Molecular mechanisms involved in plant photoprotection

Photosynthesis uses sunlight to convert water and carbon dioxide into biomass and oxygen. When in excess, light can be dangerous for the photosynthetic apparatus because it can cause photo-oxidative damage and decreases the efficiency of photosynthesis because of photoinhibition. Plants have evolved many photoprotective mechanisms in order to face reactive oxygen species production and thus avoid photoinhibition. These mechanisms include quenching of singlet and triplet excited states of chlorophyll, synthesis of antioxidant molecules and enzymes and repair processes for damaged photosystem II and photosystem I reaction centers. This review focuses on the mechanisms involved in photoprotection of chloroplasts through dissipation of energy absorbed in excess

Light-Harvesting Complex Stress-Related Proteins Catalyze Excess Energy Dissipation in Both Photosystems of Physcomitrella patens

Two LHC-like proteins, Photosystem II Subunit S (PSBS) and Light-Harvesting Complex Stress-Related (LHCSR), are essential for triggering excess energy dissipation in chloroplasts of vascular plants and green algae, respectively. The mechanism of quenching was studied in Physcomitrella patens, an early divergent streptophyta (including green algae and land plants) in which both proteins are active. PSBS was localized in grana together with photosystem II (PSII), but LHCSR was located mainly in stroma-exposed membranes together with photosystem I (PSI), and its distribution did not change upon high-light treatment. The quenched conformation can be preserved by rapidly freezing the high-light-treated tissues in liquid nitrogen. When using green fluorescent protein as an internal standard, 77K fluorescence emission spectra on isolated chloroplasts allowed for independent assessment of PSI and PSII fluorescence yield. Results showed that both photosystems underwent quenching upon high-light treatment in the wild type in contrast to mutants depleted of LHCSR, which lacked PSI quenching. Due to the contribution of LHCII, P. patens had a PSI antenna size twice as large with respect to higher plants. Thus, LHCII, which is highly abundant in stroma membranes, appears to be the target of quenching by LHCSR

Functional modulation of LHCSR1 protein from Physcomitrella patens by zeaxanthin binding and low pH

Light harvesting for oxygenic photosynthesis is regulated to prevent the formation of harmful photoproducts by activation of photoprotective mechanisms safely dissipating the energy absorbed in excess. Lumen acidification is the trigger for the formation of quenching states in pigment binding complexes. With the aim to uncover the photoprotective functional states responsible for excess energy dissipation in green algae and mosses, we compared the fluorescence dynamic properties of the light-harvesting complex stress-related (LHCSR1) protein, which is essential for fast and reversible regulation of light use efficiency in lower plants, as compared to the major LHCII antenna protein, which mainly fulfills light harvesting function. Both LHCII and LHCSR1 had a chlorophyll fluorescence yield and lifetime strongly dependent on detergent concentration but the transition from long- to short-living states was far more complete and fast in the latter. Low pH and zeaxanthin binding enhanced the relative amplitude of quenched states in LHCSR1, which were characterized by the presence of 80 ps fluorescence decay components with a red-shifted emission spectrum. We suggest that energy dissipation occurs in the chloroplast by the activation of 80 ps quenching sites in LHCSR1 which spill over excitons from the photosystem II antenna system

Single-molecule spectroscopy of LHCSR1 protein dynamics identifies two distinct states responsible for multi-timescale photosynthetic photoprotection

In oxygenic photosynthesis, light harvesting is regulated to safely dissipate excess energy and prevent the formation of harmful photoproducts. Regulation is known to be necessary for fitness, but the molecular mechanisms are not understood. One challenge has been that ensemble experiments average over active and dissipative behaviours, preventing identification of distinct states. Here, we use single-molecule spectroscopy to uncover the photoprotective states and dynamics of the light-harvesting complex stress-related 1 (LHCSR1) protein, which is responsible for dissipation in green algae and moss. We discover the existence of two dissipative states. We find that one of these states is activated by pH and the other by carotenoid composition, and that distinct protein dynamics regulate these states. Together, these two states enable the organism to respond to two types of intermittency in solar intensity-step changes (clouds and shadows) and ramp changes (sunrise), respectively. Our findings reveal key control mechanisms underlying photoprotective dissipation, with implications for increasing biomass yields and developing robust solar energy devices

Physcomitrella patens at the crossroad between algal and plant photosynthesis: a tool for studying the regulation of light harvesting

