Dynamics of Photosystem II during short and long term response to light intensity: a biochemical and biophysical study

La fotosintesi è il processo attraverso il quale le piante assorbono l'energia solare per convertirla in energia chimica e, infine, in biomassa. Durante questo processo, varie funzioni chiave sono svolte dai fotosistemi, PSI e PSII, i quali sono straordinarie macchine per l'uso dell'energia solare, combinando l'alta efficienza quantica e la presenza di meccanismi inducibili al fine di evitare fotoinibizione. L'organizzazione peculiare dei fotosistemi è determinante per la loro funzione. Entrambi i fotosistemi sono costituiti da due porzioni: un complesso core e i complessi antenna (famiglia di proteine LHC). Le proteineLHC svolgono un ruolo fondamentale nella fotosintesi , essendo coinvolte nella raccolta della luce e nella fotoprotezione. Le proteine antenna del PSII, le subunità Lhcb, sono responsabili per il meccanismo di dissipazione termica di energia di eccitazione in eccesso (NPQ, quenching non-fotochimico). Chiarire i dettagli molecolari di induzione di NPQ nelle piante superiori è una grande sfida: nel mio lavoro di dottorato di ricerca, ho studiato in particolare la riorganizzazione dei domini proteici all'interno delle membrane tilacoidali dopo trattamento con un eccesso di luce, verificando la sua importanza per il pieno funzionamento durante NPQ.

Photosynthesis is the process by which plants absorb solar energy and convert it to chemical energy and finally biomass. During this process, several key functions are carried out by Photosystems. PSI and PSII represent extraordinary machines for solar energy use, combining high quantum efficiency and the presence of inducible mechanisms in order to avoid photoinhibition which unavoidably derives from performing photosynthesis in oxygenic environment. The peculiar organization of Photosystems is determinant for function. Both Photosystems are composed by two moieties: a core complex and the antenna system (LHC protein family). LHC proteins play a key role in photosynthesis and are involved in both light harvesting and photoprotection. Among other photoprotecting mechanisms, antenna proteins of PSII, so-called Lhcb subunits, are responsible for 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 investigated the reorganization of the protein domains inside grana membranes upon high light treatment and verified its importance for full functioning of NPQ. Below the main results obtained are summarized. Section A. Zeaxanthin modulates energy quenching properties of monomeric Lhcb antenna proteins. Among photosynthetic pigments, a special role is played by zeaxanthin (Zea), which is only accumulated under excess light. We studied the dynamics of xanthophylls binding to Lhcb proteins upon exposure of leaves to excess light. We found that Lhcb6 undergoes faster Zea accumulation than any other thylakoid protein so far described. We then studied in vitro modulation of Lhcb6 (CP24) functional properties by studying the effects of binding different xanthophyll species by using several spectroscopic techniques. The results suggest for Lhcb6 a special role in binding Zea and enhancing photoprotection under excess light. The Lhcb6 subunit is a recent addition to the photosynthetic apparatus of viridiplantae, being absent in algae and first appearing in mosses, together with the adaptation to the highly stressful conditions typical of land environment. Consistently, it is involved in several regulation mechanisms, as evidenced by genetic (de Bianchi, S. et al. 2008, Kovacs, L. et al. 2006) and biochemical analysis (Ballottari, M. et al. 2007). Previously it was reported to be involved in Non-Photochemical Quenching (NPQ) (Ahn, T. K. et al. 2008, Avenson, T. J. et al. 2008). In the second part of this section we focuses on fluorescence quenching and compared the effect of aggregation, which has been proposed to occur in vivo during NPQ due to a conformational change allowing energy transfer from Chl a excited states to the short lived carotenoid S1 excited state (Ruban, A. V. et al. 2007). This aggregation-dependent quenching (ADQ) has been proposed as an alternative to charge transfer quenching (CTQ) (Ahn, T. K. et al. 2008) mechanism proposed by other groups, including our laboratory. We studied the properties, particularly dependence on zeaxanthin binding, of ADQ using time-resolved and steady state