Plaint Physiol.

(1967) 42, 1123-1130

Changes in Chlorophyll a/b Ratio and Products of '4C02 Fixation

by Algae Grown in Blue or Red Light'
J. L. Hess and N. E. Tolbert
Department of Biochemistry, Michigan State University, East Lansing, Michigan 48823
Received May 1, 1967.

Sutmmary. Chlamydomonas and Chlorella were grown for 10 davs in white light.
955 Uw/cm2 blue light (400-500 m,u) or 685 Juw/cm2 red light (above 600 mb). Rates
of growth in blue or red light were initially slow, but increased over a period of days
until normal growth rates were reestablished. During this adaptation period in blue
light, total chlorophyll per volume of algae increased 20 % while the chlorophyll a/b
ratio decreased. In red light no change was observed in the total amount of chlorophyll
or in t'he chlorophyll a/tb ratio. After adaptation to growth in blue light and upon
exposure to "CO, with either blue or white light for 3 to 10 minutes, 30 to 36 % of
the total soluble fixed 1"C accumulated in glycolate-14C which was the major product.
However, with 1 minute experiments, it was shown that phosphate esters of the photo-
synthetic carbon cycle were labeled before the glycolate. Glycolate accumulation by
algae 'grown in blue light occurred even at low light intensity. After growth of the
al,gae in red light, 1"C accumulated in malate, aspartate, glutamate and alanine, wherea;
glycolate contained less than 3 % of the soluble 14C fraction.

Several groups of insvestigators have reported an
effect of blue light upon the rate of photosvnthesis
and upon the distribution of 14C among the products
of 14CO9 fixation. Initially, Warburg et al. (24,25)
reported a stimulation of photosynthesis by Chlorella
in red light upon addition of blue light. In a recent
confirmation of this phenomenon with Acetabularia,
Tenborgh (19) provided reasons why t-his blue light
potentiation is different from the Emerson enhance-
ment effect associated with the 2 pigment systems
for electron transport. When photosynthesis was re-
stricted to blue light only, Roux et al. (17), Tyszkie-
vicz (22) and Voskresenskaya and Grishina (23)
all fotund an increased proportion of amino acids,
particularly glycine and serine, and generallv less
starch synthesis. More recently Zak (28), using
Chlorella, and Andreeva and Korzheva (1), using
sunflower leaves, have made similar observations.
Cayle and Emerson (4) in 1957 using Chlorella re-
ported that glycine was labeled in the C-2 carbon
after 5 minutes of 14CO2 fixation in blue light but
uniformly labeled from experiments in white light.
Thus, the general trend of these investigations has
been that blue light affected glycine and serine bio-
synthesis and perhaps other amino acids and protein.
However, Krotkov's group (6, 7, 21) also using
Chliorella as well as tobacco leaves, found that blue
1 Supported in part by NSF grant GB 4154 and pub-
lished as journal article No. 4064 of the Michigan Agri-
cultural Experiment Station. This investigation was re-
ported at the Fourth International Photobiology Con-
gress, Oxford, England, 1964.
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light, although stimulatory, promoted 14C labeling of
aspartate and glutamate. In some of the above ex-
periments, light intensity may have been a limiting
factor. Horvath and Szasz (10) have reported that
amino acids were major products of photosynthesis
at low light intensity and that sugars were formed at
high light intensity.
Since blue light appeared to effect glycine and
serine, we reasoned that glycolate formation would
also be affected, for in the higher plant glycolate is
a precursor for glycine and serine (16). When the
work was initiated in 1963, we could confirm with
Chlorella the stimulation of glycine and serine forma-
tion in blue light, but there was no pronounced effect
upon glycolate. Afterwards it was discovered that
algae, unlike most higher plants form serine from
P-glycerate (9). A CO pathway may be functioning
in algae (26), however, most of the free glycolate is
not metabolized to serine but excreted, since the algae
lack a normal glycolate oxidase (8. 9). However,
the algae, after growth for several days in the blue
light, altered their photosynthetic or metabolic process
in such a way that glycolate became the single major
product of short periods of 14CO, fixation. Conse-
quently, this report is concerned with algae grown
for several days in blue light in contrast to all previous
work with algae grown in white light and onlv ex-
posed to blue light during the test period.
In this investigation, we also examined the algae
for any gross spectrophotometric changes during
g.rowth in blue light. Fuj ita and Hattori (5) re-
ported that changes in chlorophyll a and b concentra-
tions in Tolypothrix responded to light quality rather
 1124 PLANT PHYSIOLOGY
tlianlight intensity. Jones andMAIN- ers( 11 ) reached- of the culturing and photosynthesis experiments in
asimiiar conclulion from work with A nacvstis. Light blue or red light N-ere performed in dark rooms.
intensitv apparently affects only th e rate of pigmnent 'rhese cultures wveregrowvn under continuous light,
COInv'rS1io01. aerated with 0.2 % CO. in air, anid maintained at
a water bath.
20 to 210 bygrow light
:hods
Materials and Met Cultures blue received

