Efficient isolation of chloroplasts from in vitro shoots of Malus and Prunus

Investigation of cellular subfraction proteomes allows the study of specific changes induced by environmental changes, stress and other conditions. Chloroplasts participate in a huge number of complex biochemical processes in plant cells by retrograde signalling, as well as by sensing and responding to cellular dysfunction. Changes in environmental conditions in a controlled way are easily achieved in in vitro model systems. However, growing plants in vitro makes it difficult to obtain sufficient material for chloroplast isolation. Therefore, the chloroplast isolation method needs to be optimised for achieving sufficient yield from a small amount of sample. We used three species of Rosaceae family that are of high agricultural interest for breeding programs in Lithuania. The method used for chloroplast isolation from Arabidopsis thaliana was optimized for Malus domestica, M. platycarpa and Prunus avium. Homogenisation of 3 g of in vitro plant material in sorbitol-based isolation medium with a laboratory blender yielded a sufficient amount of chloroplasts for proteomic analysis. The purity of the fraction was highly increased by additional step of centrifugation at 200 x g. The purity of chloroplasts was evaluated visually by microscopy, by immunoblotting with specific antibodies, as well as by using marker proteins and quantitative mass spectrometry. Although microscopy showed negligible amounts of cellular debris in all of the preparations, immunoblotting allowed detection of the presence of cytosolic marker in some of the preparations. Mass spectrometric analysis of marker proteins confirmed the presence of modest amount of non-chloroplast proteins. In conclusion, the presented method for chloroplast isolation for the Rosacea plants in vitro gives sufficient yield and purity for subcellular proteomic studies.