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Isolation and characterization of a lycopene ε-cyclase gene of Chlorella (Chromochloris) zofingiensis. Regulation of the carotenogenic pathway by nitrogen and light.

Cordero BF, Couso I, Leon R, Rodriguez H, Vargas MA - Mar Drugs (2012)

Bottom Line: A single copy of Czlcy-e was found in C. zofingiensis.High irradiance stress did not increase mRNA levels of neither lycopene β-cyclase gene (lcy-b) nor lycopene ε-cyclase gene (lcy-e) as compared with low irradiance conditions, whereas the transcript levels of psy, pds, chyB and bkt genes were enhanced, nevertheless triggering the synthesis of the secondary carotenoids astaxanthin, canthaxanthin and zeaxanthin and decreasing the levels of the primary carotenoids α-carotene, lutein, violaxanthin and β-carotene.The combined effect of both high light and nitrogen starvation stresses enhanced significantly the accumulation of these carotenoids as well as the transcript levels of bkt gene, as compared with the effect of only high irradiance stress.

View Article: PubMed Central - PubMed

Affiliation: Institute of Plant Biochemistry and Photosynthesis, CIC Cartuja, University of Seville and CSIC, Avda. Americo Vespucio, n° 49, Seville 41092, Spain. baldomero@ibvf.csic.es

ABSTRACT
The isolation and characterization of the lycopene ε-cyclase gene from the green microalga Chlorella (Chromochloris) zofingiensis (Czlcy-e) was performed. This gene is involved in the formation of the carotenoids α-carotene and lutein. Czlcy-e gene encoded a polypeptide of 654 amino acids. A single copy of Czlcy-e was found in C. zofingiensis. Functional analysis by heterologous complementation in Escherichia coli showed the ability of this protein to convert lycopene to δ-carotene. In addition, the regulation of the carotenogenic pathway by light and nitrogen was also studied in C. zofingiensis. High irradiance stress did not increase mRNA levels of neither lycopene β-cyclase gene (lcy-b) nor lycopene ε-cyclase gene (lcy-e) as compared with low irradiance conditions, whereas the transcript levels of psy, pds, chyB and bkt genes were enhanced, nevertheless triggering the synthesis of the secondary carotenoids astaxanthin, canthaxanthin and zeaxanthin and decreasing the levels of the primary carotenoids α-carotene, lutein, violaxanthin and β-carotene. Nitrogen starvation per se enhanced mRNA levels of all genes considered, except lcy-e and pds, but did not trigger the synthesis of astaxanthin, canthaxanthin nor zeaxanthin. The combined effect of both high light and nitrogen starvation stresses enhanced significantly the accumulation of these carotenoids as well as the transcript levels of bkt gene, as compared with the effect of only high irradiance stress.

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Czlcy-e gene organization. The diagram shows that the Czlcy-e gene consists of eight exons (I-VIII) and seven introns (1-7). The 5′ UTR and 3′ UTR sequences are indicated with arrows. Numbers indicate cDNA sequence coordinates (bp). UTR, unstranslated region.
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marinedrugs-10-02069-f002: Czlcy-e gene organization. The diagram shows that the Czlcy-e gene consists of eight exons (I-VIII) and seven introns (1-7). The 5′ UTR and 3′ UTR sequences are indicated with arrows. Numbers indicate cDNA sequence coordinates (bp). UTR, unstranslated region.

Mentions: Different pairs of degenerate primers were designed on the basis of the conserved motifs present in lcy-e from microalgae. A partial cDNA fragment of 669 bp was isolated by PCR amplification using degenerate primers (lcy-e-1F and lcy-e-1R) (Table 1). A complete BLAST homology searches in the Genbank database showed that this fragment had enough similarity with the lcy-e gene from other species and provided sequence information for designing specific primers for rapid amplification of 5′ and 3′ cDNA ends (RACE-PCR). This analysis generated a full-length cDNA of 2204 bp, which contained an ORF of 1965 bp, 2-nucleotides of 5′-untranslated region (UTR), and a long 3′ UTR of 237 nucleotides. A typical algal polyadenylation signal TGTAAA [26] was present in the 3′ UTR at 82 nucleotides upstream from the beginning of the poly(A) tail. The predicted protein has 654 amino acids residues, with an estimated molecular weight of 71.1 kDa, a theoretical isoelectric point of 8.45 and an instability index of 43.6 (data obtained with ProtParam program, [27]). The differences between the C. zofingiensis lcy-e gene and the cDNA sequence were compared and revealed the presence of 8 exons and 7 introns. Exon size ranged between 124 (exon III) and 488 bp (exon I), and intron size between 159 (intron 1) and 331 bp (intron 3). Approximately 56% (2.2 out of 3.9 kb) of the Czlcy-e gene corresponded to exon sequences. In all introns, the consensus GT donor and AG acceptor sequences at the 5′ and 3′ termini were found (Figure 2).


