Abstract
One- and two-dimensional electrophoresis of Nicotiana tabacum pollen and pollen tube proteins confirmed that a new protein is preferentially synthesized during pollen germination and tube growth and becomes the most abundant protein in pollen tubes. Analysis of proteins extracted with sodium dodecyl sulfate (SDS) from different pollen tube fractions showed that it is the most abundant non-covalently bound wall protein, characterized by molecular mass of 69 kDa, pI between 7.9 and 8.2, and glycosylation with glucose and/or mannose. Amino acid analysis revealed relative abundance of serine, glutamic acid and glycine, but did not show the presence of hydroxyproline. According to all these characteristics, it cannot be classified as an extensin-like protein. Another prominent wall-bound glycoprotein has a molecular mass of 66 kDa and the same pI as the 69 kDa glycoprotein. These two glycoproteins are similar also in ConA binding, rate of synthesis, and rapid incorporation into pollen tube walls. Their synthesis is strongly reduced by tunicamycin and this inhibition results in the occurrence of new polypeptides in the range of 57–61 kDa. Tunicamycin also inhibited pollen tube growth. At 10 ng ml-1 and 50 ng ml-1 the inhibitor reduced pollen tube mass after 24 h of culture by 30% and 85%, respectively. This indicates that tobacco pollen presents a system highly sensitive to tunicamycin and that cotranslational N-linked glycosylation on the rough endoplasmic reticulum is required for 66 and 69 kDa glycoprotein formation and for pollen tube growth. Although other proteins appear during pollen germination and tube growth, the new proteins occur at low levels and seem to originate through modifications of preexisting polypeptides. In contrast to 69 and 66 kDa proteins, most proteins detected by [14C]amino acid incorporation and fluorography of gels were not revealed by Coomassie blue staining.
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References
Anderson L, Anderson NG (1977) High resolution two-dimensional electrophoresis of human plasma proteins. Proc Natl Acad Sci USA 74:5421–5425
Browning DD, O'Day DH (1991) Concanavalin A and wheat germ agglutinin binding glycoproteins associated with cell fusion and zygote differentiation in Dictyostelium discoideum: effects of calcium ions and tunicamycin on glycoprotein profiles. Biochem Cell Biol 69:282–290
Calzoni GL, Speranza A (1989) Glycoproteins in germinating apple pollen. Plant Physiol Biochem 27:219–225
Čapková V, Hrabětová E, Tupý J, Říhová L (1983) Amino acid uptake and protein synthesis in cultured tobacco pollen. Biochem Physiol Pflanzen 178:511–520
Čapková V, Hrabětová E, Tupý J (1987) Protein changes in tobacco pollen culture; a newly synthesized protein related to pollen tube growth. J Plant Physiol 130:307–314
Čapková V, Hrabětová E, Tupý J (1988) Protein synthesis in pollen tubes: preferential formation of new species independent of transcription. Sex Plant Reprod 1:150–155
Cooper JB, Chen JA, Holst GJ van, Varner JE (1987) Hydroxyproline-rich glycoproteins of plant cell walls. Trends Biol Sci 12:24–27
De Klerk GJ (1985) Glycosylation and deglycosylation of storage proteins in developing Agrostemma githago L. seeds. J Plant Physiol 119:109–122
Driouich A, Gonnet P, Makkie M, Laine AC, Faye L (1989) The role of high-mannose and complex asparagine-linked glycans in the secretion and stability of glycoproteins. Planta 180:96–104
Dubois M, Gilles KA, Hamilton JK, Rebers PA, Smith F (1956) Colorimetric method of sugars and related substances. Anal Chem 28:350–356
