Polyphenoloxidase Silencing Affects Latex ... - Plant Physiology

Polyphenoloxidase Silencing Affects Latex Coagulation in Taraxacum Species1[W] Daniela Wahler2, Christian Schulze Gronover2, Carolin Richter, Florence Foucu, Richard M. Twyman, Bruno M. Moerschbacher, Rainer Fischer, Jost Muth, and Dirk Pru¨fer* Westphalian Wilhelms-University of Mu¨nster, Institute of Biochemistry and Biotechnology of Plants, 48143 Muenster, Germany (D.W., C.R., B.M.M., D.P.); Fraunhofer Institute for Molecular Biology and Applied Ecology, 52074 Aachen, Germany (C.S.G., F.F., R.F., J.M., D.P.); and Department of Biology, University of York, Heslington, York YO10 5DD, United Kingdom (R.M.T.)

Latex is the milky sap that is found in many different plants. It is produced by specialized cells known as laticifers and can comprise a mixture of proteins, carbohydrates, oils, secondary metabolites, and rubber that may help to prevent herbivory and protect wound sites against infection. The wound-induced browning of latex suggests that it contains one or more phenoloxidizing enzymes. Here, we present a comprehensive analysis of the major latex proteins from two dandelion species, Taraxacum officinale and Taraxacum kok-saghyz, and enzymatic studies showing that polyphenoloxidase (PPO) is responsible for latex browning. Electrophoretic analysis and amino-terminal sequencing of the most abundant proteins in the aqueous latex fraction revealed the presence of three PPO-related proteins generated by the proteolytic cleavage of a single precursor (prePPO). The laticifer-specific pre-PPO protein contains a transit peptide that can target reporter proteins into chloroplasts when constitutively expressed in dandelion protoplasts, perhaps indicating the presence of structures similar to plastids in laticifers, which lack genuine chloroplasts. Silencing the PPO gene by constitutive RNA interference in transgenic plants reduced PPO activity compared with wild-type controls, allowing T. kok-saghyz RNA interference lines to expel four to five times more latex than controls. Latex fluidity analysis in silenced plants showed a strong correlation between residual PPO activity and the coagulation rate, indicating that laticifer-specific PPO plays a major role in latex coagulation and wound sealing in dandelions. In contrast, very little PPO activity is found in the latex of the rubber tree Hevea brasiliensis, suggesting functional divergence of latex proteins during plant evolution.

Latex is a milky sap produced by more than 12,500 plant species spanning 20 families (Metcalfe, 1966). It is often white or colorless but can range from yellow to scarlet (e.g. in some members of the poppy family [Papaveraceae]). Latex coagulates when exposed to air and consists of an emulsion of polymers and metabolites that are often bitter or toxic. Therefore, it is proposed that natural latex has a protective function, sealing wounds, acting as a barrier to microorganisms, and discouraging herbivory (El Moussaoui et al., 2001). In addition to a wide range of lowmolecular-weight polypeptides (Nessler and Burnett, 1992; Azarkan et al., 2004), several other proteins and enzymes have been identified in the latices of laticiferous plants. These include the wound-induced proteins trypsin inhibitor, class II chitinase, and glu-

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This work was supported by the Ministry of Science and Education of Germany (grant no. FKZ 0313712). 2 These authors contributed equally to the article. * Corresponding author; e-mail [email protected]. The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Dirk Pru¨fer ([email protected]). [W] The online version of this article contains Web-only data. www.plantphysiol.org/cgi/doi/10.1104/pp.109.138743 334

taminyl cyclase in the latex of papaya (Carica papaya; Azarkan et al., 2004) as well as chitinases and b-1,3glucanase in the latex of the rubber tree Hevea brasiliensis (Martin, 1991; Subroto et al., 1996). The latex from some plants is a good source of natural rubber, and H. brasiliensis is widely cultivated for this purpose. Latex is produced in specialized cells known as laticifers, which arise in two distinct ways depending on the species (Evert, 2006). Articulated laticifers (found in the Papaveraceae, Asteraceae, and in H. brasiliensis) are organized in longitudinal chains originally laid down in the meristem, and the cell walls become perforated or completely degraded during development to form continuous channels called latex vessels. In contrast, nonarticulated laticifers (found in milkweeds [Asclepias spp.]) are organized in a branching system originating from a single precursor cell in the embryo that divides rapidly and spreads invasively during development. These are multinucleate cells that tend not to fuse into vessels (Serpe et al., 2002). Taraxacum officinale (common dandelion) and Taraxacum kok-saghyz (Russian dandelion) are members of the Asteraceae and therefore possess articulated laticifers (Esau, 1965; Evert, 2006) that secrete a latex rich in polyphenols (Schu¨tz et al., 2005; C. Schulze Gronover, unpublished data). T. kok-saghyz latex is a

Plant PhysiologyÒ, September 2009, Vol. 151, pp. 334–346, www.plantphysiol.org Ó 2009 American Society of Plant Biologists

PPO Silencing Affects Latex Coagulation in Taraxacum Species

good source of high-molecular-weight rubber (Mooibroek and Cornish, 2000; Bushman et al., 2006) and was investigated as an alternative to H. brasiliensis during World War II, when rubber supplies to Europe and the United States were interrupted. Unfortunately, the extraction of rubber from Russian dandelion latex is laborious and expensive because of rapid coagulation, and further development was abandoned when Hevea rubber became available. Coagulation of H. brasiliensis latex is caused by the major latex proteins (MLPs), which include hevein, the hevein receptor, and chitinase (Gidrol et al., 1994; Chrestin et al., 1997). A similar role has been proposed for the polyphenoloxidases (PPOs) present in the latex of certain Hevea spp. (Hanower and Brzozowska, 1977), although our data indicate that this is not the case. PPOs are found throughout the plant kingdom (Mayer and Harel, 1979; Vaughn and Duke, 1984; Mayer, 1987; Vaughn et al., 1988; Sherman et al., 1991), and they probably play a role in defense against pathogens and herbivores (Vo¨ro¨s et al., 1957; Felton et al., 1989; Duffey and Felton, 1991; Constabel and Ryan, 1998; Stout et al., 1999; Gatehouse, 2002). They are plastid-localized copper metalloenzymes that catalyze the oxidation of o-diphenols to o-diquinones (diphenolase activity; EC 1.10.3.2) and, in some species, also the o-hydroxylation of monophenols (monophenolase activity; EC 1.14.18.1; Vaughn et al., 1988; Mayer, 2006). Quinones are highly reactive electrophiles responsible for much of the oxidative browning in fruits and vegetables after wounding (Yoruk and Marshall, 2003). The wound-inducible expression of PPOs has been reported in apple (Malus domestica; Boss et al., 1995), tomato (Solanum lycopersicum; Constabel et al., 1995; Thipyapong and Steffens 1997), potato (Solanum tuberosum; Thipyapong et al., 1995), and hybrid poplar (Populus spp.; Constabel et al., 2000). In addition, the down-regulation of PPO activity by antisense RNA in tomato confers hypersusceptibility to pathogens (Thipyapong et al., 2004), whereas PPO overexpression confers enhanced resistance to bacterial diseases (Li and Steffens, 2002). It has also been suggested that PPOs evolved to protect plants against photochemical oxidation, since most PPOs characterized thus far appear to be localized in the plastids of photosynthetic cells (Sherman et al., 1995). The rapid wound-induced browning of dandelion latex suggests the presence of significant PPO activity in the laticifers. Here, we show that the PPO is the major component of the latex proteome in Taraxacum spp. and that the down-regulation of PPO activity by RNA interference (RNAi) in transgenic T. officinale and T. kok-saghyz plants inhibits browning and coagulation. This suggests that PPO may be a key factor controlling the coagulation of dandelion latex and thus its protective role. This contrasts to the situation in H. brasiliensis, where we show that PPO appears to have a negligible effect on latex coagulation. Plant Physiol. Vol. 151, 2009

