Planta (Berl.) 106, 369 373 (1972) 9 by Springer-Verlag 1972
Isoelectric Focusing of Sieve-Tube Protein P. A. J. Y a p a and D. C. Spanner Bedford College, London t~eceived March 3 / April 28, 1972 Recently a considerable a m o u n t of investigation has been carried out into the biophysical and biochemical properties of protein filaments from the sieve tube elements of angiosperms. Kleinig et al. (1971) isolated a filamentous protein from Cucurbita m a x i m a sieve tubes and reported some of its properties which in m a n y respects are similar to those of other structural proteins (such as vinblastine-preeipitated microtubule proteins, aetin, etc.). Walker and Thaine (1971) reported a soluble basic protein from Cucurbita pepo exudate which is involved in the gelling reaction on exposure to air; the gel has no organized fine structure in the electron microscope. I n addition, t h e y found a structural fraction which contains fibrils and particles. Esehrich et al. (1971) reported the occurrence in the sieve tube exudate of Cucurbita m a x i m a of a soluble fraction with diverse enzymatic properties, and a larger insoluble fraction. Since the exudate was shown b y electron microscopy to come from the sieve tubes there is reason to believe t h a t this insoluble fraction is the P-protein. Finally, Weber and Kleinig (1971) determined the molecular weights of the protein from Cucurbita m a x i m a exudate b y means of polyacrylamide gel electrophoresis obtaining five bands from 15000 to 220000. The present report is c o m p l e m e n t a r y to all this recent work. I t deals with protein extracted from isolated whole phloem tissue of Heracleum mantegazzianum b y a fraetionating technique as well as with exudate ; the latter was obtained from the cut surface of the petiolephloem b y applying 1 ~1 microeaps. The material was examined electronmicroscopically, and more interestingly, b y isoeleetric focusing on polyaerylamide gel and in a sucrose density gradient. This technique yields directly the isoeleetrie point of the protein. The fractionation of protein was based on a method described by Timpl et al. (1969). Proteins were extracted from ground phloem tissue with 0.2 M NaC1, 0.5M NaC1, 0.2M citrate buffer pH 3.0, 0.2M borate buffer pH 9.0, 8M urea (pH adjusted to 7.6 with 4M NaOH) and 0.2 M NaOH successively, at 4 ~ C. Extractions, with the exception of those in urea and alkali which were carried out overnight, lasted 30 min. The material precipitated during the dialysis of the urea and alkali fractions against tap water (4:~ C, 1-2 days), was identified as a filamentous protein by electron microscopy.
P . A . J . Yapa and D. C. Spanner:
Proteins in the NaC1, citrate and borate buffer fractions were isolated by precipitation with (NH4)2SO 4 at half saturation. The estimation of protein in each fraction was carried out by the method of Lowry et al. (1951). For electronmicroscopical examination the grids were stained with lead citrate for 8 rain. The filamentous protein fractions were submitted to isoelectric focusing in 5 % acrylamide gel with or without urea as described by Leabaek and Rntter (1968). At the same time phloem exudate collected directly in microcaps was run on the gel. Whole phloem extracts in distilled water or 0.1M phosphate buffer, pH 7.2 were subjected to electrofoeusing similarly. The gels, fixed in 10% trichloroacetie acid were stained either with 0.2% fast green (Leabuck 1971) or 0.05% Coomassie Brilliant Blue (Bours and Doorenmaalen, 1970). In addition, extract was also submitted to isoelectrie focusing on an LKB 8101 column in a sucrose density gradient. The samples were dissolved in a " l i g h t " solution which contained ampholine carrier, pH range 3-10. In order to increase the solubility of the protein and to prevent the precipitation when focused, 8M urea was used throughout the column. The solutions were transferred to the column by a peristaltic pump and the sucrose gradient was formed by a gradient mixer, LKB type 8121. A constant voltage of 350 was applied for 72 hours. After electrofocusing the column was emptied and fractions of 3 ml were collected, absorption spectra being recorded with an LKB Uvicord II absorptiometer at 280 nm.