955 ,t\v/cnl2 as measured by a recording spectro-

.i/go(I. Cultures wvere obtained fromii the Culture radiometer, model SPR fromInstrumentation Special-
C'ollection of Algae" at Indiana ,L ersitv.
niv Bloom- ties Company, Inc. This intensity of blue light also

'OSaIChick;(Enmer-
ingtol, Indiana. Chrorclla pyrenioid Weston IlluminationNlMeter,
measuredl400 ft-c bya filter.
son) ( No. 39)5) x as cultured in inorganic salt anl model 756with a quartz Values in the tables
mediumll (14 ) x ith Hoagland% miicronutrients. are mea.sure-
expressed in ft-c to reflect the actual
Chlamvdomionas r-eibhardtii Dangeat rd(-) (No. 90) ments because the spectroradiometer was not yet
was grown on the high phosphate medium described availab!e when the research was done.685 Cultuires
uv/cm'
by Orth et al. (15). Cells vere cultured in 1.5 L grown in continuousm,ured light received
between610 to Mxw/cm2 between
ot mlediuimi in 2.8 L 'Lowx form'a'
610
ernbach flasks 00 or 765

fitted w-ith air inlets and agitated on a reciprocating to 720mru. As measured by our Weston Illumination
slhaker b)v abotut 60 excursions per miniute. For cul- MIeter this intensity- was equal to 200 ft-c. The
tUringof algae inxwhite light, thes was kept biaker irn cultures weredliluted every second dav with fresh
a controlled environnment chamber aLt xvhichmain- 15° nutrient to an absorbance of 0.2 at 680my as measured
taimied a temperature in the cultur e of 200. imieditutmi in a 1 cm cuvette with a Beckman DU spectropho-
Conti11uo,LS light at the surface o fxvhlite. culture wxas
the super-high, tometer.
a-fter
Aliquotswvere removed at variousgrowtth
innoculation to
times
determine the rate of
1200 ft-c froimi WVestinghouse cool.
fluores-cent bulbs (F96T12/CW/51H -0). The cultures as expressed in increase absorbance at 680 nml.
were gassed with 0.2 % CO., v/v ir tlueair.or aAlgae l
xvere
red filter
'4CO2 Plhotosynthesis Experimnents.
remoxed in the growth
dark from the media
Cellsby wvere
cen-

also cultured in light passed by a b

svsteni wxhich transmitted light as s hoxvn in figuire 1. trifugation, 1
and a % (v/v) suspension of was cells
The red filter was Fire Red" No. Iago. [10 gelatin (Grand
Illinois), and
prepared
A 15 ml
in 1 m-t phosphate with a final pH of 6.5.
suspension, in lollipop fitted with large

Sta-e anid Lighting Company. Chic

light Nvas obtained from a bank of 15 iatt red fluo- bore stopcock for rapid removal of aliquots, was

riescent light's General Electric I 1F15T12-1R).