RESULTS Identification and Molecular Characterization of Enzymes Involved in the Rapid Browning of Dandelion Latex after Wounding

Phenol-oxidizing enzymes such as PPOs, peroxidases, and laccases often cause wound-induced browning in the plant kingdom. It is often difficult to separate the activities of these enzymes due to overlapping substrate specificities. In order to investigate the cause of rapid latex browning in dandelion (as shown for T. kok-saghyz latex in Fig. 1, A and B), T. officinale and T. kok-saghyz petioles were dissected to

Figure 1. Analysis of latex browning in Taraxacum spp. A and B, Photographs of expelling latex from T. kok-saghyz petioles immediately (A) and 30 min (B) after wounding. First browning of latex was observed directly after abscission of petioles (red arrow). C, Determination of PPO activity in the aqueous latex phase of T. officinale and T. koksaghyz in the absence (–) and presence (+) of tropolone using L-DOPA as substrate. Enzymatic activity is presented in mkat mg21 protein. D and E, PPO activity was also analyzed in L-DOPA-treated cross sections of T. officinale petioles preincubated with (E) or without (D) tropolone. PPO activity appears to be laticifer specific (black arrowheads). La, Laticifer; Ph, phloem; Xy, xylem. Bars = 100 mm. 335

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encourage the expulsion of latex, which was collected and centrifuged to obtain the aqueous phase for subsequent enzyme activity analysis (for details, see “Materials and Methods”). In the presence of 3,4dihydroxy-L-phenylalanine (L-DOPA), a PPO substrate that is also oxidized by laccases (Eisenman et al., 2007) and peroxidases (Albrecht and Kohlenbach, 1990), we observed total latex enzymatic activities of approximately 0.218 mkat mg21 (T. officinale) and approximately 0.237 mkat mg21 (T. kok-saghyz). Addition of the potent PPO inhibitor tropolone (Fuerst et al., 2006) caused a significant reduction in the total enzymatic activity to approximately 0.004 mkat mg21 for T. officinale and approximately 0.002 mkat mg21 for T. koksaghyz (Fig. 1C). The same result was evident for L-DOPA-stained laticifers in thin cross sections of T. officinale petioles in the absence (Fig. 1D) or presence (Fig. 1E) of tropolone. Here, clear signals were observed only in laticifers that had not been treated with the PPO inhibitor (Fig. 1D, arrowhead). The very weak residual browning activity (Fig. 1E, arrowhead) in tropolone-treated laticifers can be explained either by the incomplete inhibition of the PPO reaction or by the action of other enzymes such as peroxidases, which are tropolone insensitive. Taken together, the results above suggest that activity of one or more PPOs in the latex proteome is responsible for the rapid oxidation of L-DOPA. The protein composition of the latex aqueous phase from the dandelion petiole, therefore, was analyzed by SDSPAGE. As shown in Figure 2A, T. kok-saghyz latex contained five dominant protein bands that were designated MLPa (approximately 16 kD), MLPb (approximately 41 kD), MLPc (approximately 43 kD), MLPd (approximately 55 kD), and MLPe (approximately 57 kD). Bands with molecular masses equivalent to those of MLPa, MLPb, and MLPe were also

represented in the petiole latex of the closely related species T. officinale, hinting that the proteins might have conserved roles. N-terminal sequencing by Edman degradation provided confirmation by showing that corresponding bands had identical N-terminal peptides in both species: DELIPFAKDV for MLPa and DPIMAPDLTQ for both MLPb and MLPe (Fig. 2A). However, additional non-PPO-related amino sequences were generated for MLPe, indicating that MLPe is composed of more than one protein (data not shown). The PPO protein in MLPe, therefore, was designated MLPePPO. No sequences were obtained from MLPc and MLPd, indicating that the N-terminal residues were modified and unsuitable for Edman chemistry. It should be noted that the MLP pattern was identical in latices derived from stems, petioles, and roots (data not shown). Based on the above peptide sequence information, we cloned the corresponding full-length genomic sequences by inverse PCR and the cDNA sequences by 5# and 3# RACE. Comparison of these sequences revealed the presence of a 1.8-kb open reading frame in both species, uninterrupted by introns, each encoding an approximately 67-kD protein similar to PPOs. The resulting cDNA clones were designated ToPPO-1 (for T. officinale PPO1; accession no. EU154993) and TkPPO-1 (for T. kok-saghyz PPO1; accession no. pending; Supplemental Fig. S1A). MLPa, MLPb, and MLPePPO are generated by the proteolytic cleavage of ToPPO-1 or TkPPO-1 (pre-PPOs), with MLPb comprising the tyrosinase domain, MLPa comprising the C-terminal portion of the protein, and MLPePPO comprising both (Fig. 2B). Further analysis identified an additional 10-kD N-terminal bipartite transit peptide for plastid targeting (Fig. 2B). The function of the transit peptide was confirmed in the case of ToPPO-1 by joining the corresponding DNA sequence to the DsRed reporter gene

