Characterization of the Novel Photosynthetic Protein PPP7 involved in Cyclic Electron Flow around PSI

Photosynthetic organisms are able to convert light energy into chemical energy by the operation of the two photosystems, the cytochrome b6/f complex and the ATPase. The two photosystems operate in series during linear electron flow to split H2O and to generate NADP+. During electron transport, a pH gradient is generated across the thylakoid membrane which is used for the generation of ATP. In addition to the linear electron transport mode, ATP can also be produced via cyclic electron flow around photosystem I (CEF). The physiological role of CEF in vascular plants with C3-type photosynthesis is still not solved. Potential functions of CEF are (i) the dissipation of excessive light energy by increasing non-photochemical quenching (NPQ); (ii) ATP synthesis during steady-state photosynthesis; (iii) the regulation of the stromal oxidation state under stress conditions and under conditions when the Calvin cycle is not available as a sink for NADPH. With exception of the thylakoid NADPH-dehydrogenase complex and the stromal protein PGR5, the components that contribute to CEF are still unknown. Obscure is also the regulation that controls the switch from linear to cyclic flow. We have identified a novel transmembrane protein, named PPP7, which is located in thylakoids of photoautotrophic eukaryotes. Mutants lacking PPP7 exhibit the same phenotype as plants missing PGR5. These mutants show reduced NPQ, decreased P700 oxidation and perturbation of ferredoxin-dependent CEF. The work described in this thesis demonstrates that PPP7 and PGR5 interact physically, and that both co-purify with photosystem I. PPP7 does also interact in yeast assays with the cytochrome b6/f complex, as well as with the stromal proteins ferredoxin (Fd) and ferredoxin-NADPH oxido-reductase (FNR), but PPP7 is not a constitutive component of any of the major photosynthetic complexes. In consequence, the existence of a PPP7/PGR5 complex integrated in the thylakoid membrane and facilitating CEF around PSI in eukaryotes, possibly by shuttling electrons together with ferredoxin and the FNR from photosystem I to the cytochrome b6/f complex, is proposed. Moreover, CEF is enhanced in the Arabidopsis psad1 and psae1 mutants with a defect in photosystem I oxidation in contrast to the cyanobacterial psae mutant which exhibits an decreased CEF, pointing to fundamental mechanistic differences in the cyclic electron flow of cyanobacteria and vascular plants. The Arabidopsis psad1 and psae1 mutants also show higher contents of ferredoxin and of the PPP7/PGR5 complex, supporting a role of PPP7 and PGR5 in the switch from linear to cyclic electron flow depending on the redox state of the chloroplast

The extrinsic proteins of Photosystem II

In this review we examine the structure and function of the extrinsic proteins of Photosystem II. These proteins include PsbO, present in all oxygenic organisms, the PsbP and PsbQ proteins, which are found in higher plants and eukaryotic algae, and the PsbU, PsbV, CyanoQ, and CyanoP proteins, which are found in the cyanobacteria. These proteins serve to optimize oxygen evolution at physiological calcium and chloride concentrations. They also shield the Mn 4CaO 5 cluster from exogenous reductants. Numerous biochemical, genetic and structural studies have been used to probe the structure and function of these proteins within the photosystem. We will discuss the most recent proposed functional roles for these components, their structures (as deduced from biochemical and X-ray crystallographic studies) and the locations of their proposed binding domains within the Photosystem II complex. This article is part of a Special Issue entitled: Photosystem II. © 2011 Elsevier B.V. All rights reserved

Photosynthesis

Among the myriads of volumes dedicated to various aspects of photosynthesis, the current one is singular in integrating an update of the most recent insights on this most important biological process in the biosphere. While photosynthesis fuels all the life supporting processes and activities of all living creatures on Earth, from bacteria though mankind, it also created in the first place, our life supporting oxygenic atmosphere, and keeps maintaining it. This volume is organized in four sections: I) Mechanisms, II) Stress effects, III) Methods, and IV) Applications

