Alternative splicing-mediated targeting of the plant glutamate receptor AtGLR3.5 to mitochondria affects organelle morphology

Since the discovery of 20 genes encoding for putative ionotropic glutamate receptors (iGLRs) in the Arabidopsis genome, there has been considerable interest in uncovering their physiological functions. For many of these receptors neither their channel formation and/or physiological roles nor their localization within the plant cells is known. Here, we provide new information about in vivo protein localization and give insight into the biological roles of the so far uncharacterized AtGLR3.5, a member of subfamily 3 of plant glutamate receptors. Using the pGREAT vector designed for expression of fusion proteins in plants, we show that a splicing variant of AtGLR3.5 targets the inner mitochondrial membrane, while the other variant localizes to chloroplasts. Mitochondria of knockout or silenced plants showed a strikingly altered ultrastructure, lack of cristae and swelling. Furthermore, using a genetically encoded mitochondria-targeted calcium probe, we measured a slightly reduced mitochondrial calcium uptake capacity in the KO mutant. These observations indicate a functional expression of AtGLR3.5 in this organelle. Furthermore, AtGLR3.5-less mutant plants undergo anticipated senescence. Our data thus represent the first evidence of splicing-regulated, organellar targeting of a plant ion channel and they identify the first cation channel in plant mitochondria from a molecular point of view.