Genome-wide identification, molecular docking and expression analysis of enzymes involved in the primary and secondary metabolic branching points of the selenium metabolic pathway in Cardamine hupingshanensis

Cardamine hupingshanensis is a plant known for its unique selenium tolerance, making it a key model for selenium metabolism research. Adenosine phosphosulfate kinase ( APK ) and adenosine phosphosulfate reductase (APR) are widely distributed in plants and play a crucial role in selenium metabolism. While genome-wide analyses of the APK and APR families have been conducted across various plant species, a systematic identification and analysis of these gene families in Cardamine hupingshanensis is still lacking. There are 7 ChAPK and 5 ChAPR genes identified from the genome of C. hupingshanensis , which can be classified into 4 subfamilies for ChAPK and 3 subfamilies for ChAPR , respectively. All these members share similar conserved motifs and gene structures. Phylogenetic and promoter analyses suggest they are involved in environmental responses, phytohormone regulation, and light signalling. Molecular docking analysis indicated that ChAPK enzymes have a higher affinity for adenosine phosphoselenate (APSe) compared to ChAPR. In 3D interaction force analysis, residues such as His 80 , Asp 134 and Arg 137 were found to interact with the substrate APSe in ChAPK. For ChAPR, residues such as Gly 226 , Arg 313 and Leu 150 were primarily involved in the catalytic site. Leaf cells of C. hupingshanensis exhibited a reduced state under 1 µg Se L − 1 selenite concentration, but showed an oxidative state at 100 µg Se L − 1 , as indicated by antioxidant enzyme activities and metabolite assays. The gene expression level of ChAPK2-1 and ChAPR2 increased by 4.2- and 10.1-folds, respectively, in the reduced state of plant cells, with smaller increases in the oxidative state, ChAPK2-1 increased by only 1.5-fold and ChAPR2 by 5.2-fold. All members of ChAPK and ChAPR families have a strong affinity for APSe and are regulated by the redox state. However, only three members of ChAPK ( ChAPK1-1 , ChAPK1-2 , and ChAPK4-2 ) are regulated by the redox state, and these are located in the chloroplast. Furthermore, low concentrations of selenium in the nutrient solution can promote antioxidant activity in the leaves of C. hupingshanensis seedlings, whereas high concentrations of selenium exhibit the opposite effect, as confirmed by the results of oxidative metabolite and antioxidant enzyme assays.