The Inolvement Of Cytosolic Ca2+Signals In Hypocotyl Phototropism Induced High-Intensity Blue Light In Arabidopsis Thallana

Light is one of the most important environmental factors for plant growth and development.Phototropism in plants, the bending of the seedling stem toward unilateral blue light, is an adaptiveresponse that optimizes the stems and leaves of plants optimal growth orientation to capture a suitable lightsource. Blue-light (BL) receptors phototropins (PHOT1and PHOT2) regulate a wide set of physiologicaland developmental responses, including phototropism, rapid inhibition of hypocotyl growth, chloroplastrelocation movements, light-induced stomatal opening, as well as leaf expansion and positioning. In strongBL, PHOT1and PHOT2contribute redundantly to BL-induced phototropic curvature of hypocotyls. Due totheir complement functions, different mechanism about PHOT1and PHOT2regulating phototropism is lesswell understood. In recent years, physiological studies suggest that cytosolic Ca₂₊([Ca₂₊]cyt) is one of theintermediates in phototropin signaling. PHOT1and PHOT2-mediated increase in [Ca₂₊]2+cyt([Ca]cyt)differently was reported in leaves of A.thalian. However, the relationship between the BL-induced Ca₂₊signal and PHOT1/PHOT2-mediated responses remains largely unclear.To gaining further insights into Ca₂₊function in mediating BL-induced phototropic curvature ofhypocotyls in etiolated seedlings of A.thaliana, we constructed PHOT1and PHOT2overexpression vectors,and obtained PHOT-overexpression lines in the wild-type (gl1) background(PHOT1-OE gl1andPHOT2-OE gl1), PHOT2-overexpression lines in the phot1background (PHOT2-OE phot1) and vice versa(PHOT1-OE phot2). Then, we obtained transgenic A. thaliana that expressed cytosolic aequorin in gl1,phot1and phot2mutants, and phot1phot2double mutants, and above describing PHOT-overexpressionlines to analyze PHOT-mediated calcium signaling. The results demonstrated that [Ca₂₊]cytsignificantlyincreased in hypocotyls of etiolated seedlings gl1and phot1mutant in response to100μmolm-2s-1of BL for15s. However,[Ca₂₊]cytwas less in the phot2mutant than gl1, and [Ca₂₊]cytwas lower in the phot1phot2double mutants than in the phot2mutant. Furthermore, the [Ca₂₊]cytvalues in the PHOT2-OE phot1washigher than in the PHOT2-OE gl1. These results suggest that PHOT1and PHOT2maybe play a differentrole in mediating the BL-induced [Ca₂₊]cyt,and PHOT2mainly mediated the BL-induced [Ca₂₊]cytsignalin A.thaliana etiolated seedlings. Moreover, PHOT1significantly inhibiting PHOT2in strong BL-induced [Ca₂₊]cyt, maybe through interacting with PHOT2. Our results further demonstrated that PHOT2mainlyregulated Ca₂₊release from internal Ca₂₊stores, by using Ca₂₊channel blockers (LaCl2+3), Cachelating(EGTA), inhibitors of phospholipase C(U73122) and internal Ca₂₊stores inhibitors (RR). In accordancewith the pharmacological results, BL can trigger Ca₂₊influxes across plasma membrane via thePHOT1-activated plasma membrane Ca₂₊-permeable channel in hypocotyl cells of A. thaliana, by patchclamp in whole cell configuration.Subsequently, the relationship between BL-induced [Ca₂₊]cytsignal and phototropism was analyzedby comparing the hypocotyl curvatures and the rise of [Ca₂₊]cytin response to BL in gl1, phot1and phot2mutants, and phot1phot2double mutants, and PHOT-overexpression lines. These results showed that thecurvature of phot1mutant was bigger than gl1by treating with100μmol m-2s-1BL. Surprisingly,PHOT2-OE phot1transgenic lines showed stronger phototropic responses than the phot1mutant. Whereasphototropic responses of phot2mutant and gl1had not shown different. These results indicated that PHOT2maybe play a more significant role than PHOT1in mediating phototropism under unilateral strong-BL, asabove mentioned that strong-BL induced increase in [Ca₂₊]cytby PHOT2.BL-induced elevation of [Ca₂₊]cytand phototropic curvature suggested that PHOT1negatively regulatePHOT2-induced hypocotyls bending under unilateral strong-BL, maybe by directly interacting. To test thishypothesis, we performed yeast two-hybrid and bimolecular fluorescence complementation assay. Theresults showed that they did not directly interact each other. So we predict that PHOT1/PHOT2regulate[Ca₂₊]cytsignals and strong BL-induced phototropism maybe by mediating their downstream signals orother mechanism. PKS1maybe as a important candidate to provide a link between PHOT1and PHOT2.Subsequent studies demonstrated PKS1directly interacts with PHOT1and PHOT2in vivo at the plasmamembrane and in vitro. Surprisingly, PKS1also interacts with CAM4and CAM5, which are calmodulins(CAMs). Moreover, PKS proteins contribute redundantly to hypocotyl phototropism.Therefore, PKSmaybe as a intermediate link between the phototropins and Ca₂₊signals.