effects light growth and development liverwort ... · 170 rpm t1hrough a circle of 12.5 mmdiameter....

Plant Phvsiol. (1968) 43, 714-722

Effects of Light on the Growth and Development of the Liverwort,Sphaerocarpos donnellii Aust.1

David H. Miller2 and Leonard MachlisDepartment of Botany, University of California, Berkeley, California 94720

Received December 12, 1967.

A bstract. Fragments of thalli of the liverwort, Sphaetocarpos donnellii Aust., inoculatedinito liquid medium containing sucrose and mineral salts, attain a much greater dry weightafter 9 days growth in continuous 'White light than in darkness. Light causes this differenceby increasing the rate of growth of the plants. This growth response is mediated by thepigment systems of photosynthesis and phytochrome. An inhibitor of photosynthesis, DCMU,at conoentrations which inhibit light-mediated CO2 fixation, decreases the growth rate oflight-grown but not dark-grown plants. Light still slightly increases the growth rate of plantsin the presence of DCMU. This latter response is mediated by phytochrome, since it can

be effected by a 2 minute exposure to low intensity red light every 12 hours, and far-redlight reverses the effect of red. The increased growth rate effeoted by red light is re-

lated to a change in the morphology of the plants. Dark-grown plants form compact ballsof tissue consisting of lobes. These lobes are rounded and thick and exhibit an abnormalcallus-type growth, with few well-defined meristematic regions. Plants grown in red lightform fluffy ballls of tissue. The lobes of these plants have a morphology more typical ofSphaerocarpos in nature. They are 2 cell layers thick, flattened, and have numerous well-defined meristematic areas. The greater number of meristems allows for the increased growthrate of the plan(ts grown in red light.

The original discovery of a non-photosyntheticlight effect on the growth of Sphaerocarpos don-nellii wa,s made by Machli's (4). He demonstratedthat little growth, as meastured by dry weight,occurred when the plants were grown in dairkness,even thoulgh carbohydrate to support growth (1.5 %glucose) was present in the medium. When plantswere grown with glucose in continuouts wh,ite llight,however, 'their dry weight increased 4- to 5-foildover tfhait of dark-grown plants. The exogenoussugar was irequired for these growth responses.Plants maintained in conitinuous white light wilthoutsugar grew very muich less than dark-grown plantswith isugar. Mac'hli's postullated that thi's lighteffe,cit wvas mediated by phytochrome becattse asubstantial dry weight increa;se over dark control'soccurred with a's ii-ttle as 5 minutites of White lightper 24 hours and this white light response wasreversed by 45 minutes of far-red fight.

1 This work was supported in part by National Sci-ence Foundation and National Aeronautic and SpaceAdministration fellowships to D. H. Miller and Na-ti'onal Science Foundation grant GB 110)7 to L. Machlis.The material is a portion of a thesis submitted byD. H. Miller to the Graduate Division of the Universityof California, Berkeley in partial fullfillmenit of therequirements for the Ph.D. degree.

2 Present address: MSU/AEC Plant Research Lab-ora,tory, Michigan State University, East Lansing,IMichigan 48823.

714

These results were con'sildere'd by Machlils to bepreli,minary and (hi,s conclusions as ten'tative. Thepresent 'tudy is a detailled analysis of the ef'fectsof ligh,t on the growth of Sphaerocarpos.

Light effect's on the growth olf Sphaerocarposdonnellii have allso been studied by Steiner (10, 11).He found that the germination o'f spores wa's in-filuenced by phytodhrome, as was th'e developmentof the gametophyte. Red light caused the branch-inlg of the thall1i so that normal growth anid de-velopment occu,rred, whille in iblue light the thalllirema,ined unbranch'ed. Far-red light did noat re-verse the response to red, but gave a similar,thouigh lesser res,ponse.

Tihe exifstence of phytoohrome in Sphaerocarpos*has been demonstrated by Taylor and Bonner (12).T'hey 'prepared extra,cts from dark-grown plant.sgrown in this laboratory and demonstrated thepresen'ce of phytochrome in these extra,cts by dif-ferentiail 'speotrophotometry.

Methods

The OrganisIM. The organism used in all ex-peri,ments wa,s the isolate 60-25 of the femalegametophyte o'f Sphacrocarpos dontnellii Ausit. (5).

