Analysis of the molecular mechanisms of photoprotection in higher plants and green algae

The green photosynthetic organisms, plants and algae, have complex mechanisms in order to defend their photosynthetic apparatus from photoinduced oxydative damages. Such protective mechanisms work through the action of peculiar pigments, the xanthophylls, coordinated with the proteins of the photosynthetic system, which own to the Lhc super-family. In this thesis work I focused through physiological, biochemical and genetic techniques on the characterisation of the important photoprotective mechanism “Non-Photochemical quenching” (NPQ), used by the green organisms in order to control singlet excited chloropylls concentration and limit their excess through a “feedback” mechanism of thermal dissipation of the excess energy. The organisms used in this study have been Arabidopsis thaliana, model for studying higher plants, and Chlamydomonas reinhardtii, model for studying green microalgae. It has been highlighted how, even if both organisms have and use this protective mechanism, many differences are present both at quantitative level in thermal dissipation capacity and at qualitative level in the triggering and in the regulation of the dissipation. In chapter number 1 entitled “In between photosynthesis and photoinhibition: the fundamental role of carotenoids and carotenoid-binding proteins in photoprotection” a general view of photoprotection in plants and algae is given. The aim of this part of the thesis is to illustrate the events that bring to the formation of reactive oxigen species and which are the main mechanisms put in action by the photosynthetic organisms in order to detoxify reactive oxygen species or to prevent their formation. The Non-Photochemical Quenching owns to this second class of photoprotective mechanisms and is here described at general level, using the most importants informations found in literature in the last years and detailing each component of this complex mechanism. Chapter number 2 entitled “Interactions between the photosystem II subunit Psbs and xanthophylls studied in vivo and in vitro” is devoted to a detailed study of the molecular mechanism of quenching in higher plants through the in vitro analysis of the recombinant PsbS protein and the in vivo physiological characterisation of A. thaliana mutants defective in NPQ capacity. Being the thermal dissipation dependent on the presence/synthesis of xanthophylls lutein and zeaxanthin and on the photosystem II PsbS, I checked the hypothesis that the direct interaction of this subunit with xanthophylls was responsible for the phenomenon. The work demonstrated how PsbS does not interact directly with such pigments, but activates the mechanism working as a molecular sensor of the over-excited state of the system, and triggering, through conformational changes in other antenna subunits, the process of thermal dissipation which occurs on these other proteins and their coordinated pigments. The third chapter, “Non-Photochemical Quenching of chlorophyll fluorescence in Chlamydomonas reinhardtii” is a comparative characterisation of the NPQ capacity in the unicellular green alga Chlamydomonas reinhardtii using higher plants as term of comparison, in which the mechanism is more studied and better understood. In this work we report how the NPQ capacity is lower in Chlamydomonas than in higher plants and detectable at reasonable levels only in particular conditions of over-excitation of the photosynthetic electron chain. This demonstrates how such strategy is differentialy adopted by these two organisms and differentially regulated. In the fourth chapter, “The occurrence of the PsbS gene product in Chlamydomonas reinhardtii and other photosynthetic organisms and its correlation with energy quenching”, I carried on a more detailed study of the differences observed between higher plants and green algae, elucidating how the gene product PsbS, responsible fot the activation of the thermal dissipation mechanism in higher plants, is not expressed in the unicellular alga Chlamydomonas although a putative gene sequence is present. The corresponding gene sequence, when over-expressed, does not seem to code a functional polypeptide, confirming how the NPQ mechanism is radically different between plants and algae. The analysis of the presence of PsbS protein in a group of organisms representative for most of classes belonging to the Viridiplantae kingdom, coupled with the measure of their thermal dissipation, evidenced how the PsbS-dependent NPQ-mechanism: i) is clearly present as favourite photoprotective strategy among land plants or Embriophytes; ii) is exclusively conserved in those multicellular green algae adapted to environments with higher and more variable light intensities; iii) is completely absent in the unicellular microalgae, which adopt other photoprotective strategies, or other types of relevant dissipative mechanisms which are differentially regulated at molecular level .