Molecular Mechanism Of Functional Module Of Ca²⁺-CPK23-PHT1;1 For Regulation Of Arsenic Stress In Arabidopsis Thaliana

Arsenic（As）is a metalloid element with heavy metal properties,commonly found in various types of sulfides and other rock-forming minerals,which can be dissolved by rainwater and eventually penetrate and pollute the groundwater.Irrigating crops with contaminated groundwater leads to a large accumulation of arsenate in cultivated crops.Arsenic from food sources is easy to be accumulated in the human body,and organ damages,cancers and many clinical complications can be triggered through its metabolism.The metabolism of arsenic can also make the accumulation of reactive oxygen species in plants,which seriously affects the growth and development of crops,and endangers the safety of agricultural production.At present,the lack in researches on arsenic signaling pathways limits the potential application of biotechnological means,such as gene editing,to improve crop genetic improvement,which needs to be strengthened.Ca²⁺is an important secondary messenger in plants,which is widely involved in the growth and development of plants and the response to external environment stress.Calcium-dependent protein kinase（CPK）,as a Ca²⁺receptor with kinase activity,can respond to and transmit Ca²⁺signals by phosphorylation of downstream substrates.There are some evidences that arsenic stress can affect calcium signaling pathway and CPK activity,which is still in lack of direct experimental evidence to confirm the direct association between them,while the specific regulatory mechanisms also need to be further explored.For the core scientific problems mentioned above,this study,through a series of plant physiology,cell biology,molecular biology and genetics experimental techniques,found that arsenic stress induced a rapid Ca²⁺signal oscillation in Arabidopsis roots.A protein kinase CPK23 in response to arsenic stress was screened out with its phosphorylated downstream protein,a phosphate transporter PHT1;1 with arsenate transport activity.It was revealed that Ser514 residue on PHT1;1 is an important phosphorylation site which affect its subcellular localization.The molecular mechanism of plant response to arsenic stress and regulation of arsenic absorption through Ca²⁺-CPK23-PHT1;1 signaling pathway was analyzed.The main research results of this paper are as follows:1.Confirmed that arsenic stress could induce a rapid increase in cytoplasmic Ca²⁺concentration in the mature region of Arabidopsis roots by using transgenic lines expressing Ca²⁺reporter proteins YC3.6 and GCa MP6.However,phosphate treatment with the same concentration did not led to significant Ca²⁺signal in root cell.This suggests that plants respond to arsenic stress specifically and such Ca²⁺signaling pathway is independent of the regulatory pathway of nutrient signaling.2.The cpk23 in cpks mutant was screened out to be sensitive in arsenic stress by genetic means.The taproot and leaf growth of cpk23 was most significantly inhibited under arsenic stress,indicating that CPK23 is the major one of the CPKs involved in the construction of Arabidopsis arsenic tolerance.By constructing CPK23 overexpression and kinase constitutively active form mutants and detecting their arsenic-stressed growth phenotypes,we determined that the kinase activity of CPK23 plays a major role in its response to arsenic stress.3.The potential downstream protein PHT1;1 of CPK23 in response to As（V）stress was screened out by IP-MS technique,and the interaction between PHT1;1 and CPK23 was confirmed by Dual membrane system yeast two-hybridization,Bi FC and LCI experiments.Combined with the growth phenotype of the hybrid mutant under arsenic stress,PHT1;1 was identified as the downstream protein of CPK23 in arsenic stress response.On the basis of protein interaction experiments,the result of in vitro phosphorylation experiments confirmed that CPK23 could phosphorylate the Ser514 and Ser520 residues at the C-terminal of PHT1;1,while the result of in vivo phosphorylation experiments further confirmed that the phosphorylation activity of CPK23 at the C-terminal of PHT1;1 was regulated by arsenic stress.4.By constructing PHT1;1 complementary lines in the form of phosphorylation site mutations,Ser514 site was found to be the key active site of PHT1;1.Dephosphorylation at Ser514 led to an increase in arsenic uptake and the significantly arsenic-sensitive phenotypes in plants.Subcellular localization analysis of GFP fused PHT1;1 site mutations showed that phosphorylation at Ser514 could induce plasma membrane delocalization of PHT1;1.Plasma membrane delocalization under arsenic stress.Further subcellular localization analysis of PHT1;1-GFP transgenic lines in wild type and cpk23 knockout mutant under arsenic stress showed that CPK23 was necessary for the plasma membrane delocalization of PHT1;1 under arsenic stress.Based on the results above,this study revealed a new mechanism of Ca²⁺signaling involved in the regulation of heavy metal stress response.Under normal conditions（non-arsenic stress conditions）,the unstimulated Ca²⁺signal keeps the phosphorylation activity of CPK23 to PHT1;1 at a low level,and most of the PHT1;1 is maintained in a non-phosphorylated state,maintaining its normal localization on the cytoplasmic membrane,and ensuring the absorption of phosphate and normal growth and development of plants.Under arsenic stress,the concentration of Ca²⁺in the cytoplasm of plant root cells increases sharply in a short period of time,activating phosphorylation activity of CPK23 to the Ser514residue at the C-terminal of PHT1;1,causing PHT1;1 to delocalize from the plasma membrane into the cytoplasm,reducing its abundance on the plasma membrane,and thus reducing the As（V）enterence through PHT1;1 in root cells.The rapid response mechanism of Ca²⁺signal to arsenic stress can contribute to avoiding the toxic effects of arsenic stress in a short time and maintain the normal growth and development of plants before balancing nutrient absorption and stress response through long-term regulatory mechanisms.