Download Relative Requirements for Magnesium of Protein and Chlorophyll

Plant Physiol. (1978) 61, 624-625
Relative Requirements for Magnesium of Protein and Chlorophyll
Synthesis in Euglena gracilis1
Received for publication May 17, 1977 and in revised form December 12, 1977
RAYMOND E. ZIELINSKI AND CARL A. PRICE
Waksman Institute of Microbiology, Rutgers University, P. O. Box 759, Piscataway, New Jersey 08854
ABSTRACT
The relationsbips among Mg, growth, chlrophyll syntbesis, aN cytoplasmic polysome content were studied in Eugkna gracilis grown in
different levels of the metal. At all levels of magnesium from 20 to 1,600
,umolar, both protein and chlorophyll are formed with exponential kinetics.
The apparent rates of synthesis and final yields of both components are
greater at higher levels of Mg, but the rate of chlorophyll synthesis always
exceeds the rate of protein formation; ie. the most severely deficient cells
contain proportionally more chlorophyll than the sufficient cells. Cytoplasmic polysomes isolated from Mg-deficient Eugkna are indistinguishable
from those isolated from control cells. We conclude that decreased rates
of protein synthesis occur prior to and possibly are causal to decreased
rates of chlorophyll synthesis, but that the mechanism of this inhibition
remains unclear.
over of Mg, transfers of algae were made from exponentially
growing stock cultures containing 100 ,M MgSO4, which is onesixteenth the normal concentration. There was zero growth without added Mg.
Growth was monitored turbidimetrically at 540 nm. Chl was
estimated by a modification of Arnon's (1) method (3) and protein
by the Lowry method (10). All glassware was washed by soaking
in 50% (v/v) nitric acid for 2 to 3 weeks prior to use.
Polysomes were isolated from midexponential phase cultures by
the method of Avadhani and Buetow (2) with the high pH
modification of Davies et al. (4).
RESULTS AND DISCUSSION
Kinetics of Growth and Formation of Chi and protein. When
photoheterotrophic Euglena are grown with different amounts of
Mg initially present in the medium, both the rate of growth and
total yield, as measured by apparent optical density, are affected
(Fig. IA). Provided that exponentially growing cells were used to
inoculate the cultures, no lag phase was observed. Protein content
Magnesium is among the essential macronutrients of plants follows a nearly identical pattern, as shown in Figure IB. Both
(14), but the biochemical basis for this requirement has not been increase exponentially at all of the concentrations tested but with
clearly established. Indeed, the problem lies in choosing from progressively lower rate constants. The total yield of protein is
among many possible bases for a Mg requirement. The need for also progressively decreased, with the yield at 20 fLM Mg lowered
Mg has been variously ascribed to its occurrence in Chl and its to about 30%o of the control culture at 100 hr.
action as a cofactor for a variety of enzymes, notably those of
The rates of Chl accumulation are similarly exponential, with
phosphate transfer (17). Specific requirements for Mg have also decreasing rate constants at the lower levels of Mg (Fig. IC). The
been reported for the development of chloroplasts (7, 16) and Chl doubling times, however, are always slightly less than the
mitochondria (I 1). Since the complexes of Mg with enzymes have generation times of the respective cultures. Throughout the exrelatively low affinities and the metal is easily translocatable ponential stages of growth, the ratios of Chl to protein (Fig. 2)
throughout the plant, compositional studies can be difficult to vary only slightly, and then without any systematic relation to the
interpret.
concentration of Mg.
We have employed progressive Mg deficiency in Euglena
These results contrast with similar studies employing iron defigracilis (12) to inquire into the relative priorities between the ciency in Euglena (5, 13). Using virtually identical conditions,
synthesis of protein and Chl in the cell. Specifically, we asked if Funkhouser and Price (5) found that Chl synthesis was halted at
a failure in the production of Chl might precede a decrease in the low levels of iron while protein accumulation continued at normal
over-all rate of protein synthesis.
exponential rates. Those results were consistent with a rather strict
As our test organism we chose Euglena gracilis with glucose as compartmentalization of iron within the cell. In contrast, we find
the principal carbon source. Under photoheterotrophic conditions, that growth and Chl synthesis are not separable under conditions
chloroplast development is gratuitous and therefore can easily be of progressive Mg deficiency and that by inference no such
separated from global protein synthesis.
compartmentalization exists for Mg.