The Mediumn. The growth mediuLm, as devel-oped by AIachilis (4), was modified slightly. Itconsisted of 0.01 Mi KNO3, 0.01 AI KH2PO,,0.001 -r CaCl2, 0.001 ri MgSO,, 1.5 % suicrose, an(d

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MILLER AND MACHLIS-EFFECTS OF LIGHT ON SPHAEROCARPOS

a chelated trace element so;lution (9). The KH2PO4solution was adjusted to pH 5.5, autoclaved sepa-rately, and added to the rest of the medium. Thisprevent-ed precipitation of Ca3(PO4)2 and the pos-sible formation of sugar phosphates during auto-claving. Fifty ml alliquots of this medium wereplaced in 125 ml Erlenimeyer flasks. The flaskswere plugged with cotton and autoclaved at 15pounds pressure for 15 minutes.

Procedure for Inoculation. The procedure forinoculating flasks of medium was altered consider-ably from Macih'lis' original one. This substan-tially increased the reproducibility of dry weightyieldis from flask to flask wiithin any 1 treatment(121 ± SE 14.9 mg lbefore the dhanges, 150 + 2.2mg after). Tihe modified procedure is outlinedbelow.

Plants to be used as inoculum were poured fromfilasks into a sterile tea strainer, rinsed with about300 nl of 'sterile ditstillled water, anid dropped intoa sterille blender cup. About 200 ml of sterilewater was poured intto ithe cup and the blenderturned on for 25 to 30 secondis. Thi.s blendingtime assured complete fragmentation of all plants.The fragmented tissue was then poured into anothertea strainer and rinsed with albott 500 in- of sterilewalter to waish out the smala fragmenits and cellularcontents released by the (blending. The washedfragments were then dropped into a second blendercup for convenience anid an appropriate vlntime ofsterile distiilled waiter added. The suspensi-on ofwashed fragments in the blen,tder ctup was pouredinto a sterilized Erlenmeyer flask containing aTefl,on magnetiic stirrer. The flask was thenp1laced on a stirrer which maintained an even sus-pension of inotculuim throughout the period of timeinvolved in inoculating the flasks. Five ml por-tions of inocuilum were removed with a 5 ml hypo-dermic syringe with a No. 13 needle and injectedinto the flasks of medium by inserting the needlebetween the cotton plug and the neck of the flask.

In all experiments the inoculum wa's grownunder the same standard conditions (conltinuouswhite light for 9-10 days) and w'as useid at thesame point in i'ts growth curve '(from 320-380 mgdry wt). Tihe weight of inoculum per flask wasmaintained as uniiform as possible from 1 experi-ment to the next. This weight was 4 to 5 mig.

Temperature. The plants were grown at 250.Slight variations in temperature on the shakers,even those in a conitroliled tem,peralture room, causedslight di,fferences in growth rates of the plants.To eliminate such errors, dark controls were in-cluded in eacih different treatment, or boxes ofplants receiving different treatments were rotatedaround the 'thaker at 12 hour intervals.

Light. 'Continuous white light yielding about600 ft-c at the surface of the haaker was obtainedfrom a bank of Sylvania F48T12 Cool Whitefluorescent tubes.

Fluorescent tubes, ei-ther Sylvania Cool Whiteor Gro-Lux F72T12, were 'filtered through variouscom1bination's of plexigdass, plastic, or gelatin filtersto dbtain the diifferent colors of lighit describedbelow. These tubes emitted no light above about715 nm. The transmission spectra of these variousfilter combinations, as measured on a Perkin-Elmer202 UV Visible Spectrophotometer, are 'shown infigure 1.

WAVELENGTH (NM)FIG. 1. Transmission curves of various filter sys-

tems. A = blue, B = blue-green, C = green, D =yellow-green, E = red, F = short wavelength far-red,G = long wavelength far-red. See text for details.

