Journal of Microscopy, Vol. 249, Pt 1 2013, pp. 8–12 doi: 10.1111/j.1365-2818.2012.03683.x Received 5 July 2012; accepted 10 October 2012 The D3cpv Cameleon reports Ca 2+ dynamics in plant mitochondria with similar kinetics of the YC3.6 Cameleon, but with a lower sensitivity G. LORO ∗ , †, C. RUBERTI ∗ , M. ZOTTINI ∗ & A. COSTA † ∗ Dipartimento di Biologia, Universit` a degli Studi di Padova, Via U. Bassi, Padova, Italia †Dipartimento di Bioscienze, Universit` a degli Studi di Milano, Via G. Celoria, Milano, Italia Key words. Cameleon, Ca 2+ imaging, FRET, plant mitochondria. Summary Mitochondria are key organelles involved in many aspects of plant physiology and, their ability to generate specific Ca 2+ signatures in response to abiotic and biotic stimuli has been reported as one of their roles. The recent identification of the mammalian mitochondrial Ca 2+ uniporter opens a new research area in plant biology. To study the mitochondrial Ca 2+ handling, it is essential to have a reliable probe. Here we have reported the generation of an Arabidopsis transgenic line expressing the genetically encoded probe Cameleon D3cpv targeted to mitochondria, and compared its properties with the already known Cameleon YC3.6. Introduction Mitochondria are key organelles involved in many aspects of the eukaryotic cell functions, ranging from cell metabolism to stress response and programmed cell death regulation (McAinsh & Pittman, 2009; Contreras et al., 2010). Several studies carried out in mammalian cells, have revealed the ability of mitochondria to accumulate Ca 2+ upon specific stimuli (Drago et al., 2011 and reference therein). Moreover, the molecular identity of the so-called ‘mitochondrial Ca 2+ uniporter’, the inner membrane channel responsible for the mitochondrial Ca 2+ accumulation in mammalian cells, has been recently identified by two independent groups (Baughman et al., 2011; De Stefani et al., 2011). Interestingly, six predicted isoforms of this channel, with different tissue specificities, have been identified in Arabidopsis (Stael et al., 2012). To study the mitochondrial Ca 2+ handling in plant cells in vivo, we recently generated transgenic Arabidopsis plants Correspondence to: Alex Costa, Dipartimento di Bioscienze, Universit` a degli Studi di Milano, Via G. Celoria 26, 20133 Milano, Italy. Tel: +39-02-50314831; fax: +39-02-50314815; e-mail: alex.costa@unimi.it expressing the genetically encoded Ca 2+ probe Cameleon YC3.6 targeted to the mitochondria (Loro et al., 2012). The YC3.6 Cameleon probe efficiently reported mitochondrial Ca 2+ dynamics in response to different stimuli in guard and root cells (Loro et al., 2012), and it was chosen for its reported high dynamic range (changes in Ca 2+ concentration are efficiently transduced) and Ca 2+ affinity (K D for Ca 2+ is 0.25 μM in vitro; refs. Nagai et al., 2004; Palmer & Tsien, 2006). It was very recently reported that the Arabidopsis AtCML30 gene codes for a calmodulin (CaM)-like protein localized in mitochondria (Chigri et al., 2012) and that this gene is ubiquitously expressed throughout the entire plant life cycle, as indicated by published microarray data (Schmid et al., 2005, http://jsp.weigelworld.org/expviz/expviz.jsp). The presence of this mitochondrial CaM-like protein might potentially affect the Cameleon response, as reported for some of the original YCs (Yellow Cameleon), which for example failed to report Ca 2+ variations when targeted to plasma membrane (Heim & Griesbeck, 2004), where CaM can reach mM concentrations (Palmer et al., 2006). Because of this, Palmer et al. (2006) developed a new family of Cameleons, called Dcpv. Briefly, the Dcpv family derived from the classical Cameleons in which the two green fluorescent protein (GFP) variants, cyan fluorescent protein (CFP) and cpVenus (cpv; a yellow fluorescent protein (YFP) circularly permuted variant), are linked together through a mutated CaM and M13 peptide to abolish or strongly reduce the interference from endogenous CaM (Palmer et al., 2006). As in the classical Cameleon, in the Dcpv family, the Ca 2+ binding induces the conformational change of CaM and its binding to M13. The consequently reduced distance between CFP and cpv results in an increase in the fluorescence resonance energy transfer (FRET). FRET (and thus the [Ca 2+ ]) increase can be conveniently measured by the increase of the ratio between the emission intensities of cpv and CFP, respectively, upon CFP excitation. Analyses of mitochondrial Ca 2+ dynamics, carried out with the YC3.6 C  2012 The Authors Journal of Microscopy C  2012 Royal Microscopical Society