Добірка наукової літератури з теми "Antenne monomeriche"

Antenna proteins play a major role in the regulation of light-harvesting in photosynthesis. However, less is known about a possible link between their sizes (oligomerization state) and fluorescence intensity (number of photons emitted). Here, we used a microscopy-based method, Fluorescence Correlation Spectroscopy (FCS), to analyze different antenna proteins at the particle level. The direct comparison indicated that Chromera Light Harvesting (CLH) antenna particles (isolated from Chromera velia) behaved as the monomeric Light Harvesting Complex II (LHCII) (from higher plants), in terms of their radius (based on the diffusion time) and fluorescence yields. FCS data thus indicated a monomeric oligomerization state of algal CLH antenna (at our experimental conditions) that was later confirmed also by biochemical experiments. Additionally, our data provide a proof of concept that the FCS method is well suited to measure proteins sizes (oligomerization state) and fluorescence intensities (photon counts) of antenna proteins per single particle (monomers and oligomers). We proved that antenna monomers (CLH and LHCIIm) are more “quenched” than the corresponding trimers. The FCS measurement thus represents a useful experimental approach that allows studying the role of antenna oligomerization in the mechanism of photoprotection.

Zinc 20-halogenochlorins 2(20- F ), 3(20- Cl ) and 4(20- Br ) were synthesized by halogenation of a chlorophyll a derivative at the 20-position as a model for bacteriochlorophyll ( BChl )c, which possesses a methyl group at the 20-position and 20-unsubstituted BChl d. Visible spectra in a polar tetrahydrofuran ( THF ) solution showed that 2-4 were monomeric and the planarity of the chlorin ring, was distorted with increasing bulkiness of the 20-substituent. Visible, circular dichroism and IR spectra revealed that 2-4 self-aggregated to form oligomers similarly with 20-unsubstituted 1 and BChls c/d in the heterogeneous thin film as well as in homogeneous non-polar solvents (1% (v/v) THF-hexane). Therefore, the in vitro self-aggregates of 2-4 are good structural models for in vivo BChls c/d self-aggregates, the main antenna components of photosynthetic green bacteria. Fluorescence spectra showed that monomeric 3 and 4 were less emissive than 1 and 2 due to the heavy atom effect which could not be observed in the oligomeric species, indicating that the in vitro aggregates should be promising as functional (light-harvesting) models.

Diatoms play important roles in global primary productivity and biogeochemical cycling of carbon, in part owing to the ability of their photosynthetic apparatus to adapt to rapidly changing light intensity. We report a cryo–electron microscopy structure of the photosystem II (PSII)–fucoxanthin (Fx) chlorophyll (Chl) a/c binding protein (FCPII) supercomplex from the centric diatom Chaetoceros gracilis. The supercomplex comprises two protomers, each with two tetrameric and three monomeric FCPIIs around a PSII core that contains five extrinsic oxygen-evolving proteins at the lumenal surface. The structure reveals the arrangement of a huge pigment network that contributes to efficient light energy harvesting, transfer, and dissipation processes in the diatoms.

Phycobilisomes are highly organized pigment–protein antenna complexes found in the photosynthetic apparatus of cyanobacteria and rhodophyta that harvest solar energy and transport it to the reaction center. A detailed bottom-up model of pigment organization and energy transfer in phycobilisomes is essential to understanding photosynthesis in these organisms and informing rational design of artificial light-harvesting systems. In particular, heterogeneous photophysical behaviors of these proteins, which cannot be predicted de novo, may play an essential role in rapid light adaptation and photoprotection. Furthermore, the delicate architecture of these pigment–protein scaffolds sensitizes them to external perturbations, for example, surface attachment, which can be avoided by study in free solution or in vivo. Here, we present single-molecule characterization of C-phycocyanin (C-PC), a three-pigment biliprotein that self-assembles to form the midantenna rods of cyanobacterial phycobilisomes. Using the Anti-Brownian Electrokinetic (ABEL) trap to counteract Brownian motion of single particles in real time, we directly monitor the changing photophysical states of individual C-PC monomers from Spirulina platensis in free solution by simultaneous readout of their brightness, fluorescence anisotropy, fluorescence lifetime, and emission spectra. These include single-chromophore emission states for each of the three covalently bound phycocyanobilins, providing direct measurements of the spectra and photophysics of these chemically identical molecules in their native protein environment. We further show that a simple Förster resonant energy transfer (FRET) network model accurately predicts the observed photophysical states of C-PC and suggests highly variable quenching behavior of one of the chromophores, which should inform future studies of higher-order complexes.

