Planta (Berl.) 84, 295--303 (1969)

Cell-Wall-Hydrolysing Enzymes in Wall Formation as Measured by Pollen-Tube Extension* H. P. J. R. R o o o ~ and 1~. G. STA1NWLEY Forest Physiology-Genetics Laboratory, School of Forestry, University of Florida, Gainesville Received August 25/October 17, 1968

Summary. Cell-wall-softening enzymes affect the plasticity of the tip wall of pollen tubes and modify tube elongation. Length of pear pollen tubes is increased by the addition of/%l,3-glucanase at the beginning of in-vitro germination. The longer tubes after 3 hours are primarily the result of earlier germination. Application of fl-l,4-glucanase or pectinase to germimating pollen does not affect germination but enhances the growth rate of 1-hour-old pollen tubes. The stimulating effects of fi-l,4-glucanase and pectinase are additive. Denatured enzymes had no effect. Proteinase, pectin esterase, acid phosphatase and a-amylase only inhibited growth and germination. Replacing the medium 1 hour after germination begins stops pollen-tube growth; growth can be restored by adding cellulase-pectinase mixtures to the replacement medium. These results provide evidence that eellulase and pectinase are important in pollen-wall extension, and that callose-hydrolyzing enzymes are involved in pollen germination but not wall extension. Introduction Pollen-tube growth is restricted to the tip where the cytoplasmic organization differs from the rest of the tube (SAssE~I, 1964; DASI~EK and RosE~I, 1966). A high content of non-celhilosic components and the absence of callose make the tip wall the weakest and most plastic area of the pollen tube. Stress induced by turgor pressure on the tube wall results in formation of a bulge at the tip. To maintain tube formation it is necessary that the tip wall, at the time it becomes part of the tube, be sufficiently strong to withstand the transition from a longitudinal to a two times greater tangential stress (Locx~Aa~T, 1965). This strength is provided by deposition of callose and by a reorientation of the cellulose microfibrils as they are moved from the round tip to the tangential wall. During pollen-tube growth the tip does not become thinner; therefore, cell-wall synthesis must occur. Pollen-tube tips lack the cytoplasmic framework for wall synthesis described by G~E~.~ (1963) in elongating cells of Nitella. Pollen tube elongation, wall formation and extension is possible when (1) the wall is under internal stress as from turgot pressure; (2) * A contribution of the Florida Agricultural Experiment Station, Journal Series :No. 3069. 20

Plan~a

(Berl.), Bd. 84

296

H. P. J. R. RoGG~ and R. G. STA~-L:EY."

the wall is sufficiently plastic; and (3) there is a supply of constituents for wall synthesis. Since all growth occurs at the pollen tip, the rate of pollen tube growth can serve as a parameter for wall extension. One hypothesis t h a t merits experimental testing is t h a t the ratio of the pectins, hemicellulose and cellulose at the tip affects tube elongation through their influence on plasticity. Changing this ratio with the hydrolyzing enzymes fi-l,4-glucanase and pectinase should, according to this hypothesis, influence the plasticity of the tube tip and possibly the rate of pollen growth. The level of fi-l,4-glucanase has been correlated with rate of cell elongation in peas ( F ~ and MACLACHLAN, 1967). E K I ) ~ L (1957) and C o ~ C K (1956) detected only an increase in the diameter of root hairs after treatment with wall-softening enzymes. Evidence tha~ these enzymes, added in vitro, can enhance wall extension is not available. This paper reports the effect of fl-l,4glucanase, pectinase and fl-l,3-glucanase on pollen-tube elongation. Materials and Methods Pollen of pear, Pyrus communis L. cv. "Winter Nelis", collected in 1967 by the L. C. Antles Company, Wenatchee, Washington and stored at --15 ~ were used for these experiments. The germination medium consisted of a solution of 0.002 M Ca(OH)2 adjusted with H3POa to pit 5.9, 0.5 M raffinose and 0.01% It3BO 3. 5 mg pollen per 2 ml medium gave optimal germination and maximum growth with straightest tubes. The pollen were germinated and grown in test tubes (16 • 150 mm) in an incubator at 28~ the medium was gently aerated with watersaturated air of 28~ fl-l,4- and fl-l,3-glucanase (EC 3.2.1.4 ~nd EC 3.2.1.6, respectively) were kindly furnished by Dr. E.T. REuse, U.S. Army Natick Laboratories. Pectinase (EC 3.2.1.15), proteinase and pectin esterase (EC 3.1.1.11) were obtained from National Biochemicals Corp., Cleveland, Ohio; acid phosph~tase (EC 3.1.3.2) and ~-amylase (EC 3.2.1.1) from Sigma Chemical Comp., St. Louis, Mo. Pollen tube growth was stopped by adding three drops of 37% formaldehyde solution. The tube lengths were measured with a Reichert projection microscope. Results and Discussion 1. Pollen Germination. Pollen grains placed in a germination medium