Isolation and characterization of a lycopene ε-cyclase gene of Chlorella (Chromochloris) zofingiensis. Regulation of the carotenogenic pathway by nitrogen and light.

Cordero BF, Couso I, Leon R, Rodriguez H, Vargas MA - Mar Drugs (2012)

Czlcy-e gene organization. The diagram shows that the Czlcy-e gene consists of eight exons (I-VIII) and seven introns (1-7). The 5′ UTR and 3′ UTR sequences are indicated with arrows. Numbers indicate cDNA sequence coordinates (bp). UTR, unstranslated region.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3475274&req=5

marinedrugs-10-02069-f002: Czlcy-e gene organization. The diagram shows that the Czlcy-e gene consists of eight exons (I-VIII) and seven introns (1-7). The 5′ UTR and 3′ UTR sequences are indicated with arrows. Numbers indicate cDNA sequence coordinates (bp). UTR, unstranslated region.
Mentions: Different pairs of degenerate primers were designed on the basis of the conserved motifs present in lcy-e from microalgae. A partial cDNA fragment of 669 bp was isolated by PCR amplification using degenerate primers (lcy-e-1F and lcy-e-1R) (Table 1). A complete BLAST homology searches in the Genbank database showed that this fragment had enough similarity with the lcy-e gene from other species and provided sequence information for designing specific primers for rapid amplification of 5′ and 3′ cDNA ends (RACE-PCR). This analysis generated a full-length cDNA of 2204 bp, which contained an ORF of 1965 bp, 2-nucleotides of 5′-untranslated region (UTR), and a long 3′ UTR of 237 nucleotides. A typical algal polyadenylation signal TGTAAA [26] was present in the 3′ UTR at 82 nucleotides upstream from the beginning of the poly(A) tail. The predicted protein has 654 amino acids residues, with an estimated molecular weight of 71.1 kDa, a theoretical isoelectric point of 8.45 and an instability index of 43.6 (data obtained with ProtParam program, [27]). The differences between the C. zofingiensis lcy-e gene and the cDNA sequence were compared and revealed the presence of 8 exons and 7 introns. Exon size ranged between 124 (exon III) and 488 bp (exon I), and intron size between 159 (intron 1) and 331 bp (intron 3). Approximately 56% (2.2 out of 3.9 kb) of the Czlcy-e gene corresponded to exon sequences. In all introns, the consensus GT donor and AG acceptor sequences at the 5′ and 3′ termini were found (Figure 2).

Bottom Line: A single copy of Czlcy-e was found in C. zofingiensis.High irradiance stress did not increase mRNA levels of neither lycopene β-cyclase gene (lcy-b) nor lycopene ε-cyclase gene (lcy-e) as compared with low irradiance conditions, whereas the transcript levels of psy, pds, chyB and bkt genes were enhanced, nevertheless triggering the synthesis of the secondary carotenoids astaxanthin, canthaxanthin and zeaxanthin and decreasing the levels of the primary carotenoids α-carotene, lutein, violaxanthin and β-carotene.The combined effect of both high light and nitrogen starvation stresses enhanced significantly the accumulation of these carotenoids as well as the transcript levels of bkt gene, as compared with the effect of only high irradiance stress.

View Article: PubMed Central - PubMed

Affiliation: Institute of Plant Biochemistry and Photosynthesis, CIC Cartuja, University of Seville and CSIC, Avda. Americo Vespucio, n° 49, Seville 41092, Spain. baldomero@ibvf.csic.es

ABSTRACT
The isolation and characterization of the lycopene ε-cyclase gene from the green microalga Chlorella (Chromochloris) zofingiensis (Czlcy-e) was performed. This gene is involved in the formation of the carotenoids α-carotene and lutein. Czlcy-e gene encoded a polypeptide of 654 amino acids. A single copy of Czlcy-e was found in C. zofingiensis. Functional analysis by heterologous complementation in Escherichia coli showed the ability of this protein to convert lycopene to δ-carotene. In addition, the regulation of the carotenogenic pathway by light and nitrogen was also studied in C. zofingiensis. High irradiance stress did not increase mRNA levels of neither lycopene β-cyclase gene (lcy-b) nor lycopene ε-cyclase gene (lcy-e) as compared with low irradiance conditions, whereas the transcript levels of psy, pds, chyB and bkt genes were enhanced, nevertheless triggering the synthesis of the secondary carotenoids astaxanthin, canthaxanthin and zeaxanthin and decreasing the levels of the primary carotenoids α-carotene, lutein, violaxanthin and β-carotene. Nitrogen starvation per se enhanced mRNA levels of all genes considered, except lcy-e and pds, but did not trigger the synthesis of astaxanthin, canthaxanthin nor zeaxanthin. The combined effect of both high light and nitrogen starvation stresses enhanced significantly the accumulation of these carotenoids as well as the transcript levels of bkt gene, as compared with the effect of only high irradiance stress.

Show MeSH
Related in: MedlinePlus