INVOLVEMENT OF ASCORBIC-ACID AND A B-TYPE CYTOCHROME IN PLANT PLASMA-MEMBRANE REDOX REACTIONS

Higher plant plasma membranes contain a b-type cytochrome that is rapi dly reduced by ascorbic acid. The affinity towards ascorbate is 0.37 m M and is very similar to that of the chromaffin granule cytochrome b(5 61). High levels of cytochrome b reduction are reached when ascorbic a cid is added either on the cytoplasmic or cell wall side of purified p lasma membrane vesicles. This result points to a transmembrane organis ation of the heme protein or alternatively indicates the presence of a n effective ascorbate transport system. Plasma membrane vesicles loade d by ascorbic acid are capable of reducing extravesicular ferricyanide . Addition of ascorbate oxidase or washing of the vesicles does not el iminate this reaction, indicating the involvement of the intravesicula r electron donor. Absorbance changes of the cytochrome b alpha-band su ggest the electron transfer is mediated by this redox component. Elect ron transport to ferricyanide also results in the generation of a memb rane potential gradient as was demonstrated by using the charge-sensit ive optical probe oxonol VI. Addition of ascorbate oxidase and ascorba te to the vesicles loaded with ascorbate results in the oxidation and subsequent re-reduction of the cytochrome b. It is therefore suggested that ascorbate free radical (AFR) could potentially act as an electro n acceptor to the cytochrome-mediated electron transport reaction. A w orking model on the action of the cytochrome as an electron carrier be tween cytoplasmic and apoplastic ascorbate is discussed.