J. Cell Sci. 10, 315-333 (i972) 315

Printed in Great Britain

ZOOSPORE GERMINATION IN

BLASTOCLADIELLA EMERSONII

IV. ION CONTROL OVER CELL DIFFERENTIATION

D. R. SOLL» AND D. R. SONNEBORNf

Department of Zoology, Zoology Research Building,University of Wisconsin, Madison, Wisconsin, 53706 U.S.A.

SUMMARY

Zoospore germination in B. emersonii is accompanied by a series of abrupt, dramaticchanges in cell structure. Membranes appear to be variously involved in many of these changes.Germination is subject to simple manipulations of the ionic environment: swimming zoo-spores can be maintained for long periods in the buffered CaCl2 solution into which they areinitially released, whereas dilution into a solution containing KC1 and MgCls in addition toCaCl2 results in rapid, semisynchronous germination of entire zoospore populations.

The control of germination by ionic means has been characterized in the following ways:(a) Very brief (40 s to 2 min) exposure to GS, followed by replacement with buffered CaCl2,

is as effective as continuous exposure in eliciting rapid germination of the entire zoosporepopulation.

(b) The effective component of GS is KC1: GS lacking KC1 does not elicit rapid germina-tion; conversely, buffered KC1 alone is as effective as complete GS in eliciting germination.

(c) Zoospore populations are sensitive to KC1 concentration; as the KC1 concentration isreduced, the proportion of cells which undergo rapid germination is also reduced.

(d) At optimal concentration (5 x io~s M), the following salts are equally as effective as KC1in eliciting germination: KI, KBr, NaCl, CsCl, RbCl, and choline chloride.

(e) At high concentrations (2-5-5 x IO~2 M ) . CaCla and MgCl, elicit semi-synchronous con-version of zoospores to round cells, but only after sizeable delays (v. KC1). Conversion of roundcells to germlings does not occur in MgCl2 and is enormously delayed in CaCl8; when formed,the germ tubes appear abnormal.

(/) Monovalent cation salts of complex divalent anions (sulphate, tartrate, molybdate,tungstate) also exhibit decreased effectiveness (v. KC1) in eliciting germination.

(g) The monovalent cation salts NH4C1 and LiCl, the divalent cation salt MnCl2, and thenon-ionic compound sucrose are all ineffective in eliciting rapid germination. When in com-bination with an effective elicitor (KC1), LiCl totally blocks germination, MnCl2 and sucroselead to significant delays in zoospore to round cell conversion, while NH4C1 has no effect onthe population kinetics.

(h) LiCl can block germination even when added after the completion of the otherwisesufficient early exposure period to GS (see (a) above). The blocking effect of LiCl can be almostcompletely reversed by replacement with KC1.

On the basis of this characterization it is concluded that (1) rapid germination is not elicitedsimply by osmotic shock; rather, the cells are capable of responding to other (especially ionic)properties of their chemical environment; and (2) while brief exposure to KC1 is sufficient toelicit germination, there are evidently other ion-sensitive steps occurring after the completionof this initial exposure period. Implications of the results in relation to the regular ion selectivitypatterns found in other ion-dependent systems, the possible site(s) of action of the elicitingcompounds, and the newly discovered 'zoospore maintenance factor' are discussed.

• Department of Biology, Brandeis University, Waltham, Massachusetts, U.S.A.f To whom requests for reprints should be sent, at Wisconsin.

316 D.R. Soil and D. R. Sonneborn

INTRODUCTION

A variety of cells, both prokaryotic and eukaryotic, can respond rapidly anddramatically to initiating stimuli. The immediate cellular responses to such initiatingstimuli need not involve concurrent changes in gene expression at the transcriptionalor translational levels; yet the responses can involve profound changes in cell archi-tecture and function (see discussions in Jaffe, 1969, and Soil & Sonneborn, 1971a;also Moser, 1939, and Fields & Luria, 1969). With surprisingly few exceptions, verylittle is as yet known concerning even the location of the cell components involved insuch responses, much less the chemical mechanisms involved in the reception, trans-mission and/or amplification of the initiating stimuli. Nevertheless, the widespreadoccurrence of such responses, and particularly their occurrence during initial eventsof many cellular differentiations, encourage the view that elucidation of the mechan-isms involved will be necessary for complete understanding of how cells changephenotype.

Zoospore germination in the water mould Blastocladiella emersonii is characterizedby certain features which make it an experimentally convenient process for thedesired kinds of analysis. Most of the known changes accompanying the differentiationof zoospore to vegetative cell can evidently take place without concomitant RNA andprotein synthesis (Soil & Sonneborn, 1971a, b). A sequence of dramatic changes incell architecture has been described (Soil, Bromberg & Sonneborn, 1969; Lovett,1968); most of these changes appear to involve, directly or indirectly, membranes(Soil et al. 1969; Truesdell & Cantino, 1970). In view of the roles played by ions in avariety of membrane phenomena (for reviews see Hodgkin, 1964; Pressman, 1968;Whittam & Wheeler, 1970; Schwartz, 1971), it was extremely provocative to discoverthat this differentiation could be initiated and controlled by simple manipulationsof the ionic environment (Soil & Sonneborn, 1969).

