Plant Physiol. (1979) 64, 1053-10570032-0889/79/64/1053/05/$00.50/0

Influence of Fusicoccin on the Control of Cell Division by AuxinsiReceived for publication January 19, 1979 and in revised form June 4, 1979

ARMEN KURKDJiAN, JEAN-JACQUES LEGUAY, AND JEAN GUERNCentre National de la Recherche Scientifique, Laboratoire de Ginitique et Physiologie du Developpement desPlantes, I Place de l'Eglise, 91190 Gif-sur- Yvette, France

ABSTRACT

An indirect stimulation of cell divison by fusicoccin is demonstrated.Tlhe disttion of2,4dchkwopbenoxyacetic acid molecules between Acerpse5dCplatr cels and their culture medium is strngly modifli by afusicoccin treatmt, through the extrcegular acdfication induced by thetoxin. As a consequence, a stimulat of cell division is observed when theintracellular auxin concentration is sufficiently increased to reach thethreshold control level (Leguay JJ, J Guern 1977 Plant Physiol 60: 265-270).

pendently of this indirect action on cell division, the number of cellsper cluster is decreased and the volume of the celis increased in fuscocin-treated cells which show a typical enlrgement response.

A wide variety of physiological processes are influenced by thediterpene glucoside fusicoccin; they include cell enlargement, seedgermination, stomatal opening, ion exchange, etc. (9, 10). Closecorrelations have been established between the action of auxinand FC2 on cell enlargement, proton extrusion, hyperpolarizationof transmembrane potential, K+ absorption, and organic acidsynthesis (9, 10).

Only a few studies have been devoted to the action of FC oncell division. A large stimulation of growth of tobacco calluses byFC has been reported (17). However, growth was expressed interms of fresh weight increase and consequently two possibleeffects of FC, ie. stimulation of cell division and/or cell enlarge-ment, could account for the over-all stimulation of callus growthby FC. As these two possible effects ofFC were not distinguishedand as it was difficult with such a system to test the action of FCindependently of other growth regulators, no conclusion wasdrawn about the action, either direct or indirect, of FC on celldivision (17). More recently, taking into account the well-docu-mented action of auxin on the stimulation of cell division, Rolloet al. (13) have compared the action of FC and 2,4-D on thedivision of Acer pseudoplatanus cells in suspension culture. Theauthors showed that addition of 2,4-D restored cell division inresting cultures which were transferred into a medium withoutauxin, but FC failed to produce such a stimulation of cell division.The cells, however, did respond to FC by a sharp increase in K+uptake and H+ release, and consequently, the lack of stimulationof cell division could not be attributed to a lack of sensitivity ofthe cells to FC. Such an effect of FC on the ionic exchanges ofvarious strains of plant cells cultivated in liquid medium andprotoplasts has been documented recently (14).

1 This research was supported by the Centre National de la RechercheScientifique, Equipe de recherche associee No. 486.

2Abbreviations: FC: fusicoccin; DMO: 5,5-12-'4Cjdimethyloxazolidine-2,4-dione; Ci: intracellular DMO concentration; Ce: extracellular DMOconcentration.

It appears that no clear-cut picture of the influence of FC oncell division is available and the aim ofthis paper is to demonstratean indirect stimulation ofcell division by FC through the influenceof this substance on intracellular auxin concentration. This studywas initiated by our previous demonstrations that a modificationof the extracellular pH induces a modification of the distributionof auxin molecules between the cells and their culture mediumand, furthermore, that cell division is controlled by auxin througha threshold level (8). It was thought likely that FC, by loweringthe extracellular pH, could modify the intracellular concentrationof auxins and that an indirect action of cell division could beobtained in that way.

MATERIALS AND METHODS

Culture of A. psewdopkuanus Cell Suspensions. As reportedearlier (4) cells were grown in a medium where cell division waslimited either by auxin (standard medium with 0.2 pm 2,4-D) orby nitrate (standard medium with 4 AM 2,4-D). The growth curvesof cell populations were established as previously described (8).FC was sterilized by filtration through a Millipore filter with a

porosity of 0.22 ,um before adding to the culture medium to a finalconcentration of 2 pi.