Are All Plastids Created Equal? Diversity, Function, and Evolution of Plastid-targeted Genes and Chloroplast Transit Peptides in Plants

Plastids are morphologically and biochemically diverse organelles which not only perform photosynthesis but are also responsible for a wide range of metabolic, storage, regulatory, and aesthetic functions. The majority of plastid proteins are encoded by the nucleus and imported post-translationally, which allows for much greater spatiotemporal control of plastid function. Most of what is known about plastids has been studied in a relatively small group of model organisms, yet the myriad functions of plastids and significant microscopy evidence suggest that plastid function may be relatively unique in each plant species. Research in this dissertation aims to expand the field of plastid biology to non-model systems by (1) Reviewing the current state of literature to explore how the plastid proteome is shaped and how this process is regulated. How nuclear-encoded plastid-targeted proteins are imported has been enigmatic since the discovery of the main import channel, but recent evidence has helped to resolve the molecular mechanism and regulatory aspects of this process; (2) Establishing bioinformatics methods to characterize the plastid proteome quickly and efficiently in a range of plant species. A novel application of existing subcellular prediction techniques revealed that only 628-828 proteins are shared between the plastids of all assessed Angiosperms, but between 6- to 25-fold more proteins were specific to single species or taxonomic groups; (3) Analyzing mutational patterns leading to the evolution of novel chloroplast transit peptides. Insertions and deletions, particularly those that caused a shift of the transcriptional or translational start site, were responsible for the majority of novel transit peptides, implicating either exon shuffling as the dominant means of subcellular relocalization to the plastid; and, (4) Characterization of the N-terminal soluble domain of the conserved protein ALB3 when expressed from the chloroplast genome. Perturbations to chlorophyll fluorescence and ion homeostasis suggest that constitutive activation of the ALB3/SecY heterodimeric channel by substrate proteins induces permeability of the thylakoid membrane to leakage of protons and disruption of ion gradients. An overabundance of ALB3 in certain tissues may cause ion dysregulation, particularly for calcium, magnesium, and potassium

Characterizing protein compartmentalization of plant energy metabolism

[no abstract

Modeling the electron transport chain of purple non-sulfur bacteria

Purple non-sulfur bacteria (Rhodospirillaceae) have been extensively employed for studying principles of photosynthetic and respiratory electron transport phosphorylation and for investigating the regulation of gene expression in response to redox signals. Here, we use mathematical modeling to evaluate the steady-state behavior of the electron transport chain (ETC) in these bacteria under different environmental conditions. Elementary-modes analysis of a stoichiometric ETC model reveals nine operational modes. Most of them represent well-known functional states, however, two modes constitute reverse electron flow under respiratory conditions, which has been barely considered so far. We further present and analyze a kinetic model of the ETC in which rate laws of electron transfer steps are based on redox potential differences. Our model reproduces well-known phenomena of respiratory and photosynthetic operation of the ETC and also provides non-intuitive predictions. As one key result, model simulations demonstrate a stronger reduction of ubiquinone when switching from high-light to low-light conditions. This result is parameter insensitive and supports the hypothesis that the redox state of ubiquinone is a suitable signal for controlling photosynthetic gene expression

The function of monomeric Lhcb proteins ofPhotosystem II analyzed by reverse genetic

