Functional characterization of genes involved in ascorbic acid biosynthesis pathway from banana

Abstract

L-Ascorbic acid (AsA) is an antioxidant and micronutrient essential for the growth and development of plants and animals. However, many animals lack the ability to synthesize AsA due to a mutation in the L-gulono-1,4-lactone oxidase gene. In the present study, the AsA content in fruit pulp of 12 different banana cultivars was investigated and it was found that the Nendran exhibited the highest concentration of AsA at the ripe stage. This study also investigated the role of selected regulatory genes for AsA biosynthesis in banana. Among the various pathways, AsA is synthesized primarily through the Smirnoff-Wheeler pathway in plants. In this pathway, GDP-L-galactose phosphorylase (GGP) gene is known to regulate key step in AsA biosynthesis. Therefore, three MaGGP genes derived from the Nendran cultivar of banana were isolated, cloned and then overexpressed in A. thaliana. The results showed that among all three, MaGGP2 showed highest (15.58 mg/100g) AsA enhancement in the transgenic line compared to non-transformed control plants. The Myo-inositol oxygenase (MIOX) biosynthesis pathway is also known to contribute in AsA biosynthesis in plants and glucuronokinase (GlcAK) gene acts as a junction for the biosynthesis of the cell wall and AsA. To investigate its role in banana, the GlcAK gene was knocked out in cultivar Rasthali by using CRISPR/Cas9 approach. Edited plants showed 2.60 to 2.92 fold higher AsA content in ripe fruit pulp as compared to the non-edited control plants. In conclusion, this investigation sheds light on the limitations of AsA biosynthesis and offers new possibilities for AsA biofortification in plants, particularly in banana. newline