PHYSIOLOGIA PLANTARUM 73: 505-511. Copenhagen 1988

Early quantitative method for measuring germination in non-green spores of Dryopteris paleacea using an epifluorescence-microscope technique

Robert Scheuerlein, Randy Wayne and Stanley J. Roux

Scheuerlein, R., Wayne. R. and Roux. S. J. 1988. Early quantitative method formeasuring germination in non-green spores of Dryopteris paleacea using an epifluo-rescence-microscope technique. - Physiol. Plant. 73: 505-511.

A method is described to determine germination by blue-light excited red fluo-rescence in the positively photoblastic spores of Dryopteris paleacea Sw. This fluo-rescence is due to chlorophyll as evidenced from 1) a fluorescence-emission spectrumin vivo, where a bright fluorescence around 675 nm is obtained only in red light(R)-irradiated spores and 2) in vitro measurements with acetone extracts preparedfrom homogenized spores. Significant amounts of chlorophyll can be found only inR-treated spores; this chlorophyll exhibits an emission band around 668 nm. whenirradiated with 430 nm light at 2rC.Compared to other criteria for germination, such as swelling of the cell, coat splitting,greening, and rhizoid formation, which require longer periods after induction fortheir expression, chlorophyll fluorescence can be used to quantify germination aftertwo days. This result is confirmed by fluence-response curves for R-induced sporegermination; the same relationship between applied R and germination is obtainedby the evaluation with the epifluorescence method 2 days after the light treatment ascompared with the evaluation with bright-field microscopy 5 days after the inducingR.Using this technique we show for the first time that Ca^' contributes to the signal-transduction chain in phytochrome-mediated chlorophyll synthesis in spores ofDryopteris pateacea.Key words - Calcium buffers, chlorophyll formation. Dryopteris paieacea, epifluo-rescence microscope, fluorescence-emission spectrum, greening, phytochrome, sporegermination.

R. Scheuerlein (corresponding author). Institut fur Botanik und PharmazeutischeBiologie der Universitat Erlangen-Niirnherg. Staudtstrajie 5. D-S520 Ertangen. FRG(present address)- R. Wayne, Section of Plant Biology, Plant Science Building. CornellUniv.. Ithaca. NY 14853, USA (present address); S. J. Roux, Univ. of Texas at Austin,Austin, TX 78713, USA.

hibition of germination can be caused by blue lightIntroduction ^^^^^^ ,^^^ Furuya 1985, Sugai et al. 1984), ethylene

Fern spores serve as a single-celled model system to (Edwards and Miller 1972) and various environmentalstudy the regulation of germination by various factors. pollutants that may interfere with signal transductionInduction of germination can be mediated by red light (Minamikawa et al. 1987, Wada et al. 1987; see also(Furuya ct al. 1982, Haupt 1985, Kendrick and Bossen Raghavan 1980 and Furuya 1983 for review). Fern1987, Tomizawa et al. 1983, Wayne and Hepler 1984), spores are ideal for the study of signal-transductionCa-* (Fohr et al. 1987, Wayne and Hcplcr 1984, 1985), chains, since they are whole organisms yet, single cells,gibberellins (Fechner and Schraudolf 1986, Weinberg haploid, easily synchronized and readily available inand Voeller 1969) and antheridiogens (Naef 1979). In- large quantities.

Received 21 January. 1988; revised 21 March. 1988

F'hvMol PkiiK. 7. . Mm 505

Fern spores can be divided into two groups, green andnon-green, based on tbe presence or absence of signif-icant amounts of cblorophyll in tbe quiescent spore. Intbe green spore species, germination can be assayed twodays after ligbt induction by using tbe acetocarmine-cbloral bydrate metbod of Edwards and Miller (1972).In the non-green species, germination is usually deter-mined by assaying swelling of tbe cell, splitting of thespore coat (perine and exine) and tbe presence of signif-icant amounts of cbloropbyll (greening) using brigbt-field microscopy (Mobr 1956). However, due to thedifficulties of determining tbe presence of cbloropbyllwithin tbe dark-pigmented spore coat and tbe eval-uation of eoat splitting under conditions of low contrast,the assay for germination usually is undertaken 5 to 6days after induction in order to accurately score germi-nation. By tbat time, tbe rbizoid bas emerged and iseasily identified, and often tbis is tbe criterion used forgermination (Sugai et al. 1984). However, in studyingsignal-transduction chains in a developmental system itis advantageous to measure tbe response as soon afterinduction as possible in order to minimize tbe secondaryor tertiary effects, such as migration of the nucleus,mitosis or cell division. Here we report an early, quanti-tative and simple assay, wbicb enables measurement ofgermination in non-green spores as early as two daysafter tbe induction of germination with R.

