Alex Costa, Dipartimento di Bioscienze, Università degli Studi di Milano, Via G. Celoria 26, 20133 Milano, Italy. Tel: +39-02-50314831; fax: +39-02-50314815; e-mail: firstname.lastname@example.org
Mitochondria are key organelles involved in many aspects of plant physiology and, their ability to generate specific Ca2+ signatures in response to abiotic and biotic stimuli has been reported as one of their roles. The recent identification of the mammalian mitochondrial Ca2+ uniporter opens a new research area in plant biology. To study the mitochondrial Ca2+ 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.
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 Ca2+ upon specific stimuli (Drago et al., 2011 and reference therein). Moreover, the molecular identity of the so-called ‘mitochondrial Ca2+ uniporter’, the inner membrane channel responsible for the mitochondrial Ca2+ 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 Ca2+ handling in plant cells in vivo, we recently generated transgenic Arabidopsis plants expressing the genetically encoded Ca2+ probe Cameleon YC3.6 targeted to the mitochondria (Loro et al., 2012). The YC3.6 Cameleon probe efficiently reported mitochondrial Ca2+ 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 Ca2+ concentration are efficiently transduced) and Ca2+ affinity (KD for Ca2+ 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 Ca2+ 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 Ca2+ 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 [Ca2+]) 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 Ca2+ dynamics, carried out with the YC3.6 (Loro et al., 2012), could not provide clues about a possible attenuation, because of the presence of endogenous CaM, of the Cameleon-dependent response. Hence, to evaluate this possibility, in this work we have generated Arabidopsis plants expressing the D3cpv Cameleon probe targeted to mitochondria and compared its functional properties with the previously characterized YC3.6.
Materials and methods
The Arabidopsis thaliana (Columbia ecotype) plants used in this study were grown as previously reported (Loro et al., 2012).
The D3cpv coding sequence was digested from the pcDNA3-D3cpv (Palmer et al., 2006) vector with HindIII and EcoRI restriction enzymes and ligated into the 35S-CaMV cassette vector (http://www.pgreen.ac.uk/JIT/JIT_fr.htm). To generate the 4mt-D3cpv construct, the 4mt targeting peptide was isolated by digestion of pcDNA3–4mt-D1cpv (Zampese et al., 2011) with HindIII and ligated into the 35S-D3cpv linearized vector. The obtained clone was sequenced to verify the right orientation of the targeting peptide and then the entire cassette ‘35S-4mt- D3cpv-Ter’ was PCR amplified by using Phusion DNA Polymerase (Finnzymes, http://www.finnzymes.fi/). For the ‘35S-4mt-D3cpv-Ter’ cassette amplification, we used the following forward and reverse primers: 5′-CATGGGTACCGATATCGTACCCCTACTCCAAAAAT-3′ and 5′-CATGGGTACCGATATCGATCTGGATTTTAGTA-3′, where KpnI restriction sites were introduced at the 5′ and 3′ end. The amplicon of the entire expression cassette was digested with KpnI and ligated in the pGreen0179 binary vector (Hellens et al., 2000). The binary vectors were then introduced in the Agrobacterium tumefaciens GV3101 strain.
The obtained Agrobacterium strain was used to generate transgenic Arabidopsis plants by the floral-dip method (Clough & Bent, 1998). Five Arabidopsis-independent transgenic lines were selected and two independent lines were used for imaging experiments.
Confocal microscopy analyses
Confocal microscopy analyses were performed using a Leica SP5 (Leica, Germany, http://www.leica-microsystems.com) laser scanning confocal imaging system. For Cameleon-dependent-cpVenus, excitation was at 514 nm and emission between 525 and 540 nm. For tetramethylrhodamine methyl ester (TMRM) analysis, the seedlings were stained for 10 min in 5 mM KCl, 10 mM Mes, 10 mM Ca2+ pH 5.8 supplemented with 500 nM TMRM. Seedlings were washed with the same solution and analysed by means of confocal microscopy with excitation set at 543 nm and emission between 590 and 620 nm. For the colocalization analyses of cpVenus and TMRM, sequential excitation in the confocal microscope scanning configuration was adopted. Image analyses were performed by using the ImageJ bundle software (http://rsb.info.nih.gov/ij/).