Negli organismi eucaritici fotosintetici il sistema antenna \ue8 composto da subunit\ue0 codificate dalla famiglia multigenica Light harvesting complex (Lhc). Queste proteine sono coinvolte sia nella raccolta della luce che nella fotoprotezione. In particolare, le proteine antenna del PSII, le subunit\ue0 Lhcb, sembrano essere implicate nel meccanismo di dissipazione termica dell\u2019energia di eccitazione in eccesso (NPQ, Non Photochemical Quenching). Chiarire i dettagli molecolari dell\u2019induzione dell\u2019NPQ nelle piante superiori si \ue8 dimostrata essere una grande sfida. Durante il mio dottorato di ricerca, ho deciso di indagare il ruolo delle subunit\ue0 Lhcb nel quenching dell\u2019energia di eccitazione utilizzando un approccio di genetica inversa: ho ottenuto mutanti privi di ciascuna delle subunit\ue0 per capire il loro coinvolgimento nel meccanismo. Qui di seguito sono riassunti i principali risultati ottenuti. Sezione A. Mutanti per le subunit\ue0 monomeriche Lhc e fotoprotezione \uc8 stata studiata la funzione delle proteine antenna CP26, CP24 e CP29 nella raccolta della luce e nella regolazione della fotosintesi, mediante l\u2019isolamento di mutanti knockout (ko) di Arabidopsis thaliana che mancano completamente di una o due di queste subunit\ue0. In particolare nella sezione A.1 sono trattati i singoli mutanti koCP24, koCP26 e il doppio mutante koCP24/26. Tutte queste tre linee mostrano una ridotta efficienza di trasferimento di energia dai complessi trimerici di raccolta della luce (LHCII) al centro di reazione del fotosistema II (PSII) a causa della disconnessione fisica degli LHCII dal PSII. Abbiamo osservato che il trasporto di elettroni \ue8 diminuito nel genotipo koCP24, ma non nelle piante che mancano di CP26: koCP24 ha una diminuita velocit\ue0 di trasporto elettronico, un pi\uf9 basso gradiente di pH transmembrana, una ridotta capacit\ue0 di NPQ, e una crescita limitata. Inoltre, i complessi PSII di queste piante sono organizzati in array bidimensionali nelle membrane granali. Sorprendentemente, il doppio koCP24/26 mutante, mancante sia di CP24 che CP26, recupera la capacit\ue0 di trasporto elettronico, di NPQ e il tasso di crescita ai livelli del WT. Abbiamo quindi approfondito lo studio del mutante koCP24 per comprendere le ragioni di tali alterazioni fenotipiche. L\u2019analisi della cinetica di induzione di fluorescenza e di misure di trasporto di elettroni nei vari passaggi all\u2019interno della catena fotosintetica hanno suggerito che la limitazione nel trasporto degli elettroni in koCP24 \ue8 dovuta alla restrizione del trasporto degli elettroni tra i siti QA e QB del PSII, ritardando la diffusione del plastoquinone. Abbiamo concluso che la mancanza di CP24 altera l\u2019organizzazione dei PSII e limita, di conseguenza, la diffusione del plastoquinone. Tale limitazione \ue8 ripristinata in koCP24/26. Nella sezione A.2, \ue8 descritta la caratterizzazione della funzione della subunit\ue0 CP29, estendendo l'analisi alle diverse isoforme CP29. A questo scopo, ho ottenuto mutanti knock-out privi di una o pi\uf9 isoforme CP29 ed analizzato la loro capacit\ue0 fotosintetica e di fotoprotezione. La mancanza di CP29 non comporta alcuna variazione significativa del trasporto elettronico lineare/ciclico e della capacit\ue0 di transizione di stato, mentre l\u2019efficienza quantica del PSII e la capacit\ue0 di NPQ risultano alterati. L\u2019efficienza di fotoprotezione \ue8 inferiore in koCP29 rispetto sia al WT che ai mutanti che conservano una singola isoforma. \uc8 interessante notare che, mentre l\u2019espressione di una delle isoforme CP29.1 o CP29.2 ripristina la capacit\ue0 di fotoprotezione, l\u2019espressione di solo CP29.3 non porta all\u2019accumulo della proteina n\ue9 al recupero del fenotipo fotoprotettivo. Sezione B. Riorganizzazione dinamica delle membrane: dissociazione della B4 e identificazione di due siti di quenching. Le subunit\ue0 antenna sembrano essere il sito del quenching, mentre l'innesco del meccanismo \ue8 mediato da PsbS, una subunit\ue0 del PSII coinvolta nella rilevazione dell\u2019acidificazione lumenale. Abbiamo indagato il meccanismo molecolare attraverso il quale PsbS \ue8 in grado di regolare l\u2019efficienza di raccolta della luce, studiando mutanti di Arabidopsis che mancano dei singoli Lhcbs monomerici. Nella Sezione B.1 \ue8 mostrato come PsbS \ue8 in grado di regolare l'associazione/dissociazione di un complesso membrana di cinque subunit\ue0, composto dalle due proteine monomeriche CP29 e CP24 e dal complesso trimerico LHCII-M (Band 4 Complex - B4C). Abbiamo dimostrato che la dissociazione di questo supercomplesso \ue8 indispensabile per l'attivazione dell\u2019NPQ in luce alta. Coerentemente, abbiamo scoperto che mutanti knock-out mancanti delle due subunit\ue0 componenti la B4, koCP24 e koCP29, sono fortemente influenzati nella dissipazione dell\u2019energia. L'osservazione diretta mediante microscopia elettronica ha mostrato che la dissociazione della B4C porta alla ridistribuzione dei PSII all'interno delle membrane granali. Proponiamo che la dissociazione della B4C renda i due siti di