CBL1/9–CIPK23–NRAMP1 axis regulates manganese toxicity

Abstract

Summary Manganese (Mn) is an essential micronutrient in plants. However, excessive Mn absorption in acidic soils can cause Mn toxicity, which adversely affects plant growth and crop yields. At present, acidic soils cover c. 30% of the Earth's surface. However, the mechanism underpinning Mn uptake remains largely unknown. We identified cbl1/9 and cipk23 mutants exhibiting high‐Mn‐sensitive phenotype through the reverse genetics method. Furthermore, we identified the CIPK23 phosphorylated NRAMP1 through a variety of protein interaction techniques and protein kinase assays. Here, we demonstrated that two calcineurin B‐like proteins, CBL1/9, and their interacting kinase CIPK23 positively regulated the tolerance of Mn toxicity in Arabidopsis. The cbl1 cbl9 double mutant and cipk23 mutants exhibited high‐Mn‐sensitive phenotypes, which manifested as decreased primary root length, biomass, and chlorophyll concentration, and higher accumulation of Mn. In addition, CIPK23 interacted with and phosphorylated the Mn transporter NRAMP1 primarily at Ser20/22 in vitro and in vivo, and thereby induced clathrin‐mediated endocytosis of NRAMP1 to reduce its distribution on the plasma membrane and enhance plant tolerance to Mn toxicity. In summary, we found that the CBL1/9–CIPK23–NRAMP1 module regulates the tolerance to high‐Mn toxicity and provide insight into a mechanism of the tolerance of plants to Mn toxicity.

Phosphorylation by CIPK23 regulates the high‐affinity Mn transporter NRAMP1 in Arabidopsis

“…Striking is the fact that CIPK23 seems to regulate the two major Mn transporters at the plasma membrane NRAMP1 and IRT1 ([10,28] and the present work). This attests to the central role of this kinase in the control of Mn entry within the cell.…”

“…This attests to the central role of this kinase in the control of Mn entry within the cell. We and others [10,28] have shown that cipk23 and cbl1 cbl9 are sensitive to Mn excess confirming their incapacity to control Mn entry in the plant. Given that NRAMP1 and IRT1 contribute to Mn acquisition probably with different affinities, it would be interesting to further investigate the contribution of each of these transporters in the Mn hypersensitive phenotype of the mutants and more generally in the cellular response to Mn excess.…”

“…Similar to what we describe here, Zhang et al . [28] have recently also reported NRAMP1 phosphorylation by CIPK23 on Ser20. Unlike our study however, they could show that Mn‐induced internalization of NRAMP1‐GFP is abolished in cipk23 and cbl1 cbl9 mutant backgrounds.…”

“…In addition to Ser20, we identified Ser499 as the main target of CIPK23 in the C‐terminal domain of NRAMP1. Interestingly, next to Ser499, Thr498 was shown to enhance NRAMP1 activity when phosphorylated by the kinases CPK21/CPK23 [13] and was reported recently as also phosphorylated by CIPK23 [28]. If the exact function of Ser499 phosphorylation is not yet known, two hypotheses can be proposed: Ser499 phosphorylation could (a) act in synergy with Thr498 phosphorylation and further enhance NRAMP1 activity or (b) prevent Thr498 phosphorylation and downregulate NRAMP1 activity.…”

TaNRAMP3 is essential for manganese transport in Triticum aestivum

“…AtNRAMP3 and AtNRAMP4 are located on the vacuole membrane and are responsible for releasing Mn or Fe from the vacuole (Lanquar et al 2005(Lanquar et al , 2010. AtNRAMP6, located on the PM, plays an important role in the detoxification of cadmium (Cd) (Cailliatte et al 2009;Zhang et al 2023b). In rice, OsNRAMP5 is located at the far end of the outer and endodermis cells, and is the main transporter of Mn and Cd, responsible for transporting Mn and Cd from the external solution to the root cells (Sasaki et al 2012;Ueno et al 2015).…”