Plant Physiol. (1983) 71, 828-8340032-0889/83/71/0828/07/$O0.50/O

Promotion of Fern Rhizoid Elongation by Metal Ions and theFunction of the Spore Coat as an Ion Reservoir'

Received for publication September 13, 1982 and in revised form December 21, 1982

JOHN H. MILLER, THOMAS C. VOGELMANN, AND ALIX R. BASSELDepartment of Biology, Syracuse University (J. H. M., T. C. V.) and Department ofMicrobiology, UpstateMedical Center (A. R. B.), Syracuse, New York 13210

ABSTRACT

The perine, or outer coat, of spores of the fern Onocea sensibilis L. maybe chemically removed by a brief treatment with dilute NaCIO. Treatedspores germinate normally on glass-redistilled H20, but elongation of therhizoid which is differentiated during germination is severely limited.Rhizoid elongation in perine-free spores, however, is normal when thespores are germinated on Knop's mineral medium or on single-salt solutionsof Ca2+, Mn2+, or Mge+. In intact spores which retain their perine, rhizoidelongation is normal on distilled H20, and the perine serves as a source ofions which are available to the spores and can sustain rhizoid elongation,even when the external medium is deficient. Electron micrographs showthat there are structural differences in the rhizoid wall between perine-freespores germinated on distilled H20 or on nutrient solutions, and also adifference in the number of vesicles in the apical cytoplasm. Localizationof Mge+ and Ca+ in the elongating rhizoid can be visualized with chloro-tetracycline fluorescence. No concentration of these ions can be detectedby this technique in the small rhizoid initial celi before cell elongationbegins.

During spore germination in many fern species, the nucleusmigrates to an eccentric position at one end of the spore prior tocell division. The spore is partitioned by division into two cells ofunequal size. The smaller of the pair differentiates immediatelyinto a rhizoid, whereas the larger continues to divide, and itsderivatives form the prothallus. This pattern has been shown bothby light and electron microscopy in spores of Onoclea sensibilis (1,29).The initial stages of germination in this species, at least through

the completion of cell division, do not depend on an externalsupply of ions, inasmuch as the spores germinate equally well ondistilled H20 or on a mineral nutrient solution (27). Subsequentelongation of the rhizoid, however, is very limited on distilledH20, whereas on a 1 mm solution of a Ca2+ salt, rhizoid elongationis as great as on a complete nutrient solution (28). Elongation islinear for at least 72 h on either nutrient solution or distilled H20,but the rate is lower under nutrient deficiency.

Onoclea spores possess a loose brown outer coat (the perine) aswell as a thick colorless inner coat (the exine). Vogelmann (28)routinely removed the perine prior to culturing spores to see thedetails of germination; coat removal was done by exposing dor-mant spores briefly to a dilute solution of NaC1O. During thework reported in this paper, we discovered that removal of theperine was necessary to produce spores in which rhizoid elongation

'This research was supported by National Science Foundation GrantsPCM-7904593, PCM-8110079, and PCM-8109862.

was limited on distilled H20 and was promoted by Ca2". Our datashow that other metal ions in addition to Ca2+ support rhizoidelongation, that the perine is a source of ions which can maintainrhizoid elongation in the absence of an external supply in themedium, and that there is an apparent localization of metals inelongating rhizoids.

MATERIALS AND METHODS

Spore Collection and Preparation. Spore-bearing fronds ofOnoclea sensibilis were collected near Jericho, VT in early springof 1980 and were stored in plastic bags under refrigeration. Themethods of spore isolation are described by Miller and Greany(19).

Culture Conditions. Dry spores were sown on the surface ofabout 5 ml liquid medium contained in 25 x 100-mm glass culturetubes which were closed with loose fitting aluminum caps. Theculture solution was either Knop's medium (19), glass-redistilledH20, or solutions of different metal salts prepared in glass-redis-tilled H20 from reagent grade chemicals. The following salts wereused: Ca(NO3)2.4H20, MgCl2.6H20, Sr(NO3)2, MnCl2-4H20,FeCl3.6H20, NaCl, KCI, Ni(NO3)2.6H20, Co(NO3)2.H20,ZnCl2, and AlCl3-6H20. All glassware was soaked for 4 to 6 h inconcentrated HNO3 before final washing and rinsing. Cultureswere incubated under continuous cool white fluorescent light atapproximately 3 w/r2 and a temperature of 26 ± 1.C. Sporeswere kept under these conditions for approximately 55 h.Data Collection. When an experiment was terminated, a sample

