The EF-Hand Ca²⁺ Binding Protein MICU Choreographs Mitochondrial Ca²⁺ Dynamics in Arabidopsis

Abstract

Plant organelle function must constantly adjust to environmental conditions, which requires dynamic coordination. Ca 2+ signaling may play a central role in this process. Free Ca 2+ dynamics are tightly regulated and differ markedly between the cytosol, plastid stroma, and mitochondrial matrix. The mechanistic basis of compartment-specific Ca 2+ dynamics is poorly understood. Here, we studied the function of At-MICU, an EF-hand protein of Arabidopsis thaliana with homology to constituents of the mitochondrial Ca 2+ uniporter machinery in mammals. MICU binds Ca 2+ and localizes to the mitochondria in Arabidopsis. In vivo imaging of roots expressing a genetically encoded Ca 2+ sensor in the mitochondrial matrix revealed that lack of MICU increased resting concentrations of free Ca 2+ in the matrix. Furthermore, Ca 2+ elevations triggered by auxin and extracellular ATP occurred more rapidly and reached higher maximal concentrations in the mitochondria of micu mutants, whereas cytosolic Ca 2+ signatures remained unchanged. These findings support the idea that a conserved uniporter system, with composition and regulation distinct from the mammalian machinery, mediates mitochondrial Ca 2+ uptake in plants under in vivo conditions. They further suggest that MICU acts as a throttle that controls Ca 2+ uptake by moderating influx, thereby shaping Ca 2+ signatures in the matrix and preserving mitochondrial homeostasis. Our results open the door to genetic dissection of mitochondrial Ca 2+ signaling in plants.

Chloroplast-Specific in Vivo Ca2+ Imaging Using Yellow Cameleon Fluorescent Protein Sensors Reveals Organelle-Autonomous Ca2+ Signatures in the Stroma

“…2H; see Supplemental Discussion). This is opposite to the mitochondrial matrix where YC3.6 consistently indicates higher basal Ca 2+ levels (Wagner et al, 2015b) and in line with what has been observed for the relative concentrations of stromal versus cytosolic Ca 2+ in aequorin-based measurements (Sai and Johnson, 2002). Furthermore, the transient amplitude was more pronounced in the cytosol than in the plastid stroma, and the maximum 2Bam4-YC3.6 FRET-ratio of the stroma reached only slightly above the corresponding steady-state FRET ratio of the cytosol (Fig.…”

“…Extracellular ATP (eATP) acts as a signaling molecule involved in plant development and stress responses Tanaka et al, 2014;Choi et al, 2014a). eATP administration to root tip cells has been reported to trigger a cytosolic Ca 2+ transient in both aequorin expressing seedlings (Tanaka et al, 2010) and Cameleon (Tanaka et al, 2010;Krebs et al, 2012;Loro et al, 2012) with a consequent Ca 2+ accumulation in intracellular compartments including the mitochondria and the ER (Loro et al, 2012;Bonza et al, 2013;Wagner et al, 2015b). Specifically, in the root tip, eATP is initially perceived by the cells of the meristematic zone (lateral root cap cells), and afterward a Ca 2+ wave travels from this region to the elongation zone (Tanaka et al, 2010;Loro et al, 2012;Costa et al, 2013).…”

Mitochondrial Energy Signaling and Its Role in the Low-Oxygen Stress Response of Plants

“…In both mammals and plants, a uniporter complex is involved in mitochondrial Ca 2+ import (Kamer and Mootha, 2015;Wagner et al, 2015;Teardo et al, 2017), which makes use of the mitochondrial membrane potential. Ca 2+ transients in the mitochondrial matrix have been shown to occur in response to various abiotic stresses (Logan and Knight, 2003;Loro et al, 2012) that also activate an MRR.…”

“…Hypoxia has been suggested to drive Ca 2+ extrusion from mitochondrial stores in cultured maize cells (Subbaiah et al, 1998), but supporting experimental data are scarce. Conceptually, the existence of such Ca 2+ stores in the matrix under nonpathological conditions requires further scrutiny, considering a minor gradient in free Ca 2+ concentration (about 100 nM in the cytosol versus 200 nM in the matrix at baseline; Wagner et al, 2015). Considering the highly negative potential across the inner mitochondrial membrane, this strongly favors uptake rather than release of Ca…”

Techniques for the analysis of protein-protein interactions in vivo

“…One reason for the popularity of FRET is that, in addition to its use for detecting PPIs, it also can be used with genetically encoded, unimolecular sensors, and it can be used in native species in vivo (Frommer et al, 2009;Uslu and Grossmann, 2016). A range of sensor molecules have been developed or optimized for use in plants, enabling the detection of calcium (Krebs et al, 2012;Wagner et al, 2015), phosphate (Mukherjee et al, 2015), zinc (Lanquar et al, 2014), and abscisic acid (Jones et al, 2014a) as well as physiochemical states of the cell such as pH (Michard et al, 2008) and membrane voltage . One great advantage of using intramolecular FRET sensors lies in the theoretically equal stoichiometry, rendering ratiometric readouts independent of sensor concentration.…”