Planta (Berl.) 127, 163---170 (1975) 9 by Springer-Verlag 1975

Filament Formation in Vitro of a Sieve Tube Protein from Cucurbita maxima and Cucurbita pepo * Hans Kleinig Institut fiir Biologic H, Lehrstuhl ftir Zellbiologie, Universit/it Freiburg, Sch/~nzlestrai~e 9--15, D-7800 Freiburg, Federal Republic of Germany Jochen ThSnes, Inge DSrr, and l~ainer Kollmann Botanisches Institut, Lehrstuhl II, Universit~,t Kiel, Dfisternbrooker Weg 17, D-2300 Kiel, Federal Republic of Germany Received 30 July; accepted 8 August 1975 Summary. Phloem proteins of the sieve tube exudate from Cucurbita maxima Duch. and Cueurbita pepo L. were investigated as to their filament forming ability in vitro. From the two main proteins (116000 dalton, 30000 dalton) only the 116000 dalton protein was found to form reversibly distinct filaments of 6-7 nm diameter upon removal of SH-protecting agents from the buffer, whereas the 30000 dalton protein was precipitated as amorphous material under these conditions. The protein filaments were similar to the filaments ocurring within the sieve tube cells in vivo.

Introduction The mature sieve elements of angiosperm plants is characterized by the occurrence of discrete aggregations of protein(s) into several morphological forms, especially filaments (for review see Cronshaw, 1975; Kollmann, 1975). The term "P-protein" was introduced to describe these peculiar, proteinaceous structures (Cronshaw and Esau, 1967). Biochemical work on the sieve tube proteins was carried out specifically on the genus Cucurbita because of the easy manner in which the sieve tube exudate containing the proteins may be obtained from this genus (for review see Kleinig, 1975) The exudate of Cucurbita was found to contain several proteins (Kleinig et al., 1971 a; Eschrich et al., 1971; Walker and Thaine, 1971) with two major basic components each comprising about 40 % of the total protein and with very similar amino acid compositions, but with apparent molecular weights of 116000 dalton and 30000 dalton (occurring in vivo as dimer of 60000 dalton), respectively (Beyenbach et al., 1974). I t has previously been shown that the exudate from Cucurbita m a x i m a also contains filamentous proteins found in fixed sectioned plant material (Kollmann et al., 1970; Cronshaw et al., 1973), and that these filaments can be reversibly reaggregated after solubilization of the exudate proteins with thiol regents (Kleinig et al., 1971 a). The filamentous structures are predominant within the exudate and it is obvious that only a major component could be responsible for these structures. I t was not known until now, however, whether the filaments were built up of the 116000 dalton protein, the 30000 dalton protein, or of both both components. * Abbreviations: SDS = sodium dodecyl sulfate; TCA = trichloroacetic acid.

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F u r t h e r m o r e , it was u n c e r t a i n as to whether this " G - F " t r a n s i t i o n was connected with the gelling p r o p e r t y of the exudate. So far, gelling is a very characteristic feature of the Cucurbita exudate u n d e r n o n - r e d u c i n g conditions; a n d this phenom e n o n has been a t t r i b u t e d to the 116 000 d a l t o n c o m p o n e n t in Cucurbita m a x i m a (Beycnbach et al., 1974) a n d to a 136000 dalton, m i n o r p r o t e i n in Cucurbita pepo (Walker, 1972). These questions are the subject of the present investigation.