Attraverso la fotosintesi le piante usano l\u2019energia solare per produrre composti ridotti dalla CO2 e alla fine biomassa. I fotosistemi (PSI e PSII) sono complessi che legano i pigmenti responsabili della raccolta della luce, separazione di carica e hanno un ruolo essenziale nel trasporto di elettroni dall\u2019acqua al NADPH. Accoppiato al trasporto elettronico c\u2019\ue8 la formazione di un gradiente protonico che sostiene l\u2019attivit\ue0 della ATPasi per produrre ATP. PSI e PSII rappresentano straordinarie macchine per l\u2019utilizzo dell'energia solare, eppure hanno un punto debole in quanto sono riducenti monovalenti che portano alla produzione di specie reattive dell'ossigeno (ROS) in un ambiente, oggi, ricco di ossigeno creato dagli organismi fotosintetici. Inoltre, lo stato eccitato di tripletto della clorofilla reagisce con l'ossigeno molecolare per produrre ossigeno singoletto danneggiando proteine, lipidi e pigmenti presenti nei cloroplasti. Questo \ue8 il motivo per cui la luce in eccesso \ue8 dannosa e le alghe hanno evoluto meccanismi di fotoprotezione, che le piante hanno esteso e migliorato per la sopravvivenza in un ambiente terrestre ancora pi\uf9 stressato. Tra questi meccanismi di fotoprotezione, di particolare interesse \ue8 il Non-Photochemical Quenching (NPQ), che rapidamente (in pochi secondi) reagisce all\u2019 incremento degli stati eccitati della clorofilla. Il quenching porta alla dissipazione termica dell'energia assorbita in eccesso, \ue8 scatenato dal gradiente \u394pH generato attraverso la membrana tilacoidale e richiede una specifica famiglia di proteine, i Complessi di Raccolta della Luce (LHC) . LHC formano una grande superfamiglia di proteine che legano xantofille/clorofille e sono associate al PSII e PSI avendo un ruolo diretto nella raccolta della luce e/o nel quenching. Due proteine LHC-simili, PSBS e LHCSR, sono indispensabili per il NPQ rispettivamente nelle piante vascolari e alghe verdi insieme alle xantofille luteina e/o zeaxantina, che sono i ligandi delle proteine LHC. Di particolare interesse \ue8 la zeaxantina perch\ue9 viene sintetizzata in eccesso di luce a partire dalla violaxantina nel cosiddetto ciclo delle xantofille. La zeaxantina svolge un ruolo centrale nella fotoprotezione da detossificazione dei ROS, smorzando gli stati di tripletto della clorofilla (3Chl *) e, molto interessante per il mio lavoro, aumentando NPQ. Durante il mio dottorato, ho usato il muschio Physcomitrella patens come organismo modello per studiare il meccanismo di NPQ con particolare riferimento al ruolo di zeaxantina. P. patens ha una posizione strategica nell\u2019albero della vita in quanto si trova tra le alghe verdi e le piante superiori; inoltre, \ue8 stato tra i primi organismi ad emergere dall'acqua per colonizzare l'ambiente terrestre, caratterizzazione da diverse condizioni stressanti, attraverso l'evoluzione di nuovi meccanismi di fotoprotezione. Le proteine PSBS, che \ue8 apparsa per prima in P. patens, ma anche LHCSR sono ancora attive in questo organismo, offrendo cos\uec la possibilit\ue0 di studiare il NPQ sia delle alghe che delle piante nello stesso background genetico e biochimico. Questa possibilit\ue0 pu\uf2 essere sfruttata grazie ad un ulteriore caratteristica, unica, di P. patens tra gli organismi fotosintetici eucarioti, ossia la sua capacit\ue0 di fare ricombinazione omologa (HR) ad alta efficienza, rendendo il \u201cgene targeting\u201d una procedura standard. Comprendere la modulazione del NPQ durante l\u2019acclimatazione a stress abiotici \ue8 essenziale per la piena comprensione del suo ruolo. Ho iniziato il mio lavoro dopo aver osservato che P. patens risponde a moderato stress salino e osmotico aumentando la sua attivit\ue0 NPQ. Sorprendentemente, l\u2019aumento di NPQ non era dovuto sovra-accumulo delle proteine PSBS e LHCSR come nel caso dell\u2019acclimatazione ad alta luce e al freddo. Ho potuto correlare l\u2019aumento di NPQ, in seguito questi stress, con l\u2019accumulo di zeaxantina. Per verificare il ruolo della zeaxantina, abbiamo identificato un unico gene VDE nel genoma di P. patens e abbiamo fatto dei mutanti che non esprimevano questo gene (knock out, KO). Le piante vde KO non erano capaci di produrre zeaxantina e mostravano una drammatica riduzione dell\u2019NPQ cos\uec come una aumentata fotoinibizione in seguito a stress da alta luce. Abbiamo anche introdotto la mutazione VDE in genotipi che esprimevano solo la proteina LHCSR o PSBS mostrando che l\u2019NPQ dipendente da LHCSR \ue8, a sua volta, molto dipendente dalla zeaxantina rispetto all\u2019NPQ dipendente da PSBS, con un rapporto vicino a 10. In questo lavoro, per la prima volta, ho isolato LHCSR nella sua forma nativa, di proteina che lega clorofilla a/b e xantofille. Inoltre, ho mostrato che in LHCSR, a differenza di PSBS, l\u2019aumento di NPQ avviene attraverso il legame diretto della zeaxantina. Lo spettro di assorbimento e le caratteristiche dei pigmenti legati a LHCSR nativa combaciano con i dati riportati per LHCSR3 ricombinante di Chlamydomonas reinhardtii con l\u2019eccezione che LHCSR di C. reinhardtii \ue8 zeaxantina-indipendente. Precedenti studi hanno identificato due funzioni essenziali per le proteine scatenanti NPQ : i) la funzione di sensori del pH (trovato anche in PSBS) e ii) la funzione di quenching (che si trova anche in altre proteine LHCB ) come LHCB4. Nelle piante queste due funzioni sono svolte da subunit\ue0 proteiche diverse, rendendo cos\uec difficile studi in vitro. La recente scoperta di LHCSR ha reso la prospettiva di chiarire le basi molecolari del NPQ possibile: infatti, questa proteina \ue8 l'unica finora conosciuta per comprendere l'insieme delle funzioni necessarie per NPQ nella stessa unit\ue0 strutturale. Nell\u2019ultima parte di dottorato, ho provato a fare chiarezza nel meccanismo d'azione di LHCSR concentrandomi da un lato nella localizzazione sub - organello di questa proteina insieme allo studio della localizzazione di PSBS nelle membrane tilacoidali. Le membrane tilacoidi di P. patens sono organizzate in grana, ben definiti, e membrane stromatiche e sono differenzialmente esposti al compartimento stromale solubile come nelle piante vascolari. Ho sfruttato la possibilit\ue0 di frazionare le membrane grana e stroma-lamelle per verificare la loro localizzazione con detergenti e frazionamento meccanico. Sorprendentemente , ho trovato che PSBS \ue8 localizzata nelle membrane granali mentre LHCSR \ue8 localizzato in membrane stromatiche: ci\uf2 suggerisce un meccanismo d'azione diverso di NPQ . Con queste informazioni ottenute, proponiamo un modello sperimentale per l'attivazione del quenching LHCSR-dipendente: LHCSR \ue8 ricco di residui acidi nella superficie esposta al lumen; in seguito ad eccesso di luce l\u2019acidificazione potrebbe neutralizzare queste cariche e permettere la diffusione di LHCSR verso i grana grazie ad una ridotta repulsione con i PSII-LHCII supercomplessi. L\u2019isolamento di LHCSR e la sua localizzazione, mi hanno incoraggiata ad ottimizzare e sfruttare queste preparazioni. Sebbene fossi conscia della difficolt\ue0 di questo lavoro, ho cos\uec deciso di provare a purificare LHCSR con (+) e senza (-) zeaxantina legata usando P. patens WT in quanto lo studio di LHCSR nella sua conformazione \u201cquenchata\u201d e non \ue8 un ambizioso ma essenziale target per la ricerca sulla fotosintesi. Come per qualsiasi progetto a lungo termine, ho concepito diverse strategie per l\u2019isolamento di LHCSR sia dalla pianta WT di P. patens sia da creando versioni \u201ctaggate\u201d della proteina che contengono una coda di istidina per facilitare la sua purificazione. Inoltre, ho anche sovra-espresso la proteina in tabacco. I potenziali vantaggi e le insidie di questo progetto sono descritte e discusse in questa tesi insieme a risultati preliminari.Through photosynthesis plants use solar energy for producing reduced compounds from CO2 and finally biomass. Photosystems (PSI and PSII) are multisubunit pigment-binding complexes responsible for light harvesting, charge separation and play an essential role in electron transport from water to NADPH. Coupled to photosynthetic electron transport is the formation of a transmembrane pH gradient that sustains ATPase activity to produce ATP. PSI and PSII represent extraordinary machines for solar energy exploitation and yet they have a weak point in being univalent reductants which leads to production of reactive oxygen species (ROS) in the present day oxygen-rich environment that photosynthetic organisms have been creating. Moreover, chlorophyll is an excellent sensitizer and its triplet excited state reacts with molecular oxygen to yield singlet oxygen. This is why excess light is harmful and algae have evolved photoprotective mechanisms, which plants have extended and improved for survival in the even more challenging land environment. Of particular interest is Non-Photochemical Quenching (NPQ) of chlorophyll fluorescence which rapidly (within seconds) reacts to enhancement of the chlorophyll excited states. Quenching leads to the thermal dissipation of the energy absorbed in excess, is triggered by the \u394pH gradient generated across thylakoid membrane and requires specific members of the Light Harvesting Complexes (LHCs) protein family. LHCs form a large superfamily of chlorophyll-xanthophyll-binding proteins associated to PSII and PSI playing a direct role in light harvesting and/or energy quenching. Two LHC-like proteins, PSBS and LHCSR, are indispensable for NPQ respectively in vascular plants and green algae together with the xanthophylls lutein and/or zeaxanthin which are ligands for LHC proteins. Of particular interest is zeaxanthin because it is synthesized in excess light only from pre-existing violaxanthin in the so called xanthophyll cycle. Zeaxanthin plays a central role in photoprotection by scavenging of ROS quenching triplet states of chlorophyll (3Chl*) and, most interesting for my work, enhancing NPQ. During my PhD, I used the moss Physcomitrella patens as model organism to study the mechanism of NPQ with particular reference to the role of zeaxanthin. P. patens has a strategic position in the tree of life: it is an evolutionary intermediate between green algae and higher plants and was among the first organisms emerging from water to colonize the stressful land environment through the evolution of new photoprotective mechanisms. PSBS first appeared in P. patens and yet LHCSR proteins are still active yielding the possibility of studying both algal and plant NPQ in the same genetic and biochemical background. This opportunity can be exploited due to a further unique property of P. patens among eukaryotic photosynthetic organisms, i.e. its ability to perform Homologous Recombination (HR) at high efficiency, making gene targeting a standard procedure. Understanding the modulation of NPQ during acclimation to abiotic stress is essential for the full comprehension of its role. I started my work after the observation that P. patens responds to moderate salt and osmotic stress by increasing its NPQ activity. Surprisingly, NPQ enhancement was not due to over-accumulation of PSBS and/or LHCSR proteins as in the case of high light and cold acclimation. I could correlate NPQ enhancement under salt and osmotic stress with the over accumulation of zeaxanthin. When trying to verify the role of zeaxanthin we identified the unique VDE gene in P. patens genome and we knocked it out. vde KO plants were unable to produce zeaxanthin and showed a dramatic reduction in NPQ as well as an enhanced photoinhibition under excess light conditions. The introduction of the VDE mutation into LHCSR-only and PSBS-only genotypes showed that LHCSR-dependent NPQ is far more dependent on zeaxanthin than the PSBS-dependent NPQ with an activation ratio close to 10. In this work for the first time, I isolated LHCSR in the form of native chlorophyll a/b\u2013xanthophyll-binding protein and found that the NPQ enhancement actually occurs through the direct binding of zeaxanthin to the LHCSR protein, different from the case of PSBS. Absorption spectrum and pigment binding properties of native LHCSR closely fit previously data reported for recombinant Chlamydomonas reinhardtii LHCSR3 whose activity, however, is zeaxanthin independent. Previous studies have identified two essential functions associated to essential proteins triggering NPQ: i) the pH detection function (also found in PSBS) and ii) the quenching function (also found in other LHCB proteins) such as LHCB4. In plants these two functions are carried out by distinct proteic subunits, thus making difficult in vitro studies. The recent finding of LHCSR protein has made the perspective of elucidating the molecular basis of NPQ possible: in fact, this protein is the only protein so far known to comprise the whole set of functions needed for NPQ into the same structural unit. Along the last part of my PhD work, I decided to move new steps towards the understanding of the mechanism of action of LHCSR by focusing on one side on the sub-organelle localization of this protein together with the study of the localization of PSBS in thylakoid membranes. P. patens thylakoid membranes are organized into well-defined grana stacks and stroma membranes which are differentially exposed to the stromal soluble compartment as in vascular plants. I exploited the possibility to fractionate grana and stroma-lamellae membranes to verify their localization using detergents and by mechanical fractionation. Surprisingly, I found that PSBS is localized in grana membranes while LHCSR is localized in stroma exposed membranes suggesting a different action mechanism on NPQ. Here on these basis I am proposing a tentative model for the activation of LHCSR-dependent quenching, specifically located at the periphery of grana stacks. LHCSR is rich in acidic residues in its lumen-exposed surface, acidification under excess light conditions would neutralize these charges and allow diffusion towards the grana partition domains thanks to a reduced repulsion with PSII-LHCII supercomplexes. The results reported in Chapter 2 (isolation of zeaxanthin-binding LHCSR) and Chapter 3 (localization of LHCSR in the margins/stroma fraction of thylakoid membranes) encouraged me to initiate the ambitious task of optimizing and scaling up these preparations. Although I was conscious about the difficulty of this work, I decided to try the purification of LHCSR +/- zeaxanthin from WT P. patens because the differential study of LHCSR in its quenched vs unquenched conformation is an ambitious but essential target for photosynthesis research. As for any long term project, I have conceived several strategies for the isolation of LHCSR from either WT P. patens or overexpressed using WT sequence or tagged versions of the protein using a poly-Histidine tail (His-tag) to facilitate its purification. Alternatively I also have attempted overexpressing LHCSR in tobacco. The potential advantages and pitfalls of this project are described and discussed in PhD thesis together with preliminary results

