Limits...
Exploiting algal NADPH oxidase for biophotovoltaic energy

View Article: PubMed Central - PubMed

ABSTRACT

Photosynthetic microbes exhibit light‐dependent electron export across the cell membrane, which can generate electricity in biological photovoltaic (BPV) devices. How electrons are exported remains to be determined; the identification of mechanisms would help selection or generation of photosynthetic microbes capable of enhanced electrical output. We show that plasma membrane NADPH oxidase activity is a significant component of light‐dependent generation of electricity by the unicellular green alga Chlamydomonas reinhardtii. NADPH oxidases export electrons across the plasma membrane to form superoxide anion from oxygen. The C. reinhardtii mutant lacking the NADPH oxidase encoded by RBO1 is impaired in both extracellular superoxide anion production and current generation in a BPV device. Complementation with the wild‐type gene restores both capacities, demonstrating the role of the enzyme in electron export. Monitoring light‐dependent extracellular superoxide production with a colorimetric assay is shown to be an effective way of screening for electrogenic potential of candidate algal strains. The results show that algal NADPH oxidases are important for superoxide anion production and open avenues for optimizing the biological component of these devices.

No MeSH data available.


Chlamydomonas reinhardtii extracellular superoxide anion production requires RBO1. (a) Time course of O2− production by mid‐logarithmic cells of the Chlamydomonas reinhardtii cell wall‐deficient strains cw15, cw92 (both STA6RBO1) and the RBO1‐deficient sta6rbo1 mutant, determined using XTT reduction. Levels of O2− production were indistinguishable between both cell wall‐less strains cw15 and cw92. Different letters denote significant differences (one‐way ANOVA, P < 0.05). Data are mean ± SEM (n = 3). (b) DPI (20 μm) inhibited light‐dependent O2− production by all strains. Values are for 120 min. (one‐way ANOVA, P < 0.05; mean ± SEM, n = 3). There was no effect of equivalent concentration (v/v) of DMSO as the solvent for DPI. Different letters denote significant differences (one‐way ANOVA, P < 0.05). Data are mean ± SEM (n = 3).
© Copyright Policy - creativeCommonsBy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC5016757&req=5

pbi12332-fig-0004: Chlamydomonas reinhardtii extracellular superoxide anion production requires RBO1. (a) Time course of O2− production by mid‐logarithmic cells of the Chlamydomonas reinhardtii cell wall‐deficient strains cw15, cw92 (both STA6RBO1) and the RBO1‐deficient sta6rbo1 mutant, determined using XTT reduction. Levels of O2− production were indistinguishable between both cell wall‐less strains cw15 and cw92. Different letters denote significant differences (one‐way ANOVA, P < 0.05). Data are mean ± SEM (n = 3). (b) DPI (20 μm) inhibited light‐dependent O2− production by all strains. Values are for 120 min. (one‐way ANOVA, P < 0.05; mean ± SEM, n = 3). There was no effect of equivalent concentration (v/v) of DMSO as the solvent for DPI. Different letters denote significant differences (one‐way ANOVA, P < 0.05). Data are mean ± SEM (n = 3).

Mentions: We first confirmed that extracellular superoxide anion production could be detected from C. reinhardtii by assaying the reduction of cell‐impermeable XTT (2,3‐bis‐(2‐methoxy‐4‐nitro‐5‐sulfophenyl)‐2H‐tetrazolium‐5‐carboxanilide (Sutherland and Learmonth, 1997). The cell wall‐deficient strain cw92, which is wild type for both RBO1 and STA6 (STArchless6 encoding the small subunit of ADP‐Glc pyrophosphorylase; Table 1), was grown to mid‐logarithmic phase. These cells supported light‐dependent production of extracellular superoxide anion that was significantly inhibited by DPI, indicating NOX activity (Figure 3). Extracellular superoxide anion production by cw92 was indistinguishable from that of mid‐logarithmic cw15, the cell wall‐deficient STA6RBO1 strain from which the sta6rbo1 mutant is derived (Figure 4; Table 1). Both cw15 and cw92 were derived from mutagenesis of the WT 137c (Davies and Plaskitt, 1971; Pröschold et al., 2005). The genetic basis for the cw mutation remains unknown, but cw92 contains a glycoprotein in its residual cell wall that is absent from cw15 (Voigt et al., 1996). Light‐dependent extracellular O2− production by cw15 was significantly inhibited by DPI, indicating production by NOX. Critically, light‐dependent production by sta6rbo1 was significantly impaired even without DPI addition, which suggests that RBO1 was responsible for the majority of O2− production (Figure 4). The residual O2− production by sta6rbo1 that was DPI‐sensitive could be due to the continued low expression of RBO2 (Blaby et al., 2013). The DPI‐insensitive O2− production shown by all three strains suggests that alternative cell membrane redox enzymes are still in operation.