2,4-D Uptake. [l-14C]2,4-D (54 mCi/mmol 99% radiochemicalpurity) was obtained from Amersham (England). The experimentsconcerning the uptake of [14CJ2,4-D were conducted according tothe technique described elsewhere (8). Briefly, two flasks eachcontaining 100 ml of cell suspension cultivated on 4 ,UM 2,4-Dwere used. FC was injected in one flask at the beginning of theexperiment. About 4 h later [1-'4C]2,4-D was added to the twoflasks. One h later, aliquots of the suspensions were pipetted,filtered on glass fiber filters (GF/A), and the radioactivity of thecells was measured as usual (8).Changes with Time of Intracellular and Extracellular 2,4-D

Concentrtions during Growth of Population. Cells were inocu-lated into 0.32 ,UM [14C]2,4-D. The growth of the cell populationand the extracellular and intracellular concentrations of free 2,4-D molecules were measured from aliquots pipetted at intervals.The extracellular concentration of free 2,4-D was obtained bymeasuring the radioactivity of the ether-soluble fraction of thefiltered aliquots according to the previously described procedure(8). The intracellular concentration of free 2,4-D molecules wasestimated from the amount of radioactivity diffused out into 10ml of a Tricine (0.1 M) (pH 7) buffer by about 100 mg of cellscollected from the aliquots filtered with a vacuum intensity ofabout 20 cm of mercury.

In such conditions the diffusion of free 2,4-D is complete within3 h, the amount of radioactivity released during a second experi-ment being negligible. Furthermore, we verified that 2,4-D glu-coside did not diffuse out from living cells and that the 2,4-Dglucoside molecules found in the diffusion fluid originated fromdead cells. Consequently, the amount of intracellular free 2,4-Dmolecules was estimated from the radioactivity ofthe ether-solublefraction of the exsorption fluid. To calculate an intracellular

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KURKDJIAN, LEGUAY, AND GUERN

concentration of free 2,4-D, molecules were assumed to be ran-domly distributed within the over-all cell volume.Measurement of Intracellular pH. The method used is based on

the distribution of DMO molecules between the extracellularmedium and the cells. DMO (12 mCi/mmol; 99% radiochemicalpurity) was obtained from C.E.A. (France). Briefly, calculationsof intracellular pH (pHi) were made from the equation ofWaddeland Buttler (18):

1 + 1o(pi-pKa)Ci/Ce =

1+ I0pHe-p'^

where Ci and Ce are, respectively, the intracellular and extracel-lular DMO concentrations; pHi and pHe the intracellular andextracellular pH and pKa that ofDMO (6.30).The detailed procedure used to measure Ci, Ce, and pHe has

been previously described (5, 6).Distribution of Sizes of Clusters of Cells. The sizes of the

clusters of cells and their distribution were determined by filteringthe cell suspension through nylon cloth filters of different poresize. The clusters were distributed into seven classes according totheir size, and the cells from each fraction were collected byfiltration on a Whatman glass fiber filter (GF/A) and weighed.

Distribution of Sizes of the Cells. Aliquots of cell suspensionswere pipetted for observation under the light microscope andphotographs of at least 1000 cells were taken randomly at amagnification of lOx. The negative film was enlarged on Kodalithsuper ortho to a final magnification of 200x. The maximumlength of the cells was estimated by measurement of the diameterof the circumscribed circle using a particle analyzer from Zeiss.The results were registered by a counter and a calculation gavethe coordinates of the distribution curve which accounts for themorphology of a whole population of cells.For the measurement of the action of FC on the sizes of the

cells in short term experiments, we used a TV camera set. In eachcase, 150 cells were directly measured on the screen.