Blue ligh(t was obtained with one-sixteenth inchthickness of Robm and Haas Bdlue Pilexiglass 2424,supplemented by additional layers of Cinemoid blueplastic (Kleighgl Brothers, 321 West 50th Street,New York, New York) where dower 'intensitieswere required. Bilue-green light was dbtained with3 layers of green, 2 lfayers of {blue Cinemoid plastic,and 2 layers of blue gelatin. Two green Cinemoidplastilc layers were iused for green lfight. Yellow-,green light wa's obtained with 2 layers each ofgreen and orange Cinemaid plastic. Two layers ofred and 1 of orange 'Cinemoid plastic provided redlight. Layers of Whatman 3MM 'filter paper wereadded to the red and oran;ge filters to decreaseintensities as needed. The short wavelength far-redlilght was obt'ained by filtering light from SylvaniaGro-Lux 'fluorescent lights through 4 layers of redgelatin.

T'he source for far-red light was a bank of 8G-eneral Electric Showcase 40 watt incandescentbullbs fiftered through 6 cm of water containing33.33 gm/l Fe(NH4)2(SO4)2 '6H1O and thenthrough 2 one-eighth inch thicknesses of BlackPlexiglasts (Westlake Pla-stics, Lenni Mills, Penn-sylvania) and 2 layers of blue Cinemoid plastic.This complex far-red source was necessary becauseof the fact that 1 layer onuly of one-eighth inchBlack Plexiglass transmittedl enough light in therange of 700 to 710 nm to stimulate the growth ofthe plants nearly as well as red light.

The intensity of variouts filter system's wasmeasured on a standardized Eppley Tihermopile

715



PLANT PHYSIOLOGY

with a quiartz window attached to a Hewlett-PackardModel 425A DC Micro-Volt-Ammeter. For theexperiiment testing different wavelength bands fortheir activity in stimulating growth, equail inci(dentquanta of different wavelengths were calculated bythe equation X X/hc (Tr) (Tr.), where X is thenumber of qtuanta per cm2 per sec for -the differentwavelengths, X is the wavel-ength of ithe differen;tlight sources, h is Planck's constant (6.62 10-27ergs sec), c is the speed of light, T, is the thermo-pile constanit (94.3 ergs per cm2 per sec = 1 ,uvolt),anid T. is the ithermopile reading for ithe differentlightt sources in ptvolts. The wavelengths utsed inthe calctu(lation were blue = 460 nm, blue-green- 500 nm, g,reen = 520 nm, yellow-green 565unq1, red = 660 nm, and "far-red" = 710 nm.

Darkness was imposed by growing plants inA) flasks that were taped with plastic elecitricaltape or B) boxes wrapped in 4 layers of blaccksateen cloth.

Growtah Facilitics. Flasks of plants were in-cubated on rotary shakers usutally for periods of9 to 10 days. For aill continuous light and someintermittent light experiments, the shaker used wasin a 25° constant temperatutre rooom and moved at170 rpm t1hrough a circle of 12.5 mm diameter.

For most of the intermittent light experimentsthe planlts were grown in a shaker moving alt 106rpm throutgh a circle of 2.54 cm diameter. Thismachine was designed by the Department ofGrounids and Buildings of 'the University of Cali-fornia, Berkeley (6). It 'consists of 4 light-tightboxes m'ounted on the shaker. A bank of 3 Syl-vania Gro-Lux fluttorescent 'tubes was located belowthe boxes as were 2 rows of G. E. Showcase 40wvatt incanTdescenit bulbs suirrounded by BlackPlexiglass. TIhe bottom of each box consists olf2 collapsible halves. Two time clocks open theboxes and tuirn on the lights at up to hourly inter-vals for dura'tions of 1 sec to 50 minutes thusexposing the plants to intermittent light.

Dry Weight Determinaztions. At the end of anexperiment, the contents of each flask were pouredinto a 4.25 cm Buchner funnel and the mediumdrawn of'f by vacutum through layers o'f W'hatmanNo. 1 filter paper. The plants were then trans-ferred to an aluminum cup and dried for 24 hoursin a 900 oven. These dry weight measurementsare reported as mg dry weight per flask of plants.

