Photoperiodic accumulation of tetra-cis-lycopene and singlet oxygen causes leaf variegation in the rice zebra2 mutant

Abstract

In field conditions, the zebra2 (z2) mutant in rice (Oryza sativa) produces leaves with transverse pale-green/yellow stripes. We identified the z2 locus by map-based cloning using F2 population derived from a cross of the z2 mutant (indica) with Sinseonchalbyeo (japonica). ZEBRA2 encodes a carotenoid isomerase (CRTISO) which is one of the carotenoid biosynthetic enzymes. We found that the z2 mutant phenotype completely suppressed by growth under continuous light (CL; permissive) conditions, with concentrations of chlorophyll, carotenoids and chloroplast proteins at normal levels in z2 mutants. In addition, three types of reactive oxygen species (ROS; superoxide [O2-], hydrogen peroxide [H2O2], and singlet oxygen [1O2]) accumulated to high levels in z2 mutants grown under short-day (SD; alternate 10-h light/14-h dark; restrictive) conditions, but did not accumulate under CL conditions. However, the levels of lutein and zeaxanthin in z2 leaves were much lower than wild-type (WT) in both permissive CL and restrictive SD growth conditions, indicating that deficiency of these two carotenoids is not responsible for the leaf variegation phenotype. We found that the CRTISO substrate tetra-cis-lycopene accumulated during dark period under SD, but not under CL conditions. Its accumulation was also positively correlated with 1O2 levels generated during light period, which consequently altered the expression of 1O2-responsive and cell death-related genes in the variegated z2 leaves. Taking these results together, we propose that the z2 leaf variegation can be largely attributed to photoperiodic accumulation of tetra-cis-lycopene and generation of excessive 1O2 under natural day-night conditions.