The Plant Journal (2015)

doi: 10.1111/tpj.12836

Identification of a Taraxacum brevicorniculatum rubber elongation factor protein that is localized on rubber particles and promotes rubber biosynthesis € fer1,2 and Christian Schulze Gronover1,* Natalie Laibach1, Andrea Hillebrand2, Richard M. Twyman3, Dirk Pru 1 Fraunhofer Institute for Molecular Biology and Applied Ecology, Schlossplatz 8, Mu€ nster 48143, Germany, 2 Westphalian Wilhelms University of Mu€ nster, Institute of Plant Biology and Biotechnology, Schlossplatz 8, Mu€ nster 48143, Germany, and 3 TRM Ltd, York YO43 3WE, UK Received 13 January 2015; revised 18 March 2015; accepted 19 March 2015. *For correspondence (e-mail [email protected]).

SUMMARY Two protein families required for rubber biosynthesis in Taraxacum brevicorniculatum have recently been characterized, namely the cis-prenyltransferases (TbCPTs) and the small rubber particle proteins (TbSRPPs). The latter were shown to be the most abundant proteins on rubber particles, where rubber biosynthesis takes place. Here we identified a protein designated T. brevicorniculatum rubber elongation factor (TbREF) by using mass spectrometry to analyze rubber particle proteins. TbREF is homologous to the TbSRPPs but has a molecular mass that is atypical for the family. The promoter was shown to be active in laticifers, and the protein itself was localized on the rubber particle surface. In TbREF-silenced plants generated by RNA interference, the rubber content was significantly reduced, correlating with lower TbCPT protein levels and less TbCPT activity in the latex. However, the molecular mass of the rubber was not affected by TbREF silencing. The colloidal stability of rubber particles isolated from TbREF-silenced plants was also unchanged. This was not surprising because TbREF depletion did not affect the abundance of TbSRPPs, which are required for rubber particle stability. Our findings suggest that TbREF is an important component of the rubber biosynthesis machinery in T. brevicorniculatum, and may play a role in rubber particle biogenesis and influence rubber production. Keywords: rubber, Taraxacum brevicorniculatum, rubber elongation factor, latex, natural rubber.

INTRODUCTION Natural rubber, which is mainly comprised of poly(cis-1,4– isoprene), is produced in the latex of many plant species in highly specialized cells known as laticifers (Cornish, 2001a; van Beilen and Poirier, 2007). Although natural rubber is synthesized by more than 2000 plant species, only Hevea brasiliensis is used for commercial production (Cornish, 2001a). Certain species, such as guayule (Parthenium argentatum) and some dandelions (e.g. Taraxacum brevicorniculatum), are potential alternative sources of rubber (Mooibroek and Cornish, 2000; van Beilen and Poirier, 2007; Schmidt et al., 2010b). Despite the industrial relevance of natural rubber, its biosynthesis and biological function are still not completely understood. Rubber is synthesized in the cytosol of laticifers on small dispersed particles, which are surrounded by a phospholipid monolayer and contain a high-molecular-weight rubber core, thus resembling the structure of lipid droplets © 2015 The Authors The Plant Journal © 2015 John Wiley & Sons Ltd

found in seeds and other tissues (Cornish et al., 1999; Nawamawat et al., 2011; Thiam et al., 2013; Laibach et al., 2014). Rubber chain elongation is mediated by sequential addition of isopentenyldiphosphate (IPP) monomers, derived from the mevalonate pathway, to the starter molecule farnesyldiphosphate (Bandurski and Teas, 1957; Hepper and Audley, 1969; Light and Dennis, 1989; Cornish and Backhaus, 1990; Tanaka et al., 1996). This reaction is catalyzed by a rubber transferase, a specialized CPT, that is probably bound to the rubber particle (Archer et al., 1963; Madhavan et al., 1989; Cornish and Backhaus, 1990; Cornish, 1993, 2001a,b; Asawatreratanakul et al., 2003). T. brevicorniculatum CPTs are required for rubber biosynthesis, and therefore facilitate rubber chain elongation on rubber particles in this species (Post et al., 2012). Nevertheless, the most abundant proteins associated with T. brevicorniculatum rubber particles are not CPTs but 1

2 Natalie Laibach et al. small rubber particle proteins (TbSRPPs) (Hillebrand et al., 2012). Likewise, the guayule small rubber particle protein homolog (GHS) is found in the latex of P. argentatum, and a similar protein (HbSRPP) is present in the latex of H. brasiliensis (Chow et al., 2007; Schmidt et al., 2010a; Dai et al., 2013). TbSRPPs and HbSRPP are localized on the surface of rubber particles (Singh et al., 2003; Hillebrand et al., 2012). HbSRPP is a hydrophobic protein that interacts with phospholipids and therefore binds to the surface of lipid monolayers rather than integrating into them (Berthelot et al., 2012, 2014). SRPPs have been shown to influence rubber biosynthesis in vitro. In IPP incorporation assays, production of highmolecular-weight polymers was supported in the presence of both HbSRPP and GHS, but inhibited in their absence, suggesting that both proteins are involved in rubber biosynthesis (Oh et al., 1999; Kim et al., 2004). Moreover, TbSRPPs also influence rubber production and the colloidal stability of rubber particles in vivo (Hillebrand et al., 2012; Berthelot et al., 2014). SRPPs do not appear to possess enzymatic activity, and are therefore thought to promote rubber biosynthesis by maintaining the stability of the rubber particle (Hillebrand et al., 2012). Interestingly, non-rubber-producing species such as hot pepper (Capsicum annuum), avocado (Persea americana) and Arabidopsis thaliana possess SRPP-like proteins, CaSRP1 and PamLDAP1, respectively (Kim et al., 2010; Horn et al., 2013). The hot pepper and dandelion SRPPs are inducible by stress and abscisic acid, respectively, and the avocado SRRPs are localized on lipid droplets (Kim et al., 2010; Fricke et al., 2013; Horn et al., 2013). Although the function of these proteins is not completely clear, they may be important for the stabilization and organization of lipid droplets and similar structures (Laibach et al., 2014). SRPPs share a highly conserved domain with the HbREF (rubber elongation factor) protein, and this REF domain is also found in several other stress-related proteins (Dennis and Light, 1989; Gidda et al., 2013). HbREF promotes rubber biosynthesis in vitro (Dennis and Light, 1989), and its expression level in various H. brasiliensis accessions correlates with their rubber yields (Priya et al., 2006). HbREF is localized on rubber particles but cannot be immunologically distinguished from HbSRPP due to their similar sequences (Singh et al., 2003). However, the amyloid properties of HbREF and its ability to integrate into lipid monolayers indicate that this protein may promote the stability of rubber particles (Berthelot et al., 2012). Here we describe a T. brevicorniculatum REF family protein (TbREF) that is homologous to TbSRPPs, HbREF, HbSRPP and other proteins of the REF family, expressed in the latex and localized to the rubber particle surface. TbREF silencing by RNA interference (RNAi) impaired TbCPT activity and rubber production, but did not affect the colloidal stability of rubber particles. Our results

