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 Machlis
Department of Botany, University of California, Berkeley, California 94720
Received December 12, 1967.
A bstract. Fragments of thalli of the liverwort, Sphaetocarpos donnellii Aust., inoculated
inito liquid medium containing sucrose and mineral salts, attain a much greater dry weight
after 9 days growth in continuous 'White light than in darkness. Light causes this difference
by increasing the rate of growth of the plants. This growth response is mediated by the
pigment systems of photosynthesis and phytochrome. An inhibitor of photosynthesis, DCMU,
at conoentrations which inhibit light-mediated CO2 fixation, decreases the growth rate of
light-grown but not dark-grown plants. Light still slightly increases the growth rate of plants
in 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-red
light 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 balls
of tissue consisting of lobes. These lobes are rounded and thick and exhibit an abnormal
callus-type growth, with few well-defined meristematic regions. Plants grown in red light
form fluffy ballls of tissue. The lobes of these plants have a morphology more typical of
Sphaerocarpos 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 growth
rate of the plan(ts grown in red light.

The original discovery of a non-photosynthetic These results were con'sildere'd by Machlils to be

light effect on the growth of Sphaerocarpos don-
nellii wa,s made by Machli's (4). He demonstrated
that 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 plants
were grown with glucose in continuouts wh,ite llight,
however, 'their dry weight increased 4- to 5-foild
over tfhait of dark-grown plants. The exogenous
sugar was irequired for these growth responses.
Plants maintained in conitinuous white light wilthout
sugar grew very muich less than dark-grown plants
with isugar. Mac'hli's postullated that thi's light
effe,cit wvas mediated by phytochrome becattse a
substantial dry weight increa;se over dark control's
occurred with a's ii-ttle as 5 minutites of White light
per 24 hours and this white light response was
reversed by 45 minutes of far-red fight.
preli,minary and (hi,s conclusions as ten'tative. The
present 'tudy is a detailled analysis of the ef'fects
of ligh,t on the growth of Sphaerocarpos.
Light effect's on the growth olf Sphaerocarpos
donnellii 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 development
of 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 thallli
rema,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.s
grown in this laboratory and demonstrated the
presen'ce of phytochrome in these extra,cts by dif-
ferentiail 'speotrophotometry.