Our previously standardized procedure (Soil et al. 1969) involves, first, washingmature vegetative cells free of defined growth medium and incubating them inbuffered CaCl2. Zoospore release begins some 3 h later and is completed within thenext 30 min. The progeny zoospores can then be maintained at 20 °C for periodsgreater than 16 h if incubated at high cell concentrations in the buffered CaCl2 solu-tion into which they were initially released. At any time during this period, semi-synchronous and complete germination can be induced by simple dilution into asecond inorganic solution containing, in addition to buffered CaCl2, KC1 and MgCL.

In this paper, we examine the active component(s) of the germination solution,the specificity of initiating stimuli, the kinetics of initiation, and the possibility ofseparating ion-dependent steps.

METHODS

Obtaining zoospores for the assayMaintenance of stocks and growth conditions for liquid cultures were identical to those

presented by Soil et al. 1969. Briefly, 2-4 x ioe zoospores were standardly inoculated into 10 mlof liquid growth medium, DMS, in 100 x 20-mm tissue culture dishes (Falcon no. 3003) and

Ions and cell differentiation 317

grown for 12 h at 20 °C. Growth was terminated by decanting this medium, washing theplates 4 times with 3 ml of sporulation solution (SS: io~s M CaCla, io~3 M Tris-maleate buffer,pH 6-7) and then incubating the sporangia at 27 °C in SS. The release of zoospores (completionof sporulation) was complete within 3'5—40 h and the zoospores were then incubated 30—60 minin the same SS into which they had been released.

Assay of germination

Unless otherwise stated, the refined dish assay (Soil et al. 1969) was routinely employed tomonitor the conversion of zoospores to round cells. 03 ml of zoospore suspension (about8 x io6 zoospores) obtained as described above was diluted into 3-7 ml of germination solution(GS: 5 x io~* M KC1, io~a M MgCl,, io~s M CaClj, io~3 M Tris-maleate buffer, pH 67) ina series of 60 x 15-mm tissue culture dishes (Falcon no. 3002) at 27 °C. Identical procedureswere used for the variety of alternative germination solutions tested. Zero time for all the plotsexhibited represents the moment of dilution into the tested germination solution. At timeintervals, dishes were swirled to suspend zoospores; cells undergoing germination remainedfixed to the dish bottom. A sample of suspension was rapidly removed from the dish and addedto neutralized formalin solution for concentration counts. If desired, the bottom of each dishwas also fixed for measurement of the proportions of round cells and germlings. Knowledgeof the initial zoospore concentration, the zoospore concentration at the time points, and theproportion of round cells and germlings at the time points allows calculation of the concentra-tion of round cells and germlings (see Fig. 1, p. 318). In the majority of experiments presented,only zoospore to round cell conversion was monitored. The term 'germination' is used tosignify only this phase of the differentiation unless otherwise stated.

RESULTS

Brief description of morphological changes during germination

The conversion of zoospore to vegetative cell can be temporally separated into3 cell types by routine light microscopy: the zoospore, the intermediate 'round cell',and the germling. The zoospore's most prominent ultrastructural features includea posterior flagellum, a single nucleus and nucleolus, a membrane-bound 'nuclearcap' overlying the nucleus and containing virtually all of the cell's cytoplasmic ribo-somes (Lovett, 1963), a single large mitochondrion eccentrically positioned aroundthe nucleus-nuclear cap assembly, an adjoining 'lipid sac', and several cytoplasmicvesicles. The nuclear cap, nucleus, mitochondrion, lipid sac, and flagellar apparatusare regularly positioned with respect to one another (Cantino, Lovett, Leake &Lythgoe, 1963; Cantino, Truesdell & Shaw, 1968; Reichle & Fuller, 1967; Soil et al.1969). Shortly after initiation of germination, the flagellar axoneme is withdrawn intothe cell proper. At about the same time, open channels begin to appear between mem-brane-bound cytoplasmic vesicles and the plasma membrane; at least certain of thesevesicles appear to release particulate material into the channels. A new organelle, theinitial cell wall, is formed and the cell assumes a round shape, hence the name ' roundcell'. The next series of events includes: disintegration of the membrane-boundnuclear cap with concomitant release of ribosomes into the cytoplasm and appearanceof rough endoplasmic reticulum; disappearance of the typical zoospore morphologiesof the lipid sac and at least one class of cytoplasmic vesicles, the 'gamma particles';branching and finally fragmentation (Bromberg & Sonneborn, in preparation) of thepreviously single mitochondrion; and disappearance of the internal flagellar axoneme.Finally, a cellular protuberance, the germ tube (hence the name germling), is formed,

3*8 D. R. Soil and D. R. Sonneborn

presumably via a localized evagination of the plasma membrane and cell wall. Thefrequent involvement of membranes in the series of structural events is obvious fromthe above description (see Soil et al. 1969, for pictorial documentation and Soil &Sonneborn, 1971a, for quantitative documentation of the above description).

