Fig. 3: Natural rubber accumulation, rubber particle-associated protein distributions and interactions in P. sepium.

a The images in the cross-section under SCLSM bright-field showed that P. sepium stem consisted distinct layers of cortex, phloem, cambium, xylem and pith (i); Some small circular holes similar to the laticifer structure were mainly distributed in the secondary phloem adjacent to the cambium and the pith layers, and the laticifer cells were surrounded by irregular parenchyma cells in the secondary phloem (ii), but were usually surrounded by 5-6 regular parenchyma cells in the pith (iii); Red arrow, laticifers; Scale bars, 200 µm. b The cryosections were stained with Nile Red and Calcofluor White stain for the detection of cis-polyisoprene and the cell wall under SCLSM; Fluorescence images (yellow) in the cross-section revealed that cis-polyisoprenes appeared mainly in the secondary phloem and the pith (i); Most accumulated in the laticifer cells, and some dispersed in the surrounding cells (ii, iii); Fluorescence images in the tangential-section showed that cis-polyisoprenes presented disperse or nonarticulated linear shapes (iv, v, vi); Yellow, cis-polyisoprene; Blue, cell walls; Scale bars, 200 μm. c Immunostaining using primary antibodies against the rubber particle-associated proteins PsCPT, PsSRPP and PsREF, were stained with Alexa Fluor 555 and Calcofluor White stain for the detection of the secondary antibody Donkey Anti-Rabbit IgG and the cell wall via SCLSM; Fluorescence images (magenta) of the cross-sections revealed that all of the three proteins appeared in the secondary phloem and pith (i, iv, vii; Scale bars, 200 μm), almost entirely overlapping with the cis-polyisoprenes present; The fluorescence distributions in the secondary phloem revealed that PsCPT and PsSRPP were more abundant than PsREF was (iii, vi, ix; Scale bars, 100 μm), especially PsSRPP, which even extended to some radial rays in the secondary xylem (vi), whereas all proteins were present sporadically in the pith (ii, v, viii; Scale bars, 100 μm); Magenta, cis-polyisoprene; Blue, cell walls. d Protein interactions of PsCPT, PsSRPP and PsREF determined via BiFC in tobacco leaves; Compared with that of empty pSPYNE and pSPYCE coinfiltration (negative control) (i), YFP fluorescence signals (green) in coexpression of pSPYCE-PsCPT with pSPYNE-PsSRPP, pSPYCE-PsREF with pSPYNE-PsSRPP, or pSPYCE-PsCPT with pSPYNE-PsREF, were detected around the plasma membranes in the epidermal cell layers, indicating that the protein-protein interactions occur between PsCPT with PsSRPP (ii), PsSRPP with PsREF (iii), and PsCPT with PsREF (iv); Scale bars, 20 μm.