Ren-Jie Tang, Fu-Geng Zhao, Veder J. Garcia, Thomas J. Kleist, Lei Yang, Hong-Xia Zhang, and Sheng Luan

Significance

Plant growth requires a balanced supply of mineral nutrients. However, the availability of minerals varies constantly in the environment. How do plants adapt to low or high levels of minerals in the soil? The answer to this question holds the key to sustainable crop production. Mg is an essential macronutrient for plants, but high levels of Mg²⁺ can become toxic. This study uncovered a regulatory mechanism, consisting of two calcineurin B-like (CBL) Ca sensors partnering with four CBL-interacting protein kinases (CIPKs) forming a CBL–CIPK network that allows plant cells to sequester the extra Mg²⁺ into vacuoles, thereby protecting plant cells from high-Mg toxicity. To our knowledge, this report is the first that describes such a signaling mechanism for regulation of Mg homeostasis.

Abstract

Although Mg²⁺ is essential for a myriad of cellular processes, high levels of Mg²⁺ 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 Mg²⁺, implying elevated Mg²⁺ 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 Mg²⁺ sequestration. Indeed, patch-clamp analysis showed that the cbl2 cbl3 mutant exhibited reduced Mg²⁺ 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 Mg²⁺ 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 Mg²⁺, thereby protecting plants from Mg²⁺ toxicity.

See: http://www.pnas.org/content/112/10/3134.abstract.html?etoc

PNAS March 10, 2015 vol. 112 no. 10 3134-3139

Fig. 1.

The cbl2 cbl3 double-knockout mutant showed V-ATPase–dependent and –independent ionic sensitivity. (A) Western blot analysis of wild-type Col-0, the cbl2 or cbl3 single mutant, and the cbl2 cbl3 double mutant. CBL2 and CBL3 protein levels were analyzed by immunoblotting using a CBL3 antibody. The amount of β-tubulin was determined in parallel as a loading control. (B–D) Growth phenotype of wild-type Col-0 and cbl2 cbl3 and vha-a2 a3 mutant plants under different ionic stress conditions. Four-day-old seedlings were transferred onto 1/6 MS medium (B) or 1/6 MS medium supplemented with 20 mM CaCl₂ (C) or 4 mM MgCl₂ (D). Photographs were taken on the 18th day after transfer. (E) Fresh weight of seedlings on the 18th day after transfer. Data are presented as the mean ± SE of four replicate experiments. Asterisks indicate statistically significant differences compared with the Col-0 (Student’s t test, *P < 0.05).