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 psy paralogous genes, the importance of which often remains unrevealed. Thus, the aim of this work was to elaborate the roles of two psy paralogs in a model system and to reveal biochemical changes in the cell wall of psy 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 (Daucus carota) callus cells in order to induce mutations in the psy1 and psy2 genes. Gene sequencing, expression analysis, and carotenoid content analysis revealed that the psy2 gene is critical for carotenoid biosynthesis in this model and its knockout blocks carotenogenesis. The psy2 knockout also decreased the expression of the psy1 paralog. Immunohistochemical staining of the psy2 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 psy2 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.
Highlights
The role of two psy paralogs in the carotenoid biosynthesis was elucidated in the model callus system and for this purpose both genes were targeted during experiments aiming in the generation of knockout mutations
The results indicate differential roles and regulation of psy paralogs, with psy2 being critical for carotenoid biosynthesis in this model
To what extent, psy1 contributes to carotenogenesis in callus remains unclear as there was no correlation between its expression level and carotenoid content when psy2 was active
Summary
Carotenoids are a vast group of compounds that widely exist in nature. In plants, they play crucial roles in photosynthetic systems and they are precursors of phytohormones [1]. Beneficial effects of carotenoids on human health are extensive. Β-carotene and α-carotene have provitamin A activity, and others are beneficial for treatment of agerelated macular degeneration or in prevention of cardiovascular disorders and cancer [2]
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