polyphenoloxidase silencing affects latex coagulation in ......2009/07/15 · latex of papaya...

Polyphenoloxidase silencing affects latex coagulation in Taraxacum

spp.1

Prof. Dr. Dirk Prüfer

Westfälische Wilhelms-Universität Münster

Institut für Biochemie und Biotechnologie der Pflanzen

Hindenburgplatz 55

48143 Münster

Germany

Tel.: +49(0)251-83 22302

FAX: +49(0)251-82 28371

E-mail: [email protected]

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Plant Physiology Preview. Published on July 15, 2009, as DOI:10.1104/pp.109.138743

Copyright 2009 by the American Society of Plant Biologists

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Polyphenoloxidase silencing affects latex coagulation in Taraxacum

spp.1

Daniela Wahler2, Christian Schulze Gronover2, Carolin Richter, Florence Foucu, Richard M.

Twyman, Bruno M. Moerschbacher, Rainer Fischer, Jost Muth and Dirk Prüfer*

Westphalian Wilhelms-University of Münster, Institute of Biochemistry and Biotechnology of

Plants, Hindenburgplatz 55, 48143 Münster, Germany (D.W., C. R., B. M. M., D. P.)

Fraunhofer Institute for Molecular Biology and Applied Ecology (IME), Forckenbeckstrasse 6

52074 Aachen, Germany (C. S. G., F. F., R. F., J.M., D.P.)

Department of Biology, University of York, Heslington, York YO10 5DD, United Kingdom (R. M. T.)

1 This work was supported by a grant from 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].

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ABSTRACT

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 phenol-

oxidizing enzymes. Here we present a comprehensive analysis of the major latex proteins (MLPs)

from two dandelion species, Taraxacum officinale and T. kok-saghyz, and enzymatic studies

showing that polyphenoloxidase (PPO) is responsible for latex browning. Electrophoretic

analysis and N-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 (pre-PPO). 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 to wild-type controls, allowing T. kok-saghyz RNAi lines

to expel 4-5 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.

INTRODUCTION

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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 micro-organisms,

and discouraging herbivory (El Moussaoui et al., 2001). In addition to a wide range of low-

molecular-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 e.g.

the wound-induced proteins trypsin inhibitor, class-II chitinase and glutaminyl cyclase in the

latex of papaya (Azarkan et al., 2004) or chitinases and β-1,3-glucanase in the latex of the rubber

tree H. 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 for example 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 for example in milkweeds) 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 T. 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 (Schütz et al., 2005; Schulze Gronover, unpublished results).

T. kok-saghyz latex is a 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 US 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

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spp. (Hanower and Brzozowska, 1977) although our data indicate 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 (Voros 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 (Boss et al., 1995), tomato (Constabel et al., 1995; Thipyapong and Steffens

1997), potato (Thipyapong et al., 1995) and hybrid poplar (Constabel et al., 2000). In addition,

the downregulation of PPO activity by antisense RNA in tomato confers hyper-susceptibility 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 downregulation 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.

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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 Figs. 1A and 1B), T. officinale and T. kok-saghyz

petioles were dissected to 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 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 ~0.218 µkat/mg (T. officinale) and ~0.237 µkat/mg (T. kok-saghyz).

Addition of the potent PPO inhibitor tropolone (Fuerst et al., 2006) caused a significant reduction

in the total enzymatic activity to ~0.004 µkat/mg for T. officinale and ~0.002 µkat/mg 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 (see arrowhead in Fig. 1D). The very weak residual browning activity (see arrowheads

in Fig. 1E) 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 was therefore analyzed by SDS polyacrylamide gel

electrophoresis (SDS-PAGE). As shown in Fig. 2A, T. kok-saghyz latex contained five dominant

protein bands that were designated MLPa (~16 kDa), MLPb (~41 kDa), MLPc (~43 kDa), MLPd

(~55 kDa) and MLPe (~57 kDa). 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 6



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 was therefore 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, or the cDNA sequences by 5´ and 3´ RACE (rapid

amplification of cDNA ends). Comparison of these sequences revealed the presence of a 1.8-kbp

open reading frame in both species, uninterrupted by introns, each encoding a ~67 kDa protein

similar to PPOs. The resulting cDNA clones were designated ToPPO-1 (T. officinale PPO 1;

Accession no. EU154993) and TkPPO-1 (T. kok-saghyz PPO 1; 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 kDa 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 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 (Figs 3A-D and 3I-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 (Figs 3E-H and 3M-P) and

as expected, DsRed fluorescence was restricted to the cytoplasm of the protoplasts. Chloroplast

autofluorescence is represented by green pseudocolor in Fig. 3.

