Plant & Cell Physiol. 24(4): 581-586 (1983)
Steroidal Saponins are not Main Building Units of the
Prolamellar Body in Etioplasts
Satoru Murakami, Masahiko Ikeuchi and Mitsue Miyao
Department of Biology, University of Tokyo, Komaba, Meguro-ku, Tokyo 153, Japan
Key words: Etioplast (Avena) — Prolamellar body (Cucurbita) — Saponin.
Liitz (1978) prepared prolamellar body- and prothylakoid-rich fractions from Avena
etioplasts on a sucrose density gradient and argued that the prolamellar body consists mainly
of two steroidal saponins and some non-chlorophyllous pigments, while the prothylakoid contains
galactolipids and pigments. Kesselmeier and his co-workers also put forward the view that
steroidal saponins, avenacosides A and B are main building units of the prolamellar body in the
etioplasts not only of Avena but also of Hordeum and Pisum (Kesselmeier and Budzikiewicz 1979,
Kesselmeier and Ruppel 1979). Subsequently Liitz and Klein (1979) proposed that the saponin
content in a given etioplast membrane fraction can be used as a valid measure for the presence
of the prolamellar bodies. Using this marker, Lutz and his co-workers arrived at the conclusion
that protochlorophyllide (Liitz and Manning 1980) and the NADPH:protochlorophyllide
oxidoreductase (Liitz et al. 1981) are localized mostly in the prothylakoids in Avena etioplasts.
However, in our studies both protochlorophyllide and the 36,000-dalton NADPH protochlorophyllide oxidoreductase were mostly in the prolamellar bodies in Cucurbita etioplasts
(Ikeuchi and Murakami 1982a, b, 1983, Murakami and Ikeuchi 1982). We did not use
saponins as a marker for the prolamellar body because they were scarcely detected in etiolated
Cucurbita cotyledons. This led us to doubt the validity of using saponins as a marker for the
prolamellar bodies. We also questioned the concept that steroidal saponins are the essential
constituents of the prolamellar bodies. The present work was undertaken to answer these
questions.
Materials and Methods
Etioplasts were prepared from squash {Cucurbita moschata Durch. var. melonaeformis cv. Tokyo
Makino) cotyledons by the method used in the previous work (Ikeuchi and Murakami 1982b).
The homogenizing medium contained 0.5 M sucrose, 1 nut MgCl2, 1 mM Na 2 EDTA, 5 miu 2mercaptoethanol and 40 mM Tricine-NaOH, pH 7.7. The homogenate was filtered through
four layers of nylon cloth (25 yum mesh) then centrifuged at 2,000 Xg for 2 min. Intact and
581
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Sugar-containing lipids were analyzed by thin layer chromatography in various
cell fractions of etiolated Aoena leaves, Auena intact etioplasts and prolamellar bodies
isolated from Cucurbita etioplasts. We confirmed the presence of steroidal saponins,
avenacosides A and B in etiolated leaves and crude etioplast fraction of Avena, but scarcely
detected them in Avena intact etioplasts purified by Percoll density gradient centrifugation. Saponins were hardly detected in the paracrystalline prolamellar bodies from
Cucurbita etioplasts. We concluded that steroidal saponins are not main building units
of the prolamellar body in the etioplasts.
582
S. Murakami, M. Ikeuchi and M. Miyao
Results and Discussion
The P-l fraction contained cell debris, nuclei, etioplasts and their aggregates. Most of the
intact etioplasts were found in the P-2 fraction, together with a large amount of broken etioplasts
and some mitochondria (Fig. 1A). The P-4.5 fraction consisted mainly of prolamellar bodies
with attached prothylakoids, mitochondria, microbodies and other membrane structures (Fig.
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broken etioplasts in the supernatant were pelleted by centrifugation at 4,500 X^ for 5 min and
suspended in a hypotonic medium containing 1 min MgCl2, 5 HIM 2-mercaptoethanol and
40 DIM Tricine-NaOH, pH 7.7, then etioplast membranes were separated by linear sucrose
density gradient centrifugation at 30,000 Xg for 30 min. The gradient contained 0.9-1.5 M
sucrose, 1 mti MgCl2, 5 ITIM 2-mercaptoethanol and 40 DIM Tricine-NaOH, pH 7.7. The
yellow band containing the prolamellar bodies with attached prothylakoids was collected with
a syringe and the prolamellar bodies were pelleted at 30,000 Xg fr>r 30 min.
Etiolated first leaves of 9 day old oat (Avena sativa L. cv. Victory I) were homogenized for
5 s burst by Physcotron homogenizer (Nition-Irika Seisakusho) in the homogenizing medium
described above. The homogenate was filtered through four layers of nylon cloth and centrifuged successively as follows, l,OO0X£ for 1 min, 2,000 X^ for 2 min, 4,500X^ for 5 min and
30,000X£ for 30 min to give pellet P-l, P-2, P-4.5 and P-30, respectively. To obtain Avena
intact etioplasts, P-2 was resuspended in 5 ml of the homogenizing medium and centrifuged at
100 X^ for 1 min to remove large aggregates of nuclear materials and other cellular components.
The supernatant was incubated at 25°C for 20 min with 100/tg/ml deoxyribonuclease I (Sigma,
electrophoretically pure) in the presence of 10 mil MgCl2, and after addition of Na2EDTA at
a final concentration of 20 mM, layered onto 24 ml of Percoll density gradient containing 0-70%
Percoll (Pharmacia), 5% polyethyleneglycol 6,000, 1% Ficoll (Pharmacia), 1 mM MgCl2,
I mM Na2EDTA and 40 mM Tricine-NaOH, pH 7.7. After centrifugation at 8,000 X^ for
II min, two yellow bands were obtained and intact etioplasts in the lower band were collected.