Figure 2. Molecular characterization of the MLPs of T. officinale (T.o.) and T. kok-saghyz (T.k.). A, SDS-polyacrylamide gel stained with Coomassie Brilliant Blue, showing three (T.o.; MLPa, MLPb, and MLPe/ePPO) and five (T.k.; MLPa to MLPe/ePPO) dominant bands. N-terminal sequences for MLPa, MLPb, and MLPePPO are shown at right. Molecular masses are shown in kD. B, Schematic representation of the dandelion PPO-1 and its proteolytic products. Cleavage of the pre-PPO-1 protein (top) at the sites indicated with arrows produces small amounts of a nearly full-length product (MLPePPO) lacking the transit peptide (tp; green), together with larger amounts of the tyrosinase domain (MLPb; brown) and a C-terminal domain (MLPa; white). The first three amino acids from the individual cleavage products are indicated. 336

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and expressing the fusion proteins under the control of the strong constitutive cauliflower mosaic virus (CaMV) 35S promoter (Guilley et al., 1982) in T. officinale and T. kok-saghyz protoplasts (Fig. 3). The ToPPO-1 bipartite transit peptide efficiently targeted the DsRed reporter protein into the plastids, as indicated by the overall orange-red fluorescence of these organelles (Fig. 3, A–D and I–L). In contrast, DsRed fluorescence was not detected in the chloroplasts of protoplasts transfected with the same construct lacking the bipartite transit peptide sequence. Here (Fig. 3, E–H and M–P), and as expected, DsRed fluorescence was restricted to the cytoplasm of the protoplasts. Chloroplast autofluorescence is represented by green pseudocolor in Figure 3. Next, we analyzed the ToPPO-1 and TkPPO-1 mRNA levels (Fig. 4A). Reverse transcription (RT)PCR analysis with total RNAs isolated from T. officinale and T. kok-saghyz latex (Fig. 4A, lanes 2 and 4) showed that both genes were expressed strongly. Corresponding weak bands were observed in the lanes representing leaf sections lacking major vascular bundles (Fig. 4A, lanes 1 and 3), probably reflecting the presence of a small number of laticifers in leaf tissue. The expression of ToPPO-1 was studied further in transgenic T. officinale plants by expressing GUS under

the control of the ToPPO-1 promoter (for sequence, see Supplemental Fig. S1B). Nine independent transgenic lines shown to contain the pToPPO-prom-1/GUS construct (data not shown) were designated ToPPOprom1/1 to ToPPO-prom1/9. The expression pattern driven by the ToPPO-1 promoter is shown in Figure 4, B to F. In cross sections of petioles stained with the substrate 5-bromo-4-chloro-3-indolyl-b-glucuronic acid (Fig. 4B), GUS activity was restricted to cells located close to the vascular bundles (Fig. 1D). However, precise localization was not possible due to the rapid diffusion of the substrate in the expelling latex after cutting. Therefore, GUS activity was analyzed in longitudinal sections that contained unwounded laticifers. As shown in Figure 4C, reporter enzyme activity was restricted to cells showing the typical structure of articulated anastomosing laticifers. The same cell organization (Fig. 4D) can also be visualized by staining dandelion laticifers with Sudan III (Au et al., 2009). As expected from this result, GUS activity, indicated by the blue color, could be detected only in latex from the transgenic line (Fig. 4E, ToPPO-prom1/1) but not in the wild-type control (Fig. 4E, WT). In addition, immunological studies using a GUS-specific polyclonal antiserum revealed the presence of the GUS enzyme in ToPPO-prom1/1 latex (Fig. 4F). Taken together, these Figure 3. Functional analysis of the transit peptide in dandelion protoplasts. Confocal fluorescence pseudocolor images of T. officinale (A–D) and T. kok-saghyz (I–L) protoplasts expressing the ToPPO-1 transit peptide/DsRed translational fusion protein under the control of the double CaMV 35S promoter identifies DsRed fluorescence in chloroplasts 1 d after transfection. As controls, T. officinale (E–H) and T. koksaghyz (M–P) protoplasts were transfected with the same DsRed construct lacking the ToPPO-1 transit peptide. DsRed fluorescence is shown in red, and autofluorescence of chloroplasts is presented in a green pseudocolor. Images A, E, I, and M were obtained by transmission light; images B, F, J, and N represent DsRed fluorescence; autofluorescence of chloroplasts is shown in images C, G, K, and O; and images D, H, L, and P are overlays of the corresponding individual fluorescence images (DsRed and chloroplast autofluorescence). Overlay fluorescence is shown in orange. Chl, Chloroplast. Bars = 20 mm.


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Wahler et al. Figure 4. Analysis of PPO-1 expression in planta. A, Detection of ToPPO-1 and TkPPO-1 mRNA by RT-PCR, demonstrating laticifer-specific gene expression. Total RNA was isolated from leaves (lanes 1 and 3) and latex (lanes 2 and 4) of T. officinale (T.o.) and T. kok-saghyz (T.k.). The housekeeping GAPDH gene was used as a positive control. B, Thin longitudinal section of a ToPPO-prom1/1 petiole expressing the gusA gene under the control of the ToPPO-1 promoter, showing promoter activity to cells located adjacent to the vascular bundle (black arrowheads). C and D, GUS (C) and Sudan III (D) staining of articulated anastomosing laticifers. E, Detection of GUS activity in ToPPO-prom1/1 latex by staining with 5-bromo-4-chloro-3-indolyl-b-glucuronic acid. F, Immunological detection of the GUS enzyme in latex of ToPPO-prom1/1. La, Laticifer; Ph, phloem; WT, wild type; Xy, xylem. Bars = 100 mm.

results showed that the latex protein component in dandelion is represented predominantly by a laticiferspecific PPO enzyme. We next considered whether this protein was responsible for the rapid browning and subsequent coagulation of dandelion latex after wounding. Down-Regulation of PPO Expression in Transgenic Dandelion Plants and Its Impact on Latex Coagulation

The potential relationship between PPO activity and latex coagulation in T. kok-saghyz and T. officinale was investigated by RNAi. Therefore, a specific RNAi 338

construct was designed to target the PPO tyrosinase domain and was expressed under the control of the CaMV 35S promoter. Twenty-five independent T. officinale and T. kok-saghyz transgenic lines were generated by Agrobacterium tumefaciens-mediated leaf disc transformation. Transgene integration and transcription were confirmed by PCR and RT-PCR, respectively. As expected, the efficiency of ToPPO-1 and TkPPO-1 knockdown varied among the independent transformants, ranging from almost complete silencing to nearly wild-type PPO levels (data not shown). The protein composition of latex from transgenic plants showing the strongest knockdown effects (T. Plant Physiol. Vol. 151, 2009


