When light energy input exceeds the capacity for photosynthesis the plant need to dissipate the excess energy and this is done through non-photo-chemical quenching (NPQ). Photochemical quenching (photosynthesis), NPQ and fluorescence are three alternative faiths of excited chlorophylls. PsbS associates to photosystem II and is involved in NPQ. The results presented in this thesis were generated on Arabidopsis plants and mainly based on wildtype Col-0 together with a mutant deficient in PsbS (npq4) and a transgene overexpressing PsbS (oePsbS). We connect light and herbivore stress and show that the level of PsbS influences the food preference of both a specialist (Plutella) and a generalist (Spodoptera) herbivore as well as oviposition of Plutella. Level of PsbS also affects both metabolomics and transcriptomics of the plant; up-regulation of genes in the jasmonic acid (JA) -pathway and amount of JA has been found in the npq4 plants after herbivory. Since many experiments were performed in field we have also characterized the field plant and how it differs from the commonly used lab plant. We have also studied the natural variation of NPQ in Arabidopsis plants both in the field and the lab. The results show surprisingly no correlation. / Överskottsenergi kan vara skadligt för en växts membran och fotosynteskomplex. Vid överskott av solenergi blir fotosystemen mättade och växten behöver därför ett sätt för att göra sig av med all överskottsenergi, detta kallas för ”icke-fotokemisk quenching” (NPQ). Fotokemisk quenching (fotosyntes), NPQ och fluoresens är tre alternativa vägar för exalterade klorofyller. PsbS är involverad i NPQ och associerar med fotosystem II. De resultat som presenteras i denna avhandling kommer från studier av modellväxten Arabidopsis thaliana (Backtrav), i huvudsak gjorda på vildtypen i jämförelse med en mutant som saknar PsbS (npq4) och en transgen som överuttrycker PsbS (oePsbS). Vi har försökt att undersöka kopplingen mellan ljus- och herbivoristress och visar här att mängden PsbS påverkar både en specialist (Plutella) och en generalist (Spodoptera) insekt vid val av föda, samt Plutella även vid äggläggning. Växternas nivå av PsbS visade sig även påverka metabolomet och transkriptomet, och vi fann en uppreglering av gener i biosyntesen för jasmonat samt mer av själva hormonet jasmonat i npq4 växter efter herbivori. Eftersom vi har gjort många av experimenten ute i fält har vi även karakteriserat en typisk Arabidopsis växt i fält samt hur denna skiljer sig från den vanligt använda lab-växten. Dessutom har vi även undersökt naturlig variation av NPQ av Arabidopsis både i fält och på lab och resultaten visar, till vår förvåning, att det inte går att finna någon korrelation mellan dessa.

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La lumière est indispensable à la survie des plantes via le processus photosynthétique, pourtant les plantes doivent s'adapter à différentes conditions environnementales où la quantité et la qualité de la lumière peuvent être non optimales pour la photosynthèse. Cela peut provoquer des dégâts photo-induits par formation d'espèces réactives de l'oxygène (ROS), qui sont dangereux pour la plante. Pour limiter la formation des ROS, les plantes mettent en place une régulation importante qui est la dissipation thermique de l'énergie absorbée en excès, appelé Non photochemical quenching (NPQ). Il est connu que la protéine PsbS joue le rôle clé de senseur du pH bas du lumen thylacoïdal, qui est le signal initial pour activer le NPQ. Dans le contexte de cette thèse, on propose d'étudier l'hypothèse que l'absence de la protéine PsbS (diminué NPQ) pourrait augmenter la croissance et la productivité des plantes en conditions contrôlées de faible lumière par l'éminilation de la protéine PsbS chez Arabidopsis thaliana et chez la tomate. Les résultats obtenus indiquent qu'en lumière faible les plantes mutantes montrent une augmentation du rendement de photosystème II conduisant une croissance et un nombre de fleurs significativement augmentés par rapport aux plantes sauvages.De plus, une autre régulation de la photosynthèse, nommée « transitions d'état », est importante pour optimiser la photosynthèse en réponse aux variations de la quantité et de la qualité de la lumière, grâce à la migration réversible des antennes collectrices d'énergie LHCII phosphorylées du PSII au PSI, c'est aussi étudié dans ma thèse. / Light is indispensable for plant survival, but plants have to cope with different environmental situations where light quantity and quality can be not optimal for photosynthesis. This can cause photodamage due to the formation of harmful reactive oxygen species (ROS). To limit ROS formation, plants developed a mechanism important as the dissipation of excess absorbed energy as heat and is called Non Photochemical Quenching (NPQ). The PsbS protein plays the key role of sensor of the low lumenal pH, the signal to activate NPQ. In this thesis, we proposed and investigated the hypothesis that PsbS absence (NPQ decrease) would improve growth under controlled low light upon elimination of the PsbS in Arabidopsis and tomato plants. Results showed that the increase of photosystem II yield in mutant plants leaded to a significant improvement of growth and flower number in mutants as compared with wild type plants under low light, suggesting that this mutation could be useful to improve plant performances in controlled conditions where light is strongly limiting. In addition, another photosynthetic regulation, called “state transitions”, which is important to optimize photosynthesis under variable light for intensity and quality thank to reversible migration of phosphorylated light harvesting complexes LHCII from PSII to PSI also investigated in my thesis.