of spores from each condition was mounted on a microscope slidein a drop of acetocarmine-chloral hydrate stain (11). The prepa-ration was covered with a coverslip, ringed with heavy mineraloil, and heated on a warming tray at 45°C for 2 h. The slides wereexamined with a microscope equipped with a binocular drawingtube. The lengths of a random sample of 100 rhizoids/treatmentwere traced on paper at a magnification of x 310. The length ofeach traced line was measured to the nearest mm and recorded.Mean rhizoid length and the standard error of the mean for eachsample were calculated from these data.

Electron Microscopic Examination. Samples of spores whichhad been germinated either on distilled H20 or on Knop's mediumor on single-salt solutions were fixed overnight at room tempera-ture in 2.5% glutaraldehyde in a microtubule-stabilizing buffer(17). Tannic acid (0.2%) was included in the fixation of somesamples to improve visualization of the outer rhizoid wall. Post-fixation was for 1 h in 1% OS04 in 100 mm sodium-K phosphate,pH 7.2. The material was dehydrated in ethanol and embedded inSpurr's low viscosity embedding medium (26). Sections werestained with uranyl acetate and lead citrate and examined with aSiemens Elmiskop IA or 102.

Metal Gradient Visualization. Germinated spores were rinsedin distilled H20 and mounted on a microscope slide in a drop offreshly made 1 mm chlorotetracycline . HCl adjusted to pH 7.0

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METAL IONS AND RHIZOID ELONGATION

with 1 N NaOH. The spores were examined with a Zeiss Universalfluorescence microscope equipped with an epifluorescence con-denser (III RS) using filter combination UG 51/BG 3/FT 460/LP 478. The exciting wavelengths were in the near UV, and thebarrier fiters transmitted light between about 500 and 750 nm.Photographs were taken with Kodak high speed Ektachrome(ASA 400), Plus-X Pan, or Tri-X Pan, with exposure times between10 and 60 s.Chemical Removal of Perine. The outer spore coat was dissolved

with a dilute solution of NaCIO. We used a commercial hypo-chlorite bleach which was specified to be a 5.25% solution ofNaCIO. A 15% (v/v) solution of the commercial stock was pre-pared. Five to 8 mg of spores were suspended in a few ml of waterplus a drop of a wetting agent (Aerosol OT, 10lo solution, FisherScientific Co.). The suspension was put into a 15-ml centrifugetube, and the spores were collected by 10 to 30 s centrifugation(Precision Scientific Centricone). The spores were suspended for50 s in 1 ml of the 15% bleach solution. The bleach was rapidlydiluted with 10 ml water, and the spores were collected bycentrifugation. Three rinses were done with 10-ml portions ofdistilled H20, and after the final rinse, the spores were pipettedonto the surface of a 13-mm diameter polypropylene filter disc ina small Buchner funnel mounted in a suction flask. When therinse water was through the filter, the suction was left on to pullair through the spore mass and dry it. The dried spores wereshaken from the filter disc into a small Petri dish in which theywere stored under refrigeration. This procedure did not affectspore viability or change germination kinetics.

Isolation of Perine Material. The perine of Onoclea is loose andfragile and easily fragmented. A sample of spores was placedbetween two ground glass plates. The top plate was moved circu-larly over the lower with very gentle downward pressure. Thisaction broke the perine away from most of the spores, but did notrupture the spores themselves. The glass plates were separated;each was held vertically and was rapped sharply several times ona bench top. Many whole spores were dislodged, whereas thedetached perine fragments stuck to the plates, from which theywere collected by scraping with a razor blade. This mixture wasspread and rubbed gently between two polished glass plates. Whenthese were rapped on their edges, the rest of the whole spores felloff; the perine material stayed on the plates and was collected byscraping. When a sample was suspended in water and examinedwith a microscope, it was found to be free of spores.