RESULTS

Action of FC on Extracellular pH of Culture Medium. Figures1 and 2A show that the extracellular pH (pHe) of cultures (0.2,lM 2,4-D and 4 ,llM 2,4-D) exposed to 2 pM FC was stronglylowered. the influence of FC was expressed rather rapidly, and,after 280 min treatment, the decrease in pHe was about 0.9 pHunits for the cells grown on 4 ym 2,4-D and about 0.3 pH units for

I

CL

x 6.5w

100 200 300Time (minutes)

FIG. 1. Influence ofFC on extracellular pH ofA. pseudoplatanus cells.FC was added to a final concentration of 2 jLM to a population of cells

grown for 21 days on 0.2 pm 2,4-D (population density: about 5.5 x 106

cells/ml) and to a final concentration of 3 pi to a population of cellsgrown for 14 days on 4 pm 2,4-D (population density: about 2.5 x 106

cells/ml). The evolution of extracellular pH (pHe) was followed in the

flasks of treated cells and their corresponding controls with an Heito pHmeter, the electrode being plunged directly into the shaken suspensions.

x

71w

4 6Time (days)

-E

0 2 4 6 8Time (days)

FIG. 2. Influence of FC on regulation of cell division in A. pseudopla-tanus. In a first phase, cells were inoculated at the same initial populationdensit,y (about 30,000 cells/ml) into two flasks containing 250 ml of thestandard medium with 0.2 um 2,4-D. After 14 days, when the stationaryphase of growth was reached, FC was injected into one flask to 2 ,UM finalconcentration. From that time (plotted as zero time), aliquots were pipettedat intervals from the two flasks for extracellular pH measurements (A)and cell number determinations (B).

the cells grown on 0.2 ,lM 2,4-D. The difference between these twopH modifications is to be related to the fact that the populatondensities are quite different in these two conditions (about 2.5 x106 and 5.5 x 105 cells/ml).For the control and FC-treated cell populations cultivated on

4 UM 2,4-D, a rise of extracellular pH was first registered. We haveshown that it is linked to the removal of CO2 accumulated by suchdense cell population (2, 6). Despite this strong rise, the acidifi-cation induced by FC is expressed as early as 30 min.Influe of FC on "Over-all" Intracellular pH of Acer Cells.

Table I shows that FC strongly modified the distribution ofDMOmolecules between the cells and their medium when the diffusionequilibrium of these weak acid molecules was reached. the intra-cellular concentration was increased about 2.3 times. Calculationsshowed that this modification in the distribution was accountedfor by the extracellular acidification and that no significant intra-cellular pH change was apparently occurring (Table I).

Influence of FC on Distribution of Auxin Molecules betweenCells and Their Culture Medium. We have shown previously (6,8), in agreement with several authors (11, 12, 15, 16), that theintracellular concentration ofauxin molecules in A. pseudoplatanuscells is mainly governed by the relative values of the intracellularpH (pHi) and extracellular pH (pHe), the distribution of auxinmolecules between cells and medium following roughly the equa-tion ofWaddel and Buttler (18). Inasmuch as there is no importantmodification of the over-all intracellular pH induced by FC, onemust expect that the extracellular acidification results in a strongaccumulation of auxin molecules inside the cells.To check this hypothesis the short time effect (1-6 h) of FC on

the distribution of 2,4-D molecules between the cells and theirculture medium was studied first. Cells cultivated in 4 pLM 2,4-D at

a high cell population density were used to obtain a large extra-cellular acidification in a short time. Table II shows that theintracellular concentration of 2,4-D is increased about eight timesand the Ci/Ce ratio about 10 times by the 1 pH unit acidificationobtained after 5 to 6 h of FC treatment, in good agreement withwhat could be predicted from the equation ofWaddel and Buttler(18).The influence of FC on the distribution of auxin molecules

between cells and extracellular medium was studied at the criticalstage of the onset of stationary phase of growth. This situationwas selected as we have shown (8) that, in the condition used, celldivision stops when the intracellular auxin concentration fallsbelow a threshold level.

Table III shows that for the control cell population (Te), the

2xl101M 24-D X Control' o Fusicoccin

410-6M 2.4-D %0 n ControlFusacoccin,$3tdD a o O~~~~~~~~~~~~~-O

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FUSICOCCIN AND CELL DIVISON

extracellular concentration of 2,4-D decreased with time and that,as previously shown (8), about 50% of the initial dose of 2,4-Dwas still present in the medium at the onset of stationary phase(between days 14 and 15). The intracellular concentration de-creased strongly with time as expected from the extracellularconcentration decrease and the extracellular pH increase.

