Plant Physiol. (1990) 93, 141-147
0032-0889/90/93/0141 /07/$01 .00/0
Received for publication July 14, 1989
and in revised form December 28, 1989
Protein Synthesis during the Initial Phase of the
Temperature-induced Bleaching
Response in Euglena gracilis'
William Ortiz
Department of Botany and Microbiology, University of Oklahoma, Norman, Oklahoma 73019
ABSTRACT
The present work was undertaken to study the impact of
temperature on the ability of plastids to synthesize some of
their own proteins. I report that Chl accumulation during the
initial phase of the temperature treatment peaks at about 15
h. Furthermore, despite an overall increase in cellular protein
synthesis in 15 h-bleached cells, protein synthesis inside the
plastid appears to be inhibited. Synthesis of the LSU2 of the
enzyme Rubisco, in particular, was strongly impaired. It is
concluded that, despite an increase in Chl accumulation per
cell during the initial phase of the temperature response,
synthesis of plastid proteins on chloroplast ribosomes is inhibited at the elevated temperature.
Growing cultures of photoheterotrophic Euglena gracilis experience an increase in chlorophyll accumulation during the initial
phase of the temperature-induced bleaching response suggesting an increase in the synthesis of plastid components at the
bleaching temperature of 330C. A primary goal of this work was
to establish whether an increase in the synthesis of plastid
proteins accompanies the observed increase in chlorophyll accumulation. In vivo pulse-labeling experiments with [35S]sodium
sulfate were carried out with cells grown at room temperature or
at 330C. The synthesis of a number of plastid polypeptides of
nucleocytoplasmic origin, including some presumably novel polypeptides, increased in cultures treated for 15 hours at 330C. In
contrast, while synthesis of thylakoid proteins by the plastid
protein synthesis machinery decreased modestly, synthesis of
the large subunit of the enzyme ribulosebisphosphate carboxylase was strongly affected at the elevated temperature. Synthesis
of novel plastid-encoded polypeptides was not induced at the
bleaching temperature. It is concluded that protein synthesis in
plastids declines during the initial phase of the temperature
response in Euglena despite an overall increase in cellular protein
synthesis and an increase in chlorophyll accumulation per cell.
MATERIALS AND METHODS
Cell Culture
Euglena gracilis Z was grown photoheterotrophically at
23°C in gyratory shakers under cool-white fluorescent lamps
as described previously (15). The culture medium was a
modified version of Hutner's medium (14) containing 50 ng/
L of vitamin B-12. Temperature-induced bleaching was initiated by transferring the cultures to a gyratory shaker kept at
a constant temperature of 33°C ( 15).
Pulse-Labeling in Vivo
Room temperature-grown cells (230C) and 15 h-bleached
cells were harvested by centrifugation and resuspended in
fresh low-sulfate photoheterotrophic medium in which only
MgSO4 was replaced with MgCl2 (12). FeSO4, ZnSO4, and
MnSO4 are present in the medium at their normal concentrations. Radiolabeling in vivo was initiated by the addition of 4
jgCi of [35S]sodium sulfate (43 Ci/mg S; ICN Biochemicals)
per mL of cell suspension. In some instances, 50 Ag/mL
CHX, an inhibitor of protein synthesis on 80 S ribosomes,
and/or 50 ,ug/mL SPEC, an inhibitor of protein synthesis on
70 S ribosomes, were added a few minutes prior to the
addition of the radiolabeled sulfate. The cell suspension was
subsequently incubated at the original growth temperature in
the light with continuous shaking for an additional 3 h. Shortterm pulse labelings were also carried out for 30 min in the
presence of 8 gCi of [35S]sodium sulfate per mL of cell
Exposure of growing photoheterotrophic cultures of the
unicellular alga Euglena gracilis to 33°C induces a loss of total
Chl per cell (16). This phenomenon is known as heat-bleaching and results in the production of colorless cultures of the
alga with an impaired capacity to carry out photosynthesis.
Prolonged treatment at the moderately elevated temperature
brings about an irreversible bleaching of the cells.
