Quantification of energy-converting protein complexes in plant thylakoid membranes.

Knowledge about the exact abundance and ratio of photosynthetic protein complexes in thylakoid membranes is central to understanding structure-function relationships in energy conversion. Recent modeling approaches for studying light harvesting and electron transport reactions rely on quantitative information on the constituent complexes in thylakoid membranes. Over the last decades several quantitative methods have been established and refined, enabling precise stoichiometric information on the five main energy-converting building blocks in the thylakoid membrane: Light-harvesting complex II (LHCII), Photosystem II (PSII), Photosystem I (PSI), cytochrome b6f complex (cyt b6f complex), and ATPase. This paper summarizes a few quantitative spectroscopic and biochemical methods that are currently available for quantification of plant thylakoid protein complexes. Two new methods are presented for quantification of LHCII and the cyt b6f complex, which agree well with established methods. In addition, recent improvements in mass spectrometry (MS) allow deeper compositional information on thylakoid membranes. The comparison between mass spectrometric and more classical protein quantification methods shows similar quantities of complexes, confirming the potential of thylakoid protein complex quantification by MS. The quantitative information on PSII, PSI, and LHCII reveal that about one third of LHCII must be associated with PSI for a balanced light energy absorption by the two photosystems.

Physcomitrium patens Mutants in Auxin Conjugating GH3 Proteins Show Salt Stress Tolerance but Auxin Homeostasis Is Not Involved in Regulation of Oxidative Stress Factors.

Salt stress is among the most challenging abiotic stress situations that a plant can experience. High salt levels do not only occur in areas with obvious salty water, but also during drought periods where salt accumulates in the soil. The moss Physcomitrium patens became a model for studying abiotic stress in non-vascular plants. Here, we show that high salt concentrations can be tolerated in vitro, and that auxin homeostasis is connected to the performance of P. patens under these stress conditions. The auxin levels can be regulated by conjugating IAA to amino acids by two members of the family of GH3 protein auxin amino acid-synthetases that are present in P. patens. Double GH3 gene knock-out mutants were more tolerant to high salt concentrations. Furthermore, free IAA levels were differentially altered during the time points investigated. Since, among the mutant lines, an increase in IAA on at least one NaCl concentration tested was observed, we treated wild type (WT) plants concomitantly with NaCl and IAA. This experiment showed that the salt tolerance to 100 mM NaCl together with 1 and 10 µM IAA was enhanced during the earlier time points. This is an additional indication that the high IAA levels in the double GH3-KO lines could be responsible for survival in high salt conditions. While the high salt concentrations induced several selected stress metabolites including phenols, flavonoids, and enzymes such as peroxidase and superoxide dismutase, the GH3-KO genotype did not generally participate in this upregulation. While we showed that the GH3 double KO mutants were more tolerant of high (250 mM) NaCl concentrations, the altered auxin homeostasis was not directly involved in the upregulation of stress metabolites.

The structural and functional domains of plant thylakoid membranes.

In plants, the stacking of part of the photosynthetic thylakoid membrane generates two main subcompartments: the stacked grana core and unstacked stroma lamellae. However, a third distinct domain, the grana margin, has been postulated but its structural and functional identity remains elusive. Here, an optimized thylakoid fragmentation procedure combined with detailed ultrastructural, biochemical, and functional analyses reveals the distinct composition of grana margins. It is enriched with lipids, cytochrome b6 f complex, and ATPase while depleted in photosystems and light-harvesting complexes. A quantitative method is introduced that is based on Blue Native Polyacrylamide Gel Electrophoresis (BN-PAGE) and dot immunoblotting for quantifying various photosystem II (PSII) assembly forms in different thylakoid subcompartments. The results indicate that the grana margin functions as a degradation and disassembly zone for photodamaged PSII. In contrast, the stacked grana core region contains fully assembled and functional PSII holocomplexes. The stroma lamellae, finally, contain monomeric PSII as well as a significant fraction of dimeric holocomplexes that identify this membrane area as the PSII repair zone. This structural organization and the heterogeneous PSII distribution support the idea that the stacking of thylakoid membranes leads to a division of labor that establishes distinct membrane areas with specific functions.