Its threshold level reached between days 14 and 15 was about60,000 dpm/g fresh weight (ie. about 0.5 nmol 2,4-D/g).FC injected at day 13 induced an acidification of the culture

medium and, as expected, a strong modification ofthe distributionof 2,4-D molecules between the cells and their culture medium.The intracellular concentration was increased after 2 days of FCaction to a value equivalent to the one that the cells had duringthe exponential phase of growth. Consequently, as cell division

.9

u

I

-ais20

am

A B C D E F G 20 60 100 1ULength of the cells (u)

Table I. Influence ofFC on Over-all IntracellularpH Measured by DM0Method

FC was injected to a final concentration of 3 pm into a cell populationat log phase ofgrowth, cultivated on 4 pim 2,4-D. After 8 h, ['4CJDMO wasinjected in the control and FC-treated suspensions, the intracellular andextracellular concentrations ofDMO measured, and the over-all intracel-lular pH calculated from the equation of Waddel and Buttler (18) (pKa ofDMO:6.30).

Control Cells FC-treated Cells

DMO absorbed by the cells: 13,990 32,150Ci (dpm/g fresh weight)

DMO in the culture medium: 21,010 20,950Ce (dpm/ml)

Ci/Ce 0.65 1.53pHe 7.02 6.55Calculated pHi 6.79 6.80

Table II. Influence ofFC on Distribution ofAuxin Molecules between theCells and Their Culture Medium

FC was injected to a final concentration of 2 jim into a cell populationat log phase of growth cultivated on 4 jam 2,4-D. The extracellular pH wasfollowed for treated and control cells. After 270 min [14CJ2,4-D wasinjected in the two flasks. One h after, when the diffusion equilibrium wasabout to be reached (8), the amount of [14C]2,4-D absorbed by the cellsand the extracellular concentration of auxin were measured.

Control Cells FC-treated Cells

2,4-D absorbed by the cells: 7,110 56,300Ci (dpm/g fresh weight)

2,4-D in the culture medium: 8,666 6,720Ce (dpm/ml)

Ci/Ce 0.82 8.5pHe 7.09 6.04

FIG. 3. Evidence for dissociation of clusters and enlargement of cellsinduced by FC in cultures of A. pseudoplatanus cells. Experimental con-ditions were the same as those described in legend of Figure 2. Controland FC-treated cell populations were compared 9 days after the injectionof FC at the onset of the stationary phase of growth (ie. about 23 daysfrom the beginning ofthe culture). A: sizes ofclusters and their distributionwere determined according to "Materials and Methods." Ordinate repre-sents weights of cells present in each class as per cent of total weight ofcells of suspension. Class A: 0 to 100 pm; B: 100 to 200pum; C: 200 to 300pm; D: 300 to 500 pm; E: 500 to 1,000 jim; F: 1,000 to 2,000 pm; G: above2,000 pm. B: maximum lengths of cells were estimated according to"Materials and Methods' and the number of cells corresponding to a

given size was plotted against this size for control and FC-treated cells.

stops when the intracellular concentration becomes lower than thethreshold level of 60,000 dpm/g, one must expect that FC, whichincreases the intracellular concentration to a value about 150% ofthe threshold, can promote growth of cell populations at the onsetof the stationary phase of auxin-limited growth.

Inluence of FC on Growth of A. pseuadoplatus Cell Popula-tions. As shown by Figure 2B, FC injected into the cultures whencell division was blocked by a shortage of auxin molecules, theintracellular concentration being lower than the threshold level,induced a reinitiation of cell division after about 48 h. The finalnumber of cells obtained was about twice that of the controlcultures. As we have already shown (4, 7), cell division is reini-tiated 48 h after adding 2,4-D to cell populations cultivated on 0.2p.m 2,4-D and having reached the stationary phase of growth. Thisextracellular addition of 2,4-D increases the intracellular 2,4-Dconcentration above the threshold, and the simplest explanationof the FC effect on the growth of the cell population is that thiscompound, through the extracellular acidification it induces, in-creases the intracellular 2,4-D concentration above the thresold,allowing cell division to resume.

