The Photoprotective Protein PsbS from Green Microalga Lobosphaera incisa: The Amino Acid Sequence, 3D Structure and Probable pH-Sensitive Residues.

PsbS is one of the key photoprotective proteins, ensuring the tolerance of the photosynthetic apparatus (PSA) of a plant to abrupt changes in irradiance. Being a component of photosystem II, it provides the formation of quenching centers for excited states of chlorophyll in the photosynthetic antenna with an excess of light energy. The signal for "turning on" the photoprotective function of the protein is an excessive decrease in pH in the thylakoid lumen occurring when all the absorbed light energy (stored in the form of transmembrane proton potential) cannot be used for carbon assimilation. Hence, lumen-exposed protonatable amino acid residues that could serve as pH sensors are the essential components of PsbS-dependent photoprotection, and their pKa values are necessary to describe it. Previously, calculations of the lumen-exposed protonatable residue pKa values in PsbS from spinach were described in the literature. However, it has recently become clear that PsbS, although typical of higher plants and charophytes, can also provide photoprotection in green algae. Namely, the stress-induced expression of PsbS was recently shown for two green microalgae species: Chlamydomonas reinhardtii and Lobosphaera incisa. Therefore, we determined the amino acid sequence and modeled the three-dimensional structure of the PsbS from L. incisa, as well as calculated the pKa values of its lumen-exposed protonatable residues. Despite significant differences in amino acid sequence, proteins from L. incisa and Spinacia oleracea have similar three-dimensional structures. Along with the other differences, one of the two pH-sensing glutamates in PsbS from S. oleracea (namely, Glu-173) has no analogue in L. incisa protein. Moreover, there are only four glutamate residues in the lumenal region of the L. incisa protein, while there are eight glutamates in S. oleracea. However, our calculations show that, despite the relative deficiency in protonatable residues, at least two residues of L. incisa PsbS can be considered probable pH sensors: Glu-87 and Lys-196.

Chilling Upregulates Expression of the PsbS and LhcSR Genes in the Chloroplasts of the Green Microalga Lobosphaera incisa IPPAS C-2047.

Non-photochemical quenching (NPQ) of excited chlorophyll states is essential for protecting the photosynthetic apparatus (PSA) from the excessive light-induced damage in all groups of oxygenic photosynthetic organisms. The key component of the NPQ mechanism in green algae and some other groups of algae and mosses is the LhcSR protein of the light harvesting complex (LHC) protein superfamily. In vascular plants, LhcSR is replaced by PsbS, another member of the LHC superfamily and a subunit of photosystem II (PSII). PsbS also performs the photoprotective function in mosses. For a long time, PsbS had been believed to be nonfunctional in green algae, although the corresponding gene was discovered in the genome of these organisms. The first evidence of the PsbS accumulation in the model green alga Chlamydomonas reinhardtii in response to the increase in irradiance was obtained only six years ago. However, the observed increase in the PsbS content was short-termed (on an hour-timescale). Here, we report a significant (more than three orders of magnitude) and prolonged (four days) upregulation of PsbS expression in response to the chilling-induced high-light stress followed by a less significant (~ tenfold) increase in the PsbS expression for nine days. This is the first evidence for the long-term upregulation of the PsbS expression in green alga (Chlorophyta) in response to stress. Our data indicate that the role of PsbS in the PSA of Chlorophyta is not limited to the first-line defense against stress, as it was previously assumed, but includes full-scale participation in the photoprotection of PSA from the environmental stress factors.

The Effect of Chilling on the Photosynthetic Apparatus of Microalga Lobosphaera incisa IPPAS C-2047.

Photosynthetic organisms have developed a set of mechanisms aimed at preventing photo-oxidative reactions in the photosynthetic apparatus (PSA) initiated by excessively absorbed light energy. Along with high irradiance, other stressors, e.g., chilling temperatures, can lead to the absorption of the excess of light energy and hence to photo-oxidative stress. Here, we studied induction of photoprotective mechanisms in response to chilling (0°C) at a low irradiance (50 µmol PAR photons m-2·s-1) in the cells of microalga Lobosphaera incisa IPPAS C-2047. After 4 days of incubation at a low temperature, L. incisa IPPAS C-2047 cells showed a notable decrease in the photochemical activity of photosystem II (PSII) and in the efficiency of photosynthetic electron transport, as well as a significant increase in the thermal dissipation of the absorbed light energy in the light-harvesting antenna. In contrast, most conventional markers of PSA acclimation to excess light energy [total chlorophyll and carotenoid content; violaxanthin cycle pigment content and de-epoxidation state; photosynthetic antenna, PSII, and photosystem I (PSI) ratio] remained virtually unchanged. The content of major unsaturated fatty acids also remained almost unaffected, except for arachidonic acid (increased by 40%) recently assumed to activate violaxanthin de-epoxidase by adjusting its lipid microenvironment. Significant changes (4-7-fold increase) were observed in the expression of the gene encoding protective protein LhcSR. Pre-conditioning at 5°C prior to the acclimation to 0°C augmented the PSA photochemical activity. Our data show that the mid-term (4-d) acclimation of L. incisa IPPAS C-2047 to a chilling temperature at a low irradiance triggers the PSA response resembling, in part, the response to high light but relying mostly on the LhcSR protein-dependent quenching of excitation in the photosynthetic antenna.