Next, we analyzed the ToPPO-1 and TkPPO-1 mRNA levels (Fig. 4A). RT-PCR analysis with

total RNAs isolated from T. officinale and T. kok-saghyz latex (lanes 2 and 4; Fig. 4A) showed

that both genes were expressed strongly. Corresponding weak bands were observed in the lanes

representing leaf sections lacking major vascular bundles (lanes 1 and 3; Fig. 4A), probably

reflecting the presence of a small number of laticifers in leaf tissue.

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The expression of ToPPO-1 was studied further in transgenic T. officinale plants by expressing

β-glucuronidase (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 ToPPO-prom1/1 through to ToPPO-prom1/9.

The expression pattern driven by the ToPPO-1 promoter is shown in Fig. 4B–F. In cross-sections

of petioles stained with the substrate X-Gluc (Fig. 4B), GUS activity was restricted to cells

located close to the vascular bundles (see also 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 Fig. 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., 2008). As expected from this result,

GUS activity indicated by the blue color could be detected only in latex from the transgenic line

(ToPPO-prom1/1; Fig. 4E) but not the wild-type control (WT; Fig. 4E). 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 results showed that the

latex protein component in dandelion is represented predominantly by a laticifer-specific PPO

enzyme. We next considered whether this protein was responsible for the rapid browning and

subsequent coagulation of dandelion latex after wounding.

Downregulation 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 RNA interference. A specific RNAi construct was therefore

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 near wild-type PPO levels (data not shown).

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The protein composition of latex from transgenic plants showing the strongest knockdown effects

(T. officinale lines ToLP1 and ToPL2; T. kok-saghyz lines TkLP1 and TkLP2) are shown in Figs

5A and 5B (see also Supplemental Fig. S2). Latex from all the transgenic plants showed a

significant reduction in the abundance of MLPa (black arrowheads) and MLPb (red arrowheads),

but the amount of MLPePPO appeared to be the same in wild-type and transgenic plants (green

arrowheads) 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 (indicated with an asterisk in Fig. 5A).

Since the MLPb and MLPePPO proteins include the PPO catalytic domain (see Fig. 2B) but only

the smaller MLPb appears to be depleted in transgenic plants, we decided to test the latex extracts

using a semi-native 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 (red arrowheads) and a smaller amount with MLPePPO (green

arrowheads) (Figs 5C and 5D). 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 (see Figs 5A and 5B). 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 (Figs 5E and 5F). 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 to 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 Fig. 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 a considerable increase in coagulation time.

These results suggested that PPO activity has an important impact on the rate of latex coagulation

in dandelion.

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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 downregulation 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 plants. In three independent experiments, we were able to

harvest 4–5 times more latex from the RNAi lines, while no significant differences were found in

the rubber content (Table 1). In addition, the molecular weight 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

activity 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 Fig. 7A. To analyze latex

PPO activity in these plants, latex aqueous phase samples containing 2 µg 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 Fig. 7B.

The strongest enzymatic activities were found in the Asteraceae with values of 0.61 µkat/mg

protein for S. asper, 0.17 µkat/mg protein for T. officinale and 0.20 µkat/mg protein for T. kok-

saghyz. In contrast, PPO activity in H. brasiliensis latex was very weak (0.01 µkat/mg 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) (Fig. 7D). 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, 10



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

polyphenoloxidases (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 (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 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 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, e.g. the PPO enzymes found in broad bean leaf (Robinson and Dry, 1992), grape

(Rathjen and Dry, 1992) and coffee (Mazzafera and Robinson, 2000). More recently, Flurkey and

Inlow (2008) analyzed specific proteolysis target sequences in fungal and plant PPOs and showed

that N-terminal proteolysis sites were located immediately upstream of a conserved arginine

residue, whereas C-terminal proteolysis sites were located downstream of a conserved tyrosine

residue. The same amino acids are present in ToPPO-1 and TkPPO-1 (see Fig. S1A) so a similar

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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 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 (Figs 4B-C, E-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 a N-terminal plastid transit peptide (Fig. 2B) which directed a reporter protein into

plastids when expressed in T. officinale and T. kok-saghyz 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 plastid-targeting 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, 3-6 µm in

diameter, within H. brasiliensis laticifers. These so-called 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 that Frey-Wyssling complexes

may also exists within dandelion laticifers, we speculate that the mature and active forms of

