Ca²⁺ 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 Ca²⁺ signatures and their encoding and decoding machinery. Such studies have frequently focused on Ca²⁺ alone and our understanding of how Ca²⁺ 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 Ca²⁺ 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-Ca²⁺ link for distinct stimuli. Our data suggest that the association with pH may be a general feature of Ca²⁺ transients that depends on the transient characteristics and the intracellular compartment. These findings suggest a fundamental link between Ca²⁺ and pH dynamics in plant cells, generalizing previous observations of their association in growing pollen tubes and root hairs. Ca²⁺ 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. Ca²⁺ signaling may play a central role in this process. Free Ca²⁺ dynamics are tightly regulated and differ markedly between the cytosol, plastid stroma, and mitochondrial matrix. The mechanistic basis of compartment-specific Ca²⁺ 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 Ca²⁺ uniporter machinery in mammals. MICU binds Ca²⁺ and localizes to the mitochondria in Arabidopsis. In vivo imaging of roots expressing a genetically encoded Ca²⁺ sensor in the mitochondrial matrix revealed that lack of MICU increased resting concentrations of free Ca²⁺ in the matrix. Furthermore, Ca²⁺ elevations triggered by auxin and extracellular ATP occurred more rapidly and reached higher maximal concentrations in the mitochondria of micu mutants, whereas cytosolic Ca²⁺ signatures remained unchanged. These findings support the idea that a conserved uniporter system, with composition and regulation distinct from the mammalian machinery, mediates mitochondrial Ca²⁺ uptake in plants under in vivo conditions. They further suggest that MICU acts as a throttle that controls Ca²⁺ uptake by moderating influx, thereby shaping Ca²⁺ signatures in the matrix and preserving mitochondrial homeostasis. Our results open the door to genetic dissection of mitochondrial Ca²⁺ signaling in plants.

Plants expressing a chloroplast-localized Cameleon Ca2+ probe allow single-organelle analysis of chloroplast Ca2+ dynamics.

An Arabidopsis homolog of the mammalian mitochondrial calcium uniporter MCU acts as Ca2+ channel, and its absence impacts mitochondrial function and morphology.

Signaling cross talks between auxin, a regulator of plant development, and Ca²⁺, a universal second messenger, have been proposed to modulate developmental plasticity in plants. However, the underlying molecular mechanisms are largely unknown. Here, we report that in Arabidopsis roots, auxin elicits specific Ca²⁺ signaling patterns that spatially coincide with the expression pattern of auxin-regulated genes. We have identified the single EF-hand Ca²⁺-binding protein Ca²⁺-dependent modulator of ICR1 (CMI1) as an interactor of the Rho of plants (ROP) effector interactor of constitutively active ROP (ICR1). CMI1 expression is directly up-regulated by auxin, whereas the loss of function of CMI1 associates with the repression of auxin-induced Ca²⁺ increases in the lateral root cap and vasculature, indicating that CMI1 represses early auxin responses. In agreement, cmi1 mutants display an increased auxin response including shorter primary roots, longer root hairs, longer hypocotyls, and altered lateral root formation. Binding to ICR1 affects subcellular localization of CMI1 and its function. The interaction between CMI1 and ICR1 is Ca²⁺-dependent and involves a conserved hydrophobic pocket in CMI1 and calmodulin binding-like domain in ICR1. Remarkably, CMI1 is monomeric in solution and in vitro changes its secondary structure at cellular resting Ca²⁺ concentrations ranging between 10⁻⁹ and 10⁻⁸ M. Hence, CMI1 is a Ca²⁺-dependent transducer of auxin-regulated gene expression, which can function in a cell-specific fashion at steady-state as well as at elevated cellular Ca²⁺ levels to regulate auxin responses.

Plants encrypt the perception of different pathogenic stimuli into specific intracellular calcium (Ca²⁺) signatures and subsequently decrypt the signatures into appropriate downstream responses through various Ca²⁺ sensors. Two microbe-associated molecular patterns (MAMPs), bacterial flg22 and fungal chitin, and one damage-associated molecular pattern (DAMP), AtPep1, were used to study the differential Ca²⁺ signatures in Arabidopsis leaves. The results revealed that flg22, chitin, and AtPep1 induced distinct changes in Ca²⁺ dynamics in both the cytosol and nucleus. In addition, Flg22 and chitin upregulated the expression of salicylic acid-related genes, ICS1 and EDS1, whereas AtPep1 upregulated the expression of jasmonic acid-related genes, JAZ1 and PDF1.2, in addition to ICS1 and EDS1. These data demonstrated that distinct Ca²⁺ signatures caused by different molecular patterns in leaf cells lead to specific downstream events. Furthermore, these changes in the expression of defense-related genes were disrupted in a knockout mutant of the AtSR1/CAMTA3 gene, encoding a calmodulin-binding transcription factor, in which a calmodulin-binding domain on AtSR1 was required for deciphering the Ca²⁺ signatures into downstream transcription events. These observations extend our knowledge regarding unique and intrinsic roles for Ca²⁺ signaling in launching and fine-tuning plant immune response, which are mediated by the AtSR1/CAMTA3 transcription factor.