A Small Family of Chloroplast Atypical Thioredoxins      

Abstract

The reduction and the formation of regulatory disulfide bonds serve as a key signaling element in chloroplasts. Members of the thioredoxin (Trx) superfamily of oxidoreductases play a major role in these processes. We have characterized a small family of plant-specific Trxs in Arabidopsis (Arabidopsis thaliana) that are rich in cysteine and histidine residues and are typified by a variable noncanonical redox active site. We found that the redox midpoint potential of three selected family members is significantly less reducing than that of the classic Trxs. Assays of subcellular localization demonstrated that all proteins are localized to the chloroplast. Selected members showed high activity, contingent on a dithiol electron donor, toward the chloroplast 2-cysteine peroxiredoxin A and poor activity toward the chloroplast NADP-malate dehydrogenase. The expression profile of the family members suggests that they have distinct roles. The intermediate redox midpoint potential value of the atypical Trxs might imply adaptability to function in modulating the redox state of chloroplast proteins with regulatory disulfides.

A Chloroplast Light-Regulated Oxidative Sensor for Moderate Light Intensity in Arabidopsis

“…ACHT members have an atypical redox active site and a less reducing redox midpoint potential than that of the classic chloroplast Trxs (Dangoor et al, 2009). Here, our studies in vivo show that ACHT1 accepts electrons from the linear photosynthetic electron transfer reactions and, in turn, reduces the chloroplast 2-Cys Prx.…”

“…(3) Our finding that the intermolecular disulfide complexes of the catalytic active ACHT1 and 2-Cys Prx are most abundant when ACHT1 is largely oxidized (Figures 2 and 6) indicates a smaller rate of forward reaction than that required for optimal antioxidative function. (4) The higher midpoint redox potential of ACHT1 (Dangoor et al, 2009) than that of 2-Cys Prx (Horling et al, 2003) is not consistent with high rates of peroxides detoxification by the ACHT1-2-Cys Prx reaction in chloroplasts under acute oxidative stress conditions. (5) Finally, the detrimental effect on photosynthesis by the increased reduction of the ACHT1 and 2-Cys Prx pathway (Figures 5 and 8) is expected for a regulatory role and not for an antioxidative one.…”

“…The rate of 2-Cys Prx oxidation is driven by peroxides likely formed by the Mehler reaction in PSI, which diverts excessive reducing equivalents from NADPH production to O 2 reduction (Mehler, 1951). The association of both ACHT1 and 2-Cys Prx with the thylakoid membranes (König et al, 2002;Dangoor et al, 2009) might argue for localized sensing of peroxide formation. Under moderate light intensity, the reduction of ACHT1 by photosynthesis, and not its oxidation by 2-Cys Prx, is limiting and results in oxidation of ACHT1 (Figure 2).…”

“…The ACHT1 overexpressor plants also showed a corresponding decrease in photosynthetic ETR (Figure 8B), indicating a lower rate of PSII photochemistry. ACHT1 knockdown (KD) plants showed only an insignificant increase in NPQ ( Figure 8A) and decrease in ETR values compared to wild-type plants ( Figure 8B), suggesting that other ACHT members, coexpressed in Arabidopsis chloroplasts (Dangoor et al, 2009), might partially compensate for the diminished ACHT1 expression. In contrast with these specific photosynthetic defects, the maximum quantum yield values (Fv/ Fm) ( Figure 8C) and the chlorophyll A and B ( Figure 8E) and anthocyanin ( Figure 8D) content of the ACHT1 OE and KD plants were all normal.…”

“…We trapped ACHT1 disulfides in vivo by adapting to plants (Peled-Zehavi et al, 2010) the methodology that was used to study PDI and ERO1 redox state changes and reaction intermediates in yeast cells (Frand and Kaiser, 1999). Transgenic plants expressing the chloroplastic ACHT1 fused with a hemagglutinin (HA) affinity tag at its C terminus under the control of the 35S promoter (Dangoor et al, 2009) were grown under longday conditions with moderate light intensity, 80 to 100 mE/m 2 s. Protein disulfides were trapped by extracting whole plants in 10% trichloroacetic acid and then irreversibly blocking the reduced Cys residues with N-ethylmaleimide (NEM). Subsequently, the disulfide-bonded Cys residues, which are protected from NEM modification, were reduced with DTT, labeled with methoxypolyethylene glycol-maleimide (mPEG), and visualized in an immunoblot assay of fractionated denatured proteins using anti-HA specific antibodies.…”