Studies On The Carotenoid Metabolism In Porphyra Umbilicalis

Benefited from the community genome sequencing project(Joint Genome Institute,US Department of Energy)we are involved,different transcriptomes of the red alga Porphyra umbilicalis were sequenced.From the database,we firstly analyzed those transcripts for putative genes on isoprenoid and carotenoid metabolic pathways by homolog searching.Our results showed that P.umbilicalis has genes for almost all enzymes on the methylerythritol phosphate(MEP)pathway,and probably has no genes for enzymes beyond 3-hydroxy-3-methylglutaryl-CoA synthase(HMGS)on the mevalonate(MVA)pathway.It is suggested that P.umbilicalis might also exclusively use MEP pathway to supply isopentenyl diphosphate(IPP)and dimethylallyl diphosphate(DMAPP)substrates for both isoprenoid and carotenoid biosynthesis,as is in green algae.Pigment analysis revealed a relatively simple carotenoid profile,comparing with that in higher plants,in P.umbilicalis with ?-carotene,?-carotene,lutein,zeaxanthin and antheraxanthin.Bioinformatic analysis also supported this postulation as most genes on isoprenoid and carotenoid metabolic pathways are encoded by a single gene.Neither violaxanthin nor a transcript for the putative violaxanthin de-epoxidase(VDE)was found in our study.This suggested an absence of the functional xanthophyll cycle in P.umbilicalis for photoprotection.Before our studies,the molecular cloning strategies were optimized for P.umbilicalis to avoid the interference of polysaccharides which are rich in seaweeds.We identified transcripts for a putative geranylgeranyl diphosphate(GGPP)synthase(PuGGPS)and a cytochrome P450-type carotene hydroxylase(PuCHY1),respectively.Pigment complementation assays using an Escherichia coli system and HPLC analysis confirmed their catalytic activities.PuGGPS was found to synthesize GGPP from IPP and DMAPP,and PuCHY1 hydroxylated the ?-ring of ?-carotene to produce di-hydroxylated zeaxanthin and the intermediate mono-hydroxylated?-cryptoxanthin.Over-expression of PuCHY1 in the Arabidopsis thaliana carotene hydroxylase mutant(chy2)resulted in an increase in the content of violaxanthin in the rosette leaves,as a result of endogenous zeaxanthin epoxidase function onto the accumulated zeaxanthin.The content of lutein and other carotenoids were largely unchanged.This confirmed the ?-ring hydroxylation activity of PuCHY1,which was then annotated as CYP97B29.This is the first carotene hydroxylase functionally characterized in red algae.The binding capability of these two enzyme proteins with their corresponding substrates were also studied by homolog modeling.PuGGPS showed a high structure similarity with GGPS and also geranyl diphosphate(GPP)synthase(GPS)from other organisms.Although PuCHY1 was found to be a ?-carotene hydroxylase,the ?-ring of?-carotene(?,?-carotene)was found to fit the catalytic pocket,showing a possible activity towards the ?-ring.Phylogenetic trees were constructed with sequences of both GGPS and carotene hydroxylases from different organisms,ranging from prokaryotic organisms to higher plants.Analysis on both enzymes suggested that P.umbilicalis might be evolved between the two endosymbiosis events.Its GGPS had high sequence similarity with that of Cyanidioschyzon merolae,a primitive single cell red alga,followed by Phaeodactylum tricornutum.These species formed a sister group of the green lineage.Sequence comparison of PuCHY1 revealed a highly conserved oxygen-binding motif between CYP97B and C subfamilies and a highly conserved heme-binding motif between the CYP97A and B subfamilies.Members of the CYP97B subfamily were found in higher plants,green algae,red algae and also so-called heterokonts,including diatoms,brown algae,from which neither members of the CYP97A nor CYP97C subfamilies were identified from their genomes.From this,we postulated a common ancestor of the CYP97A and CYP97C subfamilies with mainly ?-ring hydroxylation activity.In heterokonts,which were evolved after the secondary endosymbiosis,the separation of such an ancestor from CYP97B did not occur.The present study showed the importance of utilizing the information from the isoprenoid and the carotenoid metabolic pathways to decipher endosymbiosis events at the early phase of the origin and evolution of eukaryotic organisms.