Material and Methods Phloem exudate from C. maxima and C. pepo was collected as described by Kollmann et al. (1970) and Beyenbach et al. (1974). Isolation and purification of the 116000 dalton and 30000 dalton fractions by ammonium sulfate precipitation and by DEAE-cellulose and Sephadex G-100 chromatography was performed essentially according to the methods previously reported (Beyenbach et al., 1974). SDS-polyacrylamide gel electrophoresis was carried out in tubes (Beyenbach et al., 1974) and for a better comparability of fractions on 0.8 mm thick vertical gel slabs. The discontinuous SDS buffer system developed by Laemmli (1970) was used with slight modification. The stacking gel contained 4.7% acrylamide, pH 7.3, the separating gel 15% acrylamide, pH 8.8. The final SDS concentration in both gels and in the electrode buffer was 0.1%. The sample buffer contained the final concentration of: 0.0625 ~r Tris-tIC1 (pit 7.3), 1.6% 2-mercaptoethanol, 2% SDS, 10% glycerol. Before electrophoresis the sample was placed in a boiling water bath for 9 rain. Electrophoresis was carried out at 20 mA for 3 to 4 h. The gels were stained with Coomassie blue 1%250 as usual. The capability of filament formation in vitro of the purified 116000 dalton and 30000 dalton fraction was tested in the following ways. The protein concentration of the eluates from the Sephadex column was brought to 2.3 or 4.0 mg/ml by use of an Amicon ultrafiltration cell (Model 52). For removal of mercaptoethanol, 2-5 ml portions of the fractions were then i) dialyzed against tap water and 0.1 Iq KC1, respectively, for 48 h or ii) oxidized by blowing air on the surface of the sample for several min to 1 h. The samples were then negatively stained, or in the case of gel formation, fixed and thin-sectioned as described below. Protein concentration was determined by the method of Lowry et al. (1951) after precipitation of proteins with 10% TCA. For electronmicroscopy samples were negatively stained with 1% sodium silicotangstate, pH 7.2. Samples of the 116000 fraction which showed a gelling reaction and samples of the 30000 fraction which showed a precipitation during dialysis were used for ultrathin sectioning. These samples were fixed in 2.5 % glutaraldehyde for 60 rain in the cold, washed with water for 4 h, postfixed in 1% OsO4 for 1 h and washed with 5 % uranylacetate for 3 h. The dehydration with ethanol was followed by an embedding in Epon-Araldite (Mollenhauer, 1964).

Results i n Fig. 1 scans of SDS-polyacrylamide gel electrophoresis of total e x u d a t e proteins from C. m a x i m a (a), of the purified 116000 d a l t o n fraction (b), a n d of the purified 30000 d a l t o n fraction (c) are shown. The samples which are represented b y scans (b) a n d (c) were used for the aggregation a n d gelling experiments. The protein p a t t e r n of C. pepo exudate was n e a r l y identical with t h a t of C. m a x i m a with, however, a higher proportion of low molecular weight proteins. The respective 116000 d a l t o n a n d 30000 d a l t o n fractions from C. pepo were also used for the following experiments. The p r o t e i n c o n c e n t r a t i o n was b r o u g h t to 2.3 m g / m l in all samples with the aid of a n Amieon u l t r a f i l t r a t i o n cell. W h e n these samples were dialyzed against 0.1 KC1 or t a p water for 48 h i n order to remove mereaptoethanol, the 116000 dal-

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/ O

J J (c) Fig. 1a--c. SDS-polyacrylamide gel electrophoresis of phloem proteins from Cucurbita maxima. (a) Total proteins, (b) purified ll6000 dalton fraction, (e) purified 30000 dalton fraction. The gel was stained with Coomassie blue and scanned at 540 nm. Arrow, direction of electrophoresis

ton samples became slightly turbid and in the 30 000 dalton samples some protein was precipitated. Gelling, however, did not occur in either sample at this protein concentration. Negative staining revealed that in the 116000 dalton sample a great quantity of fibrillar protein strands had been formed (Fig. 2). A higher magnification of the same fraction (Fig. 3) shows a single fibril consisting of several finer filaments (arrow) with a diameter of 6-7 nm. The single filaments are rather difficult to recognize, as they are either covered by staining material or they are densely aggregated and often twisted within the fibrils. The morphologieM difference between the 116 000 and the 30 000 dalton sample was striking. In negative staining the latter contained amorphous material only; filaments have not been detected so far (Fig. 4). At higher protein concentrations of r to 10 mg/ml gelling did occur during dialysis in the 116000 dalton samples, whereas the 30000 dalton protein was again precipitated. This concentration-dependence of the gelling reaction was also observed by Walker (1972) with the gelling factor of C. ioepo exudate. The gelling reaction of the t l 6 000 dalton protein was much more easily induced by blowing air on the surface of the samples. With this method gelling was also achieved at low protein concentration of 2.3 mg/ml after 10 to 30 min. I n thin sections of this gel after glutarMdehyde/osminm tetroxide fixation filaments (Figs. 5 and 6), corresponding to the negatively-stained preparations could again

