Plant Physiol. (1978) 62, 590-597
Apparent Catalase Synthesis in Sunflower Cotyledons during the
Change in Microbody Function
A MATHEMATICAL APPROACH FOR THE QUANTITATIVE EVALUATION OF DENSITY-LABELING
DATA'
Received for publication August 18, 1977 and in revised form April 5, 1978
THOMAS BETSCHE AND BERNT GERHARDT
Botanisches Institut der Universitat, D-4400 Munster, Germany
ABSTRACT
Density-labeling with 10 mM K"INOs/70% 2H2O has been used to
investigate catalase synthesis in different developmental stages of sunflower (Helanthus annuus L.) cotyledons. A mathematical approach is
introduced for the quantitative evaluation of the density-labelng data. The
method allows, in the presence of preexisting enzyme activity, calculation
of this synthesized activity (apparent enzyme synthesis) which results from
the balance between actual enzyme syntbesis and the degradation of newly
synthesized enzyme at a given time. During greening of the cotyledons,
when the catalase activity declines and the population of leaf peroxisomes
is formed, the apparent catalase synthesis is lower than, or at best equal
to, that occurring during a developmental stage when the leaf peroxisome
populaton is establised and catalase syntbesis and degradation of total
catalase are in equilibrium. TWs result suggests a formatin, in fatty
cotyledons, of the leaf peroxisomes by transformation of the glyoxysomes
rather than by de novo synthesis.
The function of microbodies in the mesophyll cells of fat-storing
and potentially photosynthetic cotyledons of plants such as the
sunflower changes during seedling development (9, 16, 17, 19, 24).
Coincident with the change from heterotrophic to autotrophic
nutrition of the seedling, the cotyledonary microbody population
loses its glyoxysomal function and acquires the enzymic characteristics of leaf peroxisomes. It is still a matter of discussion as to
how this transition in microbody function is regulated (12, 14, 21),
and whether leaf peroxisomes arise by transformation of preexisting glyoxysomes (6, 14, 22, 24), or are formed de novo (16, 18), or
developed as the ultimate stage of a microbody population which,
in the presence of continuous turnover, changes steadily in functional character from glyoxysomes over glyoxyperoxisomes to leaf
peroxisomes (21). A more recent paper on this topic (4) provided
results on density-labeling of the whole organelle during the
change in microbody function. From the difference between the
observed density increase in leaf peroxisomes and that expected
for: (a) de novo synthesis of the organelles or (b) their development
from preexisting glyoxysomes, it was suggested that the leaf peroxisomes arise by de novo synthesis. This conclusion can be
criticized on the ground that the calculation of the density increase
in the case of transformation of glyoxysomes into leaf peroxisomes
was only based upon part of the light-stimulated (leaf peroxisomal)
enzymes (19) and that no turnover of enzymes common to both
This research was supported by the Deutsche Forschungsgemeinschaft.
types of organelles was considered. Taking both facts into consideration, the density increases expected for both discussed mechanisms of leaf peroxisome formation would probably differ by the
same factor from the observed density shift. One basic result was
a leaf peroxisome population of higher density than that of
glyoxysomes labeled due to their turnover. This result can theoretically be obtained in a model in which unlabeled and labeled
glyoxysomes are proportionally transformed into leaf peroxisomes
which turn over with a rate equal to that of glyoxysome turnover.
Our studies on the developmental origin of leaf peroxisomes in
fatty cotyledons are based upon quantitative estimations of the
dynamics of microbody components. At present we are approaching this problem by examining the occurrence and the extent of
catalase synthesis during the transition of microbodies from glyoxysomal to leaf peroxisomal function (14). Catalase is a common
marker of both glyoxysomes and leaf peroxisomes. If the formation of leaf peroxisomes during greening of fatty cotyledons is due
to a de novo synthesis of the whole organelle a synthesis of leaf
peroxisomal catalase has to occur in the course of the formation
of the leaf peroxisomes.
By labeling newly synthesized enzyme molecules the label
found, under certain labeling conditions and after a certain labeling time, within the enzyme population is determined by the rate
of enzyme synthesis as well as by the rate of degradation of newly
synthesized enzyme molecules. Then, by labeling experiments,
only the difference between both processes at a given point of time
can be detected. We will denote this difference as apparent (rate
of) enzyme synthesis.
Density-labeling experiments are normally used to obtain qualitative results on the studied problem. However, quantitative
evaluation of the density-labeling data is necessary if the apparent
rate of an enzyme synthesis is to be estimated. In addition, a
synthesis of leaf peroxisomal catalase during transition in mitrobody function would occur in the presence of preexisting glyoxysomal catalase. After density-labeling of the newly synthesized
peroxisomal catalase, resolution (by their density difference) of
the two catalase populations in a CsCl density gradient is not
possible. The obtained profile of catalase activity in the density
gradient belongs to the whole enzyme population. Therefore (and
in general terms), actual measured activity profiles which are
formed by superimposition of the activity profiles of preexisting,
unlabeled, and newly synthesized, labeled enzyme have to be
resolved into their components for estimation of apparent enzyme
synthesis. In this paper results on catalase density-labeled during
different developmental stages of sunflower cotyledons are presented, and a mathematical treatment is introduced which allows
estimation, from density-labeling data, of apparent rates of enzyme synthesis in the presence of preexisting enzyme.
Downloaded from on June 15, 2017 - Published by www.plantphysiol.org
Copyright © 1978 American Society of 590
Plant Biologists. All rights reserved.