Determining the Uptake of H14C03 in thePresence of DCMU. Two grams o,f tissue wereplaced in a 50 ml Erlenmeyer flask containing10 mnl o,f standard medium with varying DCMUconcentrations. For dark uptake measurements, theflask was wrapped in 2 layers of altuminuim foil.For liighbt exiperimen;ts the flask was held 40 cmfrom a Sylvania 150 watt photoflood lamp with a16 cm water bath in between. At time 0, 20 ,ul of6 ttLvi NaHl'CO3 (1.54 uc per ml) was added tothe flask which was shaken gently for 2 minutes.The tissue was then transferred into 5 ml of 80 %

(v/v) meithanol in a ground glass homogenizer.Ilt was gro-und thoroughly, centrifuged, and thesupernatant meastured and poured into a screw capglass viall. The ground tisstsue was resuspended andcentriftuged successively in 1.5 ml aliquolts of 100 %methanol, 20 % methanol, and water. The com-bined supernatants were adjusted to equail voltumeand the radioactivity read as folilows. Fifty 1A ofeach extract was pipetted onto an aluminutm plan-chet. A few drops of 10 % acetic acid were addedand the planchets were dried in a hood underneatha photoflood lamp. The radioactivity in the sam-ples was determined with a Nucilear ChicagoGas-Flow detecting system possessing a 30 %coutniting efficiency. The aictivity is reported asthe difference between the activi'ty of extracts o flight- and dark-treated plants.

II the experiments involvinig ithe effects ofDCMU oIn growth, the D'CMU was added as anialcohol solution. AIn equail amount (4410-4 % V/V)of alcohol was added to the control medituni, becatuseeven tlhis low concentration was ilhibiitory togrowth.

Statistical Analysis of Data. The dry weightmeasturemlent of red, red folilowed by far-red, anddark-grown plants reported in ta(bles VI and VIIwere subj ected to a t test to determine if thedifferences in dry weight were statistically signifi-cant. The t test demonstrated that the differencesin dry weight between red light treated plants andeither dark-grown or red, far-red treated plantswere significant at the 1 % level, whiile the differ-ences between dark-grown and red, far-red treatedplants were not significant at the 5 % level.

Each experiment was carried out at least twice.All dry weight measuremenits reported are theaverages of at least 6 and at most 10 replicates.

Results

The Nature of the Light Requirement. Theinitial experiments tested the effect on growlth ofseverail shorit expo,sures to ilow intensity red light.If the growth stimulation by light were due com-pl-etely to phytochrome activation, then several suchexpossures per day should be as effective as con-tintuous light. Table I shows the results of a

Table I. The Effects of Intermnittent Red andContinuous White Light on Growth

Plants were grown for 9 days. The intensity ofthe red light was 188 ergs-' cm2/sec.

Treatment

Dark control2 Min red light/8 hr2 Min red light/hrContinuous white light1 Standard error.

Growth

M1g dry wt/flask100 ± 2.3'154 -- 3.1156 ± 2.7412 -- 7.8

716




typical experiment. As few as three 2 minuteexposures per day 'increased the growth of theplants by 50% over the dark controls. More fre-quent exposures, even as 'often as once per hour,did no't further increase growth, and none of theintermittent light-treated plants grew as muclh asthose given continuous light. These restiults indicatethat 2 or more pigments are involved in the growthresponse of Sphaerocarpos to continuous whitelight: a pigment saturated by a red-fight exposureevery 8 1hours and '1 or more pigments requiringlarger dosages of 'light.

Dturing the course of these experiments it be-came apparent that the length of time the plantswere aillowed to grow greatly influenced the mag-niitude of the dry weight diifferences between light-and darksgrown plants. For this reason, growthcurves were determined for plants grown in con-tinuous whilte 'light, 2 minutes of red light per ihour,and darkness (ifig 2). The arithmetiic plot (fig 2,right) clarifies the nature of the dight response.The impression had been that light was requiredfor growth. Thits idea wa's the result of the factthatt the plants had been harvested at the point intime when the differences ibetween their dry weightswere at a maximum (day 8 or 9). The fact thatdark-grown plants attain a maximum dry weightonly slightly dess than thalt of 'light-grown plants

2 min. red/hr.Continuous white light ,0-e

* A4DarkO/ aU11V

~ ~ /~ ~ ~ ~

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demonstrates that light is not required for growth.Rather, the 'difference between the sets of plantsis in the 'length of time required to reach the sametotal growth. Tthe logarithmic plot (,fig 2, left)reveal's some reassons for ,this difference. Theslope of the log phase of plants grown in whitelight is greater than the slope o'f -the plants grownin red light or darkness. Thus, white light stimu-lates the rate of growth during the log phase.White light ail-so decreases 'the lag phase of growthby about,24 hours, a's compared to the ilag phase ofdark-grown plants.

