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Efficient heterologous transformation of Chlamydomonas reinhardtii npq2 mutant with the zeaxanthin epoxidase gene isolated and characterized from Chlorella zofingiensis.

Couso I, Cordero BF, Vargas MÁ, Rodríguez H - Mar Drugs (2012)

Bottom Line: The Czzep gene was adequately inserted in the pSI105 vector and expressed in npq2.The positive transformants were able to efficiently convert zeaxanthin into violaxanthin, as well as to restore their maximum quantum efficiency of the PSII (Fv/Fm).These results show that Chlamydomonas can be an efficient tool for heterologous expression and metabolic engineering for biotechnological applications.

View Article: PubMed Central - PubMed

Affiliation: Institute of Plant Biochemistry and Photosynthesis, CIC Cartuja, University of Seville and CSIC, Avda. Américo Vespucio no. 49, 41092-Seville, Spain. inmaculada.couso@ibvf.csic.es

ABSTRACT
In the violaxanthin cycle, the violaxanthin de-epoxidase and zeaxanthin epoxidase catalyze the inter-conversion between violaxanthin and zeaxanthin in both plants and green algae. The zeaxanthin epoxidase gene from the green microalga Chlorella zofingiensis (Czzep) has been isolated. This gene encodes a polypeptide of 596 amino acids. A single copy of Czzep has been found in the C. zofingiensis genome by Southern blot analysis. qPCR analysis has shown that transcript levels of Czzep were increased after zeaxanthin formation under high light conditions. The functionality of Czzep gene by heterologous genetic complementation in the Chlamydomonas mutant npq2, which lacks zeaxanthin epoxidase (ZEP) activity and accumulates zeaxanthin in all conditions, was analyzed. The Czzep gene was adequately inserted in the pSI105 vector and expressed in npq2. The positive transformants were able to efficiently convert zeaxanthin into violaxanthin, as well as to restore their maximum quantum efficiency of the PSII (Fv/Fm). These results show that Chlamydomonas can be an efficient tool for heterologous expression and metabolic engineering for biotechnological applications.

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Effect of irradiance and nitrogen availability on the mRNA levels of the zeaxanthin epoxidase gene (zep) and cellular content of violaxanthin and zeaxanthin in Chlorella zofingiensis. (a) Low irradiance (20 µmol photons m−2 s−1) and constant nitrate concentration (LL +N); (b) low irradiance and nitrate deprivation (LL −N); (c) high irradiance (300 µmol photons m−2 s−1) and constant nitrate concentration (HL +N); and (d) high irradiance and nitrate deprivation (HL −N). Columns indicate mRNA levels of the zep gene; (♦), violaxanthin content (mg g−1 DW); (□), zeaxanthin content (mg g−1 DW). Error bars indicate the standard deviations of four independent measurements. DW, dry weight.
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marinedrugs-10-01955-f004: Effect of irradiance and nitrogen availability on the mRNA levels of the zeaxanthin epoxidase gene (zep) and cellular content of violaxanthin and zeaxanthin in Chlorella zofingiensis. (a) Low irradiance (20 µmol photons m−2 s−1) and constant nitrate concentration (LL +N); (b) low irradiance and nitrate deprivation (LL −N); (c) high irradiance (300 µmol photons m−2 s−1) and constant nitrate concentration (HL +N); and (d) high irradiance and nitrate deprivation (HL −N). Columns indicate mRNA levels of the zep gene; (♦), violaxanthin content (mg g−1 DW); (□), zeaxanthin content (mg g−1 DW). Error bars indicate the standard deviations of four independent measurements. DW, dry weight.