Plant Physiol. Vol. 62, 1978
CATALASE SYNTHESIS IN FATTY COTYLEDONS
591
heart lactate dehydrogenase (from Boehringer, 360 ,umol of
NADH oxidized/min-mg of protein) as a density marker. The
PLANT MATERIAL AND STANDARD GROWTH CONDITIONS
tubes were centrifuged in a Beckman SW 56 rotor for 66 hr at
Sunflower seeds (Helianthus annuus L., var. Seda Bel 50) were 36,000 rpm (I70,000gmax) at 5 C. After centrifugation the gradients
soaked in tap water for 10 to 12 hr at room temperature and then were fractionated into approximately 120 1-drop fractions. The
germinated in moist Vermiculite at 30 C. The counting of days of fractions were assayed alternately for catalase or glyceraldehydegermination started with the planting of the soaked seeds. After 2 3-P dehydrogenase, and lactate dehydrogenase. The recoveries of
days of growth in darkness the seedlings were transferred into catalase, glyceraldehyde-3-P dehydrogenase, and lactate dehydrogenase from gradients were 22, 34, and 32%, respectively. Every
light.
10th fraction was taken for refractive index determination. The
refractive indices were converted into density units by the equation
APPLICATION OF 2H20 AND K N03
of Bruner and Vinograd (5). The densities of catalase and glycerFor density-labeling a solution of 10 mm K'5N03 in 70% 2H20 aldehyde-3-P dehydrogenase were corrected according to the equiwas used. The length of the labeling period at each developmental librium position of the density marker.
stage was 2 days (for definition of different developmental stages
see "Results" and Fig. 1).
ASSAYS
Density-labeling during Stage I. The testae were removed from
All enzyme assays were carried out spectrophotometrically with
the seeds. The seeds were soaked in the isotope solution instead of
Gilford
two-channel automatic enzyme analyzer at 20 C. The
a
to
in
the
dark
at
30
C
tap water, and were then allowed germinate
methods employed were those described in the literature as folin Petri dishes on filter paper moistened with isotope solution.
Density-labeling during Stages II and III. Primary root and lows: catalase (13), NADP-linked glyceraldehyde-3-P dehydrodistal part of the hypocotyl were cut from the seedlings which genase (7), lactate dehydrogenase (2), isocitrate lyase (15), glycowere exposed to mild wilting conditions until distinct loss of turgor late oxidase (10). Protein was determined by the Lowry method.
was observed. Then the seedlings were immersed into the isotope
RESULTS
solution until full turgor was restored (about I hr). After this
treatment the seedlings were cultured on the isotope solution at
Development of Catalase Activity in Sunflower Cotyledons.
30o RH of the atmosphere for the first 3 hr and at 60% RH Figure
la shows the developmental pattern of catalase in sunthereafter. The counting of the labeling time started with the flower cotyledons,
and also illustrates the time course of glyoxytransfer of the seedlings to 60%o RH.
function of the cotyledonary microand
leaf
somal
peroxisomal
The seeds and seedlings of the 'H20 controls were always
as revealed by the change in marker enzyme activities.
treated identically to those which were labeled except that the bodies
of the sunflower seedlings on lO mm K'5NO3/70% 2H20
isotope solution was replaced by a solution of 10 mM K'4N03 in Growth
had no serious effect on either the developmental pattern of
'H20.
catalase activity (Fig. lb) or the particulate nature of the enzyme
(9). Similar results have been reported for glyoxysomal and leaf
PREPARATION OF EXTRACTS
peroxisomal marker enzymes of sunflower cotyledons (9, 22).
The cotyledons were ground in a chilled mortar with acidwashed sand. The homogenization medium (1 ml/cotyledon)
consisted of 50 mm Tris-HCI (pH 8.0) containing 10 mm dithioerythritol. The homogenate was filtered through cheesecloth and
centrifuged at 20,000g for 20 min. To the supernatant (crude
extract) an equal volume of acetone (- 15 C) was added. The
resultant precipitate was collected by centrifugation and dissolved
in 0.2 M K-phosphate (pH 7.0) containing 10 mm dithioerythritol.
Insoluble material was removed by centrifugation. In addition,
before using the supernatant in CsCl gradient centrifugation it
-t
was passed through an ultrafilter (Sartorius SM 11 308). The
filtrate contained 80%o of the catalase activity of the crude extract.
s
All procedures were carried out at 0 to 4 C.
When the labeling of NADP-linked glyceraldehyde-3-P dehydrogenase was studied the homogenization medium contained 0.5
M Tris-HCI (pH 8.5), 10 mm dithioerythritol, 2.5 mm EDTA, and
5 mM MgCl2 (one cotyledon/2 ml). The homogenate was filtered
<t
and centrifuged (20,000g for 20 min), and the supernatant (crude
extract) was passed through an ultrafilter. Two and two-tenths mg
of glyceraldehyde-3-P, 1.1 mg of NADPH, and 3.3 mg of dithioerythritol were added per ml of filtrate to stabilize the glyceral6
8
4
2
U
10
STAGE 1- I 1---Fm----i
dehyde-3-P dehydrogenase in the subsequent CsCl gradient cenDAYS OF GERMINATION
trifugation.
MATERIALS AND METHODS
uJ
DENSITY GRADIENT CENTRIFUGATION
CsCl (0.76 mg) (Supra grade from Merck) was added to 1.8 ml
of an enzyme solution containing 2 mg of total protein. This
solution was then mixed in a centrifuge tube with 2.7 ml of CsCl
solution of the density 1.31 kg/l which, in addition, contained 0.5
mg of NADPH/ml in the case of studies on glyceraldehyde-3-P
dehydrogenase. In general, the tubes also contained I ,ug of pig
FIG. 1. a: Changes of enzyme activities in sunflower cotyledons during
germination under standard growth conditions. Activity 100 corresponds
to 750 Amol of H202 consumed/min cotyledon for catalase (0), 165 nmol
of glyoxylate formed/min cotyledon for isocitrate lyase (0), 70 nmol of
glyoxylate formed/min cotyledon for glycolate oxidase (A). b: Developmental pattern of catalase activity in cotyledons of sunflower seedlings
grown on 10 mM K'4NO3/'H20 (
) and 10 mm K5NO3/70% 2H20
(--- ), respectively, from seeds which were already soaked in the corresponding solutions. Catalase activity: ,umol of H202 consumed/
min cotyledon.
