A bimodular oxidoreductase mediates the specific reduction of phylloquinone (vitamin K1) in chloroplasts.

Plants and certain species of cyanobacteria are the only organisms capable of synthesizing phylloquinone (vitamin K1 for vertebrates), which they use as an electron carrier during photosynthesis. Recent studies, however, have identified a plastidial pool of non-photoactive phylloquinone that could be involved in additional cellular functions. Here, we characterized an Arabidopsis bimodular enzyme--the At4g35760 gene product--comprising an integral domain homologous to the catalytic subunit of mammalian vitamin K1 epoxide reductase (VKORC1, EC 1.1.4.1) that is fused to a soluble thioredoxin-like moiety. GFP-fusion experiments in tobacco mesophyll cells established that the plant protein is targeted to plastids, and analyses of transcript and protein levels showed that expression is maximal in leaf tissues. The fused and individual VKORC1 domains were separately expressed in yeast, removing their chloroplast targeting pre-sequence and adding a C-terminal consensus signal for retention in the endoplasmic reticulum. The corresponding microsomal preparations were equally effective at mediating the dithiotreitol-dependent reduction of phylloquinone and menaquinone into their respective quinol forms. Strikingly, unlike mammalian VKORC1, the Arabidopsis enzyme did not reduce phylloquinone epoxide, and was resistant to inhibition by warfarin. The isoprenoid benzoquinone conjugates plastoquinone and ubiquinone were not substrates, establishing that the plant enzyme evolved strict specificity for the quinone form of naphthalenoid conjugates. In vitro reconstitution experiments established that the soluble thioredoxin-like domain can function as an electron donor for its integral VKORC1 partner.

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.