Germination kinetics in standard germination solution

Upon dilution of a zoospore suspension into standard GS, motility is momentarilylost and the cells assume an elongate morphology. Within 2 min, motility is regainedand the cells become progressively rounder. After an initial lag period of approxi-

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Fig. 1. Population kinetics of germination in the dish assay.• , zoospores; O, round cells; A, germlings.

mately 6 min (Fig. 1), the proportion of zoospores in the population begins decreasingand continues at a roughly constant rate for the next 15 min; thereafter, the ratediminishes. The proportion of round cells reaches a peak at about 20 min and thendecreases as the reciprocal of the germling appearance plot. Germlings begin appear-ing at 20 min and accumulate at a roughly constant rate so that by 37 min they con-stitute 80% of the population; thereafter, the rate decreases.

The zoospore lag period is a measure of the time prior to the earliest conversions tothe round cell in the population and the TM (the time necessary for 50% of the

Ions and cell differentiation 319

zoospores to become round cells) is the time of conversion for the average cell in thepopulation. The slope of the zoospore disappearance plot furnishes a measure ofpopulation heterogeneity.* If slope and extent (measured as the real or extrapolatedplateau of the zoospore disappearance plot) of germination remain constant, thendifferences between populations in either lag or TM measure differences in the rate ofcell conversion.

The central role of KCl in standard germination solution (GS). Standard germinationsolution was initially constructed to maximize synchrony and extent of germination,and consists of 5 x io~2 M KCl, io~2 M MgCl2, io~3 M CaCl2, and io~3 M Tris-maleate

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Fig. 2. Kinetics of zoospore disappearance in GS, KCl alone, or GS lacking KCl. O,GS(s x I O - ' M K C I , io- 'MMgClj , io-3MCaCl2, I O - 3 M Tris-maleate buffer, pH 6-8);A, KCl alone (5 x io"s M KCl); • , GS lacking KCl (io"s M MgCl2) io"3 M CaCla,io~3 M Tris-maleate, pH 6-8).

• The experimental points can be fitted to linear curves (see Fig. 1) and the slopes approxi-mated by these fits furnish simple, convenient indices for comparing populational heterogeneityunder different experimental conditions. Analysis of the points by the probit technique, how-ever, suggests that the times of cell transformation in the population are distributed normallyaround the mean rather than linearly (Soil et al. 1969).

320 D. R. Soil and D. R. Sonneborn

buffer (containing io~3 M NaOH for adjustment of pH). This solution differs fromsporulation solution (SS) only by the addition of KC1 and MgCl2. If all com-ponents of GS are omitted except 5 x io~2 M KC1, the population kinetics of zoosporeto round cell conversion are indistinguishable from the kinetics in complete GS(Fig. 2). This is also true for the population kinetics of germ-tube appearance. Con-versely, if KC1 is omitted from GS, only a very small proportion of the zoosporesgerminate over a time interval sufficient for essentially all zoospores to germinate incomplete GS (Fig. 2).

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Fig. 3. KC1 concentration in GS and the kinetics of zoospore disappearance. A, plottedas the percent total population, and B, plotted as the percent germinating popula-tion (measured as % germinated by 35 min). KC1 concentrations: A, 6 X I O ~ 3 M ;A, 1 x io~* M ; • , 2-6 x 10"1 M ; O, 5 x io~a "• M .

The population of zoospores is sensitive to KC1 concentration. Decreases in KC1concentration result in decreases in germination by the population (Fig. 3 A). Themain and dramatic effect is upon the proportion of cells which rapidly germinate andis not upon the rate of individual cell conversion. This point can be seen graphicallyby replotting the points of Fig. 3 A as the percent of those cells which do germinate(by 35 min) rather than as the percentage of total cells. The plot (Fig. 3B) shows that,

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Fig. 4. Kinetics of zoospore disappearance after brief exposure to GS. • , 2 min in SSand resuspension in SS (negative control); O, 2 min in GS and resuspension in GS(positive control); A, 2 min in GS and resuspension in SS (test). For this type ofexperiment, zoospores were initially concentrated by removing the upper half of theincubation medium (SS) from sporulated dish cultures. The concentrated suspensionswere then diluted 1:4 into the initial 2-rrun incubation medium (either SS or GS)and mixed. After 1 min, the cells were pelleted rapidly (45 s) and the supernatantdecanted (total time = 2 min). The pellets were gently suspended in the second incuba-tion medium and plated. Germination was monitored by an alternative method; eachdish culture was examined at successive time points under an inverted phase micro-scope and, for each time point, the % cell types were determined. This method,while less burdensome and precise than the refined dish assay, yields comparablegermination kinetics.

