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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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UPGMA tree analysis of the indicated plant, algal, cyanobacterial and bacterial lycopene cyclase amino acids sequences. Analysis was performed in MEGA5 [28]. The GenBank accession numbers for these species are shown. The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances correspond to the number of amino acid substitutions per site and were computed using the Poisson correction method. Numbers at nodes indicate bootstrap values calculated over 500 replicates.
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marinedrugs-10-02069-f004: UPGMA tree analysis of the indicated plant, algal, cyanobacterial and bacterial lycopene cyclase amino acids sequences. Analysis was performed in MEGA5 [28]. The GenBank accession numbers for these species are shown. The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances correspond to the number of amino acid substitutions per site and were computed using the Poisson correction method. Numbers at nodes indicate bootstrap values calculated over 500 replicates.

Mentions: The BlastP search results demonstrated that the cloned CzLCYe showed the highest overall homology sequence with other LCYe from green algae, such as Chlamydomonas reinhardtii and Volvox carteri (identity 66%, similarity 77%) and Auxenochlorella protothecoides and Chlorella variabilis (identity 62%, similarity 74%). The GC content of the Czlcy-e coding region was 53%, which was lower than that of C. reinhardtii and V. carteri (63%) or of A. protothecoides (61%). The phylogenetic analysis of lycopene ε- and β-cyclases from green algae, cyanobacteria, plants and bacteria is illustrated in Figure 4. Analysis was conducted in MEGA5 using UPGMA method [28]. The predicted CzLCYe forms a cluster with the LCYe of green algae, which are phylogenetically close to LCYe of plants (~44% of identity, and 61% similarity). The degree of homology was lower with the LCYb of green algae, including C. zofingiensis, and plants (about 39% identity and 55% similarity) and with both cyanobacterial cyclases (CRTLe and CRTLb) (around 35% identity and 51% similarity). As other algal LCYe, CzLCYe was distantly related to bacterial CRTY cyclases, sharing with them only a few conserved motifs and about 23% identity. The characteristic Rossmann or dinucleotide binding fold and two cyclase motifs, present in LCYe and LCYb of green algae and plants, CRTLe and CRTLb of cyanobacteria and CRTY of bacteria, were also identified in the LCYe of C. zofingiensis, between amino acids 187 and 215, 405 and 421, and 480 and 489, respectively. In addition, a leucine in a region near the C-terminus, which was demonstrated to determine ε-monocyclase activity, was also identified in LCYe of C. zofingiensis. One basic amino acid histidine or lysine in that position was found in lycopene ε-cyclases from Lactuca sativa and Prochlorococcus marinus MED4 both of them showing ε-bicyclase activity [29,30].


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)

UPGMA tree analysis of the indicated plant, algal, cyanobacterial and bacterial lycopene cyclase amino acids sequences. Analysis was performed in MEGA5 [28]. The GenBank accession numbers for these species are shown. The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances correspond to the number of amino acid substitutions per site and were computed using the Poisson correction method. Numbers at nodes indicate bootstrap values calculated over 500 replicates.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

marinedrugs-10-02069-f004: UPGMA tree analysis of the indicated plant, algal, cyanobacterial and bacterial lycopene cyclase amino acids sequences. Analysis was performed in MEGA5 [28]. The GenBank accession numbers for these species are shown. The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances correspond to the number of amino acid substitutions per site and were computed using the Poisson correction method. Numbers at nodes indicate bootstrap values calculated over 500 replicates.
Mentions: The BlastP search results demonstrated that the cloned CzLCYe showed the highest overall homology sequence with other LCYe from green algae, such as Chlamydomonas reinhardtii and Volvox carteri (identity 66%, similarity 77%) and Auxenochlorella protothecoides and Chlorella variabilis (identity 62%, similarity 74%). The GC content of the Czlcy-e coding region was 53%, which was lower than that of C. reinhardtii and V. carteri (63%) or of A. protothecoides (61%). The phylogenetic analysis of lycopene ε- and β-cyclases from green algae, cyanobacteria, plants and bacteria is illustrated in Figure 4. Analysis was conducted in MEGA5 using UPGMA method [28]. The predicted CzLCYe forms a cluster with the LCYe of green algae, which are phylogenetically close to LCYe of plants (~44% of identity, and 61% similarity). The degree of homology was lower with the LCYb of green algae, including C. zofingiensis, and plants (about 39% identity and 55% similarity) and with both cyanobacterial cyclases (CRTLe and CRTLb) (around 35% identity and 51% similarity). As other algal LCYe, CzLCYe was distantly related to bacterial CRTY cyclases, sharing with them only a few conserved motifs and about 23% identity. The characteristic Rossmann or dinucleotide binding fold and two cyclase motifs, present in LCYe and LCYb of green algae and plants, CRTLe and CRTLb of cyanobacteria and CRTY of bacteria, were also identified in the LCYe of C. zofingiensis, between amino acids 187 and 215, 405 and 421, and 480 and 489, respectively. In addition, a leucine in a region near the C-terminus, which was demonstrated to determine ε-monocyclase activity, was also identified in LCYe of C. zofingiensis. One basic amino acid histidine or lysine in that position was found in lycopene ε-cyclases from Lactuca sativa and Prochlorococcus marinus MED4 both of them showing ε-bicyclase activity [29,30].

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