Heterologous expression of AaLac1 gene in hairy roots and its role in secondary metabolism under PEG-induced osmotic stress condition in Artemisia annua L.

This study explores the Laccase gene (AaLac) family along with AaLac1 expression in hairy roots of A. annua. 42 AaLacs were identified by detecting three conserved domains: Cu-oxidase, Cu oxidase-2, and Cu oxidase-3. The physicochemical properties show that AaLacs are proteins with 541-1075 amino acids. These proteins are stable, with an instability index less than 40. Phylogenetic and motif studies have shown structural variants in AaLacs, suggesting functional divergence. 22 AaLac cis-regulatory elements were selected for their roles in drought stress, metabolic modulations, defense, and stress responses. A comparison of AtLac and AaLac proteins showed that 11 AtLacs mitigates stress reactions. In silico expression, analysis of 11 AtLacs showed that AtLac84 may function under osmotic stress. Thus, the Homolog AaLac1 was selected by expression profiling. The real-time PCR results showed that AaLac1 enhances osmotic stress tolerance in shoot and root samples. It was also used to analyze AaLac1, ADS, and CYP71AV1 gene expression in hairy roots via induction. The transformed hairy roots exhibited a greater capacity for PEG-induced osmotic stress tolerance in contrast to the untransformed roots. The gene expression analysis also depicted a significant increment in expression of AaLac1, ADS, and CYP71AV1 genes to 3.8, 6.9, and 3.1 folds respectively. The transformed hairy roots exhibited a significant increase of 2.2 and 1.4 fold in flavonoid and phenolic content respectively. Also, lignin content and artemisinin content increased by 7.05 folds and 95.6% with respect to the control. Thus, transformed hairy roots of A. annua under PEG-induced osmotic stress demonstrate the involvement of the AaLac1 gene in stress responses, lignin biosynthesis, and secondary metabolism production.

Supplementary information: The online version contains supplementary material available at 10.1007/s12298-024-01516-8.