Functional Analyses Of P2SA1in Blue Light-induced Hypocotyl Phototropism In Arabidopsis Thaliana

In plants, phototropism can guarantee the best orientation to obtain light source. Blue light receptors(PHOT1PHOT2), play an important role in this phototropism process, but the mechanism of signaltransduction and identification of new signal components still need to be a breakthrough. In Arabidopsis,PHOT1functions at both low (0.01to1mol m-2s-1) and high (>1mol m-2s-1) intensity blue light tomediate phototropic responses, but PHOT2functions only at high-intensity blue light (HBL). Thisphenomenon manifests that PHOT1and PHOT2have partially overlapping functions by their differentsensitivities to blue light. Studies have illustrated that PHOT1mediates hypocotyl phototropism via itsdownstream signal transducers NPH3, RPT2and PKS1, to regulate the activity and relocation of auxininflux and outflux carrier PIN1and AUX1. However, the mechanism is not fully understood. Geneticanalysis showed that single gene mutation of NPH3or RPT2lead to loss of hypocotyl phototropisminduced by HBL, suggested that NPH3and RPT2were involved in hypocotyl phototropism. However,RPT2interacts only with PHOT1, not with PHOT2. Recently, RCN1-1, the A1subunit of Ser/Thr proteinphosphatase2A (PP2A), has been identified to interact with PHOT2and negatively regulatePHOT2-mediated hypocotyl phototropism, but does not affect PHOT1-mediated hypocotyl phototropism.ABCB19, a newly identified auxin transporter, has been confirmed to interact only with PHOT1, not withPHOT2, to regulate hypocotyl phototropism.However, the redundant function mediated by both PHOT1and PHOT2under HBL, drasticallyrestricts the understanding of the mechanism of PHOT2signal transduction. For avoiding the interferenceof PHOT1, we screened new signal components downstream of PHOT2from EMS mutagenesis of phot1mutants. Recently, we screened and cloned two genes named P2SA1and P2SA2(Phototropin2SignalingAssociated). P2SA1gene encodes an ATP-binding cassette (ABC) transporter, which belongs to a largegene family that includes the recently isolated ABCB19gene. P2SA2was proved to be allele of NPH3(Nonphototropic hypocotyl3). Subcellular localization showed that P2SA1and P2SA2were localized inthe plasma membrane of Arabidopsis. Both P2SA1and P2SA2gene mutation lead to loss of hypocotylbend response to HBL, which is similar to the phenotype of phot1phot2double mutant. Unlike p2sa2mutant, p2sa1mutant shows the hypocotyl phototropism in response to low-intensity blue light (LBL).These results together with p2sa1pP2SA1:P2SA1and p2sa235S::P2SA2transgenic plants showed hypocotyl bend to HBL, indicated that P2SA1functions downstream of PHOT1and PHOT2, and mediateshypocotyl phototropism to HBL, but does not regulate hypocotyl bend induced by LBL in PHOT1pathway.Analysis of the biological function of P2SA1, found that the leaf surface temperature of p2sa1mutant waslower about1℃than wild typeor phot1mutant, but the p2sa1mutant displayed normal chloroplastavoidance response and root negative phototropism.Analysis of PHOT1, PHOT2and P2SA1transcripts in Arabidopsis etiolated hypocotyls by apolymerase chain reaction after reverse transcription of RNA (RT-PCR) and quantitative real-time RT-PCR,reveals that HBL enhanced the expression of P2SA1and PHOT2, but inhibited the expression of PHOT1.These results suggest that HBL-induced hypocotyls phototropism may be mediated mainly by PHOT2, andP2SA1functions in the downstream of PHOT2. Further analyses uncover that P2SA1physically interactswith PKS1, not PHOT1and PHOT2in vivo or vitro, and PKS1directly interacts with phot2, which impliesthat PKS1may function in the crosstalk between P2SA1and PHOT2as a scaffold protein.Genetic analysis showed that p2sa1, nph3, rpt2mutants, similar to phot1phot2double mutant, failedto response to HBL. Single mutant of gene PKS1、PKS2and PKS4showed no phenotype of hypocotyl bend,but double mutant behaved obviously lower hypocotyl bend, while triple mutant completely missedhypocotyl phototropism, similar to phot1phot2, p2sa1-1, nph3-6and rpt2-2mutants. Interestingly, underthe background of phot1mutant, the gene of RPT2mutation, arabidopsis restores the hypocotyl bend toHBL, but the gene of P2SA1, NPH3and PHOT2mutation, arabidopsis is still lack of hypocotyl bend toHBL. Interaction analysis showed that P2SA1does not interact with NPH3or RPT2, but can interact withPKS1, PKS2and PKS4, and PKS1can interact with NPH3in vitro. Above all, blue light receptors PHOT1and PHOT2sense blue light and transmit the signal to NPH3and P2SA1, then may produce both pathwaysof dependence and independence of RPT2. Under normal condition, the signaling pathway independence ofRPT2may be inhibited by PHOT1, when the PHOT1gene mutation, this pathway will performance. PKSsfamily proteins play important roles in transmit signal from PHOT1and PHOT2to NPH3and P2SA1.Plant phototropism is decided by the asymmetric localization of auxin, which depends on the signaltransduction pathway meditated by phototropin under blue light. Auxin distribution analysis by using p2sa1DR5rev:GFP and p2sa1DR5rev:GUS transgenic plant, we observed that auxin mainly distributed in youngleaf hook in etiolation seedling under dark, HBL stimulation from hypocotyl face induced auxin asymmetrical distribution between backlight side and light side, and from cotyledon surface induced auxindown transportation and asymmetrical distribution. The mutation of P2SA1gene resulted in loss of auxintransfer induced by HBL. Consistently, the gene of P2SA1mutation leads to loss of hypocotyl bend underside light stimulation. These results confirm that P2SA1regulates auxin transport to mediate hypocotylphototropism in response to HBL. Interaction analysis showed that P2SA1did not interact with auxintransport carrier PIN1or AUX1, but PKS1physically interacted with PIN1and CAM4in vivo or vitro.Together with our previous results that NPA (auxin outflux carrier inhibitor) inhibited HBL-inducedhypocotyl cytoplasmic Ca2+rise and hypocotyl phototropism, our results confirmed that PKS1may beinvolved in P2SA1mediating auxin transport induced by HBL, to adjust the change of cytosolic Ca2+inhypocotyl and regulate hypocotyl phototropism.In conclusion, we successfully screened and cloned two genes P2SA1and P2SA2. P2SA1geneencodes an ATP-binding cassette (ABC) transporter, and P2SA2is proved to be a NPH3alleles. BothP2SA1and P2SA2are located in the plasma membrane of Arabidopsis thaliana and involved in regulatingthe hypocotyl phototropism induced by HBL. In addition, P2SA1is essential for phototropins mediatingHBL-induced stomotal movement, but does not mediate chloroplast avoidance response and root negativephototropism. P2SA1regulates HBL-induced hypocotyl phototropism depending on interaction with PKS1to receive the signal from PHOT1or PHOT2, leading to the establishment of a differential gradient ofauxin for asymmetric growth in hypocotyls of etiolated seedlings. Our study identifies a critical componentthat is involved in HBL-induced hypocotyl phototropism and resolves a mystery about the differentialfunctions of PHOT1and PHOT2in this response.