Ca 2+ play a key role in cell signaling across organisms. The question of how a simple ion can mediate specific outcomes has spurred research into the role of Ca 2+ signatures and their encoding and decoding machinery. Such studies have frequently focused on Ca 2+ alone and our understanding of how Ca 2+ signaling is integrated with other responses is poor. Using in vivo imaging with different genetically encoded fluorescent sensors in Arabidopsis (Arabidopsis thaliana) cells, we show that Ca 2+ transients do not occur in isolation but are accompanied by pH changes in the cytosol. We estimate the degree of cytosolic acidification at up to 0.25 pH units in response to external ATP in seedling root tips. We validated this pH-Ca 2+ link for distinct stimuli. Our data suggest that the association with pH may be a general feature of Ca 2+ transients that depends on the transient characteristics and the intracellular compartment. These findings suggest a fundamental link between Ca 2+ and pH dynamics in plant cells, generalizing previous observations of their association in growing pollen tubes and root hairs. Ca 2+ signatures act in concert with pH signatures, possibly providing an additional layer of cellular signal transduction to tailor signal specificity.

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.

Phosphate (Pi) deficiency strongly limits plant growth, and plant roots foraging the soil for nutrients need to adapt to optimize Pi uptake. Ca 2+ is known to signal in root development and adaptation but has to be tightly controlled, as it is highly toxic to Pi metabolism. Under Pi starvation and the resulting decreased cellular Pi pool, the use of cytosolic free Ca 2+ ([Ca 2+ ] cyt) as a signal transducer may therefore have to be altered. Employing aequorin-expressing Arabidopsis (Arabidopsis thaliana), we show that Pi starvation, but not nitrogen starvation, strongly dampens the [Ca 2+ ] cyt increases evoked by mechanical, salt, osmotic, and oxidative stress as well as by extracellular nucleotides. The altered root [Ca 2+ ] cyt response to extracellular ATP manifests during seedling development under chronic Pi deprivation but can be reversed by Pi resupply. Employing ratiometric imaging, we delineate that Pi-starved roots have a normal response to extracellular ATP at the apex but show a strongly dampened [Ca 2+ ] cyt response in distal parts of the root tip, correlating with high reactive oxygen species levels induced by Pi starvation. Excluding iron, as well as Pi, rescues this altered [Ca 2+ ] cyt response and restores reactive oxygen species levels to those seen under nutrient-replete conditions. These results indicate that, while Pi availability does not seem to be signaled through [Ca 2+ ] cyt , Pi starvation strongly affects stress-induced [Ca 2+ ] cyt signatures. These data reveal how plants can integrate nutritional and environmental cues, adding another layer of complexity to the use of Ca 2+ as a signal transducer.

Arabidopsis thalianaglutamate receptor-like (GLR) channels are amino acid-gated ion channels involved in physiological processes including wound signaling, stomatal regulation, and pollen tube growth. Here, fluorescence microscopy and genetics were used to confirm the central role of GLR3.3 in the amino acid-elicited cytosolic Ca2+increase inArabidopsisseedling roots. To elucidate the binding properties of the receptor, we biochemically reconstituted the GLR3.3 ligand-binding domain (LBD) and analyzed its selectivity profile; our binding experiments revealed the LBD preference forl-Glu but also for sulfur-containing amino acids. Furthermore, we solved the crystal structures of the GLR3.3 LBD in complex with 4 different amino acid ligands, providing a rationale for how the LBD binding site evolved to accommodate diverse amino acids, thus laying the grounds for rational mutagenesis. Last, we inspected the structures of LBDs from nonplant species and generated homology models for other GLR isoforms. Our results establish that GLR3.3 is a receptor endowed with a unique amino acid ligand profile and provide a structural framework for engineering this and other GLR isoforms to investigate their physiology.

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