After 9 days other features of cell suspensions were also modi-fied by the FC treatment. First, the size of the cells (measured on

Table III. Changes with Time of Intracellular and Extracellular Concentrations offree 2,4-D Molecules during Growth of an Auxin-limited CellPopulation and Influence ofFC on Distribution ofAuxin Molecules

Cells were inoculated at the same initial population density (about 30,000 cells/ml) into three flasks (Te, FC1, FC2) at an initial [14CJ2,4-Dconcentration of 0.32 pM. At day 13, about a day before the cell populations were expected to reach stationary phase of growth, FC was injected intoflasks FC1 and FC2 (4 pm final concentration). Intracellular and extracellular concentrations of free 2,4-D) molecules, the extracellular pH, weremeasured from aliquots pipetted at intervals, as described under "Materials and Methods." One nmol of [14C]2,4-D is equivalent to about 125,000 dpm.

Titrmellular2,4-D Ci Extracellular2,4-D Ce Ci/Ce ExcdlllarpHTime

Te FC, FC2 Te FC, FC2 Te FC, FC2 Te FC, FC2days dpm/g x o-2 dpm/ml x 10-'3 2391 2533 2337 387 377 371 5.89 6.71 6.30 5.82 5.83 5.846 1935 2223 2041 348 335 334 5.56 6.63 6.11 6.11 6.09 6.1311 887 684 976 280 240 259 3.17 2.85 3.77 6.50 6.36 6.5213 630 609 557 243 202 215 2.60 3.01 2.59 6.60 6.50 6.6214 604 591a 739 236 193 198 2.57 3.06 3.73 6.64 6.20 6.3015 537 970 929 220 175 187 2.44 5.54 4.97 6.68 5.84 5.8016 394 735 842 187 134 153 2.11 5.48 5.96 6.81 5.65 5.79

a Italic numbers refer to measurements made after FC injection into the flasks.

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Table IV. Influence ofFC on the Shape ofA. pseudoplatanus CellsControl and FC-treated cells were compared in the same conditions as

those described for Figures 2 and 3. Length and width of cells wereestimated by measurement of diameters of circumscribed and inscribedcircles and their ratio (r) calculated. Cells of control and FC-treatedpopulations were then distributed among four classes of r values.

No. of Cellsr Classes

Control FC-treated

1-1.4 392 2681.4-1.8 33 991.8-2.2 6 502.2-2.6 2 15

1,300 cells) was strongly increased by FC, as shown by the curves

from Figure 3B, with a mean value of 43.7 ,pm (SD = 15.4) forcontrol cells and 64.8 ,um (SD = 29.6) for treated ones, thedifference being highly significant (1% level). The increase in cellvolume induced by FC was not isodiametric (Table IV), a muchhigher frequency of elongated cells being observed for treated cellpopulations. Second, Acer cells cultivated in a medium containing0.2 im 2,4-D as a limiting factor grow mostly as clusters. Oneconsequence of the treatment of the culture with FC was thedissociation of these clusters which, despite the increase in size ofthe cells, resulted in a decrease in the mean diameter of the clusters(Fig. 3A). The mean size of clusters of FC-treated populations wasabout half that of control cultures. The cells at stationary phase ofgrowth have a typical enlargement response to FC, the kineticaspects and the extent of which are described elsewhere (4).

DISCUSSION

The stimulation of cell division induced by FC is an indirecteffect of that compound. This interpretation is based on our

previous demonstration (8) that auxin controls cell divisionthrough a threshold intracellular level (Ci lim). Cell division isblocked when the intracellular concentration falls below that level,this intracellular concentration being in equilibrium with extra-cellular 2,4-D. Table III shows that an estimate of the thresholdintracellular concentration is 60,000 dpm/g fresh weight or 0.48nmol/g fresh weight. This critical value of the intracellular con-

centration of free 2,4-D molecules at the onset of stationary phasein auxin-limited growth condition is in good agreement with the0.43, 0.52, and 0.45, and 0.51 nmol/g fresh weight values obtainedin four independent experiments. Table III shows also that 48 h

after FC injection, the intracellular concentration reached (0.75nmol/g) is about the same as that reached by the cells at day(about 0.68 nmol/g). At day 1, the cell populations of the three

