A suppressor screen uncovers flu as a weak mutant and chloroplastic 1O2 triggers EX1-independent stress responses in Arabidopsis

Abstract

• Singlet oxygen (¹O₂) 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 ¹O₂ 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 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 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 ¹O₂, activating transcriptional changes, enzymatic lipid peroxidation, and programmed cell death (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.