officinale lines ToLP1 and ToLP2 and T. kok-saghyz lines TkLP1 and TkLP2) are shown in Figure 5, A and B (see also Supplemental Fig. S2). Latex from all the transgenic plants showed a significant reduction in the abundance of MLPa (black arrows) and MLPb (red arrows), but the amount of MLPePPO appeared to be the same in wild-type and transgenic plants (green arrows), reflecting the fact that MLPe is composed of several proteins. A unique additional protein band was detected in line ToLP1 (but not ToLP2), and N-terminal sequencing by Edman degradation identified this as a pectin methyl esterase (Fig. 5A, asterisk). Since the MLPb and MLPePPO proteins include the PPO catalytic domain (Fig. 2B) but only the smaller MLPb appears to be depleted in transgenic plants, we decided to test the latex extracts using a seminative gel electrophoresis method that allows the in-gel detection of PPO activity (see “Materials and Methods”). It should be noted that proteins migrate slightly faster under these conditions. As expected according to their relative abundance, most PPO activity in wild-type plants was associated with MLPb (Fig. 5, C and D, red arrows) and a smaller amount with MLPePPO (Fig. 5, C and D, green arrows). In all four of the transgenic lines, the signals for both MLPb and MLPePPO were reduced, confirming the knockdown of MLPePPO, even though this was not clear from standard SDS-PAGE experiments (Fig. 5, A and B). No PPO activity was associated with the novel protein band identified in line ToLP1. PPO activity in each of the transgenic lines was determined more precisely using an in vitro assay, confirming the strong reduction of enzyme activity in lines ToLP1, ToLP2, TkLP1, and TkLP2 (Fig. 5, E and F). To determine whether latex from the transgenic lines coagulated more slowly than wild-type latex, the leaves, stems, and roots from lines ToLP1, ToLP2, TkLP1, and TkLP2 were dissected and the flow of latex was evaluated in comparison with wild-type plants using a capillary-based test system, a technique routinely used to investigate blood coagulation (see “Materials and Methods”). The capillary suction distances (csd) were consistent over three measurements, and the comparative values recorded for wild-type and transgenic plants are shown in Figure 6. Latex from wild-type T. officinale (26.7 mm csd) and T. kok-saghyz (23.3 mm csd) plants coagulated soon after soaking up, whereas latex from the transgenic lines ToLP1 (54.7 mm csd), ToLP2 (52.0 mm csd), TkLP1 (53.3 mm csd), and TkLP2 (39.3 mm csd) showed considerable increases in coagulation time. These results suggested that PPO activity has an important impact on the rate of latex coagulation in dandelion. Since T. kok-saghyz (but not T. officinale) latex is known to contain large amounts of high-quality rubber, we also set out to determine the amount of latex that can be harvested after wounding and to analyze whether or not rubber biosynthesis was affected by the down-regulation of PPO activity. We compared the amount of latex and rubber produced in the transgenic T. kok-saghyz lines TkLP1 and TkLP2 and wild-type Plant Physiol. Vol. 151, 2009

Figure 5. Analysis of PPO abundance and activity in wild-type (wt) and RNAi-knockdown T. officinale (To) and T. kok-saghyz (Tk) plants. A and B, SDS-PAGE showing major latex proteins in wild-type and transgenic plants expressing an RNAi construct targeting the PPO tyrosinase region (ToLP1, ToLP2, TkLP1, and TkLP2). The positions of MLPa (black arrows), MLPb (red arrows), and MLPePPO (green arrows) are indicated. The asterisk in A shows a novel and uncharacterized major protein in line ToLP1. C and D, In-gel assay showing proteins with PPO activity. The MLPb (red arrows) and MLPePPO (green arrows) activities in the RNAi plants are almost completely abolished. No corresponding activity signal could be detected for MLPa, since this derivate does not contain a tyrosinase domain. E and F, Determination of the enzymatic PPO activity in wild-type and transgenic RNAi-knockdown lines. The enzymatic activity is presented as mkat mg21 protein. The values presented are means 6 SD of at least three biological samples in each case. Significant differences were found between transgenic and wild-type plants (calculated using Student’s t test; P = 0.01).

plants. In three independent experiments, we were able to harvest four to five times more latex from the RNAi lines, while no significant differences were found in the rubber content (Table I). In addition, the molecular mass of the rubber was unchanged. These data indicate that PPO activity is unnecessary for efficient rubber biosynthesis. Correlation between Latex Coagulation Rate and PPO Activity in Different Species

In order to verify whether there is a more general correlation between latex coagulation and PPO activ339

Wahler et al. Figure 6. Determination of latex fluidity in the T. officinale and T. koksaghyz RNAi-knockdown plants. Latex from wild-type (wt) and transgenic RNAi plants was harvested and analyzed as described in the legend to Figure 5. The values presented are means 6 SD of at least six biological samples in each case. Significant differences were found between transgenic and wild-type plants (calculated using Student’s t test; P = 0.01).

ity, we studied PPO activity and latex fluidity in the following laticiferous plants from three families: H. brasiliensis (Euphorbiaceae), Ficus elastica (Moraceae), and Sonchus asper as an additional Asteraceae member. The latices of T. officinale and T. kok-saghyz served as controls. Photographs of greenhouse-cultivated plants (for cultivation conditions, see “Materials and Methods”) and of latex expelled from them after injury are shown in Figure 7A. To analyze latex PPO activity in these plants, latex aqueous phase samples containing 2 mg of total protein were spotted onto a nitrocellulose membrane and incubated with L-DOPA. In parallel, PPO activity was determined by spectrophotometry. The outcome of both experiments is shown in Figure 7B. The strongest enzymatic activities were found in the Asteraceae, with values of 0.61 mkat mg21 protein for S. asper, 0.17 mkat mg21 protein for T. officinale, and 0.20 mkat mg21 protein for T. kok-saghyz. In contrast, PPO activity in H. brasiliensis latex was very weak (0.01 mkat mg21 protein), and no activity was observed in the latex of F. elastica. We next measured the latex fluidity in a glass capillary as described above. In this setup (Fig. 7C), S. asper latex had the highest PPO activity and coagulated directly after loading (7.2 mm csd; lane 1), followed by T. kok-saghyz (24 mm csd; lane 3) and T. officinale (29 mm csd; lane 2), which had very similar values, then H. brasiliensis (35.5 mm csd; lane 4) and F. elastica (113.5 mm csd; lane 5). The rate of latex coagulation correlated strongly with the level of laticifer-specific PPO activity in F. elastica and all the Asteraceae plants we studied but not in H. brasiliensis, where rapid latex coagulation occurred despite the

weak PPO activity. It should be noted that latex with high levels of PPO activity also turned brown in the capillary experiments, as shown for the two dandelion species (Fig. 7C, bottom left).