Te, FC1, and FC2 flasks are in exponential growth phase, themean number of cells per ml being about 300,000. The maximumnumber of cells reached at stationary phase by the control popu-lation (Te) is about 800,000 cells/ml. This shows that from daymore than one cell generation is produced when the intracellular2,4-D concentration decreases from 0.7 nmol to 0.5 nmol/g.Consequently, it appears that the increase of the intracellular 2,4-D concentration induced by FC is sufficiently high to account for

the increase in cell number illustrated by Figure 2B.The difference between our results and those of Robo et al. (13)

is likely due to the fact that these authors washed their cells toeliminate 2,4-D from the culture before the injection of FC. Insuch a case of a very low residual extracellular 2,4-D concentra-tion, one could expect that the intracellular auxin concentrationis not increased enough by the FC treatment to reach Ci lim. In

agreement with the results obtained by these authors, we haveshown that when 1 volume of an aliquot of a cell suspensioncultivated on 0.2 AM 2,4-D and sampled at the onset of stationaryphase of growth was inoculated into 4 volumes of 2,4-D free

medium containing FC, no growth of the cell population was

observed (unpublished results). Furthermore, an experiment sim-ilar to that described by Figure 2B, but in which FC was added 4weeks after the onset of stationary phase in order to get a very lowextracellular 2,4-D concentration at the time ofFC action, showedthat FC did not induce an increase in cell number while a 2,4-Daddition did.These results emphasize the facts that FC has no direct action

on cell division and that in order to get the FC-induced stimulationof growth the intracellular auxin concentration has to be not toofar below the threshold level.

Table III shows that if it is assumed that the intracellular pH iskept constant the distribution of 2,4-D molecules between the cellsand their culture medium follows roughly the equation ofWaddeland Buttler (18). However, some quantitative discrepancy is ob-served for the lowest and highest extracellular pH. This couldmean that a small permeability of the plasmalemma and/or thetonoplast to the anionic form of 2,4-D is involved, as suggested byRaven (11), and modifies the distribution predicted by the equa-tion of Waddel and Buttler (18). This problem of the penetrationof the anion is now under consideration.Comparison of the results of Tables II and III shows that the

FC-induced extracellular acidification appears to promote a largermodification of the distribution of 2,4-D molecules in short termexperiments (6 h, Table II) than in long term experiments (48 h,Table III). This difference is probably accounted for, to a largeextent, by the FC-induced cell enlargement. A 60 to 80%1o increasein mean cell volume has been estimated 55 h after the beginningof the FC treatment as opposed to the very small modificationsobserved after 6 h. Theoretical calculations from the equation ofWaddel and Buttler (18) show that more than 95% of intracellular2,4-D molecules are located in the cytoplasm. If it is assumed thatthe FC-induced cell enlargement is based mainly on an increasein vacuolar volume without important modification ofcytoplasmicvolume, one can see that the calculation of an over-all intracellularconcentration from a random distribution within the cell gives aCi value lowered when the cell volume is increased.An increase of intracellular pH linked to proton extrusion has

been postulated several times (3, 9), but the measurement of theover-all pHi by the DMO technique is one of the first directapproaches to this problem. Apparently, FC does not modify theintracellular pH. To account for such a result, it is reasonable tothink, at first, that the intracellular pH is stabilized in a narrowrange by the buffering power of the cell and by the pH-stat systemdescribed by Davies (1).

However, we reported earlier that a modification of the relativevolumes of the vacuole and cytoplasm markedly influences thecalculated value of pHi obtained through the DMO method (6).Consequently, the pHi value of FC-treated cells is probably higherthan that of control cells, contrary to the crude results shown inTable I. This difference is linked to the fact that the calculatedpHi value of FC-treated cells was not corrected for the cell volumeincrease. If these results are corrected for a 25% cell enlargement,assuming the cytoplasmic volume kept constant, the intracellularpH of FC-treated cells would be about 0.06 units above that ofcontrol cells. This increase appears quite small compared to thelarge extracellular acidification and negligible, in comparison withthis extracellular pH drop, with respect to the modification of thedistribution of auxin molecules (Table II). However, as it couldplay a role in the FC-induced organic acid synthesis and growthresponse, it is necessary to reinvestigate more closely the measure-

ment and kinetics of this modification.

