Nucleotide Variation In The Phytoene Synthase (ClPsy1) Gene Contributes To Golden Flesh In Watermelon (Citrullus Lanatus L.)

Vitamin A deficiency is a worldwide public nutrition problem, and β-carotene is the precursor for vitamin A synthesis. Watermelon with golden flesh (gf, due to accumulated abundance of β-carotene) is an important germplasm resource. In this study, a genetic analysis of gf segregating populations indicated that gf was controlled by a single recessive gene. BSA-seq and an initial linkage analysis placed the gf locus in a 290-Kb region on watermelon chromosome 1. Further fine mapping in a large population with over 1,000 F2 plants narrowed this region to 39.08 Kb harboring two genes, Cla97C01G008760 and Cla97C01G008770, which encode phytoene synthase (ClPsy1) and GATA zinc finger domain-containing protein, respectively. Gene sequence alignment and expression analysis between parental lines revealed Cla97C01G008760 as the best possible candidate gene for gf trait. Nonsynonymous SNP mutations in the first exon of ClPsy1 between parental lines cosegregated with the gf trait only among individuals in the genetic population but were not related to flesh color in natural watermelon panels. Promoter sequence analysis of 26 watermelon accessions revealed two SNPs in the cis-acting element sequences corresponding to MYB and MYC2 transcription factors. RNA-seq data and qRT-PCR verification showed that two MYBs and one MYC2 exhibited expression trends similar as ClPsy1 in the parental lines, which may thus play roles in the regulation of ClPsy1 expression. Our research findings indicate that the gf trait is determined not only by ClPsy1 but also by ClLCYB, ClCRTISO and ClNCED7, which play important roles in β-carotene accumulation in watermelon flesh.

Chemical inhibition of lycopene β-cyclases unmask operation of phytoene synthase 2 in ripening tomato fruits

In ripening tomato fruits, the leaf-specific carotenoids biosynthesis mediated by phytoene synthase 2 (PSY2) is replaced by a fruit-specific pathway by the expression of two chromoplast-specific genes: phytoene synthase 1 (PSY1) and lycopene-β-cyclase (CYCB). Consequently, mutations in those two and other genes contributing to intermediate steps render the ripened tomato fruits bereft of lycopene. To decipher whether PSY2-mediated pathway also operates in ripening fruits, we blocked the in vivo activity of lycopene-β-cyclases by injecting CPTA (2-(4-Chlorophenylthio) triethylamine hydrochloride), an inhibitor of lycopene-β-cyclases. The injection of CPTA induced accumulation of lycopene in leaves, immature-green and ripening fruits. Even, in tomato mutants deficient in fruit-specific carotenoid biosynthesis such as V7 and r (PSY1), and ζ-carotene isomerase (ZISO), CPTA triggered lycopene accumulation. The CPTA-treated ziso mutant fruits, where PSY1 remains functional, accumulated phytoene and phytofluene. Conversely, CPTA-treated PSY1-knockout mutant (r3756) fruits did not accumulate phytoene and phytofluene. CPTA-treated fruits were enriched in lycopene-derived volatiles and had reduced ABA levels. The lycopene accumulation was associated with the partial transformation of chloroplasts to chromoplasts bearing thread-like crystalline structures, indicating lycopene accumulation. Our study shows that inhibition of lycopene β-cyclases unmasks the operation of a parallel carotenoid biosynthetic pathway mediated by PSY2 in ripening tomato fruits.

Bioinformatics Analysis and Identification of Phytoene Synthase Gene in Microalgae

