A Tonoplast-associated Calcium Signaling Regulates Manganese Homeostasis in Arabidopsis.

Abstract Manganese (Mn) is an essential micronutrient in plants. However, excessive Mn absorption in acidic and waterlogged soils can lead to Mn toxicity. Despite their essential roles in Mn homeostasis, the transcriptional and post-transcriptional modification of Mn transporters remain poorly understood. Here, we demonstrated that high Mn stress induces an obvious Ca2+ signature in Arabidopsis. We identified four calcium-dependent protein kinases, CPK4/5/6/11, interacting with the tonoplast-localized Mn and iron (Fe) transporter MTP8 in vitro and in vivo. The cpk4/5/6/11 quadruple mutant displayed a dramatic high Mn sensitive phenotype similar to that of the mtp8 mutant. CPKs phosphorylated the N-terminal domain of MTP8 primarily at Ser31 and Ser32 residues. Transport assays combined with multiple physiology experiments on phospho-dead variant MTP8S31/32A and phospho-mimetic variant MTP8S31/32D plants under different Mn and Fe conditions suggested Ser31 and Ser32 are crucial for MTP8 function. In addition, genetic analysis showed that CPKs functioned upstream of MTP8. In summary, we identified a tonoplast-associated calcium signaling cascade, which orchestrates Mn homeostasis and links Mn toxicity, Ca2+ signaling and Mn transporters. These findings provide new insight into Mn homeostasis mechanisms and Ca2+ signaling pathways in plants and should be useful in promoting the breeding of heavy metal toxicity-tolerant plants.