Detection and quantification of vitamin K\u3csub\u3e1\u3c/sub\u3e quinol in leaf tissues

Phylloquinone (2-methyl-3-phytyl-1,4-naphthoquinone; vitamin K1) is vital to plants. It is responsible for the oneelectron transfer at the A1 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 (PhQH2). A method was developed to quantify PhQH2 and its corresponding oxidized quinone (PhQ) counterpart in a single HPLC run. PhQH2 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 PhQH2. The respective pool sizes of PhQH2 and PhQ were remarkably stable throughout the development of Arabidopsis green leaves. On the other hand, in Arabidopsis and tomato senescing leaves, PhQH2 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 PhQH2 than those kept in the dark. These data indicate that PhQH2 does not originate from the photochemical reduction of PhQ, and point to a hitherto unsuspected function of phylloquinone in plants. The putative origin of PhQH2 and its recycling into PhQ are discussed