Downloaded from on June 15, 2017 - Published by www.plantphysiol.org
Copyright © 1978 American Society of Plant Biologists. All rights reserved.
BETSCHE AND GERHARDT
592
Apparent catalase synthesis was investigated during three characteristic stages of cotyledon development (Fig. 1). Stage I includes
the time when the glyoxysomal enzymes and catalase rise to their.
maximum activities at the onset of germination. Stage II comprises
the transition in microbody function and the concomitant sharp
decrease in total catalase activity during greening of the cotyledons. Stage III, finally, denotes that developmental stage during
which catalase shows a more or less constant level of activity in
green cotyledons.
The half-life of catalase during stages II and III is about I to 2
days, as determined from decay kinetics of the enzyme activity in
inhibitor experiments (1 1). At least for these stages, the labeling
period of 2 days falls within one half-life of the enzyme (1).
Labeling of catalase during stage I occurs at a time when only
negligible amounts of preexisting catalase are present. After labeling at stage II or III the whole population of catalase molecules
within the sunflower cotyledons is composed of two populations:
unlabeled molecules which preexist from the foregone development, labeled molecules newly synthesized during the studied
developmental stage. Consequently, in the CsCl gradient loaded
with extracts from cotyledons labeled during stage II or III, the
actual measured profile of catalase activity is formed by two
catalase populations. In order to determine from the experimental
data apparent catalase synthesis at stages II and III the following
mathematical treatment was introduced.
Mathematical Treatment of Density-labeling Data. The activity
of a homogeneous population of enzyme molecules when measured after isopycnic equilibrium centrifugation of the enzyme in
a linear CsCl gradient and plotted against the density of the
gradient follows, theoretically, a Gaussian curve. The activity
profile can be described by the Gaussian equation:
f (x)
pe-M(-)
(a)
where x = the density [kg/li at any point of the profile and a =
the distance of the peak position of the profile from the y axis.
The parameter p is determined by the height (in activity units) of
the activity profile at peak position. The coordinate values of the
maximum position are p and a. The parameter a is a measure of
the bandwidth of the enzyme profile.
In the context of the present paper, equation a should describe
the activity profile of unlabeled enzyme or the 'H20 control. The
activity profile of a homogeneous population of the enzyme
molecules labeled with stable heavy isotopes is described by
equation b:
g(x)
-O (X-C) 2
qe
(b)
where c = the distance of the peak position of the labeled enzyme
profile from the y axis or c = a + b, and where b = the observed
density shift (in kg/l) with respect to the activity profile of the
unlabeled enzyme. The parameters q and /8 correspond to the
parameters p and a of equation a. Since it was observed in practice
that the bandwidths of the activity profiles obtained for the
unlabeled and the homogeneous, labeled enzyme population are
identical (23) it follows that ,B = a. However, we will not equate'
the terms a and /8 with respect to a broader range of application
of the developed mathematical approach as outlined later on.
In general, where the studied population of enzyme molecules
is heterogeneous in the sense that it is a mixture of an unlabeled
and a homogeneous, labeled enzyme population, the actual activity profile is the result of superimposition of the activity profiles
of the two homogeneous populations. The activity profile of the
composite population can be described, therefore, by equation c:
h(x) pe1 (x-a)2
(c)
Plant Physiol. Vol. 62, 1978
An activity profile described by equation c, i.e., the activity
profile of a heterogeneous enzyme population, does not follow a
Gaussian curve. It is characterized by an asymmetrical form
(except in the case where a = ,B and p = q) and also by a
bandwidth which increases with respect to the superimposing
profiles. Nevertheless, composite profiles have been frequently,
but incorrectly, drawn and treated as Gaussian curves in the
literature.
The area under the activity profile described by equation c is
the arithmetical addition of the areas under the two superimposing
profiles. The area under the activity profile of the unlabeled
enzyme (equation a) is:
F Je (x -a)2dx =
co
(d)
area under the activity profile
population (equation b) is
of the labeled enzyme
FqqC eo l ( x-c)2dx=qVt
(e)
and the
The sections of the area under the composite activity profile
which belong to the unlabeled and to the labe1ld enzyme population are then also described by equations d and e, and the total
area (F) under the composite profile is given by F Fp + Fq.
The area under an enzyme profile is proportional to the amount
of enzyme present. The ratio of the amounts of unlabeled (Ap)
and labeled (Aq) enzyme in the composite population is:
=
Fp
I
Ap
Aq Fq
_
p
(f)
Z
q
Since
Ap +Aq = total
enzyme
activity
(g)
it follows that
(h)
Aq total enzyme activity
The term p/q of equation h gives the ratio of the parameters p
and q of equations a and b in the composite activity profile
described by equation c. It can be easily estimated by differen-
tiating equation c
(Z
h' (x)= -21dS,p(Ix-a)ee~lXa2
-2 13 q
,)
(xc-c)e(C
and rearranging equation i for the condition h'(x) = 0:
n(
(Xe- C)c,2
B
q
M (x-a)e-4(xG)
Since the first derivative of a function fulfills the condition y'
= 0 only for an extreme value of this function, x in equation j
the value of x (in kg/l) at the maximum
stands only for xm.
the term pl
the
of
profile. Estimating
activity that
composite
position
of the
value
the
the
has
this
in
only xm.
advantage
way
q
is required. Dependent on the density difference
profile
composite
and labeled enzyme populations,
(b c a) between unlabeledeither
one peak, one peak and a
the composite profile shows
shoulder, or two peaks. The solution of equation j can be obtained
with every extreme value (also a minimum value) of the composite
profile, i.e. the method is independent of the value of b.