Background: Carotenoids are known as lipophilic secondary metabolites with important biological activities, which are mostly used in the food and pharmaceutical industry. They contribute to the colours of many fruits and flowers. Studies on the biosynthetic pathways of isoprenoids and carotenoids are still scarce, especially in microalgae, and are limited to specific groups Dunaliella spp. In the Chlorophyta taxon of algae, the 2-C-methyl-D-erythritol 4-phosphate/1-deoxy-D-xylulose 5-phosphate (DOXP/MEP) is the synthesis pathway of sterols and carotenoids. Objectives: In this study, we used 12 Psy gene sequences in Dunaliella sp., also Scenedesmus acutus, and Diospyros kaki to investigate a genome-wide search. The results are useful for better identification of carotenoids metabolisms and increasing the production rate of beta-carotene in pharmaceutical, food, and industrial processes. Methods: Phytoene synthase (Psy) from Dunaliella spp. was selected as the first regulatory point in the carotenoids pathway that catalysis the formation of geranylgeranyl pyrophosphate in isoprenoid biosynthesis. Structural, evolutionary, and physics-chemical characteristics were investigated using various bioinformatics tools and computer techniques. Moreover, some recently published patents were also regarded. Results: The maximum length of the conserved motif was 5167 bp for Dunaliella. sp. (DQ463306.1), and the smallest length of the conserved motif was 416 bp belong to D. salina (JQ762451.1). The average molecular weight of species was 41820.53 Da. The theoretical pI of species varied from 4.87 to 9.65, indicating vernation in the acidic nature. Two strains of D. bardawil (U91900.1 and EU328287.1) showed just a long-distance relationship with all other Dunaliella strains. Whilst, D. parva displayed the furthest vicinity with all the studied strains. Conclusion: Our study highlighted the Psy regulatory mechanism as a key factor in the carotenoids pathway to facilitate genetic and metabolic engineering studies. The obtained three-dimensional arrangement of the amino acids revealed the regional structures and folding of the diverse segments of helices, sheets, turns. This information is a key point to unveil the protein's operation mechanism. Besides, we confirmed the suitability of bioinformatic approaches for analysing the gene structures and identifying the new Psy genes in unstudied microalgal strains.

Expression of ZjPSY, A Phytoene Synthase Gene from Zoysia Japonica Leads to Leaf Yellowing and Plant Dwarfing in Arabidopsis Thaliana

Phytoene synthase (Psy) is a key limiting enzyme in the carotenoid biosynthesis pathway by regulating phytoene synthesis. In this study, ZjPSY was isolated and identified from an important lawn grass species, Zoysia japonica. ZjPSY cDNA was 1230 bp in length, corresponding to 409 amino acids. ZjPSY showed higher expression in young leaves and were down-regulated after GA3, ABA, SA, and MeJA treatments, exhibited a sensitivity to hormones. By analysis of cis-regulatory elements in ZjPSY promoter region, ZjPSY exhibited be regulated of light and multiple hormones. To investigate the functions of ZjPSY, the plant expression vector was constructed to obtain transgenic Arabidopsis thaliana. Overexpression of ZjPSY protein led to carotenoid accumulation and altered expression of genes involved in carotenoid contents. ZjPSY expressing Arabidopsis thaliana exhibited yellowing and dwarfing phenotypes and contained more carotenoids than the wild type. Yeast two-hybrid screening identified a novel interacting partner of ZjPSY, ZjJ2 (DNAJ homologue 2), which encodes heat-shock protein 40 (HSP40). Taken together, this study suggests that ZjPSY plays an important role in carotenoid synthesis, leaf color development and hormone response in transgenic plants. These results broadened the understanding of carotenoid synthesis pathways and laid a foundation for the exploration and utilization of PSY gene.

Light Harvesting-like Protein 3 Interacts with Phytoene Synthase and Is Necessary for Carotenoid and Chlorophyll Biosynthesis in Rice

Background Carotenoid biosynthesis is essential for the generation of photosynthetic pigments, phytohormone production, and flower color development. The light harvesting like 3 (LIL3) protein, which belongs to the light-harvesting complex protein family in photosystems, interacts with geranylgeranyl reductase (GGR) and protochlorophyllide oxidoreductase (POR) both of which are known to regulate terpenoid and chlorophyll biosynthesis, respectively, in both rice and Arabidopsis. Results In our study, a CRISPR-Cas9 generated 4-bp deletion mutant oslil3 showed aberrant chloroplast development, growth defects, low fertility rates and reduced pigment contents. A comparative transcriptomic analysis of oslil3 suggested that differentially expressed genes (DEGs) involved in photosynthesis, cell wall modification, primary and secondary metabolism are differentially regulated in the mutant. Protein-protein interaction assays indicated that LIL3 interacts with phytoene synthase (PSY) and in addition the gene expression of PSY genes are regulated by LIL3. Subcellular localization of LIL3 and PSY suggested that both are thylakoid membrane anchored proteins in the chloroplast. We suggest that LIL3 directly interacts with PSY to regulate carotenoid biosynthesis. Conclusion This study reveals a new role of LIL3 in regulating pigment biosynthesis through interaction with the rate limiting enzyme PSY in carotenoid biosynthesis in rice presenting it as a putative target for genetic manipulation of pigment biosynthesis pathways in crop plants.