Molecular Mechanism And Physiological Function Study Of CNGC2 And PEPR2 Regulate Intra-And Extracellular Ca Dynamics In Arabidopsis

Calcium is a macronutrient for plants and is a crucial second messenger. In this study, we explored two aspects with Ca2+ distribution and Ca2+ signaling with Arabidopsis thaliana as material. The first study is that cngc2 with inactivated CNGC2 led Ca2+ being overly accumulated at the extracellular space of the leaf cells which affected some physiologic function. The second study is that AtPEPTIDE RECEPTOR2 (AtPEPTIDE RECEPTOR2) mediates the AtPEPTIDE 1-induced cytosolic Ca2+ rise, which is required for the suppression of Glutamine Dumper gene expression in Arabidopsis roots.(1) Ca, an essential mineral element for all living organisms, is absorbed by plant root cells and transportes upward to, further redistributes in leaves through poorly defined genetic pathways. Ca2+ concentration under 0.1 mM,the mutant cngc2 with inactivated CNGC2 grew no difference with the wild type. Increasing Ca2+ supply under 10 mM to the roots caused Ca2+spillover into the extracellular space of the mutant leaf cells, which further led locally accumulated reactive oxygen species, leaf senescence, cell pinpot death, growth inhibition and cell pinpot death is mainly located in veilet encircled by mini veins. The β-glucuronidase reporter gene (GUS)-based promoter activity assay indicated that the CNGC2 specifically expressed in leaf cells surrounding mini veins. We measured Ca contents by atom absorption spectrophotomety and the result showed that the cngc2-l had significant less total Ca than the Col-0 under 10 mM Ca2+, but the cngc2-1 had significant more acetic acid-extracted Ca than the Col-0 under 10 mM Ca2+. This explained that more Ca accumulated in the extracellular space. Previously reported lack of hypersensitive response of the cngc2 mutant to an avirulent pathogen is an indirect reflection of the gene’s role in the Ca2+ spillover in our experiment system. We found that cngc2 had HR under 0.1 mM Ca2+, but cngc2 had no HR after three days under 10 mM Ca2+ followed by infiltrating PstDC3000 avrRpm1+ into the leaves. We also found that cngc2 under 10 mM Ca2+ had more starch accumulated under of dark period, and this stated Ca2+ spillovering affect the ability of starch degradation.(2) AtPEPTIDE RECEPTORl (AtPEPR1) and AtPEPR2 are two members of leucine-rich repeat receptor-like kinase family and bind to a group of AtPROPEP gene-encoded endogenous peptides, AtPeps. Previously, we found that AtPEPR2 plays a moderate role in the AtPep1-mediated innate immunity responses in Arabidopsis leaf. In this study, we found that the atpeprl and the atpepr2 mutants showed a moderate but significantly shorter root phenotype. AtPEPR1 and AtPEPR2 partially mediated AtPep1-induced root elongation inhibition. AtPep1-triggered cytosolic Ca2+ transient rise in roots showed partial dependence on AtPEPR1 and AtPEPR2, and AtPEPR2 is important than AtPEPR1. To comprehensively address the effect of AtPepl on gene expression and the roles of AtPEPR2 in mediating this expression, we compared the transcription profiling in the roots of the Col-0 and atpepr2-1 mutant under the control and 1h AtPep1 treatment conditions. Transcriptional profiling analysis found that expression of 75% of AtPep1-modulated genes in roots was fully dependent on AtPEPR2, of which two dramatically induced genes showed partial dependence on the extracellular Ca2+ concentration ([Ca2+]ext). In the process of genetic screening, Glutamine Dumpers, AtGBUs caught our attention. Arabidopsis genome contains seven Glutamine Dumpers genes (AtGDUs), encoding amino acid exporters. Three of them (AtGDU2,3,5) were among the top 10 genes that were downregulated by AtPep1 through AtPEPR2 fully dependent pathway. The GUS-based promoter activity assay indicated that treatment with AtPepl strongly suppressed promoter activity of AtGDU3 in roots, which was relieved by chelating [Ca2+]ext.Arabidopsis overexpressing AtGDU3 showed a shorter root phenotype and decreased sensitivity to the AtPepl-mediated inhibition of root elongation. Taken together, this study demonstrated a significant role of AtPEPR2 in the AtPepl-mediated signaling in the roots.Above all, on the one hand, this study explored that CNGC2 participates in the distribution of Ca2+ which lay the foundation of the relationship between Ca2+ absorption and Ca2+ distribution; On the other hand, AtPepl was identified as an active component in soluble extracts of aradbiopsis leaves and it was an endogenous peptide. AtPEPR2 played a major role in the exogenous AtPepl-mediated inhibition of root elongation. It shows that AtPep1 may be a new type of hormone involved in plant growth and development.