We have recently reported (15) that temperature-induced
Chl loss at 33°C occurs in two phases. The initial phase, which
involves an increase in total Chl per cell, is followed by a
second phase characterized by a steady decline in Chl accumulation per cell that continues for the remainder of the
treatment at 33°C. The initial phase of the temperature response has been of interest to us since the increase in Chl
accumulation per cell suggests a parallel increase in the synthesis of plastid components including proteins. A major
change in the profile of stainable polypeptides during this
initial phase is in the accumulation of presumably novel
polypeptides of 45,000 and 60,000 Mr in the thylakoid (15).
suspension.
2Abbreviations: LSU, large subunit; CHX, cycloheximide; QB,
quinone-binding protein; Rubisco, ribulose- 1 ,5-bisphosphate carboxylase; SPEC, spectinomycin; SSU, small subunit.
'This work was supported by a grant from the National Science
Foundation DCB-87 15422.
141
Downloaded from on June 16, 2017 - Published by www.plantphysiol.org
Copyright © 1990 American Society of Plant Biologists. All rights reserved.
142
ORTIZ
Total protein synthesis in control and partially bleached
cells was determined by blotting 50 ,uL aliquots of cell suspension on filter paper discs 1 h after the addition of the radiolabeled sulfate. The discs were processed according to Bollum
(2) and the amount of hot TCA-precipitable counts on the
discs was determined by scintillometry.
Isolation and Fractionation of Plastids
Plant Physiol. Vol. 93,1990
60
cn 50
40
Radiolabeled cells were harvested by centrifugation and the
[35S]plastids isolated on isoosmotic linear gradients of 10 to
50% Percoll underlaid with a cushion of isoosmotic 80%
Percoll (14, 15). The band of intact plastids was collected
from the gradient and washed by dilution with four volumes
of ice-cold gradient buffer followed by centrifugation at 5,000
rpm for 3 min (Beckman JA-20 rotor). The pellet of plastids
was resuspended and lysed in 10 mM Tricine-NaOH buffer
(pH 7.6) containing 4 mM MgCl2 and 1 mM PMSF. The
suspension of lysed plastids was immediately fractionated on
step-gradients of sucrose consisting of 0.6 and 0.93 M sucrose
in 4 mM MgC92, 10 mm Tricine-NaOH (pH 7.6) according to
Douce and Joyard (6). Centrifugation was carried out at
23,000 rpm for 1 h. Aliquots of 50 ,uL were taken from the
stroma and thylakoid suspension, blotted on filter paper discs
and prepared for counting by scintillometry according to the
method of Bollum (2).
Figure 1. Chl accumulation during the early stages of the temperature-induced bleaching in Euglena. Growing photoheterotrophic cultures of the alga were treated at 33°C in the light for up to 30 h.
Cultures were sampled at different times to determine total Chl and
cell counts.
Gel Electrophoresis and Fluorography
Polypeptides of the stroma and thylakoid fractions from
radiolabeled plastids were analyzed by electrophoresis on 10
to 15% (or 10-12.5%) linear gradients, SDS-polyacrylamide
slab gels (4) containing 2 M urea in both the stacking and
running gels. Samples (100 ,ug protein) were solubilized in
sample buffer (4) containing 2 M urea and 1 mM PMSF.
Electrophoresis was carried out overnight at 6 mAmp. The
radiolabeled polypeptides were visualized by fluorography at
-70°C according to Laskey and Mills ( 11) taking into consideration a modification introduced by Burckhardt et al. (3).
per cell at its peak constitutes a 66% increase in Chl levels
over the levels present in control cells. An increase in Chl
levels per cell could reflect an increase in the synthesis of
plastid components, including proteins, during this initial
phase of the temperature response. We have recently shown
the accumulation of presumably novel stainable polypeptides
of 60,000 and 45,000 Mr in thylakoids as early as 15 h after
the onset of the treatment at the bleaching temperature ( 15).
This observation supports the proposal that a more or less
general increase in the synthesis of plastid components could
take place during the initial phase of the temperature response.
Miscellaneous Methods
Cell numbers were determined using a hemocytometer. An
average was calculated from at least 10 independent cell
counts. Total Chl concentration was determined by the
method of Arnon (1). Determinations of total protein was
according to Larson et al. (10).