indicate that TbREF is an important component of the T. brevicorniculatum rubber particle, but is not absolutely required for rubber biosynthesis. RESULTS Identification of an atypical rubber elongation factor protein in Taraxacum brevicorniculatum To identify proteins that are directly associated with rubber particles and that may play a role in rubber biosynthesis, we isolated T. brevicorniculatum rubber particles and analyzed the proteome by SDS–PAGE and mass spectrometry. Most of the protein bands represented TbSRPPs (Schmidt et al., 2010b; Wahler et al., 2012). However, the 50 kDa protein band (Figure 1a) yielded peptides matching an expressed sequence tag in the National Center for Biotechnology Information database (http://www.ncbi.nlm.nih.gov/, accession number DR398691) which was clearly distinguishable from TbSRPPs. Nonetheless, it showed similarities to these proteins and SRPP/REF-like proteins from Arabidopsis thaliana and other plants. This expressed sequence tag was used to isolate the corresponding genomic sequence by genome walking. The full-length cDNA was amplified by reverse transcriptase–PCR using total RNA from T. brevicorniculatum latex. Sanger sequencing and subsequent analysis revealed an open reading frame of 1230 bp, encoding a protein of 409 amino acids with a predicted molecular mass of 50 kDa (Figure 1b). In silico analysis using InterPro (http://www.ebi.ac.uk/interpro/) revealed that the deduced amino acid sequence contained two conserved REF domains, and thus it was designated T. brevicorniculatum REF (TbREF). The presence of two REF domains is unusual because most REF family proteins only contain one such domain. Furthermore, the deduced TbREF amino acid sequence was only distantly related to other T. brevicorniculatum REF family proteins (maximum 18% identify with TbSRPP3 and minimum 12% identity with TbSRPP4 and TbSRPP5). T–Coffee alignment of TbREF and selected REF family proteins highlighted its unusual sequence length (Figure S1 and Table S1). Neither of the predicted REF domains in TbREF directly aligned with the corresponding domain of its homologs, but phylogenetic analysis clearly distinguished all the REF family proteins from oleosins (Figure 2a), the major lipid droplet-associated proteins found in seeds (Tzen et al., 1990; Murphy et al., 2001; Tzen, 2012; Laibach et al., 2014). Interestingly, TbREF did not cluster in REF sub-group I with proteins involved in rubber biosynthesis (e.g. PaGHS, TbSRPP3/4, HbREF and HbSRPP), nor in subgroups III or IV, comprising abiotic stress response proteins and lipid droplet-associated proteins, respectively. Instead, it clustered in group II with several REF family proteins that remain to be characterized (Dennis and Light, 1989; Kim et al., 2004, 2010; Seo et al., 2010; Hillebrand

© 2015 The Authors The Plant Journal © 2015 John Wiley & Sons Ltd, The Plant Journal, (2015), doi: 10.1111/tpj.12836

Rubber elongation factor protein in dandelion 3

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(b)

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Figure 1. Identification and spatio-temporal expression of TbREF. (a) SDS–PAGE of rubber particle proteins stained using Coomassie Brilliant Blue. Using mass spectrometry, TbREF-specific peptides were assigned to the 50 kDa protein band (indicated by an arrow). (b) Amino acid sequence of TbREF. Peptides revealed by mass spectrometry are underlined. The two REF domains are highlighted in gray. The box indicates the peptide used for production of anti-TbREF antibody. (c) Real-time PCR analysis of TbREF in total RNA from latex, roots, leaves, flowers and pedicels. (d) Real-time PCR analysis of TbREF in total RNA from T. brevicorniculatum roots in plants aged 2, 4, 8, 12, 16, 20 and 24 weeks. In (c) and (d), the values are means  SD from at least three pooled individual plants. The housekeeping gene TbEf1a (T. brevicorniculatum elongation factor 1a) was used as a control. (e) ER-targeted GFP expression driven by the TbREF promoter in longitudinal sections of roots. Scale bar = 200 lm (50 lm in the inset). (f) Immunological detection of TbREF protein in the pellet (PP), aqueous phase (AP) and rubber phase (RP) of centrifuged fresh latex. Ponceau S staining of the membrane after protein transfer is shown below the immunodetection image. (g) Back-scattered electron imaging of 10 nm gold-labeled rubber particles by immunodetection using anti-TbREF antibody. Scale bar = 400 nm.

et al., 2012; Horn et al., 2013). Figure 2(b) shows the hypothetical membrane association and integration of TbREF predicted by InterPro. Quantitative real-time PCR analysis confirmed that TbREF is expressed predominantly in the latex, but also weakly in the roots, pedicel, leaves and flowers, presumably reflecting the residual latex content of these tissues (Figure 1c, Data S1). Total RNA was extracted from roots at various time points to determine the temporal expression profile of TbREF. 2–4 weeks after germination extraction of raw latex is not yet possible so root material was used

instead since it is the tissue with the greatest latex content. Real-time PCR analysis revealed that TbREF is already expressed by 2 weeks after germination, and its expression peaks 8 weeks after germination before slowly decreasing by week 12 (Figure 1d). TbREF is localized on the surface of rubber particles in the laticifers of T. brevicorniculatum roots The latex-dominant TbREF expression profile revealed by quantitative real-time PCR was investigated in more detail by promoter analysis. We isolated 1 kb of sequence

© 2015 The Authors The Plant Journal © 2015 John Wiley & Sons Ltd, The Plant Journal, (2015), doi: 10.1111/tpj.12836

Rubber elongation factor protein in dandelion 9 Post et al., 2012), and TbREF may form part of this complex, probably by acting as a scaffold for recruitment of other proteins, including TbCPTs. TbREF may also promote rubber biosynthesis by facilitating particle biogenesis. The abundance of TbCPTs and TbSRPPs does not appear to be affected in the transgenic plants at the level of individual rubber particles, despite the lower overall level of TbCPTs in the latex, so the effect of TbREF depletion may not reflect disruption of rubber biosynthesis per se but an inability to form the number of rubber particles usually found in wild-type plants. In contrast to TbCPTs, which are exclusively located on rubber particles (Schmidt et al., 2010b), TbSRPPs are also highly abundant in the cytosol of laticifers (Hillebrand et al., 2012; Wahler et al., 2012). This may explain why the abundance of TbSRPPs is not significantly affected in the transgenic plants. In conclusion, our findings show that TbREF is an important component of the rubber biosynthesis pathway in T. brevicorniculatum root latex, and that it may function as a regulator of rubber production by coordinating the biogenesis of rubber particles. EXPERIMENTAL PROCEDURES Plant material and cultivation T. brevicorniculatum plants and seeds were purchased from the Botanical Gardens Karlsruhe (Germany), and cultivated in a greenhouse at 18°C with a 16 h photoperiod (20 klx light intensity). For all experiments, wild-type and transgenic plants were grown in the same chamber in the greenhouse, and, except for the developmental studies, were 3 months old. Plant material used for the experiments (latex, roots, leafs and flowers) was obtained from at least three individual plants per transgenic line or wild-type control unless otherwise stated.

