Plant response to abiotic stress: analysis of changes in the photosynthetic apparatus at both gene and protein level

Life on earth depends on solar energy. Photosynthesis is the biological process that allows the conversion of sunlight into chemical energy: plants, cyanobacteria and green algae capture solar radiation and convert the energy in a stable form that can be used in later times for biochemical processes, in particular to synthesise sugars starting from carbon dioxide. To carry out these reactions photosynthetic organisms need a specific apparatus which is located within the cells in dedicated organelles called chloroplasts; the photosynthetic apparatus is composed by proteins coordinating pigments, responsible for solar radiation absorption and energy transfer. Since plants cannot move far away from adverse conditions, during evolution they needed to develop adaptative mechanisms which allowed not only the survival but also to keep on growing and reproducing when climate conditions represent a source of stress. Any environmental change influences the capacity of light energy harvesting, so the photosynthetic apparatus is one of the firsts targets of changes raised to face those variations: in particular, proteins and pigments belonging to photosystems antennae are much more involved and, for this reason, their synthesis and degradation was the object of this thesis. In the first part of the work the behaviour of different light harvesting complex (Lhc) subunits, belonging to a highly conserved multigenic family, was analysed in response to different growing conditions in Zea mays. The redundancy of these sequences suggested, in fact, a possible specific role of each gene product in light harvesting and photoprotection, depending on environmental conditions. Plants were grown in different conditions of light and temperature and thylakoid membranes were isolated in order to test the accumulation of Lhc proteins. Significant differences were found in the accumulation of both major (LHCII) and minor antennae of Photosystem II (PSII) and, in particular, temperature seemed to play an important role, since the LHCII/minor antenna ratio increased with decreasing temperature. In addition, plants grown in different conditions showed different pigment composition, spectroscopic properties of antenna complexes and value of Non Photochemical Quenching. These results confirmed the suggested specific role of different antennae in the organization of the Photosystem II and photoprotection. In the second part of this thesis, the modulation of antenna polypeptides following environmental conditions was analysed using a barley (Hordeum vulgare) mutant, viridis zb63, which lacks Photosystem I, to mimic extreme and chronic over-excitation of Photosystem II. First of all, the mutant was analysed in detail, showing a reduction of PSII antenna to a minimal size, which was not further reducible. Biochemical methods and electron microscopy showed that this minimal antenna consist of a dimeric PSII reaction centre core surrounded by monomeric Lhcb4, Lhcb5 and trimeric light harvesting complex II antenna proteins, with the complete absence of Lhcb6. This minimal Photosystem II unit was shown to form arrays in vivo, possibly to increase the efficiency of energy distribution and provide photoprotection. The mutant showed a chronic reduction of plastoquinone, also at very low light intensities, but the level of oxidative stress in the cells was comparable to wild type, indicating the presence of two distinct signalling pathways activated by excess light absorbed by Photosystem II: one, dependent on the redox state of the electron transport chain, is involved in the regulation of antenna size, and the second, more directly linked to the level of photoinhibitory stress perceived by the cell, participates in regulating carotenoid biosynthesis. The effect of the plastoquinone redox state on the regulation of the light-harvesting antenna size was analysed at transcriptional and post-transcriptional levels. The mRNA level of genes encoding antenna proteins was almost unaffected in the mutant; this stability of messenger level extended to all photosynthesis-related genes, while, in contrast, analysis of protein accumulation by two-dimensional PAGE showed that the mutant undergoes strong reduction of its antenna size, with individual gene products having different levels of accumulation. Then, the plastoquinone redox state was shown to play an important role in the long term regulation of chloroplast protein expression and its modulation is active at the post-transcriptional rather than transcriptional level.