1 This work was supported in part by National Sci- Methods

ence Foundation and National Aeronautic and Space
Administration 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 by
D. H. Miller to the Graduate Division of the University
of California, Berkeley in partial fullfillmenit of the
requirements for the Ph.D. degree.
2 Present address: MSU/AEC Plant Research Lab-
ora,tory, Michigan State University, East Lansing,
IMichigan 48823.
714
The OrganisIM. The organism used in all ex-
peri,ments wa,s the isolate 60-25 of the female
gametophyte o'f Sphacrocarpos dontnellii Ausit. (5).
The Mediumn. The growth mediuLm, as devel-
oped by AIachilis (4), was modified slightly. It
consisted of 0.01 Mi KNO3, 0.01 AI KH2PO,,
0.001 -r CaCl2, 0.001 ri MgSO,, 1.5 % suicrose, an(d
 MILLER AND MACHLIS-EFFECTS OF LIGHT ON SPHAEROCARPOS
a chelated trace element so;lution (9). The KH2PO4
solution was adjusted to pH 5.5, autoclaved sepa-
rately, and added to the rest of the medium. This
prevent-ed precipitation of Ca3(PO4)2 and the pos-
sible formation of sugar phosphates during auto-
claving. Fifty ml alliquots of this medium were
placed in 125 ml Erlenimeyer flasks. The flasks
were plugged with cotton and autoclaved at 15
pounds pressure for 15 minutes.
Procedure for Inoculation. The procedure for
inoculating flasks of medium was altered consider-
ably from Macih'lis' original one. This substan-
tially increased the reproducibility of dry weight
yieldis from flask to flask wiithin any 1 treatment
(121 ± SE 14.9 mg lbefore the dhanges, 150 + 2.2
mg after). Tihe modified procedure is outlined
below.
Plants to be used as inoculum were poured from
filasks into a sterile tea strainer, rinsed with about
300 nl of 'sterile ditstillled water, anid dropped into
a sterille blender cup. About 200 ml of sterile
water was poured intto ithe cup and the blender
turned on for 25 to 30 secondis. Thi.s blending
time assured complete fragmentation of all plants.
The fragmented tissue was then poured into another
tea strainer and rinsed with albott 500 in- of sterile
walter to waish out the smala fragmenits and cellular
contents released by the (blending. The washed
fragments were then dropped into a second blender
cup for convenience anid an appropriate vlntime of
sterile distiilled waiter added. The suspensi-on of
washed fragments in the blen,tder ctup was poured
into a sterilized Erlenmeyer flask containing a
Tefl,on magnetiic stirrer. The flask was then
p1laced on a stirrer which maintained an even sus-
pension of inotculuim throughout the period of time
involved 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 injected
into the flasks of medium by inserting the needle
between the cotton plug and the neck of the flask.
In all experiments the inoculum wa's grown
under the same standard conditions (conltinuous
white light for 9-10 days) and w'as useid at the
same point in i'ts growth curve '(from 320-380 mg
dry wt). Tihe weight of inoculum per flask was
maintained 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, caused
slight di,fferences in growth rates of the plants.
To eliminate such errors, dark controls were in-
cluded in eacih different treatment, or boxes of
plants receiving different treatments were rotated
around the 'thaker at 12 hour intervals.
Light. 'Continuous white light yielding about
600 ft-c at the surface of the haaker was obtained
from a bank of Sylvania F48T12 Cool White
fluorescent tubes.
715
Fluorescent tubes, ei-ther Sylvania Cool White
or Gro-Lux F72T12, were 'filtered through various
com1bination's of plexigdass, plastic, or gelatin filters
to dbtain the diifferent colors of lighit described
below. These tubes emitted no light above about
715 nm. The transmission spectra of these various
filter combinations, as measured on a Perkin-Elmer
202 UV Visible Spectrophotometer, are 'shown in
figure 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 inch
thickness of Robm and Haas Bdlue Pilexiglass 2424,
supplemented by additional layers of Cinemoid blue
plastic (Kleighgl Brothers, 321 West 50th Street,
New York, New York) where dower 'intensities
were required. Bilue-green light was dbtained with
3 layers of green, 2 lfayers of {blue Cinemoid plastic,
and 2 layers of blue gelatin. Two green Cinemoid
plastilc layers were iused for green lfight. Yellow-
,green light wa's obtained with 2 layers each of
green and orange Cinemaid plastic. Two layers of
red and 1 of orange 'Cinemoid plastic provided red
