Cytoplasmic Calcium Is Involved In Phototropism In Arabidopsis Hypocotyls

Environmental cues have an extensive regulatory influence on the growth and the development of plant. Light is one of the most important environmental factors. Phototropins are blue light receptors controlling a range of responses such as initiate phototropism, opening of stomata, irradiance-dependent movements of chloroplasts and so on. Phototropism represents a differential growth response by which plant can change their leaf/stem position for photosynthetic light capture as effectively as possibility, especially under adverse conditions. Former research indicated that the two phototropins of Arabidopsis (PHOT1 and PHOT2) are Blue light-activated, and then interacted with its downstream signal protein NPH3 and RPT2, regulated the activation and localization of the auxin-efflux and -influx transporters, PINs and AUX1, leading to asymmetric distribution of the auxin between the illuminated and shaded side of the hypocotyls, at length led to phototropism. Physiological studies suggest that cytosolic Ca²⁺ is one of the intermediates in phototropin signaling. PHOT1 and PHOT2 increased cytosolic Ca²⁺ ([Ca²⁺]cyt) differently was reported in leaves of A.thaliana;however, the relationship between the BL-induced Ca²⁺ signal and PHOT1- and PHOT2- mediated responses remains largely unclear.Here, we examined a BL-induced increase in [Ca²⁺ ]cyt in etiolated seedlings of transgenic A.thaliana gl1,phot1,phot2,phot1phot2 expressing the Ca²⁺-sensitive luminescent protein aequorin. Aequorin luminescence did not increase after treatment of weak BL(﹤50μmol·m^-2·s^-1), but increase under strong BL (100μmol·m^-2·s^-1). When 5⁸ mm etiolated seedlings were exposed to 100μmol·m^-2·s^-1 BL for 15 s, each seedlings exhibited a transient increase in aequorin luminescence lasting 60⁸0 s. The peak [Ca²⁺ ]cyt induced by BL was 83.91±23.79 nmol. Neither low intensity BL nor RL can induce [Ca²⁺]cyt accumulation. Interestingly, the increase in [Ca²⁺]cyt in phot1 mutant was more significant than that in phot2 and phot1phot2 double mutant. Statistical analysis showed that the relative contribution of PHOT2 to the response to BL was 80%, while PHOT1 occupied 20%. Moreover, we found that the delayed time which was calculated from the moment of the end of BL irradiation to the moment of BL-induced peak [Ca²⁺]cyt was significantly difference, with a mean of 22.7±10.1 s in phot1 and 10.2±5.8 s in gl1.These indicated that the PHOT1 and PHOT2 mediated BL-induced increase in [Ca²⁺ ]cyt differently, and PHOT2 mainly mediated the BL-induced [Ca²⁺]cyt signal in A.thaliana etiolated seedlings. By using Ca²⁺ channel blockers, LaCl3; Ca²⁺ chelating, EGTA; inhibitors of PLC, U73122 and internal Ca²⁺ stores inhibitors RR, we demonstrated that PHOT2 mainly induced Ca²⁺ released to the cytosolic through PLC-dependent signaling. Patch clamp results show that BL can activate PHOT1-dependent plasma membrane Ca²⁺ channel weakly but not PHOT2-dependent, which was accordingly with the pharmacological results.BL induced phototropism and inhibited of hypocotyl elongation of etiolated seedlings.Phenotype analysis showed that both PHOT1 and PHOT2 mediated the BL-inhibited hypocotyl elongation under high intensity of BL. Because of the Ca²⁺ involved in the BL-induced hypocotyl elongation inhibition had been studied clearly, here we put our attention to the relationship between BL-induced Ca²⁺ signal and phototropism. When irradiation with higher fluence rates of BL for 12h, we found that the bend curvature of phot1 was more significant than that in wild-type (gl1) and phot2, with a mean of 75.9±12.6°in phot1 and 51.2±9.8 in gl1. Using [Ca²⁺]cyt reporter, FRET-Sensitized Emission Imaging of YC3.60, Ca²⁺ differences between illuminated and shaded sides could be found obviously after strong BL-induced phototropism. All of above indicate that Ca²⁺ involved in strong BL induced phototropism. In addition, inhibited auxin efflux by NPA strongly suppress the [Ca²⁺]cyt increase and the phototropism induced by strong BL, especially in phot1 mutant, with a proportion of 82.6% and 77.9%, respectively. So we conclude that the Ca²⁺ signal was induced by auxin polar transportation. However, yeast-two-hybrid and BiFC results showed that phototropins did not interact with the auxin efflux carrier PIN1/PIN3 in vivo and in vitro, this means PHOT1/ PHOT2 regulate auxin polar transportation by other mechanism.From all have been mentioned above, we can conclude that Ca²⁺, maybe as a intermediate downstream of auxin, involved in PHOT2-dependent strong BL-induced phototropic in Arabidopsis etiolated seedlings. More work should be done to explain the detailed mechanism.