Macro-organization of photosynthetic complexes during salt stress acclimation of green microalgae and in plant chloroplast acetyltransferase and Ser/Thr protein kinase mutants

During my doctoral work, my aim was to study the changes that occurred as a result of salt stress i) in the structure and function of the photosynthetic apparatus of Chlamydomonas reinhardtii (C. reinhardtii) ii) in the acclimatization reactions of Euglena gracilis (E. gracilis), and iii) to explore the role of protein acetylation/phosphorylation in Arabidopsis thaliana (A. thaliana) in the membrane dynamics of the photosynthetic apparatus. Salt stress had a negative effect on the growth and chlorophyll content of E. gracilis cells, as well as on the macro-organization of thylakoid membranes (TM), but had no effect on photosynthetic processes and the composition of photosynthetic complexes. At the same time, the carotenoid-chlorophyll ratio and the biotechnologically significant paramylon content increased. Accumulation of storage polysaccharide, changes in pigment composition and TM organization can help in the acclimation of E. gracilis cells to salt stress. We investigated the effect of salt stress on C. reinhardtii wild type and two stt7 and pgrl1 mutant strains. We showed that salt stress affected the repeat distance of TM; on the organization and functioning of pigment-protein complexes. The increased sensitivity of the stt7 mutant to salt stress suggests that photosynthetic state transitions play a key role in acclimation to salt stress. We investigated the role of chloroplast acetyltransferase (GNAT2) and STN7 kinase in the organization of A. thaliana TM. CD spectroscopic studies showed differences in the structure and arrangement of the PSII-LHCII supercomplex and/or LHCII in both gnat2 and stn7 mutants compared to the wild type. These results confirmed that GNAT2, like the STN7 kinase, is involved in shaping TM macroorganization. In general, it can be said that the dynamic organization of the membrane plays a key role in the stress adaptation of photosynthetic organisms, in which the post-translational modification of proteins may also play a role

Chloroplast Acetyltransferase GNAT2 is Involved in the Organization and Dynamics of Thylakoid Structure

Higher plants acclimate to changes in light conditions by adjusting the thylakoid membrane ultrastructure. Additionally, excitation energy transfer between photosystem II (PSII) and photosystem I (PSI) is balanced in a process known as state transition. These modifications are mediated by reversible phosphorylation of Lhcb1 and Lhcb2 proteins in different pools of light-harvesting complex (LHCII) trimers. Our recent study demonstrated that chloroplast acetyltransferase NUCLEAR SHUTTLE INTERACTING (NSI)/GNAT2 (general control non-repressible 5 (GCN5)-related N-acetyltransferase 2) is also needed for the regulation of light harvesting, evidenced by the inability of the gnat2 mutant to perform state transitions although there are no defects in LHCII phosphorylation. Here, we show that despite contrasting phosphorylation states of LHCII, grana packing in the gnat2 and state transition 7 (stn7) mutants possesses similar features, as the thylakoid structure of the mutants does not respond to the shift from darkness to light, which is in striking contrast to wild type (Wt). Circular dichroism and native polyacrylamide gel electrophoresis analyses further revealed that the thylakoid protein complex organization of gnat2 and stn7 resembles each other, but differ from that of Wt. Also, the location of the phosphorylated Lhcb2 as well as the LHCII antenna within the thylakoid network in gnat2 mutant is different from that of Wt. In gnat2, the LHCII antenna remains largely in grana stacks, where the phosphorylated Lhcb2 is found in all LHCII trimer pools, including those associated with PSII. These results indicate that in addition to phosphorylation-mediated regulation through STN7, the GNAT2 enzyme is involved in the organization and dynamics of thylakoid structure, probably through the regulation of chloroplast protein acetylation.</p

Chloroplast Acetyltransferase GNAT2 is Involved in the Organization and Dynamics of Thylakoid Structure

