Isolation and characterization of Nicotiana tabacum and Solanum lycopersicum lines expressing the Flavodiiron proteins of the moss Physcomitrium patens

All Photosynthetic organisms such as plants, cyanobacteria and green algae use light to produce biochemical energy. In particular, pigments associated to photosystem II (PSII) and photosystem I (PSI) transmembrane complexes are light excited and their high energy electrons transferred to downstream acceptors. Cytochrome b6f complex (cytb6f), the oxidation and reduction of the electron carriers plastoquinol (PQH2), plastocyanin (Pc) and ferredoxin-NADP(+) reductase (FNR) proteins are part of process called linear electron flow (LEF). However, photosynthetic organisms are constantly exposed to a changing environment, thus LEF might be unbalanced, and a temporary excess of excitation energy can cause a photodamage. To avoid that, photosynthetic organisms evolved systems capable to dissipate the excess energy such as cyclic electron flow (CEF) around PSI and pseudo cyclic electron flow (PCEF). PCEF can be mediated by Flavodiiron proteins (FLV), enzymes that accept electrons from PSI to reduce oxygen into water. They act as alternative electron sink downstream PSI preventing an accumulation of electrons and consequent PSI photodamage. FLV are present in all photosynthetic organisms, but they absent in Angiosperms. My thesis project was aimed to test whether the FLV of the moss Physcomitrium patens can be functional as electron sink in angiosperm. In order to do that, we expressed FLVA and FLVB of Physcomitrium patens in model crops Solanum lycopersicum and Nicotiana tabacum. All the obtained clones have been screened by measuring PSI redox kinetics and by immunoblotting in order to verify the presence and functionality of the FLV. Physiological analysis under fluctuating light conditions have also been performed to see how FLV-expressing clones respond to light conditions as compared to wild-type plants. A difference in the PSI electron transport in the low-to-high light transition has been observed. FLVs are widely present in all photosynthetic organisms but they absent in Angiosperms. However, the lack of FLVs was partially compensated by an increased cyclic electron transport. My thesis research aims to test whether FLV can be functional in angiosperm crops and if their expression could affect the activity of alternative regulatory mechanisms like CEF. For this reason, we expressed FlvA-B of Physcomitrium patens in Solanum lycopersicum and Nicotiana tabacum through a constitutive vector (pBinAR) containing the 35S promoter. All the obtained clones have been screened by PSI redox kinetics and immunoblotting in order to verify the presence and the quantity of the FLVs. Physiological analysis under fluctuating light conditions have also been performed to see how FLV-positive clones behave compared to Wild Type plant under stressing light conditions. As expected, a difference in the PSI electron transport in the low-to-high light transition has been observed. A monitoring of phenotypic traits’ changes has also been accomplished to understand how the expression of these enzymes could affect their growth.