Mitochondrial and cytosolic calcium dynamics are differentially regulated in plants.

The role of mitochondrial calcium in plant cell signaling has received little attention due to the technical difficulties in measuring changes in the mitochondrial free calcium concentration ([Ca ]m) in vivo. Here, we describe the unprecedented use of targeted aequorin to produce stably transformed Arabidopsis plants that both enable the analysis of mitochondrial calcium dynamics in planta and reveal independent regulation of [Ca ]m. The ability to monitor changes in cytoplasmic free Ca concentration ([Ca ]c) in planta using recombinant aequorin has provided new insights into our understanding of plant Ca dynamics (Knight et al., 1991, 1992, 1995; Knight and Knight, 1995). This technique has been refined to offer increased resolution of cellular Ca dynamics by targeting aequorin to specific subcellular locations within the cell. Aequorin has been successfully targeted in this way to the nucleus (Brini et al., 1993), endoplasmic reticulum (Kendall et al., 1992), and mitochondria (Rizzuto et al., 1992) of mammalian cells and to the nucleus (Van der Luit et al., 1999), chloroplasts (Johnson et al., 1995), and the tonoplast (Knight et al., 1996) of plants. This paper describes the first successful targeting of aequorin to plant mitochondria, to our knowledge, and the data presented provide the first direct measurements of the elevations in [Ca ]m resulting from physiological or environmental stimuli. The mitochondrial F1-ATPase -subunit of tobacco (Nicotiana plumbaginifolia) is nuclear encoded and contains an N-terminal extension that is cleaved upon import. Boutry et al. (1987) demonstrated that a 90-amino acid N-terminal segment of the -ATPase could efficiently target the chloramphenicol acetyl transferase protein into mitochondria of transgenic plants. Subsequent work by Boutry and colleagues demonstrated that although the first 23 amino acids of the -ATPase subunit was sufficient for targeting reporter proteins into tobacco mitochondria, the import efficiency increased significantly when 60 or 90 amino acid segments were used (Chaumont et al., 1994). In a previous study, efficient targeting of green fluorescent protein (GFP) to Arabidopsis mitochondria was achieved by engineering a chimeric construct in which the sequence encoding the first 87 amino acids of the tobacco -ATPase was ligated upstream of the mGFP5 cDNA (Logan and Leaver, 2000). To both target aequorin to mitochondria and to allow confirmation of correct targeting by fluorescence microscopy, we engineered a chimeric construct comprising the aequorin cDNA downstream of the -ATPase-mGFP5 cDNA to express a GFP-AEQ fusion protein in the mitochondria. The aequorin and mGFP5 cDNA chimera was PCR amplified from pGF-Aeq (C.A. Moore and M.R. Knight, unpublished data) with primers to add SpeI and SacI restriction sites at the 5 and 3 ends, respectively (using the primers 5 -GCCGAAACTAGTAAAGGAGAA-3 and 5 -GCGCGAGCTCGCAGAGTTTCTTAGGGGACA-3 ; restriction sites underlined). This allowed the directional cloning of the AEQ-GFP sequence, downstream of the cDNA sequence encoding the ATPase N-terminal segment, into pBIN121 (Jefferson et al., 1987) in place of the GUS cDNA. Independent plasmid clones containing the -ATPase-GFP-aequorin chimera (pBINAGA) were sequenced to confirm that the full chimera was in-frame. The construct was introduced into Arabidopsis Columbia-0 ecotype by Agrobacterium tumefaciens-mediated floral dip transformation (Clough and Bent, 1998), and Arabidopsis transformants were selected by growth on kanamycin. Analysis of positive transformants by epifluorescence microscopy demonstrated that this construct was stably inherited and correctly targeted to the mitochondria (Fig. 1). The -ATPase-GFP-aequorin fusion enabled specific measurement of the [Ca ]m in living plants and comparison with cytosolic free calcium concentration ([Ca ]c; Fig. 2). In vivo reconstitution of aequorin and Ca measurements were performed as described previously (Campbell, 1988; Knight et al., 1991, 1996). Arabidopsis plants (Columbia-0 background) expressing recombinant aequorin in the cytosol were engineered as described previously (Knight et al., 1991). The GFP-aequorin fusion protein behaves identically to unfused aequorin because 1 This work was supported by the Biotechnology and Biological Sciences Research Council. * Corresponding author; e-mail [email protected]; fax 44 –1334 – 463366. Article, publication date, and citation information can be found at www.plantphysiol.org/cgi/doi/10.1104/pp.103.026047.