Higher plants acclimate to changes in light conditions by adjusting the thylakoid membrane ultrastructure. Additionally, excitation energy transfer between photosystem II (PSII) and photosystem I (PSI) is balanced in a process known as state transition. These modifications are mediated by reversible phosphorylation of Lhcb1 and Lhcb2 proteins in different pools of light-harvesting complex (LHCII) trimers. Our recent study demonstrated that chloroplast acetyltransferase NUCLEAR SHUTTLE INTERACTING (NSI)/GNAT2 (general control non-repressible 5 (GCN5)-related N-acetyltransferase 2) is also needed for the regulation of light harvesting, evidenced by the inability of the gnat2 mutant to perform state transitions although there are no defects in LHCII phosphorylation. Here, we show that despite contrasting phosphorylation states of LHCII, grana packing in the gnat2 and state transition 7 (stn7) mutants possesses similar features, as the thylakoid structure of the mutants does not respond to the shift from darkness to light, which is in striking contrast to wild type (Wt). Circular dichroism and native polyacrylamide gel electrophoresis analyses further revealed that the thylakoid protein complex organization of gnat2 and stn7 resembles each other, but differ from that of Wt. Also, the location of the phosphorylated Lhcb2 as well as the LHCII antenna within the thylakoid network in gnat2 mutant is different from that of Wt. In gnat2, the LHCII antenna remains largely in grana stacks, where the phosphorylated Lhcb2 is found in all LHCII trimer pools, including those associated with PSII. These results indicate that in addition to phosphorylation-mediated regulation through STN7, the GNAT2 enzyme is involved in the organization and dynamics of thylakoid structure, probably through the regulation of chloroplast protein acetylation

Salt Stress Induces Paramylon Accumulation and Fine-Tuning of the Macro-Organization of Thylakoid Membranes in Euglena gracilis Cells

The effects of salt stress condition on the growth, morphology, photosynthetic performance, and paramylon content were examined in the mixotrophic, unicellular, flagellate Euglena gracilis. We found that salt stress negatively influenced cell growth, accompanied by a decrease in chlorophyll (Chl) content. Circular dichroism (CD) spectroscopy revealed the changes in the macro-organization of pigment-protein complexes due to salt treatment, while the small-angle neutron scattering (SANS) investigations suggested a reduction in the thylakoid stacking, an effect confirmed by the transmission electron microscopy (TEM). At the same time, the analysis of the thylakoid membrane complexes using native-polyacrylamide gel electrophoresis (PAGE) revealed no significant change in the composition of supercomplexes of the photosynthetic apparatus. Salt stress did not substantially affect the photosynthetic activity, as reflected by the fact that Chl fluorescence yield, electron transport rate (ETR), and energy transfer between the photosystems did not change considerably in the salt-grown cells. We have observed notable increases in the carotenoid-to-Chl ratio and the accumulation of paramylon in the salt-treated cells. We propose that the accumulation of storage polysaccharides and changes in the pigment composition and thylakoid membrane organization help the adaptation of E. gracilis cells to salt stress and contribute to the maintenance of cellular processes under stress conditions

Structural Entities Associated with Different Lipid Phases of Plant Thylakoid Membranes—Selective Susceptibilities to Different Lipases and Proteases

It is well established that plant thylakoid membranes (TMs), in addition to a bilayer, contain two isotropic lipid phases and an inverted hexagonal (HII) phase. To elucidate the origin of non-bilayer lipid phases, we recorded the 31P-NMR spectra of isolated spinach plastoglobuli and TMs and tested their susceptibilities to lipases and proteases; the structural and functional characteristics of TMs were monitored using biophysical techniques and CN-PAGE. Phospholipase-A1 gradually destroyed all 31P-NMR-detectable lipid phases of isolated TMs, but the weak signal of isolated plastoglobuli was not affected. Parallel with the destabilization of their lamellar phase, TMs lost their impermeability; other effects, mainly on Photosystem-II, lagged behind the destruction of the original phases. Wheat-germ lipase selectively eliminated the isotropic phases but exerted little or no effect on the structural and functional parameters of TMs—indicating that the isotropic phases are located outside the protein-rich regions and might be involved in membrane fusion. Trypsin and Proteinase K selectively suppressed the HII phase—suggesting that a large fraction of TM lipids encapsulate stroma-side proteins or polypeptides. We conclude that—in line with the Dynamic Exchange Model—the non-bilayer lipid phases of TMs are found in subdomains separated from but interconnected with the bilayer accommodating the main components of the photosynthetic machinery