Protein Synthesis in Control and 15 h-Bleached Euglena
and Sensitivity to Inhibitors
RESULTS
Time-Course of Chi Accumulation during the Initial Phase
of the Temperature Response in Euglena
When growing photoheterotrophic cultures of the unicellular alga are exposed to the bleaching temperature of 33°C
there is an initial rise in Chl accumulation per cell (Fig. 1).
This rise, which peaks at about 15 h, is followed by a decline
in Chl accumulation per cell. Although the figure documents
changes for up to 30 h, we have previously shown that the
decrease in Chl accumulation continues for at least 60 h (15).
It is important to note that in this instance Chl accumulation
30
0) 30
20
0
10
20
30
hours at 330C
Cultures of the alga were pulsed with [35S]sodium sulfate
and the radioactivity associated with whole cells and in the
thylakoid and stroma fractions of the plastid was determined
by measuring incorporation into hot TCA-precipitable material by the disc method of Bollum (2). Table I shows that
cultures grown at 33°C (no addition) display an increase in
total protein synthesis of about 2.8 times above the level
recorded for control cultures kept at room temperature. An
increase in cellular protein synthesis at the moderately elevated temperature was expected in view of a 1 0°C increase in
the growth temperature.
When the pulse-labeling experiment was carried out in the
presence of CHX, an inhibitor of protein synthesis on cytoplasmic ribosomes, there was a sharp decrease in total cellular
protein synthesis in control and in the 15 h-bleached culture
compared to the corresponding untreated cultures. This result
is in line with our original expectations since CHX was
Downloaded from on June 16, 2017 - Published by www.plantphysiol.org
Copyright © 1990 American Society of Plant Biologists. All rights reserved.
PROTEIN SYNTHESIS IN BLEACHING EUGLENA
Table I. Protein Synthesis in Control and 15 h-Bleached Euglena
Photoheterotrophic cultures of Euglena grown at room temperature
(control) or bleached for 15 h at 330C were pulse-labeled with [35S]Na2SO4 in the presence or in the absence of inhibitors of protein
synthesis. One h after the pulse was initiated 50 AL aliquots were
sampled and 35S-incorporation into hot TCA-precipitable material was
determined by the disc method. 35S-lncorporation into thylakoid and
stroma proteins was determined by isolating plastids from radiolabeled cells followed by subfractionation in step-gradients of sucrose
and counting by the disc method.
35S-lncorporation
Culture
Addition
Total Cella
Control
None
SPEC
CHX
CHX+ SPEC
15 h-bleached None
SPEC
CHX
CHX + SPEC
a
In cpm x 10-3. 1 06 cells-'.
Thylakoidsb Stromab
224.3
113.6
17.5
14.6
2.8
59.4
73.6
1.1
48.7
328.0
200.0
44.2
5.7
47.9
26.8
3.2
b
In cpm x 10-3. mg protein-1.
expected to eliminate the contribution of cytoplasmic ribosomes to overall protein synthesis. The remaining activity
(about 12-16%), therefore, is the result of protein synthesis
carried out in the organelle ribosomes of the plastid and the
mitochondria. Nevertheless, it is observed that organelle protein synthesis (plastid plus mitochondria) measured in the
presence of CHX increases twofold at the bleaching temperature compared to the control grown at room temperature.
This observation is consistent with an expected increase in
the activities of the organelle protein synthesis machinery
following a 10°C increase in the growth temperature.
Total cellular protein synthesis decreases 9 to 17% in control and in 15 h-bleached cells in the presence of SPEC, an
inhibitor of protein synthesis in ribosomes of chloroplasts and
mitochondria (Table I). The observed decrease is in agreement
with earlier estimates of the contribution of the protein synthesis activities in the organelles to the overall synthesis of
cellular proteins derived from experiments carried out in the
presence of CHX. Furthermore, double inhibitor incubations
with CHX and SPEC show that total cellular protein synthesis
is inhibited by about 95%. This observation suggests that
incorporation of radiolabeled sulfur into proteins depends on
the activities of cell ribosomes. Direct sulfation of cellular
proteins, therefore, can be excluded as a major contributor to
the increase in "S-incorporation at the bleaching temperature.
Since treatments involving CHX and whole cells do not
permit specific conclusions regarding the activities of the
plastid protein synthesis machinery in particular, we pulselabeled control and 15 h-bleached cells in the presence and
absence of CHX, isolated intact plastids on gradients of
Percoll, and finally subfractionated the plastids into stroma
and thylakoid fractions on step-gradients of sucrose. Hot
TCA-precipitable counts associated with these fractions were
determined by the disc method.
