Abstract

Phylloquinone is a compound present in all plants serving as cofactor for photosystem I mediated electron transport during photosynthesis. This work reports on the identification and analysis of several Arabidopsis thaliana phylloquinone absence (pha) and isochorismate synthase (ics) mutants impaired in the biosynthesis of PhQ (vitamin K1). Besides the complete lack of PhQ, these plants show a typical phenotype characterized by seedling lethality, photosynthetic defects specifically related to impaired photosystem I accumulation/activity to 5-15% of wild-type levels and partial recovery of 15% PhQ content and 50-70% PSI accumulation/activity after feeding with the metabolic precursor of vitamin K1, 1,4-dihydroxy-2-naphthoate. Map-based localization of the mutated allele in the pha plants identified a new gene, called PHYLLO. It consists of a fusion of four previously individual eubacterial genes, menF, menD, menC, and menH, required for the biosynthesis of the photosynthetic phylloquinone in cyanobacteria and the respiratory menaquinone in eubacteria. The fact that homologous men genes still reside as polycistronic units in plastomes of red algae and in eubacterial chromosomes strongly suggests that PHYLLO derived from an operon present in the proto-organelle precursor of all plastids. The principle architecture of the PHYLLO locus is conserved in the nuclear genomes of plants and the green alga Chlamydomonas reinhardtii, indicating that selective forces have been acting to maintain the cluster structure in the form of a gene fusion, presumably as an adaptation of an multifunctional association of four enzymatic activities already pre-existing in the chloroplast. In line with this finding, the data present in this work suggest that the PHYLLO composite product is part of a metabolon for the biosynthesis of phylloquinone. The menF module of PHYLLO in Chlamydomonas, encoding the isochorismate synthase activity, is full-length, whereas in higher plants this module surprisingly lacks the functional 3’ part, uncovering a recent gene splitting event during evolution. Such a gene fission event, which resulted in inactivation of the encoded ICS enzymatic activity from PHYLLO, must have been preceded by establishment of a second functional copy of the menF gene. Accordingly, double-knockouts of the ICS1 and ICS2 genes in Arabidopsis analysed during this work, were unable to synthesize PhQ, demonstrating that the activity of the menF module of PHYLLO has been replaced after the splitting of the 3’-region by at least one more ICS gene present in genomes of higher plants. The fact that ICS1 is also required for salicylic acid biosynthesis in Arabidopsis, establishes a metabolic link between photosynthesis and systemic acquired resistance. Therefore, gene fusion, duplication and fission events adapted a eubacterial multienzymatic system to the metabolic requirements of plants. Despite the essential function of PhQ for PSI stability and plant viability, analyses of ics heterozygous knockout plants, as well as complementation of the pha mutants by NA feeding and transgenic forms of PHYLLO demonstrate that the bulk of cellular phylloquinone is not associated with photosystem I, opening the possibility for additional functions of vitamin K1 in plant cell membranes.