light. Layers of Whatman 3MM 'filter paper were
added to the red and oran;ge filters to decrease
intensities as needed. The short wavelength far-red
lilght was obt'ained by filtering light from Sylvania
Gro-Lux 'fluorescent lights through 4 layers of red
gelatin.
T'he source for far-red light was a bank of 8
G-eneral Electric Showcase 40 watt incandescent
bullbs fiftered through 6 cm of water containing
33.33 gm/l Fe(NH4)2(SO4)2 '6H1O and then
through 2 one-eighth inch thicknesses of Black
Plexiglasts (Westlake Pla-stics, Lenni Mills, Penn-
sylvania) and 2 layers of blue Cinemoid plastic.
This complex far-red source was necessary because
of the fact that 1 layer onuly of one-eighth inch
Black Plexiglass transmittedl enough light in the
range of 700 to 710 nm to stimulate the growth of
the plants nearly as well as red light.
The intensity of variouts filter system's was
measured on a standardized Eppley Tihermopile
716 PLANT PHYSIOLOGY
with a quiartz window attached to a Hewlett-Packard
Model 425A DC Micro-Volt-Ammeter. For the
experiiment testing different wavelength bands for
their activity in stimulating growth, equail inci(dent
quanta of different wavelengths were calculated by
the equation X X/hc (Tr) (Tr.), where X is the
number of qtuanta per cm2 per sec for -the different
wavelengths, X is the wavel-ength of ithe differen;t
light sources, h is Planck's constant (6.62 10-27
ergs 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 different
lightt sources in ptvolts. The wavelengths utsed in
the calctu(lation were blue = 460 nm, blue-green
- 500 nm, g,reen = 520 nm, yellow-green 565
unq1, red = 660 nm, and "far-red" = 710 nm.
Darkness was imposed by growing plants in
A) flasks that were taped with plastic elecitrical
tape or B) boxes wrapped in 4 layers of blacck
sateen cloth.
Growtah Facilitics. Flasks of plants were in-
cubated on rotary shakers usutally for periods of
9 to 10 days. For aill continuous light and some
intermittent light experiments, the shaker used was
in a 25° constant temperatutre rooom and moved at
170 rpm t1hrough a circle of 12.5 mm diameter.
For most of the intermittent light experiments
the planlts were grown in a shaker moving alt 106
rpm throutgh a circle of 2.54 cm diameter. This
machine was designed by the Department of
Grounids and Buildings of 'the University of Cali-
fornia, Berkeley (6). It 'consists of 4 light-tight
boxes m'ounted on the shaker. A bank of 3 Syl-
vania Gro-Lux fluttorescent 'tubes was located below
the boxes as were 2 rows of G. E. Showcase 40
wvatt incanTdescenit bulbs suirrounded by Black
Plexiglass. TIhe bottom of each box consists olf
2 collapsible halves. Two time clocks open the
boxes and tuirn on the lights at up to hourly inter-
vals for dura'tions of 1 sec to 50 minutes thus
exposing the plants to intermittent light.
Dry Weight Determinaztions. At the end of an
experiment, the contents of each flask were poured
into a 4.25 cm Buchner funnel and the medium
drawn of'f by vacutum through layers o'f W'hatman
No. 1 filter paper. The plants were then trans-
ferred to an aluminum cup and dried for 24 hours
in a 900 oven. These dry weight measurements
are reported as mg dry weight per flask of plants.
Determining the Uptake of H14C03 in the
Presence of DCMU. Two grams o,f tissue were
placed in a 50 ml Erlenmeyer flask containing
10 mnl o,f standard medium with varying DCMU
concentrations. For dark uptake measurements, the
flask was wrapped in 2 layers of altuminuim foil.
For liighbt exiperimen;ts the flask was held 40 cm
from a Sylvania 150 watt photoflood lamp with a
16 cm water bath in between. At time 0, 20 ,ul of
6 ttLvi NaHl'CO3 (1.54 uc per ml) was added to
the 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 the
supernatant meastured and poured into a screw cap
glass viall. The ground tisstsue was resuspended and
centriftuged successively in 1.5 ml aliquolts of 100 %
methanol, 20 % methanol, and water. The com-
bined supernatants were adjusted to equail voltume
and the radioactivity read as folilows. Fifty 1A of
each extract was pipetted onto an aluminutm plan-
chet. A few drops of 10 % acetic acid were added
and the planchets were dried in a hood underneath
a photoflood lamp. The radioactivity in the sam-
ples was determined with a Nucilear Chicago
Gas-Flow detecting system possessing a 30 %
coutniting efficiency. The aictivity is reported as
the difference between the activi'ty of extracts o f