Blue maintained 20°
preillumination
in a Xx'ater bath. AfterNaH14CO3
at in a
5 minutes
designated light, a
light wvas obtained from a bank
fluoresceint lights ( General Elect F15T12-B or :ric throug-h (2-5,umole) solution was added and samples were
Sylvania F15>T8-B) and wras filter ed a blue removed at 1, 3, and 10 imiinutes. Aliquots were
celluloid filter aind 5 CI of a CuISO, solution con- dunmped directly into warm methanol and further
taining 30 g I1 (27). The transn determined nission
of bothspec-
the heate(l. The 14C products
separatedl in methanol-water
chromatography
extracts
radioautog-
blue and red commlercial filters wa.s
by and
were

trophotonmetrically by us. The cc mbination of blue raphy (3).

celluloid and CuSO, solution passe dxveen a band of einis-
400 myt and Ten
Extraction and Determiniation of Chlorophyll.
ml of 0.5 algalcentrifuged
suspension

sioIn whichl was predominately bet

500 imi/t anid contained no light al )ove 570 mM,. All in a clinical centrifuge at maximum speed for 3
minutes. The supernatant fraction was discarded and
the inverted on an absorbant surface to eliminate
tube
1001
/Red Gelatin excess Xvater.After several the cells were
placed
minutes,
resuspended in 3 ml of absolute methanol and for
c
0
/ in a stoppered centrifuge
tube in the dark at
V)
tn 1I0 least 2 hours to insure complete extraction of the
Ecn 1; pigments. After centrifugation.
the absorbance of
supernatant fluid was measured and clhloro-
50 -
.C-
12
-

the green
_II phyll a and b concentration calculated (12).
0-2
Measuremient of in vizo Absorption Spectra. The
neutral-density, filter technique of Shibata et al.
300 400 500
zl_<S700j-<-
600 with mineral oil was
which a filter paper saturated
(18) xvas employed inplaced betxveen the light source
Wavelength, mjp
and the cuvette. We found that a double thickness
Fic;. 1. Absorption spectra of filter systems used of waxed paper (Schleicher and Schuell Co. No.
for photos nthesis experiments. TI- red hrths above
gelatin600 filter B-2). placed next to the cuvette onelucidated
the face toward
transmitted onlv those wvaveleng
(
m,u the ligtht source, most effectively the fine
celluloid
only those chlorophyll spectrum.
). The blue filt
.--
insmitted structure of the in vivo The
the CuSO4 Solution filter ( - - -) tra
+-avelengths indicated atid transmit tted nio red or far ratio of chlorophyll a/b absorption was evaluated from
re(l li-ht. spectra measured oIn the Carv 15 spectrophotomiieter
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 'HESS AND TOLBERT-PHOTOSYNTHESIS BY ALGAE IN BLUE LIGHT
while using a slit idth of approximately 0.7 mm.
Under these conditions a 1 % algal suspension gave
approximatelyt 0.5 absorbancy units at 750 my.
Results
Growth of Algae. Cultures, when removed from
white light and put in either red or blue light (as
described in the Methods section) grew more slowly
for 4 or 5 days before attaining a relatively constant,
rapid grow-th rate which approximated that of the
cultures in w-hite light of similar intensities. Thus,
Chlam ydornonas and Chlorella in blue light grew
slowlv at first and then more rapidly, as measured
by the culture's absorbance at 680 m,u (fig 2). All
measurement-s were made on non-synchronized or
random cultures which initially had similar cell popula-
tions as judged from approximately similar absorbance
values. Chlauiydomonas in red light also grew more
slowly at first, but after several days in the red light,
they too grew rapidly. We think that the slow re-
covery of the growth rate in blue or red light was an
adaptation rather than a mutation, since all the results
presented in this paper were similarly reproduceable
when starting oxer again with stock cultures kept in
white light.
Rate of 14COo, Fixation. With Chlorella or
Chlamydomionas fully adapted after at least 10 days
1125
growth in either white, blue, or red light, experiments
on the rate of 14CO fixation were performed with
increasing time and intensity of red, blue or white
light. The available intensity of the blue light was
the limiting factor, but representative data in figure 3
indicated that 400 ft-c was approaching light satura-
tion. In the experiments nearly linear 14C fixation
rates over the 10 minute period were observed, and
thus CO2 availability did not become limiting. The
fixation rates with Chlorella grown in blue light were
unusual since relatively high levels of fixation
occurred in 10 minute periods at low blue light in-
tensities.
Distributtion of 14C Amtong Soluble Products of
Photosynthesis. For the purpose of presentation, the
]4C distribution among the products of CO2 fixation
has been divided into 3 groups of compounds: A) phos-
phate esters representing components of the photo-
synthetic carbon cycle, B) malate, aspartate, gluta-
mate, and alanine which are the compounds associated
with the citric acid cycle that accumulate "-C, and
C) glycolate and glycine plus serine which are asso-
ciated with the glycolate pathway (16). The percent
distribution of 14C among each product of "CO2
fixation by paper chromatography is on file along
with results from other variations of light intensity
and quality (8).
Algae grown in either white, blue or red light

