Introduction of regioselective bacterial heme oxygenases into Arabidopsis hy1-1 supports the retrograde heme signaling hypothesis

Abstract Heme is synthesized in the plastid and metabolized to phytochromobilin (PΦB) in 2 enzymatic steps: heme oxygenase (HO) and PΦB synthase (HY2). In Arabidopsis thaliana (Arabidopsis), HO1/HY1/GUN2 predominantly functions for heme catabolism among the HO isoforms. Our previous study demonstrated that HO1 localization is altered in plastids or in the cytosol due to transcriptional start-site regulation. Introduction of either plastid- or cytosol-localized HO1 into HO1-deficient mutants resulted in recovery from the long hypocotyl, low pigmentation, and genomes uncoupling (gun) phenotypes, indicating the assembly of functional phytochromes (PHYs), as well as supporting the retrograde heme signaling hypothesis. To dissect the heme signaling and PHY assembly, we introduced either of the 2 types of regioselective bacterial HOs that produce biliverdin IXα (BVIXα) or BVIXβ/δ into Arabidopsis hy1-1. Gene introduction of either plastid- or cytosol-localized BVIXα-producing HO complemented the long hypocotyl, low pigmentation, and gun phenotypes of hy1-1. Interestingly, the introduction of BVIXβ/δ-producing HO, either in the plastid or in the cytosol, failed to complement the long hypocotyl and low pigmentation phenotypes, suggesting failure of functional PHY assembly. However, these lines restored the gun phenotype, thus supporting the heme signaling hypothesis. Based on the levels of complementation of the gun phenotype, we found that the expression of photosynthesis-associated nuclear genes (PhANGs) can be separated into PHY-dependent and PHY-independent groups. Our results demonstrate that heme functions as a retrograde mobile biogenic signal from plastids, which is mediated by the cytosol, to regulate the expression of PhANGs, and this regulation is distinct in its dependency on PHY.