Preparation of T. brevicorniculatum rubber particles and latex fractionation Latex from T. brevicorniculatum was harvested into rubber extraction buffer as previously described (Schmidt et al., 2010b). The latex was fractionated to yield a pellet as well as aqueous and rubber phases (Schmidt et al., 2010b). The rubber particles were isolated and purified as described by Hillebrand et al. (2012). All experiments were performed using freshly harvested latex or freshly prepared rubber particles.

Protein identification by mass spectrometry Rubber particle proteins were purified and separated by SDS– PAGE as described by Wahler et al. (2012). The 50 kDa protein band was analyzed as described by Hillebrand et al. (2012).

Identification of the full-length TbREF gene and its promoter The full-length TbREF gene and upstream regulatory elements were isolated by genome walking on T. brevicorniculatum genomic DNA, using a GenomeWalker universal kit (Clontech, https:// www.clontech.com/) according to the manufacturer’s instructions.

We used one gene-specific primer and one nested primer as shown in Table S4, for each genome walking PCR (GSP2 nested primer, GSP1, TbREF-DP GSP3 promoter and nested TbREF-DP GSP4).

In silico analysis of the TbREF sequence Phylogenetic analysis was performed in ‘a la carte’ mode on the phylogeny.fr website, and was based on a MUSCLE alignment according to algorithms developed by Anisimova and Gascuel (2006), Chevenet et al. (2006), Dereeper et al. (2008, 2010), Edgar (2004) and Guindon and Gascuel (2003). The tree was visualized using FigTree (http://tree.bio.ed.ac.uk/software/figtree/). The multiple sequence alignment was computed using T–Coffee (Notredame et al., 2000; Di Tommaso et al., 2011). Transmembrane and domain structures were predicted using InterPro (Hunter et al., 2011; http://www.ebi.ac.uk/interpro/).

Antibody production A TbREF-specific antibody was generated by screening for suitable peptides using Protean software (Lasergene software, DNASTAR, http://www.dnastar.com/). The chosen peptide (N’CVKTKNDGIEALEDAGE) was analyzed by BLAST (http://blast.ncbi.nlm.nih.gov/Blast.cgi) but gave no significant hits. The antibody was produced by Genosphere Biotechnologies (http:// www.genosphere-biotech.com/) using this peptide conjugated to keyhole limpet hemocyanin. Antibodies specific for TbCPT and TbSRPP were produced as previously described (Hillebrand et al., 2012; Post et al., 2012). The TbSRPP antibody has been shown to recognize all five TbSRPP isoforms (Hillebrand et al., 2012).

Total RNA extraction and cDNA synthesis Total RNA was extracted from T. brevicorniculatum latex and other tissues for cDNA synthesis as previously described (Schmidt et al., 2010a). The quality of the RNA was validated by photometric detection using a NanoPhotometer UV/Vis spectrophotometer (Implen, http://www.implen.de/) and gel electrophoresis. The cDNA was synthesized using SuperScriptâ II reverse transcriptase (Life Technologies, http://www.lifetechnologies. com) according to the manufacturer’s instructions. Random hexamers (Fermentas, http://www.lifetechnologies.com) were used as primers to initiate reverse transcription prior to real-time PCR.

Preparation of the TbREF promoter–GFPER construct For promoter analysis, 1 kb of the TbREF upstream sequence was amplified using primers P–TbREF–DP HindIII fw and P–TbREF–DP XhoI rev (Table S4). The GFPER gene was isolated from pGJ1029 (kindly provided by Guido Jach, Phytowelt GreenTechnologies GmbH, Cologne, Germany) using the XhoI and XbaI restriction sites, and transferred to binary plasmid pLab12.10 (Post et al., 2014). The TbREF PCR products were digested using HindIII and XhoI, and transferred into the corresponding restriction sites of pLab12.10. The integrity of each construct was verified by Sanger sequencing using an ABI PRISMâ 3730 genetic analyzer (Life Technologies).

Confocal laser scanning microcopy GFPER expression was visualized by confocal laser scanning microscopy at an excitation wavelength of 488 nm and emission wavelengths 509–620 nm using a Leica TCS SP5 confocal microscope (Leica, http://www.leica-microsystems.com/).

© 2015 The Authors The Plant Journal © 2015 John Wiley & Sons Ltd, The Plant Journal, (2015), doi: 10.1111/tpj.12836

Rubber elongation factor protein in dandelion 5 Immunogold labeling of the T. brevicorniculatum rubber particles confirmed that TbREF is localized on the rubber particle surface (Figure 1 g). Depletion of TbREF by RNAi To investigate the detailed role of TbREF in rubber biosynthesis, we generated transgenic plants in which the levels of TbREF were reduced by RNAi. A TbREF-RNAi construct (Figure 3a) was integrated into the T. brevicorniculatum genome by Agrobacterium-mediated transformation. Quantitative real-time PCR and Western blot analysis showed that TbREF mRNA and TbREF protein levels were strongly decreased in three lines designated TbREF-RNAi #1.1, #2.2 and #4.2 (Figure 3b,c). A higher residual gene expression level was observed in line #4.2 compared to the others (Figure 3b), and this was confirmed at the protein level by Western blot analysis of total latex using the antiTbREF antibody (Figure 3c). As a representative example, the substantial loss of TbREF protein in transgenic line TbREF-RNAi #2.2 was confirmed by two-dimensional gel electrophoresis and subsequent Western blotting using the same antibody (Figure S2E). Wild-type latex yielded a distinct signal spanning a pI range of 3.5–5.5, but latex from line TbREF-RNAi #2.2 did not yield a signal. The region of membrane containing the abundant latex protein T. brevicorniculatum polyphenoloxidase 1 (Wahler et al., 2009) was stained with Ponceau S as a loading control. The depletion of TbREF had no visible phenotypic effect, and the RNAi lines were morphologically indistinguishable from wild-type plants cultivated in the same manner. The absence of TbREF affects rubber content and cisprenyltransferase activity The effect of TbREF protein depletion on rubber production was tested by comparing the rubber content and the

