Down-Regulation of Small Rubber Particle Protein Expression Affects Integrity of Rubber Particles and Rubber Content in Taraxacum brevicorniculatum

Andrea Hillebrand,David Wurbs,Christian Schulze Gronover,Daniela Wahler,Janina J Post,Dirk Prüfer,Vladislav Krzyzanek,Malte Lenders,Luis Herrera-Estrella

doi:10.1371/journal.pone.0041874

Andrea Hillebrand, David Wurbs + Show 7 more

Open Access

https://doi.org/10.1371/journal.pone.0041874

Copy DOI

Abstract

The biosynthesis of rubber is thought to take place on the surface of rubber particles in laticifers, highly specialized cells that are present in more than 40 plant families. The small rubber particle protein (SRPP) has been supposed to be involved in rubber biosynthesis, and recently five SRPPs (TbSRPP1–5) were identified in the rubber-producing dandelion species Taraxacum brevicorniculatum. Here, we demonstrate by immunogold labeling that TbSRPPs are localized to rubber particles, and that rubber particles mainly consist of TbSRPP3, 4 and 5 as shown by high-resolution two-dimensional gel electrophoresis and mass spectrometric analysis. We also carried out an RNA-interference approach in transgenic plants to address the function of TbSRPPs in rubber biosynthesis as well as rubber particle morphology and stability. TbSRPP-RNAi transgenic T. brevicorniculatum plants showed a 40–50% reduction in the dry rubber content, but neither the rubber weight average molecular mass nor the polydispersity of the rubber were affected. Although no phenotypical differences to wild-type particles could be observed in vivo, rubber particles from the TbSRPP-RNAi transgenic lines were less stable and tend to rapidly aggregate in expelling latex after wounding of laticifers. Our results prove that TbSRPPs are very crucial for rubber production in T. brevicorniculatum, probably by contributing to a most favourable and stable rubber particle architecture for efficient rubber biosynthesis and eventually storage.

Highlights

2,500 plants produce natural rubber poly, which is stored in rubber particles dispersed throughout the cytosol of either laticifers or specialized parenchyma cells [1]
There is some evidence indicating a potential role in rubber biosynthesis: In vitro-experiments revealed that the addition of recombinant HbSRPP to H. brasiliensis rubber particles suspended in latex cytosol increased the incorporation of isopentenyl pyrophosphate (IPP), while addition of antibodies directed against HbSRPP and HbREF inhibited IPP incorporation [24,25]
In this study we showed that the three small rubber particle protein (SRPP) homologs TbSRPP3, 4 and 5 are the major rubber particle proteins in T. brevicorniculatum and confirmed their association with rubber particles by immunogold labeling and scanning electron microscopy (SEM)

Summary

Introduction

2,500 plants produce natural rubber poly (cis1,4-isoprene), which is stored in rubber particles dispersed throughout the cytosol of either laticifers or specialized parenchyma cells [1]. Natural rubber is synthesized by the successive addition of isopentenyl pyrophosphate (IPP) to an initiating allylic pyrophosphate, e.g. farnesyl pyrophosphate (FPP) [1,2,3,4,5] This polymerization reaction is thought to take place on the surface of rubber particles, mediated by a particle bound cis-prenyltransferase (CPT) [1,6,7,8]. Rubber particles comprise a homogenous rubber core surrounded by a monolayer membrane containing lipids, proteins, and several minor compounds [12,14,15] This composite monolayer membrane probably mediates the colloidal stability of rubber particles by providing a net negative surface charge that prevents their coalescence in the cytosol [12,14]. It has been proposed that the rubber-producing species F. carica and F. benghalensis do not possess a SRPP homolog, suggesting that SRPP is not a general and essential protein for rubber biosynthesis [13]

Methods

Results

Conclusion