Reducing PHYTOENE SYNTHASE activity fine-tunes the abundance of a cis-carotene-derived signal that regulates the PIF3/HY5 module and plastid biogenesis

PHYTOENE SYNTHASE (PSY) is a rate-limiting enzyme catalysing the first committed step of carotenoid biosynthesis, and changes in PSY gene expression and/or protein activity alter carotenoid composition and plastid differentiation in plants. Four genetic variants of PSY (psy-4, psy-90, psy-130 and psy-145) were identified using a forward genetics approach that rescued leaf virescence phenotypes and plastid abnormalities displayed by the Arabidopsis CAROTENOID ISOMERASE (CRTISO) mutant ccr2 (carotenoid and chloroplast regulation 2) when grown under a shorter photoperiod. The four non-lethal mutations affected alternative splicing, enzyme-substrate interactions, and PSY:ORANGE multi-enzyme complex binding, constituting the dynamic posttranscriptional fine-tuning of PSY levels and activity without changing localization to the stroma and protothylakoid membranes. psy genetic variants did not alter total xanthophyll or β-carotene accumulation in ccr2 yet reduced specific acyclic linear cis-carotenes linked to the biosynthesis of a yet-to-be-identified apocarotenoid signal regulating plastid biogenesis, chlorophyll biosynthesis and photomorphogenic regulation. ccr2 psy variants modulated the ratio of PHYTOCHROME-INTERACTING FACTOR 3/ELONGATED HYPOCOTYL 5 (PIF3/HY5), displayed a normal PLB formation in etioplasts, and chlorophyll accumulation during seedling photomorphogenesis. Thus, suppressing PSY activity and impairing PSY:ORANGE protein interactions reveals how cis-carotene abundance can be fine-tuned through holoenzyme-metabolon interactions to control plastid development.