Exploiting algal NADPH oxidase for biophotovoltaic energy
Chlamydomonas reinhardtii extracellular superoxide anion production requires RBO1. (a) Time course of O2− production by mid‐logarithmic cells of the Chlamydomonas reinhardtii cell wall‐deficient strains cw15, cw92 (both STA6RBO1) and the RBO1‐deficient sta6rbo1 mutant, determined using XTT reduction. Levels of O2− production were indistinguishable between both cell wall‐less strains cw15 and cw92. Different letters denote significant differences (one‐way ANOVA, P < 0.05). Data are mean ± SEM (n = 3). (b) DPI (20 μm) inhibited light‐dependent O2− production by all strains. Values are for 120 min. (one‐way ANOVA, P < 0.05; mean ± SEM, n = 3). There was no effect of equivalent concentration (v/v) of DMSO as the solvent for DPI. Different letters denote significant differences (one‐way ANOVA, P < 0.05). Data are mean ± SEM (n = 3).
© Copyright Policy - creativeCommonsBy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC5016757&req=5

pbi12332-fig-0004: Chlamydomonas reinhardtii extracellular superoxide anion production requires RBO1. (a) Time course of O2− production by mid‐logarithmic cells of the Chlamydomonas reinhardtii cell wall‐deficient strains cw15, cw92 (both STA6RBO1) and the RBO1‐deficient sta6rbo1 mutant, determined using XTT reduction. Levels of O2− production were indistinguishable between both cell wall‐less strains cw15 and cw92. Different letters denote significant differences (one‐way ANOVA, P < 0.05). Data are mean ± SEM (n = 3). (b) DPI (20 μm) inhibited light‐dependent O2− production by all strains. Values are for 120 min. (one‐way ANOVA, P < 0.05; mean ± SEM, n = 3). There was no effect of equivalent concentration (v/v) of DMSO as the solvent for DPI. Different letters denote significant differences (one‐way ANOVA, P < 0.05). Data are mean ± SEM (n = 3).
Mentions: We first confirmed that extracellular superoxide anion production could be detected from C. reinhardtii by assaying the reduction of cell‐impermeable XTT (2,3‐bis‐(2‐methoxy‐4‐nitro‐5‐sulfophenyl)‐2H‐tetrazolium‐5‐carboxanilide (Sutherland and Learmonth, 1997). The cell wall‐deficient strain cw92, which is wild type for both RBO1 and STA6 (STArchless6 encoding the small subunit of ADP‐Glc pyrophosphorylase; Table 1), was grown to mid‐logarithmic phase. These cells supported light‐dependent production of extracellular superoxide anion that was significantly inhibited by DPI, indicating NOX activity (Figure 3). Extracellular superoxide anion production by cw92 was indistinguishable from that of mid‐logarithmic cw15, the cell wall‐deficient STA6RBO1 strain from which the sta6rbo1 mutant is derived (Figure 4; Table 1). Both cw15 and cw92 were derived from mutagenesis of the WT 137c (Davies and Plaskitt, 1971; Pröschold et al., 2005). The genetic basis for the cw mutation remains unknown, but cw92 contains a glycoprotein in its residual cell wall that is absent from cw15 (Voigt et al., 1996). Light‐dependent extracellular O2− production by cw15 was significantly inhibited by DPI, indicating production by NOX. Critically, light‐dependent production by sta6rbo1 was significantly impaired even without DPI addition, which suggests that RBO1 was responsible for the majority of O2− production (Figure 4). The residual O2− production by sta6rbo1 that was DPI‐sensitive could be due to the continued low expression of RBO2 (Blaby et al., 2013). The DPI‐insensitive O2− production shown by all three strains suggests that alternative cell membrane redox enzymes are still in operation.

View Article: PubMed Central - PubMed

ABSTRACT

Photosynthetic microbes exhibit light&#8208;dependent electron export across the cell membrane, which can generate electricity in biological photovoltaic (BPV) devices. How electrons are exported remains to be determined; the identification of mechanisms would help selection or generation of photosynthetic microbes capable of enhanced electrical output. We show that plasma membrane NADPH oxidase activity is a significant component of light&#8208;dependent generation of electricity by the unicellular green alga Chlamydomonas reinhardtii. NADPH oxidases export electrons across the plasma membrane to form superoxide anion from oxygen. The C.&nbsp;reinhardtii mutant lacking the NADPH oxidase encoded by RBO1 is impaired in both extracellular superoxide anion production and current generation in a BPV device. Complementation with the wild&#8208;type gene restores both capacities, demonstrating the role of the enzyme in electron export. Monitoring light&#8208;dependent extracellular superoxide production with a colorimetric assay is shown to be an effective way of screening for electrogenic potential of candidate algal strains. The results show that algal NADPH oxidases are important for superoxide anion production and open avenues for optimizing the biological component of these devices.

No MeSH data available.