molecular mass of poly(cis-1,4–isoprene) in TbREF-RNAi plants and wild-type controls. 1H-NMR analysis of freezedried and powdered root material indicated that the rubber content of TbREF-RNAi lines #1.1 and #2.2 was significantly lower than that of wild-type plants (Table 1). However, the rubber content of TbREF-RNAi line #4.2 was similar to that of wild-type plants due to the residual TbREF expression in this line (Figure 3c). Although the rubber content of TbREF-RNAi lines #1.1 and #2.2 was reduced by the depletion of TbREF protein, the rubber molecular mass determined by size-exclusion chromatography did not differ significantly between TbREF-RNAi and wild-type plants (Table 1). Next we performed in vitro (1–14C)-IPP incorporation assays using rubber particles purified from TbREF-RNAi and wild-type plants to determine whether the lower rubber content of the transgenic lines was caused by the loss of CPT activity (Post et al., 2012). When the reaction was complete, we extracted small isoprenoids using butanol and larger ones using toluene/hexane, and measured the amount of (1–14C)-IPP incorporated by scintillation counting. We found that incorporation of (1–14C)-IPP was significantly lower in both fractions from the transgenic lines, indicating a loss of TbCPT activity (Figure 4a). Analysis of TbCPT1, TbCPT2 and TbCPT3 expression levels showed that TbREF silencing had a negligible effect on mRNA levels (Figure 4b), but that the protein levels were reduced substantially, as shown by Western blotting using an antibody recognizing all three TbCPTs (Figure 4c and Table S2). More TbCPT proteins were present in the latex of TbREF-RNAi line #4.2, in agreement with the higher residual TbREF protein level. This indicates a correlation between the TbCPT1-3 and TbREF protein amount in TbREF-RNAi plants. We also analyzed the quantity of TbCPTs present on purified rubber particles, and found no

Figure 2. In silico analysis of TbREF. (a) Phylogenetic tree showing the relationships among REF domain proteins. The tree was computed in ‘a la carte’ mode on the phylogeny.fr website, and is based on a Muscle alignment according to algorithms developed by Anisimova and Gascuel (2006), Chevenet et al. (2006), Dereeper et al. (2008, 2010), Guindon and Gascuel (2003) and Edgar (2004). The GenBank accession numbers and species for the sequences are given below: oleosin_Helianthus_annuus, Helianthus annuus, P29529.1; Tc_Oleosin, Theobroma cacao, XP_007012203.1; Oleosin1_Ricinus_communis, Ricinus communis, XP_002511014.1; oleosin4_Arabidopsis_thaliana, Arabidopsis thaliana, NP_189403.1; SRP3_Arabidopsis_thaliana, A. thaliana, NP_182299.1; SRP2_Arabidopsis_thaliana, A. thaliana, NP_176904.1; SRP1_Arabidopsis_thaliana, A. thaliana, NP_187201.1; SRP1_Capsicum_annuum, Capsicum annuum, ADI60300.1; SRPP_Hevea_brasiliensis, Hevea brasiliensis, O82803.1; SRPP5_Taraxacum_brevicorniculatum, Taraxacum brevicorniculatum, AGE89410.1; SRPP4_Taraxacum_brevicorniculatum, T. brevicorniculatum, AGE89409.1; SRPP3_Taraxacum_brevicorniculatum, T. brevicorniculatum, AGE89408.1; SRPP2_Taraxacum_brevicorniculatum, T. brevicorniculatum, AGE89407.1; SRPP1_Taraxacum_brevicorniculatum, T. brevicorniculatum, AGE89406.1; LDAP1_Persea_americana, Persea americana, AGQ04593.1; LDAP2_Persea_americana, P. americana, AGQ04594.1; REF2_Theobroma_cacao, T. cacao, XP_007030997.1; REF_Hevea_brasiliensis, H. brasiliensis, P15252.2; GHS_Parthenium_argentataum, Parthenium argentatum, AAQ11374.1; SRP_Vitis_riparia, Vitis riparia, Q9SW70.1; REF_Populus_trichocarpa, Populus trichocarpa, XP_002319520.1; REF_Morus_alba, Morus alba, ACV90044.1; REF1_Theobroma_cacao, T. cacao, XP_007030996.1; REF_Copaifera_officinalis, Copaifera officinalis, AEX97052.1; SRP_Ipomoea_batatas, Ipomoea batatas, ABP35522.1; SRP1_Zea_mays, Zea mays, ACG39345.1; SRP2_Zea_mays, Z. mays, NP_001149834.1; SRP_Oryza_sativa_japonica, O. sativa japonica, NP_001054672.1; SRP_Oryza_sativa_indica, O. sativa indica, A2Y0H2.1; SRP1_Phaseolus_vulgaris, Phaseolus vulgaris, XP_007148173.1; SRP2_Phaseolus_vulgaris, P. vulgaris, AGV54312.1; REF_Ricinus_communis, R. communis, XP_002512427.1; SRPP1_Ricinus_communis, R. communis, XP_002514917.1; SRPP2_Ricinus_communis, R. communis, XP_002531884.1; REF1_Arabidopsis_lyrata, Arabidopsis lyrata, XP_002888592.1; REF2_Arabidopsis_lyrata, Arabidopsis lyrata, XP_002882419.1; REF1_Medicago_truncatula, Medicago truncatula, XP_003593563.1; REF2_Medicago_truncatula, M. truncatula, XP_003601787.1; CTD-REF_Medicago_truncatula, M. truncatula, XP_003601789.1; SRP_Hevea_brasiliensis, H. brasiliensis, AAP46159.1; REF_Eucommia_ulmoides, Eucommia ulmoides, AHX74091.1; REF_Taraxacum_brevicorniculatum, T. brevicorniculatum, DR398691. Numbers I–IV indicate hypothetical sub-groups. (b) Hypothetical transmembrane structure of TbREF, predicted by InterPro (Hunter et al., 2011; http://www.ebi.ac.uk/interpro/).

© 2015 The Authors The Plant Journal © 2015 John Wiley & Sons Ltd, The Plant Journal, (2015), doi: 10.1111/tpj.12836

6 Natalie Laibach et al.

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Figure 3. TbREF gene and protein expression in wild-type plants and three independent transgenic TbREF-RNAi lines designated TbREF-RNAi #1.1, #2.2 and #4.2. (a) Schematic depiction of the TbREF-RNAi construct. (b) Relative TbREF mRNA levels in the latex of wild-type (WT) and TbREF-RNAi plants determined by quantitative real-time PCR. Relative mRNA levels were normalized against the constitutive TbEf1a gene encoding T. brevicorniculatum elongation factor 1a. Values are means  SE of three technical replicates of pooled latex material from at least three individual plants per line. (c) Protein analysis of latex from TbREF-RNAi lines compared to a wild-type control. Latex samples from at least three individual plants per line were pooled and used for protein separation by SDS–PAGE and subsequent Western blot analysis. The nitrocellulose membrane was used for immunodetection of TbREF using a TbREF-specific antibody. Ponceau S staining of the membrane after protein transfer is shown below the immunodetection image.