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: in- sights 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 eciency 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 and formation of PSII reaction center depleted domains in grana partitions. We observed that photosynthetic electron transport is aected 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 diusion. We conclude that CP24 absence alters PSII organization and consequently limits plastoquinone diffusion. The limitation in plastoquinone diusion is restore in koCP24/26. In Section A.2 I characterized the function of CP29 subunits, extending the analyses to the dierent 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 signicant alteration in linear/cyclic electron transport rate and maximal extent of state transition, while PSII quantum eciency and capacity for NPQ were aected. Photoprotection eciency 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 quench- ing events: B4 dissociation and identication 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 lumenal acidication. In this section we investigate the molecular mechanism by which PsbS regulates light harvesting eciency by studying Arabidopsis mutants specically devoid of individual monomeric Lhcbs. In Section B.1 we showed that PsbS controls the association/dissociation of a ve-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 uorescence 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 aected in heat dissipation. Direct observation by electron microscopy and image analysis 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 uorescence analysis. Recent results based on uorescence lifetime analysis \in vivo" (Holzwarth et al., 2009) 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 (Miloslavina et al., 2008). These two quenching events could well originate in each of the two physical domains of grana revealed by electron microscopy analysis previously reported (see Section B.1). We thus proceeded to investigate the modulation of energy quenching in knock out mutants by comparing the uorescence 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 organiza- tion: role of PSII antenna subunit. 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 (Section A). Thylakoid membranes from wild-type and three mutants lacking lightharvesting complexes CP24, CP26 or CP29 respectively, were studied by picosecond- uorescence spectroscopy on thylakoids, using dierent 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, as evidenced by very similar excitons migration times in both wild-type and koCP26 samples. In contrast, the absence of CP29 and especially CP24 lead to substantial changes in the PSII organization as evidenced by a signicant increase of the apparent migration time, demonstrating a bad connection between a signicant part of the peripheral antenna and the RCs. Section D. A mutant without minor antenna proteins: towards a solution of the dierential role of monomeric Lhcb proteins vs major LHCII in Non Photochemical Quenching. In this section we attempt to answer to the question on the localization of the quenching site(s) in the major LHCII or in monomeric Lhcb proteins. In order to verify the implication of monomeric antenna CP24, CP26 and CP29 in quenching we tried to isolate a mutant knocked out for all the three subunit. Lhcb5 (CP26) and Lhcb6 (CP24) are coded by a single gene while three isoforms of Lhcb4 (CP29) are present in the Arabidopsis genome, we thus looked for a ve-gene mutant. We tried to isolate such a mutant from the screening of two dierent populations but without success. In particular, we found that is not possible to obtain a mutant lacking both CP26 and CP29 at the same time. We veried that is not a problem of seed germination but instead a defect of the embryo development. This unexpected genetic evidence shows that, for a correct and functional organization of the PSII supercomplexes, either Lhcb5 or Lhcb4.1 must be present: probably these two subunits can replace themselves each other when one lacks, while none of the other monomeric antenna (Lhcb6, Lhcb4.2, Lhcb4.3) are able to takes their place in PSII. Two mutants obtained in these screenings allowed us to make some considerations about the role of monomeric antenna proteins in NPQ: mutants koCP29/24 which dier for the condition at the Lhcb5 locus, either homozygous WT or heterozygous. We observed a dose eect for the expression of Lhcb5, that is lower in the heterozygous since one allele only is functional. Interestingly, the amount of CP26 on thylakoid membrane strictly correlated with the quenching amplitude measured at 1200 mol photons m��2s��1 of actinic light. These results suggest that the residual quenching in koCP29/24 plants is largely due to the capacity of CP26 subunit to mediate energy quenching. This is a further indication of the primary role of monomeric Lhcb in NPQ, as recently stated by CT (charge transfer) quenching models (Avenson et al., 2008; Ahn et al., 2008).
