Résultat de la recherche
Engineering Light Harvesting Complex II
To sustain the life on Earth solar energy has to be converted into chemical energy, this is possible thanks to the process called photosynthesis. Through a set of interconnected pigment-binding proteins, collectively called “light harvesting complex” (LHC), photon energy is collected and funneled towards the reaction centers (RCs) of the two photosystems. The RCs feed a series of redox reactions that ultimately allow the production of reducing power (NADPH) and ATP, used for the synthesis of organic molecules. Despite having a highly conserved core structure, the light harvesting complex II (LHCII) is capable to acclimate to a wide range of environmental conditions. LHCII can functionally associate to both photosystems thus allowing a fine tuning of the electron transport chain, or act as dissipators of excess light energy protecting the photosystems from photodamage. This dynamic regulation is crucial for the adaptation of plants to different light conditions. LHCII is mostly composed of homo and hetero trimers of three isoforms: LHCB1, LHCB2 and LHCB3. The first two, through the phosphorylation of a threonine present in the N-terminal domain, are crucial for the regulation of the dynamics of the LHCII network. LHCB2 phosphorylation plays a central role in LHCII association to photosystem I and is thus regarded as the regulatory isoform of LHCII. The role of LHCB1 phosphorylation is less obvious; however, it has an impact in vivo allowing a partial adaptive response also in absence of LHCB2. Thanks to the CRISPR/Cas9 technique, we produced multiple mutants for the clustered genes coding for LHCB1 and LHCB2, thus allowing the production of complete null mutants for these two LHCII isoforms. These mutant lines constitute an ideal platform to study the impact of targeted modifications on the LHCII network via the production of complemented lines. LHCB1 is the most abundant isoform of LHCII and, consequently, a multiple mutation of the five genes encoding this protein results in a pale phenotype, reduced PSII antenna cross-section, altered thylakoid structure along with lower Photosystem I over Photosystem II reaction center ratio. Interestingly, the loss of one of these two major isoforms results in compensatory effects at the phosphorylation level of the remaining. Loss of LHCB1 results in a de-phosphorylation of the remaining LHCB2, while loss of LHCB2 results in an over-phosphorylation of LHCB1. The complete knock out plants for both LHCB1 and LHCB2 were tested under prolonged fluctuating light, moderate temperature stress and their combination. This revealed an increased susceptibility to only the combined stress for the complete LHCB1 knock out, visible as a clear growth delay, combined with a decrease in the photosynthetic efficiency. Surprisingly, the loss of LHCB2, which impairs the antenna re-allocation between the two photosystems, did not result in any major defect under the combined stress condition. Modification of the threonine of the phosphorylation site to alanine (non phosphorylable) or aspartate (constitutive negative charge "phospho-mimic») for both LHCB1 and LHCB2 reveals the impact of such irreversible modification on photosynthetic acclimation and on the dynamics of the photosynthetic complexes. We demonstrated that the complete removal of LHCB2 protein or the substitution of its phosphorylation site by alanine or aspartate largely, result in a physiologically overlapping phenotype with sharp reduction of state transitions and decreased LHCII-PSI-LHCI supercomplex formation. These defects result in slower acclimation to fluctuating light. Our results show that only with the phospho-threonine group LHCB2 can fully accomplish state transitions and that the negative charge of the aspartate substitution has no impact on short-term photosynthetic acclimation. Disentangling the defined role of each antenna isoform, LHCB1 and LHCB2, could shed light in short and long term acclimatory processes. Leading to a better comprehension on how each isoform contributes to LHCII network organization and results in an optimal balance between light capture and photoprotection. The multiple null lines produced during this project are a milestone along this path and open future perspectives towards the design of innovative LHCII complementation studies.