Synthesis of thylakoid and stroma proteins increases when
the growth temperature is increased to 33°C (Table I). The
143
increase, however, is more modest compared to the steep
increase in total cellular protein synthesis observed at the
elevated temperature. Synthesis of thylakoid proteins increases 1.5 times at the bleaching temperature while synthesis
of stromal proteins increases 1.75 times. Since these numbers
are drawn from cultures not treated with CHX, synthesis of
thylakoid and stroma proteins under these conditions represents the total contribution of the cytoplasmic protein synthesis machinery plus the contribution of the plastid machinery.
When the cultures are treated with CHX to inhibit protein
synthesis on cytoplasmic ribosomes the contribution of the
plastid protein synthesis machinery can be established. A
decrease in protein synthesis in plastids is evident in the 15
h-bleached culture with regard to the synthesis of thylakoid
and stroma proteins (Table I). Temperature, however, does
not affect equally the synthesis of thylakoid and stroma proteins inside the organelle. In fact, synthesis of thylakoid proteins in the plastid decreases by about 20% while synthesis of
stroma proteins experiences a sharper decline of about 65%
at the bleaching temperature. In view of these results, the
observed overall increase in the synthesis of proteins destined
for the plastid at 33°C can be attributed to the contribution
of the nucleocytoplasmic compartment. Furthermore, in the
broader context of an increase in total cellular protein synthesis at the elevated temperature, the results indicate a specific
inhibitory effect of temperature on protein synthesis in
plastids.
Synthesis of Thylakoid Proteins
Thylakoids were prepared from Euglena grown at room
temperature (0) and at 33°C for 15 h (15) and pulse-labeled
in vivo in the presence (+) and in the absence (-) of CHX.
The synthesis of specific proteins destined for the thylakoid
was analyzed by gel electrophoresis followed by fluorography
for the detection of the radiolabeled polypeptides (Fig. 2). In
cultures with no antibiotic added, synthesis of proteins destined for the thylakoid increases after a 15 h incubation at the
bleaching temperature (Fig. 2; lanes 0 and 15). Particularly
evident is the synthesis of polypeptides of 45,000 and 60,000
Mr (large arrowheads) which we identified earlier as presumably novel polypeptides in stained profiles of bleaching thylakoids (15). Although the 45,000 Mr does not appear to be
present in thylakoids from control cultures, it constitutes one
of the major synthesis products destined for the thylakoids in
15 h-bleached cells. The 60,000 Mr polypeptide, on the other
hand, is synthesized at low levels in control cultures but its
synthesis greatly increases at the elevated temperature. Likewise, some thylakoid polypeptides experience an increased
synthesis at the elevated temperature (open stars), while others
actually decrease (solid stars). Nevertheless, polypeptides
whose synthesis increases at the bleaching temperature, including the 45,000 and 60,000 Mr species, appear to be
products of nucleocytoplasmic origin since their synthesis is
inhibited in the presence of CHX (in Fig. 2, cf. lane 15,
-CHX with lane 15, +CHX). In support of this conclusion,
Figure 3 shows that synthesis of the 45,000 and 60,000 Mr
species will take place in the presence of SPEC, an inhibitor
of protein synthesis on chloroplast ribosomes. The addition
of CHX and SPEC to the cell suspension, as expected, com-
Downloaded from on June 16, 2017 - Published by www.plantphysiol.org
Copyright © 1990 American Society of Plant Biologists. All rights reserved.
144
ORTIZ
6O'~~~
60_-
_
_ ffi-
:.
64
-
~-!
*
Plant Physiol. Vol. 93, 1990
l,
..
iL.L.
i
f.
4.
_28
( -_
.F*it
*:I
_0
.-w
_A"
19
...