=
=
-n(x-c) from on June 15, 2017 - Published by www.plantphysiol.org
Downloaded
Copyright © 1978 American Society of Plant Biologists. All rights reserved.
CATALASE SYNTHESIS IN FATTY COTYLEDONS
Plant Physiol. Vol. 62, 1978
To solve equation j as well as equation h the parameters a, c,
a, and 13 must be known. The parameters a and a have to be
estimated from the unlabeled enzyme profile, the parameters c
and ,8 from the activity profile of homogeneous, labeled enzyme.
The latter profile can be experimentally obtained by densitylabeling during a time when no unlabeled, preexisting enzyme is
present. In detail, we determined the four parameters as follows.
Transforming equation a to a':
Vlnp - In f (x)' =V;' (x-a)
(a')
a linear expression of the Gaussian equation is obtained, and on
a graph the bell-shaped activity profile is converted into a straight
line (Fig. 2). Since ,Anip7-jIniJ(x) = y* is positive for x > a and
negative for x < a the line intersects the x axis at x = a. The slope
of the linearized activity profile gives the value of a.
To transform the y values (= activity values measured in the
CsCl gradient) or equation a into the y* values of equation a' the
value of the parameter p has to be determined directly from the
measured activity profile of unlabeled enzyme. Thereby the value
of a will be defined since p and a are the coordinate values at
peak position.
The peak position of an activity profile can be determined from
a graph within a certain error limit only. In the case of a Gaussian
curve, the error limit can be narrowed if regression lines are
calculated separately for the points (y*/x > a) and (y*/x < a) of
its linearized form. Because the experimental data never follow a
perfect Gaussian distribution, the two calculated regression lines
form an angle (in case of a perfect Gaussian distribution the two
regression lines have identical slopes) and intersect at x = a. The
value of the intersection point is taken as the most correct estimate
of the parameter a. The new, corrected value of the parameter a
implies a somewhat altered value of the parameter p. The values
of y* may be also corrected for the error due to the initial
determination of the peak position from the graph.
The slopes of the two calculated regression lines give the a
values for the corresponding half-sections of the activity profile.
Values of the parameters c and ,1 can be estimated in analogy
to these of the parmeters a and a using equation b':
Vinq - tn g (x)' =Vi (x -c)
(b')
10
593
A more usual method for the estimation of the parameters a, c,
a, and 13 is the probit plot of the experimental data (23, 25). With
this method the linear expression of the Gaussian curve is obtained
in practice by a graphical procedure. Using equations a' and b'
has the advantage that the four parameters can be calculated
solely by an arithmetical treatment. In addition, by the outlined
method one may be better aware of an asymmetry, which eventually occurs, of an activity profile, and one determines more
accurately the peak position of an asymmetrical profile which a
probit plot locates at 50% profile area.
The mathematical treatment of the activity profile of a composite enzyme population, as outlined so far, is based on the
presupposition that the composite profile is formed by superimposition of the profiles of homogeneous enzyme populations since
only for symmetrical activity profiles in a CsCl gradient is a
mathematical expression, the Gaussian equation, known to begin
with. The mathematical treatment can be extended to such cases
where the superimposing profiles show an asymmetrical form
provided that each branch of the profiles is describable by a
Gaussian equation (only then is the simple approach applicable
to solving the problem by starting from Gaussian equations).
Equation j was derived from equation i under the condition
h'(x) = 0 which is fulfilled only by the x value at maximum
position of the composite profile. However, the maximum position
of a composite profile is determined only by these branches of the
two superimposing profiles which face one another. The parameters of a Gaussian equation for these branches are solely important
for the solution of equation j. In the following we will consider
this case where only the labeled enzyme profile should be asymmetrical. According to the above mentioned presupposition, each
branch of this profile is that of an imaginary Gaussian curve. Both
curves have the same values of the parameters q and c, the
coordinate values at maximum position. They differ only with
respect to the bandwidth, i.e. the value of the parameter ,1 is solely
different. Then, 813 should be denoted the parameter /3 of this
Gaussian equation which describes the branch of the labeled
enzyme proie reaching into lower density with respect to the
peak position. The parameter ,/ of the other branch of the labeled
enzyme profile should be denoted 32. Solving equation j for the
outlined case, only the term 1B has to be substituted by the term
,13. Since equation e has to be rewritten in the form:
F=.
q
RI +
q
2
W
(e')
equation h changes to:
50
y
100
10
1,30
1,25
DEN SI T Y
[kg l]
Aq =tot. enzyme act.($V , +1)
(h')
Determination of Parameters a and a. The parameters a and a
of the activity profile of an unlabeled catalase population were
determined from unlabeled enzyme profiles obtained at different
developmental stages in parallel runs to those of labeled enzyme
(Fig. 2). Regression lines were calculated for each branch of the
activity profiles and gave r2 values between 0.97 and 0.99 (r is the
correlation coefficient of a regression line and is estimated statistically; the more r2 approaches 1, the more closely is the real-life
enzyme profile described by a Gaussian equation). From the
intersection points of the regression lines a most correct estimate
of 1.280 [kg/l1 for a was obtained. The slopes of the regression
lines gave a mean a value of 1720.2 No single a value showed a
deviation from the mean value of more than ± 11%. The deviations
were randomly distributed, ie. there was no tendency in the a
FIG. 2. Linear expression of catalase profiles of Figure 5 (1). Shown
are the regression lines calculated for each half-section of linearized
activity profiles of catalase from unlabeled (0) and density-labeled (0)
cotyledons. Relative activity: expressed as a percentage of maximum value
2 The parameters a and f have the dimension: (dimension of
of corresponding activity profile.