light- and dark-treated plants.
II the experiments involvinig ithe effects of
DCMU oIn growth, the D'CMU was added as ani
alcohol solution. AIn equail amount (4410-4 % V/V)
of alcohol was added to the control medituni, becatuse
even tlhis low concentration was ilhibiitory to
growth.
Statistical Analysis of Data. The dry weight
measturemlent of red, red folilowed by far-red, and
dark-grown plants reported in ta(bles VI and VII
were subj ected to a t test to determine if the
differences in dry weight were statistically signifi-
cant. The t test demonstrated that the differences
in dry weight between red light treated plants and
either dark-grown or red, far-red treated plants
were significant at the 1 % level, whiile the differ-
ences between dark-grown and red, far-red treated
plants were not significant at the 5 % level.
Each experiment was carried out at least twice.
All dry weight measuremenits reported are the
averages of at least 6 and at most 10 replicates.
Results
The Nature of the Light Requirement. The
initial experiments tested the effect on growlth of
severail shorit expo,sures to ilow intensity red light.
If the growth stimulation by light were due com-
pl-etely to phytochrome activation, then several such
expossures per day should be as effective as con-
tintuous light. Table I shows the results of a
Table I. The Effects of Intermnittent Red and
Continuous White Light on Growth
Plants were grown for 9 days. The intensity of
the red light was 188 ergs-' cm2/sec.
Treatment Growth
M1g dry wt/f lask
Dark control 100 ± 2.3'
2 Min red light/8 hr 154 -- 3.1
2 Min red light/hr 156 ± 2.7
Continuous white light 412 -- 7.8
1 Standard error.
 MILLER AND MACHLIS-EFFECTS OF LIGHT ON SPHAEROCARPOS 717
typical experiment. As few as three 2 minute demonstrates that light is not required for growth.
exposures per day 'increased the growth of the Rather, the 'difference between the sets of plants
plants by 50% over the dark controls. More fre- is in the 'length of time required to reach the same
quent exposures, even as 'often as once per hour, total growth. Tthe logarithmic plot (,fig 2, left)
did no't further increase growth, and none of the reveal's some reassons for ,this difference. The
intermittent light-treated plants grew as muclh as slope of the log phase of plants grown in white
those given continuous light. These restiults indicate light is greater than the slope o'f -the plants grown
that 2 or more pigments are involved in the growth in red light or darkness. Thus, white light stimu-
response of Sphaerocarpos to continuous white lates the rate of growth during the log phase.
light: a pigment saturated by a red-fight exposure White light ail-so decreases 'the lag phase of growth
every 8 1hours and '1 or more pigments requiring by about,24 hours, a's compared to the ilag phase of
larger dosages of 'light. dark-grown plants.
Dturing the course of these experiments it be- Demonstration of the Involvemient of the Photo-
came apparent that the length of time the plants synthetic Pigments. Experiments were done to de-
were aillowed to grow greatly influenced the mag- termine what high energy pigment system(s)
niitude of the dry weight diifferences between light- shorten the lag phase and increase the growth rate
and darksgrown plants. For this reason, growth o'f the plants. To see whether the photosynthetic
curves were determined for plants grown in con- pigments were mediating these effects of white
tinuous whilte 'light, 2 minutes of red light per ihour, light, aIn inihibiltor of photosynthesis, 3,4-dichloro-
and darkness (ifig 2). The arithmetiic plot (fig 2, iphenyl,l,1-dimethylurea ('DCMU) was utsed (13).
right) clarifies the nature of the dight response. The fir'st experiment was designed to see if DCMU
The impression had been that light was required woul'd, in fact, 'inhibit photosynthesis in Sphaero-
for growth. Thits idea wa's the result of the fact carpos. Various concentrations of D1CMU were
thatt the plants had been harvested at the point in mixed in 'the regu(lar growth medium. Any re-
time when the differences ibetween their dry weights sultant chanige in photosynthetic activity wa's meas-
were at a maximum (day 8 or 9). The fact that ured as changes in th'e ability to fix CO2 in the
dark-grown plants attain a maximum dry weight light (fig 3). Increasing DCMU concentrations
only slightly dess than thalt of 'light-grown plants decrease CO2 fixation until at aibotut 5 kM DCMU