=L 10th day~~~~~~~~~~~~~~~~~~~0h
E dth
dyy
0.6|

~~~~~~~~~~5th day

.0
0.4-
0

Time (hours)
FIG. 2. Rate of growth of Chlorella pyrciioidosa and Chlaniydomonas reinhardtii after designate days in blue
light. Vertical litnes designate the variation of rate of growth from repeated experiments after 10 days culture
in blue light.
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Copyright © 1967 American Society of Plant Biologists. All rights reserved.
1126 PLANT PHYSIOLOGY

c
.2 30- A B C
40..
a

0
0

0~~~~
c 20-
t min
0 0
01 10 min

10 mi
b 10
E
N.
o
0
3min
0 13 1m 3m
min
x-)
I v
I0. I I I I I I I I I I -F I I
I a

0.5 4 0.5 2 4 0.5 4

Blue light intensity (ft-c x 10-2)
FIG. 3. Rate of total 14C fixation by algae in increasing intensities of blue light. In part A, C/ilo cll//a pyrenoi-
dosa were grown in white light and in part B in blue light for 10 days before the experimiient. In part C,
Chlamydomonas reinihardtii were adapted for 10 days in blue light. Ordiniate values are total 14C in the soluble
fraction from 15 ml of a 1 % algal suspension.

and allowed to fix 14C02 in the same tyrpe of light
incorporated initially the highest percentage of 14C
into the phosphate esters of the photosynthetic carbon
cycle. Typical data for Chlamydomonas are sum-
marized in figure 4. As indicated by the rapid re-
duction in the percent of the total 14C fixed which
accumulated in these esters, the pool sizes in the blue
adapted algae appeared smaller than in the algae
grown in red light.
Chlamydomonas grown in blue light rapidly accu-
mulated 31 % of the newly fixed 14C into glycolate
during photosynthesis with 350 ft-c of blue light. If
the algae were grown in red light, they accumulated
only a trace of 14C labeled glycolate (fig 4). Similar
For both algae the percent of the total 14C incor-
porated into malate, aspartate, glutamate. and alanine
was somewhat greater with aligae grown in red light
than white light and much greater than with algae
grown in blue light (fig 4). Thus, grovth in blue
light promoted 14C accumulation during the initial
minutes of 14C0. fixation into glvcolate while growth
in red light resulted in 14C accunmlation in malate,
aspartate, glutamate, and alanine.
If blue light were used for 14CO2 fixation experi-
ments with algae grown in white light or during the
first to third day after initiation of their growth in
blue light, the large significant changes in the per-
centage of 14C incorporated into glycolate ere not x

results were obtained with Chlorella except that the observed. Since accumulation of a large percentage
total percentage of fixed 14C in glycolate was only of the 14C in glycolate in blue light did not occur with
about half that found with Chlamydomonas. algae growrn in white light, glycolate accumulation
For Chlamydomonas the percent '4C in glycine and was probably not related to possible alterations in
serine was not greatly altered by their growth in assimilatory power produced by the blue liglht.
either blue or red light. Chiorella, however, showed Algae grown for 10 days in blue or red light
a 1 to 2 fold increase in the percent of the total 14C produced about the same products regardless of
in serine immediately after culture in blue light was whether 14C02 fixation was measured in white, blue