RESULTS

Hypochlorite treatment of spores caused the subsequent elon-gation of rhizoids to be dependent upon an external supply ofmineral nutrients. In the experiment shown in Table I, bothuntreated and hypochlorite-treated spores were germinated oneither glass-redistilled H20 or on Knop's medium. The lengths ofthe rhizoids of untreated spores were the same on either medium,

Table I. Effect ofNaC1O Treatment of Spores on Subsequent RhizoidElongation

Treated spores were exposed to 15% commercial bleach solution for 50s, rinsed, and dried before culture as described in "Materials andMethods." All spores were germinated under standard conditions for 50h.

Average Rhizoid LengthaTreatment

Distilled H20 Knop's solution

,umNone 163.1 ±- 8.9 155.8 + 6.4NaCIO 78.4 + 6.1 161.9 + 9.5

a±SE.

about 160 pm. The rhizoids of the treated spores were only abouthalf this length on water, whereas on Knop's medium they wereas long as those of control spores. We interpreted this to meanthat NaCIO treatment removed from the spores ions which wererequired for rhizoid elongation, but that the required ions couldbe supplied exogenously from a nutrient solution.One visible result of treating Onoclea spores with NaCIO is

removal of the perine. Several experiments showed that the perinewas the source of the ions which were removed by hypochloritetreatment.

Samples of dry spores were treated for various times with 15%bleach. The spores were rinsed, as described in "Materials andMethods," and most were dried, but some spores from eachtreatment were examined to determine the extent of perine re-moval. Samples of spores from each time of exposure to NaC1Owere sown on both distilled water and Knop's medium and weregrown for 64 h. The results are shown in Figure 1 in which theaverage rhizoid length is plotted against the time that the sporeswere exposed to NaCIO. Spores grown on Knop's medium hadthe same length rhizoids, regardless of the duration of exposure toNaCIO. As little as 10-s exposure to NaCIO greatly reducedrhizoid length if the spores were grown on water, and any exposure20 s or longer produced the maximal limitation of rhizoid elon-gation. This pattern was correlated with the time at which theperine was removed completely from the spores. In the presenceof NaCIO, the perine began to expand and became filmy within10 s; after 20 s, it was gossamer and faint, and it dissolvedcompletely between 20 and 40 s. Thus, a 20 to 40 s treatment withNaCIO which caused rhizoid elongation to show maximal depen-dence on inorganic nutrient ions was the same duration as theshortest treatment which completely removed the perine. Longerexposures of the spores produced no further reduction in rhizoidelongation.The substances which supported rhizoid elongation were

leached easily from whole spores. This was shown by growingNaC1O-treated and untreated spores together in the same culturetube. Untreated spores retained the perine after germination, andcould be distinguished easily from the perine-free, bleach-treatedspores (Table II). The medium was distilled H20, and cultureswere established of treated spores alone, untreated spores alone,or an equal mixture of the two. The average rhizoid length ofuntreated spores was the same in separate or mixed culture,whereas the limited rhizoid elongation of treated spores alone wasincreased about 3-fold when they were grown together with un-treated spores still bearing their perines.The role of the perine as supplier of the rhizoid-promoting

factor(s) was confirmed in the experiment presented in Table III.NaC1O-treated spores were sown on distilled H20, on Knop'smedium, or on distilled H20 upon which had been sprinkledabout 1 mg isolated perine material. Addition ofthis small amountof material to distilled H20 produced a medium on which rhizoidelongation was as great as on Knop's solution, and much greaterthan the very limited elongation on water alone.Vogelmann (28) showed that Ca2+ promoted rhizoid elongation,

but we found that Ca2+ is not unique in this property. NaC1O-treated spores were sown on solutions of single salts. The variousions and concentrations used are given in Table IV. Figure 2shows data collected in a single experiment on the effects of Ca2+,Mg2+, and Mn2 . All three of these cations at their optimalconcentrations increased rhizoid length 5- to 6-fold compared withdistilled H20. The optimal concentration for Ca2" varied between100 and 1000 jiM in replicate experiments, neither being signifi-cantly different from the other. Mg2+, however, had an optimalconcentration of 100 jiM, and 1000 luM clearly was supra-optimal.Mn2+ was characterized by a broader curve with an optimalconcentration at 10 jiM and significant promotion by lower con-centrations than either Ca2+ or Mg2+. Weak but significant pro-

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SECONDS EXPOSURE TO NaCIO

FIG. 1. Effect of time of exposure of dormant spores to NaClO on subsequent rhizoid elongation. Spores were treated for different time with NaC1Oas described in "Materials and Methods." They were sown on either Knop's mineral medium (0) or on glass-redistilled water (0) and were grown for64 h. The average rhizoid length for each condition was determined from a sample of 100 rhizoids. Error bars indicate ± 1 SE.