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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 (Sánchez-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 (Coupé et al., 1972) and is 5–34-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 e.g. 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 micro-organisms). 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

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latex. These limitations could be circumvented through the use of T. kok-saghyz plants with

reduced PPO activity, since the latex could be harvested by low-speed centrifugation from

mechanically-macerated material as is currently the case for guayule latex (Cornish, 1996;

Cornish, 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. Analysis of PPO activity

was significant 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 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 4–5 times more latex could be harvested compared to the wild-

type accessions. However, all currently available accessions are wild-growing isolates, i.e. major

breeding programs are needed to produce elite T. kok-saghyz varieties for use as a cash crop.

MATERIALS AND METHODS

Plant material and cultivation conditions

Seed and plant material were obtained from the Botanical Gardens Karlsruhe, Germany (T. kok-

saghyz) and the Botanical Gardens of the Westphalian Wilhelms-University of Münster,

Germany (H. brasiliensis, F. elastica, S. asper and T. 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 Münster (‘Alte

Tropen’) 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

14



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 (15,700 x g, 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 instructions, 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´-AAA CTC GAG GAG TTC ATC GGC TCG GGC AAC-

3´, Tk-PPO-1rev 5´-GAC ATT CCA GCA GGT GGT ATA GT-3´, GAPDHfwd 5´-TCA TCG

TCA CAT CCA TTC CCA TAC-3´ and GAPDHrev 5´-CAC CAG TGC TGC TAG GGA TGA

TGT-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 L-DOPA (3,4-dihydroxy-

L-phenylalanine, 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

(є478 = 3313 M-1cm-1) 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 pre-incubated with the aqueous latex phase for 30

15



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 pre-warmed 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 PVDF 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% SDS-PAGE

and the gel was washed 3x 20 min in 100 mM sodium phosphate buffer (pH 6) containing 1%

Triton X-100, and 3x 20 min in 100 mM sodium phosphate buffer (pH 6). The gel was then

incubated in 100 mM sodium phosphate buffer (pH 6) containing 20 mM 3,4-dihydroxy-L-

phenylalanine (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 µg 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, semi-thin 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-β-D-glucuronide 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 1x PBS and incubated in a 1:10000 dilution of a

polyclonal goat anti-rabbit-serum (alkaline phosphatase conjugate), followed by detection using 16



NBT/BCIP (Roche).

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 CTAB method (Doyle

and Doyle, 1990). For the inverse polymerase chain reaction (IPCR), 1 µg 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 0.3-0.5 µg ml-1 in the presence of 3 U ml-1 T4 DNA ligase (Promega) overnight

at 15°C. The ligation mixture was extracted with phenol:chloroform, precipitated with ethanol,

and resuspended in water to a concentration of 20 ng µl-1. In order to amplify ToPPO-1

fragments, we designed two degenerate primers based on the peptide amino acid sequences.

These were named LZ50 (5′-CAA MCA MCC TMC GTG TCC GGC C-3′) and LZ45 (5′-TGG

TGI AIG GRG GIC AAC AAT C-3′). The IPCR reactions were prepared using 200 ng of

recircularized genomic DNA in a final volume of 50 µl containing 50 mM MgCl2, 0.25 mM of

each dNTP, 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, 50-60°C) and

extension (3 min, 72°C), with a final extension step at 72°C for 10 min. IPCR 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′-GCA AAG GGT ATG

AGT TCA TCG G-3′) and LZ62 (5′-AAG ATG TTT TTC CGG CGA GTC T-3′), LZ55 (5′-CAT

CAT GCG AAT GTC GAT AGA ATG-3′) and LZ56 (5′-CCA CCT GCT GGA ATG TCG GC-

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′-AAA CTC

GAG ATA TGG CAT CCC TTG CAC CAT C-3′) and LZ73 (5′-CAA TCC TCA TAC TCG

ATT TTG ATC-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′-CAG GTG AAC AAT ATT CAT CAG TGG AGC CAC-3′ (5′ RACE)

and 5′-AAA GAT GTT TTT CCG GCG AGT CTT GA-3′ (3′ RACE) according to the

manufacturer’s recommendations. The clone TkPPO-1 was obtained by RT-PCR on total RNA 17



from T. kok-saghyz using the primers 5′-AAA ACT CGA GAT ATG GCA TCC CTT GCA CCA

TCT-3′ (Tkfw) and 5′-AAA ATC TAG ATC AAT CCT CAT ACT CGA TTT TGA TC-3′

(Tkbw).