DISCUSSION

Latex is a milky sap produced by a large number of diverse plants. It typically comprises a mixture of proteins, carbohydrates, oils, secondary metabolites, and gums that may protect injured plants from infection and herbivory. The rapid browning of dandelion latex after wounding argues for the presence of one or more phenol-oxidizing enzymes, specifically PPOs, peroxidases, and/or laccases. Our data indicate that PPOs are responsible for the browning of dandelion latex, since the browning process can be almost completely prevented both in vitro and in vivo following treatment with tropolone, a PPO inhibitor (Fig. 1). The residual browning activity in these experiments might reflect either the incomplete inhibition of PPO activity or the activity of peroxidases and/or laccases. However, genomic and proteomic approaches have thus far failed to identify these latter enzymes in T. officinale and T. kok-saghyz latex (D. Wahler, unpublished results), whereas we found that PPO is a major constituent of the aqueous latex fraction. Therefore, we propose that residual browning activity reflects incomplete inhibition of PPO by tropolone. T. kok-saghyz latex contained five dominant protein bands, three of which were also observed in T. officinale. N-terminal sequencing by Edman degradation

Table I. Amount of latex harvested after wounding, as well as rubber quantity and quality from wild-type T. kok-saghyz plants and transgenic lines TkLP1 and TkLP2 Each value represents the mean 6 SD from at least three biological samples. Significant differences were found between transgenic and wild-type plants for the amount of expelled latex (calculated using Student’s t test; P = 0.01). mw, Molecular mass; Rc, rubber content. Plant

Wild type TkLP1 TkLP2 340

Amount of Latex Harvested after Wounding

Rubber Quantity Rc

Rubber Quality mW

mg

mg mL21 latex

D 3 106

0.5 1.9 2.3

60.4 60.3 60.1

139.93 155.13 166.17

616.90 619.14 617.08

4.5 5.7 4.0 Plant Physiol. Vol. 151, 2009


Figure 7. Latex and PPO activity in different laticiferous plants. A, Photographs of S. asper, T. officinale, T. kok-saghyz, H. brasiliensis, and F. elastica and of droplets of latex expelled from each plant. Differences in latex fluidity and coagulation are apparent from the size and flow of the droplets. B, Top, PPO activity visualized by dot-blot L-DOPA staining for all the plants listed above (2 mg of total protein from the latex aqueous phase was spotted onto a nitrocellulose membrane and placed on 1% agarose gels containing 20 mM L-DOPA). Bottom, Enzymatic activity is presented in mkat mg21 protein. C, Latex from S. asper (1), T. officinale (2), T. kok-saghyz (3), H. brasiliensis (4), and F. elastica (5); all the plants listed above were harvested after wounding by drawing into 10-mL microglass capillaries. The inset (lower left) shows latex browning in the capillary system for T. officinale (2) and T. kok-saghyz (3). D, After no further movement of the latex, the distance from the end of the capillary to the stopping point was determined in mm csd. All values are presented as means 6 SD of at least six biological samples in each case. No significant differences existed in enzyme activity (B) and csd (D) between T. officinale and T. kok-saghyz, as calculated using Student’s t test (P = 0.01).

showed that the matching bands shared the same N-terminal peptides, strongly indicating that they are structurally and functionally conserved. The peptide sequences were used to isolate corresponding PPO genes and cDNA sequences, which were named ToPPO-1 and TkPPO-1. The genes encode proteins with two copper-binding motifs found in many other PPOs. Further analysis confirmed that three of the proteins, MLPa, MLPb, and MLPePPO, are generated Plant Physiol. Vol. 151, 2009

by proteolytic cleavage of ToPPO-1/TkPPO-1, with MLPb comprising the tyrosinase domain, MLPa comprising the C-terminal domain, and MLPePPO comprising both (Fig. 2). Proteolytic processing of many other PPOs has been described, such as the PPO enzymes found in broad bean leaf (Vicia faba; Robinson and Dry, 1992), grape (Vitis vinifera; Rathjen and Dry, 1992), and coffee (Coffea arabica; Mazzafera and Robinson, 2000). More recently, Flurkey and Inlow (2008) analyzed 341

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specific proteolysis target sequences in fungal and plant PPOs and showed that N-terminal proteolysis sites were located immediately upstream of a conserved Arg residue, whereas C-terminal proteolysis sites were located downstream of a conserved Tyr residue. The same amino acids are present in ToPPO-1 and TkPPO-1 (Supplemental Fig. S1A), so a similar proteolysis mechanism is probably involved. Transgenic T. officinale plants expressing GUS under the control of the ToPPO-1 promoter showed that PPO-1 expression is restricted to laticifers, which are characterized by an articulated anastomosing cell structure (Esau, 1965) similar to those found in opium poppy (Papaver somniferum; Thureson-Klein, 1970). However, in cross sections of transgenic petioles, clear histochemical localization of GUS activity to the laticiferous system was not possible because of the rapid diffusion of the metabolized GUS substrate after wounding. Therefore, additional studies were performed, including RT-PCR experiments, GUS staining of intact laticifers in longitudinal petiole sections, and direct detection of GUS activity in latex. Both PPO-1 mRNA (Fig. 4A) and GUS activity in transgenic plants (Fig. 4, B, C, E, and F) were exclusively detected in the latex, indicating strict laticifer-specific activity of the corresponding promoter elements. In contrast, the transcription of three key genes of the benzylisoquinoline alkaloid biosynthetic pathway in opium poppy was shown to be restricted to the parietal region of sieve elements adjacent or proximal to laticifers, even though the products of this pathway accumulated specifically in the laticifers (Bird et al., 2003). A similar situation cannot be excluded for ToPPO-1 and TkPPO-1 mRNAs, so further experiments involving immunofluorescence labeling and/or in situ hybridization are needed to gain further insight into dandelion PPO-1 gene expression. PPOs typically carry an N-terminal transit peptide that is cleaved from the precursor proteins to yield a mature protein (Sommer et al., 1994). This transit peptide is bipartite, for initial translocation to the chloroplast and subsequent transfer to the thylakoid lumen, indicating a two-step maturation process (Soll and Schleiff, 2004). The immature forms of ToPPO-1 and TkPPO-1 also carry an N-terminal plastid transit peptide (Fig. 2B) that directed a reporter protein into plastids when expressed in T. officinale and T. koksaghyz protoplasts (Fig. 3). This was possible even though localization experiments indicated that ToPPO-1 is expressed specifically in laticifers, which lack conventional plastids. The presence of plastidtargeting peptides on a laticifer-specific protein suggests that there may be structures similar to plastids in laticifers, a hypothesis supported by electron microscopy data revealing spherical complexes, 3 to 6 mm in diameter, within H. brasiliensis laticifers. These socalled Frey-Wyssling complexes are highly modified plastids enclosed within a double membrane that accumulate carotenes and PPOs (Frey-Wyssling, 1929; Dickenson, 1969; Archer, 1980). Anticipating 342