= (,umol/min cotyledon). (kg/l)-.
Downloaded from on June 15, 2017 - activity)*
Published(kg/I)-2
by www.plantphysiol.org
Copyright © 1978 American Society of Plant Biologists. All rights reserved.
Plant Physiol. Vol. 62, 1978
BETSCHE ANID) GERHARDT
594
values of one of the two branches of the unlabeled enzyme profiles
to lower or raise values beyond the
mean
value.
of cotyledonary catalase in the CsCl gradient with respect to the
'H20 control (Fig. 5) (9). If the labeling experiments were carried
out at stage III, an incorporation of label into catalase was
obtained (Fig. 5). The density increase of the enzyme averaged
Determinatlon of Parameters c and I. The parameters c and
of the labeled catalase population were determined from the
activity profile of catalase labeled during stage I (Fig. 2). At the 0.0 10 [kg/lI.
The peak position of composite, non-Gaussian profiles has to
beginning of stage I only negligible amounts of unlabeled catalase
were present. Imbibition and germination of sunflower seeds in
be determined directly from a plot of measured enzyme activity
the presence of 10 mm K5N03/70% 2H20 up to day 3 resulted in against gradient density (or against collected gradient fractions if
the density gradient shows linearity in the corresponding region).
an increase in the density of cotyledonary catalase with respect to
the '1H20 control. Using equation b' a most correct estimate of Since gradient fractions have a finite size and assays a certain
1.300 [kg/l) for c was obtained. Labeling of cotyledonary catalase
during stage I resulted in a density increase in the enzyme averc
aging 0.020 [kg/l (1.6% density shift).
100
Calculations of the parameter ,8 gave the result that this branch
of the activity profile had constantly lower , values (P1) which
reached into lower density with respect to the peak position. In
the average, had a value of 1158 and the value of # was 1.7
50
times higher. Thus, the activity profile of catalase labeled during
stage I had an asymmetrical form which indicates a heterogeneous
enzyme population. The single branch on each side of the peak
d
I
position could be described quite well by a Gaussian equation as
documented by the single regression lines for the linearized profile
A
-
p
I
UJ
(I
>r2>0.97).
Experimental Proof of Applicabiity of Mathematical Aproach.
If unlabeled catalase and catalase labeled during stage I (Fig. 3A)
were mixed in the ratio 1:1 the mixture showed the activity profile
of Figure 3B after isopycnic equilibrium centrifugation in a CsCl
gradient. From the experimental data a mixing ratio of the two
catalase populations of 1:0.84 was calculated by substituting fi1
for ,B in equation j and by using equation h' (Table I). In a
corresponding experiment in which the two catalase populations
were mixed in the ratio 1:3, a mixing ratio of 1:2.6 was calculated
from the experimental data (Table I). Figure 4 shows that in a
composite enzyme population, there exists no linear relation between the ratio of unlabeled (preexisting) to labeled (newly synthesized) molecules and the density shift (b = c - a) observed
with respect to the unlabeled enzyme populations.
Densky Labeln of Catalase during Stages 11 and III. Treatment of sunflower seedlings with the isotope solution during stage
II did not result in a significant density shift of the activity profile
'
WU
100
1i.
0
1,25
Xx 130
DENSITY t kgl i1
FIG. 3. A: activity profiles of isopycnically banded unlabeled catalase
(0) and catalase density-labeled during stage I (*). Marked in the graph
are parameters a and p of equation a as well as parameters c and q of
equation b. B: activity profile of isopycnically banded catalase population
composed of unlabeled and density-labeled (stage 1) catalase in the ratio
1: 1. Xm. in the graph corresponds to parameter x of equation j. Relative
activity: expressed as a percentage of maximum value of corresponding
activity profile.
Table I. Application of the introduced mathematical approach to catalase populations containing
known amounts of labeled enzyme.
Mathematical and experimental details are outlined in the text. The values of the parameters a,
c, >, B1, and 22 were determined from the activity profiles shown in Fig. 3A and transformed
into linearized form by equation (a') (unlabeled catalase profile) and equation (b') (labeled
catalase profile): a = 1.281 (kg/l), c = 1.302 (kg/l), o = 1796 (for dimension see footnote 2),
2 = 2126.
1 = 1091,
Prepared Catalase Population
Total
Activity
Unlabeled Cat./
Labeled Cat.
Density2)
Solution of
Equation (J)
p/q
Calculated by
Equation (h')
Contained in
the Population
.moles H202/min.ml
kg/l
1)
Amount of Labeled Catalase in
the Prepared Catalase Population
376
1 : 1
1.2873)
1.266
172
188
376
1 : 3
1.295
0.410
272
282
pmoles
H202/min-ml
Density at peak position of the catalase profile in the CsCl gradient
= x
cf. Fig. 3B.
2 of equation
4) The parameter Downloaded
substituted
was 15,
by 1.by www.plantphysiol.org
from (j)
on June
2017 - Published
Copyright © 1978 American Society of Plant Biologists. All rights reserved.
of equation
(j).
Plant Physiol. Vol. 62, 1978
CATALASE SYNTHESIS IN FATTY COTYLEDONS
595
DISCUSSION
100w
The mathematical treatment presented in this paper allows the
determination, from density-labeling data, of the amount of newly
synthesized enzyme present at a given point of time in an enzyme
population composed of newly synthesized and preexisting molecules. The calculation of this apparent enzyme synthesis requires
the knowledge of certain parameters of the preexisting and the
newly synthesized enzyme populations which can be determined
experimentally. The calculation is then based on the sole presupposition that the values of these parameters do not change in the
loo
4<
4
4
600
w
w
m 40
2&-
course
0,002
0,01
DENSITY SHIFT
of the studies.
