Faculty Opinions recommendation of ROR1/RPA2A, a putative replication protein A2, functions in epigenetic gene silencing and in regulation of meristem development in Arabidopsis.

We screened for suppressors of repressor of silencing1 ( ros1 ) using the silenced 35S promoter- neomycin phosphotransferase II (Pro 35S : NPTII ) gene as a marker and identiﬁed two allelic mutants, ror1-1 and ror1-2 (for suppressor of ros1 ). Map-based cloning revealed that ROR1 encodes a 31-kD protein similar to DNA replication protein A2 (RPA2A). Mutations in ROR1 reactivate the silenced Pro 35S : NPTII gene but not RD29A promoter- luciferase in the ros1 mutant. DNA methylation in rDNA , centromeric DNA, and RD29A promoter regions is not affected by ror1 . However, chromatin immunoprecipitation data suggest that histone H3 acetylation is increased and histone H3K9 dimethylation is decreased in the 35S promoter in the ror1 ros1 mutant compared with ros1 . These results indicate that release of silenced Pro 35S : NPTII by ror1 mutations is independent of DNA methylation. ROR1/RPA2A is strongly expressed in shoot and root meristems. Mutations in ROR1/ RPA2A affect cell division in meristems but not ﬁnal cell sizes. Our work suggests important roles of ROR1/RPA2A in epigenetic gene silencing and in the regulation of plant development. kanamycin-resistant and developmental phenotypes, named ror1-1ros1 and ror1-2ros1 . Forgeneticsanalysis,thetwomutants were backcrossed to ros1 , and the resulting F1 seedlings exhibited the ros1 phenotypes. F2 progeny from self-fertilized F1 plants showed an ; 3:1 segregation of kanamycin sensitivity to resistant phenotypes. The resistant ones also showed the similar developmental phenotypes. We also crossed ror1-1 ros1 or ror1-2 ros1 to C24, and F2 progeny exhibited ; 3:1 of normal plants to development defect ones, which is consistent with the developmental phenotypes in ror1-1 ros1 or ror1-2 ros1 mutants being independent of ros1 mutations. The combined results indicate that ror1 mutants are caused by single nuclear recessive mutations. A T-DNA insertion line ( ror1-3 ), SALK_129173 was obtained from the Arabidopsis Stock Center, and the insertion site were conﬁrmed by PCR and sequencing. In this study, we mainly used ror1-1 ros1 as experimental materials; if not specially stated in the text, ror1-1 ros1 is ror1 ros1 . Seedlings grown on MS medium for 2 weeks were subjected to luciferase imaging analysis after being treated or not with 100 m M ABA for 5 h as described (Gong et al., 2002). We used the growth defect phenotypes of ror1 mutants to isolate the correspondinggene.The ror1-1ros1 mutantwascrossedtothewild-type gl1 (Columbia accession). The F2 population was screened for ror1 mutants on the basis of ror1 growth phenotypes. We used simple sequence length polymorphism markers to map ROR1 ﬁrst to chromo- some2betweenthemarkersF3K23(usingtheprimerpair5 9 -GGGCCGA-TACAGAGAGACCCTAAC-3 9 and5 9 -ACTCTCCGCCATGTCTCCGATC-3 9 ) and F3N11 (using the primer pair 5 9 -ATGCACGCACCCTTCTACTC-3 9 and 5 9 -TTCGCACATGTGAGATTATGGG-3 9 ). Then, markers F26B6 (us- ing the primer pair 5 9 -TAGGGCTTTGAGAACCCACGTG-3 9 and 5 9 -AAA-GTACCGTCGCGCACAG-3 9 ), F27D4 (using the primer pair 5 9 -CGA- GTCACATTCGCGCAATGATG-3 9 and 5 9 -GGTAGCAAAGAAGAGAA-GACCTCTC-3 9 ), and F25P17 (using the primer pair 5 9 -CGACA- CCAAATGTCGGCTCTGAG-3 9 and 5 9 -TAGTGACTTGATGAATGATTGC-CGC-3 9 ) were used to narrow down the ror1 mutation to within the BAC clone T28I24. To identify the ror1 mutation, candidate genes from wild-type and ror1 ros1 mutant plants were sequenced. The sequences were compared to ﬁnd the mutation.