Studies Of Carotenoid Metabolism In Bangiophyceae(Rhodophyta)

Carotenoids are the most important pigments on earth,participated in various biological processes in plants,such as photosynthesis,photomorphogenesis and photoprotection.According to the structure,carotenoids can be divided into ?-branch and?-branch.The ?-branch carotenoids are widely distributed in algae and land plants.And the ?-branch are not found in some of red algae and Chromista.Bangiophyceae(Rhodophyta)is the first group that accumulate ?-branch carotenoids,including the only two genus Bangia and Porphyra.The carotenoids of Bangiophyceae contained lutein,zeaxanthin,?-carotene,and ?-carotene as major carotenoids,and minor carotenoids of lutein-5,6-epoxide,antheraxanthin and zeinoxanthin were also detected by HPLC.Carotenoids are derived from isopentenyl diphosphate(IPP)and its allylic isomer dimethylallyl diphosphate(DMAPP),and condensed two geranylgeranyl pyrophosphate(GGPP)with GGPP synthase(GGPPS).Bangiophyceae had only one GGPPS,and maybe originated from cyanobacteria in the primary endosymbiotic event.Through the E.coli color complementation assay and enzyme activiity in vitro,Bf GGPPS showed a specific GGPPS activity with IPP and DMAPP as substrates.Bf GGPPS also showed its activity after transformed into Arabidopsis ggpps11 mutants.And we also found Bf GGPPS can interact with phytoene synthase of Bangia(Bf PSY),to control the carotenoid metabolism,which comfired by the yeast two-hybrid(Y2H)and co-immunoprecipitation(Co-IP)experiments.The ?-branch carotenoids differ from the ?-branch in that cyclization and hydroxylation of the ?-ring.We found two lycopene cyclases in the Porphyra genome.Pu LCYB had ?-cyclase activity,and Pu LCYE had ?-cyclase activity,proved by E.coli pigment system.When combined,the ? and ? cyclases convert lycopene to ?-carotene,a carotenoid with one ?-ring cyclization and one ?-ring cyclization.The Arabidopsis cyclase mutant complementation experiments also demonstrated Pu LCYB and Pu LCYE had ?-cyclization and ?-cyclization activities in vivo,respectively.In the evolutionary relationship,LCYE from red algae and green plants had different origins,LCYE of red algae was derived from LCYB of the original red algae by gene duplication.For carotene hydroxylase,the previous work has found an active beta-hydroxylase of P450 family(CYP97B)in P.umbilicalis.And we also found only one Bf CHY from Bangia,which was an CYP97B56 located in the plastid.Therefore,we checked if Bf CHY had both ?-hydroxylation and ?-hydroxylation activities.Our results demonstrated Bf CHY can hydroxylate ?-rings with the substrates of ?-carotene and?-carotene.But Bf CHY didn't show ?-hydroxylation activity in three systems,including E.coli,Synechocystis and Arabidopsis.Based on the results of GGPPS,LCYB and LCYE,we elucidated the separation of?-branch and ?-branch carotenoids in Bangiophyceae,and found their different modes of evolution.And with three methods,the ?-hydroxylation of CYP97 B was not found,and made it clear that the CYP97 B of red algae didn't participate the ?-hydroxylation of lutein synthesis.