Demonstration of the Involvemient of the Photo-synthetic Pigments. Experiments were done to de-termine what high energy pigment system(s)shorten the lag phase and increase the growth rateo'f the plants. To see whether the photosyntheticpigments were mediating these effects of whitelight, aIn inihibiltor of photosynthesis, 3,4-dichloro-iphenyl,l,1-dimethylurea ('DCMU) was utsed (13).The fir'st experiment was designed to see if DCMUwoul'd, in fact, 'inhibit photosynthesis in Sphaero-carpos. Various concentrations of D1CMU weremixed in 'the regu(lar growth medium. Any re-sultant chanige in photosynthetic activity wa's meas-ured as changes in th'e ability to fix CO2 in thelight (fig 3). Increasing DCMU concentrationsdecrease CO2 fixation until at aibotut 5 kM DCMU

a)- 4000iU0

a)E.- 300

cO0

N

200

b.0)

C1 100E

Continuous white light

Dark

2 3 4 5 6 7 8 9 10 11 12 13 14 15

Days DaysFIG. 2. Logarithmic plot (left) and arithmetic plot (right) of plants grown in continuous .white light, 2

minutes red light (200 ergs cm-2 sec13) per hour, and darkness.

717

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300CO

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PLANT PHYSIOLOGY

1%\

0\

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0\ -. &

-6

LOG CONC DCMU (M)FIG. 3. The effect of DCMU on light-mediated

CO2 fixation. Radioactivity reported as counts fixedin light minus counts fixed in darkness.

4001t

300

2001-

100 1 - --- --- DARK

// I.0 -7 - 6 - 5

LOG CONC DCMU ( MA)FIG. 4. The effect of DCMU on growth of plants

for 9 days in continuous white light and darkness.

light Ino longer increases the rate of CO., fixation.Thi's concentration of DCMU, then, completely in-hibits photosynthetic activity.

The next experiment was designed to see ifthese samne concentrations of DCMU whioh inhibitphotosynthesi,s also inhibit growth. Pilan,ts wereinoculated into flasks with medium containing dif-ferent concentrations of DCMU and were grownin conitinuouts white light or darkness (fig 4).With increasing concentrations of DCMU, growthin continuous 'light is increasingly inhibited uinitilthe degree of inhibition (levels off at ablout thesame concentration that completeily inhibits photo-synthesis. Growth in 'the dark, however, is tinl-a,ffected by any concentration of DCMU. Thus,concentrations of DCMU which inhibit photosyn-thesis also inhibit the growth of Sphaerocarpos inthe light 'but not in tthe dark, indicating that thehigh energy light response is mediated by thephotosynthetic pigment system.

In ald the experiments where DCMU was addedto the medium, no conicentraltion of -the in'hibitorwvas effective in reducing the growth of light-grownplan,ts to the same levell as that of dark-igrownplants. The next experiment was done to deter-mine how this DCMU-independent light reesponsemight be related to the growth response caused byintermittent red light (table II). Two important

Table II. The Effect of DClMU o01 the Growtlt ofPlants in Light and Dark

The intensity of red light was 122 ergs-' cm2/sec.Plants were grown for 9 days.

GrowthWith 4,um

Treatment Without DCMU DCMU

.Mg dry wt/flaskDark control 85 ± 4.61 83 ± 4.1Continuous white light 380 + 6.8 140 ± 5.07 Sec red light/ hr 136 ± 4.1 135 + 4.51 Standard error.

results are to be noted. First, DCMU affectsneither dark-grown plants nor the growth responseo-f plants to intermittent red light. Second, andmore important, the short, red-il'ight treatments areas eiffective a's continuous white light in stimulatingthe growth of D,CMU-treated plants.

Demonstration of the Involvenment of Phvto-chrome. The frequency oif light 'exposures neededto saturate the red-light reisponse was determinednext. Four groups of plants were given exposturesto red light of varying frequency and du-ration suchtha,t the total exposure to li,ght was 12 minutesevery 24 hours (taible III). The increases in dryweight oif light-treated plants over dark controlsare about the same with dark intervals oif 4 to 12hours. With an interval of 24 hours, however, a

maximum response is not obtaiined. Red light at

718

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3ty



MILLER AND MACHLIS-EFFECTS OF LIGHT ON SPHAEROCARPOS71

Table III. The Effect of Various Red Light Treat-inents on Growth

Red light initensity o-f 188 ergs-' CM2/sec. Pl-antswere grown for 9 days.

