Fusicoccin enhanced the growth of collocytes from Apium Gravtolens petioles and modified the rheological parameters tested. But these changes do not sufficiently explain the variations of the cell wall extension. Such effects are discusse

Jaccard, M.

Pilet, P. E.

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Plant & Cell Physiol. 20(1): 1-7 (1979)Growth and rheological changes of collenchyma ce l l s :The fusicoccin e£fectM. Jaccard and P. E. PiletInstitute of Plant Biology and Physiology of the University,Place de la Riponne 6, CH-1005 Lausanne, Switzerland(Received March 7, 1978)Fusicoccin enhanced the growth of collocytes from Apium Gravtolens petioles andmodified the rheological parameters tested. But these changes do not sufficiently explainthe variations of the cell wall extension. Such effects are discussed.Key words: Cell wall — Fusicoccin — Growth — pH Effect — Rheology — Wallextension.Fusicoccin (FC), a diterpene glucoside, promoted the elongation of variousorgans within minutes of application (12, 13, 26). Such action was similar to theeffect of low pH and auxin on cell growth. FC has been reported to increase theproton concentration in the cell wall. The similar action of FC, low pH, and auxinon growth (3, 4, 24) may explain the primary action of auxin; the hypothesispresented (7, 23) is discussed in references {16, 27, 29).Collenchyma is a choice material for testing cell wall extension and rheologyduring growth processes (8, 9). The rheology (including rheological parametersand the effect low pH on extension rate) of isolated collocyte bundles has been shownto change during growth and differentiation of the tissues. In order to test only theinteractions between growth and the rheology of the collocyte walls, we decided touse FC. This substance can stimulate growth, while biphasic auxin responsessuggest that the auxin action may consist of at least two stages (16, 17, 28). It hasalso been shown that the FC effect is not reduced by some inhibitors, e.g., abscisicacid (19, 21). Changes of extensibility and induced growth have been also reportedfor isolated collocyte bundles incubated 4 hr in a solution containing auxin (20).But collenchyma can undergo passive growth, like the epidermis (25), being extendedby the other tissues of the organs. Thus, the growth behavior of collocytes isolatedfrom the petiole or naturally associated with it could be different. Collocyte wallsprepared from intact collenchyma bundles, inside the petiole itself, were used in thisstudy.Materials and methodsPlants of Apium Graveolens were cultivated in a greenhouse (under controlledtemperature and humidity). High- and low-growing petioles (see 9) were detachedfrom the shoot and washed in a beaker with distilled water. After alcohol sterili-zation, the basal part of the petiole (9) was cut, the initial length of the collocyteM. Jaccard and P. E. PiletFig. 1. Diagram showing the frame used for basalpart segments and indicating several types of possibleH mm r—| mm assays. 1. Basal part segment. 2. Upper agarI \ I \ I \ block. 3. Lower agar block. I. Control' "" "~t segments. II. FC application on two segmentsections. I l l and IV: tests analyzing effectsof basipetal or acropetal FC transport in relationto collocytc growth.bundles being 3 cm±0.5 mm. Next, high-growing petiole segments were fixedfor several hours (relative humidity 100%, temperature 24°C) on a special frame(Fig. 1). Agar blocks (4 mm long and 10 mm in diameter) containing 5 nfflphosphate buffer (pH 6)+0.5% sucrose±FC (at various concentrations) coveredthe two cut sections. This method was preferred to incubation in aqueous solutionfor many reasons (it was closest to the natural conditions); many types of experi-ments were done (Fig. 1), but only assays I and II are reported here.After the selected incubation time, the length of the petiole segments and thecollenchyma bundles was measured then the collenchyma bundles were carefullyexcised, quickly frozen and stored at -20°C. The growth of the basal part segmentsand the elongation of collenchyma bundles attached to them were similar. Whenbundles were to be used, they were thawed at room temperature and washed withphosphate buffer. Rheological parameters were measured as previously reported(8): a collocyte bundle was introduced between the pincers of a creep apparatusand submitted to a 10 g force applied instantaneously. The extension observed(reported to the initial length of the sample) could be separated into an instantaneousstrain ei and a time dependent strain et (viscoelastic extension: creep of the speci-men) ; the sum of these two parameters giving the total strain £tot- After time