Figure 1.
Association of TbSRPPs with rubber particles. A.
Fifty micrograms of purified rubber particle proteins were separated by isoelectric focussing (pH 3–10 IPG strips) and subsequent SDS-PAGE. The gel was stained with colloidal Coomassie Brilliant Blue. B. Backscattered electron imaging of rubber particles labeled with 10 nm gold particles by immunodetection using the TbSRPP-antibody. C. Gold labeling of rubber particles was not appreciably detected in backscattered electron images, when the corresponding pre-immune serum was used as primary antibody. Micrographs are shown as inverted images. Scale bars = 400 nm.
Figure 2.
Analysis of TbSRPP1–5 expression in TbSRPP-RNAi transgenic and wild-type plants. A.
TbSRPP transcription levels in the latex of three independent TbSRPP-RNAi transgenic lines (designated S3, S76 and S85) were quantified by RT-PCR. Relative transcription of TbSRPPs was normalized to the constitutively expressed control gene TbACTIN. The ratios of TbSRPPs/TbACTIN transcription levels in wild-type (wt) served as a reference and were set to the value of one. Values represent mean ± SE (n = 3). B, C. Fifty microgram of rubber particle proteins from wild-type (B) and TbSRPP-RNAi lines (C, exemplarily shown for line S3) was separated by isoelectric focussing (pH 3–6 IPG strips) and subsequent SDS-PAGE. Proteins were transferred to nitrocellulose membranes and TbSRPPs were detected using an anti-TbSRPP antibody and a secondary antibody conjugated with horse radish peroxidase. D, E. Ponceau-S staining of the nitrocellulose membranes confirmed equal loading of gels prepared for wild-type (D) and line S3 (E), by showing that proteins spots of the C-terminus of the Polyphenoloxidase-1 (TbPPO-1) were present in similar amounts [29], [30].
Table 1.
Table 1. Content, molecular mass and polydispersity of rubber from exuded latex and freeze-dried roots and corresponding long-chain prenyltransferase activity.
Figure 3.
In situ analysis of the ultra-structure of rubber particles from TbSRPP-RNAi transgenic plants and wild-type plants by transmission electron microscopy.
A. Micrograph of ultra-thin cross sections of roots from TbSRPP-RNAi transgenic plant (S3) at low and B. higher magnification. C. Micrograph of ultra-thin cross sections of roots from a wild-type plant at low and D. higher magnification. Enlarged areas are indicated with dashed boxes. Key: RP, rubber particle; V, vacuole; CW, cell wall; PC, parenchyma cell. Scale bars = 1 µm.
Figure 4.
In vitro analysis of the colloidal stability of rubber particles from TbSRPP-RNAi transgenic plants and wild-type plants.
A. Measurement of the zeta (ζ)-potential of rubber particles from wild-type plants (filled circles) and TbSRPP-RNAi transgenic plants (open circles) suspended in modified rubber extraction buffer (REB) at given pH values. Values represent mean ± SD of three independent plant lines (S3, S76 and S85) respectively of six wild-type plants (wt). No significant differences were detected among the plants using Students t-test (P, 0.05). B. Measurement of the z-average diameter of rubber particles from wild-type plants (filled circles) and TbSRPP-RNAi transgenic plants (open circles) suspended in modified REB with the indicated pH values. Values represent mean ± SD of three independent plant lines (S3, S76 and S85) respectively of six wild-type plants (wt). Significant differences have been detected among TbSRPP-RNAi lines and wild-type plants using Students t-test (P, 0.01). C, F. Confocal laser scanning microscopic analysis of isolated rubber particles from TbSRPP-RNAi transgenic plants (C) and wild-type plants (F) that were stained with nile red. D, E and G, H. Transmission electron microscopic analysis of isolated rubber particles from TbSRPP-RNAi transgenic and wild-type plants. D. Micrograph of isolated rubber particles from TbSRPP-RNAi transgenic plant line S3 suspended in modified rubber extraction buffer (REB) pH 7.2 at low and E. higher magnification. G. Micrograph of rubber particles from wild-type suspended in modified REB pH 7.2 at low and H. higher magnification. Enlarged areas are indicated with dashed boxes. Scale bars = 1 µm.