quenching, possibilmente CP29 e CP24, disponibili per lo switch a una conformazione quenchiata. Questi cambiamenti sono reversibili e non richiedono la sintesi/degradazione proteica, consentendo in tal modo cambiamenti di dimensione dell'antenna PSII e l'adattamento a rapide variazioni delle condizioni ambientali. Nella sezione B.2 abbiamo studiato questo meccanismo di quenching mediante analisi di fluorescenza ultra-rapida. Recenti risultati sui tempi di vita di fluorescenza in vivo propongono l\u2019attivazione di due siti indipendenti di quenching durante l\u2019NPQ: Q1 si localizza nei complessi LHCII, funzionalmente staccati dal PSII/RC (centro di reazione) con un meccanismo che richiede PsbS ma non Zea; Q2 si trova ed \ue8 collegato al complesso PSII, e dipende dalla formazione di Zea. Questi due eventi di quenching potrebbero originarsi in ciascuno dei due domini fisici granali rivelati dall\u2019analisi di microscopia elettronica come precedentemente riportato. Abbiamo quindi studiato la modulazione del quenching in mutanti knock out confrontando i tempi di vita di fluorescenza in condizioni di quenching e non quenching in foglie intatte: abbiamo ottenuto risultati coerenti con il modello di due siti di quenching situati, rispettivamente, nel dominio C2S2 e nel dominio arricchito in LHCII. I dati indicano che il sito Q1 manca nel koCP24 mentre il Q2 \ue8 attenuato nel koCP29. Sulla base dei risultati di questa sezione, possiamo concludere che durante l\u2019induzione dell\u2019NPQ in vivo il supercomplex del PSII si dissocia in due frazioni, separate in domini distinti della membrana granale e protetti ciascuno dalla sovra-eccitazione grazie all\u2019attivit\ue0 di siti di quenching localizzati in CP24 e CP29. Sezione C. Trasferimento di energia di eccitazione e organizzazione della membrana: ruolo delle subunit\ue0 antenna del PSII. In questa sezione \ue8 riportato lo studio del ruolo dei singoli complessi antenna fotosintetici di PSII sia nell\u2019organizzazione di membrana che nel trasferimento dell\u2019energia di eccitazione, utilizzando i mutanti knock out precedentemente isolati. Membrane tilacoidali wild-type e dei tre mutanti mancanti dei complessi CP24, CP26 o CP29, sono stati studiati con spettroscopia di fluorescenza rapida, utilizzando combinazioni differenti di lunghezze d'onda di eccitazione e di detection, al fine di separare le cinetiche del PSI e PSII. Tali misurazioni spettroscopiche hanno rivelato che la mancanza di CP26 non ha modificato l'organizzazione del PSII. Al contrario, l'assenza di CP29 e soprattutto di CP24 porta a cambiamenti sostanziali dell'organizzazione del PSII come evidenziato da un aumento significativo del tempo di migrazione apparente, dimostrando una cattiva connessione tra una parte significativa dell\u2019antenna periferica e i RC. Sezione D.In eukaryotes the photosynthetic antenna system is composed by subunits encoded by the light harvesting complex (Lhc) multigene family. These proteins play a key role in photosynthesis and are involved in both light harvesting and photoprotection. In particular, antenna protein of PSII, the Lhcb subunits, have been proposed to be involved in the mechanism of thermal dissipation of excitation energy in excess (NPQ, non-photochemical quenching). Elucidating the molecular details of NPQ induction in higher plants has proven to be a major challenge. In my phD work, I decided to investigate the role of Lhcbs in energy quenching by using a reverse genetic approach: I knocked out each subunit in order to understand their involvement in the mechanism. Here below the major results obtained are summarized. Section A. Mutants of monomeric Lhc and photoprotection: insights on the role of minor subunits in thermal energy dissipation. In this section I investigate the function of chlorophyll a/b binding antenna proteins, CP26, CP24 and CP29 in light harvesting and regulation of photosynthesis by isolating Arabidopsis thaliana knockout (ko) lines that completely lacked one or two of these proteins. In particular in Section A.1 I focused on single mutant koCP24, koCP26 and double mutant koCP24/26. All these three mutant lines have a decreased efficiency of energy transfer from trimeric light-harvesting complex II (LHCII) to the reaction center of photosystem II (PSII) due to the physical disconnection of LHCII from PSII. We observed that photosynthetic electron transport is affected in koCP24 plants but not in plants lacking CP26: the former mutant has decreased electron transport rates, a lower pH gradient across the grana membranes, a reduced capacity for non-photochemical quenching, and a limited growth. Furthermore, the PSII particles of these plants are organized in unusual two-dimensional arrays in the grana membranes. Surprisingly, the double mutant koCP24/26, lacking both CP24 and CP26 subunits, restores overall electron transport, non-photochemical quenching, and growth rate to wild type levels. We further analysed the koCP24 phenotype to understand the reasons for the photosynthetic defection. Fluorescence induction kinetics and electron transport measurements at selected steps of the