initiated. This result was similar to earlier experi- or red light. Thus, the same accumulatioil of 14C-
ments cited in the introduction with Chlorella. Simul- glvcolate bv algae grown in blue light occurred when
taneously with increase serine-14C, we observed that the 10 minute '4C0 fixation period waS run in either
the percent of 14C accumulating in P-glycerate de- blue or white light. These facts support the hypo-
creased about half. thesis that the cultures growin in blue light actually
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 HESS AND TOLBERT-PHOTOSYNTHESIS BY ALGAE IN BLUE LIGHT
experienced a period of slow cellular adaptation which
was not rapidly reversed by white light.
The data in figure 5 emphasizes the effectiveness
that growth of Chlamilydomonas in blue light has on
glycolate production. It is established that the amount
of glycolate production by algae increases at higher
light intensities (2, 13, 20,26). In the present ex-
periments glycolate production at 1 10 ft-c by Chlamnv-
donmonas grown in either red or white light was less
than 3 % after 10 minutes (see fig 5B for algae
grown in red light), but increasing the light intensity
to 1200 ft-c resulted in considerable formation of
glvcolate-14C. However, if the Chlamnydomionas were
grown in blue light for 10 days, then they formed in
110 ft-c of blue (fig 5A) or white light (data not
shown) nearly the same amount of glycolate-14C which
the algae grown in white or red light could only pro-
duce at 1200 ft-c. For Chla;nndomonas grown in blue
light '4C in glycolate amounted to 15 to 36 % of the
total 14C fixed between 3 and 10 minutes, and con-
sequently glycolate-'4C was the major soluble product
of CO,, fixation. This amount was 3 to 4 times more
14C than in any other single product, yet as seen from
figure 4, during the first minute of 14CO2 fixation,
there was more 14C in P-glycerate or sugar phos-
1121,
phates than in glycolate. Thus, algae grown in blue
light accumulated large amounts of glycolate-14C even
at low light intensities and high light intensity further
increased the amount of glycolate which was formed.
It is also apparent in figure 5, that glycine and serine
accumulation was not affected by growing the
Chla-mi,domuonas in blue light. This is consistent with
the fact that serine and glycine synthesis is independent
of glycolate formation in algae (9), in contrast to
plants in which serine and glycine are formed from
glycolate ( 16).
Chlorophyll Coniteiit. Using a Carv 15 recording
spectrophotometer, absorption spectra were measured
on cell suspensions as described in the 'Methods sec-
tion. The spectra in figure 6 have equal absorbance
values at 550 my, but, for clarity of presentation, the
curves have been separated in the figure. The change
of the 680 mrp/7655 mu absorbance ratio, which repre-
sent in vivo maxima for chlorophylls a and b re-
spectively, indicated a significant decrease in the
chlorophyll a/b ratio as the period of culture in blue
light increased. No significant changes in these re-
gions of the spectrum were observed for algae grown
in red light. Other portions of the spectrum did
reflect absorption changes caused by growing the cells

o 60 \ Phosphate Esters - * Glycolate Malate, Aspartate,

0 Glutomate + Alanine
a Glycine +

40- Red _.

.0 - -

