Screening And Functional Analyses Of Proteins That Interact With RPT2 In The Response To Blue Light In Arabidopsis

As an significant environmental factor,light plays an important role in various growth and developmental processes in plants,such as seed germination,flowering,fruit and so on.Among them,blue light is very important for plants to mediate the photomorphism,the movement of plants,the biological clock and so on.For a long time,researchers have been working on analyzing mechanisms of the phototropism and the chloroplast movement of plants and have made great progress.It has been found that phototropism,chloroplast accumulation/avoidance movement,stomatal opening,leaf expansion,and petiole/leaf orientation are controlled by blue light photoreceptors,PHOT1(Phototropin 1)and PHOT2(Phototropin 2).At present,although there is a certain understanding of molecular mechanisms of phototropism mediated by blue light,however,it is still necessary to identify some new blue light regulatory factors to further analyze molecular mechanisms of phototropism to provide theoretical guidance for the genetic improvement of crops by means of modern biotechnology.These results showed that RPT2(Root phototropism 2)is a key component in the pathway of the hypocotyl curvature mediated by high blue light,and PHOT1 has a function in inhibiting hypocotyls phototropism in the upstream of RPT2.However,the specific mechanism of RPT2 is still unclear.Therefore,we used RPT2 as the bait protein to screen yeast library,to obtain key components of phototropism mediated by blue light,and to set up the signal transduction pathway of phototropism of plants.Through yeast library screening,we successfully screened 33 proteins(RIPs,RPT2 Interacting Proteins)which might interact with RPT2,including JAC1(J-domain protein required for chloroplast accumulation response 1)and PHOT1.We selected proteins which might be relate to the reaction mediated by blue light.The yeast two-hybrid experiments showed that five proteins,including JAC1 and PHOT1,could interact with RPT2.The phenotypic analysis showed that these corresponding genes of 13 T-DNA insertion mutants,including jacl,could not regulate the function mediating the inhibitory action of PHOT1.JAC1 could respectively interact with PHOT 1 and RPT2 in vivo.We also certified that PHOT 1,JAC1 and RPT2 were involved in the chloroplast accumulation movement.The chloroplast movement analysis showed that,when irradiated 30 min under 1 ?molm-2s-1 blue light,WT,photl and rpt2-2 showed the chloroplast accumulation movement,but jacl did not show the chloroplast accumulation movement.These results indicated that JAC1 can effectively regulate the chloroplast accumulation movement.When we increased the blue light intensity to 3 ?molm-2s-1,jacl obviously showed the chloroplast avoidance movement,but this time WT,photl and rpt2-2 still showed the chloroplast accumulation movement.Interestingly,mutation of PHOT1 and RPT2 could partially inhibit the chloroplast avoidance movement of jac1 under low blue light.When irradiated to 100 ?molm·2s-1 blue light,all of these six mutants showed the chloroplast avoidance movement,and showed a certain amount of time dependence.In conclusion,through yeast library screening,we screened and validated 5 proteins including JAC1 and PHOT1 which can interaction with RPT2.The genetic analysis showed that these 13 T-DNA insertion mutants including jacl could not regulate the function mediating the inhibitory action of PHOT1.The chloroplast movement analysis showed that JAC1 can effectively regulate the chloroplast accumulation movement.However,mutation of PHOT1 and RPT2 could partially inhibit the chloroplast avoidance movement of jacl under low blue light.