The subcellular localization of LbHLH was determined using transformed onion epidermal cells harboring a GFP expression vector [26]. The pCAMBIA1300 vector was digested with SalI to form a linear vector. To obtain LbHLH cDNA, primers LbHLH OE-S and LbHLH OE-A were designed with SalI digestion sites (Table S1). The full-length coding sequence (CDS) of LbHLH carrying a SalI digestion site was introduced into the pCAMBIA1300 vector under the control of the CaMV 35S promoter by homologous recombination using a ClonExpress II One Step Cloning Kit (Vazyme Biotech Co., Ltd., China). Agrobacterium tumefaciens GV3101 was used to transform the pCAMBIA1300-LbHLH recombinant vector into onion epidermal cells [27]. After two days of cultivation in the light, fluorescent signals of GFP-labeled LbHLH were detected under a TCS S8 MP two-photon laser-scanning confocal microscope (Leica, Germany). DAPI was used to locate the nucleus and was observed under excitation at 358 nm [28]. FM4 − 64 (N-(3-Triethylammoniumpropyl)-4-(6-(4-(Diethylamino)Phenyl) Hexatrienyl) Pyridinium Dibromide, Invitrogen) was used to locate the plasma membrane and observed under excitation at 559 nm [29].

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