swell immediately and after 10--20 min a callose deposition takes place (Cu~RI~, 1957). I n pear pollen this occurs in the intine below the pore from which the pollen tube protrudes. If pollen grains fail to germinate, formation of callose goes on until it fills the whole grain. Meanwhile, m a n y enzymes present in the wall (ZINGER and P]~T~OVSKAYA-BARA~OVA, 1961) are released into the medium. Some of these are hydrolyzing, cellwall softening (CWS) enzymes like fi-1,3 and fl-l,4-ghieanase, and pectinase. The latter two are released immediately after the pollen is placed into the medium (ST~L]~Y and THOMAS, 1967). These enzymes can then enter the grain, presumably, through the germ pores; it is at

Cell-WM1-HydrolysingEnzymesin Wall Formation

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this region that the intine wall is first softened. The pollen tube emerges from the callose-containing pore area of the intine. The callose disappears from the tip and the tube starts growing. Generally only one tube protrudes, but in about 0.1% of the pollen grains 2 tubes come out. The number of tubes per grain was not increased by treatment with high concentrations of CWS enzymes. Even strong hydrolyzing agents, such as tI2S04, only gave rise to one so cMled "instant pollen tube" in triporate Petunia pollen (LI~sK]n~s and MVT,LV,~m~S, 1967). The preference for one pore may depend on its position in the tetrad. Some multiporate pollen form several tubes in vitro, probably because the softening proceeds equally rapidly at two or more pores. Addition to the medium of fi-l,3-glucanase at 0.1 to 0.2 mg/ml at the start of germination significantly increases pollen tube length after 3 hrs growth (Fig. 1 ; Table). This increase only occurs when the enzyme is applied at the beginning of germination; no growth stimulation resulted when 1-hour-old tubes were treated. Thus, the longer tubes obtained by treatment with/~-l,3-glucanase are primarily the result of accelerated germination resulting from the enzyme dissolving the callose inside the pore intine and initial pollen tube. fl-l,3-glucanase did not soften the tube tip or affect growth when added after germination. This confirms our observations and those of G6~s~-B~u (1965) that callose is absent in the tip region of a growing pollen tube. Callose can be very rapidly synthesized (G6~sKA-BnYLASS, 1965). In pollen tubes, callose forms just behind the tip, immediately after extension of the newly-formed tube wall. One function of ca]lose is to strengthen the tube wall (WAT~nK]~Y~, 1965; G6~SKA-BR~ZLASS,1965), thus assuring continuation of tube formation. Cessation of tube growth is usually coupled with callose deposition at the tip wall. When germinated pollen remains in the medium for a prolonged period a high level of callose forms in the wall and inside the tubes. Disturbance of pollen-tube growth in vivo after incompatible pollination is also coupled with increased callose deposition (TtlP~, 1959; LINS~ENS, 1964; ROGGV.~ and LI~sKva~s, 1967). The observation of TlSCltLER (1910) that diastase added to pollen medium caused an ejection of pollen tubes of Cassia was not confirmed by applications of amylase in this study of Pyrus communis pollen. 2. Pollen-Tube Growth. The growth rate of pear pollen tubes is practically uniform during the first 3 hours (Fig. 2). Previous authors who studied the effect of cellulase and pectinase on pollen-tube growth (BI~,w]3AKva~ and KwAcK, 1964) and root-hair elongation (CoI~MACK, 1956; ECKI)Am% 1957) reported only inhibition. In our experiments fl-l,4-glucanase and pectinase at 0.10 to 0.25 mg/ml significantly increased tube lengths, by 22 and 30%, respectively (Table; Fig. 1). 20*

298

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Fig. 1. The effect of different concentrations of cell wall softening enzymes on pollen tube growth 3 hours after start of gel~nination Table. E//ect o/ cell-wall hydrolytic enzymes on pollen-tube growth One relative unit = 3.8 ~x Enzyme added

Concerttration (mg/ml)

