Lesion mimic mutants commonly display spontaneous cell death in pre-senescent green leaves under normal conditions, without pathogen attack. Despite molecular and phenotypic characterization of several lesion mimic mutants, the mechanisms of the spontaneous formation of cell death lesions remain largely unknown. Here, we examined the rice lesion mimic mutant spotted leaf3 (spl3). In mutants grown under a light/dark cycle, spl3 mutants appeared similar to wild type at early developmental stages, but lesions gradually appeared in the mature leaves close to heading stage. By contrast, in mutants grown under continuous light, severe cell death lesions formed in developing leaves, even at the seedling stage. Histochemical analysis showed that hydrogen peroxide accumulated in the mutants, likely causing the cell death phenotype. By map-based cloning and complementation, we showed that a 1-bp deletion in the first exon of Oryza sativa Mitogen-Activated Protein Kinase Kinase Kinase1 (OsMAPKKK1)/OsEDR1/ OsACDR1 causes the spl3 mutant phenotype. We found that the spl3 mutants were insensitive to abscisic acid (ABA), showing normal root growth in ABA-containing media and delayed leaf yellowing during dark-induced and natural senescence. Expression of ABA signaling-associated genes was also less responsive to ABA treatment in the mutants. Furthermore, the spl3 mutants had lower transcript levels and activities of catalases, which scavenge hydrogen peroxide, probably due to impairment of ABA-responsive signaling. Finally we discuss a possible molecular mechanism of lesion formation in the mature leaves of spl3 mutants.

In field conditions, the zebra2 (z2) mutant in rice (Oryza sativa) produces leaves with transverse pale-green/yellow stripes. It was recently reported that ZEBRA2 encodes carotenoid isomerase (CRTISO) and that low levels of lutein, an essential carotenoid for non-photochemical quenching, cause leaf variegation in z2 mutants. However, we found that the z2 mutant phenotype was completely suppressed by growth under continuous light (CL; permissive) conditions, with concentrations of chlorophyll, carotenoids and chloroplast proteins at normal levels in z2 mutants under CL. 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 conditions (SD; alternate 10-h light/14-h dark; restrictive), but do not accumulate under CL conditions. However, the levels of lutein and zeaxanthin in z2 leaves were much lower than normal 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 the dark periods under SD, but not under CL conditions. Its accumulation was also positively correlated with 1O2 levels generated during the 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.