Abbreviations - EGTA. ethyleneglycol-bis(/i-aminoethylether)-N,N.N',N'-tetraacetic acid; PIPES, piperazine-N,N'-bis(2-ethanesulfonic acid); R, red light; X^. transmission maxi-mum of the interference filter, measured at the center of thehalfband width; /!„, wavelength used for excitation; A , wave-length used to measure emitted fluorescence.

Materials and methods

Plant material

Spores of Dryopteris paleacea Sw. (Dryopteridaceae;Tryon and Tryon 1982) were collected from plants grow-ing in tbe Botanical Garden at tbe Univ. of Erlangen-Nurnberg (Eriangen, FRG) during the summer of 1985and they were stored in a desiccator at 5°C in darkness.

IVeatments

Spores were sown on an aqueous medium containing7.4 mM Ca(NO,)., 3.45 mM KNO,, 1.01 mM MgSO4,10 mM EGTA and 20 mM PIPES at pH 6.0±0.1 (stan-dard medium). The free Ca'* concentration was 0.1mM, tbe free Mg-* concentration 1 mM (R. Wayne,1985. Thesis, Univ. of Massachusetts, Amherst, MA,USA). Additionally, a medium was used witb a freeCa- * concentration <1() "M and witb a free Mg-* con-centration of 1 mM; 3.45 mM KNO,, 1.03 mM MgSOj,10 mM EGTA, 20 mM PIPES at pH 6.0±0.1 (Ca-* freemedium). The media were prepared with milli-0 water(Millipore Corp,, Bedford, MA, USA) witb a resistance> 18MQ.

Spores remained in darkness for 20 b at 22°C. Tbere-after, tbey were R-irradiated. Red ligbt was obtained bypassing ligbt from a 5(X)W projector bulb (DAY/DAK,General Electric, USA) through a beat absorbing filterand an A1666 interference filter (A , = 666 nm, balfbandwidtb = 23 nm, Scbott, Mainz. The irradiance was 5.5or 2.7 W m % as measured witb a Li-Cor ligbt meter (Ll185; Lincoln, NE, USA) and a quantum sensor. Darkcontrols were run parallel to eacb irradiation experi-ment. Afterwards, spores were stored in darkness at22°C until evaluation. All manipulations were carriedout under dim green light.

Evaluation

Spores were examined with a Zeiss Univ. Microscope,equipped with either brigbt field or epifluorescence op-tics. In the epifluorescence mode, spores were illumi-nated witb a 1(X)W mercury vapor lamp. Tbe excitinglight was passed through either a 1) BP-490 excitationfilter combined with a dicbroic mirror that allows excita-tion ligbt of wavelengths less than 500 nm to pass and anLP520 barrier filter (blue excitation) or 2) a G365 exci-tation filter combined witb a dicbroic mirror that allowsexcitation ligbt of wavelengths less than 395 nm to passand an LP429 barrier filter (UV excitation). Germina-tion was assayed witb eitber 16X/0.4 or a 40x/().75 NANeofluar objective combined witb an optovar set at1.25X and lOx eye-pieces. Spores were counted in lotsof 50 or 100 spores.

Pbotograpbs were taken witb a Zeiss 35 Camera usingEktacbrome 400 daylight film. Exposures lasted from 1to 30 s, wben the Zeiss automatie exposure meter wasset at reciprocity = 3.

