Inhibition of Carotenoid Biosynthesis by CRISPR/Cas9 Triggers Cell Wall Remodelling in Carrot

Abstract

Recent data indicate that modifications to carotenoid biosynthesis pathway in plants alter the expression of genes affecting chemical composition of the cell wall. Phytoene synthase (PSY) is a rate limiting factor of carotenoid biosynthesis and it may exhibit species-specific and organ-specific roles determined by the presence of <i>psy</i> paralogous genes, the importance of which often remains unrevealed. Thus, the aim of this work was to elaborate the roles of two <i>psy</i> paralogs in a model system and to reveal biochemical changes in the cell wall of <i>psy</i> knockout mutants. For this purpose, Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR associated (Cas9) proteins (CRISPR/Cas9) vectors were introduced to carotenoid-rich carrot (<i>Daucus carota</i>) callus cells in order to induce mutations in the <i>psy1</i> and <i>psy2</i> genes. Gene sequencing, expression analysis, and carotenoid content analysis revealed that the <i>psy2</i> gene is critical for carotenoid biosynthesis in this model and its knockout blocks carotenogenesis. The <i>psy2</i> knockout also decreased the expression of the <i>psy1</i> paralog. Immunohistochemical staining of the <i>psy2</i> mutant cells showed altered composition of arabinogalactan proteins, pectins, and extensins in the mutant cell walls. In particular, low-methylesterified pectins were abundantly present in the cell walls of carotenoid-rich callus in contrast to the carotenoid-free <i>psy2</i> mutant. Transmission electron microscopy revealed altered plastid transition to amyloplasts instead of chromoplasts. The results demonstrate for the first time that the inhibited biosynthesis of carotenoids triggers the cell wall remodelling.