Le microphytobenthos (MPB) des latitudes tempérées est dominé par les diatomées. Deux grands groupes se distinguent, les épipéliques (mobiles) des sédiments vaseux, et les épipsammiques (fixées) des sédiments sablo-vaseux. Afin de mieux comprendre la production des vasières, le MPB a été étudié par des approches du niveau physiologique au niveau écologique. Dans un premier temps, l’étude s’est focalisée sur des expérimentations en laboratoire. La réponse des différents groupes à la lumière a montré que la forme de vie et la mobilité sont en lien étroit avec la capacité de photoprotection physiologique. Ainsi, les diatomées non-mobiles présentent une meilleure photoprotection physiologique que les diatomées mobiles qui peuvent fuir les excès de lumière. Dans une deuxième partie, le travail s’est effectué sur des échantillons ramenés en laboratoire. Des profils de migrations ont été réalisés par mesure continue de la fluorescence. Il a été établi que le MPB présente un rythme de migration interne pouvant être modulé par la lumière. De plus la qualité de la lumière modifie les profils de migration. Il est communément admis que les phases de division cellulaire se dérouleraient en profondeur. La cytométrie en flux permet de vérifier cette hypothèse. Finalement les mesures effectuées en laboratoire ont été comparées à des mesures effectuées directement sur le terrain à l’échelle de la communauté. Il a ainsi pu être vérifié que la photoprotection sous lumière fluctuante est fonction de la population. Pour les populations épipéliques, la photoprotection physiologique ne varie pas au cours des fluctuations lumineuses, laissant supposer que la migration module ces fluctuations. Les populations épipsammiques, quant à elles, modifient leur réponse physiologique en fonction des fluctuations lumineuses. / Microphytobentos (MPB) from temperate latitude is mainly composed of diatoms. Those microorganisms can be separated in two groups: the epipelic one from muddy sediments (composed of mobile diatoms) and the epipsammic one from sandy-muddy sediments (composed of diatoms living attached to their substrate). In order to investigate mudflats’ primary production, the MPB compartment was studied through diverse approaches from the physiological level to the ecological one. In the first place, laboratory experiments (in vitro experiments), focusing on light reaction of epipelic and epipsammic diatoms, showed that their life form and their mobility were strongly connected to their physiological photoprotection ability. Thereby, the motionless diatoms were characterized by higher physiological photoprotection abilities than the mobile ones, which could avoid excess of light. In the second place, the fluorescence of collected samples (in vivo experiments) was measured to acquire diatoms’ migration profiles. The results pointed out an internal and light-regulated migration pattern of the MPB and furthermore highlighted the effect of light quality on migration profiles. Besides, the commonly accepted hypothesis of deep cell division phases was tested and confirmed through flow cytometry experiments. Eventually, laboratory measurements were compared to in situ ones realized at the scale of the whole community. These comparisons revealed that diatoms photoprotection in fluctuating light depended on the targeted populations. Epipelic organisms were indeed characterized by an unvarying photoprotection, diatoms migration regulating alone the effect of light fluctuations. On the contrary, motionless epipsammic populations required a light-regulated photoprotection.