photosynthetic chain suggested that koCP24 limitation in electron transport was due to restricted electron transport between QA and QB, which retards plastoquinone diffusion. We conclude that CP24 absence alters PSII organization and consequently limits plastoquinone diffusion. The limitation in plastoquinone diffusion is restore in koCP24/26. In Section A.2 I characterized the function of CP29 subunits, extending the analyses to the different CP29 isoforms. To this aim, I have constructed knock-out mutants lacking one or more Lhcb4 isoforms and analyzed their performance in photosynthesis and photoprotection. We found that lacks of CP29 did not result in any significant alteration in linear/cyclic electron transport rate and maximal extent of state transition, while PSII quantum efficiency and capacity for NPQ were affected. Photoprotection efficiency was lower in koCP29 plants with respect to either WT or mutants retaining a single Lhcb4 isoform. Interestingly, while deletion of either isoforms Lhcb4.1 or Lhcb4.2 get into a compensatory accumulation of the remaining subunit, photoprotection capacity in the double mutant Lhcb4.1/4.2 was not restored by Lhcb4.3 accumulation. Section B. Membrane dynamics and re-organization for the quenching events: B4 dissociation and identification of two distinct quenching sites. Antenna subunits are hypothesized to be the site of energy quenching, while the trigger of the mechanism is mediated by PsbS, a PSII subunit that is involved in detection of luminal acidification. In this section we investigate the molecular mechanism by which PsbS regulates light harvesting efficiency by studying Arabidopsis mutants specifically devoid of individual monomeric Lhcbs. In Section B.1 we showed that PsbS controls the association/dissociation of a five-subunit membrane complex, composed of two monomeric Lhcb proteins, CP29 and CP24 and the trimeric LHCII-M (namely Band 4 Complex - B4C). We demonstrated that the dissociation of this supercomplex is indispensable for the onset of non-photochemical fluorescence quenching in high light. Consistently, we found that knock-out mutants lacking the two subunits participating to the B4C, namely CP24 and CP29, are strongly affected in heat dissipation. Direct observation by electron microscopy showed that B4C dissociation leads to the redistribution of PSII within grana membranes. We interpret these results proposing that the dissociation of B4C makes quenching sites, possibly CP29 and CP24, available for the switch to an energy-quenching conformation. These changes are reversible and do not require protein synthesis/degradation, thus allowing for changes in PSII antenna size and adaptation to rapidly changing environmental conditions. In Section B.2 we studied this quenching mechanism by ultra-fast Chl fluorescence analysis. Recent results based on fluorescence lifetime analysis in vivo proposed that two independent quenching sites are activated during NPQ: Q1 is located in the major LHCII complexes, which are functionally detached from the PSII/RC (reaction centre) supercomplex with a mechanism that strictly requires PsbS but not Zea; Q2 is located in and connected to the PSII complex and is dependent on the Zea formation. These two quenching events could well originate in each of the two physical domains of grana revealed by electron microscopy analysis previously reported. We thus proceeded to investigate the modulation of energy quenching in knock out mutants by comparing the fluorescence lifetimes under quenched and unquenched conditions in intact leaves: we obtained results that are consistent with the model of two quenching sites located, respectively, in the C2S2 domain and in the LHCII-enriched domain. Data reported suggest that Q1 site is released in the koCP24 mutant while Q2 is attenuated in the koCP29 mutant. On the bases of the results of this section, we conclude that during the establishment of NPQ in vivo the PSII supercomplex dissociates into two moieties, which segregates into distinct domain of the grana membrane and are each protected from over-excitation by the activity of quenching sites probably located in CP24 and CP29. Section C. Excitation energy transfer and membrane organization: role of PSII antenna subunits. In this section we investigated the role of individual photosynthetic antenna complexes of PSII both in membrane organization and excitation energy transfer, by using the knock out mutants previously isolated. Thylakoid membranes from wild-type and three mutants lacking light harvesting complexes CP24, CP26 or CP29 respectively, were studied by ps-fluorescence spectroscopy on thylakoids, using different combination of excitation and detection wavelengths in order to separate PSI and PSII kinetics. Spectroscopic measurements revealed that absence of CP26 did not alter PSII organization. In contrast, the absence of CP29 and especially CP24 lead to substantial changes in the PSII organization as evidenced by a significant increase of the apparent migration time, demonstrating a bad connection between a significant part of the peripheral antenna and the RCs. Section D

