A functional zeaxanthin epoxidase from red algae shedding light on the evolution of light‐harvesting carotenoids and the xanthophyll cycle in photosynthetic eukaryotes

Abstract

The epoxy-xanthophylls antheraxanthin and violaxanthin are key precursors of light-harvesting carotenoids and participate in the photoprotective xanthophyll cycle. Thus, the invention of zeaxanthin epoxidase (ZEP) catalyzing their formation from zeaxanthin has been a fundamental step in the evolution of photosynthetic eukaryotes. ZEP genes have only been found in Viridiplantae and chromalveolate algae with secondary plastids of red algal ancestry, suggesting that ZEP evolved in the Viridiplantae and spread to chromalveolates by lateral gene transfer. By searching publicly available sequence data from 11 red algae covering all currently recognized red algal classes we identified ZEP candidates in three species. Phylogenetic analyses showed that the red algal ZEP is most closely related to ZEP proteins from photosynthetic chromalveolates possessing secondary plastids of red algal origin. Its enzymatic activity was assessed by high performance liquid chromatography (HPLC) analyses of red algal pigment extracts and by cloning and functional expression of the ZEP gene from Madagascaria erythrocladioides in leaves of the ZEP-deficient aba2 mutant of Nicotiana plumbaginifolia. Unlike other ZEP enzymes examined so far, the red algal ZEP introduces only a single epoxy group into zeaxanthin, yielding antheraxanthin instead of violaxanthin. The results indicate that ZEP evolved before the split of Rhodophyta and Viridiplantae and that chromalveolates acquired ZEP from the red algal endosymbiont and not by lateral gene transfer. Moreover, the red algal ZEP enables engineering of transgenic plants incorporating antheraxanthin instead of violaxanthin in their photosynthetic machinery.

Lipid Dependence of Xanthophyll Cycling in Higher Plants and Algae

“…In the red algae Graciliaria gracilis or G. multipartite an absence of Vx was detected and Ax seems to be the only substrate which can be converted to Zx (Bertrand and Schoefs, 1999). Despite the ongoing uncertainties about the presence of a xanthophyll cycle in Rodophyta, a recent study has presented clear evidence that at least a Zx epoxidase, which is able to convert Zx to Ax, is present in the red algae (Dautermann and Lohr, 2017).…”

An algal enzyme required for biosynthesis of the most abundant marine carotenoids

“…Therefore, we validated the predictions for VDL by also analyzing the VDE sequences from 17 chromalveolates and 4 green plants/algae, with the expected targeting elements for luminal localization predicted in all investigated proteins (data file S1). (27) led to a much lower accumulation of neoxanthin in tobacco leaves compared with luminal targeting (table S2), indicating a higher activity of the enzyme at the more acidic pH in the thylakoid lumen. In leaves transformed with PtVDL1, the concentration of 9′cisneoxanthin also increased significantly by about 26 mmol/ mol Chl a for lumentargeted PtVDL1 (P = 2 × 10 −7 ) and by about 16 mmol/mol Chl a for stromatargeted PtVDL1 (P = 3 × 10 −6 ), suggesting that part of the neoxanthin generated by PtVDL1 may have been converted to 9′cisneoxanthin by an endogenous enzyme ( Fig.…”

“…us/#/projects/104), and the Online Resource for Community Annota tion of Eukaryotes (http://bioinformatics.psb.ugent.be/orcae/) were searched with the BLAST tool (45) using the amino acid sequences of the VDE family members from P. tricornutum (17) as input. MMETSP mining, protein alignments, and phylogenetic analyses using maximum likelihood methods with bootstrapping were performed as previously described (27). Accessions of sequence data used for cloning of algal genes and for in silico analyses are compiled in data file S2.…”

“…Gene fragments encoding the mature proteins were placed inframe behind a gene fragment encoding the Nterminal targeting sequence of the VDE from N. tabacum (tp NtVDE = amino acid positions 1 to 134) for targeting of the expression prod ucts to the thylakoid lumen of tobacco plastids. In the case of the VDL1 from P. tricornutum, the gene fragment was also placed behind a gene fragment encoding the targeting sequence of the zeaxanthin epoxidase from A. thaliana (tp AtZEP ) for targeting of the protein to the plastid stroma as previously described (27). Further details on PCR templates and primers, cloning strategies, and protein fragments en coded in the final expression constructs are given in data file S3.…”

Comparative Analysis of MicroRNA Expression in Three Paulownia Species with Phytoplasma Infection

“…Another DEM, MiR5368g-5p, was down-regulated in PTI and PTFI seedlings. miR5368g-5p was predicted to target ZEP, which is the key enzyme responsible for the accumulation of antheraxanthin and violaxanthin [66]. Yang, et al [67] reported that a high expression of ZEP increased the stored carotenoid pigments in transgenic tomato plants.…”