Continuous white light

a)
- 400
0
iU
1000
900 0
800
700
600 2 min. red/hr.
0) 500 Continuous white light ,0-e a)
O 400
0 300
E Dark
CO
O)
.- 300
200
E * A4Dark 0
4- 100 O/ aU
11V cO
*_ 90
80 N
o 70
c,, 60
o 50
40 200
~ ~ /
30 ~ ~ ~ ~

cn
o 20 -'V~ ~ /

b.
0)
O 10
8 ~ ~ ~ E
CM 7
Z 6 C1 100
3: 5
4
1- 3
-o
2
cr
E
c 2 3 4 5 6 7 8 9 10 11 12 13 14 15
Days Days
FIG. 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.
718 PLANT PHYSIOLOGY

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 if
these samne concentrations of DCMU whioh inhibit
photosynthesi,s also inhibit growth. Pilan,ts were
inoculated into flasks with medium containing dif-
ferent concentrations of DCMU and were grown
1%\ in conitinuouts white light or darkness (fig 4).
With increasing concentrations of DCMU, growth
0 in continuous 'light is increasingly inhibited uinitil
I.- the degree of inhibition (levels off at ablout the
0 same concentration that completeily inhibits photo-
0\
synthesis. Growth in 'the dark, however, is tinl-
Id a,ffected by any concentration of DCMU. Thus,
concentrations of DCMU which inhibit photosyn-
\0 thesis also inhibit the growth of Sphaerocarpos in
the light 'but not in tthe dark, indicating that the
high energy light response is mediated by the
photosynthetic pigment system.
0\ -. & In ald the experiments where DCMU was added
to the medium, no conicentraltion of -the in'hibitor
wvas effective in reducing the growth of light-grown
plan,ts to the same levell as that of dark-igrown
-6 plants. The next experiment was done to deter-
mine how this DCMU-independent light reesponse
LOG CONC DCMU (M) might be related to the growth response caused by
FIG. 3. The effect of DCMU on light-mediated intermittent red light (table II). Two important
CO2 fixation. Radioactivity reported as counts fixed
in light minus counts fixed in darkness. Table II. The Effect of DClMU o01 the Growtlt of
Plants in Light and Dark
The intensity of red light was 122 ergs-' cm2/sec.
4001t Plants were grown for 9 days.
Growth
With 4,um
Treatment Without DCMU DCMU
.Mg dry wt/flask
300 Dark control 85 ± 4.61 83 ± 4.1
Continuous white light 380 + 6.8 140 ± 5.0
7 Sec red light/ hr 136 ± 4.1 135 + 4.5
1 Standard error.

I-

3 results are to be noted. First, DCMU affects

2001-
ty neither dark-grown plants nor the growth response
o-f plants to intermittent red light. Second, and
more important, the short, red-il'ight treatments are
as eiffective a's continuous white light in stimulating
the growth of D,CMU-treated plants.
100 1 - --- --- DARK Demonstration of the Involvenment of Phvto-
chrome. The frequency oif light 'exposures needed
to saturate the red-light reisponse was determined
next. Four groups of plants were given expostures
to red light of varying frequency and du-ration such
tha,t the total exposure to li,ght was 12 minutes
// I. every 24 hours (taible III). The increases in dry
7 - 6 -5
0 - weight oif light-treated plants over dark controls
LOG CONC DCMU ( MA) are about the same with dark intervals oif 4 to 12
FIG. 4. The effect of DCMU on growth of plants hours. With an interval of 24 hours, however, a

