Department of Plant Biology and Biotechnology, Faculty of Biotechnology and Horticulture, University of Agriculture in Krakow, 31-425 Krakow, Poland.
Institute of Biology, Biotechnology and Environmental Protection, Faculty of Natural Sciences, University of Silesia in Katowice, 40-032 Katowice, Poland.
Int J Mol Sci. 2021 Jun 17;22(12):6516. doi: 10.3390/ijms22126516.
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 paralogous genes, the importance of which often remains unrevealed. Thus, the aim of this work was to elaborate the roles of two paralogs in a model system and to reveal biochemical changes in the cell wall of 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 () callus cells in order to induce mutations in the and genes. Gene sequencing, expression analysis, and carotenoid content analysis revealed that the gene is critical for carotenoid biosynthesis in this model and its knockout blocks carotenogenesis. The knockout also decreased the expression of the paralog. Immunohistochemical staining of the 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 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.
最近的数据表明，植物类胡萝卜素生物合成途径的修饰会改变影响细胞壁化学成分的基因的表达。八氢番茄红素合酶（PSY）是类胡萝卜素生物合成的限速因子，它可能表现出物种特异性和器官特异性的作用，这取决于同源基因的存在，而这些基因的重要性往往尚未揭示。因此，本工作的目的是阐述两个同源基因在模式系统中的作用，并揭示敲除突变体细胞壁的生化变化。为此，将成簇规律间隔短回文重复序列（CRISPR）和 CRISPR 相关（Cas9）蛋白（CRISPR/Cas9）载体引入富含类胡萝卜素的胡萝卜（）愈伤组织细胞中，以诱导和基因的突变。基因测序、表达分析和类胡萝卜素含量分析表明，在该模型中基因对于类胡萝卜素生物合成至关重要，其敲除会阻断类胡萝卜素的生物合成。基因的敲除也降低了同源基因的表达。突变细胞的免疫组织化学染色显示，突变细胞的细胞壁中阿拉伯半乳聚糖蛋白、果胶和伸展蛋白的组成发生了改变。特别是，富含类胡萝卜素的愈伤组织细胞壁中存在大量低甲酯果胶，而不含类胡萝卜素的突变体则相反。透射电子显微镜显示质体向淀粉体的转变发生了改变，而不是向有色体的转变。这些结果首次证明，抑制类胡萝卜素的生物合成会触发细胞壁的重塑。