Table II. Effect on Rhizoid Length of Growing NaC10-Treated andUntreated Spores Together in a Common Culture Tube

NaClO-treated spores were exposed to 15% commercial bleach solutionfor 50 s, rinsed, and dried before culture as described in "Materials andMethods." All cultures were done on glass-redistilled H20. NaC1O-treatedspores were sown both separately and in mixed culture. Spores weregerminated under standard conditions for 52 h.

Average Rhizoid Length'Treatment

Separate sowing Mixed sowing

,umNone 145.5 ± 9.8 140.7 ± 8.7NaC1O 38.5 ± 4.5 117.7 ± 7.4

a SE.

motion was produced by Sr2" at 10 and 100 uM and by Fe3" at0.01 and 0.1 ,IM (data not shown). Fe3+ was toxic and severelyinhibited germination at concentrations above 1 ,LM. Na+, K+, andAP3' did not affect rhizoid length (data not shown), although Al3+inhibited germination at 10 ,UM and higher concentrations. Co2+,Ni2', and Zn2+ inhibited germination at most concentrations, andrhizoid lengths were not measured for the small number of sporeswhich did germinate. It was apparent by comparing the effects ofthe different salts that the large promotion of rhizoid elongationproduced by Ca2+, Mg2+, and Mn2+ was not attributable to theanions N03- or Cl- which accompanied them.Inasmuch as either Ca2+, Mg2+, or Mn2+ could support maximal

Table III. Effect on Rhizoid Length ofAdding Perine Material to DistilledWater Cultures ofNaC10-Treated Spores

All spores were treated with 15% commercial bleach solution for 50 s,rinsed, and dried before culture as described in "Materials and Methods."Spores were sown on either glass-redistilled H20, glass-redistilled H20plus perine materiaL or Knop's solution. Spores were germinated understandard conditions for 52 h.

Culture Medium Average Rhizoid Length'

JimH20 33.2 ± 2.4H20 plus perine 201.2 ± 9.6Knop's solution 206.9 + 8.5

a SE.

rhizoid elongation, it was a question whether one, two, or all ofthem were involved in rhizoid growth under normal circum-stances. CTC2 fluorescence has been used to demonstrate apparentCa2+ gradients in a variety oftip-growing cells (23). CTC, however,binds a number of metal ions; some ions, such as Zn2+, Ca2 ,Mg2+, or Sr2+, greatly enhance the fluorescence of the complex,whereas others, such as Mn2+, quench the fluorescence (5). Welooked for CTC fluorescence in NaCIO-treated spores which hadbeen grown for 52 h under various nutrient conditions. Table Vlists the media on which spores were cultured and the averagerhizoid length after 52 h under each condition. Similar rhizoid

2 Abbreviation: CTC, chlorotetracycline.

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METAL IONS AND RHIZOID ELONGATION

i I I I I pI I I

f -3 -2 -1 0 1 2 3

LOG CONCENTRATION--M

FIG. 2. Rhizoid growth of NaClO-treated spores cultured on single-salt solutions of different concentrations. Dormant spores were treated with 15%commercial NaClO bleach for 50 s to remove the spore perine. They were then cultured for 50 h on solutions of MnC12 (0), Ca(NO3)2 (x), or MgC12(0). Average rhizoid lengths were determined for each condition from a sample of 100 rhizoids. The error bars indicate 1 SE.

Table IV. Ions and Concentrations TestedEach ion was tested throughout the indicated concentration range in

10-fold steps from the lowest to the highest concentration.Concentration Range

IonLowest Highest

'LMCa2+, Mg2+, Sr2+, Na+, K+ 1 1000Mn2+ 0.001 1000Fe3+ 0.001 100Co2+, Ni2+, Zn2+, A13+ 0.01 100

Table V. Rhizoid Lengths of Spores Grown on Various Media and Testedfor CTC Fluorescence

Spores were treated with NaClO to remove the perine and were grownfor 52 h on the indicated medium.