for 9 days in continuous white light and darkness. maximum response is not obtaiined. Red light at
 MILLER AND MACHLIS-EFFECTS OF LIGHT ON SPHAEROCARPOS71 719
Table III. The Effect of Various Red Light Treat- plant's was less than the maximum. At these very
inents on Growth l-ow intensities the response i-ncreased with in-
Red light initensity o-f 188 ergs-' CM2/sec. Pl-ants creasing intensity up to -saturation.
were grown for 9 days. The effect of different wavelength band-s on th-e
low intensity 'light respon-se of Sph~aerocarpos i-s
Red light treatment Growth simil-ar to t-hat of phytochrome. This i's shown by
Mg dry wt/fkask the results of -an experiment in whic-h plants were
Dark controlI 105 ±2.4' given intermittent light of 5 different wavelengths
2 Min/4 hr 156 ±2.1 (ta-ble V). The intensities of the 5 quallities of
4 Min,/8 hr 1,55 ±2.5 light were adjutsted so that they wer-e at equalI
6 Min/12 hr 159 ±2.6 incident quanta. Red light 'is by -far the most
12 Min/24 hr 144 ±4.0 effecotive wavelenigth.
1Standar-d error. T-he definitive proof 'that a red-ilight re-sponse
is mediated by phytochrome is a demonstration that
least once every 12 houirs is necessary for a maxi- the response is repeated-ly reversible by aliternating
mulm -response. exposures to 'red and far-red light. Taible VI
Another characteristic of phytoch'ro-me-mediated demonstra-tes th-is characteristic 'in t-he -system uinder
responses i:s that th-ey can be elicited 'by very low study.
light intensities (ta:'ble IV). I-t is -apparent that Table VI. Repeated Reversibility of the Growth
the plants are 'sensitive to very low initensities of
red 'light. Furth-er, the response is independenIt of Response
Light wa-s given onoe perday f-or 9 days. The
initensities of th-e red and far-red were 4000 and 1410
T-able IV. The Effect of Red Light Intensity ergs-' CM2/seC, respectively. R = 2 min red, FR=
on Growth 40 min far-red.
Plants were grown for 9 days. Exposures of 2
minutes per 12 hours. Intensities given in ergs-' CM2/ Treatment Growth
sec.
Mg dry wt/flask
Inten,sity Growth Dark control 108 ±+ 1.51
R 124±--1.9
mg dry wt/flask RI, FR 110 ±+ 2.2
0 (dark control) 122 ±- 5 2' R, FR, R 124±4-1.4
38 182±4-4.5 R, FR, R, FR 114 ±- 2.4
189 185 ±- 3.3 1 Standard error.
3,77 179 ±2.4
3018 184 ±3.6
Standard error. Morphological Differences. The plants grow in
the form of fluffy balls of ti,ssue with each ball
composed of many lobes. The type of lobes -formed
intensity over a wide 'range. In other experim~ents in dark-grown a) and red-light-grown b) plants
t-he -total light energy given per exposure wa's de- are 'shown in -figure 5.
c-rea:sed to th-e point where the pigment sys-temn was Dark-grown plants form very compact bail's o-f
no-t sa-tu-rated a-s evidenced by the fact thalt the dry tissuie. Their lobes are solid masse-s of cell's re-
wei-ght di'fferenc-e between light- and dark-grown sem'b'lin'g c-al(lus tiissu.e. Th-ere are very few well-
defined meri,stemat-ic areas.
Table V. The Effect of Different Wavelength Bands Plants grown in 'red 'light -for-m much looser
on the Low Intensity Light-Mediated ball's whi-ch consi,st of fla-ttened Aseets of tis.,sue.
Growth Response Their lobes are u-sualily 2 celil layers t-hick and
Light given 2 min/12 hr. Intensities r-anged from exhibit a typical growth habit for Sph-aerocarpos.
1,88 in the far-r-ed to 28,2 ergs"' cm2/s:ec in th-e blue. The marginis are meri'stematic areas of small1 com-
Plants were grown for 9 days. pact cells which gradually entlarge towa:rd th-e
center of the sh-eet of ti'ssue. There aTe many
R-esponse a-reas of meristematic activity in each ba'l'l of tissue.
Treatment Growth (light-dark) T-t is believed that the gre-ater nu-m'ber of meri,stems
Mg dry wt per flask ail-lows -for a greater rate of growth. 'Plants grown
Dark control 86 ±- 2.0' 0 in blue light exhibit a morphology 'similar to that
Blue (460 mm) 97±4-2.7 11 of plants grown in red light, but a much higher
Blue-green (520 nm) 96 ±4 2.9 12 intensity of blue light i's ne-eded to satUrate thi's
Yellow-green (565 nm) 94 ±4 2.7 8 response '(ca. 800 ergs/cM2/sec).
Red (660 mm) 1,26±::3.4 40 Th-e 'less c-ompact growth halbit of plants grown
Far-red (715 nm) 1,09 ±- 3.3 23
in red lig-ht resuilt's in a considerably higher fresih
1 Standard error. weight to dry weight ratio a-s comipared to dark-
 s. -
720 PLANT PHYSIOLOGY

'4.
_,2. ..:..: ..
'A
*;s<,.M:1; ::.
_s iu
...
:.:

.:. :.::
::..:.::.: ::..:.
w ..............
.., ..........
::: .::

