Mechanism Of MfERF049 Inhibiting H₂O₂ Production Induced By Low Calcium In Arabidopsis Thaliana

Ca is an essential mineral element for plant growth and development.As a second messenger,it is the most important signal molecule to sense the inside and outside environment of the cell,and plays an important role in the cell.NIK1?NSP-interacting kinase?is a receptor-like kinase and the virulence target of the Geminivirus nuclear shuttle protein.NIK1 belongs to the Leucine-Rich Repeat Receptor-Like protein Kinase?LRR-RLK?family,which is the largest subfamily of the Receptor-Like protein Kinases?RLKs?,these kinases play an important role in plant growth and development,environmental stress response and so on.At present,the role of NIK1 in defense signal transmission and anti-viral infection has been deeply studied.However,the function of NIK1 in Ca²⁺-dependent growth of plants is still unclear.In this study,nik1 mutants were used to study the function of NIK1 gene and related proteins,and the mechanism of NIK1 inhibiting H₂O₂ production induced by low calcium in Arabidopsis cells was preliminarily revealed.The main results of this paper are as follows:?1?The phenotype analysis of different Ca²⁺concentrations showed that the mutant nik1 was smaller and the leaves were curly yellower than the wild-type Col-0under low Ca²⁺(0?M Ca²⁺)conditions.The root length of nik1 mutant was shorter than that of wild-type plant,and the fresh weight was lower than that of wild-type plant.The results of mutant complement test showed that the low calcium sensitive phenotype could be recovered,which indicated that the mutated NIK1 gene resulted in the low calcium sensitive phenotype of nik1 mutant.?2?In order to study whether NIK1 gene affects the accumulation of calcium,using Inductively Coupled Plasma Emission Spectrometer to determine the content of Ca in plants.It was found that the content of Ca in leaves and roots of nik1 mutant was not significantly different from that of wild-type under CK and low calcium conditions.The results showed that NIK1 did not affect the accumulation of calcium in plants.?3?Construction of plant material that drives the GUS reporter gene by the NIK1 promoter and stable T₃ generation plants were obtained.Through GUS histochemical staining,it was found that the NIK1 promoter was expressed in different tissues of Arabidopsis thaliana during different growth periods.?4?Through the analysis of the transcriptome,46 related-gene were screened and the variation of gene expression in wild-type plants by mutants is more than 5,and most of them were related to the production of hydrogen peroxide and salicylic acid.RT-PCR results show that six of them?At1g21250,At2g24850,At2g18660,At5g61160,At1g02450,At4g23150?were up-regulated in the nik1 mutant,which verified the reliability of transcriptome data.The results of DAB?Diaminobenzine?histochemical staining showed that the accumulation of H₂O₂ in nik1 mutation was higher than that in wild-type Col-0 at low calcium concentration.The results of fluorescence dye DHR?Dihydrorhodamine-123?treatment showed that the accumulation of H₂O₂ in the root of nik1 mutant was higher than that of wild-type Col-0 under low calcium condition.?5?GST-NIK1 Kinase Domain was obtained by in vitro expression and purification.The binding properties of GST-NIK1 Kinase Domain protein to ions were determined by microcalorimetry?MicroCal iTC200?.Results showed that GST-NIK1 Kinase Domain protein could specifically bind to Ca²⁺,Mg²⁺and Mn²⁺.