Identification and functional analysis of nucleoside hydrolases involved in the degradation of nicotinamide ribose to nicotinamide in kale (Brassica oleracea L. var. acephala DC.)

Nicotinamide riboside (NR), a precursor of nicotinamide adenine dinucleotide (NAD + ), plays a vital role in cellular metabolism and exhibits promising pharmacological properties. While its significance as a nutraceutical is well established, the metabolic pathway responsible for NR degradation in plants remains poorly understood. In this study, we identified and characterized nucleoside hydrolase genes in kale ( Brassica oleracea L. var. acephala DC.) that catalyze the hydrolysis of NR to nicotinamide (NAM). Using a combination of HMMER searches and BLASTp analyses, we identified seven candidate genes, of which five were cloned ( BolNSH1 - BolNSH5 ). In vitro assays demonstrated that BolNSH1, BolNSH2, and BolNSH3 efficiently hydrolyze NR producing NAM. Transient expression in Nicotiana benthamiana confirmed the in vivo functionality of these enzymes. Virus-induced gene silencing (VIGS) of BolNSH1 , BolNSH2 , BolNSH3 , and BolNSH4 in kale resulted in elevated NR levels, further validating their role in NR metabolism. Additionally, the nucleoside hydrolases exhibited broad substrate specificity, hydrolyzing a range of pyridine, pyrimidine and purine nucleosides. These findings provide new insights into the NR metabolic pathway in plants and lay the groundwork for developing kale varieties with enhanced NR content. Our work highlights the potential for modulating NR metabolism in plants to create high-value kale germplasm. • Nicotinamide riboside (NR) plays a vital role in cellular metabolism and exhibits promising pharmacological properties. • A combination of HMMER searches and BLASTp analyses identified the candidate nucleoside hydrolase genes. • BolNSH1, BolNSH2, BolNSH3, and BolNSH4 in kale were demonstrated that can catalyze the hydrolysis of NR to nicotinamide. • BolNSH1, BolNSH2, and BolNSH3 exhibited broad substrate specificity, hydrolyzing pyridine, pyrimidine and purine nucleosides. • These findings enhance our understanding of NR metabolism and lay a foundation for developing high-value kale germplasm.