Particle bombardment

The CDS of AtRZFP, without its stop codon, was ligated in-frame to the N-terminus of the green fluorescent protein (GFP) under the control of CaMV 35S promoter (35S:ZFP-GFP). GFP under control of 35S promoter was also generated (35S:GFP). All primers used to make these constructs are shown in Supporting Information Supplementary Table S1. For subcellular location analysis, the constructs 35S:AtRZFP-GFP and 35S:GFP were introduced separately into onion epidermal cells using particle bombardment (Bio-Rad, Hercules, CA, USA). The transformed cells were analyzed under an LSM700 confocal laser microscope (Zeiss, Jena, Germany). GUS activity analysis was performed (Jefferson et al., 1987 (link)). For western blotting analysis, proteins were isolated from onion epidermal cells expressing 35S:AtRZFP-GFP transformed via particle bombardment or non-transformed onion epidermal cells (control). Western blot was performed according to the procedures described by Han et al. (2005) (link), and an anti-GFP antibody (Beyotime, Shanghai, China) was used to detect 35S:AtRZFP-GFP.

The full-length PfHSP17.2 coding region (without the stop codon) was ligated to the 3’ end of a green fluorescent protein (GFP) to generate a PfHSP17.2–GFP fusion construct, which was driven by the cauliflower mosaic virus (CaMV) 35S promoter. An empty pCAMBIA 1302 was used as a control. The PfHSP17.2–GFP construct and 35S-GFP (control) were introduced into onion epidermal cells by particle bombardment (Bio-Rad, CA, USA). Following bombardment, the cells were incubated in the dark at 25°C for 2–3 days prior to observation. Transiently transformed cells were analyzed using a confocal laser scanning microscope.

The subcellular localization of PsnMYB108 protein was performed according to the previous study (Wang et al., 2019a (link), b (link)). The coding sequence of PsnMYB108 gene without termination codon was linked to the 5’ terminus of the GFP gene, in order to construct the pBI121-PsnMYB108-GFP fusion expression vector. The primers and restriction sites used to construct the vector are listed in Supplementary Table S1. Then we transferred the gene fusion expression vector (pBI121-PsnMYB108-GFP) and the control vector (pBI121-GFP) into the onion epidermal cells using particle bombardment (Bio-Rad, Hercules, CA, United States). The bombarded onion epidermises were cultured in the dark for 24–36 h, then they were observed under the confocal laser scanning microscope (LSM 700, Zeiss, Germany) after staining with 100 ng/mL DAPI in the dark for 15 min (staining the nucleus).

The cDNA sequence of BplMYB46 was obtained from the birch transcriptome (Wang et al., 2014). The CDS of BplMYB46 without the stop codon fused in‐frame to the N‐terminus of green fluorescent protein (GFP) was transformed into the pROK2 vector under the control of the CaMV 35S promoter (35S : MYB‐GFP; primer sequences are shown in Table S2). The GFP protein under the control of the 35S promoter was used as a control (35S : GFP). The 35S : MYB‐GFP and 35S : GFP constructs were introduced into onion epidermal cells by particle bombardment (Bio‐Rad laboratories, Inc. Hercules, California, USA). After incubation for 48 h, the transformed onion epidermal cells were stained with DAPI (100 ng/mL) and visualized under an LSM700 confocal laser microscope (Zeiss, Jena, Germany).

The coding sequence of BpGRF3 was fused in frame to the amino terminus of the enhanced green fluorescent protein (eGFP) coding sequence under control of the CaMV35S promoter, as previously described (Liu et al., 2020 (link), 2022a (link)). As a control, GFP transcribed using the CaMV 35S promoter (35S::GFP) was used. Particle bombardment (Bio‐Rad, Hercules, CA, USA) was used to introduce the constructs into onion epidermal cells. Confocal laser‐scanning microscopy (LSM700, Zeiss, Jena, Germany) was then used to analyse the transformed cells. The primers are listed in Table S1.

The pBI121-gene-GFPs and pBI121-GFP (control) were separately transformed into onion epidermal cells using particle bombardment (BioRad). After incubation on 1/2MS medium for 24 h in the dark, the transformed onion epidermal cells were stained with DAPI (100 ng/mL) and visualized under a confocal laser-scanning microscope (A1, Nikon, Japan).

The coding sequence (CDS) of ThNAC7 with no termination codon was fused with the C-terminus of the GFP following the CaMV 35S promoter. 35S::GFP was used as control. All primer used are listed in Table S1. The 35S::ThNAC7-GFP and 35S::GFP plasmids was respectively imported into onion epidermal cells by particle bombardment (Bio-Rad, Hercules, CA, USA). The nuclei were stained using 4′,6-diamidino-2-phenylindole (DAPI) (10 μg·mL⁻¹) with phosphate buffered saline for 5 min. The transformed onion epidermal cells were visualised by LSM700 confocal laser scanning microscopy (Zeiss, Jena, Germany).