that Frey-Wyssling complexes may also exist within dandelion laticifers, we speculate that the mature and active forms of ToPPO-1 and TkPPO-1 would be stored in this cellular compartment and would be released after wounding by a yet unknown mechanism. However, localization experiments are needed to verify this hypothesis. PPOs are known to possess diphenolase activity, which catalyzes the oxidation of o-diphenols to o-quinones (Sańchez-Ferrer et al., 1995). Quinones are highly reactive molecules that are responsible for wound-induced browning in many plants. Quinones could react with a variety of substances in the cytosol of dandelion laticifers (e.g. proteins and oligosaccharides) to form a complex brown polymer. The direct availability of a functional mature PPO (by direct release from Frey-Wyssling complexes) would facilitate immediate wound sealing after injury (e.g. by herbivores) and therefore minimize the risk of secondary infection with facultative pathogens. We proposed that PPO activity is responsible for coagulation and browning in dandelion latex, and the RNAi knockdown of this enzyme strongly supports this hypothesis by prolonging the liquid phase. PPO activity in H. brasiliensis is localized to latex lutoids and Frey-Wyssling complexes (Coupe´ et al., 1972) and is 5- to 34-fold higher in lutoids than in Frey-Wyssling complexes (Wititsuwannakul et al., 2002). It has also been proposed that PPOs play a role in the coagulation of H. brasiliensis latex, since the exogenous application of substances interfering with PPO activity, such as reducing agents or a nitrogen atmosphere, prolongs the latex flow (Hanower et al., 1976; Hanower and Brzozowska, 1977; Brzozowska-Hanower et al., 1978). In contrast to the situation in dandelion, we showed that PPO is a minor component of the H. brasiliensis latex we studied, although we cannot exclude the possibility that PPO activities differ among H. brasiliensis accessions. Nevertheless, the rate of latex coagulation in H. brasiliensis (with low PPO activity) and T. kok-saghyz (with high PPO activity) is very similar, indicating that other factors contribute to the coagulation of H. brasiliensis latex. Some of these factors have recently been identified (Gidrol et al., 1994; Silpi et al., 2006). The latex from certain plants is a good source of natural rubber, but rubber extraction is made more complex by the tendency of latex to coagulate in contact with air (a property that allows it to seal open wounds, forming a barrier to microorganisms). The rubber tree H. brasiliensis is the predominant commercial source of natural rubber, but the Russian dandelion T. kok-saghyz also contains large amounts of rubber and has been investigated as a potential alternative source (van Beilen and Poirier, 2007). One challenge that must be overcome before T. kok-saghyz can be considered as a commercial rubber source is the rapid coagulation and browning of extruded latex. These limitations could be circumvented through the use of T. kok-saghyz plants with reduced PPO activity, Plant Physiol. Vol. 151, 2009


since the latex could be harvested by low-speed centrifugation from mechanically macerated material, as is currently the case for guayule (Parthenium argentatum) latex (Cornish, 1996, 1998; Cornish and Brichta, 2002), and the rubber could be extracted with the process currently used for H. brasiliensis. To demonstrate this principle, we generated transgenic T. officinale and T. kok-saghyz plants in which the pre-PPO1 mRNA was targeted by RNAi. Among 50 transformed lines, 25 for each species, two T. officinale and two T. kok-saghyz transgenic lines were selected based on their strong knockdown activity. PPO activity was significantly depleted in these lines, which correlated well with increased coagulation times when latex was extracted and tested. In contrast, latex fluidity was not affected in transgenic T. officinale or T. kok-saghyz lines showing no or very weak PPO silencing, where coagulation times were very similar to wild-type values (data not shown). This indicates that PPO enzymatic activity must be strongly depleted in order to increase latex fluidity and suggests that there might be a threshold above which the normal coagulation phenotype is displayed. The introduction of T. kok-saghyz varieties with PPO silencing could provide an opening for the development of alternative rubber sources, since four to five times more latex could be harvested compared with the wild-type accessions. However, all currently available accessions are wild-growing isolates (i.e. major breeding programs are needed to produce elite T. koksaghyz varieties for use as a cash crop).

MATERIALS AND METHODS Plant Material and Cultivation Conditions Seed and plant materials were obtained from the Botanical Gardens in Karlsruhe, Germany (Taraxacum kok-saghyz), and the Botanical Gardens of the Westphalian Wilhelms-University of Mu¨nster, Germany (Hevea brasiliensis, Ficus elastica, Sonchus asper, and Taraxacum officinale). For all subsequent experiments, plants were cultivated in greenhouses: S. asper, T. officinale, and T. kok-saghyz in the institute greenhouse at 16°C with a 16-h photoperiod, and H. brasiliensis and F. elastica in the tropical greenhouse of the Botanical Gardens of the Westphalian Wilhelms-University of Mu¨nster at 30°C with relative air humidity of 80% under natural light conditions. For molecular characterization of the T. officinale and T. kok-saghyz major latex proteins, latex was harvested from 9-month-old plants by abscising petioles (or roots) with a razor blade. For the PPO activity/coagulation experiments, latex was harvested at 2 PM in summer (August 2008) from all plants cultivated under the conditions described above by scoring the bark or abscising petioles (or roots) with a razor blade.

Latex Collection and Purification of the Aqueous Latex Phase Freshly tapped latex was collected in ice-cold Eppendorf tubes containing 50 mM sodium phosphate buffer (pH 6). The latex was centrifuged (15,700g, 10 min, 4°C) to separate the aqueous latex phase from the pellet and (in some species) from an upper viscous layer. In some species, the centrifugation step was repeated to remove impurities from the aqueous phase.

RNA Isolation and RT-PCR Total RNA was isolated from T. officinale and T. kok-saghyz tissue and latex using the RNAmagic kit (Biobudget) following the manufacturer’s instruc-


tions, and 500 ng of total RNA was used as the template for SuperScript II reverse transcriptase (Invitrogen) using an oligo(dT18) primer. PCR amplification was performed with Thermoprime DNA Polymerase (Thermo) and gene-specific primers Tk-PPO-1fwd (5#-AAACTCGAGGAGTTCATCGGCTCGGGCAAC-3#), Tk-PPO-1rev (5#-GACATTCCAGCAGGTGGTATAGT-3#), GAPDHfwd (5#-TCATCGTCACATCCATTCCCATAC-3#), and GAPDHrev (5#-CACCAGTGCTGCTAGGGATGATGT-3#).

