Detection and quantification of vitamin K(1) quinol in leaf tissues

Phylloquinone (2-methyl-3-phytyl-1,4-naphthoquinone; vitamin K(1)) is vital to plants. It is responsible for the one-electron transfer at the A(1) site of photosystem I, a process that involves turnover between the quinone and semi-quinone forms of phylloquinone. Using HPLC coupled with fluorometric detection to analyze Arabidopsis leaf extracts, we detected a third redox form of phylloquinone corresponding to its fully reduced - quinol-naphthoquinone ring (PhQH(2)). A method was developed to quantify PhQH(2) and its corresponding oxidized quinone (PhQ) counterpart in a single HPLC run. PhQH(2) was found in leaves of all dicotyledonous and monocotyledonous species tested, but not in fruits or in tubers. Its level correlated with that of PhQ, and represented 5-10% of total leaf phylloquinone. Analysis of purified pea chloroplasts showed that these organelles accounted for the bulk of PhQH(2). The respective pool sizes of PhQH(2) and PhQ were remarkably stable throughout the development of Arabidopsis green leaves. On the other hand, in Arabidopsis and tomato senescing leaves, PhQH(2) was found to increase at the expense of PhQ, and represented 25-35% of the total pool of phylloquinone. Arabidopsis leaves exposed to light contained lower level of PhQH(2) than those kept in the dark. These data indicate that PhQH(2) does not originate from the photochemical reduction of PhQ, and point to a hitherto unsuspected function of phylloquinone in plants. The putative origin of PhQH(2) and its recycling into PhQ are discussed.