0,02
[kg/ l]
FIG. 4. Theoretical plot for relationship of percentage of labeled catalase in a composed catalase population to density shift which results for
composed enzyme population with respect to density of unlabeled catalase.
Calculations were based on values of parameters a, c, a, fi1, and 12 given
in Table 1.
error limit, the peak position cannot exactly be located, by drawing
the plot, within the limit of 1 to 2 gradient fractions or 0.002 units
of density. Consequently, the apparent catalase syntheses, given
by the value of Aq at the end of a certain developmental stage, can
be determined only with this limiting error for estimating the peak
position of composite enzyme profiles. The values given in Table
II were calculated considering the technical limitations of the
method. It follows that during stage II also an apparent catalase
synthesis could have occurred. Its upper limit would correspond
to a density shift of 0.002 [kg/l1.
Density Labeling of NADP-linked D-Glyceraldehyde-3-P Dehydrogenase. To compare in relative terms the labeling of the
amino acid pool available for catalase synthesis during stages II
cm
X
and III, the labeling of NADP-linked glyceraldehyde-3-P dehyI1drogenase was studied in a control experiment. Since there is
Istrong evidence that this chloroplast enzyme, like catalase, is
L.)
4
synthesized on cytoplasmic ribosomes (8), glyceraldehyde-3-P dew
z
hydrogenase and catalase are probably synthesized from the same
amino acid pool. During stage II the activity of glyceraldehyde-3c]
C
4
P dehydrogenase rises in sunflower cotyledons from a very low
-J
w
value to about 0.4 ,umol of NADPH oxidized/min cotyledon. In
labeling experiments the rise in glyceraldehyde-3-P dehydrogenase
activity was accompanied by a density increase of 0.0 16 [kg/l]
(1.1%). If sunflower seedlings were grown in darkness up to the
beginning of stage III the activity of glyceraldehyde-3-P dehydrogenase increased only to 0.07 ,tmol of NADPH oxidized/
min cotyledon. Transfer of such etiolated seedlings into light
resulted in a rise of the enzyme activity during stage III to about
0.5 ,umol of NADPH oxidized/min -cotyledon, and a density shift
of 0.010 [kg/l] (0.08%) was obtained in the isotope treatment.
Based on these results with glyceraldehyde-3-P dehydrogenase,
the degree of labeling of the amino acid pool available for catalase
synthesis seems not be less at stage II than at stage III. However,
in regard to cotyledon development, the stage III for which
labeling of the control enzyme was studied differed from that of
the standard seedling development. Labeling of the amino acid
pool at stage III had to be compared for both growth conditions.
The activity changes of catalase occurring during standard development (Fig. 1) were not substantially affected by the altered
growth conditions (3, 14, 16, 19). Delay of irradiation of the
seedlings up to stage III also did not influence the apparent
FRACTION NUMBER
catalase synthesis with respect to stage III of standard seedling
FIG. 5. Activity profiles of isopycnically banded catalase from unladevelopment (Table II). This result on catalase indicates that
similar labeling of the amino acid pool available for this enzyme beled (0) and density-labeled (-) sunflower cotyledons. Density-labeling
synthesis occurred during stage III whether the seedlings devel- during developmental stage 1, stage 11, and stage Ill. Activity profiles have
been aligned such that profiles of control enzyme (A, A; lactate dehydrooped under standard or altered growth conditions. Thus, the genase)
coincide. Relative activity: all points of each individual activity
interpretation of the results on the control enzyme glyceraldehydeare expressed as a percentage of its maximum. (Ol, ): density of
3-P dehydrogenase may be correct.Downloaded from on June 15, 2017 -profile
Published
by www.plantphysiol.org
CsCI gradients.
Copyright © 1978 American Society of Plant Biologists. All rights reserved.
596
BETSCHE AND GERHARDT
Plant Physiol. Vol. 62, 1978
Table II. Apparent catalase synthesis at certain developmental stages of sunflower cotyledons as
calculated from the results of density-labeling experiments.
Developmental
Stage
Density Increase
of Catalase
Total Catalase
Apparent Catalase
Ratio of Apparent
Activity5)
Synthesis6)
Catalase Syntheses
(Stage X : Stage III)
(kg/l)4)
)imoles H202/min.cotyledon
9807)
18.1 : 1
I
0.020 +
II
0.000 + 0.002
268
0(+)52
(0)
II/III2)
0.009 + 0.002
124
+ 9
68 9
(58)
III
0.010 + 0.002
93
54 +
7
(47)
III3)
0.007 + 0.002
119
56 - 10
(47)
40.001
1007
11.0
:
1
1.3 : 1
1.0 : 1
1)
The definitions of the developmental stages I,II, and III are given in the text.
2) Stage II/III denotes a developmental stage intermediary between stage II and stage III.
3) Seedlings etiolated up to stage III and then transferred into light.
4) Stage I: density increase = c - a; all other developmental stages: density increase = xmax - a.
5) Measured in the crude extract.
6) Shown are the values of apparent catalase syntheses with their limits due to the error limit
in the estimation of density increases (cf. sec. column). The values in parentheses are calculated on the basis 3 = X. (see Discussion).
Increase in catalase activity measured during stage I.
Apparent rates of catalase synthesis were calculated for different ond, the point of physiological saturation labeling itself can differ
developmental stages of sunflower cotyledons following strictly for catalase populations labeled at different developmental stages.
the outlined mathematical treatment and based solely on the Both conclusions mean that values of the parameters ,B (I, #2)
experimental data (Table II). Constancy of the parameters of the and c cannot be considered to be constant during cotyledon
unlabeled catalase population was proved for the different devel- development.
