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Homeostasis of the essential micronutrient manganese (Mn) is crucially determined through availability and uptake efficiency in all organisms. Mn deficiency of plants especially occurs in alkaline and calcareous soils, seriously restricting crop yield. However, the mechanisms underlying the sensing and signaling of Mn availability and conferring regulation of Mn uptake await elucidation. Here, we uncover that Mn depletion triggers spatiotemporally defined long-lasting Ca

Manganese (Mn) is an essential micronutrient for all living organisms. However, excess supply of Mn as occurring in acid or waterlogged soils has toxic effects on plant physiology and development. Although a variety of Mn transporter families has been characterized, our understanding of how these transporters are regulated to uphold and adjust Mn homeostasis in plants remains rudimentary. Here, we identify two calcineurin-B-like proteins, CBL2/3, and their interacting kinases, CIPK3/9/26, as key regulators for plant Mn homeostasis. We found that Arabidopsis mutants lacking CBL2 and 3 or their interacting protein kinases CIPK3/9/26 exhibit remarkable high-Mn tolerance. Intriguingly, CIPK3/9/26 interact with and phosphorylate the tonoplast-localized Mn and iron (Fe) transporter MTP8 primarily at Ser35, which is conserved among MTP8 proteins from various species. Mn transport complementation assays in yeast combined with multiple physiological assays indicate that CBL-CIPK-mediated phosphorylation of MTP8 negatively regulates its transport activity from the cytoplasm to the vacuole. Moreover, we report that sequential phosphorylation of MTP8 initially at Ser31/32 by the calcium-dependent protein kinases CPK5 and subsequently by CIPK26 at Ser35 provides an activation/deactivation fine-tuning mechanism for differential Mn transport regulation. Collectively, our studies define a two-tiered calcium-controlled mechanism for dynamically orchestrating Mn homeostasis under conditions of fluctuating Mn supply.

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, transcriptional and post-transcriptional modifications 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, that interact 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 the Ser31 and Ser32 residues. Transport assays combined with multiple physiological experiments on phospho-dead variant MTP8S31/32A and phospho-mimetic variant MTP8S31/32D plants under different Mn and Fe conditions suggested that 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 that 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, providing potential targets for engineering heavy metal toxicity-tolerant plants.

Nitrogen (N) is a limiting nutrient for plant growth and productivity. The phytohormone abscisic acid (ABA) has been suggested to play a vital role in nitrate uptake in fluctuating N environments. However, the molecular mechanisms underlying the involvement of ABA in N deficiency responses are largely unknown. In this study, we demonstrated that ABA signaling components, particularly the three subclass III SUCROSE NON-FERMENTING1 (SNF1)-RELATED PROTEIN KINASE 2S (SnRK2) proteins, function in root foraging and uptake of nitrate under N deficiency in Arabidopsis thaliana. The snrk2.2snrk2.3snrk2.6 triple mutant grew a longer primary root and had a higher rate of nitrate influx and accumulation compared with wild-type plants under nitrate deficiency. Strikingly, SnRK2.2/2.3/2.6 proteins interacted with and phosphorylated the nitrate transceptor NITRATE TRANSPORTER1.1 (NRT1.1) in vitro and in vivo. The phosphorylation of NRT1.1 by SnRK2s resulted in a significant decrease of nitrate uptake and impairment of root growth. Moreover, we identified NRT1.1Ser585 as a previously unknown functional site: the phosphomimetic NRT1.1S585D was impaired in both low- and high-affinity transport activities. Taken together, our findings provide new insight into how plants fine-tune growth via ABA signaling under N deficiency.