Metal Binding in Photosystem II Super- and Subcomplexes from Barley Thylakoids

Abstract

Metals exert important functions in the chloroplast of plants, where they act as cofactors and catalysts in the photosynthetic electron transport chain. In particular, manganese (Mn) has a key function because of its indispensable role in the water-splitting reaction of photosystem II (PSII). More and better knowledge is required on how the various complexes of PSII are affected in response to, for example, nutritional disorders and other environmental stress conditions. We here present, to our knowledge, a new method that allows the analysis of metal binding in intact photosynthetic complexes of barley (Hordeum vulgare) thylakoids. The method is based on size exclusion chromatography coupled to inductively coupled plasma triple-quadrupole mass spectrometry. Proper fractionation of PSII super-and subcomplexes was achieved by critical selection of elution buffers, detergents for protein solubilization, and stabilizers to maintain complex integrity. The applicability of the method was shown by quantification of Mn binding in PSII from thylakoids of two barley genotypes with contrasting Mn efficiency exposed to increasing levels of Mn deficiency. The amount of PSII supercomplexes was drastically reduced in response to Mn deficiency. The Mn efficient genotype bound significantly more Mn per unit of PSII under control and mild Mn deficiency conditions than the inefficient genotype, despite having lower or similar total leaf Mn concentrations. It is concluded that the new method facilitates studies of the internal use of Mn and other biometals in various PSII complexes as well as their relative dynamics according to changes in environmental conditions.

The Evolutionarily Conserved Protein PHOTOSYNTHESIS AFFECTED MUTANT71 is Required for Efficient Manganese Uptake at the Thylakoid Membrane in Arabidopsis

“…The OEC is an inorganic Mn 4 CaO 5 cluster, and several lines of evidence suggest a depletion of Mn 2+ ions in thylakoids of pam71. Manganese deficiency is associated with the reduction in PSII supercomplexes in barley (Hordeum vulgare) (Schmidt et al, 2015), and this feature is readily observed in pam71 (Figures 2A). Second, Mn 2+ ions were differentially distributed, in favor of the stroma, in pam71 chloroplasts during illumination ( Figure 7C).…”

“…Next, the intactness of the inorganic Mn 4 CaO 5 cluster in pam71 was studied by fractionation of thylakoid protein complexes using size-exclusion chromatography (SEC) combined with inductively coupled plasma mass spectrometry (ICP-MS) to analyze the ion intensity of manganese and sulfur (Schmidt et al, 2015). This allowed us to analyze manganese binding in various PSII superand subcomplexes in wild-type and pam71-1 thylakoids.…”

“…This allowed us to analyze manganese binding in various PSII superand subcomplexes in wild-type and pam71-1 thylakoids. Manganese binding to PSII super , PSII dimer , and PSII monomer complexes was assigned according to Schmidt et al (2015) (Figure 3C). A marked decrease in the ion intensity of manganese was observed in pam71-1 samples compared with the wild type ( Figure 3C, left and middle panels).…”

“…(C) Size exclusion profiles were recorded for the nonoxide ion 55 Mn + and the oxide ion 48 SO + in the wild type (left panel) and pam71-1 (middle panel) and given as ion intensities (counts 10 5 s 21 ). Individual 55 Mn + fractions were assigned to PSII dimers/monomers = PSII di/mono and PSII supercomplexes = PSII super according to Schmidt et al (2015). Quantification of the Mn:S stoichiometric ratio (as described in Methods) is shown for a representative sample (right panel) out of five samples derived from independent thylakoid preparations.…”

“…Thylakoid membranes were isolated according to Schmidt et al (2015). Protein concentration was determined using the Pierce BCA Protein Assay PAM71 Is Required for Thylakoid Manganese Uptake(Thermo Fisher Scientific).…”