Chlorophyll determination in vivo

Chlorophyll fluorescence was measured using a Zeisspbotomicroscope II equipped with a Zeiss microspec-trophotometer and a Zeiss Zonax microprocessor. Tbeoptical path consisted of tbe following components;40X/0.5 NA Neofluar objective; BP4,5()-490 excitationfilter; dicbroic mirror <5()() nm, LP52() barrier filter;optovar, 1.25x; eye-pieces lOx; aperature at the imageplane, KM) nm. The voltage to the photomultiplier tubewas set to 8(K)V and the gain to UK). The V. 821211Lambda sean program was used and tbe errors due totbe wavelength dependence of sensitivity of tbe photo-multiplier tube and tbe chromatic aberration of tbeoptic system were corrected witb tbe V I.O Black BodyRadiation correction program.

Chlorophyll determination in vitro

Chlorophyll was extracted from 80 mg spores (dryweigbt). Spores were prewasbed in 20 ml of 10 mMEGTA at pH 6.0±0.1 for 20b, thereafter tbey weresown on 20 ml culture medium. Two days after sat-

506 Physiol. Plant 7.1. 19SS

u rat ing R, spores were separated from the culture me-d ium by filtration through a Millipore filter (8 |xm poresize; Millipore Corp., Bedford, MA, USA) and homo-genization was performed in a glass-bead homogenizer(B . Braun, Melsungen) with 9 ml of 100% acetone(Merck, Art. 822251, Darmstadt). After centrifugationfor 10 min at 1 5(K) g. the chlorophyll contents in thesupernatant was determined by measuring the absorb-ance in a 10 mm quartz cuvette at 663 and 645 nm withan Uvikon 860 spectrophotometer (Kondron, AnalytikG m b H , Munchen, FRG) according to Arnon (1949).

T h e in vitro fluorescence-emission spectrum of chlo-rophyll in 1(X)% acetone was determined with a Perkin-E l m e r 650-105 fluorescence spectrophotometer, mea-sured in a 10 mm quartz cuvette. A^ was 430 nm; both

slits controlling excitation as well as emission were set to5 nm.

Statistics

In all figures regarding germination the 68% confidenceinterval is given as estimated from the equation

±o =

where p = germinated fraction and n = number ofcounted spores. Otherwise, data are presented as themean ± SE.

Fig. 1. Photomicrographs of non-irradiated(A, C) or R-irradiatcd (B, D to F) spores.taken 3 days after imbibition in standardmedium. A and B (the same as E but withhigher magnification) show fluorescence.induced by blue excitation. C and D (thesame as F but with higher magnification)show fluorescence induced by IJV excita-t ion. R is saturating (60 s. 5.5 V^m^). Tri-angles indicate the site of coat splitting; | ,lacsura; bars correspond to 50 |im. Thevisu;il impression of the weak fluorescencelit non-germinated spores, observedthrough the microscope, is equal in A andB. In order to show this weak tluoreseencein A in more detail the exposure time fortaking the photomicrographs was pro-longed. Photomicrographs of R-irradiatedspores show selected areas containing ahigh amount of cells with eoal splitting.

36 PhyMol. Plani. 73. 1988 507

1.0

0)

ud)o1/10)

^ 0.501

cr.

500 550 600 650

Emission wavelength, nm

700

Fig. 2. In vivo fluorescence-emissionspectrum of ehlorophyll at 2 r C mea-sured in single spores 3 days afterimbibition and after continuousdarkness ( • ) or saturating R (O),60s. .S.5Wm ^ 10 •'Af Ca^^ A,60 s, .S.5Wm--. Ca'* < 1 0 " Af). Ex-citation: blue light (n = 14). Inset: Invitro fluorescence-emission spec-trum of chlorophyll in 1(X)% acetoneat 21°C. Eighty mg spores were sownon 20 ml of culture medium; 3 daysafter imbibition and after continuousdarkness (U)' M Ca*-) or saturatingR (3(X) s, 2.7 W m -, 10 ^ Af Ca'* orCa^*<10 "M) chlorophyll was ex-tracted with acetone. A ., = 4.30 nm.Relative fluorescence is given asphotons nm ' m"' s '.