Table 1 Poly(cis-1,4–isoprene) content and molecular mass distribution of rubber from the roots of wild-type (WT) T. brevicorniculatum plants and TbREF-RNAi lines Rubber molecular mass distribution

Plant line

Poly(cis-1,4–isoprene) content of dry roots (mg g 1)

WT TbREF-RNAi #1.1 TbREF-RNAi #2.2 TbREF-RNAi #4.2

2.53151 1.52958 0.78372 1.89365

   

0.13462 0.57772* 0.18553* 0.55981

weight average molecular mass (106 Da) 1.63 1.58 1.72 1.76

   

0.03 0.16 0.17 0.08

number average molecular mass (106 Da) 1.30 1.27 1.36 1.29

   

0.02 0.11 0.12 0.11

weight average molecular mass / number average molecular mass 1.25 1.24 1.27 1.37

   

0.01 0.02 0.03 0.15

The poly(cis-1,4-isoprene) levels in dry root material were determined by 1H–NMR spectroscopy. Values are means  SD of four independent plants per line. The molecular mass distribution of rubber from exuded latex was determined by size-exclusion chromatography. Values are means  SD of three independent plants per line. * Statistically significant differences between the TbREF-RNAi plants and wild-type controls (P < 0.05, ANOVA with the Tukey–Kramer test).

significant differences between the transgenic TbREF-RNAi plants and wild-type plants (Figure S2B and Table S2). We also separated latex fractions from TbCPT-RNAi plants that contain almost no rubber particles and therefore yield a pellet and the aqueous phase but no rubber phase (Post et al., 2012). The TbREF protein was found mostly in the aqueous phase but also in the pellet phase, in contrast to wild-type plants, where TbREF was found mostly in the rubber phase but also in the aqueous phase (Figure S2D). Having confirmed that depletion of TbREF inhibits TbCPT activity and thus causes a decrease in rubber pro-

duction, we investigated the effect of TbREF depletion on rubber particle stability to determine whether TbREF stabilizes the rubber particle, as already shown for TbSRPPs. We therefore measured the z–average diameter of purified rubber particles from TbREF-RNAi and wild-type plants by dynamic light scattering at various pH values as previously described (Hillebrand et al., 2012). There were no significant differences between wild-type and transgenic plants, and similarly there were no differences in other indicative parameters such as the polydispersity index (Figure 5a and Table S3), indicating that rubber particle stability is not

© 2015 The Authors The Plant Journal © 2015 John Wiley & Sons Ltd, The Plant Journal, (2015), doi: 10.1111/tpj.12836

Rubber elongation factor protein in dandelion 7

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affected in the transgenic plants. We also compared the expression of TbSRPP1 and TbSRPP3–5 in transgenic and wild-type plants by quantitative real-time PCR analysis, and found that all four transcripts were twofold more abundant in the transgenic lines (Figure 5b, Data S1) but the corresponding protein levels were no different when comparing total latex extracts or purified rubber particles (Figure 5c and Table S4). DISCUSSION

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Figure 4. Gene and protein expression of TbCPT1–3 in wild-type plants and three independent transgenic TbREF-RNAi lines designated TbREF-RNAi #1.1, #2.2 and #4.2. (a) (1–14C)-IPP incorporation assay using exuded latex. After the reaction, small and large isoprene molecules were extracted using butanol and toluene, respectively. Values are means  SD for (1–14C)-IPP incorporation measured in three independent experiments, each with three or four technical replicates. Asterisks indicate statistically significant differences between the TbREF-RNAi plants and wild-type controls (P < 0.05, ANOVA with the Tukey– Kramer test). (b) Relative TbCPT1–3 mRNA levels in latex from wild-type (WT) and TbREF-RNAi plants determined by quantitative real-time PCR. Relative mRNA levels were normalized against the constitutive TbEf1a gene encoding T. brevicorniculatum elongation factor 1a. Values are means  SE of three technical replicates of pooled latex material from at least three individual plants per line. (c) Protein analysis of latex from TbREF-RNAi lines compared to a wild-type control. Latex samples from at least three individual plants per line were pooled and used for protein separation by SDS–PAGE and subsequent Western blot analysis. The nitrocellulose membrane was used for immunodetection of TbCPTs using an anti-TbCPT antibody that recognizes all three isoforms. Ponceau S staining of the membrane after protein transfer is shown below the immunodetection image.

Two major proteins are associated with rubber particles in the latex of H. brasiliensis: HbREF and HbSRPP (Dennis and Light, 1989; Oh et al., 1999). These homologous proteins influence rubber biosynthesis in a positive manner, and HbSRPP orthologs identified in the latex of guayule (PaGHS) and dandelion (TbSRPPs and TkSRPPs) also have positive effects on rubber biosynthesis (Kim et al., 2004; Collins-Silva et al., 2012; Hillebrand et al., 2012). We have isolated a 50 kDa T. brevicorniculatum protein (TbREF) based on the peptides identified by mass spectrometry that is homologous to HbREF (Figure 1a,b, Data S1). Unlike all the other REF family proteins discovered so far, TbREF contains two REF domains and its molecular mass is twice that of its homologs. Despite these differences, phylogenetic analysis (Figure 2a) allocated TbREF to sub-group II, which contains uncharacterized REF family proteins such as the stress-related protein AtSRP2, rather than sub-group I, which contains REF family proteins known to be associated with rubber biosynthesis (Dennis and Light, 1989; Oh et al., 1999; Kim et al., 2004; Hillebrand et al., 2012). TbREF mRNA was restricted almost entirely to the latex (Figure 1c), a property that is shared with the transcripts of other rubber biosynthesis genes, i.e. all three TbCPT genes and all five TbSRPP genes except TbSRPP2 (Schmidt et al., 2010a). The temporal expression profile of TbREF was also similar to that of some TbSRPP transcripts (Schmidt et al., 2010a), with increasing expression at 4–8 weeks followed by a decrease (Figure 1d). The latex-specific expression of TbREF was supported by the laticifer-specific activity of its upstream sequence (Figure 1e). GFPER expression was detected in laticifers (clearly identified by their anastomoses) in the vascular tissue of the roots (Hagel et al., 2008; Wahler et al., 2009). Furthermore, in fractionated latex, the TbREF protein was detected in the rubber particle fraction and the aqueous fraction (Figure 1f); the aqueous fraction mainly comprises the cytosolic component of the laticifers (Schmidt et al., 2010b). The TbREF signal in the aqueous phase may represent contamination with small rubber particles or the presence of TbREF in the cytosol, although the latter is unlikely because of the strong aggregation behavior of TbREF. Immunogold labeling confirmed that TbREF is localized on the surface of rubber particles isolated from

