ABSTRACT 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. Here we identified four genetic variants of PSY ( psy −4 , psy −90 , psy −130 and psy −145 ) using a forward genetics approach that rescued leaf virescence phenotypes 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 overall total xanthophyll or cis-carotene accumulation in ccr2 yet reduced specific acyclic linear cis -carotenes linked to the biosynthesis of a yet-to-be-identified apocarotenoid signal. 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 threshold specific cis -carotene levels can be fine-tuned through holoenzyme-metabolon interactions to control plastid development. Highlights Manipulation of the PHYTOENE SYNTHASE catalytic activity in concert with its regulatory protein, ORANGE, reduces threshold levels of acyclic linear cis -carotenes that signal control over plastid biogenesis in dark and light grown Arabidopsis seedlings

Reducing PSY activity fine tunes threshold levels of acis-carotene-derived signal that regulates the PIF3/HY5 module and plastid biogenesis