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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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Effect of irradiance and nitrogen availability on the mRNA levels of the phytoene synthase (psy), phytoene desaturase (pds), lycopene β-cyclase (lcy-b), lycopene ε-cyclase (lcy-e), carotene β-hydroxylase (chyB) and β-carotene oxygenase (bkt) genes in C. zofingiensis. Culture conditions: low irradiance (20 µmol photons m−2 s−1) and nitrate replete (black line); low irradiance and nitrate deprivation (red line); high irradiance (300 µmol photons m−2 s−1) and nitrate replete (green line); high irradiance and nitrate deprivation (blue line). Error bars indicate the standard deviations of four independent measurements.
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marinedrugs-10-02069-f006: Effect of irradiance and nitrogen availability on the mRNA levels of the phytoene synthase (psy), phytoene desaturase (pds), lycopene β-cyclase (lcy-b), lycopene ε-cyclase (lcy-e), carotene β-hydroxylase (chyB) and β-carotene oxygenase (bkt) genes in C. zofingiensis. Culture conditions: low irradiance (20 µmol photons m−2 s−1) and nitrate replete (black line); low irradiance and nitrate deprivation (red line); high irradiance (300 µmol photons m−2 s−1) and nitrate replete (green line); high irradiance and nitrate deprivation (blue line). Error bars indicate the standard deviations of four independent measurements.

Mentions: C. zofingiensis cells were grown photoautotrophically at low irradiance (20 μmol photon m−2 s−1) and a nitrogen replete concentration (nitrate 20 mM), as indicated in Experimental Section, until the middle of the exponential phase. Cells were then kept in the dark for 18 h, in order to make the mRNA levels come down to basal values. After this dark period, cells were subjected to either low (20 μmol photon m−2 s−1) or high irradiance (300 μmol photon m−2 s−1), under either nitrogen replete or nitrogen-deprivation conditions. Under conditions of nitrogen replete, nitrate concentration was measured daily (data not shown) and the nitrate consumed by the cells was added to the cultures. The evolution with time of transcriptional expression of the carotenogenic gene lcy-e as well as psy, pds, lcy-b, chyB and bkt as affected by irradiance and nitrogen availability was monitored by qRT-PCR. Changes in content of both the primary carotenoids lutein and α-carotene, β-carotene and violaxanthin and the secondary carotenoids astaxanthin, canthaxanthin and zeaxanthin were also determined in order to correlate transcript levels with the biosynthesis of them. As shown in Figure 6, under nitrogen replete concentrations and both at low and high irradiance, the relative transcript levels of lcy-e increased significantly, attaining 14-fold higher values than basal ones after 48 h, decreasing thereafter. On the contrary, nitrogen starvation did not affect the transcript levels of lcy-e, registering similar values to basal ones at both low and high irradiance. As previously described [25], the transcriptional expression of the Czlcy-b gene increased under nitrogen starvation (4-fold higher than basal levels), attaining a maximum after 24 h at low irradiance and decreasing later, similar maximal values being reached later, at 48 h, at high irradiance and being kept with time. Under nitrogen replete, no difference in maximal transcript levels either at high or low irradiance was observed. In the case of psy, mRNA levels at low irradiance and nitrogen replete were similar to those in the dark, increasing by about 5-fold when irradiance was raised from 20 to 300 µmol photons m−2 s−1, both under nitrogen starvation and nitrogen replete, attaining a peak after 24 h and decreasing slightly with time. At low irradiance, nitrogen starvation enhanced psy transcript levels by 3-fold with respect to the basal level after 96 h. The pds transcript levels, regardless of nitrogen availability, were two-fold higher at low irradiance after 24 h and three-fold higher at high irradiance after 5 h, when compared to basal levels, decreasing with time in both cases. The relative mRNA levels of chyB were about 4- and 7-fold higher than basal levels at low and high irradiance, respectively, under nitrogen replete, attaining a peak after 24 h. Nitrate deprivation increased slightly the transcript levels of chyB at low irradiance, showing similar values at high light intensity. In the case of bkt, mRNA levels at low irradiance and nitrogen replete were similar to basal values, increasing by about 4-fold when irradiance was raised from low to high light intensity, attaining a peak after 24 h and remaining constant with time. At low irradiance, nitrogen starvation enhanced bkt transcript levels by 3-fold with respect to the basal level after 24–48 h, remaining constant thereafter. The highest values were attained at high irradiance and nitrogen deprivation, reaching mRNA levels of bkt 7-fold higher as compared to the basal ones after 24 h, decreasing with time.