T,the force applied was quickly removed and the stabilization of the residual strainer caused a permanent strain ep, which has been reported (8) to be a plastic strain(having strain-hardening properties). Substraction ep from etot gave the elasticstrain eei- A time T of 2 min was chosen because frozen-thawed bundles (especial-ly the FC-treated samples) showed a time-dependent strain et that stabilized after2 min.The extension rate of collocytes was traced with a creep apparatus and the low-pH effects on it were calculated as previously reported (9). The extension rate atpH 6 (ER6) and pH 4 (ER4) were measured, then the low-pH effect on extensiongrowth was calculated (JER4—ERe—ER4) and expressed in logarithmic values.Results and discussionFig. 2A shows the growth of the collocyte bundles treated with or without FC.Differences between FG treatment at 5 X 10~7 M concentration and control treatmentswere significant after 6 hr of incubation. Other data {20) expressed the growth ofcollocytes in relation to their initial mean length in the tissues. But these data werenot very sensitive because of the large variation in the initial length of the collocytesin the bundles. Thus, elongation was better expressed as a function of the initiallength of the collocyte bundles. Another paper (5) has reported that when workingFusicoccin effect on collenchyma cells6 9 12TIME IN HOURS9 12TIME IN HOURSFig. 2. Kinetic elongation and rheological parameters of collenchyma bundles treated with {FC) or without (C)fusicoccin (JxV0-' M). A: elongation in mm (^standard deviation). B: rheological parameter£tot in % (±standard deviation).with the usual material, enhancement of the growth rate was observed some minutesafter the FG application. However, the FC concentration used was higher thanours and consequently, the biological properties of the collocytes could have beendifferent. Our selected concentration (5X10~ 7 M) was not the optimal FC levelas can be seen in Fig. 3. Also, some endogenous substances may have formed duringthe petiole excision to inhibit the FC growth effect.The FC effect on the rheological parameter £tot was not significant (Studenttest P=0.05) before 12 hr, as can be seen in Fig. 2B. This indicates that changes ofthe rheology of the collocyte walls could occur after growth promotion. FC isknown to have a low transport rate in organs (18); thus, the elongation observedmay be limited during the first hours of treatment to the extremities of the collen-chyma bundles. In creep relaxation tests, such an effect could not be detected withthe ends of the collocyte samples being inserted between the pincers of a creepapparatus. However, most extensibility results of short-time experiments done with200 -ISOFig. 3. Relative elongation in % {^standard deviation) ofcollenchyma bundles measured after 12 hr of incubation in relationto FC concentration.4U= 102 X (idLTH-^Lc)/^LcJLTR: elongation of collenchyma bundles treated withFC. 4Lc: elongation of control collenchyma bundles.100s 50ozoX - -54 M. Jaccard and P. E. Piletauxin have shown that extensibility changed after growth stimulation (14, 17);also, during the beginning of the response, the growth observed may have occurredin the ends of the segments as reported for the effect of low pH on extension (6).The control tissues exhibited a stiffening of the cell walls during the kinetics ofgrowth. It is generally assumed that this phenomenon is a result of the cell wallextension appearing without loosening, because of the strain-hardening behaviorof collocytes or other primary cell walls (2, 8). The present data agree with thismodel, except that the total strain decreased during 3 to 6 hr of the treatment,while growth did not statistically change.Several concentrations of FC were tested and the results of the growth ofcollocyte bundles, after 12 hr of incubation, are presented in Fig. 3. As can benoted, stimulation was effective from a concentration of 10~7M and increasedlinearly according to a logarithmic scale concentration. But growth values werenot statistically different (Student test P=0.05) between 10~6 and 10~5 M as alreadyreported for maize roots {19).In contrast, from the analysis of creep tests (Fig. 4 and 5), no significant changesof the rheological parameters etot> et> sel a nd «p at 10~7M of FC were found.The parameter values presented no linear increase according to a logarithmic scaleof FC concentration; rheological parameters indicated significant differences betweenconcentrations of 10~5 and 10~6 M.The extensibility properties of the collocyte walls, estimated by changes of therheological parameters, do not exactly describe the in vivo modifications of the wallsduring the FC-induced growth. No close correlations have been found betweenrheological parameter data and collocyte bundle elongation. Although frozen-thawed and living tissues exhibited similar rheological behavior (8, 15), rheologicaltests reduced the cross area of the samples while normal growth did not (11). Onthe other hand, the stress state in the creep test was not identical to that appearingduring normal growth. These facts could explain the low correlation observed.3.0w 2.0_i<° 1.51.0\Co-12- 7 - 6 - 5AU CoX -IFC IN 1 0 ' x M- 7 - 6 - 5FC IN IO"X M0.8 z!m0.7 °m06 10.50.40 . 