The rise and fall of Light-Harvesting Complex Stress-Related proteins as photoprotection agents during evolution

Photosynthesis depends on light. However, excess light can be harmful for the photosynthetic apparatus because it produces reactive oxygen species (ROS) that cause photoinhibition. Oxygenic organisms evolved photoprotection mechanisms to counteract light-dependent ROS production, including preventive dissipation of excited states of chlorophyll (1Chl*) into heat in the process termed non-photochemical quenching (NPQ). This consists in the activation of 1Chl* quenching reactions when the thylakoid luminal pH drops below 5.2. In turn, acidification occurs when the rate of the CO2 reducing cycle is saturated and cannot regenerate ADP+Pi, thus inhibiting ATPase activity and the return of protons (H+) to the stromal compartment. The major and fastest component of NPQ is energy quenching, qE, which in algae depends on the Light-Harvesting Complex Stress-Related (LHCSR) proteins. In mosses, LHCSR proteins have remained the major catalysts of energy dissipation, but a minor contribution also occurs via a homologous protein, Photosystem II Subunit S (PSBS). In vascular plants, however, LHCSR has disappeared and PSBS is the only pH-sensitive trigger of qE. Why did PSBS replace LHCSR in the later stages of land colonization? Both PSBS and LHCSR belong to the Light Harvesting Complex superfamily (LHC) and share properties such as harboring protonatable residues that are exposed to the chloroplast lumen, which is essential for pH sensing. However, there are also conspicuous differences: LHCSR binds chlorophylls and xanthophylls while PSBS does not, implying that the former may well catalyse quenching reactions while the latter needs pigment-binding partners for its quenching function. Here, the evolution of quenching mechanisms for excess light is reviewed with a focus on the role of LHCSR versus PSBS, and the reasons for the redundancy of LHCSR in vascular plants as PSBS became established

