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

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.