Mentions: C. zofingiensis cells were grown photoautotrophically at low irradiance and constant nitrate, as indicated in the Experimental Section, until the middle of the exponential phase. Then, cells were kept in the dark for 18 h, in order to make the transcript levels come down to basal values. After this dark period, cells were subjected to either low or high irradiance, under either constant nitrogen or nitrogen-deprivation conditions. The evolution with time of mRNA levels of the Czzep gene as affected by irradiance and nitrogen availability was monitored by quantitative real time PCR (qRT-PCR). In addition, changes in zeaxanthin and violaxanthin cellular contents were also determined in order to correlate mRNA levels with the biosynthesis of these carotenoids. As shown in Figure 4a,b, at low irradiance the levels of transcripts did not start to increase until 24 h after the dark period, regardless of nitrate availability. Under high irradiance and constant nitrogen conditions, the transcript levels of Czzep decreased between 10% and 60% after 5 h and 10 h from the beginning of the experiment, respectively (Figure 4c). Although there was also a high increase of the transcripts levels after 24 h under these conditions, they did not reach the levels registered at low irradiance. However, an additive response was detected in the nitrogen-free culture under high light conditions (Figure 4d), transcript levels after 24 h of induction being higher than those exhibited under low irradiance. With regards to the cellular carotenoid content, zeaxanthin was only registered at high irradiance, the levels of this carotenoid increased with time after 10 h, with a much higher accumulation of this carotenoid taking place under nitrogen depletion conditions (Figure 4c,d). Antheraxanthin was only detected at high irradiance as well, its levels ranging from 0.12 mg∙g−1 DW at 10 h to 0.09 mg g−1 DW at 72 h of high light exposure. Both zeaxanthin and antheraxanthin contents seem to be correlated with the increased mRNA levels of Czzep. The concentration of violaxanthin decreased with time at high irradiance, especially in the case of nitrogen-free conditions, while in the case of low light conditions it remained almost constant and only a little increase was detected at the 10 h or 24 h under nitrogen deprivation. Under high irradiance, astaxanthin was also present both in nitrogen depleted and in nitrogen repleted conditions, reaching values of 0.21 mg g−1 DW and 0.5 mg g−1 DW after 48 h, respectively.


Efficient heterologous transformation of Chlamydomonas reinhardtii npq2 mutant with the zeaxanthin epoxidase gene isolated and characterized from Chlorella zofingiensis.

Couso I, Cordero BF, Vargas MÁ, Rodríguez H - Mar Drugs (2012)

Effect of irradiance and nitrogen availability on the mRNA levels of the zeaxanthin epoxidase gene (zep) and cellular content of violaxanthin and zeaxanthin in Chlorella zofingiensis. (a) Low irradiance (20 µmol photons m−2 s−1) and constant nitrate concentration (LL +N); (b) low irradiance and nitrate deprivation (LL −N); (c) high irradiance (300 µmol photons m−2 s−1) and constant nitrate concentration (HL +N); and (d) high irradiance and nitrate deprivation (HL −N). Columns indicate mRNA levels of the zep gene; (♦), violaxanthin content (mg g−1 DW); (□), zeaxanthin content (mg g−1 DW). Error bars indicate the standard deviations of four independent measurements. DW, dry weight.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC3475266&req=5