..,
Figure 2. Synthesis of thylakoid proteins in partially bleached photoheterotrophic Euglena. Cultures grown at room temperature (0) or
at 330C for 15 h (15) were pulse-labeled in vivo with [35S]sodium
sulfate in the presence (+) or in the absence (-) of CHX. Plastids
were isolated in isoosmotic gradients of Percoll and immediately
fractionated in step-gradients of sucrose to obtain the thylakoid
fraction. Polypeptides of the thylakoid were analyzed by gel electrophoresis followed by fluorography for the detection of the radiolabeled
products. Protein standards: bovine serum albumin, ovalbumin, trypsinogen, and lysozyme are identified with small arrowheads. Numbers
on the left and right hand side of the figure reflect Mr in thousands.
pletely inhibited synthesis of the novel polypeptides. Furthermore, since the radiolabeling experiments in Figure 3 were
carried out for only 30 min instead of the usual 3 h, the results
indicate that synthesis of the major novel polypeptides is not
brought about by a prolonged nutritional shock in the lowsulfate incubation medium.
In terms of the activity of the plastid protein synthesis
machinery and its contribution to the synthesis of thylakoid
proteins which can be assessed in the presence of CHX, it is
observed that the synthesis of some thylakoid proteins decreases at the elevated temperature (in Fig. 2, cf lane 0 with
lane 15). Among the products of the plastid protein synthesis
machinery experiencing a decreased level of synthesis are the
major chloroplast-encoded polypeptides of 64,000 Mr associated with PSI in Euglena (5) and a Chl-a-binding polypeptide
of 41,000 Mr associated with PSII (5). Synthesis of QB, which
in Euglena has a Mr of 28,000 (17), was not affected. Two
other polypeptides of 19,000 and 22,000 Mr do not appear to
be synthesized in 15 h-bleached plastids. Synthesis of a second
Chl-a-binding polypeptide of 54,000 Mr associated with PSII
(5) appears to increase in plastids from 15 h-bleached cells.
Overall, the results presented in Figure 2 indicate that plastids
from partially bleached cells continue to import polypeptides
from the nucleocytoplasmic compartment (some of them
novel) for incorporation into the thylakoids despite a modest
decrease in protein synthesis in the plastid. This result suggests
that early during growth at the bleaching temperature a gradual transformation of the photosynthetic membranes of the
plastid begins to take place. In addition, it is significant that
synthesis of some of the Chl-binding polypeptides decreases
Figure 3. Synthesis of the major 60,000 and 45,000 Mr novel polypeptides of the thylakoid in the presence of spectinomycin. Cultures
grown at 330C for 15 h were pulse-labeled in vivo with [35S]sodium
sulfate for 30 min in the presence (SPEC) or absence (0) of SPEC
and in the presence of SPEC plus CHX (CHX + SPEC). Plastids were
isolated in isoosmotic gradients of Percoll and immediately fractionated in step-gradients of sucrose. Polypeptides of the thylakoid were
analyzed by gel electrophoresis followed by fluorography for the
detection of the radiolabeled products. Novel polypeptides are identified by their Mr in thousands.
at a time when Chl accumulation per cell has greatly increased. This observation is rather puzzling at the moment
since it runs contrary to our original expectations of an
increased synthesis of plastid components particularly of Chlbinding polypeptides at the elevated temperature.
Synthesis of Stroma Proteins
A similar strategy was followed to study the impact of
temperature on the synthesis of stroma proteins (Fig. 4).
Synthesis of stroma proteins determined in the absence of
CHX increases at the elevated temperature (Fig. 4, lanes 0
and 15). In most cases, however, the increase involves the
synthesis of polypeptides that are already evident in the
stroma from control plastids (lane 0) but whose synthesis is
greatly increased at the elevated temperature (lane 15). Synthesis of novel polypeptides is also evident (open stars) including the synthesis of a polypeptide of 63,000 Mr (large
arrowhead). A stainable novel polypeptide of 63,000 Mr was
reported to accumulate in the stroma of bleaching plastids
(15). Despite the apparent similarity in size between this
polypeptide of the stroma and the 60,000 Mr species of the
thylakoid (Fig. 2), we have no evidence to indicate that the
two polypeptides are related. It is important to note, however,
that the 63,000 Mr polypeptide, like the 60,000 Mr species of
the thylakoid, is a product of the nucleocytoplasmic com-
Downloaded from on June 16, 2017 - Published by www.plantphysiol.org
Copyright © 1990 American Society of Plant Biologists. All rights reserved.
