GEOapplication/xmlftp://ftp.ncbi.nlm.nih.gov/geo/series/GSE292nnn/GSE292110/primaryOK200TranscriptomicsArabidopsis thalianaExpression profiling by high throughput sequencinghttps://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE292110GEOGSEfalseA suppressor screen uncovers flu as a weak mutant and chloroplastic 1O2 triggers EX1-independent stress responses in Arabidopsis• Singlet oxygen (1O2) is the main ROS produced in chloroplasts, particularly under environmental stress conditions. Studies using the Arabidopsis conditional flu (flu1-1) mutant have shown that chloroplastic 1O2 acts as a signal, activating retrograde signaling that eventually leads to photoinhibition or cell death. • Through a forward genetic screen for suppressors using EMS-mutagenized Arabidopsis flu1-1/ex1 mutants, we isolated two suppressor mutants (mut1 and mut2) that restored 1O2-induced stress responses. Using combined methods of map-based cloning and high-throughput sequencing, we mapped the causal mutations in both mut1 and mut2 to the FLU gene again. • We verified that the widely used flu1-1 is not the long-recognized null mutant, but rather a weak allele, encoding a variant that binds weakly to GluTR1 and leads to partial shutdown of tetrapyrrole biosynthesis in darkness. We further found that the FLU-knockout mutants produce more 1O2, activating transcriptional changes, enzymatic lipid peroxidation, and PCD even in the absence of EX1. Overexpression of SAFE1 suppressed 1O2-induced PCD in mut2 in a dosage-dependent manner. • Collectively, our findings redefine the nature of the widely used flu1-1 mutant and reveal that excessive 1O2 can overwhelm the inhibitory effect of endogenous SAFE1 and induce stress responses independently of EX1.2026/05/02GSE292110GSM8849365GSM8849366GSM8849367GSM8849368GSM8849361GSM8849362GSM8849363GSM8849364GSM8849360GSM8849369GSM8849358GSM884935931102292110Arabidopsis thaliana