Our data do not support a regulation of the synthesis of the Chl
MATERIALS AND METHODS
molecule by Mg per se. The correlation between total protein
content and Chl accumulation (Fig. 2 inset) is consistent with the
Our strain of E. gracilis (Klebs) is derived from the z strain of notion
that protein synthesis controls the accumulation of Chl in
Pringsheim, but differs from it in the morphology of its thylakoids. Mg-deficient
Euglena. This conclusion is furThe cells were cultured photoheterotrophically on a modified ther supportedphotoheterotrophic
by
evidence
that
protein
synthesis is required for
Hutner medium as described previously (5). Mg was added as the normal Chl production when etiolated plant
tissues are exposed
sulfate salt. Constant levels of sulfate were maintained by adding to light (6, 9). In view of this seeming dependency,
is surprising
appropriate amounts of KHSO4. In order to minimize the carry- to find that the rate of Chl accumulation, while itparallel
to, is
always greater than the rate of protein synthesis.
1 Supported in part by Grant HD-05602 and by a biomedical research
The decrease in protein and Chl synthesis caused by Mg depsupport grant from the National Institutes of Health to Rutgers University. rivation is a reversible one. The addition of 1,600 tIM Mg to cells
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Copyright © 1978 American Society of Plant Biologists. All rights reserved.
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FIG. 1. Kinetics of (A) cell growth, (B) protein formation, and (C) Chl
synthesis in E. gracilis grown under photoheterotrophic conditions. The
,umolar concentration of Mg initially present in each culture is indicated.
The apparent rate constants of exponential increase (values in parentheses)
were calculated for each culture between 40 and 78 hr after inoculation
according to the equation N = Noexp(kt).
j60
.
0
C4Q~
L.
625
Mg REQUIREMENTS OF EUGLENA
Plant Physiol. Vol. 61, 1978
Protein
.20
nWffi)
2~~~~~~0
6
40
80
120 17O
Time (1)
FIG. 2. Chl to protein ratios against time in photoheterotrophically
cultured Euglena Initial ,lmolar concentrations of Mg for the respective
cultures are indicated. Inset shows the proportional relationship of Chl
and protein in the same cultures. The solid line was drawn through the
control (1,600 uM) points. Open squares indicate the values for the 20 /LN
Mg2+ culture. The 100 and 50,UM Mg2+ cultures did not differ significantly
from the control values.
that had grown to stationary phase in 20 ttM Mg stimulated cell
density and the yield of Chl to nearly normal levels (data not
presented). We take this to mean that the changes observed in the
cultures were attributable to the level of Mg initially present in
the growth medium and not to irreversible changes in the cells.
The stationary phase is usually glucose-limited and the photoheterotrophic cells become auxotrophic. During early stationary
phase, Chl synthesis continues at a rate that is sharply diminished
(Fig. IC) but nonetheless more rapid than protein synthesis. The
Chl to protein ratios of low Mg cultures increase more slowly, to
a lesser extent, and at a later time after inoculation than with Mgsufficient cultures. A proportional plot of Chl against protein (Fig.
2 inset) indicates, however, that on the basis of the protein
accumulation, the cells grown in 20 uM Mg are slightly CM-rich.
Polysome and RNA Levels. Since we found that Mg deficiency
affects the apparent rate and extent of protein synthesis in Euglena,
we wondered if the lesion could be at the level of the polysomes.
We isolated polysomes from midexponential phase cultures of
Euglena grown in different concentrations of Mg. The sedimentation profiles for different cultures were compared with respect
to two criteria: the ratio of polysomes to total ribosomes; and the
size distribution of the polysomes. Our results (not shown) indicate
that polysome profiles obtained from Mg-deficient Euglena do not
differ significantly from Mg-sufficient cultures. Polysomes extracted from all cultures tested represented about 10%1o of the total
cellular RNA and about 50o of the total ribosomes, with pentamers or hexamers the most abundant.