Fig. 5. The effect of growth density on the kinetics of zoospore disappearance in10"' M KC1 germination solution (io~s M KC1, icr2 M MgCla, io~3 M Tris-maleatebuffer, pH 6-8). O, zoospores originating from low sporangial density dish cultures(1 x io8 cells/io ml DM2); • , zoospores from high-density cultures (7 x io" cells/10 ml DM,). Supernatants from settled suspensions of 'low origin' zoospores werepartially removed so that cell densities of 'high' and 'low' origin zoospores duringgermination would be roughly equivalent.

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3 2 2 D. R. Soil and D. R. Sonneborn

for the rapidly germinating portion of the population, lag and slope remain unaffectedby KC1 concentration.

The kinetics of initiation. Under standard germination conditions, the cell populationremains in GS throughout transformation. However, zoospores need only briefexposure to GS for rapid, synchronous and complete germination.

To test this point, zoospores were exposed to GS for various times and were thenrapidly centrifuged and resuspended in either GS (control) or SS (test) - refer toFig. 4 legend for method. Fig. 4 shows that for a total original exposure (includingcentrifugation time) of 2 min, zoospores resuspended in SS germinate with kinetics

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Fig. 6. Zoospore disappearance kinetics in the chloride salts of a variety of monovalentcations in GS (5 x io~* M of the test chloride, io~2 M MgClj, io~3 M CaCU, io~3 MTris-maleate, pH 6-8). O, K+ control: • , tested monovalent cation.

indistinguishable from zoospores continuously exposed to GS. Other experimentshave indicated that as little as 40 s exposure to GS at 24 °C is sufficient to elicit nearlynormal germination kinetics (Soil, 1969). The mechanics of the brief exposure pro-cedure do not per se elicit significant germination, since other portions of the zoosporepopulation sent through the procedure but exposed only to SS, exhibit no more thana small percent germination (Fig. 4). (Note. It is especially important in this type ofexperiment to use aged zoospores (at least 1 h old) because freshly released zoosporesexhibit significant levels of germination when pelleted and resuspended in fresh SS(see Discussion).)

The surprising and important result here, then, is that exposure times to GS whichare only a fraction of the time required for the first zoospores to convert to round cellsare sufficient to elicit rapid, synchronous conversion of the entire population.

Biological control of ion sensitivity: effects of zoospore origin. As the cell density ofgrowth cultures is increased, the progeny zoospores display an increasing asynchronyof germination in standard germination solution. It was demonstrated that thisdensity-dependent effect arises by the time progeny zoospores are harvested and

Ions and cell differentiation 323

that it is not determined by cell density during subsequent germination (Soil &Sonneborn, 1969).

An additional property of 'high origin' zoospores can now be documented. Highorigin zoospore populations (originating from dish cultures containing 7 x io8 sporan-gia per dish) germinate less completely in sub-maximal KCl concentration than do ' loworigin' zoospores (originating from dish cultures containing io6 sporangia per dish),

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Fig. 7. Effects of inactive monovalent cations on zoospore disappearance in thepresence of KCl. All solutions contained i c r ' M Tris-maleate, pH 6-8. A, NH4C1: O,2-5 x io"1 M KCl; • , 25 x io"1 M KCl, 2-5 x io"2 M NH,C1; A, 2-5 x io"2 M NH4C1.B, LiCl: O, 25 x I O - 2 M KCl;io-2 M LiCl.

2-5 x io~2 M KCl, 2-5 x io~a M LiCl; A, 2-5 x

e.g. at io~2 M KCl, 42 and 83% of 'high' and 'low' origin zoospores respectivelygerminated by 45 min (Fig. 5). Moderately low origin zoospores (originating fromcultures containing 2-4 x io6 sporangia per dish) are used throughout this paper, butthe ability to alter predictably the sensitivity of zoospore populations to KCl concen-tration may provide a useful tool for certain kinds of experiments (Soil, 1969).

Specificity of initiating ions

Monovalent cations. After KCl was identified as the active ingredient in standardgermination solution, other monovalent cations were tested as possible substitutes.At 5 x io~2 M the chloride salts of Na+, Cs+, Rb+ and choline+ were found to sub-stitute perfectly for K+ in GS (Fig. 6). Both Cs+ and choline+ have been examinedover a concentration range; in both cases, concentration dependencies were exhibited.

324 D. R. Soil and D. R. Sonneborn

A mixture of 2-5 x io~z M each of Na+ and K+ yielded germination kinetics identicalto 5 x io~2 M of either ion alone; in other words, the effects of the 2 cations in mixturewere additive (Fig. 6).

