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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Slow anion channels (SLAC/SLAH) are efflux channels previously shown to be critical for stomatal regulation. However, detailed analysis using the β-glucuronidase reporter gene showed that members of the SLAC/SLAH gene family are predominantly expressed in roots, in addition to stomatal guard cells, implicating distinct function(s) of SLAC/SLAH in the roots. Comprehensive mutant analyses of all slac/slah mutants indicated that slah3 plants showed a greater growth defect than wild-type plants when ammonium was supplied as the sole nitrogen source. Ammonium toxicity was mimicked by acidic pH in nitrogen-free external medium, suggesting that medium acidification by ammonium-fed plants may underlie ammonium toxicity. Interestingly, such toxicity was more severe in slah3 mutants and, particularly in wild-type plants, was alleviated by supplementing the media with micromolar levels of nitrate. These data thus provide evidence that SLAH3, a nitrate efflux channel, plays a role in nitrate-dependent alleviation of ammonium toxicity in plants.