Analysis of PPO Activity and Localization, DsRed and GUS Assays, and Western Blots In order to test the involvement of PPO in latex browning, the substrate (Sigma-Aldrich) and the potent PPO inhibitor tropolone (Fuerst et al., 2006) were used in a photometric assay. L-DOPA was dissolved in 100 mM citrate buffer (pH 5) and applied at a final concentration of 20 mM. Its conversion to dopaquinone was followed at 478 nm (478 = 3,313 M21 cm21) using an Uvikon943 spectrophotometer (Kontron Instruments). All values were obtained by subtraction of the blank value. Tropolone was used at a final concentration of 0.1 mM to arrest PPO activity. Tropolone was preincubated with the aqueous latex phase for 30 min at room temperature prior to photometric measurements and was included at the stated concentrations in substrate solutions. All photometric measurements were carried out at 30°C with prewarmed substrate solutions. For MLP characterization, latex from T. officinale and T. kok-saghyz plants was harvested and processed as described above and the aqueous phase was fractionated by 10% SDS-PAGE (Laemmli, 1970), so that proteins could be visualized by standard Coomassie Brilliant Blue staining. N-terminal and internal peptide sequences were obtained by blotting proteins onto a polyvinylidene difluoride membrane, followed by staining with sulforodamin B (Sigma) and analysis using an ABI Procise 491 Protein Sequencer (Applied Biosystems). Homology searching was carried out using the advanced BLAST program (Altschul et al., 1997). For the detection of PPO activity, unheated latex proteins were separated by 10% SDSPAGE and the gel was washed three times for 20 min each in 100 mM sodium phosphate buffer (pH 6) containing 1% Triton X-100 and three times for 20 min each in 100 mM sodium phosphate buffer (pH 6). The gel was then incubated in 100 mM sodium phosphate buffer (pH 6) containing 20 mM L-DOPA (Sigma). When a brown color developed, the reaction was stopped by replacing the L-DOPA solution with 100 mM sodium phosphate buffer (pH 6). For dot-blot analysis, 2 mg of latex protein was spotted onto a nitrocellulose membrane (Schleicher & Schuell) and placed onto an agarose gel containing 100 mM sodium phosphate buffer (pH 6) and 20 mM L-DOPA. For in situ PPO assays, semithin sections of T. officinale petiole were cut with a razor blade and incubated in 20 mM L-DOPA solution. For inhibition of the PPO enzyme in laticifers, thin sections were preincubated for 30 min with 0.1 mM tropolone prior to the L-DOPA stain. Histochemical localization of GUS activity in transgenic dandelion plants and detection of GUS activity in the aqueous latex phase was performed using the substrate 5-bromo-4-chloro-3-indolyl-b-Dglucuronide without an oxidative catalyst, according to established protocols (Jefferson et al., 1987). The stained thin sections were visualized with an Axioskop 40 microscope (Zeiss). Detection of DsRed fluorescence (excitation, 556 nm; emission, 586 nm) in the chloroplasts or cytoplasm of transfected protoplasts was carried out using a TCS SP confocal microscope (Leica Microsystems). DsRed fluorescence was displayed in red, and chloroplast autofluorescence was rendered in green. For the immunological detection of GUS, proteins of the aqueous latex phase were separated by 10% SDS-PAGE and electroblotted onto nitrocellulose membranes. Membranes were incubated with a 1:600 dilution of a polyclonal rabbit anti-GUS serum (Invitrogen), washed three times in 13 phosphate-buffered saline, and incubated in a 1:10,000 dilution of a polyclonal goat anti-rabbit serum (alkaline phosphatase conjugate), followed by detection using nitroblue tetrazolium/5-bromo4-chloro-3-indolyl phosphate (Roche). L-DOPA

Identification and Cloning of ToPPO-1 and TkPPO-1, and Assembly of pToPPO-1, pToPPO-prom1/GUS, 35STP/DsRed, and 35S/RNAi Constructs Genomic DNA was extracted from young T. officinale leaves using the cetyltrimethyl-ammonium bromide method (Doyle and Doyle, 1990). For inverse PCR, 1 mg of T. officinale genomic DNA was digested with different restriction enzymes at 37°C for 2 h. Following phenol:chloroform extraction and ethanol precipitation, the digested DNA was self-ligated at a concentration of 0.3 to 0.5 mg mL21 in the presence of 3 units mL21 T4 DNA ligase (Promega) overnight

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at 15°C. The ligation mixture was extracted with phenol:chloroform, precipitated with ethanol, and resuspended in water to a concentration of 20 ng mL21. In order to amplify ToPPO-1 fragments, we designed two degenerate primers based on the peptide amino acid sequences. These were named LZ50 (5#-CAAMCAMCCTMCGTGTCCGGCC-3#) and LZ45 (5#-TGGTGIAIGGRGGICAACAATC-3#). The inverse PCRs were prepared using 200 ng of recircularized genomic DNA in a final volume of 50 mL containing 50 mM MgCl2, 0.25 mM of each deoxyribonucleotide triphosphate, 10 pmol of each primer, and 1 unit of Taq DNA polymerase. Undigested genomic DNA was used as the control. PCR was initiated by a denaturation step at 94°C for 15 min, followed by 36 cycles of denaturation (30 s, 94°C), primer annealing (30 s, 50°C–60°C), and extension (3 min, 72°C), with a final extension step at 72°C for 10 min. Inverse PCR fragments were cloned in the pGEM-T vector (Promega) and sequenced. To clone the entire coding region, we used the ToPPO-1 primers LZ60 (5#-GCAAAGGGTATGAGTTCATCGG-3#), LZ62 (5#-AAGATGTTTTTCCGGCGAGTCT-3#), LZ55 (5#-CATCATGCGAATGTCGATAGAATG-3#), and LZ56 (5#-CCACCTGCTGGAATGTCGGC-3#). Total RNA was isolated from T. officinale latex using Trizol Reagent (Invitrogen) and processed using the OneStep RT-PCR system (Qiagen) and the primers LZ74 (5#-AAACTCGAGATATGGCATCCCTTGCACCATC-3#) and LZ73 (5#-CAATCCTCATACTCGATTTTGATC-3#) following the manufacturer’s instructions. The missing 5# and 3# ends of the ToPPO-1 mRNA were identified using the BD SMART RACE cDNA amplification system (Clontech) and primers 5#-CAGGTGAACAATATTCATCAGTGGAGCCAC-3# (5# RACE) and 5#-AAAGATGTTTTTCCGGCGAGTCTTGA-3# (3# RACE) according to the manufacturer’s recommendations. The clone TkPPO-1 was obtained by RT-PCR on total RNA from T. kok-saghyz using the primers 5#-AAAACTCGAGATATGGCATCCCTTGCACCATCT-3# (Tkfw) and 5#-AAAATCTAGATCAATCCTCATACTCGATTTTGATC-3# (Tkbw). A 1,525-bp promoter fragment was isolated using a gene-specific primer (5#-CAGGTGAACAATATTCATCAGTGGAGCCAC-3#) and the Universal GenomeWalker Kit (Clontech). This fragment was named pToPPO-1 (Supplemental Fig. S1B) and represents the sequence immediately upstream of the ToPPO-1 translational start codon. For the construction of pToPPO-prom-1/ GUS, the uidA (gusA) gene was placed under the control of pToPPO-1 and ligated into the pTRAk vector. The pTP/DsRed plasmid was obtained after PCR amplification of the leader peptide with the primers LZ74 and LZ75 (5#-AAACCATGGCGATCGGATCTGCAAAGGCCAG-3#) and translational fusion to the DsRed coding sequence in the pDsRed cloning vector where the DsRed gene is under the control of a double CaMV 35S promoter (C. Schulze Gronover, unpublished data). For the RNAi construct, a 141-bp PCR fragment was amplified using the RNAi-dicer optimized primers RNAi_for (5#-TTTGGTACCATCCATTGGCCTTTGCAG-3#) and RNAi_rev (5#-TTTGCGGCCGCGTGGTGGAAGTTTGAAGTC-3#). The PCR fragment was inserted into the KpnI and NotI sites of the Gateway vector pENTR4 (Invitrogen) and transferred to the destination plasmid pFGC5941 (www.chromDB.org) containing the chalcone synthase intron. The integrity of all constructs was verified by sequencing. Database analysis was carried out with the Wisconsin Package (Genetics Computer Group). Chloroplast transit and thylakoid lumen targeting signals were analyzed using the ChloroP algorithms (Emanuelsson et al., 1999).