The above observations could be interpreted to mean that either those monovalentcations tested work equally well at the particular concentrations used, or that in factthe monovalent anion Cl~ is the specific ion responsible for the effect. However, notall chloride salts are effective in eliciting germination. Neither NH4+, Li+, nor the

Fig. 8. LiCl-elicited inhibition of germination; effect of delayed addition and reversi-bility. All salts were 5 x io~a M in io~3 M Tris-maleate, pH 6-8. O, 5 min in KC1 andresuspension in KC1 (control); A, 5 min in KC1 and resuspension in LiCl (delayedaddition); • , 15 min in LiCl and resuspension in KC1 (reversibility). The methodsof rapid pelleting of zoospores and rapid scoring under the inverted microscope wereused (see Fig. 4 legend). Percentage germination was counted and rounded off to thenearest 5 %.

divalent cation Mn2+ as chloride salts are effective (see Fig. 6 for NH4C1 and LiCl;Fig. 12, p. 327, for MnCl2). The effects of NH4C1 particularly argue against the possi-bility that chloride per se is the biologically effective ion. Not only does NH4C1 alone(or in GS lacking KC1) fail to elicit rapid germination, but also, when combined inequimolar (2-5 x io~2 M each) concentration with KC1, NH4C1 has no effect, i.e. theequimolar mixture elicits germination with kinetics indistinguishable from 2-5 x IO~2M

KC1 alone (Fig. 7A). NH4C1 alone in GS is not lethal since, as is the case with GSlacking monovalent cations, germination can eventually be completed after very long

Ions and cell differentiation 325

time intervals (several hours). The failure of NH4C1 to elicit rapid germination alsospeaks against the effect being due simply to osmolarity (see below).

The chloride salt of Li+ also elicited no germination when substituted for K+ inGS (Figs. 6 and 7B). In the presence of equimolar concentrations of KC1 and LiCl nogermination was detectable (Fig. 7B) even after 8h incubation. Therefore, unlikeNH4C1, LiCl inhibited the effectiveness of KC1 when in combination. Inhibition byLiCl can, however, be at least partially reversed. When zoospores were incubated in5 x io~2 M LiCl-GS (i.e. standard GS in which KC1 was replaced by LiCl) for 15 min,washed, and then incubated in 5 x icr2 M KC1-GS, germination did begin, but onlyafter a 20-min lag period (Fig. 8). Germination then proceeded synchronously butincompletely, plateauing at about 20% zoospores. The cells which did germinate(about 80%) formed abnormally thickened germ tubes.

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Fig. 9.The effect of divalent cations on zoospore disappearance kinetics in 5 x 1 o~3 M KC1germination solution. • , 5 x icr3 M KC1, io~" M MgCl2, io~3 M CaClB, and icr3 MTris-maleate, pH 6-8; O, 5 x io"3 M KC1, io"3 M Tris-maleate.

Since only a short exposure of a population to a high concentration of K+ wassufficient to elicit maximal germination kinetics (Fig. 4), an attempt was made to testwhether both K+ and Li+ affected the same event. After exposure to 5 x io~2 M KC1for 5 min (i.e., at about the termination of the standard lag period), the cells wererapidly pelleted and resuspended in either 5 x io~2 M KC1 (control) or 5 x io~2 M LiCl(test). The control population had germinated completely by 20 min (Fig. 8). Only 5 %of the test population germinated. Therefore, Li+ blocks the germination processeven after a 5-min exposure to K+. That portion of the population (5%) which didgerminate is presumed to have completed the Li+-sensitive event(s) prior to sub-stitution of LiCl for KC1.

Divalent cations. In standard GS, varying the concentrations of the divalent cationsalts CaCl2 or MgCl2 has no effect on germination kinetics, although concentrationsequal to or above 5 x io~2 M result in significant cell death. An effect is observed, how-

326 D. R. Soil and D. R. Sonneborn

ever, if the concentration of KC1 is submaximal. At 5 x icr3 M KC1, the difference ingermination kinetics in the presence versus absence of icr3 M CaCl2 plus icr2 M MgCl2

is substantial (Fig. 9). This indicates that at low, but not at maximal, KC1 concentra-tion the divalent cations either enhance the effect of the monovalent or express anadditive effect of their own.

The latter is probably the case since CaCl2 and MgCl2 at concentrations higherthan in GS exhibit observable effects upon germination when in combination withbuffer alone. Each can initiate synchronous germination, but with interesting dif-ferences from corresponding concentrations of KC1. At 5 x io~2 M CaCU the syn-chrony (slope) and extent of round cell formation are indistinguishable from that in

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Fig. 10. Zoospore disappearance kinetics in high-concentration CaCl2 alone and incombination with KC1. All solutions contained io~3 M Tris-maleate, pH 6-8.O, 5 x io~* M KC1; A, 5 x icr1 M CaCls; • , 2-5 x io~a M KC1, 2-5 x io"1 M CaCl2.Fig. 11. Zoospore disappearance kinetics in MgCl2 alone compared to CaCl, alone andKC1 alone. All solutions contained io~3 M Tris-maleate, pH 6 7 . All salt solutionswere 2-5 x io"a M since 5 x 10"' M MgCls results in significant cell lysis. O, KC1;• , MgCl2; A, CaCl,.