145
PROTEIN SYNTHESIS IN BLEACHING EUGLENA
0
15
0
15
0.
LU
en
x
(Q
0
-
63m_
I
L0
w
0m
C,,
- LSu
O~ww
-
63 _
*mm
.,
At
.m
ssu.
-CHX
+CHX
Figure 4. Proteins of the stroma synthesized in partially bleached
photoheterotrophic Euglena. Cultures were grown at room temperature (0) or at the bleaching temperature for 15 h (15). In vivo pulselabeling was carried out with [35S]sodium sulfate in the presence (+)
or in the absence (-) of CHX. Plastids were isolated in isoosmotic
gradients of Percoll and immediately fractionated in sucrose stepgradients to obtain the stroma. Polypeptides of the stroma were
analyzed by gel electrophoresis followed by fluorography for the
detection of the radiolabeled products. Protein standards: bovine
serum albumin, ovalbumin, trypsinogen, and lysozyme are identified
with small arrowheads. Numbers on the left hand side of the figure
reflect Mr in thousands.
partment since synthesis of the polypeptide is sensitive to
CHX (Fig. 4, lanes 15). In this regard, synthesis of the novel
63,000 polypeptide will take place in the presence of SPEC
but not when CHX and SPEC are included in the radiolabeling mixture (Fig. 5). This observation supports the conclusion
that the major novel polypeptide of the stroma is a product
of nucleocytoplasmic origin. Furthermore, based on this
short-term radiolabeling experiment of 30 min, it is possible
to conclude that synthesis of the novel polypeptide does not
represent a response to a prolonged exposure to incubation
medium containing limited amounts of sulfate.
Pulse-labeling studies carried out in the presence of CHX
with cells grown at room temperature and at 330C for 15 h
show reduced synthesis of the LSU of Rubisco at the bleaching
temperature (Fig. 4, lanes 0 and 15). This result was expected
based on the results summarized in Table I indicating a sharp
decrease in the synthesis of stroma proteins in plastids at the
elevated temperature. It is significant, however, that this decrease in the LSU of Rubisco does not parallel the behavior
of the SSU. In fact, synthesis of the SSU actually increases at
the higher temperature (Fig. 4, -CHX: lanes 0 and 15). It
appears, then, that synthesis of the LSU and SSU is not tightly
coordinated during the initial phase of the bleaching response.
DISCUSSION
An increase in the accumulation of total Chl per cell during
the early phases of the temperature-induced bleaching re-
Figure 5. Synthesis of the major 63,000 Mr novel polypeptide of the
stroma in the presence of spectinomycin. Partially bleached cultures
(15 h) were pulse labelled in vivo with radiolabeled sodium sulfate for
30 min in the presence (SPEC) or absence (0) of SPEC and in the
presence of SPEC plus CHX (CHX + SPEC). Plastids were isolated
in isoosmotic gradients of Percoll and fractionated in sucrose stepgradients. Polypeptides of the stroma were analyzed by gel electrophoresis followed by fluorography for the detection of the radiolabeled
products. The novel polypeptide of the stroma is identified by the Mr
in thousands.
sponse in Euglena was of interest to us because it suggested
an acceleration in the synthesis of plastid components particularly proteins. Our expectations were based on previous
studies of profiles of stainable polypeptides of thylakoids and
stroma which showed the accumulation of presumably novel
polypeptides at the bleaching temperature (15). The results
presented in this report show that the novel polypeptides
destined for the thylakoid or the stroma are synthesized on
cytoplasmic ribosomes and imported by plastids at the bleaching temperature. For the most part, the import of these
polypeptides accounts for the overall increase in the synthesis
of plastid proteins at 330C (Table I). In contrast, protein
synthesis in the plastid actually decreases at the elevated
temperature. Specifically affected are some of the Chl-abinding polypeptides associated with PSI and II. It was of
interest to note, however, that synthesis of a second Chl-abinding polypeptide associated with PSII (54,000 Mr) actually
increases at the elevated temperature. The observed inhibition
in the synthesis of thylakoid polypeptides inside the plastid is
clearly not uniform since the synthesis of some polypeptides
appears to be more strongly affected by temperature than
others. The molecular mechanism that underlies this difference is not understood at the present time.
