| Abstract |
The photosynthetic apparatus in oxygenic photosynthesis consists of four major complexes (photosystem I, photosystem II, cytochrome b6f and ATPase). The vectorial integration of these complexes in the thylakoid membrane makes possible the transport of electrons across the membrane and the release of protons in lumen. The thylakoid membrane is energized through proton accumulation and ATP is formed though the pmf-driven ATPase rotation. Hence, steady state photosynthetic electron transport is coupled to ATP synthesis. When light saturates photosynthesis the reaction centers are under excitation pressure (especially PSII) and major photoprotective mechanisms are activated (non photochemical quenching and state transition quenching). Sustainable autoregulation of the photosynthetic apparatus maximizes quantum yield and increases photophotection. This work has focused on the role of polyamines (putrescine, Put; spermidine, Spd; and spermine, Spm) during biogenesis and functional assembly of the photosynthetic apparatus. They are ubiquitous low molecular weight aleiphatic amines with 2, 3 and 4 amino moieties, respectively. Polyamines are considered as hormone-like molecules although the underlying mechanism is not known. Due to their elevated levels during stress, they are corelated with tolerance of cells/organisms. Knowledge concerning the relationship between polyamines and the photosynthetic apparatus is rather fragmentary. During chloroplast biogenesis the plastidal levels of polyamines are reduced within hours to a new set point but the reason is not well understood. The missing underlying molecular mechanism of polyamine action in the photosynthetic apparatus was both the major obstacle and the main subject of this study. In this work a top-down approach through an in vivo functional monitoring of the biogenesis of the photosynthetic apparatus, allowed a better understanding of the role of polyamines. Further investigation of photosynthesis regulatory mechanisms, the use of the mutants C-2A΄ and Wt-LHC of Scenedesmus obliquus and in vitro experiments allowed the proposal of two novel molecular mechanisms concerning polyamine actions. Results are presented in four chapters. In the 1rst chapter comparative analysis of light dependent and light independent biogenesis of the photosynthetic apparatus revealed why down-regulation of polyamines is a prerequisite of this developmental stage. Namely, a key process like chlorophyll biosynthesis and the subsequent assemply of chlorophyll binding proteins (photosystems and light harvesting complexes) are hindered by high levels of polyamines (>2mM Spm), because both light dependent protochlorophyllide oxidoreductase (LPOR) and light independent protochlorophyllide oxidoreductase (DPOR) are inhibited. In addition, use of the mutant C- 2A΄ made possible to reveal a novel phase of photosynthetic activation. Namely, the already known light independent chloroplast biogenesis reach a functional threshold of photosynthetic efficiency (unknown stable intermediate phase of suboptimum efficiency, Fv/Fm=0.31). Hence, the crucial distinction between intact-active and intact-inactive is suggested. Activation is possible through distinct events that among others include a 50% increase of Spm bound to LHC of PSII, a dramatic increase of oligomerization degree of LHC monomers (from 10% to 90%) and an optimization of photosynthetic efficiency (from 0.31 to more than 0.70). This phase lasts only 300 s when light intensity is high enough (1000 μmolquanta.m-2s-1). Studies with the mutant Wt-LHC demonstrated that polyamines are involved in the regulation of light energy utilization. A detailed study (2nd chapter) of the regulation of photosynthesis indicated that Put has different effects than Spd and Spm. The common action of Spd and Spm in functional terms, distinct from the role of Put was verified by in vitro studies (3rd chapter). Put treatment inhibits early NPQ rise, indicating inactivation of PsbS and alters membrane relaxation kinetics (delays membrane de-energization). Interestingly, Spd- and Spm-treatment caused an up-regulation of the energy dissipated nonradiatively as heat by the photosynthetic apparatus (350% and 420% respectively) and also induced a sustainable form of non photochemical quenching. The details of the underlying molecular mechanism were revealed by isolation of the thylakoid membranes and subsequent isolation of the native monomers of light harvesting complex of PSII. Namely, it was found that the iminogroup(s) of Spd and Spm were responsible for the quenching of chlorophyll fluorescence. Furthermore, activation of the quenching mechanism is possible through a pHdependent step (protonation of a secondary amine group of polyamines). Put was unequivocally excluded as a quencher and Spd and Spm were the first in vivo and in vitro quenchers of chlorophyll fluorescence. The evaluation of these results allowed the proposal of a novel mechanism of non-photochemical quenching. The traits of the proposed mechanism (activation through protonation, inactivation through deprotonation, direct interaction between polyamines and light harvesting complex) improve our understanding concerning qE without conflicting with the xanthophyll cycle model, that is already correlated with NPQ. The latter is more slow process due to the enzymatic step that requires, than the proposed protonation of the iminogroup of polyamines and thus it might have a role in stabilizing and enhancing quenching. In line with these results, it was demonstrated that in LHC-lacking mutants (Wt-LHC) polyamines can not participate in energy dissipation. Additionally, a beneficial role of Spm in activation of reaction centers of PSII was shown. In the 4th chapter this was confirmed in isolated chloroplasts (Spm increase 55% Fv, mainly due to activation of PSIIα reaction centers) further studied with low temperature (77K) spectroscopy. Activation of reaction centers is probably possible through a functional assembly of CP43 with PSII core. Moreover, experiments under low salt media revealed the cationic effects of polyamines in the photosynthetic apparatus. Polyamines proved more efficient than inorganic cations which are normally found in chloroplast and a detailed study (concerning linear electron flow, PSII activity, overall efficiency to name a few) is illustrated. This study allowed the in-depth analysis of polyamine bionergetic role in photophosphorylation, attributed to non-cationic action. Put was proved as an efficient stimulator of photophosphorylation (provoked up to 80% increase). Furthermore, small increase or decrease of Put levels (in the range of 0-1.5 mM) leads to subsequent up or down regulation of chemiosmotic ATP synthesis, suggesting that for the same light energy input plants have a mechanism to increase profit by fine tuning levels of Put. The effect of Put treatment on relaxation kinetics of thylakoid membrane energization, the simulation of in vivo conditions in in vitro experiments and Put biochemical properties indicate, that ion trapping could be responsible for the stimulation mechanism. The two proposed mechanisms are in line with the increase of polyamines during stress and they may exlain why inability for rise leads to sensitivity.
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