3 ^Fig. 4. Rheological parameters (in %, ^standard deviation) of collenchyma bundles measured after 12 hr ofincubation in relation to the FC concentration tested. A: total strain etot- B: time-dependent strain et.Co and C12: control tissues after 0 and 12 hr of incubation, respectively.Fusicoccin effect on collenchyma cellsX - c o- 7201.51.00.5- 6 - 5FC IN1O-"MFig. 5. Rheological parameters (in %, ^standard deviation) of collenchyma bundles measured after 12 hr ofincubation in relation to the FC concentration. A: elastic strain eei- B: permanent strain ep. Co andCi»: control tissues after 0 and 12 hr of incubation, respectively.Total strain etot is composed of instantaneous ei strain and time-dependentet strain (<9); such a definition being subject to discussion (/). In the rheologicaltests, the instantaneous strain ei was that which appeared prior to about 2 sec.Any extension occurring after 2 sec was designated as creep and the time-dependentstrain Et was the measurement of a viscoelastic strain of 2 min. However, visco-elastic extension did not begin at 2 sec and part or all of the instantaneous straing[ could be a viscoelastic extension which occurred too rapidly to be estimated ascreep. If this can be verified and if the FC treatment similarly affects the visco-leastic relaxation times tested, the instantaneous and time-dependent strains couldshow the same relative changes to FC treatment; in the present case this propositionis accepted because etot a n d =u changed in the same relative ratio upon FCtreatment.These results do not exclude changes of parts of the creep curve for some re-laxation times, as observed with relaxation tests on other materials treated with FC(30).Table 1 Rheological parameters and low-pH effect on the extension rate offusicoccin-treated (FC) control (Co,C12) and low growing (LG) collocytesCo*C12*F CLG4. t o , '18.9±2.613.2±1.129.6±4.69.2±1.28.38±0.806.38 ±0.2810.85±1.174.62 ±0.67« p '10.3 ±1.886.34 ±0.918.68+3.84.50±0.604.23 ±0.753.06 ±0.37.7 ±1.411.68±0.12logl0(JER4)<2.7 ±0.162.52 ±0.133.13 ±0.090.0* See legends of Fig. 4 and 5.° FC-treated bundles (at 10~5M concentration); incubation, 12 hr.* Low-growing collocytes (see 9).c Strains expressed in %c.d Expressed in %-10-» min-l.The standard deviation is given for each value.6 M. Jaccard and P. E. PiletThe permanent strain ep was enhanced more by FC than the elastic straineei; this agrees with other results (/, 2, 11, 14) which showed that the "plasticity"of the tissues increased more with increasing growth than the "elasticity".Rheological parameters and the low-pH effect on extension rate change withgrowth and differentiation of collocytes (9). A close relationship may exist betweenrheological parameter values and the low-pH effect on extension rate values. Thishypothesis was tested (Table 1). After 12 hr of incubation, both rheological para-meters and the low-pH effect on extension rate were different for control and FC-treated tissues; however, rheological parameters were significantly different betweencontrol bundles after 0 and 12 hr of incubation, while the low-pH effect did notchange. Also, collocytes of low-growing material exhibited rheological parametervalues near those of control tissues after 12 hr of incubation. In contrast, the pHeffect on the extension rate was not significant for low-growing tissues. No closerelationships were found between the low-pH effect on the extension rate and rheo-logical parameters.ConclusionExtensibility and minimum relaxation time (22, 30) change after acidic treat-ment. Most results obtained have shown that FC-induced growth is a consequenceof proton extrusion from the plasmalemma to the cell wall. In addition, theextensibility has been found to change when measured plasmometrically after FCtreatment (10).If such a situation is the same for collocytes, this could explain the change ofrheological parameters, but not the enhancement of the low-pH effect on theextension, which must be related to a possible secondary effect of fusicoccin. Thishypothesis should be studied by experimentally testing the effect of auxin on rheologyand the growth of collenchyma bundles.References( 1 ) Cleland, R.: The mechanical behaviour of isolated Avena coleoptile walls subjected to constantstress. Properties and relation to cell elongation. Plant Physiol. 