Two hour pretreatment with exo-β-D-(l,3)-glucanase cause (at a concentration of 0.4 unit/ml) significant stimulation of the georeaction of maize apical root segments, horizontally placed for 7 hr. Implications of this enzyme effect on cell wall extension are discusse

Pilet, Paul-Emile

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Plant & Cell Physiol. 16: 1175-1178 (1975)Short communicationRoot georeaction affected by exo-£-D-(l,3)-glucanasePaul-Emile PiletInstitute of Plant Physiology and Biology of the University, 1005 Lausanne, PI. de la Riponne,Switzerland(Received September 8, 1975)Two hour pretreatment with exo-/3-r>-(l,3)-glucanase cause (at a concentrationof 0.4 unit/ml) significant stimulation of the georeaction of maize apical root segments,horizontally placed for 7 hr. Implications of this enzyme effect on cell wall extensionare discussed.The action of the cell wall extension of X-/3G, which occurs in many plants(2, 3, 6, 8), is still discussed. X-/JG may induce elongation of segments from Pisumstem and Avena coleoptile (9, 24, 25). This enzyme, as well as auxin (10, 20), in-creases reversible extensibility of the cell walls (9, 11). But the X-/?G effect is notadditive to the IAA action when given together (10) and it has been postulated thatIAA induces both activation and formation of X-/JG (12). In contrast, there hasbeen a number of reports in which the role of glucanases in growth responses hasbeen questioned (4, 5, 22). Recently, nojirimycin was reported as being a potentinhibitor of IAA-induced growth of excised Avena coleoptile and Pisum epicotylsegments. Since nojirimycin is an inhibitor of X-/JG, this enzyme may act inIAA-induced changes of non-cellulosic wall glucan (14). These observationssupport the hypothesis that IAA-induced wall extension may be mediated by X-/3G(13).On the other hand, the root georeaction has to be related to the changes of rootelongation (/, 75). It seems clear now, at least for Zea roots, that the downwardbending of horizontally placed root segments is due to an asymmetrical extensionof the walls of the cortical cells from the extending zone of the root (19).The aim of the work reported in this paper was to determine, by physiologicalexperiments, whether X-/9G could act on maize root georeaction and, consequently,on the extension of the cell wall of the cortical cells from the elongating zone of theroots. Effects of X-/SG on root growth will be discussed in relation to light anddarkness in another paper.The conditions for germination of maize (Zea mays L. cv. Kelvedon 33)fruits have been described previously (17). When the primary roots reached alength of 15 ± 3 mm, seedlings were placed vertically with their roots immersed (2hr,dark, 25±1°C) in a buffered solution (pH 5.6) with or without X-£G. Then, afterAbbreviations: X-£G, exo-/?-t>-(l,3)-glucanase; IAA, indole-3-acetic acid.11751176 P. E. Pilet70SO500.4 0.6 0.8 1.0OLUCANASE UNIT PER mlFig. 1.12 16 20TIME IN HOURSFig, 2.Fig. 1 The effect of different concentrations of X-fiG (2 hr pretreatment of intact roots) on the georeaction {cur-vature in degrees ± standard error) of maize apical root segments horizontally placed for 7 ± 0.5 hr. The bend-ing (53.7±4.6) of the controlled segments, not pretreated for 2 hr with the solution, was found tobe not significantly different from those of the control assay (0.0 unit).Fig. 2 Georeaction (curvature in degrees ± standard error) of maize apical root segments in relation to time(0 to 20 hr). Two-hour pretreatment of intact roots with a solution without (A) or with (B) 0.4 unitof X-/JG.careful washing with tap water, only rectilinear segments were mounted horizontallyin plastic frames with their basal cut ends covered with moist filter paper (pH 6.1,76').The frames were kept in closed boxes (90^5% relative humidity; white light of0.9±0.006 J m~2 s-1; 22±i°C). Bending of the root segments was recorded (after7±0.5hr) by shadow photographs. Each result represents the mean response ofat least 45 roots. Standard errors of the mean have been calculated and significantdifferences assessed by the 2-test.The enzyme preparation containing exo-/J-D-(l,3)-glucanase (EC 3.2.1.6)was kindly supplied by Dr. M. Horisberger (Soc. Ass. Techn. Nestle, La Tour dePeilz, Switzerland). X-/?G was isolated from the culture of Basidiomycete speciesQM 806 and purified (7). Its activity was referred to as 1 unit/ml when 1 mole ofglucose was produced from 0.05% laminarin incubated (15 min) in the erizyme solu-tion (50 HIM acetate buffer, pH 4.8; 37°C; final volume: lml). The specificactivity of X-/JG was 90.0 units per mg protein.The georeaction (after 7^0.5 hr) of the horizontal root segments prepared fromintact roots pretreated 2 hr with X-/SG was then analyzed. The changes of thedownward curvature, in relation to several concentrations of X-/JG, are reported inFig. 