abstract: Most of the sunlight powering natural photosynthesis is absorbed by antenna arrays that transfer, and regulate the delivery of excitation energy to reaction centers in the chloroplast where photosynthesis takes place. Under intense sunlight the plants and certain organisms cannot fully utilize all of the sunlight received by antennas and excess redox species are formed which could potentially harm them. To prevent this, excess energy is dissipated by antennas before it reaches to the reaction centers to initiate electron transfer needed in the next steps of photosynthesis. This phenomenon is called non-photochemical quenching (NPQ). The mechanism of NPQ is not fully understood, but the process is believed to be initiated by a drop in the pH in thylakoid lumen in cells. This causes changes in otherwise nonresponsive energy acceptors which accept the excess energy, preventing oversensitization of the reaction center. To mimic this phenomenon and get insight into the mechanism of NPQ, a novel pH sensitive dye 3'6'-indolinorhodamine was designed and synthesized which in a neutral solution stays in a closed (colorless) form and does not absorb light while at low pH it opens (colored) and absorbs light. The absorption of the dye overlaps porphyrin emission, thus making energy transfer from the porphyrin to the dye thermodynamically possible. Several self-regulating molecular model systems were designed and synthesized consisting of this dye and zinc porphyrins organized on a hexaphenylbenzene framework to functionally mimic the role of the antenna in NPQ. When a dye-zinc porphyrin dyad is dissolved in an organic solvent, the zinc porphyrin antenna absorbs and emits light by normal photophysical processes. Time resolved fluorescence experiments using the single-photon-timing method with excitation at 425 nm and emission at 600 nm yielded a lifetime of 2.09 ns for the porphyrin first excited singlet state. When acetic acid is added to the solution of the dyad, the pH sensitive dye opens and quenches the zinc porphyrin emission decreasing the lifetime of the porphyrin first excited singlet state to 23 ps, and converting the excitation energy to heat. Under similar experimental conditions in a neutral solution, a model hexad containing the dye and five zinc porphyrins organized on a hexaphenylbenzene core decays exponentially with a time constant of 2.1 ns, which is essentially the same lifetime as observed for related monomeric zinc porphyrins. When a solution of the hexad is acidified, the dye opens and quenches all porphyrin first excited singlet states to <40 ps. This converts the excitation energy to heat and renders the porphyrins kinetically incompetent to readily donate electrons by photoinduced electron transfer, thereby mimicking the role of the antenna in photosynthetic photoprotection. / Dissertation/Thesis / Ph.D. Chemistry 2012

京都大学 / 新制・課程博士 / 博士(理学) / 甲第23048号 / 理博第4725号 / 新制||理||1677(附属図書館) / 京都大学大学院理学研究科生物科学専攻 / (主査)教授 鹿内 利治, 准教授 竹中 瑞樹, 教授 長谷 あきら / 学位規則第4条第1項該当 / Doctor of Science / Kyoto University / DFAM

<p>In photosynthesis, sunlight is converted into chemical energy that is stored mainly as carbohydrates and supplies basically all life on Earth with energy.</p><p>In order to efficiently absorb the light energy, plants have developed the outer light harvesting antenna, which is composed of ten different protein subunits (LHC) that bind chlorophyll a and b as well as different carotenoids. In addition to the light harvesting function, the antenna has the capacity to dissipate excess energy as heat (feedback de-excitation or qE), which is crucial to avoid oxidative damage under conditions of high excitation pressure. Another regulatory function in the antenna is the state transitions in which the distribution of the trimeric LHC II between photosystem I (PS I) and II is controlled. The same ten antenna proteins are conserved in all higher plants and based on evolutionary arguments this has led to the suggestion that each protein has a specific function.</p><p>I have investigated the functions of individual antenna proteins of PS II (Lhcb proteins) by antisense inhibition in the model plant Arabidopsis thaliana. Four antisense lines were obtained, in which the target proteins were reduced, in some cases beyond detection level, in other cases small amounts remained.</p><p>The results show that CP29 has a unique function as organising the antenna. CP26 can form trimers that substitute for Lhcb1 and Lhcb2 in the antenna structure, but the trimers that accumulate as a response to the lack of Lhcb1 and Lhcb2 cannot take over the LHC II function in state transitions. It has been argued that LHC II is essential for grana stacking, but antisense plants without Lhcb1 and Lhcb2 do form grana. Furthermore, LHC II is necessary to maintain growth rates in very low light.</p><p>Numerous biochemical evidences have suggested that CP29 and/or CP26 were crucial for feedback de-excitation. Analysis of two antisense lines each lacking one of these proteins clearly shows that there is no direct involvement of either CP29 or CP26 in this process. Investigation of the other antisense lines shows that no Lhcb protein is indispensable for qE. A model for feedback de-excitation is presented in which PsbS plays a major role.</p><p>The positions of the minor antenna proteins in the PS II supercomplex were established by comparisons of transmission electron micrographs of supercomplexes from the wild type and antisense plants.</p><p>A fitness experiment was conducted where the antisense plants were grown in the field and seed production was used to estimate the fitness of the different genotypes. Based on the results from this experiment it is concluded that each Lhcb protein is important, because all antisense lines show reduced fitness in the field.</p>