Red light treatment Growth

Mg dry wt/fkaskDark controlI 105 ±2.4'2 Min/4 hr 156 ±2.14 Min,/8 hr 1,55 ±2.56 Min/12 hr 159 ±2.612 Min/24 hr 144 ±4.0

1Standar-d error.

least once every 12 houirs is necessary for a maxi-mulm -response.

Another characteristic of phytoch'ro-me-mediatedresponses i:s that th-ey can be elicited 'by very lowlight intensities (ta:'ble IV). I-t is -apparent thatthe plants are 'sensitive to very low initensities ofred 'light. Furth-er, the response is independenIt of

T-able IV. The Effect of Red Light Intensityon Growth

Plants were grown for 9 days. Exposures of 2minutes per 12 hours. Intensities given in ergs-' CM2/sec.

Inten,sity Growth

mg dry wt/flask0 (dark control) 122 ±- 52'38 182±4-4.5189 185 ±- 3.33,77 179 ±2.43018 184 ±3.6Standard error.

intensity over a wide 'range. In other experim~entst-he -total light energy given per exposure wa's de-c-rea:sed to th-e point where the pigment sys-temn wasno-t sa-tu-rated a-s evidenced by the fact thalt the drywei-ght di'fferenc-e between light- and dark-grown

Table V. The Effect of Different Wavelength Bandson the Low Intensity Light-Mediated

Growth ResponseLight given 2 min/12 hr. Intensities r-anged from

1,88 in the far-r-ed to 28,2 ergs"' cm2/s:ec in th-e blue.Plants were grown for 9 days.

R-esponseTreatment Growth (light-dark)

Mg dry wt per flaskDark control 86 ±- 2.0' 0Blue (460 mm) 97±4-2.7 11Blue-green (520 nm) 96 ±4 2.9 12Yellow-green (565 nm) 94 ±4 2.7 8Red (660 mm) 1,26±::3.4 40Far-red (715 nm) 1,09 ±- 3.3 231 Standard error.

plant's was less than the maximum. At these veryl-ow intensities the response i-ncreased with in-creasing intensity up to -saturation.

The effect of different wavelength band-s on th-elow intensity 'light respon-se of Sph~aerocarpos i-ssimil-ar to t-hat of phytochrome. This i's shown bythe results of -an experiment in whic-h plants weregiven intermittent light of 5 different wavelengths(ta-ble V). The intensities of the 5 quallities oflight were adjutsted so that they wer-e at equalIincident quanta. Red light 'is by -far the mosteffecotive wavelenigth.

T-he definitive proof 'that a red-ilight re-sponseis mediated by phytochrome is a demonstration thatthe response is repeated-ly reversible by aliternatingexposures to 'red and far-red light. Taible VIdemonstra-tes th-is characteristic 'in t-he -system uinderstudy.

Table VI. Repeated Reversibility of the GrowthResponse

Light wa-s given onoe per day f-or 9 days. Theinitensities of th-e red and far-red were 4000 and 1410ergs-' CM2/seC, respectively. R = 2 min red, FR=40 min far-red.

Treatment Growth

Mg dry wt/flaskDark control 108 ±+ 1.51R 124±--1.9RI, FR 110 ±+ 2.2R, FR, R 124±4-1.4R, FR, R, FR 114 ±- 2.41 Standard error.

Morphological Differences. The plants grow inthe form of fluffy balls of ti,ssue with each ballcomposed of many lobes. The type of lobes -formedin dark-grown a) and red-light-grown b) plantsare 'shown in -figure 5.

Dark-grown plants form very compact bail's o-ftissuie. Their lobes are solid masse-s of cell's re-sem'b'lin'g c-al(lus tiissu.e. Th-ere are very few well-defined meri,stemat-ic areas.