© 2015 The Authors The Plant Journal © 2015 John Wiley & Sons Ltd, The Plant Journal, (2015), doi: 10.1111/tpj.12836

8 Natalie Laibach et al.

(a)

(b)

(c)

Figure 5. TbSRPP1–5 gene and protein expression in wild-type plants and three independent transgenic TbREF-RNAi lines designated TbREF-RNAi #1.1, #2.2 and #4.2. (a) Measurement of the z–average diameter of rubber particles from wildtype plants and TbREF-RNAi transgenic plants suspended in modified rubber extraction buffer with the indicated pH values. Values are means  SD of three independent plants per line (#1.1, #2.2 and #4.2) or three wild-type plants (WT), with three technical replicates in each case. No significant differences at P < 0.05 were detected between the TbREF-RNAi and wild-type control plants using ANOVA with the Tukey–Kramer test. (b) Relative TbSRPP1–5 mRNA levels in latex from wild-type (WT) and TbREF-RNAi plants quantified by real-time PCR. The relative mRNA levels were normalized against those of the constitutive TbEf1a gene encoding T. brevicorniculatum elongation factor 1a. Values are means  SE of three technical replicates of pooled latex material from at least three individual plants per line. (c) Protein analysis of latex from TbREF-RNAi lines compared to a wild-type control. Latex samples from at least three individual plants per line were used for protein separation by SDS–PAGE and subsequent Western blot analysis. The nitrocellulose membrane was used for immunodetection of TbSRPP1–5 using a TbSRPP-specific antibody that recognizes all five isoforms (Hillebrand et al., 2012). Ponceau S staining of the membrane after protein transfer is shown below the immunodetection image.

root latex. Cross-reaction with TbSRPPs (as reported for the immunological detection of HbSRPP and HbREF) was avoided by choosing an unshared epitope for production of the anti-TbREF antibody (Singh et al., 2003). The function of TbREF was investigated by generating transgenic plants in which the TbREF gene was silenced by RNAi, and three independent lines with severely reduced TbREF transcription and TbREF protein levels were analyzed to determine their ability to produce rubber (Figure 3). The poly(cis-1,4–isoprene) content was lower in the TbREF-RNAi plants than in wild-type controls, and this was caused by a reduction in the activity of TbCPTs, which are required for rubber synthesis in T. brevicorniculatum (Post et al., 2012). Expression analysis revealed that all three TbCPT genes were induced to different extents in the three transgenic lines (Figure 4b), potentially reflecting a feedback mechanism triggered by the lower TbCPT protein levels, enzyme activities and/or poly(cis-1,4–isoprene) content (Figure 4a,c and Table 1). However, the molecular mass of the rubber did not differ significantly among the TbREFRNAi and wild-type plants (Table 1), indicating that the rubber chain elongation process was unaffected by the lower TbREF protein levels. Similar results were reported for TbSRPP-RNAi plants, and TbSRPPs were shown to facilitate rubber biosynthesis by promoting the stability of rubber particles and thus preventing their agglomeration (Hillebrand et al., 2012). HbSRPP and HbREF may have similar functions because they can interact with membrane lipids and attach to the surface of lipid monolayers or even integrate into them (Berthelot et al., 2012). We considered the possibility that TbREF, like the TbSRPPs, also contributes to rubber production by maintaining the integrity of rubber particles. We therefore determined the z–average size of rubber particles produced by the TbREF-RNAi plants, but found no significant difference compared to those produced by wild-type plants (Figure 5a). TbREF silencing induced a twofold increase in the abundance of TbSRPP mRNA levels, but this was not translated into an increase in protein levels, perhaps reflecting the existence of feedback regulation (Figure 5b,c). Therefore, even if TbREF does help to stabilize rubber particles, loss of the protein appears to be compensated by the presence of TbSRPPs. The lower TbCPT protein levels in TbREF-RNAi plants suggest that TbREF may play a more important role in the regulation of rubber biosynthesis, rather than in stabilization of rubber particles. It is unlikely to be a signaling molecule because it is directly associated with the rubber particles, and it is also unlikely to influence TbCPT activity directly because the molecular mass of the small amount of rubber produced in transgenic plants is no different to that produced in wild-type controls. It has already been proposed that rubber chain elongation requires a protein complex rather than individual enzymes (Cornish, 2001a,b;

© 2015 The Authors The Plant Journal © 2015 John Wiley & Sons Ltd, The Plant Journal, (2015), doi: 10.1111/tpj.12836

Rubber elongation factor protein in dandelion 9 Post et al., 2012), and TbREF may form part of this complex, probably by acting as a scaffold for recruitment of other proteins, including TbCPTs. TbREF may also promote rubber biosynthesis by facilitating particle biogenesis. The abundance of TbCPTs and TbSRPPs does not appear to be affected in the transgenic plants at the level of individual rubber particles, despite the lower overall level of TbCPTs in the latex, so the effect of TbREF depletion may not reflect disruption of rubber biosynthesis per se but an inability to form the number of rubber particles usually found in wild-type plants. In contrast to TbCPTs, which are exclusively located on rubber particles (Schmidt et al., 2010b), TbSRPPs are also highly abundant in the cytosol of laticifers (Hillebrand et al., 2012; Wahler et al., 2012). This may explain why the abundance of TbSRPPs is not significantly affected in the transgenic plants. In conclusion, our findings show that TbREF is an important component of the rubber biosynthesis pathway in T. brevicorniculatum root latex, and that it may function as a regulator of rubber production by coordinating the biogenesis of rubber particles. EXPERIMENTAL PROCEDURES Plant material and cultivation T. brevicorniculatum plants and seeds were purchased from the Botanical Gardens Karlsruhe (Germany), and cultivated in a greenhouse at 18°C with a 16 h photoperiod (20 klx light intensity). For all experiments, wild-type and transgenic plants were grown in the same chamber in the greenhouse, and, except for the developmental studies, were 3 months old. Plant material used for the experiments (latex, roots, leafs and flowers) was obtained from at least three individual plants per transgenic line or wild-type control unless otherwise stated.

Preparation of T. brevicorniculatum rubber particles and latex fractionation Latex from T. brevicorniculatum was harvested into rubber extraction buffer as previously described (Schmidt et al., 2010b). The latex was fractionated to yield a pellet as well as aqueous and rubber phases (Schmidt et al., 2010b). The rubber particles were isolated and purified as described by Hillebrand et al. (2012). All experiments were performed using freshly harvested latex or freshly prepared rubber particles.

