Comparative analysis of MAMP-induced calcium influx in arabidopsis seedlings and protoplasts

Abstract

Rapid transient elevation of cytoplasmic calcium (Ca2+) levels in plant cells is an early signaling event triggered by many environmental cues including abiotic and biotic stresses. Cellular Ca2+ levels and their alterations can be monitored by genetically encoded reporter systems such as the bioluminescent protein, aequorin. Employment of proteinaceous Ca2+ sensors is usually performed in transgenic lines that constitutively express the reporter construct. Such settings limit the usage of these Ca2+ biosensors to particular reporter variants and plant genetic backgrounds, which can be a severe constraint in genetic pathway analysis. Here we systematically explored the potential of Arabidopsis thaliana leaf mesophyll protoplasts, either derived from a transgenic apoaequorin-expressing line or transfected with apoaequorin reporter constructs, as a complementary biological resource to monitor cytoplasmic changes of Ca2+ levels in response to various biotic stress elicitors. We tested a range of endogenous and pathogenderived elicitors in seedlings and protoplasts of the corresponding apoaequorin-expressing reporter line. We found that the protoplast system largely reflects the Ca2+ signatures seen in intact transgenic seedlings. Results of inhibitor experiments including the calculation of IC50 values indicated that the protoplast system is also suitable for pharmacological studies. Moreover, analyses of Ca2+signatures in mutant backgrounds, genetic complementation of the mutant phenotypes and expression of sensor variants targeted to different subcellular localizations can be readily performed. Thus, in addition to the prevalent use of seedlings, the leaf mesophyll protoplast setup represents a versatile and convenient tool for the analysis of Ca2+ signaling pathways in plant cells.