Plants grown in 'red 'light -for-m much looserball's whi-ch consi,st of fla-ttened Aseets of tis.,sue.Their lobes are u-sualily 2 celil layers t-hick andexhibit a typical growth habit for Sph-aerocarpos.The marginis are meri'stematic areas of small1 com-pact cells which gradually entlarge towa:rd th-ecenter of the sh-eet of ti'ssue. There aTe manya-reas of meristematic activity in each ba'l'l of tissue.T-t is believed that the gre-ater nu-m'ber of meri,stemsail-lows -for a greater rate of growth. 'Plants grownin blue light exhibit a morphology 'similar to thatof plants grown in red light, but a much higherintensity of blue light i's ne-eded to satUrate thi'sresponse '(ca. 800 ergs/cM2/sec).

Th-e 'less c-ompact growth halbit of plants grownin red lig-ht resuilt's in a considerably higher fresihweight to dry weight ratio a-s comipared to dark-

719



PLANT PHYSIOLOGY

a

'4.'A

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*;s<,.M:1; ::.

_s iu...

:.:

.:. :.::::..:.::.: ::..:.

w ..............s . . -.., ..........

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FIG. 5. Morphology of lobes o-f Sphacrocarpos grown in a) darkness and b) intermittentred light.

720




Table VII. The Effect of Red and Far-Red Light onthe Fresh Weight, Dry Weight, and the Fresh

Weight to Dry Weight RxatioMeassurement made after 9 days of growth. Inten,

sities: red = 200 ergs-' cm2/sec, far-red = 1410 ergs'cm2/sec.

Treatment Response per flask

Fr wt Dry wt Ratio

g mgDark control 2.30 + 0.501 143 ± 1.4 16.11 Miin red/24 hr 3.79 ± 0.41 176 ± 1.9 21.51 Min red + 50 minfar-red/24 hr 2.56 ± 0.32 147 ± 1.9 17.4

Standard error.

grown plants. This increased ratio is an accuratemeasure of the change in morphology ca'used byred 'light an'd i's reversible by far-red ilight (tableVII).

Discussion

The resuilts of the DCMU experiments demon-strate that the photosynthetic pigments are involvedin in,creasing the growth rate of the plant's. Thenecessity of exogenous sucrose for osbtaining thesegrowth responses (4) indiicates that the effect o-fphotosynthesis i.s prolbably mediated through some

synthetic capacity other than carbohydraxte prodtuc-tion. Further 'support for 'the noninvolvement ofcarbohydrate proiduction i's provilded by Machlis(4). He demonstrated t'hat 3 % 002, bubbledthrough the medium, stimulated the growth ofplants in the absence of 'stgar, but had n'o effecton gro'wth when 'stugar was present in the meditum.Photosynthetic amino acid (2) or ATP (1) pro-

duction may be involved in the stimulation of thegrowt'h rate by the photosynthetic pigments. Asimilar phenomenon has been demonstrated in-volvinog light-indutced antho'cyanin 'synzthesis in appleskin (3). Even though the skin was supplied wi'thsucrose in the externail medium, the photoreceptorwas the photosynthetic system.

Photosynthesis has been implicated in otherlight-induced growbh responses in liverworts.Steiner (110) demonstrated an effect o'f photosyn-thesis on the growth rate of sporeolings of Sphaero-carpos. Stange (7) 'showed t'hat the rate of re-

generation of organized meristems in ,the liverwort,Riella, is dependent on photosynthesis. Cytologicalchanges related to meristem formation (increase inntuclear 'size, celil division) occur (mtuch sooner inplants kept in light than in those kept in dark (8).The necessi'ty of 'high dosages of light led her tobelieve that photo'synthesis wa.s the process causingthe hastening of meristem formation. It is un-

*clear, however, 'how mucoh of these effects may bedue simnply to carbohydrate production since therewas no external carbon source in the growth media.

It iis likely that the phytochrome-mediated in-crease in growth rate is rela,ted to the changes inmorphdlogy induced by red light because of theclose correlation of these 2 responses. Althougha causal relationship is as yet unclear, it seemsprolbalble that the change in morphology, involvinga greater numiber of organized meristems, cautses am(ore rapid rate of cell division and hence growth.

