GUN2 and GUN3 Coordinate Heme and Chlorophyll Metabolism Thereby Impacting Singlet Oxygen Production and Photooxidative Responses.

Tetrapyrrole biosynthesis is an absolute essential metabolic pathway in plants that predominantly gives rise to chlorophyll, heme, and phytochromobilin. Dysregulation of tetrapyrrole biosynthesis severely impairs plant growth and development when heme and chlorophyll synthesis are not adjusted to the needs in the respective plant organ and when excessive accumulation of light-absorbing tetrapyrrole intermediates cause oxidative damage. However, how chlorophyll and heme synthesis are properly balanced during early development of greening seedlings remains largely elusive. In this study, we performed a suppressor screen on the photosensitive Arabidopsis thaliana pif1 pif3 (for phytochrome-interacting factors) double mutant, which exhibits excessive singlet oxygen (1O₂) accumulation, and identified four different point mutants of the GENOMES UNCOUPLED 3 (GUN3, also known as HY2) gene encoding phytochromobilin synthase. Further genetic analysis revealed that either mutation of GUN3 or also GUN2 (encoding heme oxygenase, also known as HY1/HO1), but not FC1 (encoding ferrochelatase), rescues the cell death phenotype of pif1 pif3, lowers 1O₂ levels, and suppresses 1O₂-responsive gene expression. Furthermore, gun2 and gun3 mutations lead to heme-mediated feedback inhibition of 5-aminolevulinic acid synthesis and consequently reduced protochlorophyllide accumulation in the pif1 pif3 background. Notably, GUN2 and GUN3 physically interact with GUN4, while their mutations markedly correspond with decreased content of GUN4 and GUN5, which are involved in Mg chelation at the beginning of chlorophyll biosynthesis. Our studies point to an important regulatory role of GUN2 and GUN3 for the mutual link of heme and chlorophyll synthesis.