Identification of Key Residues for pH Dependent Activation of Violaxanthin De-Epoxidase from Arabidopsis thaliana

Plants are often exposed to saturating light conditions, which can lead to oxidative stress. The carotenoid zeaxanthin, synthesized from violaxanthin by Violaxanthin De-Epoxidase (VDE) plays a major role in the protection from excess illumination. VDE activation is triggered by a pH reduction in the thylakoids lumen occurring under saturating light. In this work the mechanism of the VDE activation was investigated on a molecular level using multi conformer continuum electrostatic calculations, site directed mutagenesis and molecular dynamics. The pKa values of residues of the inactive VDE were determined to identify target residues that could be implicated in the activation. Five such target residues were investigated closer by site directed mutagenesis, whereas variants in four residues (D98, D117, H168 and D206) caused a reduction in enzymatic activity indicating a role in the activation of VDE while D86 mutants did not show any alteration. The analysis of the VDE sequence showed that the four putative activation residues are all conserved in plants but not in diatoms, explaining why VDE in these algae is already activated at higher pH. Molecular dynamics showed that the VDE structure was coherent at pH 7 with a low amount of water penetrating the hydrophobic barrel. Simulations carried out with the candidate residues locked into their protonated state showed instead an increased amount of water penetrating the barrel and the rupture of the H121–Y214 hydrogen bond at the end of the barrel, which is essential for VDE activation. These results suggest that VDE activation relies on a robust and redundant network, in which the four residues identified in this study play a major role

Intraspecific Variation in Pinus Pinaster PSII Photochemical Efficiency in Response to Winter Stress and Freezing Temperatures

As part of a program to select maritime pine (Pinus pinaster Ait.) genotypes for resistance to low winter temperatures, we examined variation in photosystem II activity by chlorophyll fluorescence. Populations and families within populations from contrasting climates were tested during two consecutive winters through two progeny trials, one located at a continental and xeric site and one at a mesic site with Atlantic influence. We also obtained the LT50, or the temperature that causes 50% damage, by controlled freezing and the subsequent analysis of chlorophyll fluorescence in needles and stems that were collected from populations at the continental trial site

The Hetero-Hexameric Nature of a Chloroplast AAA+ FtsH Protease Contributes to Its Thermodynamic Stability

FtsH is an evolutionary conserved membrane-bound metalloprotease complex. While in most prokaryotes FtsH is encoded by a single gene, multiple FtsH genes are found in eukaryotes. Genetic and biochemical data suggest that the Arabidopsis chloroplast FtsH is a hetero-hexamer. This raises the question why photosynthetic organisms require a heteromeric complex, whereas in most bacteria a homomeric one is sufficient. To gain structural information of the possible complexes, the Arabidopsis FtsH2 (type B) and FtsH5 (type A) were modeled. An in silico study with mixed models of FtsH2/5 suggests that heteromeric hexamer structure with ratio of 4∶2 is more likely to exists. Specifically, calculation of the buried surface area at the interfaces between neighboring subunits revealed that a hetero-complex should be thermodynamically more stable than a homo-hexamer, due to the presence of additional hydrophobic and hydrophilic interactions. To biochemically assess this model, we generated Arabidopsis transgenic plants, expressing epitope-tagged FtsH2 and immuno-purified the protein. Mass-spectrometry analysis showed that FtsH2 is associated with FtsH1, FtsH5 and FtsH8. Interestingly, we found that ‘type B’ subunits (FtsH2 and FtsH8) were 2–3 fold more abundant than ‘type A’ (FtsH1 and FtsH5). The biochemical data corroborate the in silico model and suggest that the thylakoid FtsH hexamer is composed of two ‘type A’ and four ‘type B’ subunits

A substrate-independent, 14:3:3 protein-mediated plastid import pathway of NADPH:protochlorophyllide oxidoreductase A

Plastids are semiautonomous organelles that contain only limited coding information in their own DNA. Because most of their genome was transferred to the nucleus after their endosymbiotic origin, plastids must import the major part of their protein constituents from the cytosol. The exact role of cytosolic targeting factors in the regulation of plastid protein import has not been determined. Here, we report that the nucleus-encoded NADPH:protochlorophyllide (Pchlide) oxidoreductase A plastid precursor (pPORA) can use two different plastid import pathways that differ by the requirements for cytosolic 14:3:3 proteins and Hsp70. pPORA synthesized in a wheat germ lysate segregated into different precursor fractions. While import of free pPORA and only Hsp70-complexed pPORA was Pchlide-dependent and involved the previously identified Pchlide-dependent translocon, 14:3:3 protein- and Hsp70-complexed pPORA was transported into Pchlide-free chloroplasts through the Toc75-containing standard translocon at the outer chloroplast membrane/translocon at the inner chloroplast membrane machinery. A 14:3:3 protein binding site was identified in the mature region of the 35S-pPORA, which governed 14:3:3 protein- and Hsp70-mediated, Pchlide-independent plastid import. Collectively, our results reveal that the import of pPORA into the plastids is tightly regulated and involves different cytosolic targeting factors and plastid envelope translocon complexes