Dunbar BF (1987) Two dimensional electrophoresis and immunological techniques. Plenum Press, New York
Faye L, Mouatassim B, Ghorbel A (1986) Cell wall and cytoplasmic isozymes of radish β-fructosidase have different N-linked oligosaccharides. Plant Physiol 80:27–33
Heslop-Harrison J (1987) Pollen germination and pollen tube growth. In: Giles KL, Prakash J (eds) Pollen: cytology and development (Int Rev Cytol 107). Academic Press, Orlando, pp 1–78
Hori H, Elbein AD (1981) Tunicamycin inhibits protein glycosylation in suspension cultured soybean cells. Plant Physiol 67:882–886
LaFayette PR, Travis RL (1989) Effect of inhibition of glycosylation on the appearance of a 60 kD membrane glycopolypeptide in endomembrane fractions of soybean root. Plant Physiol 89:299–304
Li Yi-qin, Linskens HF (1983) Wall-bound proteins of pollen tubes after selfand cross-pollination in Lilium longiflorum. Theor Appl Genet 67:11–16
Li Yi-qin, Croes AF, Linskens HF (1983) Cell-wall proteins in pollen and roots of Lilium longiflorum: extraction and partial characterization. Planta 158:422–427
Li Yi-qin, Tsao TH, Linskens HF (1986) Dependence of Lilium pollen germination and tube growth on protein synthesis and glycosylation after inhibitor treatments. Proc Kon Nederl Akad Wet C 89:61–73
Ochiai SK, Katagira YU, Ochiai H (1985) Analysis of N-linked oligosaccharide chains of glycoproteins on nitrocellulose sheets using lectin-peroxidase reagents. Anal Biochem 147:222–229
Petrů E, Hrabětová E, Tupý J (1964) The technique of obtaining germinating pollen without microbial contamination. Biol Plant 6:68–69
Rae AL, Harris PJ, Bacic A, Clarke AE (1985) Composition of the cell walls of Nicotiana alata Link et Otto pollen tubes. Planta 166:128–133
Rodrigo PA (1984) The structure of the glycopeptide of human gamma G-myeloma. J Biol Chem 246:3259–3268
Rodriguez-Rosales MP, Ferrol N, Roldán M, Belver A, Donaire JP (1990) Cell wall acyl-lipids, proteins and polysaccharides in mature and germinated olive pollen. Rev Española Fisiol 46:371–377
Smith E, Gooley AA, Hudson GC, Williams KL (1989) Glycoproteins that exhibit size polymorphism in Dictyostelium discoideum. Genetics 122:59–64
Sreekrishna K, Jones CE, Guetzow KA, Prasad MR, Joshi VC (1980) A modified method for amino acid analysis with ninhydrin of Coomassie-stained proteins from polyacrylamide gel. Anal Biochem 103:55–57
Steer MW, Steer JM (1989) Tansley Review No. 16. Pollen tube tip growth. New Phytol 111:323–358
Tupý J, Říhová L (1984) Changes and growth effect of pH in pollen tube culture. J Plant Physiol 115:1–10
Tupý J, Hrabětová E, Balatková V (1977) A simple rapid method of determining pollen tube growth in mass culture. Plant Sci Lett 9:285–290
Tupý J, Süss J, Říhová L (1986) RNA synthesis and ribosome status in pollen tube growth of Nicotiana tabacum L.; Effects of external pH. J Plant Physiol 123:467–476
Tupý J, Říhová L, Čapková V, Žárský V (1992) Differentiation and maturation of tobacco pollen in situ and in suspension culture. In: Ottaviano E, Mulcahy DL, Sari Gorla M, Bergamini Mulcahy G (eds) Angiosperm pollen and ovules. Springer, Berlin Heidelberg New York, pp 309–314
Varner JE, Lin LS (1989) Plant cell wall architecture. Cell 56:231–239
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Čapková, V., Tupý, J. & Zbrožek, J. Protein synthesis in tobacco pollen tubes: preferential synthesis of cell-wall 69-kDa and 66-kDa glycoproteins. Sexual Plant Reprod 7, 57–66 (1994). https://doi.org/10.1007/BF00241888
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DOI: https://doi.org/10.1007/BF00241888