Molecular Mechanism Of The Ca²⁺-CPK21/23-NRAMP1 Module Response To Mn Deficiency In Arabidopsis Thaliana

Manganese（Mn）is an essential micronutrient for plant growth and development and plays a pivotal role in photosynthesis,protein and lipid synthesis,enzyme activity regulation and oxidative stress responses.Soil p H is the most critical factor in soil Mn availability.In alkaline and calcareous soils,high soil p H causes a decrease on Mn absorbed and utilized in plants,leading to Mn deficiency in plants,severely limiting crop yield and quality.However,there are still few researches on the regulation of Mn uptake and utilization,limiting the potential application of techniques such as gene editing to enhance Mn efficiency in crops.Therefore,in-depth studies on the molecular mechanisms of Mn uptake,transport,and utilization in plants are crucial to improve crop yield and quality.Ca²⁺is one of the most important second messengers in eukaryotic cells.It has been a hot spot but a challenge for phycological research.Calcium-dependent protein kinase（CPK）,a specific class of Ca²⁺receptors,respond to and transfer Ca²⁺signals by phosphorylating downstream substrates.However,studies on whether Ca²⁺signaling is involved in Mn deficiency and how it regulates Mn deficiency are still unknown and need to be solved.To address these core scientific issues,we uncovered that Mn deficiency excites long-lasting Ca²⁺signaling oscillations in Arabidopsis roots and proposes a theoretical model for the regulation of Mn deficiency by the Ca²⁺-CPK21/23-NRAMP1 module through using a series of experimental techniques in plant physiology,cell biology,biochemistry,molecular biology and genetics.The main findings of the thesis are as follows:1.To investigate whether Mn deficiency can induce Ca²⁺signaling in plants,we first measured Ca²⁺signaling in plants using the optimized Ca²⁺indicator GCa MP6f-m Cherry and found that Mn deficiency induced a long-lasting Ca²⁺oscillations signal in Arabidopsis roots,and the Ca²⁺signals were mainly concentrated in the elongation zone of Arabidopsis roots and were most strongly expressed in the pericycle region of the roots.Intriguingly,unlike Mn deficiency,high Mn stress only induced a short-term Ca²⁺signal in Arabidopsis roots.2.In view of NRAMP1 is the key Mn transporter for plant Mn uptake and Mn deficiency can excite long-lasting Ca²⁺signaling.We performed Bi FC experiments to screen which CPK family members interact with NRAMP1 and found that CPK21/23 interacted with NRAMP1.Further screening by reverse genetics phenotype analysis revealed that only the cpk21/23double mutant was significantly sensitive to Mn deficiency,display a significant reduction in root length,fresh weight,and Mn concentration in shoot.GUS staining experiments and subcellular localization analysis further demonstrated that CPK21 and CPK23 share the same expression pattern to NRAMP1.3.To further confirm the interaction between CPK21/23 and NRAMP1,we investigated the protein interaction by LCI,Pull-down and Co-IP assays.LCI experiments in tobacco leaves revealed that CPK21/23 interacted with NRAMP1 in vitro.Pull-down assay demonstrated that the NRAMP1-N/C interacted with CPK21/23 directly in vitro.Co-IP assay proved that NRAMP1 interacted with CPK21/23 in vivo through transgenic lines.4.CPK,as a protein kinase,transfer Ca²⁺signals by phosphorylating downstream substrates.We found that CPK21/23 phosphorylated the NRAMP1-N/C and mainly phosphorylated the S20 and T498 sites through phosphorylation assay in vitro,and Mn deficiency enhanced the phosphorylation of NRAMP1 by CPK21/23.In addition,the T498site was found crucial for NRAMP1 transport activity by phenotypic assays,yeast transport activity assays and phosphorylation assays in vivo.5.Genetic analysis was performed by hybridizing the CPK23 overexpression line and nramp1.Then the phenotype analysis of transgenic lines under Mn deficiency displayed a sensitive phenotype with significantly reduced root length,consisting with nramp1.This demonstrates that CPK21/23 act upstream of NRAMP1 under Mn deficiency.Based on the above main findings,we propose a theoretical model of the Ca²⁺-CPK21/23-NRAMP1 functional module in response to Mn deficiency.We found that Mn deficiency can stimulate Ca²⁺signaling in plants,elucidated the biological functions of CPK family in response to Mn deficiency.Systematically revealed the molecular mechanism respond to Mn deficiency by CPK21/23 through phosphorylation the plasma membrane-localized Mn transporter NRAMP1,resolved the molecular process and key site of Mn transport activity on NRAMP1,and broadened the Ca²⁺signaling pathway.It lays theoretical foundation and technical support for the use of NRAMPs family to create crop varieties with super-enrichment of beneficial heavy metals and low accumulation of toxic heavy metals,and provided molecular targets and new ideas to solve the problem of Mn deficiency in alkaline soils in China.