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Identification of an ascorbate-dependent cytochrome b of the tonoplast membrane sharing biochemical features with members of the cytochrome b561 family

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Abstract

Two membrane-bound, ascorbate-dependent b-type cytochromes were identified in etiolated bean (Phaseolus vulgaris L.) hypocotyls. Following solubilization of microsomal membranes and anion-exchange chromatography at pH 8.0, two major cytochrome peaks (P-I and P-II) were separated. Both cytochromes were reduced by ascorbate and re-oxidized by monodehydroascorbate, but P-I reduction by ascorbate was higher and saturated at far lower concentrations of ascorbate with respect to P-II. The α-band was symmetrically centered at 561 nm in P-I, but it was asymmetric in P-II with a maximum at 562 nm and shoulder at 557 nm. Ascorbate reduction of P-II, but not P-I, was inhibited by diethyl pyrocarbonate. Reduced P-II but not P-I was readily oxidized by certain ferric chelates, including FeEDTA and Fe-nitrilotriacetic acid. Purified P-I, associated with the plasma membrane, showed up as a 63-kDa glycosylated protein during sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) and behaved as a monomer of about 70 kDa during size-exclusion chromatography. P-I identified with a previously purified ascorbate-dependent b-type cytochrome of bean hypocotyl plasma membranes [P. Trost et al. (2000) Biochim Biophys Acta 1468:1–5]. Partially purified P-II, on the other hand, correlated with a heme-protein of 27 kDa in SDS–PAGE gels, was dimeric (60 kDa) during size-exclusion chromatography, and was associated with the tonoplast marker V-ATPase in sucrose gradients. The sequence of a peptide of 11 residues obtained by tryptic digestion of P-II was found to be identical to a segment of a putative cytochrome b561 of Zea mays and highly conserved in other related plant sequences, including that of Arabidopsis thaliana cytochrome b561-1 (CAA18169). The biochemical features fully support the assignment of P-II cytochrome to the family of cytochrome b561, ascorbate-dependent (CYBASC) cytochromes, which also includes cytochrome b561 of animal chromaffin granules. The presence of a cytochrome reducing ferric chelates on the tonoplast is consistent with the role of plant vacuoles in iron homeostasis.

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Abbreviations

cyt b561 :

Cytochrome b561

DEPC :

Diethyl pyrocarbonate

GS-II :

Glucan synthase II

MDA :

Monodehydroascorbate

OG :

Octyl-β-d-glucopyranoside

PM :

Plasma membrane

TMBZ :

3,3′,5,5′-Tetramethylbenzidine

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Acknowledgements

We are grateful to Prof. Masayoshi Maeshima (Nagoya University, Japan) for donating the antibodies against Arabidopsis thaliana V-ATPase. This work was supported by MIUR (PRIN “Bioenergetica: aspetti genetici, biochimici e fisiopatologici”).

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Correspondence to Valeria Preger.

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Preger, V., Scagliarini, S., Pupillo, P. et al. Identification of an ascorbate-dependent cytochrome b of the tonoplast membrane sharing biochemical features with members of the cytochrome b561 family. Planta 220, 365–375 (2005). https://doi.org/10.1007/s00425-004-1360-0

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  • DOI: https://doi.org/10.1007/s00425-004-1360-0

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