The Real-time Labeling Of Ca²⁺ In BY2 Cells And Its Response In Auxin Signal Transduction

The calcium is an essential element of life.The calcium ion(Ca²⁺),bivalent cation of the element,is an indispensable component in biological organism.Ca²⁺transmits cell signals in cell signal transduction as a second messenger.Thus it is also an important object in cell signal transduction study.In order to observation in real time of Ca²⁺in plant cell that we transformed the aequorin(AEQ)gene into tobacco BY2 cell which had been labeled by green fluorescent protein(GFP)to the SCMP2(secretive carrier membrane proteins 2).The bivalent transformed BY2 cell lines were obtained by screening and molecular detection of the transformed cells.The bivalent transformed cells were treated with coelenterazine and observed in real time under microscope.Fluorescence obtained after treatment of EGTA,ethanol and La Cl₃ proved that the fluorescence signal of cells was directly related to calcium ions.The control BY2 cells with single AEQ transformed could not directly observe fluorescence under microscope.That indicated the AEQ responded to calcium ions and emit the weak fluorescence could further stimulate GFP fluorescence and effectively enhance the weak fluorescence of AEQ responded to Ca²⁺.Thus,bivalent labeled calcium ion response BY2 cells were constructed for the real-time observation of Ca²⁺in the cells.Compared with the fluorescence intensity the bivalent was collected at about three times exposure in response Ca²⁺than the direct excitation GFP fluorescence(external excitation fluorescence).The auxin and ABP1 correlation with calcium signal was studied in calcium-labeled BY2 cells.An estradiol inducible p ER16 Ti plasmid vector was used for the ABP1 inducing.The tobacco ABP1 c DNA was cloned under the inducible promoter in sense overexpression(ABP1-ox)or antisense inhibited expression(ABP1-anti)and transformed into calcium-labeled BY2 cells.The quantitative and semi-quantitative analysis of ABP1 gene expression after estradiol induction verified the ABP1 gene significantly increased expression in ABP1-ox cells and significantly decreased in ABP1-anti cells.The Ca²⁺signal was observed combined with the auxin treatment and ABP1 expression regulation.The results showed that intracellular Ca²⁺signals in ABP1-ox cells could rapidly respond to the action of extracellular auxin and transfer the signals into cells.There is intensifying signal in the cytoplasmic membrane and near the nucleus.However,ABP1-anti cells with weak signal in cell seems unable to effectively transfer the signal to the vicinity of the cell nuclear membrane.If the estradiol treatment was extended ABP1-anti cells was excessively inhibited with the still weak Ca²⁺signal.The cell growth was also significantly affected with elongation and division blocked even cell death.In consideration of the ABP1 is still a controversial as auxin signaling receptor.This study can only prove that ABP1 is involved in auxin signaling and Ca²⁺response transduction.The overexpression and inhibition of this gene significantly affect the signal transduction of Ca²⁺in BY2 cells.If ABP1 is auxin receptor,it can be directly associated with Ca²⁺signal transduction.If ABP1 is not receptor in auxin fast response,it is estimate that the ABP1 can participate the homeostasis of IAA to affect auxin function.Because ABP1 has the strong IAA affinity and with the high gene expression activity in cell,it can combine of the auxin in steady state affects the freedom auxin concentration levels in the cell.And auxin concentration level will affect auxin action including the series of processes of Ca²⁺response.