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be identified (Figs. 2 and 3). In this sectioned material too the filaments often appeared in an aggregated form as bundles. These bundles were often surrounded by a "fluffy" material, which was due to the random orientation of the filaments and bundles whose arrangement was only rarely seen exactly parallel to the section plane. The diameter of a single filament in sectioned material was about 7-10 nm (maxim. 13 nm), which corresponds to the negatively stained protein filaments. Again in contrast to the 116 000 dalton protein, thin sections of the precipitated 30000 dalton protein did not show any discrete structures, only masses of amorphous protein were observed (Fig. 7). Both, gel and filament formation of the 116 000 dalton protein as well as the precipitation of the 30 000 dalton protein were reversible upon renewed addition of 2-mercaptoethanol. The solubilization, however, was often not quantitative. Some of the protein remained insoluble, certainly being due to irreversible denaturation of the proteins.

Discussion As has been shown above, the purified 116000 dalton component of the sieve tube exudate from C. m a x i m a and C. pepo can be reversibly induced to form filamentous structures and, upon higher oxidation, gels which likewise contain these filaments. I t seems to be obvious from the experimental results t h a t filament formation and gelling is due to a conformational change of the proteins by the formation of disulfide bonds. I t can not be decided at the moment as to whether the gelhng reaction is simply the expression of a higher concentration of filaments or whether another mechanism is involved which leads to a construction of a three-dimensional network (principally not distinguishable in fixed sectioned material) which itself is dependent upon a higher degree of disulfide bond formation and/or a higher protein concentration. The other major component of the exudate, the 30000 dalton protein (about 40 % of total protein in the sieve tubes of C. m a x i m a ) does not form any similar structures under the experimental conditions described but tends to precipitate upon removal of 2-mercaptoethanol as amorphous material. The reaggregated filaments are morphologically very similar to the filaments seen in fixed and thinsectioned sieve elements and there seems to be little doubt t h a t the structures are, in principle, the same. On the other hand, it seems to be very difficult to transfer any in vitro properties or mechanisms to the in vivo situation and vice versa due to the special environmental conditions within an in vivo sieve element (high hydrostatic pressure, high p i t etc.). This means e.g.

Fig. 2. 116000 dalton sieve tube protein of Cucurbita maxima showing numerous fibrillar strands. Magn. 10000:1 Fig. 3. The enlargement of single fibre from :Fig. 2 demonstrating a separation into small, identical, protein filaments. Diameter 6-7 nm. Magn. 55000:1 Fig. 4. 30000 dalton sieve tube protein of Cucurbita maxima (negatively stained) showing an amorphous form. Magn. 10000:1

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Fig. 5. 116000 dalton sieve tube protein of Cucurbita pepo. Filaments are frequently arranged in loose bundles. Diameter of the filaments 7 10 nm. Magn. 55000:1 Fig. 6. The same protein as in Fig. 5 seen at lower magnification. Magn. 30000:1 Fig. 7. Precipitation of 30000 dalton sieve tube protein of Cucurbita pepo consisting of clumps of amorphous material. Magn. 30000:1