Results and discussion

Spores contain endogenous compounds that fluorescebiue, yellow-green and red (Fig. lA-F). The dim yel-low-green fluorescence (e.g. Fig. lA), which can bequenched by the addition of KI and the blue fluo-rescence (e.g. Fig. IC) is found in the spore coat; more-over, the yellow-green fluorescence may also be local-ized in the intine, as concluded from the bright yellow-green fluorescence shown around the laesura (Fig. lAand B) and by the borders of the split spore coat (Fig.IB). Both types of fluorescence seem to be independentof the light treatment. The blue fluorescence may bedue to the presence of phloroglucinols (Tryon and Tryon1982, Tryon et al. 1973); whereas the yellow-green fluo-rescence may be at least partly due to the presence ofcellulose (Pettitt 1979). The autofluorescence color andpattern (Fig. IE, F) may be of some taxonomic impor-tance.

The red fluorescence two days after the inducing R(Fig. IB and D-F) is localized in the chloroplasts and isdue to chlorophyll, as evidenced from the followinginvestigations.

1) The in vivo fluorescence-emission spectrum shows apeak around 675 nm (Fig. 2). Comparable results werereported for fluorescence measurements of chlorophyllat room temperature in bean leaves (Thorne 1971),maize leaves and in isolated, light treated etioplasts (H.L. Kraak 1986. Thesis, Agricultural Univ., Wagenin-gen. The Netherlands). By contrast, this bright fluo-rescence cannot be observed in non-irradiated spores

sown on standard medium or in R-irradiated sporessown on a Ca^*-free medium. Both these conditions areknown to inhibit germination (Wayne and Hepler1984). Thus, this fluorescence seems to be correlatedwith the germination response. This latter observationis nicely confirmed in Tab. 1. Bright red fluorescence(relative fluorescence >0.1) can only be observed inR-treated spores. Under these conditions some sporeswith reduced fluorescence (relative fluorescence <0.1)can also be found. As a result, ca 85% show the brightfluorescence and this is in agreement with the germina-tion response obtained for R-irradiated spores in thefollowing experiments (see below, e.g. Fig. 3). By con-trast, no bright fluorescence is obtained in darkness. Forthe expression of this R effect, Ca'* ions are required in

Tab. 1. Relative red fluorescence (A,, = blue,A,^ = 670nm)ofnon irradiated and R-irradiatcd spores, sown on standard me-dium (+|Ca'*|) or Ca-* free medium (- |Ca- ' | ) . Measurementon a Zeiss photomicroscope 3 days after imbibition. For eachtreatment n = 14. ' Calculated only with germinated spores, asdetermined by bright-field microscopy 6 days after imbibition;cf. also Fig. 4. 'Calculated only with non germinated spores.

Treatment

Dark.Red,

Red.

+ Ca'*+ Ca2*

-Ca'*

Relative redfluorescence

O.O3±O.(X)2

1.06+0.23(1.2210.23)'

0.08+O.O.S(0.04+0.(K)4)-

Germination (%)

0

85.719.4

7.116.9

308 Physiol. Plant. 7.1. 1988

Tab. 2 . Contents of chlorophyll, extracted with 100% acetonefrom 80 mg spores (dry weight). Three days after imbibition on20 ml culture medium and after continuous darkness (standardmedium, + Ca-*|) or saturating R (3(K) s. 2.7Wm -, standardmedium, + Ca-*| or Ca-* free medium, -[Ca-*]) spores wereseparated from the culture medium and chlorophyll was ex-tracted. Each treatment was repeated 3 times. For comparisontbe germination response, obtained for the various treatments,is given.

Treatment Chlorophyll,(80 mg spores)

Germination,%

Dark,Red,Red,

0.83±0.259.55+0.801.55±0.19

056.1 ±1.67.7±0.9

the culture medium. Only 7% brigbt fluorescence isobserved after saturating R in spores sown on aCa-*-free medium. Using tbis technique we show forthe first time tbat Ca-* contributes to tbe signal-trans-duction chain in pbytocbrome-mediated cbloropbyllsynthesis in Dryopteris spores; but it cannot be decidedyet what step in tbe transduction chain is involved.