~~~~lue\
0/ %_
20-
4
0

I 3 10 1 3 I0 I 3
Time (min)
FIG. 4. Percent distribution of 14C among soluble products formed by Chlaiamydomlonias after adaptation to
blue or red light for 10 days: (0- )) Cells grown in blue light anid 14CO. photosynthesis was performed in 350
ft-c blue light; ( 0----0) Cells grown in red light and 14CO, photosynthesis was performed in 200 ft-c of red
light ( >600 mu).
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1128 PLANT PHYSIOLOGY

c __

4-

O 1200 ft-c u

340

'~~~~~
20 200 ft-c white

1101f
' 3 10 I 3 10
Time (minutes)
FIG. 5. Total 14C in glycolate or glycine plus serine formed by 0.5 % (v/v) suspension of Chlaiiiydomonas reini-
hardtii grown in (A) Blue light or (B) red light; ( * 0 ) after 14CO. fixation in 350 ft-c blue light oi
(* -U) in 1200 ft-c white light.

in red light, but these were not consistent and too
complicated for a careful evaluation by this technique.
The above in vivo measurements were verified by
results from spectral measurements of chlorophyll in
extracts from the algae. The total chlorophyll con-
tent on the basis of the cell volume increased about
20 % during the first 6 days of culture in blue light,
and a significant decrease appeared in the chlorophyll
a/b ratio (fig 7). Although the ratio of chlorophyll
a/b varied from 1 to 2.8 for different starting cul-
tures which had been grown in white light, a con-
sistent trend was a decrease in the chlorophyll a/b
ratio during culture of the algae in blue light. Similar
results were obtained for Chlorella. The data from
Chlamydomonas extracts, however, were more con-
sistent than with Chlorella, perhaps, because the
pigments from Chlorella were extracted less qulantita-
tively.
Discussion
Three major changes were observed when Chlanty-
domonas or Chlorella were grown in 955 ILw/Cm2 of
continuous blue light (425-540 mu) or in 765 uw/cm2
red light (above 600 mu). A) Growth rate slowed
for 3 to 5 days, but returned after 5 to 10 days to
rates equal to those with similar amounts of white
light. B) After 5 to 10 days adaptation to blue
light the algae incorporated about 30 % of the total
'4C fixed in 10 minutes into glycolate at low (110
ft-c) blue or white lilght intensities and even more at
1200 ft-c light. This phenomena did not occur until