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

red light.
 MILLER AND MACHLIS-EFFECTS OF LIGHT ON SPHAEROCARPOS
Table VII. The Effect of Red and Far-Red Light on
the Fresh Weight, Dry Weight, and the Fresh
Weight to Dry Weight Rxatio
Meassurement 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 mg
Dark control 2.30 + 0.501 143 ± 1.4 16.1
1 Miin red/24 hr 3.79 ± 0.41 176 ± 1.9 21.5
1 Min red + 50 min
far-red/24 hr 2.56 ± 0.32 147 ± 1.9 17.4
Standard error.
grown plants. This increased ratio is an accurate
measure of the change in morphology ca'used by
red 'light an'd i's reversible by far-red ilight (table
VII).
Discussion
The resuilts of the DCMU experiments demon-
strate that the photosynthetic pigments are involved
in in,creasing the growth rate of the plant's. The
necessity of exogenous sucrose for osbtaining these
growth responses (4) indiicates that the effect o-f
photosynthesis i.s prolbably mediated through some
synthetic capacity other than carbohydraxte prodtuc-
tion. Further 'support for 'the noninvolvement of
carbohydrate proiduction i's provilded by Machlis
(4). He demonstrated t'hat 3 % bubbled
002,
through the medium, stimulated the growth of
plants in the absence of 'stgar, but had n'o effect
on 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 the
growt'h rate by the photosynthetic pigments. A
similar phenomenon has been demonstrated in-
volvinog light-indutced antho'cyanin 'synzthesis in apple
skin (3). Even though the skin was supplied wi'th
sucrose in the externail medium, the photoreceptor
was the photosynthetic system.
Photosynthesis has been implicated in other
light-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. Cytological
changes related to meristem formation (increase in
ntuclear 'size, celil division) occur (mtuch sooner in
plants kept in light than in those kept in dark (8).
The necessi'ty of 'high dosages of light led her to
believe that photo'synthesis wa.s the process causing
the hastening of meristem formation. It is un-
*clear, however, 'how mucoh of these effects may be
due simnply to carbohydrate production since there
was no external carbon source in the growth media.
721
It iis likely that the phytochrome-mediated in-
crease in growth rate is rela,ted to the changes in
morphdlogy induced by red light because of the
close correlation of these 2 responses. Although
a causal relationship is as yet unclear, it seems
prolbalble that the change in morphology, involving
a greater numiber of organized meristems, cautses a
m(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 rarely
branc;hed. Ten minutes of red light administered
4 times a day caused the thaqli to branch profusely.
Far-red 'light did not reverse this effect of red but
gave a similar though lesser response. For 2
reasons, this morphological change observed by
Steiner does not seem to be related to ,the morpho-
logical changes observed in this study. First, bltle
light -can induice the same morphological devdlop-
ment in our plants as red fight, though a higher
dosage of blue (800 ergs-' cm2/sec) than that of
red is necessary to do thils. In Steiner's system,
however, blue light is without effect. Second, the
morphology of blule flight-grown thalli that S,tein,er
ob'served wa's typical of Sphaerocarpos in all re-
spects except for the absence of branching. In
this 'sttdy, however, dark-grown plants exhibited
an atypical calilu-s-Ilike morphology completely dif-
ferent from that of plants grown in red qight. A
fturther di.fference between the 2 systems is t'he
a!bsence of any far-red reversal of the morpho-
logical change observed by Steiner. This may be
dtue to the 'same problem encountered here, namely,
'the high sensitivity o'f the response to light in the
region of 700 to 715 n'm. A 'small amo-untt of light
in thi's wavelength band in Steiner's far-red soturce
couild be re-sponsilble for 'hi's far-red treatments
giving a 'respon'se similar to red light.
The diifferences in resuilts obtained here and
those reported by Steiner may also be dule to
differences in growth conditions and the plants
used. His plants were grown on a mineral salts
medium solidified with agar an'd ours were in
submerged agitated liquiid cultu1re with sucrose, as
well1 as minerail sal,ts, in the me-ditum. Also, hi's
plant material was sporelings whille oturs were
fragments of mature thaili.
Literature Cited
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3. DowNs, R. J., H. W. SIEGELMAN, W. L. BUTLER,
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pigments for anthocyanin synthesis in apple skin.
Nature 205: 909-10.
722 PLANT PHYSIOLOGY
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