T h e b e h a v i o u r of t h e p r o t e i n s in g e l - e l e e t r o f o c u s i n g is i n d i c a t e d in Fig. 1. P l a i n p h l o e m e x u d a t e (A) s h o w e d 2 b a n d s n e a r t h e a n o d e . T w o c o m p a r a b l e b a n d s w e r e o b t a i n e d f r o m p h l o e m e x u d a t e (B) on gel
~ ine of application II IA I
JJ ii ii !i iJ I iPiJ I II
Fig. 1 A - - E . Isoelectric focusing patterns of protein from the phloem of Heracleum: A, phloem exudate without urea; B, exudate in 8/r urea (the dotted bands may be artifacts due to the ampholine); C, aqueous extract from whole phloem tissue; D, 8M urea fraction of whole phloem extract, run in urea; E, NaOH fraction of whole phloem extract, run in urea
Isoeleetrie Focusing of Sieve-Tube Protein
Fig. 2. Electron micrographs of "peak" fractions defined (a) in Fig. 3; b) in Fig. 4. Stained with lead citrate, x 120000
containing 8M urea, with other minor bands. Whole phloem tissue extracted in dist,illed water (C) gave two bands, which again interestingly were comparable with bands obtained with phloem exudate. The behaviour of urea (D) and NaOH (E) fractions on gel containing 8M urea are again similar to the exudate and whole tissue extracts so far as the two main bands are concerned. Electron microscopical examination (Fig. 2) showed that both these fractions contained filamentous material, with occasional non-filamentous material in the NaOH fraction. The similarity of the separation patterns obtained with both exudate and extracts suggests that the common components came from ~he sieve tubes. However the identification of the main bands with P-protein cannot be regarded as more than very likely.
P.A.J.Yapa and D.C. Spanner: 109 8
Fig. 3. UV absorption pattern at 280 nm of 8M urea fraction resulting from fractionating whole phloem tissue; run in sucrose density gradient with 8M urea. The peak is at pH 4.9. Compare D, Fig. 1
~ 7 ~
UV absorption (arbitrQryscare)
Fig. 4. UV absorption pattern at 280nm of whole phloem tissue extract run in sucrose density gradient with 8M urea. The peak is at pH 4.95. Compare C, Fig. 1
Q u a n t i t a t i v e d e t e r m i n a t i o n of p r o t e i n s showed t h a t 47 % of t h e t o t a l p r o t e i n s e x t r a c t e d (excluding t h e small filamentous fraction in the c o m b i n e d NaC1 extracts) was composed of p r o t e i n a p p e a r i n g f i l a m e n t o u s in t h e electron microscope. Eleetrofocusing on t h e L K B column r e v e a l e d t h e isoeleetric p o i n t of the filamentous p r o t e i n (Fig. 2a) in t h e urea fraction to be 4.9 a n d t h e single p e a k (Fig. 3) is i n d i c a t i v e of t h e p u r i t y .
Isoelectric Focusing of Sieve-Tube Protein
The isoeleetric focusing in 8M urea of whole phloem tissue extract on the L K B column gave its highest peak (Fig. 4) at p I 4.95 which again represents a filamentous fraction (Fig. 2b) and confirms t h a t this is the major protein component of the phloem tissue. This suggests t h a t it is the P-protein. U n f o r t u n a t e l y we had only t e m p o r a r y use of the L K B sucrose density gradient apparatus, otherwise the work would have been carried further. The results obtained however, b y both methods, suggest t h a t the P-protein has an isoelectric point of slightly below 5. At the alkaline p H of the sieve tube sap it would therefore be negatively charged, fulfilling one of the requirements of the electroosmotic (potassium) theory of sieve tube mechanism. To this modest extent the present results support the theory. References
Boars, J., van Doorenmaalen, J. : Gel isoelectric focusing of lens crystallins. Science Tools 17, 36-38 (1970). Eschrich, W., Evert, R.F., Heyser, W.: Proteins of the sieve tube exudate of Cucurbita m a x i m a Planta (Berl.) 100, 208-221 (1971). Klcinig, H., Dorr, L, Weber, C., Kollmann, R.: Filamentous proteins from plant sieve tubes. Nature (Lond.) New Biol. 229, 152-153 (1971). Leaback, D. H. : Private communication (1971). Leaback, D. H., Ratter, A. C. : Polyacrylamide isoelectric focusing. A new technique for electrophoresis of proteins. Biochem. biophys, l~es. Commun. 32, 447-453 (1968). Lowry, O. H., I~osebrough, N. J., Farr, A. L., Randall, R. J. : Protein measurement with the folin-phenol reagent. J. biol. Chem. 193, 265-275 (1951). Timpl, R., Wolff, T., Weister, M. : Acidic structural proteins of connective tissue. I Solubilization and preliminary chemical characterization. Biochem. biophys. Acta (Amst.) 194, 112-120 (1969). 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., Kleinig, It. : )~Iolecular weights of Cucurbita sieve tube proteins. Planta (Berl.) 99, 179-182 (1971). P. A. J. u D. C. Spanner Botany Dept. Bedford College London, NW1 4NS England