2) Significant amounts of ehloropbyll can be extractedfrom R-irradlated spores as shown in Tab. 2; by con-trast , considerably reduced quantities were found innon-irradiated spores or in R-irradiated spores sown onCa-*-free medium. Furthermore, the fluorescence-emission spectrum of cblurophyll, extracted fromDryopteris spores, is shown in Fig. 2 (inset). Again,significant fluorescence with a maximum around 668 nmis measured only in the extract obtained from R-irradia-ted spores sown on culture medium witb a free Ca-*concentration of 11) •* M. In general, coincidence isfound for the fluorescence-emission spectrum in vivoand in vitro. This further confirms that the red fluo-rescence measured results from light emission fromchlorophyll. Botb the slight bypsochromic shift of thefluorescence-emission maximum and the more con-densed form of tbe fluorescence-emission band, foundunde r in vitro conditions, may be due to tbe alteredhydration of chlorophyll, as well as to changes in thechlorophyll alb ratio during extraction (Bruisma 1963).

The kinetics of tbe formation of tbe red fluorescence ascompared to other characters used as indicators for thegermination response, are given in Figs 3 and 4. Inspores that have been synchronized, germination can bedetermined as early as 2 days from irradiation by usingthe chlorophyll-fluorescence metbod (Fig. 3a). At tbattime other indications of germination are still lacking.Coat splitting can be detected after anotber day's delay(Figs 3a, 4), and greening after at least 2 days delay.However, all 3 metbods sbow a corresponding germina-tion response of ca 80%. By contrast, a reduced value isindicated by rhizoid formation, and in darkness none of

tbe germination criteria can be observed up to 5 daysafter irradiation (Figs 3a, 4). Red ligbt causes a gradualspore swelling (Figs 3b, 4) during the first 5 days. Aftertbe fifth day a rapid increase is obtained, indicating theformation of a multicellular gametophyte (data notshown). By contrast, dark controls sbow a continuousdecrease in cell size during tbe first 5 days (Fig, 3b).Tbese observations characterize chlorophyll fluores-cence as an early criterion to quantify germinationwitbin two days in non-green fern spores. Tbis result isconfirmed in Fig. 5, showing tbe fluence-response curvefor the induction of spore germination with R: eval-uation with the epifluorescence metbod 2 days after thelight treatment as well as evaluation witb bright-fieldmicroscopy 5 days after the ligbt treatment sbow thesame relationship between applied R and germinationrespKjnse. Moreover, since light requirement for sporegermination can be replaced by gibberellins, e.g. in

', 60

1 2 3 4

Time from irradiation, doys

Time from irradiafion, doys

Fig. 3. Various criteria of germination scored daily after asaturating R-irradiation (60 s, 5.5 Wm-, standard medium)(O). For comparison dark controls at different time intervalsare given (•) .a) Chlorophyll fluorescence, coat splitting, rhizoid formationand visible greening were used as criteria for germination.b) Spore length was used as a criterion for germination; sporelength was determined as the distance from intine to intinealong the longest axis.

36* Physioi. Planl. 73. 1988 509

dark red time fromirradiation

2 days

100

I 60o

20

0*7/ ^ •0 2 10 20 50

R-Fluence. Jm'

100 200 500

Fig. 5 . Fluence-responce curve for the induction of spore ger-mination with R (2.7 W m ^ 1 s to 2 min) on standard medium.Germination was evaluated either with the fluoreseencemethod 2 days after R (•) or with bright-field microscopy 5days after R (O).

Anemia phyttitidis, (Schraudolf 1962, 1985) or in Lygo-dium japonicum (Manabe et al. 1987) the new methodmay also be used for chemical induction of germinationby antheridiogens.

Acknowledgements - The generous support with laboratoryfacilities and the interest of Drs R. M. Brown Jr. (Austin). J.Brand (Austin) and K. Knobloeh (Erlangen) and the membersof these laboratories is gratefully aeknowledged. The authorsthank Mrs U. Mader for the eareful preparation of the draw-ings. This research was supported in part by the DeutscheForschungsgemeinschaft (Sche 276/1-1).

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Edited by L. O. Bjorn

Fig. 4. Photomicrographs of non-irradiated orsaturating (60 s. 5.5 Wm \ standard medium), m me ni iiuriuuiv. uiavriiig..T. inju>,u .». .••..

spores, rhizoid formation (r) and the site of coat splitting (triangle) is indicated. Bar = 25

R-irradiated spores taken at different intervals after light treatment. R wasIn the schematic drawings, added to the photomicrographs with R-irradiated

Physiol. Planl. 7.1. 511