimental protocol of curve generation. The model provided a very good fit to experimental and published data, coping with the large variability in curve characteristics. The model was further used to quantitatively compare the light responses of NPQ and of PSII electron transport rate, ETR, through the use of indices combining parameters of the models describing the two types of light–response curves. Their application to experimental and published data showed a systematic large delay of the buildup of NPQ relatively to the saturation of photochemistry. It was found that when ETR reaches saturation, NPQ is on average still below one fifth of its maximum attainable level, which is only reached at irradiances about three times higher. It was also found that organisms having the DD–DT type of XC appeared to be able to start operating the XC at lower irradiances than those of the VAZ type.

Phytoplankton Pigments: Pigments and photoacclimation processes

This chapter reviews the nature of pigment variations in phytoplankton in response to changes in light regime (irradiance, spectral composition and daylength). These changes belonging to processes called acclimation and/or adaptation maximize the evolutionary fitness of a species, within the constraints set by the environmental conditions (Raven and Geider, 2003). In general, adaptation indicates long-term evolutionary outcome based on the genes a given species obtains (genetic adaptation) while acclimation denotes adjustments in response to variation in key-environmental variables (physiological acclimation). Photo-acclimation corresponds to a mosaic of processes involving many cellular components and occurring over a broad range of time scales, from seconds to days. These processes, covering many physiological, biochemical, biophysical and biological changes, allow the optimization of cell activities, such as photosynthesis, respiration, growth and division when faced with changing irradiance (e.g., Herzig and Dubinsky, 1993; Anning et al., 2000; Raven and Geider, 2003). This is an important issue in phytoplankton ecology because of the fluctuating light environment experienced by pelagic algae, related to daylight variations together with the exponential decrease of light and the vertical – active or passive – movements of algae along the water column. In order to cope with these never-ending fluctuations in light-regime, marine phytoplankton can adjust their pigment pool, which is mainly constituted by two functional categories, namely pigments used for light harvesting and for photoprotection. Many accessory pigments constituting the light-harvesting complexes are photosynthetically active i.e. they are able to transfer the energy absorbed from sunlight to the photosynthetic reaction centers (RC) of photosystems (PS) II and I. They are called light harvesting pigments and include the photosynthetic carotenoids. However, some carotenoids are not involved in photosynthesis and do not transfer the absorbed energy to the RC. These non-photosynthetically active carotenoids are also called photoprotective carotenoids (PPC). The function and dynamics of long-term (hours-days) and short-term (minutes-hours) photo-acclimation are described in the following two sections (4.4.1 and 4.4.2, respectively). The long-term photo-acclimation response mainly consists in changes of structure and composition of the photosystems while the short-term photo-acclimation process mainly concerns the xanthophyll cycle (XC) activation and the associated non-photochemical fluorescence quenching (NPQ). In the third section (4.4.3), the ecophysiological variability of XC and its use as a biological tracer in oceanographic studies is reported.

Growth form defines physiological photoprotective capacity in intertidal benthic diatoms

In intertidal marine sediments, characterized by rapidly fluctuating and often extreme light conditions, primary production is frequently dominated by diatoms. We performed a comparative analysis of photophysiological traits in 15 marine benthic diatom species belonging to the four major morphological growth forms (epipelon (EPL), motile epipsammon (EPM-M) and non-motile epipsammon (EPM-NM) and tychoplankton (TYCHO)) found in these sediments. Our analyses revealed a clear relationship between growth form and photoprotective capacity, and identified fast regulatory physiological photoprotective traits (that is, non-photochemical quenching (NPQ) and the xanthophyll cycle (XC)) as key traits defining the functional light response of these diatoms. EPM-NM and motile EPL showed the highest and lowest NPQ, respectively, with EPM-M showing intermediate values. Like EPL, TYCHO had low NPQ, irrespective of whether they were grown in benthic or planktonic conditions, reflecting an adaptation to a low light environment. Our results thus provide the first experimental evidence for the existence of a trade-off between behavioural (motility) and physiological photoprotective mechanisms (NPQ and the XC) in the four major intertidal benthic diatoms growth forms using unialgal cultures. Remarkably, although motility is restricted to the raphid pennate diatom clade, raphid pennate species, which have adopted a non-motile epipsammic or a tychoplanktonic life style, display the physiological photoprotective response typical of these growth forms. This observation underscores the importance of growth form and not phylogenetic relatedness as the prime determinant shaping the physiological photoprotective capacity of benthic diatoms.