A 1525-bp promoter fragment was isolated using a gene-specific primer (5′-CAG GTG AAC

AAT ATT CAT CAG TGG AGC CAC-3′) and the Universal GenomeWalker™ Kit (Clontech).

This fragment was named pToPPO-1 (Supplementary Fig. 1B) 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′-AAA CCA TGG CGA TCG GAT CTG CAA AGG

CCA G-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 (Schulze Gronover,

unpublished). For the RNAi construct, a 141-bp PCR fragment was amplified using the RNAi-

dicer optimized primers RNAi_for (5′-TTT GGT ACC ATC CAT TGG CCT TTG CAG-3′) and

RNAi_rev (5′-TTT GCG GCC GCG TGG TGG AAG TTT GAA GTC-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 spp. and dandelion protoplast

preparation and transfection

A. tumefaciens strain LBA4404, carrying the binary constructs, was cultured in 100 ml induction

broth (5 g l-1 sucrose, 5 g l-1 peptone, 5 g l-1 casein hydrolysate, 1 g l-1 yeast extract, 10 mM MES,

2 mM MgSO4, pH 7) containing the appropriate antibiotics (50 mg l-1 kanamycin, 100 mg l-1

rifampicin and 300 mg l-1 streptomycin). The bacteria were cultured at 28°C to the end of the log-

phase, then centrifuged, and the pellet was resuspended in co-culture medium (4.4 g l-1

Murashige-Skoog salt solution including vitamins, 10 mM MES, 18 g l-1 glucose, pH 5.6),

18



supplemented with 200 µM acetosyringone. Leaf discs (~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 l-1 Murashige-Skoog salt solution, 10 g l-1 glucose, 8 g l-1 agar,

pH 5.8) and inoculated in the co-culture medium with A. tumefaciens for 30 min. The leaf discs

were then placed on wet filter paper for 14–20 h at 26°C. To induce regeneration, the leaf discs

were placed on regeneration medium (4.4 g l-1 Murashige-Skoog salt solution including vitamins,

20 g l-1 glucose, 8.5 g l-1 agar, 250 mg l-1 Claforan, pH 5.8) supplemented with 1 mg l-1 6-

benzyladenine and 0.2 mg l-1 naphthalenacetic acid for callus and shoot induction. The elongation

of shoots was maintained by the addition of 2 mg l-1 zeatin, 20 µg l-1 naphthalenacetic acid and 20

µg l-1 gibberellic acid GA3. Rooting was induced on regeneration medium without hormones,

whereas all regeneration media contained 3 mg l-1 phosphinothricin for selection. All chemicals

and reagents were purchased from Duchefa (Haarlem). 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 x 105 protoplasts and 10 µg 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. kok-saghyz 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 µl, Hirschmann Laborgeräte). The coagulation

rate of the latex was determined by measuring the capillary suction distance.

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 µl latex was dissolved in 100 µl sodium phosphate buffer (pH 6) and centrifuged at 13,500 x

g, 20 min, 4°C. The upper rubber fraction was coagulated by adding 50 µl glacial acetic acid. The

coagulated rubber was dried and weighed. The molecular size distribution of rubber was 19



determined by size exclusion chromatography (SEC) using an Agilent Series 1100 with a

column system comprising three PSS SDV 5µ columns (100-100000 Å) (Polymer Standard

Service GmbH, Mainz, Germany). Samples were prepared by centrifuging 100 µl latex in sodium

phosphate buffer (pH 6) at 13,500 x g, 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 min-1.

The SEC system was calibrated with standard polyisoprenes in the range 1090-1040000 Da

(Polymer Standard Service GmbH).

ACKNOWLEDGEMENTS

The authors would like to thank Dr. Kai Müller and Dr. Gerhard Schwall for helpful discussions. The

technical assistance of Eva Aguado, Christiane Fischer, Raphael Soeur and Julia Boike is gratefully

acknowledged.

