Aiping Gao

Introduction: The color of Clausena lansium L. Skeels cv. Jixin fruit peel is brown (BP), while the mutant cv. Zijin had purple fruit peels (PP). The coloration of the peels was attributed to significant differences in chlorophyll, carotenoid, and anthocyanin content between BP and PP.Methods: This study investigates the biosynthetic metabolic activities in the brown and purple peels of Clausena lansium L. Skeels using metabolomics and transcriptomics. It aims to identify metabolic pathways and differentially expressed genes related to flavonoids and anthocyanins biosynthesis.Results: The PP (purple peel) has higher levels of a-carotene and b-carotene but lower levels of chlorophyll a, chlorophyll b, and lutein compared to BP. Zeaxanthin was absent from both peels, suggesting that the b-carotene hydroxylase enzyme is not active. Both peels contain delphinidin-based (Dp) and cyanidin-based (Cy) anthocyanins, but not pelargonidin-based (Pg). The total anthocyanin content and the Dp/Cy ratio are higher in PP than in BP. The delphinidin, cyanidin, and mallow derivatives in the PP were significantly higher than in the BP. The increase of total anthocyanin content and Dp/Cy ratio may be the main reason for the peel color changing from brown to purple. The significant increase of F3H expression in purple peels suggested a higher efficiency of catalyzing the conversion of naringenin into dihydroflavonols in the PP, leading to the higher content of total anthocyanin. Despite the significant increase of FLS expression in PP, the contents of kaempferol, quercetin, and myricetin significantly decreased, suggesting that the increase of FLS expression did not lead to an increase in flavonol biosynthesis.Discussion: The competition between F3’H and F3’5’H may determine the ratio of Dp/Cy, the higher levels of F3’H, F3’5’H, and UFGT expression, lead to the increase accumulation of total anthocyanin and Dp/Cy in PP. The deficiency of Pg in both peels resulted from the substrate specificity of the DFR enzyme. The research also describes the transition in color from BP to PP and details of the biosynthetic pathways for carotenoids and anthocyanins, elucidating the molecular processes underlying anthocyanin production.

Mangoes (Mangifera indica L.) are an important kind of perennial fruit tree, but their biochemical testing method and transformation technology were insufficient and had not been rigorously explored. The protoplast technology is an excellent method for creating a rapid and effective tool for transient expression and transformation assays, particularly in plants that lack an Agrobacterium-mediated plant transformation system. This study optimized the conditions of the protoplast isolation and transformation system, which can provide a lot of help in the gene expression regulation study of mango. The most beneficial protoplast isolation conditions were 150 mg/mL of cellulase R-10 and 180 mg/mL of macerozyme R-10 in the digestion solution at pH 5.6 and 12 h of digestion time. The 0.16 M and 0.08 M mannitol in wash solution (WI) and suspension for counting (MMG), respectively, were optimal for the protoplast isolation yield. The isolated leaf protoplasts (~5.4 × 10⁵ cells/10 mL) were transfected for 30 min mediated by 40% calcium-chloride-based polyethylene glycol (PEG)-4000-CaCl₂, from which 84.38% of the protoplasts were transformed. About 0.08 M and 0.12 M of mannitol concentration in MMG and transfection solutions, respectively, were optimal for protoplast viability. Under the florescence signal, GFP was seen in the transformed protoplasts. This showed that the target gene was successfully induced into the protoplast and that it can be transcribed and translated. Experimental results in this paper show that our high-efficiency protoplast isolation and PEG-mediated transformation protocols can provide excellent new methods for creating a rapid and effective tool for the molecular mechanism study of mangoes.