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.

Carotene hydroxylases catalyze the hydroxylation of a-and b-carotene hydrocarbons into xanthophylls. In red algae, b-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 b-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 b-hydroxylation activity of PuCHY1, and also suggested a possible e-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.Keywords: Bangiales; carotene hydroxylase; carotenoid metabolism; CYP97B; cytochrome P450; Porphyra umbilicalis; red algae Citation: Yang LE, Huang XQ, Hang Y, Deng YY, Lu QQ, Lu S (2014) The P450-type carotene hydroxylase PuCHY1 from Porphyra suggested the evolution of carotenoid metabolism in red algae. J Integr Plant Biol 56: 902-915.

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Carotenoids are essential phytonutrients synthesized by all photosynthetic organisms. Acyclic lycopene is the first branching point for carotenoid biosynthesis. Lycopene βand 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 β,β-branch (e.g., β-carotene) are found in all photosynthetic organisms, but those of the β,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 β-carotene and lutein biosynthesis. Our phylogenetic analysis suggests that LCYEs of the green lineage and the red algae originated separately during evolution.

Lycopene cyclases cyclize the open ends of acyclic lycopene (ψ,ψ-carotene) into β- or ε-ionone rings in the crucial bifurcation step of carotenoid biosynthesis. Among all carotenoid constituents, β-carotene (β,β-carotene) is found in all photosynthetic organisms, except for purple bacteria and heliobacteria, suggesting a ubiquitous distribution of lycopene β-cyclase activity in these organisms. In this work, we isolated a gene (BfLCYB) encoding a lycopene β-cyclase from Bangia fuscopurpurea, a red alga that is considered to be one of the primitive multicellular eukaryotic photosynthetic organisms and accumulates carotenoid constituents with both β- and ε-rings, including β-carotene, zeaxanthin, α-carotene (β,ε-carotene) and lutein. Functional complementation in Escherichia coli demonstrated that BfLCYB is able to catalyze cyclization of lycopene into monocyclic γ-carotene (β,ψ-carotene) and bicyclic β-carotene, and cyclization of the open end of monocyclic δ-carotene (ε,ψ-carotene) to produce α-carotene. No ε-cyclization activity was identified for BfLCYB. Sequence comparison showed that BfLCYB shares conserved domains with other functionally characterized lycopene cyclases from different organisms and belongs to a group of ancient lycopene cyclases. Although B. fuscopurpurea also synthesizes α-carotene and lutein, its enzyme-catalyzing ε-cyclization is still unknown.

Cytological observations and sequence analyses of genes for the plastid RUBISCO LSU (rbcL) and nuclear SSU ribosomal RNA (nrSSU) were carried out on filamentous Bangiales from China, including 11 marine samples and one freshwater sample. Our results showed that the marine samples of M2S, M(6-8)J, M(9-10)F and M11G have both sexual and asexual reproduction, while the M1L, M(3-5)S and FS samples only reproduce asexually. All marine and freshwater samples have the consistent chromosome numbers (n=4). DNA sequence analysis showed that M1L, M3S, M(6-8)J, M(9-10)F and M11G samples shared high similarities of more than 97.8 and 99.6 % for rbcL and nrSSU, respectively, whereas those of M(2, 4, 5)S samples shared lower similarities with other marine samples (89.9-90.1 %; 92.0-93.2 %). Phylogenetic analysis revealed that the marine samples were clustered in "Bangia" 1 and "Bangia" 2 with strong bootstrap support, suggesting there are at least two marine species of filamentous Bangiales in China. The freshwater FS sample had significant low sequence similarity in the two genes studied with the marine samples (89.4-92.5 %; 88.3-90.9 %), but was well resolved in the clade of Bangia with European freshwater samples. Although the chromosome number of FS was different from other freshwater populations from North America and Europe (n=3), the FS sample is a member of Bangia according to its habitat, reproductive mode and phylogenetic analyses.

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