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TABLESTable 1: 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 TkLP 2. Each value represents the

mean ± SD (standard deviation) 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)). Rc = rubber content, mw = molecular weight

Plant Amount of latex harvested after wounding [mg]

Rubber quantity Rc [mg/ml latex]

Rubber quality mW [da 106]

Tkwt 0.5 ± 0.4 139.93 ± 16.90 4.5

TkLP1 1.9 ± 0.3 155.13 ± 19.14 5.7

TkLP2 2.3 ± 0.1 166.17 ± 17.08 4.0

FIGURE LEGENDS

25



Figure 1. Analysis of latex browning in Taraxacum spp. Photographs of expelling latex from T.

officinale petioles immediately (A) and 30 min (B) after wounding. First browning of latex was

observed directly after abscission of petioles (indicated by a red arrow). C, Determination of PPO

activity in the aqueous latex phase of T. officinale and T. kok-saghyz in the absence (–) and

presence (+) of tropolone using L-DOPA as substrate. Enzymatic activity is presented in µkat/mg

protein. PPO activity was also analyzed in L-DOPA-treated cross-sections of T. officinale petioles

pre-incubated with (E) or without (D) tropolone. PPO activity appears to be laticifer-specific

(black arrow). Xy = Xylem, Ph = Phloem, La = Laticifer, Scale bars = 100 µm.

Figure 2. Molecular characterization of the major latex proteins (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/MLPePPO) and five (T.k.; MLPa-e/ePPO) dominant

bands. N-terminal sequences for MLPa, MLPb and MLPePPO are shown in bold letters. Molecular

weights are shown in kDa. 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 near full length product (MLPePPO) lacking the transit peptide (tp,

green), together with larger amounts of the tyrosinase domain (MLPb, brown) and C-terminal

domain (MLPa, white). The first three amino acids from the individual cleavage products are

indicated.

Figure 3. Functional analysis of the signal peptide in dandelion protoplasts. Confocal

fluorescence pseudocolor images of T. officinale (A-D) and T. kok-saghyz (I-L) protoplasts

expressing the ToPPO-1 signal peptide/DsRed translational fusion protein under the control of the

double CaMV 35S promoter identifies DsRed fluorescence in chloroplasts 1 days post

transfection. As controls T. officinale (E-H) and T. kok-saghyz (M-P) protoplasts were transfected

with the same DsRed construct lacking the ToPPO-1 signal peptide. DsRed fluorescence is shown

in red and autofluorescence of chloroplasts is presented in a green pseudocolor. Pictures A, E, I,

M were obtained by transmission light, pictures B, F, J, N represent DsRed fluorescence,

autofluorescence of chloroplasts is shown in pictures C, G, K, O, pictures D, H, L, P are overlays

of the corresponding individual fluorescence pictures (DsRed and chloroplast autofluorescence).

26



Overlay fluorescence is shown in orange. Chl = Chloroplast. Scale bars = 20 µm.

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 genes 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 locate adjacent to the vascular bundle (black

arrows). GUS (C) or Sudan III (D) staining of articulated anastomosing laticifers. E, Detection of

GUS activity in ToPPO-prom1/1 latex by staining with X-gluc. F, Immunological detection of the

GUS enzyme in latex of ToPPO-prom1/1. Xy = Xylem, Ph = Phloem, La = Laticifer. Scale bars =

100 µm.

Figure 5. Analysis of PPO abundance and activity in wild-type and RNAi-knockdown T.

officinale (To) and T. kok-saghyz (Tk) plants. A and B, SDS-PAGE showing major latex proteins

in wild-type (wt) and transgenic plants expressing a 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. Asterisk shows 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 (wt) and transgenic RNAi-knockdown lines. The enzymatic activity is

presented as µkat/mg protein. The values presented are means ± SD (standard deviation) 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)).

Figure 6. Determination of latex fluidity in the T. officinale and T. kok-saghyz RNAi-knockdown

plants. Latex from wild-type (wt) and transgenic RNAi plants was harvested and analyzed as

described in the legend to Fig. 5. The values presented are means ± SD (standard deviation) of at

least six biological samples in each case. Significant differences were found between transgenic 27



and wild-type plants (calculated using Student’s t test (P, 0.01)).

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 µg of total protein from the latex aqueous phase were spotted onto nitrocellulose

membrane and placed on 1% agarose gels containing 20 mM L-DOPA). B, bottom, enzymatic

activity is presented in µkat/mg 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 plants was harvested

after wounding by drawing into 10 µl microglass capillaries. Inset figure (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 capillary suction distance (mm csd). All values are presented as means ±

standard deviation of at least six biological samples in each case. No significant differences

existed in enzyme activity (B) and capillary suction distance (D) between T. officinale and T. kok-

saghyz, as calculated using Student’s t test (P, 0.01).