Although there is much evidence demonstrating the need for
high levels of Mg by several components of the protein-synthesizing machinery (2, 8, 15) and our results point to a decline in
protein synthesis during progressive Mg deficiency, the results
obtained from the isolation of polysomes from Euglena are inconclusive. We note that our methods recovered only a small fraction
of the total ribosomes of the cell and that Mg itself is required to
isolate polysomes; so that substantial differences in the polysome
content in vivo might have been missed.
Although our experiments were performed with photoheterotrophically grown organisms, we see no reason to expect that these
priorities for Mg would differ within an autotrophic organism.
Apart from the advantages of working with a microorganism
under controlled conditions, photoheterotrophic Euglena provides
a better test for studying the role of Mg than would an obligately
autotrophic higher plant. If the production of Chl were essential
to the maintenance of normal growth rates, a tight coupling
between protein and Chl accumulation, such as we observed,
could well have been interpreted to mean that protein synthesis
requires Chl formation rather than the other way around.
LITERATURE CITED
1. ARNON DI 1949 Copper enzymes in isolated chloroplasts. Polyphenol oxidase in Beta vulgaris.
Plant Physiol 24: 1-15
2. AVADHANI NG, DE BUETOW 1972 Isolation of active polyribosomes from the cytoplasm.
mitochondria and chloroplasts of Euglena gracilis. Biochem J 128: 353-365
3. BRUINSMAJ 1961 A comment on the spectrophotometric determination of chlorophyll. Biochim
Biophys Acta 52: 576-578
4. DAVIEs E. BA LARKINS, RH KNIGHT 1972 Polyribosomes from peas. An improved procedure
for their isolation in the absence of ribonuclease inhibitors. Plant Physiol 50: 581-584
5. FUNKHOUSER EA, CA PRICE 1974 Chloroplast RNA: possible site of an early lesion in iron
deficiency. Plant Cell Physiol 15: 883-889
6. GASSMAN M, L BOGORAD 1967 Control of chlorophyll production in rapidly greening bean
leaves. Plant Physiol 42: 774-780
7. HALL JD, R BARR, AH AL-ABBAs, FL CRANE 1972 The ultrastructure of chloroplasts in
mineral-deficient maize leaves. Plant Physiol 50: 404409
8. HASELKORN R, LB ROTHMAN-DANES 1973 Protein synthesis. Annu Rev Biochem 42: 397-438
9. KIRK JTO, RL ALLEN 1965 Dependence of chloroplast pigment synthesis on protein synthesis:
effect of actidione. Biochem Biophys Res Commun 21: 523-530
10. LOWRY OH. NJ RoSEBROUGH, AL FARR, RJ RANDALL 1951 Protein measurement with the
Folin phenol reagent. J Biol Chem 193: 265-275
11. MARINos NG 1963 Studies on submicroscopic aspects of mineral deficiencies. II. Nitrogen,
potassium, sulfur, phosphorus and magnesium deficiencies in the shoot apex of barley. Am
J Bot 50: 998-1005
12. ONDRATSCHEK K 1941 Uber den Mineralsalzbedarf heterotropher Flagellaten. Arkiv Mikrobiol 12: 241-253
13. PRICE CA, EF CARELL 1964 Control by iron of growth and chlorophyll formation in Euglena
gracilis. Plant Physiol 39: 862-868
14. SALM-HORSTMAR P 1849 Versuche ueber die nothwendigen Aschenbestandtheile einer Pflanzen-species. J Prakt Chem 46: 193-211
15. SCHWARZBACH SD, G FREYSSINET, JA SCHIFF 1974 The chloroplast and cytoplasmic ribosomes of Euglena. I. Stability of chloroplast ribosomes prepared by an improved procedure.
Plant Physiol 53: 533-542
16. THOMSON WW, TE WEIER 1962 The fine structure of chloroplasts from mineral deficient
leaves of Phaseolus vulgaris. Am J Bot 49: 1047-1055
17. ZIMMERMAN M 1947 Magnesium in plants. Soil Sci 63: 1-12
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Copyright © 1978 American Society of Plant Biologists. All rights reserved.