Synthesis of the LSU, a product of the chloroplast protein
synthesis machinery, is sharply reduced in 15 h-bleached
Downloaded from on June 16, 2017 - Published by www.plantphysiol.org
Copyright © 1990 American Society of Plant Biologists. All rights reserved.
ORTIZ
146
plastids while the synthesis of the SSU, a product of nucleocytoplasmic origin, actually increases at 33°C. This situation
suggests a rather loose coordination in the activities of the
cytoplasmic protein synthesis apparatus and the plastid protein synthesis machinery in the synthesis of the two subunits
of Rubisco at this early stage of the bleaching response. It is
possible to propose, however, that in view of the reduction in
the synthesis of the LSU, the levels of imported, newly synthesized SSU are regulated at a posttranslational level through
an increase in polypeptide turnover. This type of regulation
has been shown to occur in Chlamydomonas (18) and results
in the rapid and selective degradation of the SSU when
synthesis of SSU is in excess of the LSU. It is not known
whether high levels of SSU synthesis are sustained in Euglena
on prolonged treatments at 33°C when synthesis of LSU is
expected to drop even further.
Much interest has surfaced in recent years regarding the
specific synthesis of chloroplast proteins in response to an
increase in growth temperature. The question often raised is
how does a semiautonomous genetic entity like the chloroplast cope with increases in temperature and protect its integrity against the damaging effects of the high temperature. In
this respect, comparisons with the bleaching phenomenon in
Euglena should be considered with some caution since there
are obvious differences in the range of temperatures under
study. Bleaching, for example, is carried out at 33°C while,
for the most part, other studies with higher plants and algae
involve temperatures in the40°C range. Nevertheless, changes
have been reported in the synthesis of chloroplast proteins
resulting in the import of novel polypeptides of nucleocytoplasmic origin into the chloroplast (7, 19, 20). Depending on
the plant species under consideration, the publications report
the accumulation in chloroplasts of polypeptides of 21,000 to
24,000 Mr under heat-shock conditions. Although the function of these polypeptides is not known, they presumably
protect the integrity of the organelle against stress damage. It
is of interest to note that treatment at the bleaching temperature leads to an increased synthesis of polypeptides in the
21,000 to 24,000 Mr range which accumulate in the stroma
of 15 h-bleached Euglena (Fig. 4, lane 15, -CHX, just below
the trypsinogen standard). Minor polypeptides in this Mr
range are already evident in the stroma of control cells and
the increased synthesis at33°C can be attributed to a general
increase in the synthesis of chloroplast proteins of nucleocytoplasmic origin (Table
I). The relationship between the minor
polypeptides in the 21,000 to 24,000Mr of Euglena and those
described in other plant systems (7, 19, 20) is not known.
Since chloroplasts constitute a separate genetic entity in
plant cells, it has been suggested that chloroplasts may respond
to elevated temperatures by synthesizing chloroplast-encoded
heat-shock proteins on the chloroplast protein synthesis machinery. Two recent reports appear to support this contention.
Kloppstech et at. (8) have presented evidence for the synthesis
of a 70,000 Mr heat-shock protein of chloroplast origin in
Acetabularia. Furthermore, Krishnasamy et at. (9) reported
an induction in the synthesis of four polypeptides of 85,000,
70,000, 60,000 and 23,000
Mr when isolated plastids of Vigna
sinensis are incubated at temperatures above 35C. This group
cautions, however, that induction depends on the gradual
Plant Physiol. Vol. 93,1990
application of the heat-shock treatment to the leaves. In
contrast, Nieto-Sotelo and Ho (13) have shown that isolated
plastids and mitochondria from maize do not synthesize heatshock proteins at 40°C. Their results were supported with
pulse-labeling studies carried out in vivo in the presence of
CHX with essentially the same results. Our study with bleaching Euglena is in line with the results of Nieto-Sotelo and Ho.
Therefore, it is concluded that bleaching in Euglena does not
induce synthesis of novel chloroplast-encoded polypeptides in
the time frame considered in this study. Shorter as well as
longer times of incubation must be studied first before a clear
picture emerges in this respect. The involvement of the chloroplast genome in the temperature response evidently requires
further study to sort out some of these differences.
ACKNOWLEDGMENTS
The author wishes to thank Ms. B. Richey for typing the
manuscript.
