MCU proteins dominate in vivo mitochondrial Ca2+ uptake in Arabidopsis roots

Abstract

Ca2+ signaling is central to plant development and acclimation. While Ca2+-responsive proteins have been investigated intensely in plants, only a few Ca2+-permeable channels have been identified, and our understanding of how intracellular Ca2+ fluxes are facilitated remains limited. Arabidopsis thaliana homologues of the mammalian channel-forming mitochondrial calcium uniporter (MCU) protein showed Ca2+ transport activity in vitro. Yet, the evolutionary complexity of MCU proteins, as well as reports about alternative systems and unperturbed mitochondrial Ca2+ uptake in knockout lines of MCU genes, leave critical questions about the in vivo functions of the MCU protein family in plants unanswered. Here, we demonstrate that MCU proteins mediate mitochondrial Ca2+ transport in planta and that this mechanism is the major route for fast Ca2+ uptake. Guided by the subcellular localization, expression, and conservation of MCU proteins, we generated an mcu triple knockout line. Using Ca2+ imaging in living root tips and the stimulation of Ca2+ transients of different amplitudes, we demonstrated that mitochondrial Ca2+ uptake became limiting in the triple mutant. The drastic cell physiological phenotype of impaired subcellular Ca2+ transport coincided with deregulated jasmonate-related signaling and thigmomorphogenesis. Our findings establish MCUs as a major mitochondrial Ca2+ entry route in planta and link mitochondrial Ca2+ transport with phytohormone signaling.

Perturbation of mitochondrial Ca2+ homeostasis activates cross-compartmental proteostatic response in Arabidopsis

“…Although both Arabidopsis MCU1 and MCU2 are sufficient to generate mitochondrial Ca 2+ influx when expressed in yeast, their roles in controlling mtCa 2+ responses in planta had not been established 34,35 . A recent study reported the significantly reduced mtCa 2+ basal level and the mitochondrial Ca 2+ uptake in the root cell of Arabidopsis mcu1 2 3 triple mutant 38 , which matches the expression pattern of MCU1, MCU2 and MCU3 in root cells near the root tip. And the residual Ca 2+ uptake activity in the mcu1 2 3 line is likely due to the predominant expression of MCU6 in roots (Supplementary Fig.…”

“…Stress stimuli evoke cytosolic Ca 2+ transient and mitochondrial Ca 2+ uptake in plants 16,17,[36][37][38][39][40][41][42] . To study the function of MCUs in the control of mtCa 2+ homeostasis, we generated transgenic Arabidopsis Col-0 lines in which Ca 2+ reporter Aequorin was targeted to mitochondrial matrix under the control of ubiquitin promoter (Col:mtAq).…”

“…Stress stimulus-specific Ca 2+ dynamics in the chloroplast stroma ( cs Ca 2+ ) are correlated with the transcript levels of MCU6 36 . A recent study reported that mitochondrial Ca 2+ uptake became limiting in the root cell of mcu1 mcu2 mcu3 triple mutant, demonstrating that MCU proteins mediate mitochondrial Ca 2+ uptake in vivo 38 .…”

The integration of reactive oxygen species (ROS) and calcium signalling in abiotic stress responses

“…Mitochondrial Ca 2+ ‐ROS crosstalk might be facilitated by the Ca 2+ signalling kinase CIPK9 and VDAC3 in a phosphorylation‐dependent manner. The MCU complex also participates in Ca 2+ uptake at the inner mitochondrial membrane (Ruberti et al, 2022; Wagner et al, 2015; Wang and Teng, 2018). The MCU complex and VDACs act as ROS sensors during hypoxia and inflammation‐mediated oxidative stress in animals (Dong et al, 2017).…”

Calcium Signaling in Plant-Insect Interactions

“…In plant studies, the bioluminescent protein aequorin (derived from the jellyfish Aequorea victoria ) was first used in 1967 as a genetically encoded Ca 2+ biosensor [ 126 ]. However, despite the use of aequorin to detect Ca 2+ changes in response to various stresses in plants [ 127 ], including temperature [ 128 ], pathogens [ 129 ], salt stress [ 127 ] and wounding [ 130 ] was successful, this system it is not well-suited for real-time imaging due to the extremely low luminescent signal it produces [ 131 ]. Anyway, aequorin-expressing transgenic plants have been used in the study of diverse microbe- or damage-associated molecular patterns (MAMPs/DAMPs) where comparative analyses between the elicitors flg22 and elf18 revealed differences in Ca 2+ signaling and cellobiose exposure which generated an intracellular Ca 2+ elevation [ 129 ].…”