The parameter ,B of a labeled enzyme population is only a
opmental stages. Values of the parameters of the newly synthesized
catalase population, however, can at the moment only be deter- function of its heterogeneity which can be expected to decrease
mined at stage I and their constancy from stage I through stage during cotyledon development. Any development-dependent diIII was presupposed. The correctness of this presupposition has to vergence in the real value of the parameter ,B from the estimated
value (stage I) would then be directed towards the limit #1 = a
be checked from the physiological point of view.
In a storage tissue such as the sunflower cotyledon, metabolites and #2 = a since a homogeneous, labeled enzyme population
derived from storage compounds may be used in protein synthesis. shows the same bandwidth as the unlabeled population (23).
With a constant contribution of unlabeled metabolites to enzyme Apparent rates of catalase synthesis which were calculated on the
synthesis occurring under labeling conditions, a homogeneous basis fi = a (f' = ia, 82 = a) are given in Table II in parentheses.
population of newly synthesized, labeled enzyme molecules will It shows that in our case any change of ,B -. a does not have an
be obtained (provided that labeling of the relevant amino acid essential influence on the calculated apparent rates of catalase
pool is rapid compared to enzyme synthesis). The newly synthe- synthesis and especially not on their ratios.
The actual value of the parameter c of a labeled enzyme
sized enzyme population is characterized by certain physiological
saturation labeling (23) which determines the density increase population depends on the heterogeneity of the population and
with respect to the unlabeled enzyme population. The failure to on its point of physiological saturation labeling. Since the former
achieve a homogeneous, labeled catalase population during stage is expected to decrease and the latter probably increases during
I can then be explained on the ground that the contribution of cotyledon development, the combined effect could lead toward c
unlabeled precursors to protein synthesis changed during the 2- values for older developmental stages greater than that determined
at stage I. However, if the c value is theoretically raised within a
day labeling period.
Unlabeled metabolites derived from storage compounds will reasonable limit no seriously altered results are obtained for the
contribute less to protein synthesis as the storage protein and fat calculated apparent catalase synthesis at stages II and III. Assumdisappear from the cotyledons during their development. The ing that the density difference (b = c - a) between unlabeled and
most rapid breakdown of the storage compounds occurs during labeled catalase observed at stage I would increase by 50%o to
0.030 [kg/l] for stage II or stage III, the calculated apparent
the first 2 to 3 days of development (9, 19, 20).
From these outlined considerations the following can be con- catalase syntheses would differ only by !l:0o from the values
cluded (provided that labeling of the relevant amino acid pool is given in Table II. A density difference of 0.030 [kg/l1 corresponds
not otherwise seriously altered by cotyledon development). First, to a considerable density shift of 2.3%, a value not reported, to
catalase populations synthesized under constant labeling condi- our knowledge, in the many papers on density-labeling of enzymes
tions at developmental stages later than stage I may differ from considering comparable labeling conditions.
No significant density increase in catalase was obtained with
the catalase population labeled during stage I with respect to their
heterogeneity. They may come closer to or reach the point of respect to the 'H20 control by labeling sunflower cotyledons
during the labeling period. Sec- during developmental stage II. However, a density shift could
physiological saturation labeling
Downloaded from on June 15, 2017 - Published by www.plantphysiol.org
Copyright © 1978 American Society of Plant Biologists. All rights reserved.
Plant Physiol. Vol. 62, 1978
CATALASE SYNTHESIS IN FATTY COTYLEDONS
have occurred within the error limit of the technique (see under
"Results"), and an apparent catalase synthesis cannot be excluded
unequivocally for stage II. None, or up to one-fifth, of the catalase
existing at the end of stage II could theoretically be newly synthesized enzyme (Table II and Fig. 4). On the other hand, catalase
synthesis was demonstrated for stage II by labeling experiments
using [14Claminolevulinic acid of [3Hlleucine as precursor of either
the heme or the protein moiety of the catalase molecule (3, 14).
The purity of the isolated catalase was based on its almost constant
specific radioactivity and on its electrophoretic homogeneity (3).
Since radioactive precursors are incorporated into catalase during stage II, the density-labeling data of stage II cannot be
interpreted in the sense that no apparent catalase synthesis occurs
during this developmental stage. Apparent catalase synthesis at
stage II amounts to a value > 0 but c that of the apparent catalase
synthesis at stage III (Table II). It can result from turnover of
glyoxysomal catalase (4) and from synthesis of catalase housed
within leaf peroxisomes. Whether the catalase of the leaf peroxisomes is synthesized in the course of a de novo formation of the
organelles, or during its turnover in microbodies transformed from
the glyoxysomal into the leaf peroxisomal state cannot be decided
unequivocally from the presented data. The apparent catalase
synthesis at stage II may be an indicator of the relative size of a
leaf peroxisome population formed de novo and existing at the end
of this stage. However, the transformation model is at present
favored since the apparent catalase synthesis during the formation
of the leaf peroxisomes (stage II) is only c that occurring during
the established state of this population (stage III), a result which
is easier to deduce from a general catalase turnover than from a
catalase synthesis at stage II due to a de novo formation of leaf
peroxisomes and catalase turnover during stage III. An apparent
catalase synthesis at stage II c that of stage III was also suggested
by radiolabeling data, but interfering metabolic processes impair
a quantitative evaluation of these data (14).
As indicated by the density-labeling data the actual catalase
synthesis at stages II and III is considerably lower than that of
stage I which itself can be - the apparent catalase synthesis. An
actual catalase synthesis during stage II or III equal to that at
stage I may be assumed. To reach the activity levels of catalase
(measured at the end of these stages) from the activity levels
existing at the beginning of these stages and raised by the assumed
actual catalase syntheses a definite degradation rate of the enzyme
is required. If the degradation of the unlabeled, preexisting catalase (at the beginning of stages II or III) and the degradation of
newly synthesized, labeled enzyme are not substantially different,
the necessary high degradation rate would remove so much of the
unlabeled catalase that relative proportions of unlabeled and
labeled enzyme would result which correspond to density shifts
considerably higher than those observed.
