C2 er confocal laser scanning microscope

Based on the tree peony genome sequence resource, specific primers were designed to obtain the sequence of PsSPL2 (Supplementary Table 1). The cDNA was synthesized using total RNA of the petals from “High Noon” at five blooming stages. Neighbor-joining method was used for constructing phylogenetic tree from evolutionary distance data by MEGA 6. Multiple sequence alignment was conducted using DNAMAN. The open reading frame (ORF) region of PsSPL2 without the termination codon was cloned into the pCAMBIA1302-GFP vector, and in-fusion cloning primers were listed in Supplementary Table 1. Subsequently, the recombinant plasmid was transformed into Agrobacterium tumefaciens strain GV3101 through freeze-thaw method. The positive colonies were selected and incubated at 28°C to OD₆₀₀ of 0.3 with the culture medium containing kanamycin. The Agrobacteria containing the target plasmids were resuspended in equal volume of infiltration buffer (10 mM MES, 10 mM MgCl₂, and 100 mM acetosyringone) and stationarily cultured for 4–6 h at room temperature. The onion epidermis was immersed in a sterile dark environment for 6–12 h and cultured on MS solid medium for 3–4 days. Finally, the fluorescence was observed under a Nikon C2-ER confocal laser scanning microscope (Nikon, Japan). All transient expression assays were repeated three times.

The full-length cDNA without the termination codon of PsMYB114L/PsMYB12L was amplified with special primers (PsMYB114L-GFPF/PsMYB12L-GFPF and PsMYB114L-GFPR/PsMYB12L-GFPR) (Table S1) with restriction sites (Xba I and Kpn I) and subcloned into the pCAMBIA1301-GFP vector between the Xba I and Kpn I sites to create the PsMYB114L-GFP/PsMYB12L-GFP fusion construct. The recombinant vectors (PsMYB114L-GFP/PsMYB12L-GFP) and control vector (pCAMBIA1301-GFP) were then introduced into tobacco leaves by agroinfiltration. These infiltrated plants were grown for over 72 h in a growth chamber, and the GFP fluorescence of samples was observed under a Nikon C2-ER confocal laser scanning microscope (Nikon, Tokyo, Japan) [67 (link)].

Full-length cDNA with the termination codon removed from PlWRKY13 was used as a template, and specific primers with restriction sites (XbaI and KpnI) were designed for PCR amplification (Table 1). After enzyme digestion, the obtained product was ligated to the pROKII-GFP vector that was also double digested, and the fusion expression vector pROKII-PlWRKY13-GFP was verified by sequencing. The recombinant vector (pROKII-PlWRKY13-GFP) and control vector (pROKII-GFP) were introduced into tobacco leaves through Agrobacterium infection. After culture for 3 days, the GFP fluorescence of samples was observed under a Nikon C2-ER confocal laser scanning microscope (Nikon, Tokyo, Japan).

The full-length ORF of PsMYB111 without the termination codon was cloned into the pCAMBIA1302-GFP vector. The primers are listed in Supplementary Table 7. Subsequently, the recombinant plasmid and control pCAMBIA1302-GFP plasmid were transferred into Agrobacterium strain GV3101 by the freeze–thaw method. The Agrobacterium containing the target plasmid was resuspended in infiltration buffer (with 10 mM MES, 10 mM MgCl₂, and 100 mM AS) to an OD600 of 0.4 and stationarily cultured for 2 h until infiltration. The Agrobacterium mixture was then injected into two young leaves of each N. benthamiana plant from the lower epidermis via a syringe without a needle. The infiltrated plants were grown in a growth chamber under the same conditions described above for 72 h, and GFP and 4′,6-diamidino-2-phenylindole fluorescence was observed under a Nikon C2-ER confocal laser scanning microscope (Nikon, Tokyo, Japan). All transient expression assays were repeated three times.

The open reading frame (ORF) sequence of CaSPDS was cloned into the pBWA(V)HS-GLosgfp vector using the BsaⅠ and Eco3IⅠ restriction endonucleases to form pBWA(V)HS-CaSPDS-GLosgfp. This fusion construct was transformed into tobacco epidermal cells using Agrobacterium-mediated transient transformation [52 (link)]. pBWA(V)HS-GLosgfp was used as a control. After 36–72 h of darkness, a Nikon C2-ER confocal laser scanning microscope (Nikon Instruments, Tokyo, Japan) was used to collect fluorescence images.

The MsYSL6 ORF without stop codes was cloned into the pBWA(V)HS-GLosgfp vector to construct a fusion plasmid. Then, the fusion plasmid was introduced into Agrobacterium tumefaciens GV3101 by freeze–thaw method and injected into tobacco leaf through the epidermis with a syringe. After cultivation for 2 d, fluorescence signals were recorded using a confocal laser scanning microscope (Nikon C2-ER Laser Scanning Confocal Microscope (Tokyo, Japan)). The green fluorescent protein was excited at 488 nm, and emissions were collected at 510 nm. The chlorophyll autofluorescence was excited at 640 nm, emissions were collected at 675 nm, and overlay images were created.