Regulation of chloroplast pigment-binding proteins turnover in Arabidopsis thaliana

Plants continually experience fluctuations in the conditions of their environment, particularly in the quantity and quality of incident light. Since devoid of motility, plants have evolved a plethora of molecular mechanisms aimed to accommodate changes in irradiance, in order to enable efficient light harvesting in limited light while avoiding photoinhibition under excess light. A number of works have investigated the modifications occurring in various plant species when grown under limiting or excess irradiances. In particular, changes in light intensity elicit major responses at the level of chloroplast, which can be distinguished in short- and long-term response based on their timescale of activation. In particular the long-term responses to irradiance at the chloroplast level are known as acclimation, and consist in the selective synthesis and degradation of functional components in order to optimize the photosynthetic efficiency. Regulation of proteins turnover is surely involved in acclimation: it allows a fine tuning of the proteins quality and quantity, thus defining the metabolic processes active in the cell. However, the regulatory mechanisms which underlie the molecular rearrangement of chloroplast, in particular those elicited during acclimation to excess light, are still poorly defined. This thesis is aimed at gaining new insight on timing and factors affecting turnover of pigment-binding complexes, and how these modulate the acclimatory response in Arabidopsis thaliana leaves. Several efforts have been made in order to measure the turnover rate of photosynthetic complexes, both in low and high light conditions. Indeed, with the exception of D1 subunits, whose turnover rate has been assessed accurately, no information are available regarding the half-life of the other chlorophyll-binding complexes, such as PSI, LHCI and LHCII. The turnover of LHCII complexes is of particular interest, since degradation of this antenna is triggered by the high light response and represents a strategy to regulate the PSII functional antenna size. As first trials, both radioactive and non-radioactive pulse-chase experiments were performed. Pulse-chase with 35S, supplied either as Na2SO4 or methionine, showed that the plant cell actively recycles sulphur, as well as stores it probably in the vacuole, thus it leads to an over-estimation of protein turnover rate. A second approach resorted to deuterium oxide as a labelling system, coupled to a mass spectrometry analysis. 2H2O is totally invasive, rapidly equilibrates with the water environment, and therefore does not distort turnover rate estimation due to over-labelling of the system. In this case, a too low labelling efficiency was reached, thus making the assessment of turnover rate unreliable even with a very sensitive techniques such as mass spectrometry. Altogether, these results confirmed that determination of LHCII turnover rate was an hard task, indeed efficient labelling cannot be achieved by means of a classic pulse-chase approach, due to the long half-life of these complexes. A different strategy was thus pursued, namely purification of recombinant LHC isoforms to be used as internal standards in an mass-spectrometry analysis, in order to evaluate LHC abundance in thylakoids from low- vs. high-light acclimated plants and to assess which subunits are regulated during acclamatory responses. Both cloning and expression trials were carried out in this thesis work. Regarding the molecular mechanisms underlying acclimation, the role of chlorophylls degradation pathway on the high light dependent re-organization of the photosynthetic machinery was studied. By analyzing mutants devoid of chlorophylls catabolism enzymes (nolnyc and pph), we assessed that these enzymes are responsible for chlorophylls degradation not only during senescence, as previously shown, but even upon exposure to high light conditions. Both mutants were less effective than the wild type in regulating photosystems composition and abundance once challenged with HL, thus indicating that chlorophylls removal is one of the early events triggering acclimation of the photosynthetic apparatus. Nevertheless, chlorophylls catabolism was shown not essential for acclimation, since both mutants were effective in preserving PSII quantum yield in high light. In addition to chlorophylls, the function of xanthophylls in the structural stability of photosynthetic supercomplexes was assessed. Besides the well-known role in the stabilization of LHCII complexes, xanthophylls abundance was shown to modulate the PSI relative content: indeed, a positive correlation was assessed between xanthophylls/carotenoids and PSI/PSII ratios, within a wide range of values. These results were obtained by analyzing wild type plants whose xanthophylls/carotenoids content was modulated by the light intensity to which they were acclimated. Moreover, we isolated and studied the nox mutant, completely devoid of xanthophylls and unable to grow on soil since specifically depleted in PSI. Such a characterization lead to the conclusion that xanthophylls have a fundamental role in the biogenesis of PSI, thus making biosynthesis of these molecules crucial for sustaining autotrophic growth. The last part of the thesis aimed at finding new candidate proteins involved in the acclimation response to HL. A number of genes were selected on the strength of their annotation, in which an ubiquitin/proteasome-like domain was identified and the localization was indicated as chloroplastic. Among the tested loci, we selected 3 genes whose corresponding knock-out mutants yielded into impaired PSII photoprotection in high light: AT2G22890, AT4G27030 and AT4G32250. The first two encode for isoforms of fatty acid desaturase 4 (FAD4), thus suggesting a role for lipids biosynthesis in the response to photoxidative stress. The third gene identified encodes for a protein kinase, bound to the chloroplast envelope, whose lack yield into a severe photosensitivity. No homologous proteins are present in the Arabidopsis thaliana genome, while ortholog were found in different plant species, thus indicating that its function has been conserved through evolution. We hypothesized it could be related to some signal transduction pathways, linking chloroplast to nucleus, or might regulate protein import thought the chloroplast envelope, or phosphorylation of stromatic target proteins.