Comparative evaluation of extraction methods for apoplastic proteins from maize leaves

The plant apoplast comprises the cell wall matrix and the intercellular spaces, and plays a major role in a wide range of physiological processes, including water and nutrient transport [1], plant-pathogen interactions, and perception and transduction of environmental signals [2,3]. Proteins present in the plant apoplast reflect this broad functional diversity. Studies on the dynamic change of apoplast protein composition revealed new insights into plant responses to abiotic stress [4-7], nutrient supply [8-10], wounding [11], water deficiency [12,13], pathogen response [14-16] and xylem composition [17,18]. The selection of a suitable extraction protocol is a crucial step in proteomics surveys as proteins reveal a high degree of biochemical heterogeneity and investigated plant materials can be characterized by the presence of non-protein components interfering with subsequent analytical techniques, e.g. two-dimensional gel electrophoresis (2-DE) or liquid chromatography-mass spectrometry (LC-MS). These biological realities led to the establishment of sample preparation methods for numerous plant species and tissues, such as Arabidopsis leaves [19], papaya leaves [20], sunflower leaves [21], cotton seedlings [22], apple and strawberry fruit [23], potato tuber [24], grapevine leaves and roots [25], grape berry cell wall [26], rubber latex [27], cotton fibers [28], banana meristem [29] and chloroplast [30], among others. Despite their biological significance, investigations on apoplastic proteins are hampered due to their low abundance compared to intracellular protein concentrations. The extraction of proteins from the leaf and root apoplast is mainly based on the principle of vacuum infiltration with an extraction solution, followed by a mild centrifugation step to collect the apoplastic washing fluid. The composition of the infiltration solution is essential as it has to fulfil certain prerequisites, such as maintenance of osmotic pressure to prevent collapsing of