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)

Effect of irradiance and nitrogen availability on the mRNA levels of the phytoene synthase (psy), phytoene desaturase (pds), lycopene β-cyclase (lcy-b), lycopene ε-cyclase (lcy-e), carotene β-hydroxylase (chyB) and β-carotene oxygenase (bkt) genes in C. zofingiensis. Culture conditions: low irradiance (20 µmol photons m−2 s−1) and nitrate replete (black line); low irradiance and nitrate deprivation (red line); high irradiance (300 µmol photons m−2 s−1) and nitrate replete (green line); high irradiance and nitrate deprivation (blue line). Error bars indicate the standard deviations of four independent measurements.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

marinedrugs-10-02069-f006: Effect of irradiance and nitrogen availability on the mRNA levels of the phytoene synthase (psy), phytoene desaturase (pds), lycopene β-cyclase (lcy-b), lycopene ε-cyclase (lcy-e), carotene β-hydroxylase (chyB) and β-carotene oxygenase (bkt) genes in C. zofingiensis. Culture conditions: low irradiance (20 µmol photons m−2 s−1) and nitrate replete (black line); low irradiance and nitrate deprivation (red line); high irradiance (300 µmol photons m−2 s−1) and nitrate replete (green line); high irradiance and nitrate deprivation (blue line). Error bars indicate the standard deviations of four independent measurements.
Mentions: C. zofingiensis cells were grown photoautotrophically at low irradiance (20 μmol photon m−2 s−1) and a nitrogen replete concentration (nitrate 20 mM), as indicated in Experimental Section, until the middle of the exponential phase. Cells were then kept in the dark for 18 h, in order to make the mRNA levels come down to basal values. After this dark period, cells were subjected to either low (20 μmol photon m−2 s−1) or high irradiance (300 μmol photon m−2 s−1), under either nitrogen replete or nitrogen-deprivation conditions. Under conditions of nitrogen replete, nitrate concentration was measured daily (data not shown) and the nitrate consumed by the cells was added to the cultures. The evolution with time of transcriptional expression of the carotenogenic gene lcy-e as well as psy, pds, lcy-b, chyB and bkt as affected by irradiance and nitrogen availability was monitored by qRT-PCR. Changes in content of both the primary carotenoids lutein and α-carotene, β-carotene and violaxanthin and the secondary carotenoids astaxanthin, canthaxanthin and zeaxanthin were also determined in order to correlate transcript levels with the biosynthesis of them. As shown in Figure 6, under nitrogen replete concentrations and both at low and high irradiance, the relative transcript levels of lcy-e increased significantly, attaining 14-fold higher values than basal ones after 48 h, decreasing thereafter. On the contrary, nitrogen starvation did not affect the transcript levels of lcy-e, registering similar values to basal ones at both low and high irradiance. As previously described [25], the transcriptional expression of the Czlcy-b gene increased under nitrogen starvation (4-fold higher than basal levels), attaining a maximum after 24 h at low irradiance and decreasing later, similar maximal values being reached later, at 48 h, at high irradiance and being kept with time. Under nitrogen replete, no difference in maximal transcript levels either at high or low irradiance was observed. In the case of psy, mRNA levels at low irradiance and nitrogen replete were similar to those in the dark, increasing by about 5-fold when irradiance was raised from 20 to 300 µmol photons m−2 s−1, both under nitrogen starvation and nitrogen replete, attaining a peak after 24 h and decreasing slightly with time. At low irradiance, nitrogen starvation enhanced psy transcript levels by 3-fold with respect to the basal level after 96 h. The pds transcript levels, regardless of nitrogen availability, were two-fold higher at low irradiance after 24 h and three-fold higher at high irradiance after 5 h, when compared to basal levels, decreasing with time in both cases. The relative mRNA levels of chyB were about 4- and 7-fold higher than basal levels at low and high irradiance, respectively, under nitrogen replete, attaining a peak after 24 h. Nitrate deprivation increased slightly the transcript levels of chyB at low irradiance, showing similar values at high light intensity. In the case of bkt, mRNA levels at low irradiance and nitrogen replete were similar to basal values, increasing by about 4-fold when irradiance was raised from low to high light intensity, attaining a peak after 24 h and remaining constant with time. At low irradiance, nitrogen starvation enhanced bkt transcript levels by 3-fold with respect to the basal level after 24–48 h, remaining constant thereafter. The highest values were attained at high irradiance and nitrogen deprivation, reaching mRNA levels of bkt 7-fold higher as compared to the basal ones after 24 h, decreasing with time.

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