A suppressor screen uncovers flu as a weak mutant and chloroplastic ¹ O ₂ triggers EX1 ‐independent stress responses in Arabidopsis

Summary Singlet oxygen ( 1 O 2 ) is the main reactive oxygen species produced in chloroplasts, particularly under environmental stress conditions. Studies using the Arabidopsis conditional flu ( flu1‐1 ) mutant have shown that chloroplastic 1 O 2 acts as a signal, activating retrograde signaling that eventually leads to photoinhibition or cell death. Through a forward genetic screen for suppressors using ethyl methanesulfonate (EMS)‐mutagenized Arabidopsis flu1‐1/ex1 mutants, we isolated two suppressor mutants ( mut1 and mut2 ) that restored 1 O 2 ‐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 FLUORESCENT ( 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 the dark. We further found that the FLU ‐knockout mutants produce more 1 O 2 , activating transcriptional changes, enzymatic lipid peroxidation, and programmed cell death (PCD) even in the absence of EX1. Overexpression of SAFE1 suppressed 1 O 2 ‐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 1 O 2 can overwhelm the inhibitory effect of endogenous SAFE1 and induce stress responses independently of EX1.