28



+- +-

C

A B

D E

Ph

Xy

La

Xy

Ph

La

Wahler_et_al_Fig_1

Figure 1. Analysis of latex browning in Taraxacum spp. Photographs of expelling latex from T. officinale petioles immediately (A) and 30 min (B) after wounding. First browning of latex was observed directly after abscission of petioles (indicated by a red arrow). C, Determination of PPO activity in the aqueous latex phase of T. officinale and T. kok-saghyz in the absence (–) and presence (+) of tropolone using l-DOPA as substrate. Enzymatic activity is presented in µkat/mg protein. PPO activity was also analyzed in l-DOPA-treated cross-sections of T. officinale petioles pre-incubated with (E) or without (D) tropolone. PPO activity appears to be laticifer-specific (black arrow). Xy = Xylem, Ph = Phloem, La = Laticifer, Scale bars = 100 µm.



Wahler_et_al_Fig_2

[kDa] T.o. T.k.

A

MLPe/MLPePPO

MLPdMLPc

MLPb

MLPa

DPIMAPDLTQ

DPIMAPDLTQ

DELIPFAKDV

Btyrosinase C-termtp

DPI…

DPI…

DEL…

100 -

50 -

37 -

20 -

pre-

PPO

Figure 2. Molecular characterization of the major latex proteins (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/MLPePPO) and five (T.k.; MLPa-e/ePPO) dominant bands. N-terminal sequences for MLPa, MLPb and MLPePPO are shown in bold letters. Molecular weights are shown in kDa. 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 near full length product (MLPePPO) lacking the transit peptide (tp, green), together with larger amounts of the tyrosinase domain (MLPb, brown) and C-terminal domain (MLPa, white). The first three amino acids from the individual cleavage products are indicated.



Wahler_et_al_Fig_3

Figure 3. Functional analysis of the signal peptide in dandelion protoplasts. Confocal fluorescence pseudocolor images of T. officinale (A-D) and T. kok-saghyz (I-L) protoplasts expressing the ToPPO-1 signal peptide/DsRed translational fusion protein under the control of the double CaMV 35S promoter identifies DsRed fluorescence in chloroplasts 1 days post transfection. As controls T. officinale (E-H) and T. kok-saghyz (M-P) protoplasts were transfected with the same DsRed construct lacking the ToPPO-1 signal peptide. DsRed fluorescence is shown in red and autofluorescence of chloroplasts is presented in a green pseudocolor. Pictures A, E, I, M were obtained by transmission light, pictures B, F, J, N represent DsRed fluorescence, autofluorescence of chloroplasts is shown in pictures C, G, K, O, pictures D, H, L, P are overlays of the corresponding individual fluorescence pictures (DsRed and chloroplast autofluorescence). Overlay fluorescence is shown in orange. Chl = Chloroplast. Scale bars = 20 µm.



A

PPO-1

GAPDH

T.o. T.k.

1 2 3 4

B

Wahler_et_al_Fig_4

E F WT

D

C

WT ToPPO-prom1/1

ToPPO-prom1/1

La

Ph

Xy

La

100

75

50

37

[kDa]



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. The housekeeping GAPDH genes from T. officinale (T.o.) and T. kok-saghyz (T.k.) served 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 locate adjacent to the vascular bundle (black arrows). GUS (C) or Sudan III (D) staining of articulated anastomosing laticifers. E, Detection of GUS activity in ToPPO-prom1/1 latex by staining with X-gluc. F, Immunological detection of the GUS enzyme in latex of ToPPO-prom1/1. Xy = Xylem, Ph = Phloem, La = Laticifer. Scale bars = 100 µm.



Wahler_et_al_Fig_5

Figure 5. Analysis of PPO abundance and activity in wild-type and RNAi-knockdown T. officinale (To) and T. kok-saghyz (Tk) plants. A and B, SDS-PAGE showing major latex proteins in wild-type (wt) and transgenic plants expressing a 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. Asterisk shows 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 (wt) and transgenic RNAi-knockdown lines. The enzymatic activity is presented as µkat/mg protein. The values presented are means ± SD (standard deviation) 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)).



Wahler_et_al_Fig_6

Figure 6. Determination of latex fluidity in the T. officinale and T. kok-saghyz RNAi-knockdown plants. Latex from wild-type (wt) and transgenic RNAi plants was harvested and analyzed as described in the legend to Fig. 5. The values presented are means ±SD (standard deviation) 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)).



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