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 post-transcriptional 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 ß-carotene accumulation in ccr2, yet they reduced specific acyclic linear cis-carotenes linked to the biosynthesis of a currently unidentified apocarotenoid signal regulating plastid biogenesis, chlorophyll biosynthesis, and photomorphogenic regulation. ccr2 psy variants modulated the PHYTOCHROME-INTERACTING FACTOR 3/ELONGATED HYPOCOTYL 5 (PIF3/HY5) ratio, and displayed a normal prolamellar body formation in etioplasts and chlorophyll accumulation during seedling photomorphogenesis. Thus, suppressing PSY activity and impairing PSY:ORANGE protein interactions revealed how cis-carotene abundance can be fine-tuned through holoenzyme-metabolon interactions to control plastid development.

A Comprehensive Phylogenetic Analysis of the Serpin Superfamily.

Serine protease inhibitors (serpins) are found in all kingdoms of life and play essential roles in multiple physiological processes. Owing to the diversity of the superfamily, phylogenetic analysis is challenging and prokaryotic serpins have been speculated to have been acquired from Metazoa through horizontal gene transfer due to their unexpectedly high homology. Here, we have leveraged a structural alignment of diverse serpins to generate a comprehensive 6,000-sequence phylogeny that encompasses serpins from all kingdoms of life. We show that in addition to a central "hub" of highly conserved serpins, there has been extensive diversification of the superfamily into many novel functional clades. Our analysis indicates that the hub proteins are ancient and are similar because of convergent evolution, rather than the alternative hypothesis of horizontal gene transfer. This work clarifies longstanding questions in the evolution of serpins and provides new directions for research in the field of serpin biology.