Acknowledgment-The authors wish to thank H. Petersson, Mathematical Institute, University
of Munster for his advice on mathematical problems.
597
LITERATURE CITED
1. ACTON GJ, H DRUMM, H MOHR 1974 Control of synthesis de novo of ascorbate oxidase in the
mustard seedling (Sinapis alba L.) by phytochrome. Planta 121: 39-50
2. BFRO.MFYER HU, K GAWFHN. M GRASSI. 1974 Enzyme als biochemische Reagentien. In HU
Bergmeyer, ed, Methoden der enzymatischen Analyse. Verlag Chemie, Weinheim, pp
512-513
3. BFTSciiE T 1975 Zur Funktionsanderung von Microbodies in den Kotyledonen von Helianihus
annuus L. Untersuchungen an Katalase. Thesis, Univ Heidelberg
4. BROWN RH, MJ MERRETT 1977 Density labelling during microbody development in cotyledons.
New Phytol 79: 73-81
5. BRUNFR R. J VINOGRAD 1965 The evaluation of standard sedimentation coefficients of sodium
RNA and sodium DNA from sedimentation velocity data in concentrated NaCI and CsCI
solutions. Biochim Biophys Acta 108: 18-29
6. BURKE JJ. RN TRELEASE 1975 Cytochemical demonstration of malate synthase and glycolate
oxidase in microbodies of cucumber cotyledons. Plant Physiol 56: 710-717
7. FEIRRAREND J 1969 Der Einflufi von Cytokininen auf die Bildung von Photosyntheseenzymen
in Roggenkeimlingen. Planta 84: 11-29
8. FEiERAREND J. U SCHRADER-REICIMARDT 1976 Biochemical differentiation of plastids and
other organelles in rye leaves with high-temperature-induced deficiency of plastid ribosomes.
Planta 129: 133-145
9. GERHiARDT B 1973 Untersuchungen zur Funktionsanderung der Microbodies in den Keimblittern von Helianihus annuus L. Planta 110: 15-28
10. GERHARDT B 1974 Studies on the fQrmation of glycolate oxidase in the developing cotyledons
of Helianthus annuus L. Z Pflanzenphysiol 74: 14-21
11. GERHARDT B 1974 The change in microbody function in developing sunflower cotyledons.
Studies on the regulatory mechanism. Int Symp Plant Cell Different, Lisboa Port Acta Biol
Ser A 14: 3-19
12. GERHARDT B 1978 Microbodies/Peroxisomen pflanzlicher Zellen. Morphologie. Biochemie,
Funktion und Entwicklung eines Zellorganells. Cell Biology Monographs, Vol 5. SpringerVerlag, Wien
13. GERHARDT B. H BFEVERS 1970 Developmental studies on glyoxysomes in Ricinus endosperm.
J Cell Biol 44: 94-102
14. GERHARDT B, T BETSCHE 1976 The change of microbodies from glyoxysomal to peroxisomal
function within fatty, greening cotyledons: hypotheses, results, problems. Ber Deutsch Bot
Ges 89: 321-334
15. HOCK B, H BEFVERS 1966 Development and decline of the glyoxylate cycle enzymes in
watermelon seedlings (Citrullus vulgaris Schrad.). Effects of dactinomycin and cycloheximide. Z Pflanzenphysiol 55: 405414
16. KAGAWA T. H BEEVERS 1975 Development of microbodies (glyoxysomes and leaf peroxisomes)
in cotyledons of germinating watermelon seedlings. Plant Physiol 55: 258-264
17. KAGAWA T. JM LORD H BEEVERS 1973 The origin and turnover of organelle membranes in
castor bean endosperm. Plant Physiol 51: 61-65
18. KAGAWA T. JM LORD, H BEEVERS 1975 Lecithin synthesis during microbody biogenesis in
watermelon cotyledons. Arch Biochem Biophys 167: 45-53
19. SCIINARRENREROER C. A OFQSER, NE TOi.RERT 1971 Development of microbodies in sunflower
cotyledons and castor bean endosperm during germination. Plant Physiol 48: 566-574
20. SCHNARRENRERRER C. A OESER, NE TOi.RERT 1972 Isolation of protein bodies on sucrose
gradients. Planta 104: 185-194
21. SCHOPFER P, D BAJRACHARYA. R BERGFEiD. H FAL.K 1976 Phytochrome-mediated transformation of glyoxysomes into peroxisomes in the cotyledons of mustard (Sinapis alba L.)
seedlings. Planta 133: 73-80
22. THEIMER RR, G ANDING. B SCHMIDT-NEuHAUS 1975 Density-labeling evidence against a de
novo formation of peroxisomes during greening of fat-storing cotyledons. FEBS Lett 57:
89-92
23. TONCo W-F, P SCHOPFER 1976 Phytochrome-mediated de novo synthesis of phenylalanine
ammonia-lyase: an approach using pre-induced mustard seedlings. Proc Nat Acad Sci USA
73: 4017-4021
24. TREI.FASE RN, PJ GRURFER. EN NEWCOMR 1971 Microbodies (glyoxysomes and peroxisomes)
in cucumber cotyledons. Correlative biochemical and ultrastructural study in light- and
dark-grown seedlings. Plant Physiol 48: 461-475
25. ZIEI.KE HR. P FII.NER 1971 Synthesis and turnover of nitrate reductase induced by nitrate in
cultured tobacco cells. J Biol Chem 246: 1772-1779
Downloaded from on June 15, 2017 - Published by www.plantphysiol.org
Copyright © 1978 American Society of Plant Biologists. All rights reserved.