The open reading frame (ORF) region of LbSAD2 was cloned into the vector pCAMBIA1300, containing a CaMV 35S promoter, hygromycin resistance gene, and GFP reporter gene, by homologous recombination with the primers LbSAD2 1300-S and LbSAD2 1300-A (Supplementary Table 1) using the In-Fusion HD Cloning Kit (Clontech Laboratories, Inc.). The resulting p1300-LbSAD2 vector was transformed into onion epidermal cells using Agrobacterium tumefaciens strain GV3101 (Sun et al., 2007). Fluorescence signals of labeled LbSAD2 were detected by microscopy (TCS S8 MP two-photon laser scanning confocal microscope, Leica, Germany). Simultaneously, the nucleus was positioned using DAPI staining observed under 358 nm excitation. The plasma membrane was stained using N-(3-triethylammoniumpropyl)-4-(6-(4-(diethylamino)phenyl)hexatrienyl (FM4-64, pyridinium dibromide, Invitrogen) and excited under 559 nm.

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