Root tip Ca2+ imaging
For root cell imaging, the vertically grown 12-day-old seedlings were gently transferred to microscope cover glasses in a drop of the imaging solution. To keep the root in a fixed position during the experiments, a 1% agar block was gently placed over the seedling, and the shoot was not submerged by the solution. For the experiments performed in seedling roots, the solution was 5 mM KCl, 10 mM Mes, 10 mM Ca2+, pH 5.8 adjusted with Tris. ATP was added as disodium salt (Sigma-Aldrich, St. Louis, MO, U.S.A.) to the chamber by perfusion with the same solution supplemented with ATP, which was not removed from the medium throughout the entire experiment. The pH of the solution was readjusted to 5.8 after Adenosine 5'-triphosphate (ATP) addition. Seedling roots expressing the fluorescent probes were imaged in vivo by an inverted fluorescence microscope (Leica DMI6000 B, Germany, http://www.leica-microsystems.com/) with 20x, numerical aperture (NA) 0.5, dry objective. Excitation light was produced by a fluorescent lamp at 440 nm (436/20 nm, dichroic 455DCXR). The light emitted was collected through a beam-splitter (OES s.r.l., Padua, Italy; emission filters ET 480/40 M for CFP and ET 535/30 M for cpVenus) and a dichroic mirror (515 DCXR). Filters and dichroic mirrors were purchased from Omega Optical and Chroma. Images were acquired using a cooled CCD camera (OES s.r.l, Padua, Italy) attached to a 12-bit frame grabber. Exposure time was 500 ms with a 4 × 4 charge-coupled device (CCD) binning. The acquired images were analysed by ImageJ software. As regards time course experiments, fluorescence intensity was determined over regions of interest (ROIs) which correspond to a root region or which cover single or small groups of mitochondria. Regions corresponding to the background for each wavelength were analysed and subtracted from values measured for the different ROIs of the two channels (CFP and cpVenus). The background-subtracted values for the selected ROIs were used for the ratio (R) calculation and normalized to the initial ratio (R0) and plotted versus time (ΔR/R0).
Measurement of D3cpv and YC3.6 dynamic ranges in mitochondria
To determine the in vivo maximum response of the two probes (ΔRmax/R0) within mitochondria, leaf epidermal cells were permeabilized with 0.2 mM digitonin in an intracellular-like medium containing 100 mM potassium-gluconate, 1 mM MgCl2, 10 mM Hepes, pH 7.5 and 5 mM ethylene glycol tetraacetic acid (EGTA) for 4 min. At the end of this period the digitonin was removed and the cells were kept in the same medium with 5 mM EGTA for 5 min first, and then maintained in 1 mM EGTA. To measure the ΔRmax/R0, 10 mM Ca2+ was added to the medium. The ratio measurements were performed looking at single immobile mitochondria with the same microscope acquisition system and parameters used for root cell imaging, but with a 63x, N.A. 1.40 immersion oil objective. The ratios were then averaged and reported ±SE (n= 15 for 4mt-D3cpv and n= 10 for 4mt-YC3.6).
All data are representative of at least 9 analysed different cells or roots. Reported traces are the most representative.
Results and discussion
We have recently reported the use of Cameleon YC3.6 for monitoring of Ca2+ dynamics in plant mitochondria (Loro et al., 2012). Because of the discovery of a mitochondrial localized CaM-like protein (AtCML30) (Chigri et al., 2012), we wondered if its ubiquitous presence (Fig. S1) can affect the mitochondrial Ca2+ measurements in terms of kinetic and response resolution. Thus, we decided to perform a comparison between YC3.6 response properties and D3cpv, the CaM insensitive Cameleon probe (Palmer et al., 2006).
To this aim, we first generated stable Arabidopsis plants expressing the D3cpv (Palmer et al., 2006) targeted to mitochondria. The mitochondrial localization of D3cpv was achieved following the same strategy adopted for YC3.6. We fused to the N-terminal of the probe the mitochondrial targeting sequence from subunit VIII of human cytochrome c oxidase repeated four time (4mt; Palmer et al., 2006; Fig. 1a) and, to ensure the ubiquitous expression in plant, the probe was placed under the control of a single 35S-CaMV promoter (Fig. 1a) in the backbone of pGreen0179 binary vector (Hellens et al., 2000). The construct was then introduced in Arabidopsis by floral dip method (Clough & Bent, 1998), and several independent lines were selected. We confirmed the mitochondrial localization of 4mt-D3cpv probe using the TMRM dye, a cationic potentiometric probe that specifically accumulates in mitochondria with a hyperpolarized inner membrane potential (Figs. 1b–d; Schwarzländer et al., 2012). The sequential confocal microscope acquisitions of cpv and TMRM fluorescences (Fig. 1b–c) showed a clear merge of the two fluorescences in mitochondria of the analysed root cells (Fig. 1d).