1. As can be seen, the reaction is significantly enhanced with 0.4 unit of X-/JGbut significantly inhibited with 1.0 unit of X-/?G. A similar concentration effect(except for the values of X-/SG doses which are difficult to compare) has been obtain-ed for the elongation of oat coleoptile segments (10) and pear pollen tubes (21).Root georeaction affected by glucanase 1177When the georeaction is studied in relation to time (Fig. 2A), note that thecurving is very rapid for the first few hours, as previously observed (16, 23).Pretreatment with X-/3G (at the maximum activating concentration of 0.4 unit)only acts, as shown in Fig. 2B, on the first phase of the georeaction, significantlyincreasing (except for the first 2 hr, which is difficult to explain) the downwardbending.From the present data, we can assume that, exogenous X-/JG, at certainconcentrations, stimulates the georeaction of maize roots and, consequently, may acton the extension of the wall of the upper cortical cells located in the elongatingpart of the root (19). But it remains to be proved whether endogenous /S-(.l,3)-glucanase really controls root cell elongation.References( / ) Audus, L. J. and M. E. Brownbridge: Studies on the geotropism of roots I. Growth ratedistribution during response and the effects of applied auxin. J. Exp. Bot. 8: 105-124 (1957).( 2) Buchala, A. J. and H. Meier: /3-Glucanases and non-cellulosic glucans in the developingoat plant. Planla 111: 245-252 (1973).( 3) Clarke, A. E. and B. A. Stone: /3-1,3-Glucan hydrolases from the grape vine (Vitis vinifera)and other plants. Pliytochem. 1: 175-188 (1975).(4) Cleland, R.: Auxin and wall extensibility: Reversibility of auxin-induced wall-looseningprocess. Science 160: 192-194 (1968).( 5 ) Datko, A. H. and G. A. Maclachlan: Indoleacetic acid and the synthesis of glucanases andpectic enzymes. Plant Physiol. 43: 735-742 (1968).(6") Heyn, A. N. J.: Glucanase activity in coleoptiles of Avena. Arch. Biochem. Biophys. 132:442-449 (1969).( 7) Huotari, F. I., T. E. Nelson, F. S. Smith and S. Kirkwood: Purification of an exo-/3-D-(l— 3)-glucanase from Basidiomycete species QM 806. J. Biol. Chem. 243: 952-956 (1968).( 8) Kivilaan, A., R. S. Bandurski and A. Schulze: A partial characterization of an autolyticallysolubilized cell wall glucan. Plant Physiol. 48: 389-393 (1971).( 9) Masuda, Y.: Role of cell-wall-degrading enzymes in cell-wall loosening in oat coleoptiles.Planla 83: 171-184 (1968).(10) Masuda, Y., S. Oi and Y. Satomura: Further studies on the role of cell-wall-degrading en-zymes in cell-wall loosening in oat coleoptiles. Plant & Cell Physiol. 11: 631-638 (1970).(11) Masuda, Y. and S. Wada: Effect of ^-1,3-glucanase on the elongation growth of oat coleoptile.Bot. Mag. Tokyo 80: 100-102 (1967).(12) Masuda, Y. and R. Yamamoto: Effect of auxin on j3-1,3-glucanase activity in Avenacoleoptile. Development, Growth and Differentiation 11: 287-296 (1970).(13) Nevins, D.J.: The effect of nojirimycin on plant growth and its implications concerninga role for exo-0-glucanases in auxin-induced cell expansion. Plant & Cell Physiol. 16: 347-356 (1975).(14) Nevins, D. J.: The in vitro stimulation of IAA-induced modification of Avena cell wall polysac-charides by an exo-glucanase. ibid. 16: 495-503 (1975).(75) Pilet, P. E.: Root cap and root growth. Planla 106: 169-171 (1972).(16) Pilet, P. E.: Control by the root cap on growth and georeaction of roots. In Plant GrowthSubstances 1973, Proc. 8di Intern. Conf., p. 1104-1110, Hirokawa Publ. Co. Tokyo, 1974.(17) Pilet, P. E.: Effects of light on the georeaction and growth inhibitor content of roots. Physiol.Plant. 33: 94-97 (1975).1178 P. E. Pilet(18) Pilet, P. E.: Abscisic acid as a root growth inhibitor: physiological analyses. Planla 122:299-302 (1975).(73) Pilet, P. E. and A. Nougarede: Root cell georeaction and growth inhibition. Plant Sci.Letters 3: 331-334 (1974).(20) Pilet, P. E. and J. Cl. Roland: Growth and extensibility or collenchyma cells, ibid. 2: 203-207 (1974).(21) Roggen, H. P. J. R. and R. G. Stanley: Cell-wall-hydrolysing enzymes in wall formationas measured by pollen-tube extension. Planla 84: 295-303 (1969).(22) Ruesink, A. W.: Polysaccharidases and the control of cell-wall elongation, ibid. 89: 95-107(1969).(23) Shaw, S. and M. B. Wilkins: The source and lateral transport of growth inhibitors in geo-tropically stimulated roots of Zea mays and Pisum sativum. ibid. 109: 11—26, (1973).(24) Tanimoto, E. and Y. Masuda: Effect of auxin on cell wall degrading enzymes. Physiol.Plant. 21: 820-826 (1968).(25) Wada, S., E. Tanimoto and Y. Masuda: Cell elongation and metabolic turnover of the cellwall as affected by auxin and cell wall degrading enzymes. Plant & Cell Physiol. 9: 369-376(1968).

Root georeaction affected by exo-β-D-(l,3)-glucanase

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