Medium Average Rhizoid Lengtha,um

Distilled H20 31.5 ± 3.0Knop's medium 177.7 ± 28.6Ca2+, 1000 AM 120.4 ± 9.4Mg2+, 100 UM 129.3 ± 9.3Mn2+, 100loM 118.9 12.6Distilled H20, spores with perine 115.6 ± 10.1

SE.

fluorescence was exhibited by spores which were cultured oneither Knop's medium, Ca(NO3)2, or MgCl2 and in intact spores(with perine) on water (illustrated for Ca2' and Knop's, Fig. 3).The entire length of the rhizoid fluoresced, with the most intenseemission at the tip (Fig. 3, a and e). We did not observe CTCfluorescence in ungerminated spores, or in the small, lens-shapedrhizoid cell immediately after it was formed; fluorescence devel-oped only when the rhizoid was visibly outgrown and was elon-

gating. Although spores cultured on MnCl2 had rhizoids as longas those cultured on Ca2+ or Mg2+, the rhizoids showed no

fluorescence (Fig. 3g). This arees with the observation of Caswelland Hutchinson (5) that Mn + quenches CTC fluorescence. Hy-pochlorite-treated spores grown on distilled H20 had short rhi-zoids which generally showed no fluorescence after 52 h in culture(Fig. 3c), although weak fluorescence at the tip could sometimesbe observed. Protonemal cells did not give CTC fluorescence.Observation of plasmolyzed rhizoids showed that CTC fluores-cence was associated with the protoplast and not the cell wall.When spores cultured on Ca2+ were rinsed and treated for 1 hwith 1 mm ethylene glycol bis(,8-aminoethyl ether)-N,N,N',N'-tetraacetic acid, rhizoid fluorescence in CTC was greatly reducedor abolished.

Rhizoids ofNaC1O-treated spores which were cultured on CaMg2+, or Mn2+ were similar in ultrastructure to those grown onKnop's medium (Fig. 4, a, b, and d). Rhizoids of NaCIO-treatedspores grown on distilled H20 were shorter (Tables I and V) andhad thicker walls (Fig. 4, c, e, and f) than rhizoids grown in thepresence of ions. The wall thickness at the rhizoid apex averaged0.9 ,um for 52-h-old spores grown on distilled H20. The compa-

rable thicknesses for rhizoids grown on Knop's medium, Ca2 ,

and either Mg2+ or Mn2+ were 0.2, 0.3, and 0.5 P,m, respectively.When tannic acid was present in the fixative, an electron-dense,thickened outer layer was visible in the walls of distilled-H20-grown rhizoids (Fig. 4, c and f).The size of vesicles in the apical 5 pm of rhizoids ranged from

0.05 to 0.2 ,um under all growth conditions, but rhizoids in distilledH20 contained fewer vesicles (compare Fig. 4, e and d). Near thecell membrane of the rhizoid tip (the apical 0.5 pm) there werehalf as many vesicles in water-grown spores as in ion-grown spores

(average number: 4/pm2 in water-grown, 8/pm2 in ion-grown).The numbers of other organelles, such as mitochondria and RER,were not correlated with the composition of the medium. Micro-tubules were observed under all conditions except in the apical 1

pm of the rhizoid.When rhizoids elongate in the presence of divalent cations, the

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FIG. 3. Chlorotetracycline fluorescence in rhizoids of spores grown under various nutrient conditions. Paired photographs are presented of the samefields (a-b, c-d, e-f, g-h) in which the first of each pair is the fluorescence micrograph and the second a light micrograph of the same spores. Spores weregrown for 52 h on (a-b) Knop's medium, (c-d) distilled water, (e-f) 100 mm Ca2 , (g-h) 100 mm Mn2 . Magnification; x 100.

nucleus moves from the base of the rhizoid, near the protonemalcell, and maintains a position near the growing tip. In water-grown spores, the initiation of nuclear migration occurs in shorterrhizoids than in ion-grown spores (Fig. 4, a and c). Figure 4ashows a nucleus remaining at the base of the rhizoid from a sporecultured for 46 h on Knop's medium, whereas Figure 4c showsthat nuclear migration has begun in the rhizoid of a spore culturedfor 52 h on distilled H20, even though the rhizoid is substantiallyshorter.

