Blue-light-induced PIN3Polarization For Root Negative Phototropic Response In Arabidopsis

Root negative phototropism is one of the important tropic responses for plants to adapt to the changing environments, which can avoid the damage of light damage and other stressful stimulus from the upper layers of soil, and facilitate water and nutrient absorption from the soil. In the decade, progress has been made in understanding the role of blue light in the regulation of these critical processes, especially phototropism. It has been demonstrated that the asymmetric distribution of auxin plays the major role in phototropism, particularly the polarization of PIN1and PIN3. But the mechanism of root negative phototropism remains elusive, which needs further research.We elucidate the role of auxin in root negative phototropism by using the roots of Arabidopsis thaliana. The main results including:1Root negative phototropism is regulated by blue light receptor photlFirst of all, we examine root negative phototropic response of wild type by using different quality of light. Our results show that only blue light can function in root negative phototropism. In Arabidopsis, there are two blue light receptor families: cryptochrome and phototropin. Thus, our results demonstrate that root negative phototropism is regulated by photl particularly.2The asymmetric distribution of auxin is required for root negative phototropismWe assayed differential auxin response using the auxin-responsive marker DR5rev::GFP, founding that an increase DR5activity is detected in the illuminated side of roots under unilateral blue light illumination. In photl mutant, which have defective in root negative phototropism, no asymmetric DR5activity is detected. Similar in pin3-4mutants, reduced asymmetric DR5activity is detected, which is consistent with its reduced root negative phototropic response. Our results reveal that the polar auxin transport is required for root negative phototropism, especially regulated by PIN3.3PIN3is involved in root negative phototropismThe polarization of PIN1can transport the auxin to the root tip, but do not change the lateral flow of auxin, which indicating that other auxin efflux carriers may be involved in this process. Since PIN3can change the auxin flow to the lower side of roots in root gravitropism, PIN3is a good candidate for asymmetric auxin distribution in root negative phototropism.We used the PIN3::PIN3-GFP marker line to test the localization of PIN3under unilateral blue light illumination. In the dark, none polarization of PIN3is detected, then under unilateral blue light illumination for2h, an increase activity of PIN3is detected on the lateral outer PM in the illuminated side of roots, which can direct the auxin flow to the illuminated side of roots, where promoting growth and causing the root bend away from the light.4de novo protein synthesis and protein degradation is not involved in blue-light-induced PIN3polarization in root negative phototropic responseUsing the protein synthesis inhibitor CHX and protein degradation inhibitor MG132, our results show that the normal blue-light-induced PIN3polarization is detected in wild-type plants under these pharmacological treatments. These results indicate that de novo protein synthesis and protein degradation does not participate in blue-light-induced PIN3polarization in root negative phototropism5Blue-light-induced PIN3polarization is mediated by GNOM-dependent, BFA-sensitive trafficking pathwayThe wild type exhibits defective in root negative phototropism under BFA treatment and blue-light-induced PIN3polarization is disturbed, which indicates that the BFA-sensitive trafficking pathway is involved in this process. Then we use BFA-resistent line:GNOMM696L treatment with BFA and unilateral blue light illumination. Our results show that the GNOMM696L exhits the normal root negative phototropic resposne and blue-light-induced PIN3polarization even under BFA treatment. These results indicate that blue-light-induced PIN3polarization is regulated by a BFA-sensitive, GNOM-dependent trafficking pathway.6PID and PP2A antagonistically mediate blue-light-induced PIN3polarizationIn Pro35:PID mutant, the blue-light-induced PIN3polarization and root negative phototropic response was affected upon blue light illumination for2h. In addition, the pp2aal mutant showed reduced root negative phototropic response and blue-light-induced PIN3polarization. These results reveal that blue-light-induced PIN3polarization is regulated by PID/PP2A.7The expression of both PID and PP2A is regulated by blue light receptor PHOTl In order to test whether blue light can regulate the activity of PID and PP2A, we use the PID::PID-YFP and PP2AA1::PP2AA1-GFP marker lines. Our results indicate that the fluorescence signal intensity resulting from PID-YFP was reduced in roots exposed to blue light compared with that of plants grown in the dark. In contrast, while the PP2AA1-GFP signal was weak in the dark, blue light illumination can increase the PP2AA1-GFP signal intensity. These data demonstrate that the expression of both PID and PP2AA1is regulated by blue light.Inphotl mutant, no visible change of PID-YFP and PP2AA1-GFP was observed. These results demonstrate that the polarization of PINl is mediated by the activity of PID and PP2A. These observations were further supported by quantitative real-time PCR (qRT-PCR) analysis of PID and PP2AA1expression in plants grown first in the dark and then exposed to blue light.