Hours germinated

Number Tube length of tubes (relative units) measured minus enenzymes zymes

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* Significant at the 1% level. C o n c e n t r a t i o n s a b o v e 1 m g / m l i n h i b i t e d pollen t u b e growth. I n c o n t r a s t t o t h e effect of fl-l,3-glucanase, t h e s t i m u l a t i n g effect of fl-l,4-glucanase a n d p e c t i n a s e t a k e s place w h e n t h e e n z y m e s are a p p l i e d to 1-hour-old p o l l e n t u b e s ; t h e effect is visible w i t h i n 0.5 hours a f t e r t r e a t m e n t . D e n a t u r i n g t h e e n z y m e b y 5 m i n boilLrtg e l i m i n a t e d b o t h t h e s t i m u l a t i n g a n d i n h i b i t i n g effects. Thus, s t i m u l a t i o n is n o t caused b y i n o r g a n i c

Cell-Wall-Hydrolysing Enzymes in Wall Formation

299

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Fig. 2. Growth rate of pear pollen tubes. Shaded area represents standard deviations. One relative unit = 3.8 tz impurities in. the enzyme, as JAcxso~ (1959) had observed in root hairs. The enzymes proteinase, pectin esterase, acid phosphatase and ~-amylase, tested over a wide concentration range, had no effect at low concentrations and inhibited pollen tube growth at concentrations over 1 mg/ml. The balance existing between rigidity and plasticity in the tube-tip wall is controlled primarily by cell-wall-synthesizing and ceU-wallsoftening enzymes. This must be a well-regulated system since the growth rate of the pollen tubes is uniform under our growth conditions during the first 2.5 hours (Fig. 2). Addition of exogenous fl-l,4-glucanase and pectinase to the medium results presumably in a higher degree of plasticity in the tip region and thus in an increase in the observed growth rate. Unlike many compounds reviewed by L r N s x ~ s (1964) and Ros]~N (1968) enzyme treatments at growth-stimulating concentrations do not increase the total length of pollen tubes. To detect the enzyme effect, it is necessary to determine the growth rate of elongating pollen tubes and not just their length after a given time. BR]~W~ER and KWACK (1964), investigating only the final effect of CWS enzymes on pollen tube growth, found total tube length decreased, i.e. elongation was inhibited. They used a concentration 20 times greater than was used in our experiments. Pollen-tube growth is indeed inhibited at high enzyme concentrations and the tubes start to disintegrate. The stimulatory effects of fl-l,4-glucanasc and pectinase are additive (Table). This sugg6sts that the two different components of the tip wall which are hydrolyzed by these enzymes, independently contribute to

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~'ig. 3. The effect of medium replacement on pollen tube growth. ~Iedia were removed by cen%rifuging2 rain at 500 g the total growth. Since fl-l,3-glucanase only affects callose and initial germination, it did not increase the effect of pectinase on total growth. External CWS enzymes stimulated pollen tube growth. If the hypothesis that the presence of externM, as well as internal, cytoplasmic CWS enzymes is essential for rapid wall extension is correct, then removing these enzymes must inhibit the rate of pollen tube growth. In fact, replacement of the medium in which the pollen germinated with fresh medium, represented by curve 0.0--0.0 in Fig. 3, caused a cessation of pollen tube growth after 0.5 hour. The endogenous level of internal and surface-bound CWS enzymes was presumably sufficient to maintain growth for the 0.5-hour period. The growth rate prior to removal of the enzyme-containing medium is restored by applying fresh medium containing a fl-l,4-glucanase-pectinase mixture. The control in which the medium was not replaced (Fig. 3) shows uniform growth, indicating that the procedure used to replace the media (legend, Fig. 3) had no influence on growth. Pollen tubes in replaced medium withou% added CWS enzymes reinitiated growth after 1 hour, probably as the result of renewed synthesis and restoration of essential CWS enzymes. The ratio pectinase/fl-l,4-glucanase of 0.1/0.3 mg-m1-1 causes the greatest growth stimulation. These results provide evidence that//-1,4glucanase and pectinase play an important role in wall extension of the pollen-tube tip. CWS enzymes also affect wall extension when added externally to pollen in growth medium. Our experiments, with apically