A systems-wide understanding of photosynthetic acclimation in algae and higher plants

The ability of phototrophs to colonise different environments relied on the robust protection against oxidative stress in phototrophs, a critical requirement for the successful evolutionary transition from water to land. Photosynthetic organisms have developed numerous strategies to adapt their photosynthetic apparatus to changing light conditions in order to optimise their photosynthetic yield, crucial for life to exist on Earth. Photosynthetic acclimation is an excellent example of the complexity of biological systems, in which highly diverse processes, ranging from electron excitation over protein protonation to enzymatic processes coupling ion gradients with biosynthetic activity interact on drastically different timescales, ranging from picoseconds to hours. An efficient functioning of the photosynthetic apparatus and its protection is paramount for efficient downstream processes including metabolism and growth. Modern experimental techniques can be successfully integrated with theoretical and mathematical models to promote our understanding of underlying mechanisms and principles. This Review aims to provide a retrospective analysis of multidisciplinary photosynthetic acclimation research carried out by members of the Marie Curie Initial Training Project “AccliPhot”, placing the results in a wider context. The Review also highlights the applicability of photosynthetic organisms for industry, particularly with regards to the cultivation of microalgae. It aims to demonstrate how theoretical concepts can successfully complement experimental studies broadening our knowledge of common principles in acclimation processes in photosynthetic organisms, as well as in the field of applied microalgal biotechnology