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: in- sights 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 eciency 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 and formation of PSII reaction center depleted domains in grana partitions. We observed that photosynthetic electron transport is aected 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 diusion. We conclude that CP24 absence alters PSII organization and consequently limits plastoquinone diffusion. The limitation in plastoquinone diusion is restore in koCP24/26. In Section A.2 I characterized the function of CP29 subunits, extending the analyses to the dierent 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 signicant alteration in linear/cyclic electron transport rate and maximal extent of state transition, while PSII quantum eciency and capacity for NPQ were aected. Photoprotection eciency 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 quench- ing events: B4 dissociation and identication 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 lumenal acidication. In this section we investigate the molecular mechanism by which PsbS regulates light harvesting eciency by studying Arabidopsis mutants specically devoid of individual monomeric Lhcbs. In Section B.1 we showed that PsbS controls the association/dissociation of a ve-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 uorescence 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 aected in heat dissipation. Direct observation by electron microscopy and image analysis 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 uorescence analysis. Recent results based on uorescence lifetime analysis \in vivo" (Holzwarth et al., 2009) 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 (Miloslavina et al., 2008). These two quenching events could well originate in each of the two physical domains of grana revealed by electron microscopy analysis previously reported (see Section B.1). We thus proceeded to investigate the modulation of energy quenching in knock out mutants by comparing the uorescence 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 organiza- tion: role of PSII antenna subunit. 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 (Section A). Thylakoid membranes from wild-type and three mutants lacking lightharvesting complexes CP24, CP26 or CP29 respectively, were studied by picosecond- uorescence spectroscopy on thylakoids, using dierent 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, as evidenced by very similar excitons migration times in both wild-type and koCP26 samples. In contrast, the absence of CP29 and especially CP24 lead to substantial changes in the PSII organization as evidenced by a signicant increase of the apparent migration time, demonstrating a bad connection between a signicant part of the peripheral antenna and the RCs. Section D. A mutant without minor antenna proteins: towards a solution of the dierential role of monomeric Lhcb proteins vs major LHCII in Non Photochemical Quenching. In this section we attempt to answer to the question on the localization of the quenching site(s) in the major LHCII or in monomeric Lhcb proteins. In order to verify the implication of monomeric antenna CP24, CP26 and CP29 in quenching we tried to isolate a mutant knocked out for all the three subunit. Lhcb5 (CP26) and Lhcb6 (CP24) are coded by a single gene while three isoforms of Lhcb4 (CP29) are present in the Arabidopsis genome, we thus looked for a ve-gene mutant. We tried to isolate such a mutant from the screening of two dierent populations but without success. In particular, we found that is not possible to obtain a mutant lacking both CP26 and CP29 at the same time. We veried that is not a problem of seed germination but instead a defect of the embryo development. This unexpected genetic evidence shows that, for a correct and functional organization of the PSII supercomplexes, either Lhcb5 or Lhcb4.1 must be present: probably these two subunits can replace themselves each other when one lacks, while none of the other monomeric antenna (Lhcb6, Lhcb4.2, Lhcb4.3) are able to takes their place in PSII. Two mutants obtained in these screenings allowed us to make some considerations about the role of monomeric antenna proteins in NPQ: mutants koCP29/24 which dier for the condition at the Lhcb5 locus, either homozygous WT or heterozygous. We observed a dose eect for the expression of Lhcb5, that is lower in the heterozygous since one allele only is functional. Interestingly, the amount of CP26 on thylakoid membrane strictly correlated with the quenching amplitude measured at 1200 mol photons m��2s��1 of actinic light. These results suggest that the residual quenching in koCP29/24 plants is largely due to the capacity of CP26 subunit to mediate energy quenching. This is a further indication of the primary role of monomeric Lhcb in NPQ, as recently stated by CT (charge transfer) quenching models (Avenson et al., 2008; Ahn et al., 2008).