Algae, a New Biomass Resource

Algae are oxygenic photoautotrophs, offering a very high level of biodiversity and thus suitable for different practical applications. Today, they are mainly cultivated for human/animal food or to extract high-value chemicals and pharmaceuticals. However, their exploitation could be extended. Algae are attractive as high-yield biomass producers, because of the short life cycle, the ability to grow up to very high cell densities, and the easy large-scale cultivation that does not compete with other demands such as those of conventional crops agriculture, notably arable land. Algae can be a resource of renewable, sustainable biofuels. In addition, they can be transformed into \u201ccell factories\u201d to produce recombinant proteins of interest for pharmaceutical companies

Photoprotective Excess Energy Dissipation. Chapter11

This book introduces the basic physical, chemical, and biological principles underlying the first steps in photosynthesis: light absorption, excitation energy transfer, and charge separation. In Part 1, we introduce pigments and their spectroscopic/ redox properties. In Part 2, pigment-proteins as they occur in various natural systems (plants, algae, photosynthetic bacteria) are described, including the regulation of light harvesting. Part 3 deals with the physics underlying light harvesting: energy transfer and electron transport. Part 4 introduces basic and advanced spectroscopic methods, including data analysis. In Part 5, we discuss artificial and natural photosynthetic systems, how they are assembled, and what the energy transfer properties are