Acknowledgment-FC was a generous g from Prof. E. Marre.

LITERATURE CITED

1. DAVIES DD 1973 Control of and by pH. Symp Soc Exp Biol 27: 513-5292. DoREE M, JJ LEGUAY, C TERRINE 1972 Flux de CO2 et modulations de permeabilite cellulaire

chez ls cellules d'Acerpseudoplatanus L. Physiol Veg 10: 115-131

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3. HASCHICE HP, U LurroGE 1977 Auxin action on K+-H+-exchange and growth, 14COrfixation,and malate accumulation in Avena coleoptile segments. In E Marre, 0 Ciferri, eds, Regu-lation of Cell Membrane Activities in Plants. Elsevier, Amsterdam, pp 261-265

4. KuRsnAN A 1979 Action de la fusicoccine sur la morphologie et le grandissement de cellulesd'Acerpseudoplalanus cultivees in vitro. Physiol Veg, 17: 303-313

5. KuRx.rusN A, J GuEIW 1978 Intracellular pH in higher plant cells. I. Improvements in the useof the 5,5-dimethyloxazolidine-2("C], 4-dione distribution technique. Plant Sci LettI 1: 337-344

6. KuRuDn.&N A, JJ LEGUAY, J GuERN 1978 Measurement of intracellular pH and aspects of itscontrol in higher plant cells cultivated in liquid medium. Resp Physiol, 33: 75-89

7. LEGUAY JJ, J GUERN 1975 Quantitative effects of 2,4-dichlorophenoxyacetic acid on growth ofsuspension-cultured Acer pseudoplatanus cells. Plant Physiol 56: 356-359

8. LEGUAY JJ, J GUERN 1977 Quantitative effects of 2,4-dichlorophenoxyacetic acid on growth ofsuspension-cultured Acer pseudoplatanus cells. II. Influence of 2,4-D metabolism and intra-cellular pH on the control of cell division by intracellular 2,4-D concentration. Plant Physiol60: 265-270

9. MARRE E 1977 Effects of fusicoccin and hormones on plant cell membrane activities: obser-vations and hypotheses. In E Marre, 0 Ciferri, eds, Regulation of Cell Membrane Activitiesin Plants. Elsevier, Amsterdam, pp 185-202

10. MARRaE E, P LADo, F RAsI CALDOGNO, R CoLomwo, M Cocucci, MI DE MscstmTs 1975Regulation of proton extrusion by plant hormones and cell elongation. Physiol Veg 13: 797-

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81111. RAVEN IA 1975 Transport of indoleacetic acid in plant cells in relation to pH and electrical

potential gradient and its significance for polar IAA transport. New PhytoL 74: 163-17212. RAVEN JA, FA SMTrrH 1977 Characteristics, functions and regulation of active proton extrusion.

In E. MarrE, 0 Ciferri, ods, Regulation of Cell Membrane Activities in Plants. Elsevier,Amsterdam, pp 25-40

13. RoLLo F, E NIELsEN, R CEULA 1977 Cell division and ion transport as tests for the discrimi-nation between the actions of 2,4-D and fusicoccin. In E Marre, 0 Ciferri, eds, Regulationof Cell Membrane Activities in Plants. Elsevier, Amsterdam, pp 261-265

14. RoLLO F, E NIESEN, F SALLA, R CELPA 1977 Effect of fusicoccin on plant cell cultures andprotoplasts. Planta, 135: 199-201

15. RuBERY PH 1978 Hydrogen ion dependence of carrier-mediated auxin uptake by suspensioncultured crown-gall cells. Planta 142: 203-206

16. RuBERY PH, AR SHELDRAE 1973 Effect of pH and surface charge on cell uptake of auxin.Nature 244: 285-288

17. SPARAPANo L 1976 The action of fusicoccin alone and with some plant growth substances ontobacco tissue cultures. Physiol Plant 38: 323-326

18. WADDEL WJ, TC BumTnER 1959 Calculation of intracellular pH from the distribution of 5,5-dimethyl-2,4-oxazolidinedione (DMO). Application to skeletal muscle of the dog. J ClinInvest 38: 720-729

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