Subcellular Localization,Calcium Binding Specificity And Physiological Function In Calcium Dependent Growth Of Arabidopsis EDR2

Calcium is an essential mineral element for plant growth and development.Currently,the plant biology research on Ca is mainly concentrated on two aspects.One is the molecular mechanism of Ca²⁺ absorption and storage and the other is the molecular mechauism of signal transduction mediated by Ca²⁺,which worked as a second messenger during endogenous and environmental stimuli.The mechanism of Ca²⁺ regulating plant growth and development directly has not yet been in-depth studied.Arabidopsis EDR2?ENHANCED DISEASE RESISTANCE2?contains three predicted domains which are PH?Pleckstrin Homology?,START?STAR Transfer?and DUF1336?Domain of Unknown Function 1336?.Studies have proved that atedr2 can show powdery mildew resistance phenotype on the leaves mediated by the salicylic acid defense pathway.However,the exact subcellular location and the function in Ca dependent growth of AtEDR2 have not been reported.In this paper,the main experimental results on these aspects are as follows.?1?We found that atedr2 showed significantly inhibition phenotype on leaves and roots only in Ca²⁺ deficiency medium by comparing the growth of Col and atedr2 in mediums with different mineral elements and different concentration.It revealed that AtEDR2 had a specific regulation on Ca²⁺ dependent growth in Arabidopsis leaves and roots.Total and water-soluble calcium in leaves and roots show no difference between atedr2 and Col in full-nutrition or Ca²⁺-lack medium.It infers that the sensitive phenotype to low Ca²⁺ of atedr2 may due to Ca²⁺ distribution other than calcium absorption or accumulation.We expressed and purified AtEDR2 domains with GST?Glutathione-S-Transferase?tag.Then we used MicroCal iTC200 to inject CaCl2 into solution containing the purified AtEDR2 domains and there was a significant and Ca²⁺ concentration dependent exothermic reaction,revealing that the AtEDR PH domain can bind Ca²⁺.Studies have proved that AtEDR2 PH domain can bind PI4P?phosphatidylinositol 4-phosphate?.Immunofluorescence result in this paper showed that whether under low Ca²⁺ treatment or CK,there is no difference of PI4P distribution between atedr2 and Col.It reveals that AtEDR2 does not regulate the distribution of PI4P.AtEDR2 regulation on Ca²⁺ dependent growth in Arabidopsis leaves and roots may due to the binding of Ca²⁺ by PH domain.?2?To analyze the tissue-specificity of AtEDR2 promoter,we use the fusion gene formed byAtEDR2 promoter and a GUS reporter gene to transform Arabidopsis thaliana.GUS staining shows that AtEDR2 expresses in roots,leaves,flowers and silique stalks.AtEDR2 promoter also had strong activity in trichome,but AtEDR2 deletion had no obvious influence on the density and character of trichome.We use the fusion gene formed by AtEDR2 coding sequence and GFP reporter gene to transform tobacco.AtEDR2::GFP can co-localize with ER::mCherry accounting for AtEDR2 location in endoplasmic reticulum.In the same way,we found the PH domain was necessary for AtEDR2 located to endoplasmic reticulum.?3?Based on proteome profiling of atedr2 with Col,we analyzed the phenotype of the mutants corresponding to the differentially expressed proteins between Col and atedr2 under low Ca²⁺ treatment and found that atstt3b was sensitive to low Ca²⁺treatment.AtSTT3B is a putative oligosaccharides glycosyl transferase in endoplasmic reticulum and is responsible for carbohydrate loading process but not for mannose trimming of glycosylated proteins.We found that low Ca²⁺ treatment indues significant accumulation of glycosylated proteins.However,there was no significant difference between Col and atedr2 mutant on the bundance of the total glycosylated proteins.It reveals that AtEDR2 does not participate in the regulation of protein glycosylation with low calcium stress.?4?Microarray-based transcripion analysis found that low Ca²⁺ indues expression of genes involved in reative oxygen species and salicylic acid accumulation.Under low Ca²⁺ condition,the atedr2's leaves had more H2O2 than that in Col.The fold change of these genes expression in the atedr2 under low Ca²⁺condition was significantly higher than those in Col.It demonstrated that AtEDR2 negatively regulated the low Ca²⁺ induced gene expression.Blocking entylele signaling cascade with atedr2-6/ein2-1 double mutant had no effect on the growth inhibition phenotype of the atedr2-6.However,expressing salicylic acid hydrolase-encoding gene NahG in atedr2-6?atedr2-6/35S:NahG?could partially rescue the growth inhibition phenotype of atedr2-6 on the low Ca²⁺ growth condition.It demonstrated that salicylic acid biosynthesis pathway partially takes part in the AtEDR2-mediated Ca dependent growth process.