Cell-Wall-Hydrolysing Enzymes in Wall Formation

301

growing pollen tubes, support the suggestion ( B o ~ E R , 1961; FA~ and MACT,Ac~rLAN, 1967; THOMAS and MVLLINS, 1967) that CWS enzymes are involved in cell-wall extension. We estimated the level of fl-l,4glucanase released by germinating pear pollen equals about 20 % of the concentration added experimentally to yield maximum tube growth. DATKO and MACLACHL~'~ (1968) found that treating pea seedlings with indoleacetie acid (IAA) increased CWS enzymes. In-vitro addition of IAA to pollen germination media stimulates extension of some, but not all tubes (Jon~I and VAsm, 1961). In our experiments, addition of IAA over a range of non-inhibiting concentrations did not stimulate pollen-tube extension or change the response to the added CWS enzymes. Optimum germination and pollen growth requires a certain minimum concentration of grains, namely, 0.5 mg.m1-1 of medium. Calcium has been suggested as the cause of this "population" or "crowding" effect (BlCEW]~AK]~ and KWACK, 1964). In the medium-replacement experiments (Fig. 3) the replacement solution contained the optimum Ca concentration; yet, pollen growth only continued at the normal rate when CWS enzymes were added. Ca probably plays an indirect role in the "population" effect. The concentration of CWS enzymes is the primary initiating factor of tube extension. Preliminary attempts to detect a chemotropic response of pear pollen to CWS enzymes by the well-agar assay (RosEN, 1964) failed. Nevertheless, since CWS enzymes are involved in tube growth, they may facilitate the chemotropic response in vivo without being the cause. MASVDA and WADA (1967) reported that a crude preparation of fl-l,3-glucanase stimulated extension of oat eoleoptfles. CLELAND (1968), using the same, highly purified fl-glueanases which Dr. E. T. REESE supplied for our pollen experiments, failed to confirm their results. On the basis of this finding and his own inhibitor studies on auxin-induced extension of Avena eoleoptiles, CLELAN]) concludes that cell wall loosening is probably not mediated by CWS enzymes. However, the increased wall extensibility after treatment with auxin was not completely reversed by cyanide or dinitrophenol. The irreversible change in cell-wall extension may represent the effect of CWS enzymes on wall extension. This study was supported by U.S.D.A., Forest Service Grant WO-Pll. The skillful assistance of Mrs. S~A MEsx is appreciatively acknowledged. References

BON~EI~, J.: On the mechanics of auxin induced growth. In: Plant Growth Regulation, p. 307--328. Ames: Iowa State Univ. Press 1961.

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BREWBAKV.R,J. L., and B. H. KWAOK:The calcium ion and substances influencing pollen growth. In: Pollen Physiology and Fertilization (H. F. LINSKE~S, ed.), p. 143--151. Amsterdam: North-Holland Pnbl. Co. 1964. CLELA~D, R. : Auxin and wall extensibility: Reversibility of auxin induced wallloosening process. Science 160, 192--194 (1968). CORMACK,R. G. H. : A further study on the growth of Brassica roots in solutions of pectic enzymes. Canad. J. Bot. 36, 983--989 (1956). CURRIER, H. B. : Callose substance in plant cells. Amer. J. Bot. 44, 478--488 (1957), DAS~EK, W. V., and W. G. ROSE~: Electron microscopical localization of chemical components in the growth zone of lily pollen tubes. Protoplasma 61, 192--204 (1966). DATKO, A.H., and G. A. MACT~C}~A~: Indoleaeetic acid and the synthesis of glucanases and pectic enzymes. Plant Physiol. 43, 735--742 (1968). E}~DAnT~,I.: On the growth mechanism of root hairs. Physiol. Plantarum (Cph.) 19, 708--806 (1957). FAIr, D. F., and G. A. M~CLAC~A~: Studies on the regulation of eellulase activity and growth in excised pea epieotyle sections. Canad. J. Bot. 45, 1837--1844 (1967). G5~SKA-BRYLASS,A.: Cy~omorphologieal studies of callose in pollen tubes. Acta Soe. Bot. Pol. 34, 757--762 (1965). GREE~, P. B. : On mechanisms of elongation. In: Cytodifferentiation and Macromolecular Synthesis (M. LOCKE, ed.), p. 203--234. New York and London: Acad. Press 1963. JAs}~soiv, W. T. : Effect of pectinase and cellulase preparations on the growth and development of root hairs. Physiol. Plantarum (Cph.) 12, 502--510 (1959). Jom~I, B. M., and I. K. VAsm: Physiology of pollen. Bot. Rev. 27, 325--381 (1961). Ln~sxENs, H. F. : Pollen physiology. Ann. Rev. Plant Physiol. 15, 255--270 (1964). - - , and J.M.L. MULLENEERS: Formation of "instant pollen tubes". Acta bot. neerl. 16, 132--142 (1967). LOCX~AI~W,J. A.: Cell extension. In: Plant Biochemistry (J. BO~ER and J. E. VAR~En, eds.), p. 826--849. New York and London: Acad. Press 1965. M-~SUDA,Y., and S. WADA: Effect of fl-l,3-glucanase on the elongation growth of oat coleoptile. Bot. Mag. (Tokyo) 80, 100--102 (1967). RoaGn~, H. P. J. R., and H. F. LINSXENS: Pollen tube growth and respiration in incompatible intergeneric crosses, l~aturwissensehaften 20, 542--543 (1967). Rosin