DISCUSSION

We have shown that rhizoid elongation in Onoclea spores isseverely limited on distilled H20 when the perine is removed(Table I and Fig. 1). Elongation attains normal levels when thespores are grown either on Knop's mineral medium or on single-salt solutions of Ca2+, Mg2+, or Mn2+ (Figs. 1 and 2). Weakpromotion of elongation is observed with Fe+ or Sr2+. Maximalrhizoid elongation clearly depends on the cationic composition ofthe medium. The perine itself, when it is present, supplies sufficientions to sustain rhizoid elongation, even when the external mediumis deficient (Tables II and III). The natural significance of thisfunction of the perine is uncertain. It seems improbable that aspore in nature would fall into a moist spot, otherwise suitable forgermination, in which the nutrient conditions were so poor as tomimic distilled H20. Still, one must leave open the possibility thatthe perine, with its apparent supply of ions, may enhance thesurvivability of young gametophytes under certain conditions.

Our finding does suggest some caution in interpreting labora-tory studies on ionic requirements for spore germination and earlygrowth. The generalization which has been drawn from the liter-ature is that spore germination is indifferent to the mineral nutrientcontent of the medium, whereas subsequent prothallus develop-ment depends on adequate nutrition (18). Some authors specifi-cally note instances in which germination is the same on distilledH20 and on a mineral medium (16, 27), and Kato (16) draws theconclusion that germination can occur at the expense solely ofendogenous reserves. We believe that this conclusion is correct,but that it can best be demonstrated with spores from which theperine has been removed to deprive them of this possible ionsource. Otherwise, on nutritionally deficient media, spores maydraw on a reservoir of ions which is not truly endogenous, in thesense of within the protoplast.Our data demonstrate that rhizoid elongation in Onoclea is

relatively nonspecific in its cation requirement since Ca2+, Mg2+,and Mn + are equally effective at the proper concentrations. Kato(16) reported that both Ca2+ and Mg + promoted rhizoid elonga-tion in Pteris vittata. Divalent cations are required to sustainelongation in some other tip-growing plant cells, such as pollentubes. In some species, Ca2+ is specifically required for tube growth(3), whereas in others, metals besides Ca2+ may stimulate elonga-tion (7, 8). Calcium localization in pollen tubes has been demon-strated by autoradiography ofwhole pollen incubated in 45Ca (15).The localization of Ca2+ in a variety of tip-growing cells has beeninferred from CTC fluorescence (21, 23), and its physiological

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METAL IONS AND RHIZOID ELONGATION

I dFIG. 4. Electron micrographs of rhizoids from spores grown under various nutrient conditions. All spores had the perine removed with NaClO

treatment. a, Rhizoid of spore grown for 46 h on Knop's medium. b, Rhizoid of spore grown for 52 h on Knop's medium and fixed in glutaraldehydecontaining tannic acid. c, Spore germinated on distilled H20 for 52 h and fixed in glutaraldehyde containing tannic acid. d, Enlargement of tip of therhizoid shown in b. e, Tip of rhizoid from spores grown on distilled H20 for 53 h. f, Rhizoid tip from spore grown on distilled H20 for 52 h and fixedin glutaraldehyde containing tannic acid. Magnification bar = 10 ,um in a, b, and c; I um in d, e, and f. G, Golgi; M, mitochondria; N, nucleus.

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Plant Physiol. Vol. 71, 1983

importance has been inferred from the effects of ionophoreA23187 (14, 22).

Because the fern rhizoid elongates rapidly at its tip, it has beenpictured as a metabolically active cell in which wall synthesis isintense. Several ultrastructural studies are consistent with thisview, showing the apical cytoplasm of the rhizoid to be rich inGolgi bodies and vesicles, to which the authors usually ascribe afunction in wall synthesis (2, 10, 12, 13). Our pictures (Fig. 4, dand e) show that the number of vesicles is much smaller in rhizoidsgrown on water than in nutrient-grown rhizoids. Another featureof rhizoids is the high activity which can be demonstrated for anumber of phosphatases (25). Acid and alkaline phosphatases,ATPase, and 5'-nucleotidase are located almost exclusively in therhizoid. Smith (25) favors the view that these enzymes are involvedin the transport of materials by the rhizoid. We suggest thatanother function may be in reactions which are part of wallsynthesis and assembly, and that the metal ions which supportrhizoid elongation may have a common role as activators for someof these enzymes. Although great variation is found among en-zymes isolated from different sources, examples can be found ofeach of the types mentioned above which are activated by one ormore of Ca2e, Mg2+, or Mn2+ (9, 20, 30).The localization of Ca2+ and Mg2e in rhizoids by CTC fluores-

