2.10. BSMV Inoculation and Treatment

BSMV analysis was conducted according to He et al. [38]. RT-PCR was performed using primers containing NheI sites (Table S2) to obtain a 286-bp cDNA fragment of the barley phytoene desaturase gene (HvPDS) and a 254-bp cDNA fragment of HvSAMS3. The inoculation was conducted on the second leaf of XZ5 plants at the two-leaf stage. For BSMV:HvPDS VIGS, the leaf samples were chosen based on the observed phenotype, and photos of the leaf were acquired using a camera (EOS 7D, Canon, Japan). To confirm the VIGS effectiveness and function of HvSAMS3 in XZ5, eight independent sets of treatments were performed: mock-inoculated with BSMV:γ; mock-inoculated with BSMV:γ and treated with drought, salinity, and D+S; BSMV:HvSAMS3-inoculated seedlings; and BSMV:HvSAMS3-inoculated seedlings treated with drought, salinity, and D+S. Every treatment contained 5 biological replicates, and each had 5 plants. Additionally, the transcript levels of HvPDS and HvSAMS3 were measured using quantitative RT-PCR and semiquantitative RT-PCR, respectively. Ten days after the treatments, the second leaves from the upper of barley seedlings were harvested for measurements of the following parameters: potassium (K⁺) concentration; endogenous polyamide (PA) and 1-aminocyclopropane-1-carboxylic acid (ACC) (ethylene) contents; polyamine oxidase (PAO), and diamine oxidase (DAO), and antioxidant enzyme activities.