La fosforilazione reversibile di proteine tilacoidali negli organismi fotosintetici è un meccanismo per far fronte a condizioni di luce variabili. Nelle monocotiledoni, contrariamente a quanto avviene nelle dicotiledoni, è stato dimostrato che la fosforilazione dell’antenna minore CP29, in seguito ad esposizione ad alte intensità luminose, induce un incremento in NPQ e diminuisce la produzione di specie reattive dell’ossigeno (ROS). La chinasi del complesso di antenne maggiori (LHCII), denominata STN7, e la relativa fosfatasi TAP38/PPH1, non partecipano in questo meccanismo, come in precedenza dimostrato nel nostro laboratorio, suggerendo l’ipotesi che un set di chinasi/fosfatasi differente fosse coinvolto nella regolazione di questo fenomeno. Recentemente, abbiamo analizzato un mutante knockout OsSTN8, gentilmente concessoci dal laboratorio di CH Lee, dimostrando che in aggiunta alla fosforilazione del core del Fotosistema II (PSII), anche quella di CP29 era soppressa, così provando che STN8 è la chinasi coinvolta nella fosforilazione di CP29 nelle monocotiledoni. Per meglio analizzare l’attività di OsSTN8 abbiamo trasformato linee mutanti di A. thaliana, dove il meccanismo di fosforilazione di CP29 in alta luce è assente, data la presenza di una libreria di mutanti molto vasta e dalla facilità con cui questa specie è geneticamente manipolabile rispetto a riso. I mutanti di A. thaliana stn8 e stn7stn8 complementati con OsSTN8 mostravano un recupero della fosforilazione del PSII core, come dimostrato tramite analisi Western blot. In aggiunta, la chinasi di riso fosforilava anche CP29 in condizioni di alta luce, contrariamente a quanto osservato nelle linee wild type. Misure di NPQ sono state eseguite sulle linee trasformate al fine di valutare l’effetto della fosforilazione dell’antenna minore sulla fotoprotezione. Un lieve incremento è stato osservato nelle linee trasformate con la chinasi di riso, indicando un possibile contributo di P-CP29 nella fotoprotezione in condizioni di di alta luce. Per meglio discernere il contributo di P-CP29 dalla fosforilazione del PSII core, mutanti Atlhcb4 sono stati co-trasformati con OsSTN8 e CP29 di riso o Arabidopsis, sia nella forma nativa che mutata al sito di fosforilazione Thr-83, ossia il sito identificato in riso come target della chinasi STN8. Un 6X-histag è stato addizionato alle proteine espresse per facilitare i processi di purificazione e permette analisi spettroscopiche delle forme fosforilate e non di CP29. Linee transgeniche sono state recentemente ottenute e misure fisiologiche saranno eseguite, sia in vivo che in vitro a seguito della purificazione della proteina. In A. thaliana la fosfatasi PBCP (Photosystem II Core Phosphatase) è stata dimostrata essere responsabile della defosforilazione del PSII core, controbilanciando l’attività di STN8. La proteina ricombinante OsPBCP da noi ottenuta era in grado di defosforilare in vitro sia le proteine del core che CP29 presenti nei tilacoidi e nelle preparazioni di complessi isolati da gradiente di saccarosio. Alla luce di questi risultati, abbiamo dimostrato che STN8 e CP29 sono, rispettivamente, la chinasi e fosfatasi coinvolte nella fosforilazione di CP29 nelle monocotiledoni, e OsSTN8 conserva la sua attività quando espressa in una dicotiledone come Arabidopsis thaliana.
Reversible phosphorylation of thylakoid proteins in photosynthetic organisms is a way to cope with changing light conditions. It has been demonstrated that in monocots, as opposed to dicots, upon high light exposure the minor antenna CP29 is phosphorylated enhancing NPQ and reducing singlet oxygen production. The major light-harvesting complex II (LHCII) kinase STN7 and its related phosphatase PPH1/TAP38 have been proven not to be involved in this mechanism in monocots, indicating that a different set of kinases/phosphatases act in regulating this acclimatory response. Recently, we have analyzed an OsSTN8 knockout mutant, kindly provided by the laboratory of CH Lee, in which we determined that in addition to that of the PSII core proteins, CP29 phosphorylation was suppressed as well, thus proving that STN8 is the kinase involved in CP29 phosphorylation in monocots. To further investigate OsSTN8 activity we transformed A.thaliana mutant lines, where CP29 phosphorylation is absent in high light, given the availability of mutant libraries and the ease with which this species is manipulable compared to rice. A.thaliana stn8 and stn7stn8 mutants transformed with OsSTN8 restored phosphorylation of the PSII core proteins, as confirmed through immunoblot analysis. Furthermore, the kinase was able to phosphorylate CP29 under high light conditions, as opposed to the wild type strain. Non-Photochemical Quenching (NPQ) measurements were performed on the transformed lines to assess the effect of the minor antenna phosphorylation on photoprotection, showing a mild increase in NPQ. To better understand the individual contribution of CP29 phosphorylation in transgenic Arabidopsis apart from that of PSII core phosphorylation in high light, knockout lines for Lhcb4 of A.thaliana were co-transformed in order to express OsSTN8 and CP29 either from rice or A.thaliana, both in its native and mutated form at Thr-83, site of phosphorylation in rice. A 6X-Histag was added for improved purification in order to conduct spectroscopic analyses on phosphorylated and unphosphorylated forms of CP29. Transgenic lines were recently obtained and physiological analyses will be performed in the near future, both in vivo and in vitro through purification of the protein. In A.thaliana the phosphatase PBCP was determined to be involved in PSII core dephosphorylation and counteract the effect of STN8. Our recombinant OsPBCP was capable of dephosphorylating in vitro both PSII core proteins and CP29, in thylakoids as well as isolated complexes from a sucrose gradient. In light of these results, we have determined that STN8 and PBCP are respectively the kinase and phosphatase involved in CP29 phosphorylation in monocots, and OsSTN8 retains its activity when expressed in a dicot such as Arabidopsis thaliana.