cence is consistent with a role in elongation. When spores aregerminated on distilled H20, no CTC fluorescence is observed,but growth on either Ca2' or Mg2e alone leads both to rhizoidelongation and the appearance of bright fluorescence in the rhi-zoid. Although Mn2+ promotes rhizoid elongation, it cannot belocalized by this technique, because it quenches CTC fluorescence(5). CTC seems usually to be employed when it is desired todemonstrate Ca2' localization, but clearly the observation ofCTCfluorescence alone is not sufficient. Because the Mg-CTC chelateis also strongly fluorescent, its contribution must be considered.Caswell and Warren (6) recognized the possibility of confoundingsignals from Ca2e and Mg2+ with CTC. Caswell (4) measured thecomplete fluorescence emission spectra for CTC-treated mito-chondria which were either coupled or uncoupled and was able todistinguish between fluorescence from the different complexedions. Saunders and Hepler (24) observed CTC fluorescence incytokinin-treated Funaria hygrometrica filaments through narrowband-pass interference filters chosen near the peak emission wave-lengths of the Mg- and Ca-CTC complexes and stated that theywere able to separate the two signals and ascribed more than halfthe observed fluorescence to Ca2+. By whatever method, it isnecessary to confirm which metal is responsible for CTC fluores-cence. Our experiments in which spores were grown on single-saltsolutions demonstrate unequivocally the localization of both Ca2+and Mg2e in elongating rhizoids. Manganese, which is physiolog-ically active in our system, is impossible to localize by this tech-nique, because the Mn-CTC complex does not fluoresce. Thebright fluorescence of rhizoids from intact spores grown on dis-tilled H20 supports our conclusion that ions from the perine(probably Ca2+ or Mg2+) can be accumulated and utilized by therhizoids.No CTC fluorescence was observed before actual outgrowth of

the rhizoid. Thus, we cannot associate, by this technique, an iongradient with the path of nuclear movement or the position of theasymmetric cell division which initiates rhizoid differentiation.However, we are preparing a report which will show that a

sensitive stain for heavy metals reveals the local accumulation ofmetals in a portion of the exine very early in germination, in apattern which is correlated with spore polarity.

LITERATURE CITED

1. BASSEL AR, CC KUEHNERT, JH MILLER 1981 Nuclear migration and asymmetriccell division in Onoclea sensibilis spores: an ultrastructural and cytochemicalstudy. Am J Bot 68: 350-360

2. BEISVAG T 1970 An electronmicroscopic investigation of the young gametophyteof the fern Blechnum spicant (L) Roth. Grana 10: 121-135

3. BREWBAKER JL, BH KWACK 1963 The essential role of calcium ion in pollengermination and pollen tube growth. Am J Bot 50: 859-865

4. CAswELL AH 1972 The migration of divalent cations in mitochondria visualizedby a fluorescent chelate probe. J Membr Biol 7: 345-364

5. CAswELL AH, JD HUTCHINSON 1971 Visualization of membrane bound cationsby a fluorescent technique. Biochem Biophys Res Commun 42: 43-49

6. CAswELL AH, S WARREN 1972 Observation of calcium uptake by isolatedsarcoplasmic reticulum employing a fluorescent chelate probe. Biochem Bio-phys Res Commun 46: 1757-1763

7. DE BRUYN JA 1966 The in vitro germination of pollen of Setaria sphacelata 2.Relationships between boron and certain cations. Physiol Plant 19: 322-327

8. DE BRuYN JA 1966 The in vitro germination of pollen of Setaria sphacelata. 1.Effects ofcarbohydrates, hormones, vitamins and micronutrients. Physiol Plant19: 365-376

9. DRUMMOND GI, M YAMAMOTO 1971 Nucleotide phosphomonoesterases. In PDBoyer, ed, The Enzymes, Ed 3, Vol IV. Academic Press, New York, pp 337-354

10. DYER AF, DG CRAN 1976 The formation and ultrastructure of rhizoids onprotonemata of Dryopteris borreri Newm. Ann Bot 40: 757-765

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