spectroscopy. We obtained evidence that monomeric Lhcb proteins undergo ADQ even better than trimeric LHCII for which this mechanism was originally proposed. In these proteins the amplitude of the process is enhanced by zeaxanthin, while this is not the case for LHCII. Nevertheless, when LHCII is mixed with Lhcb6, this provides zeaxanthin-deppendent enhancement. This result complements previous studies of CTQ, which localized the quenching site to Chl 603, Chl 609, and Zea in carotenoid-binding site L2 (Ahn, T. K. et al. 2008) and suggests that two different types of quenching may occur in Lhcb proteins. Section B. Membrane dynamics during NPQ: PsbS and zeaxanthin-dependent reorganization of Photosystem II is controlled by dissociation of a pentameric supercomplex. Antenna subunits heve been shown to host the site of energy quenching, while the trigger of the mechanism is mediated by PsbS (Bonente, G. et al. 2008), a PSII subunit involved in transducing the signal of over-excitation consisting into lumen acidification (Li, X. P. et al. 2002, Li, X. P. et al. 2004). In this section we investigate the molecular mechanism by which PsbS regulates light harvesting efficiency. We showed that PsbS controls the association/dissociation of a five-subunit membrane complex, composed of two monomeric Lhcb proteins, Lhcb4 and Lhcb6 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. Direct observation of grana membranes upon treatment with excess light for different timelengths by electron microscopy and image analysis showed that B4C dissociation leads to the redistribution of PSII within grana membranes, reducing average distances between PSII core complexes. This phenomenon was reversible upon dark relaxation. We interpret these results proposing that the dissociation of B4C makes quenching sites, possibly Lhcb4 and Lhcb6, available for the switch to an energy-quenching conformation. Section C. New insights on the role of the monomeric Lhcb4 antenna subunit. PSII is surrounded by a external antenna system composed by trimeric LHCII and monomeric minor antenna complexes. Several evidences suggests that Lhcb4, in particular, is a key factor in both light harvesting and photoprotection (Ballottari, M. et al. 2007) or under chronic excitation of PSII (Morosinotto, T. et al. 2006), b) is able to perform charge transfer quenching (Ahn, T. K. et al. 2008). We characterized the function of Lhcb4 subunits in Arabidopsis thaliana. In order to determine the function of Lhcb4 in A. thaliana, we have constructed knock-out mutants lacking one or more Lhcb4 isoforms and analyzed their performance in photosynthesis and photoprotection. The absence of Lhcb4 also caused a destabilization of PSII supercomplexes, modifying antenna system organization. The distribution of PSII complexes within grana membranes is affected in koLhcb4 and LHCII enriched domains are formed. PSII quantum efficiency and NPQ activity were affected, and photoprotection efficiency under high light conditions was impaired in koLhcb4 plants with respect to either WT or mutants depleted of any other Lhcb subunit. Electron microscopy analysis reveal that PSII supercomplex from koLhcb4 plants bears a hole in its structure. We conclude that Lhcb4 is a fundamental component of PSII which is essential for maintenance of both the function and structural organization of this photosystem. Section D. Chlorophyll b reductase affected the regulation of antenna complexes during light stress. The molecular mechanism of antenna complexes breakdown is largely unknown and it is still unclear whether chlorophyll degradation precedes the degradation of the protein moiety or whether protein degradation is the first event. Recently chlorophyll b reductase mutant has been isolated (Kusaba, M. et al. 2007). This enzyme is responsible for the first step of chlorophyll degradation pathway, the conversion of Chlorophyll (Chl) b in Chl a and the mutant is called “stay-green”, because of PSII antenna retention upon leaf senescence induction (Horie, Y. et al. 2009). We characterized the response of Chl b reductase ko mutant to acclimation in high light. The mutant showed a slower antenna size reduction with respect to WT. This enzyme is upregulated during HL acclimation. In vitro assay with recombinant Chl b reductase demonstrated that its activity is higher when zeaxanthin, which accumulate during stress, is bound to PSII antenna complexes.