C2 er

The coding sequence of PlPM19L without a stop codon was amplified via gene-specific primers (forward 5′-CAGTGGTCTCACAACATGGCTGCAGTTGGGAAGAC-3′; reverse 5′-CAGTGGTCTCATACAAATCCTAGTGCCAGTGTCAC-3′) and was fused into the green fluorescent protein (GFP) region of the pBWA(V)HS-GLosgfp vector. Then, the fusion constructs of pBWA(V)HS-PlPM19L-GLosgfp and empty pBWA(V)HS-GLosgfp were transformed into Agrobacterium tumefaciens strain GV3101 via the freeze–thaw method. Overnight Agrobacterium cultures containing pBWA(V)HS-PlPM19L-GLosgfp and empty pBWA(V)HS-GLosgfp vectors were resuspended in resurrection buffer (10 mM MES, 10 mM MgCl₂, and 0.2 mM acetosyringone) and then were injected into 4~5-week-old Nicotiana benthamiana leaves. After 2 days of weak light cultivation, the GFP signals of pBWA(V)HS-PlPM19L-GLosgfp and empty pBWA(V)HS-GLosgfp were observed via confocal laser microscopy (Nikon C2-ER, Tokyo, Japan) to determine the subcellular localization of PlPM19L.

The full-length coding region without the stop codon (1,416 bp) of CaPIF8 (XM_016689836.1) was cloned into the Super1300-GFP vector between the Xba I and Kpn I sites. The pSuper1300:CaPIF8:GFP vector was transformed into Agrobacterium tumefaciens strain GV3101. The pSuper1300:CaPIF8:GFP and pSuper1300:GFP (as control) were grown overnight, re-suspended in the induction medium (10 mM MgCl₂, 10 mM MES, and 200 μmol L^–1 acetosyringone), and injected into Nicotiana benthamiana. GFP fluorescence was photographed using a confocal laser-scanning microscope (Nikon C2-ER, Tokyo, Japan).

The specific primers (AhTPS9-F/R) were utilized to amplify the complete cDNA of AhTPS9 from tobacco leaves. The coding sequence data for AhTPS9 from PeanutBase (https://www.peanutbase.org/) were used as a reference. The resulting PCR products were connected to the pBWA (V) HS-GLosgfp vector, generating the pBWA (V) HS- AhTPS9-GFP vector which incorporates the green fluorescent protein (GFP) reporter gene. After confirmation through sequencing, the positive clones were transferred into Agrobacterium tumefaciens (EHA105) using electrotransformation. Young seedlings (30 days old) were carefully chosen, and injections were performed into the lower epidermis of leaves. Subsequently, these seedlings were cultured under low light conditions for 2 days. Observation and imaging took place using a laser confocal microscope (Nikon C2-ER, Tokyo, Japan), with the corresponding empty vector serving as a control.

To verify the subcellular localization of WD40 genes in tobacco, primers were designed for segment-length coding sequences (CDS) of NtWD40-389 (which named as NtTTG1 for its particularly high sequence similarity with TTG1 gene in Arabidopsis and other plants). The subcellular localization vector NtTTG1-PC1300s-GFP was constructed by homologous recombination (Fig. S7A). Generally, the plasmids (NtTTG1-PC1300s-GFP and PC1300s-GFP) were transformed into Agrobacterium GV3101, and positive colonies were verified and expanded to OD value 1 at 28 °C. These colonies were re-suspended with MgCl₂ + As + MES. After 3 h in darkness, they were injected into leaves of Nicotiana benthamiana. After injection, tobacco plants were cultivated in darkness for 1 d, and in normal condition for 1 d. Finally, GFP signals were detected using a confocal microscopy system (Nikon C2-ER, Japan).

The amplified AhBBX6 coding sequence was first ligated into the pBWA(V)HS-GFP vector using the in-fusion method and transfected into E. coli-competent cells (5α) to construct the pBWA(V)HS-AhBBX6-GFP vector plasmid. The constructed vector plasmid generated was transferred into the Agrobacterium tumefaciens strain GV3101 and cultivated at 30 °C for 2 d. The suspension OD value was adjusted to 0.6, and then the cultured strain was injected into the lower epidermis of vigorously growing tobacco leaves. After cultivation, under low light conditions for 2 d, the injected leaf samples were taken to make slides and then observed under a laser confocal microscope (Nikon C2-ER, Tokyo, Japan). The GFP vector without AhBBX6 was used as a control.

The CDS sequence of IbBBX28 was constructed into the pSm35s-nYFP vector and named NY-IBBBX28. The CDS sequences of the interacting protein coding genes were constructed into the pSm35s-cYFP vector and named IbHOX11-CY and IbZMAT2-CY, respectively. The primers required for the BiFC assay are shown in Supplementary Table S3. BamHⅠ and XbaⅠ were selected as the restriction sites, and the constructed vector plasmid was transformed into Agrobacterium GV3101 for the BiFC assay. Agrobacterium tumefaciens was inoculated into 10 ml LB liquid medium with spectinomycin and incubated in a shaker (200 rpm) at 28°C for 1 h. Then, it was centrifuged at 4,000 rpm for 10 min, the supernatant was discarded, and the bacterial cell was resuspended (10 mM MgCl₂ and 120 μM AS). The OD₆₀₀ was adjusted to about 0.6. The Agrobacterium solutions of the two genes were mixed in a ratio of 1:1, and left at room temperature for 3 h, and then injected with four to five leaf stage tobacco leaves. The injected tobacco was incubated at 26°C (16 h light/8 h dark) for 2 days; then, it was observed and photographed with a laser scanning confocal microscope (Nikon C2-ER). The excitation wavelength of yellow fluorescent protein YFP was 511 nm, and the emission wavelength was 525 nm.

A subcellular localization expression vector (pCAMBIA1301-osgfp-ky::VvDREB2c::GFP) was constructed using the pCAMBIA1301-osgfp-ky vector (Figure S1A), primers XbaDREB2c-F1 (5′-GCTCTAGAATGGATACCTGCGTTCAAG-3′) and BamDREB2c-R1 (5′-CGGGATCCGAACCCCATATCTGATA -3′), and the enzymes XbaI and BamHI. The constructed vector plasmid was then transferred into Agrobacterium GV3101. Finally, Agrobacterium suspension was injected into tobacco seedlings (Nicotiana rustica var Pavonii), following which the plants were cultured under low-light conditions for 2 days, after which the injected tobacco leaves were observed and photographed using a confocal microscope (C2-ER; Nikon, Minato City, Japan). The excitation and emission wavelengths of the chloroplast fluorescence signal were 640 and 675 nm, respectively, whereas those of the GFP fluorescent proteins were 488 and 510 nm.