Functional Characterization of the First Bona Fide Phytoene Synthase in Red Algae from Pyropia yezoensis.

The formation of phytoene by condensing two geranylgeranyl diphosphate molecules catalyzed by phytoene synthase (PSY) is the first committed and rate-limiting step in carotenoid biosynthesis, which has been extensively investigated in bacteria, land plants and microalgae. However, this step in macroalgae remains unknown. In the present study, a gene encoding putative phytoene synthase was cloned from the economic red alga Pyropia yezoensis-a species that has long been used in food and pharmaceuticals. The conservative motifs/domains and the tertiary structure predicted using bioinformatic tools suggested that the cloned PyPSY should encode a phytoene synthase; this was empirically confirmed by pigment complementation in E. coli. This phytoene synthase was encoded by a single copy gene, whose expression was presumably regulated by many factors. The phylogenetic relationship of PSYs from different organisms suggested that red algae are probably the progeny of primary endosymbiosis and plastid donors of secondary endosymbiosis.

Redefining Pyropia (Bangiales, Rhodophyta): Four New Genera, Resurrection of Porphyrella and Description of Calidia pseudolobata sp. nov. From China.

The cosmopolitan red algal genus Pyropia sensu lato is the most speciose of the bladed Bangiales genera. In a major revision of the Bangiales, Pyropia was resurrected from Porphyra, although there was evidence at the time that species of Pyropia could be separated into several genera. Subsequent global phylogenetic analyses continued to resolve species assigned to Pyropia into several major clades with strong support, and the latest biogeographic analyses indicated that species distribution was also a pointer to the underlying phylogeny of Pyropia sensu lato. Therefore, in the present study, we have redefined the genus Pyropia, resurrected Porphyrella, and proposed four new genera: Calidia, Neoporphyra, Neopyropia, and Uedaea. Based on a molecular phylogenetic study of the bladed Bangiales of China, a species which did not match any known taxa was resolved in the new genus Calidia. The species, Calidia pseudolobata sp. nov., is described based on both morphological and molecular data. Molecular sequence data for rbcL, 18S, and COI-5P were amplified for 15 samples in the present study. All the obtained rbcL sequences were identical to each other except for one (LYCN117) with one base pair difference. Two haplotypes of 18S (V9 region) were observed with one base pair difference (C/T30 ). All the obtained COI-5P sequences were identical. Morphological comparisons were conducted not only with species in Calidia, but also with generically uncertain species currently assigned to Porphyra.

A molecular phylogeny of the bladed Bangiales (Rhodophyta) in China provides insights into biodiversity and biogeography of the genus Pyropia.

A molecular taxonomic study was undertaken for the first time of the bladed Bangiales of the mainland coast of China (Northwest Pacific) based on sequence data of 201 plastid rbcL and 148 nuclear 18S sequences of historical and contemporary specimens. The results revealed that only one genus of bladed Bangiales, Pyropia, was present along Chinese coast. Species delimitation was determined using two empirical methods: the Automatic Barcode Gap Discovery (ABGD) and General Mixed Yule Coalescence (GMYC) coupled with detection of monophyly in tree reconstruction. At least fourteen species of Pyropia were recovered. Six species were confirmed that had been recorded previously based on morphology (Py. suborbiculata, Py. yezoensis, Py. haitanensis, Py. katadae, Py. tenera and Py. acanthophora), three species were recorded from China for the first time (Py. kinositae, Py. pseudolinearis and Py. tanegashimensis), and five cryptic species that did not match any molecular sequences were also discovered. The phylogeny of the concatenated rbcL and 18S dataset resolved three singletons and four clades. Each clades has a strong trend towards occupying a biogeographic region, but they are not confined to them. A transoceanic and antitropical pattern of distribution was found for Pyropia at both the subgeneric and species level. This together with high biodiversity (ca. 30% of all known Pyropia species) indicates that the Northwest Pacific might act as a centre of origin for modern distribution of Pyropia since the early Cenozoic.

The P450-type carotene hydroxylase PuCHY1 from Porphyra suggests the evolution of carotenoid metabolism in red algae.

Carotene hydroxylases catalyze the hydroxylation of α- and ß-carotene hydrocarbons into xanthophylls. In red algae, ß-carotene is a ubiquitously distributed carotenoid, and hydroxylated carotenoids such as zeaxanthin and lutein are also found. However, no enzyme with carotene hydroxylase activity had been previously identified in red algae. Here, we report the isolation of a gene encoding a cytochrome P450-type carotene hydroxylase (PuCHY1) from Porphyra umbilicalis, a red alga with an ancient origin. Sequence comparisons found PuCHY1 belongs to the CYP97B subfamily, which has members from different photosynthetic organisms ranging from red algae to land plants. Functional complementation in Escherichia coli suggested that PuCHY1 catalyzed the conversion from ß-carotene to zeaxanthin. When we overexpressed PuCHY1 in the Arabidopsis thaliana chy2 mutant, pigment analysis showed a significant accumulation of hydroxylated carotenoids, including neoxanthin, violaxanthin, and lutein in the leaves of transgenic plants. These results confirmed a ß-hydroxylation activity of PuCHY1, and also suggested a possible ϵ-hydroxylation function. The pigment profile and gene expression analyses of the algal thallus under high-light stress suggested that P. umbilicalis is unlikely to operate a partial xanthophyll cycle for photoprotection.

Functional Characterization of Lycopene Cyclases Illustrates the Metabolic Pathway toward Lutein in Red Algal Seaweeds.

Carotenoids are essential phytonutrients synthesized by all photosynthetic organisms. Acyclic lycopene is the first branching point for carotenoid biosynthesis. Lycopene ß- and ε-cyclases (LCYB and LCYE, respectively) catalyze the cyclization of its open ends and direct the metabolic flux into different downstream branches. Carotenoids of the ß,ß-branch (e.g., ß-carotene) are found in all photosynthetic organisms, but those of the ß,ε-branch (e.g., lutein) are generally absent in cyanobacteria, heterokonts, and some red algae. Although both LCYBs and LCYEs have been characterized from land plants, there are only a few reports on LCYs from cyanobacteria and algae. Here, we cloned four LCY genes from Porphyra umbilicalis and Pyropia yezoensis (susabi-nori) of Bangiales, the most primitive red algal order that synthesizes lutein. Our functional characterization in both Escherichia coli and Arabidopsis thaliana demonstrated that each species has a pair of LCYB and LCYE. Similar to LCYs from higher plants, red algal LCYBs cyclize both ends of lycopene, and their LCYEs only cyclize a single end. The characterization of LCYEs from red algae resolved the first bifurcation step toward ß-carotene and lutein biosynthesis. Our phylogenetic analysis suggests that LCYEs of the green lineage and the red algae originated separately during evolution.