Ions and cell differentiation 327

5 x io~2 M KCl (Fig. 10). However, the lag period is significantly extended, i.e. theCaCl2 curve is displaced by more than 10 min to later times. The results are similarwhen the salts are compared at 2-5 x io~2 M (Fig. 11). A mixture of equimolar (2-5 xicr2 M each) KCl and CaCl2 resulted in a lag period closer to that of 5 x io~2 M KClalone (Fig. 10). Germ-tube formation was even more dramatically delayed in CaCl2-induced germination; the TM values for germ-tube formation were 30 and 100 min

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Fig. 12. The effect of MnCls in the presence and absence of KCl on zoospore dis-appearance kinetics. All solutions contained io~3 M Tris-maleate, pH 6 7 . O, 5 x io~J MKCl; • , S X I O - ' M K C I , I O " ' M MnCl2; A, io"a M MnClj, or 5 x io"1 M MnCl2;A, io~s M Tris-maleate, pH 6-7, alone.

in KCl and CaCl2, respectively (5 x io~2 M). MgCl2 also initiated synchronous butdelayed round-cell formation when assayed at high concentration (in this case,2-5 x io~2 M). However, the delay was less severe than with equimolar CaCl2 (Fig. 11).On the other hand, the effect on germ-tube formation is much more severe. No morethan 5-10% of the round cells convert to germlings in 2 h.

While both buffered CaCl2 and MgCl2 at high concentrations initiate synchronous

328 D. R. Soil and D. R. Sonneborn

round cell formation, MnCl2 totally fails to elicit germination (Fig. 12). However,when in combination with KCl, MnCl2 affects germination by increasing the lagperiod without affecting either synchrony or extent (Fig. 12).

Anions. The facts (a) that NH4C1 at concentrations equal to KCl did not initiategermination, and (b) that NH4C1 and KCl at equimolar concentrations initiatedgermination kinetics identical to KCl alone, argue against the possibility that it is themonovalent anion Cl~ which initiates the transformation. In fact, the Cl~, I~, andBr~ salts of K+ initiate identical germination kinetics (Fig. 13). However, monovalentcation salts of divalent anions such as SO4

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decreased effectiveness in initiating germination (Fig. 13).

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Fig. 13. Zoospore disappearance kinetics in monovalent cation salts of various anions.The monovalent cation concentration was 5 x io~* M in all cases, A, The halide saltsof K+ were tested in solutions containing io~* M MgClj, io~3 M CaCl2, and io~3 MTns-maleate, pH 6'8. Similar results were obtained when tested with buffer alone.B, The sulphate salt of K+ was tested in buffer alone. Similar results were obtained withio~s M MgClj and io~3 M CaCl,. c, The Na+ salts of molybdate and tungstate, andthe K+, Na+ salt of tartrate were tested in solutions containing io~a M MgCU,io~3 M CaClj, and io~s M Tris-maleate, pH 6-8.

The lack of a simple osmotic effect. Rapid germination is not elicited simply byosmotic shock since, at osmolarities which elicit rapid germination with KCl, neitherLiCl, NH4C1, MnCl2 nor sucrose (a non-ionic compound) elicits germination (seeTable 1 for osmolarities of NH4C1 and sucrose solutions). LiCl blocks germinationeven in the presence of KCl (Fig. 7 B), but the other 3 compounds do not (see Fig. 7 Afor NH4C1, Fig. 12 for MnCl2, Fig. 14 for sucrose).

Both MnCl2 and sucrose, when in combination with KCl, do cause increases inboth lag and Tg,, (Fig. 12 for MnCl2; Fig. 14 for sucrose). These latter effects alsocannot be simply accounted for on the basis of osmolarity. For example, bufferedKCl (5 x io~2 M) and GS differ in osmolarity by 30 mosmol, yet they elicit identical

Ions and cell differentiation 329

germination kinetics. On the other hand, the addition of io~2 M MnCl2 to buffered5 x icr2 M KC1 yields a solution of nearly identical osmolarity to GS (see Table 1); yetthis solution brings about a radical increase in both lag and T^. Similarly the increasesin lag and T^ brought about by using high concentrations of CaCl2 or MgCl2 in placeof KC1 cannot be accounted for just by osmolar effects (see Table 1 for CaCl2).

