Characterization of chloroplast phosphoproteins controlling manganese use efficiency using quantitative proteomics

Manganese is important for molecular functions in plants, i.e. as a co-factor in enzymes and in the oxygen evolving complex of photosystem II, located like most of the photosynthetic machinery, in the thylakoid membranes of chloroplasts. Soils that lack plant available micronutrients such as manganese are generally not suitable for crop production. Fortunately, various plant genotypes differ in their ability to grow in soil with low amounts of micronutrients, providing an opportunity to identify strains that tolerate low manganese levels.
We want to identify and quantified phosphoproteins involved in manganese use efficiency, focusing on the phosphoproteome from thylakoid preparations from two barley genotypes, manganese efficient (Vanessa) and inefficient (Antonia) genotype.
Experimental: By monitoring the photosynthetic efficiency (Fv/Fm) a decline in activity is observed as a consequence of manganese deficiency. Barley leaves will be harvested at three specific (Fv/Fm) values after manganese depletion, and again after resupply of manganese. Protein from the purified thylakoids will be digested using a modified spin filter-based protocol that includes trypsin digestion in 0.5 % SDC that enables better protein solubilization. Phosphopeptides will be purified using titaniumdioxide beads. Peptides will be analyzed and label free quantified using a 1D-LC UPLC system coupled to an Orbitrap Velos mass spectrometer.