LITERATURE CITED
1. Arnon DI (1949) Copper enzymes in isolated chloroplasts. Polyphenol oxidases in Beta vulgaris. Plant Physiol 24: 1-15
2. Bollum FJ(1966) Filter paper disk technique for assaying radioactive macromolecules. In GL Cantoni, RD Davies, eds, Procedures in Nucleic Acid Research. Harper and Row, New
York, pp 296-300
3. Burckhardt J, Telford J, Birnstiel ML (1979) Detection of
labelled RNA species by contact hybridization. Nucleic Acid
Res6: 2963-2971
4. Chua NH(1980) Electrophoretic analysis of chloroplast proteins.
Methods Enzymol 69: 434-446
5. Cunningham FX, Schiff JA (1986) Chl-protein complexes from
Euglena gracilis and mutants deficient in Chl b.II. Polypeptide
composition. Plant Physiol 80: 231-238
6. Douce R, Joyard J (1982) Purification of the chloroplast envelope. In M Edelman, RB Hallick, NH Chua, eds, Methods in
Chloroplast Molecular Biology. Academic Press, New York,
pp 239-256
7. Kloppstech K, Meyer G, Schuster G, Ohad I (1985) Synthesis,
transport and localization of a nuclear coded 22-kd heat-shock
protein in the chloroplast membranes of peas and Chlamydomonas reinhardi. EMBO J 4: 1901-1909
8. Kloppstech K, Ohad I, Schweiger HG (1986) Evidence for an
extranuclear coding site for heat-shock protein in Acetabularia.
Eur J Cell Biol 42: 239-245
9. Krishnasamy
S, Mannan RM, Krishnan M, Gnanam A (1988)
Heat shock response of the chloroplast genome in Vigna
sinensis. J Biol Chem 263: 5104-5109
10. Larson E, Howlett B, Jagendorf A (1986) Artificial reductant
enhancement of the Lowry method for protein determination.
Anal Biochem 155: 243-248
11. Laskey RA, Mills D (1975) Quantitative film detection of 3H
and
in polyacrylamide gels by fluorography. Eur J Biochem
56: 335-341
12. Monroy AF, McCarthy SA, Schwartzbach SD (1987) Evidence
for translational regulation of chloroplast and mitochondrial
biogenesis in Euglena. Plant Sci 51: 61-76
13. Nieto-Sotelo J, Ho THD (1987) Absence of heat shock protein
synthesis in isolated mitochondria and plastids from maize. J
Biol Chem 262: 12288-12292
14. Ortiz W Reardon EM, Price CA (1980) Preparation of chloroplasts from Euglena highly active in protein synthesis. Plant
Physiol 66: 291-294
15. Ortiz W, Wilson
CJ (1988) Induced changes in chloroplast
protein accumulation during heat bleaching in Euglena gracilis. Plant Physiol 86: 554-561
16. Pringsheim EG, Pringsheim 0 (1952) Experimental elimination
14C
Downloaded from on June 16, 2017 - Published by www.plantphysiol.org
Copyright © 1990 American Society of Plant Biologists. All rights reserved.
PROTEIN SYNTHESIS IN BLEACHING EUGLENA
of chromatophores and eye-spot in Euiglena gracilis. New
Phytol 51: 65-76
17. Reardon EM, Price CA (1984) Synthesis ofthe psbA gene product
in Eluglena. In organello and in vitro. Plant Physiol 75: 246248
18. Schmidt GW, Mishkind ML (1983) Rapid degradation of unassembled ribulose-1,5-bisphosphate carboxylase small subunits
in chloroplasts. Proc Natl Acad Sci USA 80: 2632-2636
147
19. Suss KH, Yordanov IT (1986) Biosynthetic cause of in vivo
acquired thermotolerance of photosynthetic light reactions and
metabolic responses of chloroplasts to heat stress. Plant Physiol
81: 192-199
20. Vierling E, Mishkind ML, Schmidt GW, Key JL (1986) Specific
heat shock proteins are transported into chloroplasts. Proc Natl
Acad Sci USA 83: 361-365
Downloaded from on June 16, 2017 - Published by www.plantphysiol.org
Copyright © 1990 American Society of Plant Biologists. All rights reserved.