Agrobacterium-Mediated Transformation of Taraxacum Species and Dandelion Protoplast Preparation and Transfection Agrobacterium tumefaciens strain LBA4404, carrying the binary constructs, was cultured in 100 mL of induction broth (5 g L21 Suc, 5 g L21 peptone, 5 g L21 casein hydrolysate, 1 g L21 yeast extract, 10 mM MES, and 2 mM MgSO4, pH 7) containing the appropriate antibiotics (50 mg L21 kanamycin, 100 mg L21 rifampicin, and 300 mg L21 streptomycin). The bacteria were cultured at 28°C to the end of the log phase and then centrifuged, and the pellet was resuspended in coculture medium (4.4 g L21 Murashige and Skoog salt solution including vitamins, 10 mM MES, and 18 g L21 Glc, pH 5.6), supplemented with 200 mM acetosyringone. Leaf discs (approximately 1 cm2) were punched from the leaves of 6- to 10-week-old T. officinale and T. kok-saghyz plants that had been grown under sterile conditions on solid medium (2.2 g L21 Murashige and Skoog salt solution, 10 g L21 Glc, and 8 g L21 agar, pH 5.8) and inoculated in the coculture medium with A. tumefaciens for 30 min. The leaf discs were then placed on wet filter paper for 14 to 20 h at 26°C. To induce regeneration, the leaf discs were placed on regeneration

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medium (4.4 g L21 Murashige and Skoog salt solution including vitamins, 20 g L21 Glc, 8.5 g L21 agar, and 250 mg L21 Claforan, pH 5.8) supplemented with 1 mg L21 6-benzyladenine and 0.2 mg L21 naphthaleneacetic acid for callus and shoot induction. The elongation of shoots was maintained by the addition of 2 mg L21 zeatin, 20 mg L21 naphthaleneacetic acid, and 20 mg L21 gibberellic acid. Rooting was induced on regeneration medium without hormones, whereas all regeneration media contained 3 mg L21 phosphinothricin for selection. All chemicals and reagents were purchased from Duchefa. Transgenic dandelion plants were transferred to soil and cultivated at 16°C with a 16-h photoperiod. Protoplasts were isolated from T. officinale and T. kok-saghyz according to established protocols (Negrutiu, 1981). Ca(NO3)2/polyethylene glycol-mediated DNA transfer was performed as described (Negrutiu et al., 1987) using 3.3 3 105 protoplasts and 10 mg of pTP/DsRed or pDsRed (basic cloning vector) plasmid DNA per transfection experiment.

Measurement of Latex Coagulation For the measurement of latex coagulation, roots from T. officinale and T. koksaghyz wild-type and RNAi plants as well as bark from H. brasiliensis, roots from S. asper, and petioles from F. elastica were dissected and the latex allowed to exude from the cut. After 10 s, the latex was collected in a glass capillary (ringcaps, 1.2.3.4.5 mL; Hirschmann Laborgera¨te). The coagulation rate of the latex was determined by measuring the csd.

Measurement of Latex Quantity, Rubber Content, and Molecular Weight The quantity of harvested latex was determined by collecting the latex extruded from abscised pedicels and measuring its weight using a fine balance. For the measurement of rubber content, 100 mL of latex was dissolved in 100 mL of sodium phosphate buffer (pH 6) and centrifuged at 13,500g, 20 min, 4°C. The upper rubber fraction was coagulated by adding 50 mL of glacial acetic acid. The coagulated rubber was dried and weighed. The molecular size distribution of rubber was determined by size exclusion chromatography (SEC) using an Agilent Series 1100 with a column system comprising three PSS ˚ ; Polymer Standard Service). Samples were SDV 5m columns (100–100,000 A prepared by centrifuging 100 mL of latex in sodium phosphate buffer (pH 6) at 13,500g, 20 min, 4°C to separate the rubber from soluble material. The upper rubber fraction was transferred to a fresh vial and dissolved in tetrahydrofuran, which was also used as the solvent and eluent in subsequent SEC steps with a flow rate of 1 mL min21. The SEC system was calibrated with standard polyisoprenes in the range 1,090 to 1,040,000 D (Polymer Standard Service). Sequence data from this article can be found in the GenBank/EMBL data libraries under accession number EU154993 (ToPPO-1).

Supplemental Data The following materials are available in the online version of this article. Supplemental Figure S1. Amino acid sequence of ToPPO-1 and TkPPO-1 and promoter sequence of ToPPO-1. Supplemental Figure S2. Semiquantitative RT-PCR analysis of RNAiknockdown T. officinale and T. kok-saghyz plants.

ACKNOWLEDGMENTS We thank Dr. Kai Mu¨ller and Dr. Gerhard Schwall for helpful discussions. The technical assistance of Eva Aguado, Christiane Fischer, Raphael Soeur, and Julia Boike is gratefully acknowledged. Received March 18, 2009; accepted July 10, 2009; published July 15, 2009.

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