possible aggregation-disaggregation processes of proteins, or equilibrium relationships and their possible functions in translocation. The latter possibility seems to be supported b y the present findings, since not the whole exudate proteins but only a distinct component is able to form these morphologically distinct filaments (for another view, however, see Weber et al., 1974). The 30000 dalton protein is obviously not a structural component which means t h a t it is not observable, using microscopic techniques, as discrete aggregations within the sieve elements. I t may, however, be present in a soluble form. This raises the terminological problem discussed earlier (Beyenbach et al., 1974) t h a t at least in the genus Cucurbita only about 40 % of the total sieve tube protein, i.e. the 116000 dalton component, satisfies the term " P - p r o t e i n " , since "P-proteins" are defined as structural components (Cronshaw and Esau, 1967). The 30000 dalton protein as well as the numerous minor components, which are mainly acidic proteins, m a y be termed "soluble sieve tube proteins". One final point we like to discuss briefly concerns the categorization of the filament-forming protein as a structural protein in a broader sense. F r o m the present knowledge it is obvious t h a t this protein is not very similar in its chemical propertiest to the actins sens. fat. and tubulins. We would also like to stress the view of Hepler and Palevitz (1974) t h a t "P-protein" should not be confused with the relatively well characterized microfilaments and microtubnles. On the other hand, the filaments-forming property and perhaps the precipitability with vinblastine sulfate (Kleinig et al. , 1971b) m a y warrant the inclusion of this protein, as a new and somewhat exotic type, into the so-called structural protein category of biological systems. Definite statements on this subject, however, m u s t await further research. The investigations were supported by Deutsche Forschungsgemeinschaft. We wish to thank Mr. J. A. Thompson for reading and correcting the manuscript. References Beyenb~ch, J., Weber, C., Kleinig, H. : Sieve-tube proteins from Cucurbita maxima. Planta (Berl.) 119, 113-124 (1974) Cronshaw, J. : P-porteins. In: Phloem transport (S. Aronoff, S.J. Dainty, L. Srivastava, and C.A. Swanson, eds.). New York: Plenum, 1975 (in press) Cronshaw, J., Esau, K. : Tubular and fibrillar components of mature and differentiating sieve elements. J. Cell Biol. 34, 801-815 (1967) Cronshaw, g., Gilder, J., Stone, D. : Fine structural studies of P-proteins in Cucurbita, Cucumis, and Nicotiana. J. UItrastruct. Res. 4&, 192-205 (1973) Eschrich, W., Evert, R.F., Heyser, W.: Proteins of the sieve tube exudate of Cucurbita maxima. Planta (Berl.) 100, 208-221 (1971) Hepler, P. K., PMevitz, B.A.: 1Vficrotubules and microfilaments. Ann. Rev. Plant Physiol. ~5, 309-362 (1974)

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Kleinig, H. : Biochemistry of phloem proteins. In: Phloem transport (S. Aronoff, S. J. Dainty, L. Srivastava, and C.A. Swanson, eds.). New York: Plenum 1975 (in press) Kleinig, It., D6rr, I., Weber, C., Kollmann, R. : Filamentous proteins from plant sieve tubes. Nature (Lond.) l~ew Biol. 229, 152-153 (1971a) Kleinig, H., D5rr, I., Kollmann, R.: Vinblastine-induced precipitation of phloem proteins in vitro. Protoplasma (Wien) 711,293-302 (1971 b) Kollmann, R., D5rr, I., Kleinig, H. : Portein filaments--structural components of the phloem exudate. L Observations with Cucurbita and Nicotiana. Planta (Berl.) 95, 86 94 (1970) Kollmann, R. : Sieve element structure in relation to function. In: Phloem transport (S. Aronoff, S.J. Dainty, L. Srivastava, and C.A. Swanson, eds.). New York: Plenum 1975 (in press) Laemmli, N. : Cleavage of structural proteins during assembly of the head of bacteriophage T~. Nature (Lond.) 227, 680 684 (1970) Lowry, D. H., Rosebrough, 1~. J., Farr, N. L., l~andall, R. L. : Protein measurement with the Folin phenol reagent. J. biol. Chem. 1911,265-275 (1951) Mollenhauer, It. It.: Plastic embedding mictures for use in electron microscopy. Stain Technol. 119, 111-114 (1964) Walker, T. S.: The purification and some properties of a protein causing gelling in phloem sieve tube exudate from Cucurbita pepo. Biochim. biophys. Acta (Amst.) 257, 433444 (1972) Walker, T.S., Thaine, R. : Proteins and fine structural components in exudate from sieve tubes in Cucurbita pepo stems. Ann. Bot. 35, 773-790 (1971) Weber, C., Franke, W. W., Kartenbeek, J. : Structure and biochemistry of phloem-proteins isolated from Cucurbita maxima. Exp. Cell Rcs. 87, 79-106 (1974)

Filament formation in vitro of a sieve tube protein from Cucurbita maxima and Cucurbita pepo.

Phloem proteins of the sieve tube exudate from Cucurbita maxima Duch. and Cucurbita pepo L. were investigated as to their filament forming ability in ...
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