Gene cloning and expression profile of a novel carotenoid hydroxylase (CYP97C) from the green alga Haematococcus pluvialis

Abstract

A full-length complementary DNA (cDNA) sequence of ε-ring CHY (designated Haecyp97c) was cloned from the green alga Haematococcus pluvialis by reverse transcription polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends methods. The Haecyp97c cDNA sequence was 1,995 base pairs (bp) in length, which contained a 1,620-bp open reading frame, a 46-bp 5′-untranslated region (UTR), and a 329-bp 3′-UTR with the characteristic of the poly (A) tail. The deduced protein had a calculated molecular mass of 58.71 kDa with an estimated isoelectric point of 7.94. Multiple alignment analysis revealed that the deduced amino acid sequence of HaeCYP97C shared high identity of 72-85 % with corresponding CYP97Cs from other eukaryotes. The catalytic motifs of cytochrome P450s were detected in the amino acid sequence of HaeCYP97C. The transcriptional levels of Haecyp97c and xanthophylls accumulation under high light (HL) stress have been examined. The results revealed that Haecyp97c transcript was strongly increased after 13-28 h under HL stress. Meanwhile, the concentrations of chlorophylls, carotenes, and lutein were decreased, and zeaxanthin and astaxanthin concentrations were increased rapidly, respectively. These facts indicated that HaeCYP97C was perhaps involved in xanthophyll biosynthesis, which plays an important role in adaption to HL for H. pluvialis.

Evolutionary origins, molecular cloning and expression of carotenoid hydroxylases in eukaryotic photosynthetic algae

“…To further study genes encoding CYP97 homologs in green algae, three full-length cDNA sequences of cyp97 homologs, including Haecyp97a , Haecyp97b , and Haecyp97c [84] were isolated from commercial green alga H. pluvialis strain Flotow 1844. Briefly, 1,017- and 984-bp cDNA fragments encoding HaeCYP97A/B were generated by RT-PCR with degenerate primers [see Additional file 5: Table S3].…”

“…A similar, although less pronounced trend was observed under white HL: the highest bch transcriptional level occurred at 54 h of exposure, with 3.3-fold higher compared with the control. Our previous study has demonstrated that zeaxanthin concentrations under blue and white HL treatments were increased markedly and reached their highest levels at 34 h (blue) and 48 h (white) of exposure [84], which is listed in Figure S5 for details [see Additional file 9: Figure S5]. BCH is the mainly enzyme catalyzing the hydroxylation of β -carotene, which produces zeaxanthin associated with the xanthophylls cycle [40,87].…”

“…The contradiction between low bch expression levels and marked increase of zeaxanthin concentration during the early stage of treatments was explained by the fact that when photoprotection is required, violaxanthin is rapidly converted via antheraxanthin to zeaxanthin by violaxanthin de-epoxidase [88]. The astaxanthin concentration under blue or white HL stress was higher than that of the control, reaching a maximum level at 34 h and 54 h of exposure, respectively [84], which is listed in Figure S5 for details [see Additional file 9: Figure S5]. Accumulation of astaxanthin may be responsible for the rapidly increasing levels of bch and sharply decreasing concentrations of zeaxanthin at later treatment stages.…”

“…Studies on mutant of higher plants showed that CYP97A3 and CYP97C1 are the enzymes primarily responsible for catalyzing hydroxylation of β - and ϵ -ring of α -carotene, respectively, producing α -branch xanthophylls (lutein), the most abundant carotenoids in light-harvesting complexes (LHCs) which is key structural and functional components of light harvesting [6,8,26,33,37,39,59,72]. During early stages (0–4 h) of HL (blue and white) treatments, lutein concentrations were lower than that of the control [84], which is listed in Figure S5 for details [see Additional file 9: Figure S5]. This result is consistent with the decreased transcriptional levels of Haecyp97a and Haecyp97c observed in H. pluvialis (Figure 6B and D).…”

Sequencing and Characterization of Novel PII Signaling Protein Gene in Microalga Haematococcus pluvialis

“…Driven by their nutrition condition, H. pluvialis cells have two different physiological traits: (1) in favorable conditions they are in a green motile stage; (2) under stress conditions (especially nitrogen depletion) the green cells will transform into a reddish non-motile resting stage, coupled with astaxanthin accumulation [19,20,21]. Several key genes related to astaxanthin biosynthesis and stress responding have been cloned and characterized, such as pds [22], CYP97C [23], MnSOD [24], and TR1 [25]. Despite many reports on astaxanthin accumulation of H. pluvialis upon nitrogen depletion [26,27,28], there is no information of its PII protein and associated genetic transcription information on this unicellular microalga.…”