In photosynthesis, sunlight is converted into chemical energy that is stored mainly as carbohydrates and supplies basically all life on Earth with energy. In order to efficiently absorb the light energy, plants have developed the outer light harvesting antenna, which is composed of ten different protein subunits (LHC) that bind chlorophyll a and b as well as different carotenoids. In addition to the light harvesting function, the antenna has the capacity to dissipate excess energy as heat (feedback de-excitation or qE), which is crucial to avoid oxidative damage under conditions of high excitation pressure. Another regulatory function in the antenna is the state transitions in which the distribution of the trimeric LHC II between photosystem I (PS I) and II is controlled. The same ten antenna proteins are conserved in all higher plants and based on evolutionary arguments this has led to the suggestion that each protein has a specific function. I have investigated the functions of individual antenna proteins of PS II (Lhcb proteins) by antisense inhibition in the model plant Arabidopsis thaliana. Four antisense lines were obtained, in which the target proteins were reduced, in some cases beyond detection level, in other cases small amounts remained. The results show that CP29 has a unique function as organising the antenna. CP26 can form trimers that substitute for Lhcb1 and Lhcb2 in the antenna structure, but the trimers that accumulate as a response to the lack of Lhcb1 and Lhcb2 cannot take over the LHC II function in state transitions. It has been argued that LHC II is essential for grana stacking, but antisense plants without Lhcb1 and Lhcb2 do form grana. Furthermore, LHC II is necessary to maintain growth rates in very low light. Numerous biochemical evidences have suggested that CP29 and/or CP26 were crucial for feedback de-excitation. Analysis of two antisense lines each lacking one of these proteins clearly shows that there is no direct involvement of either CP29 or CP26 in this process. Investigation of the other antisense lines shows that no Lhcb protein is indispensable for qE. A model for feedback de-excitation is presented in which PsbS plays a major role. The positions of the minor antenna proteins in the PS II supercomplex were established by comparisons of transmission electron micrographs of supercomplexes from the wild type and antisense plants. A fitness experiment was conducted where the antisense plants were grown in the field and seed production was used to estimate the fitness of the different genotypes. Based on the results from this experiment it is concluded that each Lhcb protein is important, because all antisense lines show reduced fitness in the field.

Nonphotochemical quenching and state transitions are an important photoprotective mechanism against excessive irradiation. In this work I studied changes in the size of the effective crosssection of photosystem II antennae in regard to the level of nonphotochemical quenching (state transitions) under different levels of light induced stress.

Strong light can cause damage and be lethal for photosynthetic organisms. An increase of thermal dissipation of excess absorbed energy at the level of photosynthetic antenna is one of the processes protecting against deleterious effects of light. In cyanobacteria, a soluble photoactive carotenoid binding protein, Orange Carotenoid Protein (OCP) mediates this process. The photoactivated OCP by interacting with the core of phycobilisome (PB; the major photosynthetic antenna of cyanobacteria) triggers the photoprotective mechanism, which decreases the energy arriving at the reaction centres and PSII fluorescence. The excess energy is dissipated as harmless heat. To regain full PB capacity in low light intensities, theFluorescence Recovery Protein (FRP) is required. FRP accelerates the deactivation of OCP.In this work, I present my input in the understanding of the mechanism underlying the OCPrelated photoprotection. I further characterized the FRP of Synechocystis PCC6803, the model organism in our studies. I established that the Synechocystis FRP is shorter than what it was proposed in Cyanobase and it begins at Met26. Our results also revealed the great importance of a high OCP to FRP ratio for existence of photoprotection. The most remarkable achievement of this thesis is the in vitro reconstitution of the OCPrelated mechanism using isolated OCP, PB and FRP. I demonstrated that light is only needed for OCP photoactivation but OCP binding to PB is light independent. Only the photoactivated OCP is able to bind the PB and quench all its fluorescence. Based on our in vitro experiments we proposed a molecular model of OCP-related photoprotection. The in vitro reconstituted system was applied to examine the importance of a conserved salt bridge (Arg155-Glu244) between the two domains of OCP and showed that this salt bridge stabilises the inactive form of OCP. During photoactivation this salt bridge is broken and Arg155 is involved in the interaction between the OCP and the PB. The site of OCP binding in the core of a PB wasalso investigated with the in vitro reconstituted system. Our results demonstrated that the terminal energy emitters of the PB are not needed and that the first site of fluorescence quenching is an APC trimer emitting at 660 nm. Finally, we characterised the properties of excited states of the carotenoid in the photoactivated OCP showing that one of these states presents a very pronounced charge transfer character that likely has a principal role in energy dissipation. Our results strongly suggested that the OCP not only induces thermal energy dissipation but also acts as the energy dissipator.