[32] Metabolite levels in specific cells and subcellular compartments of plant leaves

This chapter focuses on the metabolite levels in specific cells and the subcellular compartments of plant leaves. Leaf material is difficult to fractionate because a typical plant cell contains several different, mechanically fragile subcellular organelles and is surrounded by a mechanically strong plant cell wall. Rather than attempting to isolate whole organelles or cells, leaves are frozen in liquid N2 and then broken to small fragments that are enriched in material from a given compartment. These fragments are physically separated under conditions when the metabolic activity or redistribution of metabolites is prevented and subsequently their metabolism is quenched. The exclusion of water, or the use of extremely low temperatures, prevents metabolic activity during the fractionation procedures. The chapter describes the silicone oil centrifugation that allows chloroplasts to be separated from the remainder of the protoplast and to be quenched within 2–3 sec of disrupting the protoplast.