marinedrugs-10-01955-f004: Effect of irradiance and nitrogen availability on the mRNA levels of the zeaxanthin epoxidase gene (zep) and cellular content of violaxanthin and zeaxanthin in Chlorella zofingiensis. (a) Low irradiance (20 µmol photons m−2 s−1) and constant nitrate concentration (LL +N); (b) low irradiance and nitrate deprivation (LL −N); (c) high irradiance (300 µmol photons m−2 s−1) and constant nitrate concentration (HL +N); and (d) high irradiance and nitrate deprivation (HL −N). Columns indicate mRNA levels of the zep gene; (♦), violaxanthin content (mg g−1 DW); (□), zeaxanthin content (mg g−1 DW). Error bars indicate the standard deviations of four independent measurements. DW, dry weight.
Mentions: C. zofingiensis cells were grown photoautotrophically at low irradiance and constant nitrate, as indicated in the Experimental Section, until the middle of the exponential phase. Then, cells were kept in the dark for 18 h, in order to make the transcript levels come down to basal values. After this dark period, cells were subjected to either low or high irradiance, under either constant nitrogen or nitrogen-deprivation conditions. The evolution with time of mRNA levels of the Czzep gene as affected by irradiance and nitrogen availability was monitored by quantitative real time PCR (qRT-PCR). In addition, changes in zeaxanthin and violaxanthin cellular contents were also determined in order to correlate mRNA levels with the biosynthesis of these carotenoids. As shown in Figure 4a,b, at low irradiance the levels of transcripts did not start to increase until 24 h after the dark period, regardless of nitrate availability. Under high irradiance and constant nitrogen conditions, the transcript levels of Czzep decreased between 10% and 60% after 5 h and 10 h from the beginning of the experiment, respectively (Figure 4c). Although there was also a high increase of the transcripts levels after 24 h under these conditions, they did not reach the levels registered at low irradiance. However, an additive response was detected in the nitrogen-free culture under high light conditions (Figure 4d), transcript levels after 24 h of induction being higher than those exhibited under low irradiance. With regards to the cellular carotenoid content, zeaxanthin was only registered at high irradiance, the levels of this carotenoid increased with time after 10 h, with a much higher accumulation of this carotenoid taking place under nitrogen depletion conditions (Figure 4c,d). Antheraxanthin was only detected at high irradiance as well, its levels ranging from 0.12 mg∙g−1 DW at 10 h to 0.09 mg g−1 DW at 72 h of high light exposure. Both zeaxanthin and antheraxanthin contents seem to be correlated with the increased mRNA levels of Czzep. The concentration of violaxanthin decreased with time at high irradiance, especially in the case of nitrogen-free conditions, while in the case of low light conditions it remained almost constant and only a little increase was detected at the 10 h or 24 h under nitrogen deprivation. Under high irradiance, astaxanthin was also present both in nitrogen depleted and in nitrogen repleted conditions, reaching values of 0.21 mg g−1 DW and 0.5 mg g−1 DW after 48 h, respectively.

Bottom Line: The Czzep gene was adequately inserted in the pSI105 vector and expressed in npq2.The positive transformants were able to efficiently convert zeaxanthin into violaxanthin, as well as to restore their maximum quantum efficiency of the PSII (Fv/Fm).These results show that Chlamydomonas can be an efficient tool for heterologous expression and metabolic engineering for biotechnological applications.

View Article: PubMed Central - PubMed

Affiliation: Institute of Plant Biochemistry and Photosynthesis, CIC Cartuja, University of Seville and CSIC, Avda. Américo Vespucio no. 49, 41092-Seville, Spain. inmaculada.couso@ibvf.csic.es

ABSTRACT
In the violaxanthin cycle, the violaxanthin de-epoxidase and zeaxanthin epoxidase catalyze the inter-conversion between violaxanthin and zeaxanthin in both plants and green algae. The zeaxanthin epoxidase gene from the green microalga Chlorella zofingiensis (Czzep) has been isolated. This gene encodes a polypeptide of 596 amino acids. A single copy of Czzep has been found in the C. zofingiensis genome by Southern blot analysis. qPCR analysis has shown that transcript levels of Czzep were increased after zeaxanthin formation under high light conditions. The functionality of Czzep gene by heterologous genetic complementation in the Chlamydomonas mutant npq2, which lacks zeaxanthin epoxidase (ZEP) activity and accumulates zeaxanthin in all conditions, was analyzed. The Czzep gene was adequately inserted in the pSI105 vector and expressed in npq2. The positive transformants were able to efficiently convert zeaxanthin into violaxanthin, as well as to restore their maximum quantum efficiency of the PSII (Fv/Fm). These results show that Chlamydomonas can be an efficient tool for heterologous expression and metabolic engineering for biotechnological applications.

Show MeSH
Related in: MedlinePlus