ABSTRACT: BACKGROUND: Xanthophylls are oxygenated carotenoids playing an essential role as structural components of the photosynthetic apparatus. Xanthophylls contribute to the assembly and stability of light-harvesting complex, to light absorbance and to photoprotection. The first step in xanthophyll biosynthesis from alpha- and beta-carotene is the hydroxylation of epsilon- and beta-rings, performed by both non-heme iron oxygenases (CHY1, CHY2) and P450 cytochromes (LUT1/CYP97C1, LUT5/CYP97A3). The Arabidopsis triple chy1chy2lut5 mutant is almost completely depleted in beta-xanthophylls. RESULTS: Here we report on the quadruple chy1chy2lut2lut5 mutant, additionally carrying the lut2 mutation (affecting lycopene epsilon-cyclase). This genotype lacks lutein and yet it shows a compensatory increase in beta-xanthophylls with respect to chy1chy2lut5 mutant. Mutant plants show an even stronger photosensitivity than chy1chy2lut5, a complete lack of qE, the rapidly reversible component of non-photochemical quenching, and a peculiar organization of the pigment binding complexes into thylakoids. Biochemical analysis reveals that the chy1chy2lut2lut5 mutant is depleted in Lhcb subunits and is specifically affected in Photosystem I function, showing a deficiency in PSI-LHCI supercomplexes. Moreover, by analyzing a series of single, double, triple and quadruple Arabidopsis mutants in xanthophyll biosynthesis, we show a hitherto undescribed correlation between xanthophyll levels and the PSI-PSII ratio. The decrease in the xanthophyll/carotenoid ratio causes a proportional decrease in the LHCII and PSI core levels with respect to PSII. CONCLUSIONS: The physiological and biochemical phenotype of the chy1chy2lut2lut5 mutant shows that (i) LUT1/CYP97C1 protein reveals a major beta-carotene hydroxylase activity in vivo when depleted in its preferred substrate alpha-carotene; (ii) xanthophylls are needed for normal level of Photosystem I and LHCII accumulation.