S-teiner has allso demonstrated a red 'light-medi-ated morphologicall change in sporelings of Sphaero-carpos (10). Plants grown in blue light rarelybranc;hed. Ten minutes of red light administered4 times a day caused the thaqli to branch profusely.Far-red 'light did not reverse this effect of red butgave a similar though lesser response. For 2reasons, this morphological change observed bySteiner does not seem to be related to ,the morpho-logical changes observed in this study. First, bltlelight -can induice the same morphological devdlop-ment in our plants as red fight, though a higherdosage of blue (800 ergs-' cm2/sec) than that ofred is necessary to do thils. In Steiner's system,however, blue light is without effect. Second, themorphology of blule flight-grown thalli that S,tein,erob'served wa's typical of Sphaerocarpos in all re-spects except for the absence of branching. Inthis 'sttdy, however, dark-grown plants exhibitedan atypical calilu-s-Ilike morphology completely dif-ferent from that of plants grown in red qight. Afturther di.fference between the 2 systems is t'hea!bsence of any far-red reversal of the morpho-logical change observed by Steiner. This may bedtue to the 'same problem encountered here, namely,'the high sensitivity o'f the response to light in theregion of 700 to 715 n'm. A 'small amo-untt of lightin thi's wavelength band in Steiner's far-red soturcecouild be re-sponsilble for 'hi's far-red treatmentsgiving a 'respon'se similar to red light.

The diifferences in resuilts obtained here andthose reported by Steiner may also be dule todifferences in growth conditions and the plantsused. His plants were grown on a mineral saltsmedium solidified with agar an'd ours were insubmerged agitated liquiid cultu1re with sucrose, aswell1 as minerail sal,ts, in the me-ditum. Also, hi'splant material was sporelings whille oturs werefragments of mature thaili.

Literature Cited

1. ARNON, D. I., M. B. ALLEN, AND F. R. WHATLEY.1954. Photosynthesis by ilsolated chloroplasts.Nature 174: 394-96.

2. CALVIN, M. AND I. A. BASSHAM. 1962. The pho-tosynthesis of carbon compounds. W. A. BenjaminInc., New York.

3. DowNs, R. J., H. W. SIEGELMAN, W. L. BUTLER,AND S. B. HENDRICKS. 1965. Photoreceptivepigments for anthocyanin synthesis in apple skin.Nature 205: 909-10.

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4. MACHLIS, L. 1962. The effects of mineral salts,glucose, and light o.n the growth of the liverwort,Sphaerocarpos donnellii. Physiol. Plantarum 15:354-62.

5. MACHLIS, L. AND W. T. DOYLE. 1962. Submergedgrow.th of pure cultureLs of the liverwort, Sphaero-carpos donnellii. Physiol. Plantarum 15: 351-53.

6. AILLER, D. H. 1967. The effects of light on thegrowth and development of the liverwort, Sphae-rocarpos donnellii Aust. Ph. D. dissertation. Uni-versity of California, Berkeley, California.

7. STANGE, L. 1957. Untersuchungen fiber Umnstim-mungs-und Differenzierungsvorginge in regener-

ierenden Zellen des Leb-ermooses Riella. Z. Botan.45: 197-244.

8. STANGE, L. 1960. Die Abhiingigkeit der Regene-rationsvorginge bei Riella von der Dauer unddem Zeitpunkt der Photosynthese. Z. Botan.48: 143-52.

9. STEIN, J. R. 1958. A morphologic and geneticstudy of Goniunz pectorale. Am. J. Botany 45:665-72.

10. STEINER, A. M. 1963. Der Einfluss des Lichtsauf Morphogenese und Chloroplastenentwicklungder Gametophyten von Sphaerocarpus donnelliiAust. Z. Botan. 51: 399-423.

11. STEINER. A. M. 1964. Der Einfluss von Licht undTem,peratur auf die Sporenkeimung bei Sphaero-carpus donnellii Aust. (Hepaticae). Z. Botan.52: 245-82.

12. TAYLOR, A. 0. AND B. A. BONNER. 1967. Isoda-tion of phytochrome from the alga Mesotaeniumand liverwort Sphaerocarpos. Plant Physiol. 42:762-66.

13. WESSELS, J. S. C. AND R. VAN DER VEEN. 1956.The action of some derivatives of phenylurethanand of 3-pheiiyl-1,1-dimethylurea on the Hill re-

action. Biochim. Biophys. Acta. 19: 548-49.

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effects light growth and development liverwort ... · 170 rpm t1hrough a circle of 12.5 mmdiameter....

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