To compare the 4mt-D3cpv properties with the 4mt-YC3.6, we first treated Arabidopsis seedling root tips with 2 mM external ATP (eATP) and monitored the mitochondrial Ca2+ accumulation and response kinetics in both transgenic lines. From the traces shown in Figure 2(a), it was evident that both probes allowed the monitoring of [Ca2+]m variations but they reported different maximum ratio changes and recovery times (t/2, 665 s ± 0.26 SE for YC3.6 and 455 s ± 0.52 SE for D3cpv) to the same stimulus, with the D3cpv showing the smaller amplitude and faster recovery compared with the YC3.6 (Fig. 2a). Hence, to better define in vivo the differences between the two probes, we availed ourselves of the observation that root tip cells treated with different eATP concentrations respond in a dose-dependent fashion both in cytoplasm and mitochondria (Loro et al., 2012). We treated the Arabidopsis seedling expressing the 4mt-D3cpv Cameleon probe with 2, 0.5, and 0.01 mM eATP, and compared the results with those previously obtained with the 4mt-YC3.6 plants (Loro et al., 2012). The data thus obtained demonstrate that the 4mt-D3cpv probe (Fig. 2b, grey bars) reports the mitochondrial Ca2+ accumulation in response to all tested concentrations, but with smaller peak amplitudes compared to 4mt-YC3.6 (Fig. 2b, black bars). In particular, the maximum ratio variation of 4mt-D3cpv in response to 0.01 mM eATP was four times smaller than that of 4mt-YC3.6 (Fig. 2b). Besides, the 4mt-D3cpv did not report differences in the [Ca2+]m peak in response to 2 and 0.5 mM eATP (p value < 0.5). We then tested the 4mt-D3cpv response, in plant mitochondria, in permeabilized leaf epidermal cells (Costa et al., 2010; Loro et al., 2012). The mitochondria were first bathed in 1 mM EGTA buffer (to chelate free Ca2+) and then perfused with 5 mM Ca2+. We measured the Cameleon ratios before and after the addition of Ca2+ and calculated the maximum ratio variations in both transgenic lines (Fig. 2c). In this condition (from 0 to 5 mM Ca2+), the maximum mitochondrial Ca2+ accumulation is supposed to occur (Palmer & Tsien, 2006), and therefore the maximum probe/s response/s observed. In the experiment shown in Figure 2(c), the maximum response (ΔRmax/R0) of the 4mt-YC3.6 (1.23 ± 0.051 SE) was double compared to the 4mt-D3cpv (0.575 ± 0.061 S.E.), demonstrating that in mitochondria the latter has a smaller dynamic range. Interestingly, the smaller dynamic range of D3cpv, compared with YC3.6, was previously reported by Hendel et al. (2008) in presynaptic boutons of Drosophila larvae neurons. In their experimental conditions, the in vivo maximum ratio change, in the cytoplasm, was 1.36 for YC3.6 and 0.9 for D3cpv, close to our observations in mitochondria. In the same work, Hendel et al. also reported the in vivo KD for both probes, which was 0.36 μM for YC3.6 and 0.49 μM for D3cpv (Hendel et al., 2008), values close to those measured in vitro (0.25 μM for YC3.6 and 0.6 μM for D3cpv) in the original papers (Nagai et al., 2004; Palmer et al., 2006), but slightly different. The different KD value of the two probes, both in vivo and in vitro, is probably the reason of the lower D3cpv responsiveness. Hence, the different responses we observed between the two analysed probes in mitochondria, seem mainly to depend on the intrinsic probe properties and not to other factor/s, such as endogenous mitochondrial CaM.
In conclusion, the comparison between the two different Cameleon probes here reported demonstrates that both 4mt-YC3.6 and 4mt-D3cpv allow monitoring of mitochondrial Ca2+ dynamics, but the former offers a better sensitivity, due mainly to its higher dynamic range and lower KD. Last but not least, the comparison between these probes allows to assert that the endogenous mitochondrial CaM levels seem not to affect the Ca2+ measurements performed with 4mt-YC3.6 probe, making the latter the preferred one for monitoring Ca2+ dynamics in plant cells.
We thank Dr. Claudio Olivari for critical comments on the paper. Work partially supported by the Italian Ministero dell’Istruzione, dell’Università e della Ricerca (MIUR) through the grant FIRB 2010 (RBFR10S1LJ_001) to A.C.