Table 1. Osmolarity of medium in relation to germination of Blastocladiella

Solution

SS io"3 M CaCl2; io~3 M Tris-maleateGS 5 x io"1 M KC1; io"1 M MgCls; io"3 M CaCl,, io"3 M

Tris-maleate5 x io"1 M KC1; io"3 M Tris-maleate25 x io"1 M KC1; io~3 M Tris-maleate5 x io"s M sucrose; io"3 M Tris-maleate25 x io"2 M KC1; 2-5 x io"a M sucrose; io~3 M Tris-

maleateS x io"1 M NH4CI; i o - ' M MgCl2; io"3 M CaCl2;

io"3 M Tris-maleate5 x io-8 M KC1; io-8 M MnClj, io~3 M Tris-maleate5 x io~a M CaClj; io"3 M Tris-maleate

Osmolarity*

6129

9954SS77

130

131123

Germinationf

_

+ +

+ ++ +—+

—

++

• mosmol kg"1.f + + , Rapid germination of the GS type; + , germination kinetics of the delayed type,

exhibiting an increase in both lag and T60, but no change in extent and slope; —, very low orinsignificant germination within the time necessary for complete germination in GS; of theSS type.

DISCUSSION

Ionic control of germination

Rapid conversion of zoospores to vegetative cells can be rigidly controlled bymanipulation of the ionic environment. The initial conversion of zoospores to roundcells is dramatically influenced by the ionic strength of the environment, but onlycertain ions are effective. This latter fact, together with the fact that sucrose is alsoineffective, indicate that the conversion is not controlled simply by the osmolarity ofthe environment. The possibility has not been ruled out, however, that some veryearly event in zoospore conversion might be sensitive to osmolarity and that theineffective chemicals then do not permit the successful completion of later events inthis conversion. Indeed, one of the ineffective salts, LiCl, can block the completionof the conversion after initial events permitted by KC1 have been completed (seebelow).

In standard germination solution, 5 x io2 M KC1 is the effective initiator, but at thesame concentration the chloride salts of certain other monovalent cations (Rb+, Cs+,Na+, choline+) are equally as effective. The indistinguishable response of cell popula-tions to these various cations, together with the lack of response to LiCl and NH4C1,differs markedly from the regular selectivity patterns found in other ion-dependentsystems and is difficult to reconcile with the theories formulated to explain such

33° D. R. Soil and D. R. Sonneborn

100 r<k fc A

90

80

70

60

s o

40

30

20

10

10 20 30Time, mm

40 50

Fig. 14. Zoospore disappearance in KCl alone, in sucrose alone, and in an equimolarmixture of KCl and sucrose. 'Low origin' zoospores (1 x IO°/IO ml DM4) wereemployed because of their increased sensitivity to lower concentrations of KCl. Allsolutions contained io"3 M Tris-maleate, pH 6-8. O, 2-5 x io"2 M KCl; A, 2-5 xio~* M sucrose, • , 2-5 x io~a M KCl, 2-5 x io~2 M sucrose.

systems (Diamond & Wright, 1969). A further complication which must be taken intoaccount is that, in the presence of an effective initiator (KCl), the ineffective chemicalsmay have neutral (NH4C1), intermediate (MnCl2, sucrose), or totally inhibitory(LiCl) effects. In the series of experiments described in this paper, cationic specificityis tested at concentrations which elicit maximal or near-maximal response (2-5-5 xio~2 M). The possibility must remain that selectivities among the effective monovalentcations might be revealed at submaximal concentrations only. Unfortunately, germi-nation kinetics at very low salt concentrations are more variable and thus reliabledifferences between monovalent cations are difficult to determine. In this respectit should be recalled, however, that concentration dependencies have indeed beenobserved for KCl, CsCl, and choline chloride. For KCl it is interesting that concentra-tion dramatically influences the proportion of cells which germinate rapidly, but that

Iom and cell differentiation 331

the average cell which germinates rapidly appears to do so at the same rate irrespec-tive of KCl concentration.

Initiation by exogenous KCl is completed at very early times - long before the firstseries of morphologically detectable events occur in the earliest cells to convert.KCl need be present for only a fraction of the lag time prior to flagellar retraction; yetthe entire population germinates with kinetics indistinguishable from populationsexposed continuously to KCl. Therefore, either stimulation is completed within theshort exposure interval, or only ion accumulation is sufficiently completed during thisperiod and stimulation may be accomplished at later times. If LiCl is added just priorto flagellar retraction (i.e. after the completion of the KC1-'sensitive' period),germination is reversibly blocked. However, it is not yet clear whether LiCl acts toreverse (or inhibit) an actual effect of KCl or whether it blocks some independentmetabolic or structural event(s). These results do suggest, nevertheless, that theremay be more than one ion-sensitive step during the conversion of zoospore to roundcell.