47: 805-811 (1971).( 2) Cleland, R.: Cell wall extension. Ann. Rev. Plant Physiol. 22: 197-222 (1971).( 3) Cleland, R.: Auxin-induced hydrogen ion excretion: correlation with growth and controlby external pH and water stress. Planta 127: 233-242 (1975).(4) Cleland, R. E.: Kinetic of hormone-induced H+excretion. Plant Physiol. 58: 210-213 (1976).( 5 ) Cleland, R. E.: Fusicoccin-induced growth and hydrogen ion excretion of Avena coleoptile:relation to auxin responses. Planta 128: 201-206 (1976).( 6) Evans, M. L.: Rapid responses to plant hormones. Ann. Rev. Plant Physiol. 25: 195-223 (1974).( 7 ) Hager, A., H. Menzel and A. Krauss: Versuche und Hypothese zur Primarwirkung desAuxins beim Streckungwachstum. Planta 100: 47-75 (1971).( 8) Jaccard, M. and P. E. Pilet: Extensibility and rheology of collenchyma cells I. Creep relax-ation and viscoelasticity of young and senescent cells. Plant & Cell Physiol. 16: 113-120 (1975).( 9 ) Jaccard, M. and P. E. Pilet: Extensibility and rheology of collenchyma cells II. Low pHeffect on the extension of collocytes isolated from high- and low-growing material, ibid. 18:883-891 (1977).(10) Lado, P., F. Rasi-Caldogno and R. Colombo: Effect of cycloheximide on IAA or FC inducedcell enlargement in pea internode segments. Plant Sci. Lett. 9: 93-101 (1977).Fusicoccin effect on collenchyma cells 7(11) Lockhart, J. A., G. Bretz and R. Kenner: An analysis of cell wall extension. Ann. New YorkAcad. Sri. 144: 19-33 (1967).(12) Marred E., R. Colombo, P. Lado and F. Rasi-Caldogno: Correlation between proton extrusionand stimulation of cell enalrgement. Effects of fusicoccin and of cytokinins on leaf fragmentsand isolated cotyledons. Plant Sri. Lett. 2: 139-150 (1974).(13) Marre\ E., P. Lado, F. Rasi-Caldogno, R. Colombo and M. I. de Michelis: Evidence for thecoupling of proton extrusion to K+ uptake in pea internode segments treated in fusicoccin orauxin, ibid. 3: 365-379 (1974).(14) Masuda, Y.: Auxin-induced cell expansion in relation to cell wall extensibility. Plant & CellPhysiol. 10: 1-9 (1969).(15) Olson, A. C , J. Bonner and D. J. Morr6: Force extension analysis of Avena coleoptile cell walls.Planta 66: 127-133 (1965).(16) Parrish, D. J. and P. J. Davies: On the relationships between extracellular pH and the growthof excised pea stem segments. Plant Physiol. 59: 574-578 (1977).(17) Penny, P., D. Penny, D. Marshall and J. K. Heyes: Early responses of excised stem segmentsto auxin. J. Exp. Bot. 23: 23-36 (1972).(18) Pernet, J. J. and P. E. Pilet: in preparation(19) Pilet, P. E.: Fusicoccin and auxin effects on root growth. Plant Sri. Lett. 7: 81-84 (1976).(20) Pilet, P. E. and J. Cl. Roland: Growth and extensibility of collenchyma cells, ibid. 2: 203-207 (1974).(21) Pitman, M. G., N. Schaefer and R. A. Wildes: Stimulation of H+ efflux and cation uptake byfusicoccin in barley roots, ibid. 4: 323-329 (1975).(22) Rayle, D. L. and R. Cleland: Enhancement of wall loosening and elongation by acid solution.Plant Physiol. 46: 250-253 (1970).(23) Rayle, D. L. and R. Cleland: The in vitro acid growth response: relation to in-vivo growthresponses and auxin action. Planta 104: 282-296 (1972).(24) Rayle, D. L.: Auxin induced hydrogen-ion secretion in Avena coleoptiles and its implications.ibid. 114: 63-73 (1973).(25) Roelofeen, P. A.: Ultrastructure of the wall in growing cells and its relation to the direction ofgrowth. Adv. Bot. Research 2: 69-149 (1965).(26") Servettaz, O., F. Cortesi and C. P. Longo: Effect of benzyladenine on some enzymes of mito-chondria and microbodies in excised sunflower cotyledons. Plant Physiol. 58: 569—572 (1976).(27) Sakurai, N., D. J. Nevinsand Y. Masuda: Auxin-and hydrogen-ion induced cell wall looseningand cell extension in Avena coleoptile segments. Plant & Cell Physiol. 18: 371-380 (1977).(28) Tietze-Hass, E. and K. Dorffling: Initial phases of indoleacetic acid induced growth in col-eoptile segments of Avena sativa L. Planta 135: 149-154 (1977).(29) Van der Hoef, L. N., J. S. Findley, J. J. Burke and W. E. Blizzard: Auxin has no effect onmodification of external pH by soybean hypocotyl cells. Plant Physiol. 59: 1000-1003 (1977).(30) Yamagata, Y. and Y. Masuda: Comparative studies on auxin and fusicoccin actions on plantgrowth. Plant & Cell Physiol. 16: 41-52 (1976).

Growth and rheological changes of collenchyma cells: The fusicoccin effect

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