Roles of CRWN-family proteins in protecting genomic DNA against oxidative damage.

CROWDED NUCLEI (CRWN) family in Arabidopsis consists of four members, CRWN1 to CRWN4. It has been previously reported that the CRWN proteins are involved in the control of nuclear morphology and degradation of ABI5. In this study, however, we discover that CRWN-family proteins are not only involved in attenuating responsiveness to abscisic acid (ABA), but also implicated in inhibiting reactive oxygen species (ROS) production and DNA damage induced by genotoxic agent methyl methanesulfonate (MMS). Our results demonstrate that three crwn double mutants, i.e. crwn1 crwn3, crwn2 crwn3, and crwn2 crwn4, show slightly earlier leaf senescence, enhanced leaf cell death, and obvious overaccumulation of ROS under regular growth conditions. When treated with 0.15 μM ABA or 0.01% MMS, two double mutants, crwn1 crwn3 and crwn2 crwn3, exhibit significant decreased germination rates as well as leaf opening and greening rates. Moreover, subsequent investigations indicate that the MMS treatment strongly inhibits the growth of crwn mutant seedlings, while this inhibition is substantially relieved by imidazole (IMZ); by contrast, DNA methylation inhibitor 5-aza-2'-deoxycytidine (5-aza-dC) has no effect on relief of the growth inhibition. Further studies reveal that under 0.01% MMS treatment conditions, crwn mutants, especially the three double mutants, accumulate more ROS compared to Col-0, and their genomic DNA suffers from more severe DNA damage relative to Col-0, which is indicated by significantly higher 8-oxo-7-hydrodeoxyguanosine (8-oxo dG) content as observed in the crwn mutants. Altogether, these data clearly demonstrate that the CRWN-family proteins play important roles in diminishing ROS accumulation and protecting genomic DNA against excessive oxidative damage caused by MMS.