The anion Cl~ is neither sufficient nor specific in initiating germination. It is notsufficient in that the chloride salts of Li+, NH4+, and Mn2+ do not elicit rapid germina-tion. It is not specific in that K+ salts of other halide anions are equally as effective asthe chloride salt in initiating germination. More complex anions such as SO4

2~ permitgermination but decreases in conversion rate and/or synchrony of germination result.This may be due to metabolic side-effects of these anions, but, more interestingly,it may also be the result of decreased accessibility of the more complex anions to thesite of action. In this latter case the cation could experience greater difficulty inapproaching the site of action because of retardation due to a generated electricalfield. This phenomenon has been used in accounting for the generation of electricalpotential across cell membranes (Woodbury, 1965) and might also suggest that themonovalent cations move across membrane(s) to reach their site(s) of action. Indeed,preliminary release and uptake experiments (Soil, unpublished) indicate that theinitiation of germination can be accompanied by rapid ion movements. 45Ca is releasedfrom pre-labelled zoospores beginning immediately upon dilution in GS (germina-tion) but not upon dilution into SS (zoospore maintenance); Conversely, 137Cs israpidly accumulated upon initiation of germination.

At high concentrations (2-5-5 x I0~2 M)> t n e chloride salts of the divalent cationsCa2+ and Mg2+ are effective initiators but, in contrast to the effective monovalentcations, these divalent cations permit synchronous and complete round cell forma-tion only after extended lag periods. Thus the time at which the average cell convertsfrom zoospore to round cell is increased and this increase is different between Ca2+

and Mg2+. A third divalent cation, Mn2+, does not by itself (as a chloride salt) elicitgermination. In contrast to its monovalent counterpart, NH4+, it increases theaverage conversion time when in combination with KCl.

Preliminary information exists with respect to the ionic control of events beyondround cell formation. On the one hand, brief exposure to exogenous KCl sets in motionthe entire sequence of events involved in germling formation. On the other hand,Ca2+ and Mg2+, at concentrations which permit synchronous but delayed round cell

332 D. R. Soil and D. R. Sonneborn

formation, adversely affect conversion of round cells to germlings. In CaCl2 alone,germ-tube formation is enormously delayed and asynchronous; when formed, thegerm tubes appear morphologically aberrant. In MgCl2 alone, an as yet irreversibleblock to germ-tube formation occurs. These results suggest, then, that there maybe additional ion-sensitive steps occurring after initiation during the conversion ofround cells to germlings.

The several results reviewed above indicate that whether the successive steps ingermination are elicited rapidly, only after significant delays, or not at all is notdetermined simply by the osmolarity of the medium. Rather, the cells must at least inpart recognize differing properties of the chemicals themselves. What these propertiesmight be and how the cells recognize such properties remain intriguing unansweredquestions.

Relation of ionic control of germination to zoospore maintenance factor. Most of theexperiments reported in this paper involve diluting moderately aged zoospores 13-foldinto various test solutions; dilution into SS results in a very low level of germinationat most, whereas dilution into GS results in complete germination. If, instead ofdilution, zoospores are thoroughly washed by centrifugation, significant germinationcan occur even when such zoospores are incubated in SS. Moreover, a factor can berecovered from the supernatant of centrifuged zoospores which prevents germinationof washed zoospores in SS ('zoospore maintenance factor'; Soil and Sonneborn,unpublished).

Ion-induced initiation of germination as it has been studied in this paper must, insome way, counteract the effect of this factor. The model currently under considera-tion is that maintenance factor is loosely (electrostatically?) bound to specificreceptor sites in the zoospore. Either by repeated washing or by the addition ofcertain cations in high concentration, the receptor sites are freed of factor and re-placed by either initiating ions or specific endogenous molecules. While such receptorsites have not as yet been identified, it would not surprise us if they were to be foundin the plasma membrane and/or in many of the organelle and vesicle membraneswhich participate in the series of ultrastructural events accompanying germination.

The excellent technical assistance of Joseph Brennan, Renate Bromberg, and Renate Meuserduring various phases of this work is gratefully appreciated. The work was supported by GrantGB 6030 from the National Science Foundation and by Grant GM 13832 from the NationalInstitutes of Health. The senior author was supported by a predoctoral fellowship on TrainingGrant GM 01435 from the National Institutes of Health.

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CANTINO, E. C , TRUESDELL, L. C. & SHAW, D. S. (1968). Life history of the motile spore ofBlastocladiella emersonii: a study in cell differentiation. J. Elisha Mitchell scient. Soc. 84,125-146.

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SOLL, D. R., BROMBERG, R. & SONNEBORN, D. R. (1969). Zoospore germination in the watermold, Blastocladiella emersonii. I. Measurement of germination and sequence of subcellularmorphological changes. Devi Biol. 20, 183-217.

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SOLL, D. R. & SONNEBORN, D. R. (1971a). Zoospore germination in Blastocladiella emersonii:Cell differentiation without protein synthesis? Proc. natn. Acad. Sci. U.S.A. 68, 459-463.

SOLL, D. R. & SONNEBORN, D. R. (19716). Zoospore germination in Blastocladiella emersonii.III . Structural changes in relation to protein and RNA synthesis. J. Cell Sci. 9, 679-699.

TRUESDELL, L. C. & CANTINO, E. C. (1970). Decay of y particles in germinating zoospores ofBlastocladiella emersonii. Arch. Mikrobiol. 70, 378-392.

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(Received 6 September 1971)