Protein identification by mass spectrometry Rubber particle proteins were purified and separated by SDS– PAGE as described by Wahler et al. (2012). The 50 kDa protein band was analyzed as described by Hillebrand et al. (2012).

Identification of the full-length TbREF gene and its promoter The full-length TbREF gene and upstream regulatory elements were isolated by genome walking on T. brevicorniculatum genomic DNA, using a GenomeWalker universal kit (Clontech, https:// www.clontech.com/) according to the manufacturer’s instructions.

We used one gene-specific primer and one nested primer as shown in Table S4, for each genome walking PCR (GSP2 nested primer, GSP1, TbREF-DP GSP3 promoter and nested TbREF-DP GSP4).

In silico analysis of the TbREF sequence Phylogenetic analysis was performed in ‘a la carte’ mode on the phylogeny.fr website, and was based on a MUSCLE alignment according to algorithms developed by Anisimova and Gascuel (2006), Chevenet et al. (2006), Dereeper et al. (2008, 2010), Edgar (2004) and Guindon and Gascuel (2003). The tree was visualized using FigTree (http://tree.bio.ed.ac.uk/software/figtree/). The multiple sequence alignment was computed using T–Coffee (Notredame et al., 2000; Di Tommaso et al., 2011). Transmembrane and domain structures were predicted using InterPro (Hunter et al., 2011; http://www.ebi.ac.uk/interpro/).

Antibody production A TbREF-specific antibody was generated by screening for suitable peptides using Protean software (Lasergene software, DNASTAR, http://www.dnastar.com/). The chosen peptide (N’CVKTKNDGIEALEDAGE) was analyzed by BLAST (http://blast.ncbi.nlm.nih.gov/Blast.cgi) but gave no significant hits. The antibody was produced by Genosphere Biotechnologies (http:// www.genosphere-biotech.com/) using this peptide conjugated to keyhole limpet hemocyanin. Antibodies specific for TbCPT and TbSRPP were produced as previously described (Hillebrand et al., 2012; Post et al., 2012). The TbSRPP antibody has been shown to recognize all five TbSRPP isoforms (Hillebrand et al., 2012).

Total RNA extraction and cDNA synthesis Total RNA was extracted from T. brevicorniculatum latex and other tissues for cDNA synthesis as previously described (Schmidt et al., 2010a). The quality of the RNA was validated by photometric detection using a NanoPhotometer UV/Vis spectrophotometer (Implen, http://www.implen.de/) and gel electrophoresis. The cDNA was synthesized using SuperScriptâ II reverse transcriptase (Life Technologies, http://www.lifetechnologies. com) according to the manufacturer’s instructions. Random hexamers (Fermentas, http://www.lifetechnologies.com) were used as primers to initiate reverse transcription prior to real-time PCR.

Preparation of the TbREF promoter–GFPER construct For promoter analysis, 1 kb of the TbREF upstream sequence was amplified using primers P–TbREF–DP HindIII fw and P–TbREF–DP XhoI rev (Table S4). The GFPER gene was isolated from pGJ1029 (kindly provided by Guido Jach, Phytowelt GreenTechnologies GmbH, Cologne, Germany) using the XhoI and XbaI restriction sites, and transferred to binary plasmid pLab12.10 (Post et al., 2014). The TbREF PCR products were digested using HindIII and XhoI, and transferred into the corresponding restriction sites of pLab12.10. The integrity of each construct was verified by Sanger sequencing using an ABI PRISMâ 3730 genetic analyzer (Life Technologies).

Confocal laser scanning microcopy GFPER expression was visualized by confocal laser scanning microscopy at an excitation wavelength of 488 nm and emission wavelengths 509–620 nm using a Leica TCS SP5 confocal microscope (Leica, http://www.leica-microsystems.com/).

© 2015 The Authors The Plant Journal © 2015 John Wiley & Sons Ltd, The Plant Journal, (2015), doi: 10.1111/tpj.12836

10 Natalie Laibach et al. Scanning electron microscopy Rubber particles were fixed in 2.5% glutaraldehyde in 0.05 M sodium cacodylate buffer (pH 7.3) for 1 h at room temperature. After three washes with 0.05 M sodium cacodylate buffer (pH 7.3) each for 5 min, the particles were centrifuged (5000 g) and post-fixed using 1% osmium tetroxide for 1 h. All washing steps were carried out at room temperature. Then the particles were washed three times with 0.05 M sodium cacodylate buffer (pH 7.3) and then three times with PBS (pH 7.4), each for 5 min. For immunogold labeling, 10 ll aliquots of each suspension were placed on a freshly glow-discharged 10 nm carbon layer covered Pioloform film (AGAR SCIENTIFIC, http://www.agarscientific.com) supported by a copper mesh. The particles were allowed to sediment for 5 min, and were then washed with PBS (pH 7.4) for 10 min, and incubated in PBS (pH 7.4) containing 5% bovine serum albumin (BSA) for 15 min, followed by three washes in PBS (pH 7.4), each for 5 min. The grids were then incubated for 3 h with either primary anti-TbREF antibody (diluted 1:5 in PBS, pH 7.4) or pre-immune serum (diluted 1:5 in PBS, pH 7.4) as a negative control. The grids were washed five times for 5 min each with PBS (pH 7.7), and then for 5 min using Tris/BSA buffer (pH 8.2). The samples were then incubated with a secondary goat-anti rabbit IgG antibody coupled to 10 nm colloidal gold particles (Sigma-Aldrich, https://www.sigmaaldrich.com/) diluted 1:20 in Tris/BSA buffer (pH 8.2) for 2 h. Finally, the samples were washed six times with Tris/BSA buffer (pH 8.2) for 5 min each, and once with distilled water for 5 min. After air drying, the sample was covered with 2 nm platinum/carbon. Images were recorded on an S–5000 field-emission scanning electron microscope (Hitachi, www.hitachi-hta.com) at an electron energy of 20 kV. The secondary and back-scattered electron signals were recorded.

Preparation of the TbREF-RNAi construct A 350 bp fragment of the TbREF cDNA sequence (nucleotides 761–1110) was amplified by PCR using primers REF RNAi 2 KpnI fw and REF RNAi 2 XhoI rev (Table S1). The cDNA fragment was inserted into the KpnI and XhoI sites of Gateway vector pENTR4 (Life Technologies, www. lifetechnologies.com), and transferred to pFGC5941 (Wahler et al., 2009) using Gatewayâ LR Clonaseâ II Enzyme mix (Life Technologies). The integrity of the construct was verified by Sanger sequencing using an ABI PRISMâ 3730 genetic analyzer (Life Technologies).

