Although Mg2+ is essential for a myriad of cellular processes, high levels of Mg 2+ in the environment, such as those found in serpentine soils, become toxic to plants. In this study, we identified two calcineurin B-like (CBL) proteins, CBL2 and CBL3, as key regulators for plant growth under high-Mg conditions. The Arabidopsis mutant lacking both CBL2 and CBL3 displayed severe growth retardation in the presence of excess Mg 2+ , implying elevated Mg 2+ toxicity in these plants. Unexpectedly, the cbl2 cbl3 mutant plants retained lower Mg content than wild-type plants under either normal or high-Mg conditions, suggesting that CBL2 and CBL3 may be required for vacuolar Mg 2+ sequestration. Indeed, patch-clamp analysis showed that the cbl2 cbl3 mutant exhibited reduced Mg 2+ influx into the vacuole. We further identified four CBL-interacting protein kinases (CIPKs), CIPK3, -9, -23, and -26, as functionally overlapping components downstream of CBL2/3 in the signaling pathway that facilitates Mg 2+ homeostasis. The cipk3 cipk9 cipk23 cipk26 quadruple mutant, like the cbl2 cbl3 double mutant, was hypersensitive to high-Mg conditions; furthermore, CIPK3/9/23/26 physically interacted with CBL2/3 at the vacuolar membrane. Our results thus provide evidence that CBL2/3 and CIPK3/9/23/26 constitute a multivalent interacting network that regulates the vacuolar sequestration of Mg 2+ , thereby protecting plants from Mg 2+ toxicity.magnesium toxicity | calcium sensor | vacuole | magnesium transport P lants absorb essential mineral nutrients from the soil and translocate them to different organs for specific physiological processes. Most of these minerals are in the ionic forms and require a wide array of transporters to move them across the cell membranes and sort them into subcellular compartments (1). Although plants rely on a sufficient supply of mineral nutrients for proper growth and development, an excess of minerals often causes toxicity to plant cells. To adapt to the constantly changing availability of minerals in the environment, plants have evolved mechanisms that enhance ion uptake under low-nutrient conditions and sequester excessive ions in the vacuole when external levels are high. Such mechanisms enable plant cells to maintain a steady level of each nutrient ion, namely, ionic homeostasis. At the molecular level, this homeostasis entails the coordinated functions of a large number of regulatory molecules that constitute elaborate signaling networks to control the affinities and activities of numerous ion transporters. In these signaling networks, Ca 2+ serves as a central messenger (2). A number of external ionic stresses can evoke stimulus-specific cellular Ca signals that are represented by the distinct spatiotemporal patterns of Ca 2+ fluxes between cytosol and Ca 2+ stores (3, 4). These "Ca 2+ signatures" can be detected and relayed into diverse downstream signaling events by plant Ca 2+

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Plant responses to developmental and environmental cues are often mediated by calcium (Ca 2+ ) signals that are transmitted by diverse calcium sensors. The calcineurin B-like (CBL) protein family represents calcium sensors that decode calcium signals through specific interactions with a group of CBL-interacting protein kinases. We report functional analysis of Arabidopsis CBL2 and CBL3, two closely related CBL members that are localized to the vacuolar membrane through the N-terminal tonoplast-targeting sequence. While cbl2 or cbl3 single mutant did not show any phenotypic difference from the wild type, the cbl2 cbl3 double mutant was stunted with leaf tip necrosis, underdeveloped roots, shorter siliques and fewer seeds. These defects were reminiscent of those in the vha-a2 vha-a3 double mutant deficient in vacuolar H + -ATPase (V-ATPase). Indeed, the V-ATPase activity was reduced in the cbl2 cbl3 double mutant, connecting tonoplast CBL-type calcium sensors to the regulation of V-ATPase. Furthermore, cbl2 cbl3 double mutant was compromised in ionic tolerance and micronutrient accumulation, consistent with the defect in V-ATPase activity that has been shown to function in ion compartmentalization. Our results suggest that calcium sensors CBL2 and CBL3 serve as molecular links between calcium signaling and V-ATPase, a central regulator of intracellular ion homeostasis.