Molecular Mechanism Of Ca²⁺ Signaling Modulates Manganese Homeostasis In Arabidopsis Thaliana

Manganese（Mn）is one of the essential microelements for plant growth and development.Soil pH is the most critical factor affecting the availability of Mn.In alkaline and calcareous soils with elevated pH,plants absorb and use less Mn prone to Mn deficiency.However,in acidic soils with decreased pH,Mn availability increased,and plants absorb excessive Mn,resulting in Mn toxicity.Mn toxicity can cause brown spots,gangrene,and leaf curling symptoms in plants,destroying the normal growth and development of plants,resulting in yield and harvests reduction.At present,about 30%of the world’s soils are acidic soils.With the application of chemical nitrogen fertilizer and acid rain precipitation,soil acidification is further intensified.Mn toxicity has become one of the key factors limiting crop yield and quality in acidic soils.Therefore,it is of great significance to explore the molecular mechanism of uptake,transport,and distribution of Mn in plants,especially the study of Mn nutrient efficiency in alkaline soils and adaptation mechanism in acidic soils,which is of great importance to improve crop yield and quality.Ca²⁺is a key regulator of plant growth,development and stress response.As a unique Ca²⁺sensors in plants,calcineurin-like B protein CBL can form a complex regulatory network with CBL-interacting protein kinase CIPK,decode and transmit Ca²⁺signals by phosphorylation and modification of downstream substrates,and regulate the absorption and utilization of various mineral elements.However,the study of how Ca²⁺signals are generated,sensed,decoded and transmitted in the environment is still elusive,and its molecular mechanism needs to be further explored.To address these core scientific issues,we used a series of experimental techniques in plant physiology,biochemistry,molecular biology,and cell biology.Strikingly,we found that high Mn stress induced an obvious Ca²⁺signature in Arabidopsis root cells,and systematically revealed the functional and molecular mechanisms of the CBL2/3-CIPK3/9/26 module regulating Mn homeostasis.We elucidated the mechanisms of Ca²⁺signaling to fine-tune plant Mn homeostatic biology process and the"gas/brake"mechanism through the tonoplast localized Mn transporter MTP8 phosphorylated differentially by CBL2/3-CIPK/3/9/26 and Ca²⁺dependent protein kinase CPK4/5/6/11.The main results were as follows:（1）Based on fluo-4 AM staining and jellyfish luminescent protein Aequorin methods,we found that high Mn stress promoted[Ca²⁺]_cyt elevation in Arabidopsis root cells and induced an obvious Ca²⁺signature in plants.（2）We pursued a reverse genetics strategy to screen mutants of Arabidopsis CBL and CIPK family members that were significantly sensitive or tolerant to high Mn stress,and found that cbl2 cbl3 double mutant and its interaction proteins cipk3 cipk9 cipk26 triple mutant were significantly tolerant to high Mn stress,showing longer primary root length and increased fresh weight.However,CBL2/3 overexpression lines were significantly sensitive to high Mn stress,which showed that the primary root length was shorter,the fresh weight and chlorophyll content were significantly reduced,and the leaves chlorosis and necrosis.Further studies showed that the Mn content of cbl2 cbl3 and cipk3 cipk9 cipk26 mutants increased significantly in roots while decreased remarkably in shoots under high Mn stress.Genetic analysis showed that CBL2/3 act upstream of MTP8.（3）BiFC,LCI,Pull-down and Co-IP assays showed that CIPK3/9/26 interacted with MTP8.In vitro phosphorylation assays showed that CIPK3/9/26 primarily phosphorylated MTP8 at Ser35.In addition,we used bioinformatics methods to analyze the conservation of phosphorylation sites and found that Ser35 of MTP8 was conserved in dicotyledons.（4）Yeast transport activity,vacuolar Mn content determination and high Mn stress phenotype analysis,showed that CIPK3/9/26-mediated phosphorylation of MTP8^Ser35inhibited MTP8 transport activity and was involved in the regulation of Mn homeostasis in Arabidopsis.Further experiments,combining western blot and tobacco protein expression assays showed that CIPK3/9/26-mediated phosphorylation of MTP8^Ser35 did not affect MTP8protein stability and subcellular localization.（5）In vivo phosphorylation assay,liquid chromatography-tandem mass spectrometry（LC-MS/MS）analysis,protoplast expression,and high Mn stress phenotype analysis displayed that the phosphorylation of MTP8 by CPKs and CIPKs differed in temporal dynamics.CPK4/5/6/11 regulated the initial response to high Mn stress and segregated excess Mn into vacuoles to alleviate Mn toxic effect.In the later stage,to achieve the optimal balance between growth and stress,CBL2/3 recruits CIPK3/9/26 to the vacuolar membrane and phosphorylates MTP8,reducing MTP8 activity to act as a"brake"mechanism.In conclusion,we find that high Mn stress induces Ca²⁺signature in plants,and reveal the function of CBL2/3 and CIPK3/9/26 in response to high Mn stress,elucidate the molecular mechanism of CBL2/3-CIPK3/9/26 module in response to high Mn stress through phosphorylation tonoplast localized Mn transporter MTP8,and clarify the key phosphorylation site of MTP8.We systematically illustrate the biological process and molecular mechanism of differential phosphorylation of MTP8 by CIPK3/9/26 and CPK4/5/6/11 to fine-tune Mn homeostasis in plants.The research results provide a new perspective and molecular target for solving the problem of Mn toxicity in crops in acid soils,and lay a theoretical foundation and technical support for accurately creating new varieties with high concentration of beneficial heavy metals and low accumulation of toxic heavy metals by using MTP protein family.