Agrobacterium-mediated transformation of T. brevicorniculatum Plants were transformed as previously described (Wahler et al., 2009) using Agrobacterium tumefaciens strain EHA105 carrying the binary vector described above. Roots of the T. brevicorniculatum polyphenoloxidase 1 knockdown line LP1 (Wahler et al., 2009) were transformed using Agrobacterium rhizogenes strain NCPPB2659 carrying the TbREF promoter–GFPER construct as previously described (Collier et al., 2005).

(10 pmol each). Samples used for real-time PCR represented three technical replicates of pooled latex material from at least three individual plants per line. The primers used to amplify the TbREF, TbCPT and TbSRPP transcripts are listed in Table S4 (TbREF qRT fwd and rev, TbSRPP1–5 qRT fw and rev, TbCPTfwd and TbCPT1–3-rev). The transcript of the housekeeping gene TbEf1a (T. brevicorniculatum elongation factor 1a) was used as a reference. TbEf1a has been shown to be suitable as a reference gene in Nicotiana tabacum (Schmidt and Delaney, 2010), and has previously been used for real-time PCR analysis in T. brevicorniculatum (Fricke et al., 2013). Melting curves were analyzed to exclude false amplifications. CT values were then determined, and the relative expression level of each gene was calculated using the relative expression software tool (REST) (Pfaffl et al., 2002) based on previously determined primer efficiencies (Table S5, Data S1).

SDS–PAGE and Western blotting Latex was harvested from the roots of at least three individual adult T. brevicorniculatum plants per line as previously described (Schmidt et al., 2010b) and then pooled. SDS–PAGE was performed using equal amounts of protein from whole latex in each lane. Protein concentrations were determined as described by Bradford (1976). Proteins were separated on a polyacrylamide gel containing 10% SDS, and transferred to nitrocellulose membranes (Towbin et al., 1979). The membranes were stained with 0.1% Ponceau S in 5% acetic acid to confirm equal loading, and were then incubated with TbREF-specific antibody (diluted 1:500), TbSRPP-specific antibody (diluted 1:500) or TbCPT-specifc antibody (diluted 1:250) for 2 h. After washing, bound primary antibody was detected using a secondary mouse anti-rabbit IgG conjugated to horseradish peroxidase according to the manufacturer’s instructions concerning dilution (Sigma-Aldrich). TbREF, TbSRPPs and TbCPTs were visualized on X–ray films or by chemiluminescence detection using a FUSION–SL Advance 4.2 MP camera (Peqlab).

Two-dimensional gel electrophoresis of rubber particle proteins Fresh latex aliquots (100 ll) were suspended in ice-cold rubber extraction buffer, and the dense SDS buffer protocol previously described by Wang et al. (2003) was used for protein extraction. After solubilizing proteins in rehydration buffer [7 M urea, 2% CHAPS, 50 mM dithiothreitol and 0.2% Bio-Lyteâ 3/10 ampholytes (Bio–Rad)] for at least 4 h, 100 mg protein was loaded onto 7 cm ReadyStripTM immobilized pH gradient strips, pH range 3–10 (Bio– Rad). The strips were rehydrated overnight, and isoelectric focusing was performed at 20°C with a maximum current of 50 mA using the following program: 300 V for 30 min, 1000 V for 30 min, then 5000 V until 12 kVh were achieved. Proteins focused on the strips were separated by 12% SDS–PAGE, and either stained with PageBlue (Thermo Scientific, http://www.thermoscientific.com) or transferred to nitrocellulose membranes for Western blotting as described above.

Determination of rubber particle z–average size

Quantitative real-time PCR Quantitative real-time PCR was performed using a CFX96TM realtime PCR detection system (Bio–Rad, http://www.bio-rad.com/) as described by Bustin et al. (2009). Each reaction comprised 2.5 ll cDNA (1:20) added to 5 ll KAPA SYBR fast reaction mix (Peqlab, http://www.peqlab.de) and 2.5 ll primer mixture

The z–average size was determined as described by Hillebrand et al. (2012) but using disposable 1 ml polystyrene cuvettes. Only measurements classified as appropriate, mostly characterized by a constant count rate, by Zetasizer Nano software (Malvern, http://www.malvern.com) were included in further analysis.

© 2015 The Authors The Plant Journal © 2015 John Wiley & Sons Ltd, The Plant Journal, (2015), doi: 10.1111/tpj.12836

Rubber elongation factor protein in dandelion 11 Size-exclusion chromatography Rubber samples prepared as described above were dissolved at 60°C for 16 h in toluene to a final concentration of 0.75 mg/ml. The samples were passed through a Cameo 3N 0.22 lm filter (Roth, http://www.carlroth.com) and transferred to new glass vials. Size-exclusion chromatography was performed using a SECcurity GPC system with a PSS SDV 20 l 8 9 50 mm precolumn, a PSS SDV Linear XL 20 l 8 9 300 nm analytical column, and a PSS SDV 100  A 20 8 9 300 mm analytical column (Polymer Standards Service, http://www.pss-polymer.com).

Quantitative determination of poly(cis-1,4–isoprene) levels by 1H-NMR spectroscopy Poly(cis-1,4-isoprene) levels were determined by 1H-NMR spectroscopy as previously described (Post et al., 2012).

[1–14C] IPP incorporation assay The [1–14C] IPP incorporation rate was determined as previously described without measuring the molecular mass of the products by size-exclusion chromatography (Post et al., 2012).

ACKNOWLEDGEMENTS The technical assistance of Daniela Ahlert (Institute of Plant Biol€ nster, Germany) and Ulrike Keller ogy and Biotechnology, Mu € nster, Germany) (Institute for Medical Physics and Biophysics, Mu is kindly acknowledged. We thank Claudia Huber and Wolfgang Eisenreich (Department of Biochemistry, Technische Universita€t € nchen, Germany) for 1H-NMR analysis of T. brevicorniculatum Mu root material. This work was supported by a grant from European Union Framework Program 7 (project number 212827, EUPEARLS) and the Bundesministerium fu¨r Bildung und Forschung of Germany (grant number FKZ 0315971C).

SUPPORTING INFORMATION Additional Supporting Information may be found in the online version of this article. Figure S1. Alignment of the amino acid sequences of TbREF, TbSRPP2, TbSRPP3, HbREF, HbSRPP, PaGHS, AtSRP1, CaSRP1 and PamLDAP1. Figure S2. Expression of TbREF, TbCPT1–3 and TbSRPP1–5 in wild-type plants and three independent transgenic TbREF-RNAi lines. Table S1. Sequence length and source species of peptides used for the alignment shown in Figure S2. Table S2. Relative protein content of specific antibody signals in TbREF-RNAi transgenic lines compared to wild-type plants. Table S3. Rubber particle parameters and z–average size. Table S4. Sequences of oligonucleotides. Table S5. Efficiencies of real-time PCR primer pairs. Data S1. Real-time CFX manager reports.

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