Functional characterization of moss flavodiiron protein activity in Hordeum vulgare and Nicotiana tabacum

Photosynthesis is the process by which photosynthetic organisms, such as cyanobacteria, green algae and plants, use light energy to fix CO2 into organic compounds. In eukaryotes, the light-dependent reactions take place in the thylakoid membranes of chloroplasts, where the electron transport chain is located. In the linear electron flow (LEF), several factors are involved: photosystem II (PSII), plastoquinone (PQ), the cytochrome b₆f complex, plastocyanin (Pc), photosystem I (PSI), ferredoxin (Fd) and ferredoxin-NADP+ reductase (FNR), which catalyzes the reduction of NADP⁺ to NADPH. During LEF, a proton gradient across the thylakoid membrane is generated, which is exploited by ATP synthase to produce ATP. These two products (NADPH and ATP) will then be used by the Calvin-Benson cycle to fix CO2. However, rapid changes in light conditions can cause an imbalance between the light energy absorbed and the ability of the photosynthetic system to use it. This can cause an accumulation of electrons and increase the risk of photodamage. To avoid this problem, there are two main alternative pathways downstream of PSI: cyclic electron flow (CEF) and pseudocyclic electron flow (PCEF), which allow ATP production without reducing NADP+ to NADPH. In PCEF, flavodiiron proteins (FLVs) play a fundamental role. These enzymes are able to accept electrons from PSI and reduce molecular oxygen to water. This process helps to prevent the accumulation of reducing power and the consequent photodamage to PSI, especially under fluctuating light conditions or rapid increases in light intensity. FLVs are present in all photosynthetic organisms, but they were lost during the evolution of angiosperms. In this study, the FLVA and FLVB proteins from Physcomitrium patens were studied in two species of interest, the model crop Nicotiana tabacum and the agronomically relevant species Hordeum vulgare. In barley, the aim was to identify lines in which the proteins were functional. In tobacco, instead, lines in which FLV expression had already been confirmed were used. These plants were exposed to fluctuating light, and it was observed that the transgenic plants had both higher photosynthetic efficiency and improved growth compared to WT plants. This suggests that FLVs in Nicotiana tabacum can be active and important for the protection of the photosynthetic system under fluctuating light conditions.