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Copyright © 1967 American Society of Plant Biologists. All rights reserved.
 HESS AND TOLBERT-PHOTOSYNTHESIS BY ALGAE IN BLUE LIGHT
Y The accumulation of such large amounts of 14C in
1-
s
A, m,u
FIG. 6. In vivo spectra and chlorophyll a/b ratio
for Chlamydomonas reinhardtii grown for designated
number of days in blue light. T(he spectra were mea-
sured on approximately 1 % algal suspensions and re-
corded with a Cary 15 Spectrophotometer. All absor-
bance values at 550 mA were approximately equal. The
absorbance ratio a/b equals the absorbance ratio 680
mnu/655 mg which was indicative of the chlorophyll a/
chlorophyll b ratio.
= 3.0-
cg \ Chlomydomonas
0
3%2.0 0
06
0
Days
FIG. 7. Changes in the chlorophyll content of
Chlartydomiionias reinhardtii as determined by the amount
of chlorophyll extracted with methanol. The change
in the chlorophyll a/b ratio is plotted as a function of
the increasing nuimber of days of growth in blue light.
after the initial period of adaptation. After growth
in blue light, 14CO2, fixation in white light produced
the same "lC distribution among products as in blue
light. Algae grown in red light incorporated more
1"C into malate, aspartate, glutamate and alanine and
only trace amounts into glycolate. C) During growth
in blue light, chlorophyll content increased 20 % while
the chlorophyll a/b ratio decreased. The results were
reproduceable when starting again with fresh cultures
which had been grown in white light. The results
suggest a slow environmental adaptation over several
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Copyright © 1967 American Society of Plant Biologists. All rights reserved.
1129
generations to the specific light quality. The data
invite speculation that both CO, fixation and the
pigments for electron transport in photosynthesis are
so intimately interdependent that complementary
changes in both systems compensate for environmental
alterations. These changes could maximize the
capacitv of an organism to utilize light and CO,.
glycolate by algae grown in blue light does not indi-
cate that glycolate was being formed by a separate
pathway of CO, fixation. In all experiments P-glyc-
erate and sugar phosphates contained the most 14C
during the first minute of 14CO2 fixation, while
samples taken at 3 minutes contained much more
glycolate-14C than P-glycerate-"-C or any sugar phos-
phate (fig 4). The results suggest that glycolate-14C
was accumulating as an end product of photosynthesis,
and probably it was being excreted (9, 20).
Our experiments do not prove that the increased
chlorophvll b content with respect to chlorophyll a is
linked to the altered 14CO2 fixation. Both changes
occurred upon growing algae in blue light, but they
need not be associated. The decrease chlorophyll a/b
ratio during growth in blue light might be expected
from the data of Fujita and Hattori (5). The Soret
band absorption for chlorophyll b is approximately
5so % greater than the absorption for chlorophyll a
between 400 to 500 m/u. Plants grown in shade also
seem to have more chlorophyll b.
Unlike glycolate-14C formation, the rate of label-
ing of glycine and serine did not increase when
Chlamydomonas were adapted oil blue light. When
Chlorella were placed in blue lighlt 14C-label in serine
increased immediately and before similar changes
occurred for glycolate-_4C. With Chlorella, increased
serine-14C was accompanied by decrease 'IC in
P-glycerate. These results appear consistent with the
absence of a typical glycolate oxidase in algae and
with the formation of serine from P-glycerate rather
than from glycolate as occurs in higher plants (16).
Movement of 14C from P-glycerate to serine may
occur due to limited availability in low blue light of
NADPH and ATP which is needed to convert
P-glycerate to triose phosphate. Although not in-
vestigated, one might predict with higher plants. which
rapidly metabolize glycolate to serine and then to
sucrose, that growth in blue light would also increase
the pool size of glycine and serine and sugars. Thus
glycine and serine accumulation by higher plants in
blue light could arise from both P-glycerate and
glycolate.
Cavle and Emerson (4) ran "-CO, fixation ex-
periments with Chlorella pyrenoidosa in the presence
of 8 microeinstein/cm2 /min of blue or red light.
Their algae had been grown in white light and only
the subsequent 5 minute experiment was done in
monochromatic light. As we also observed, they
found no significant difference in total amount of
"CO, fixation or distribution of 1"C between amino
acids and phosphate esters. However, they observed
that the specific activity of the alanine, glycine and
1130 PLA.NT I'HYSIOLOGY
serine XXhlicil WaCs prod(uTced in ; minutes. 0o blue light
xv as greater than that fromii red liglht. Further, the
distribution of 14C in gIlvcine, but not in alanine, was
altered b)v the blue light. ft would appear that
metabolic clhanges in blue lighlt begin immediately.
The large clhange in 1 4C distribution which leads to
an accumila6tiot of glvcolate b1 algae in blue light,
as. observed 1b us, seemis to orcctr only after e-everal
dav-s of grovth in blue liglt.
In a series of palp)ers frolmi Krotkov's laboratory.
it hals been reporte(I that the addition of blue light
to red light stimulated 1 4C(O, inicorporationi into
aspartate. malate anti glitamllate duir-inig 30 iminlute
experimilenlts and (lecreased 14(7 in glycine and glvcolate
6, 7, 21). Although1 these resUlts appear the opposite
fromii ours, the 2 tyl)es of experimenits are not colm-
parab'e witlh respect to grovth of the algae or type
of im0onoclrromatic light emliloved. Krotkov's groul)
use(d algae gro\Xn in white light with 5 % CO., rathel-
than in the blue or redl light vith 0.2 % CO. Fur-
tlher, they used a (lark plretreatment p)eriod which
varie(l fromii 1 .5 to 20 hours. l171eir blue light only
supplemente(l red lighlt and( even wh en their 1lue
filter alonie wXas used, it tralusmittecl timuclh light be-
twveen 500 to 600 muw, some at 680 M/1, an(d all liglit
about 710 minu (21 ). Our- u1se of a1 (uSO4 Solution,
as emiiphasizdcd by \Withrov ( 27), comipletely elimi-
nates red light witl blue p)igment filters.
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