Lipids were extracted successively by suspending the pellets (P-l, P-2, P-4.5, P-30, Avena
intact etioplasts and isolated Cucurbita prolamellar bodies) in methanol, acetone and chloroformmethanol (1 : 1). Lipids were extracted also from etiolated Avena leaves in the same solvents
with a glass homogenizer. The extracts in different solvents were combined, evacuated to
dryness, then dissolved in a small volume of chloroform-methanol (1 : 1). Lipids were applied
on a silica gel plate (Silica Gel 60, Merck) to give a color intensity equal to that of monogalactosyl
diglyceride upon spraying with an anthron reagent and developed with the solvent system,
chloroform : methanol : water (70 : 30 : 4) according to Liitz (1978). Sugar-containing lipids
were detected with an anthron reagent. Steroidal lipids were identified with several spray
reagents such as sulfuric acid-acetic acid (Fujino 1978); ferric chloride-acetic acid-sulfuric acid
(Fujino 1978) and anisaldehyde-antimony trichloride (Touchstone and Dobbins 1978). A large
amount of sucrose, present in the isolation medium, was found near the origin on the chromatograms.
For transmission electron microscope observation the samples were fixed for 2 h with 2%
glutaraldehyde in the isolation medium, followed by 2 h fixation with 2% OsOa in 100 mM
phosphate buffer, pH 7.0. Ultrathin sections were stained successively with 2% uranyl acetate
for 10 min and lead citrate for 5 min. For scanning electron microscopy, isolated squash
prolamellar bodies were fixed with 2% OsO4 for 20 h, dehydrated with ethanol, then frozenand-cleaved and dried by a critical point method. The samples were coated with a thin layer of
platinum/carbon using Balzers Freeze-Etch Unit, BAF 301, according to the procedure described
by Nagano et al. (1982) and observed with a high resolution scanning electron microscope,
HFS 2S, equipped with a field emission electron gun (Yamada et al. 1982).
Prolamellar bodies and saponins
583
r
-;,
.;
v
5,
V ^..;
'jj
C
Fig. 1 Electron micrographs of cell and intact etioplast fractions prepared from etiolated Avena leaves.
fraction; B, P-4.5 fraction; C, P-30 fraction; D, intact etioplast fraction. Bar, 1 /an.
A, P-2
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IB). The P-30 fraction contained mostly small vesicles of unknown origin and nature (Fig. 1C).
Small amounts of tubular elements of the prolamellar bodies were also included in this fraction.
The intact etioplast fraction which had been prepared from the P-2 fraction by Percoll density
gradient centrifugation contained a very small amounts of broken etioplasts and other cell
organelles (Fig. ID). The paracrystalline structure of the prolamellar bodies in the etioplasts
was slightly disordered due to the low concentration (1 mM) of MgCl2 in the isolation medium.
Thin-layer chromatography of lipids extracted from Avena etiolated leaves (Fig. 2, lane 1)
gave essentially the same result as that presented by Kesselmeier and Ruppel (1979) and Lutz
(1981a). In addition to monogalactosyl, digalactosyl and sulfoquinovosyl diglycerides, two
colored bands (Fig. 2, marked by A and B) which had been identified, respectively, as avenacoside
A and B by Kesselmeier and Budzikiewicz (1979) were visualized not only with an anthron reagent, but also with other spray reagents for detecting steroidal lipids, such as sulfuric acid-acetic
acid, ferric chloride-acetic acid-sulfuric acid and anisaldehyde-antimony trichloride (data not
S. Murakami, M. Ikeuchi and M. Miyao
584
MGDG -
- MGDG
DGDG
SQDG
A
B
X
S
- DGDG
- SQDG
•
A
1
- s
Fig. 2 Sugar-containing lipids in Avena leaf extract (1), Avena cell fractions (2, P-l; 3, P-2; 4, P-4.5; 5, P-30),
Avena intact etioplast fraction (6) and Cucurbita prolamellar body fraction (7).
Lipids were extracted, chromatographed and visualized with an anthron reagent as described in Materials and Methods. A, avenacoside A;
B, avenacoside B; MGDG, monogalactosyl diglyceride; DGDG, digalactosyl diglyceride; S, sucrose; SQ.DG,
sulfoquinovosyl diglyceride; X, unidentified.
shown). The results presented in this work confirmed the occurrence of a substantial amount
of steroidal saponins in Avena etiolated leaves.
Among the four cell fractions (P-l, P-2, P-4.5 and P-30), saponins, though present in all,
were most abundant in P-30, which included many unidentified small vesicles and a small
amount of fragments of prolamellar bodies. Saponins were also found in the P-2 and P-4.5
fractions which contained, respectively, the etioplasts and the prolamellar bodies.
Fig. 3 Electron micrographs of prolamellar body fraction isolated from Cucurbita etioplasts.
A, transmission
electron micrograph; B, High resolution scanning electron micrograph. Bar; A, 1 /on; B, 0.5 /jm.
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- B
-X
Prolamellar bodies and saponins
585
The authors wish to thank Mrs. N. Yamada, Miss M. Nagano and Dr. M. Osumi, Department of Biology,
Japan Women's University, for their collaboration in scanning electron microscopy. This work was supported
in part by Grants-in-Aid for Science Research to S. M. (No. 511212 and 511614) from the Ministry of Education,
Science and Culture in Japan.
References
Fujino, Y. (1978) Shishilsu Bunseki-ho Nyumon. p. 96. Gakkai Shuppan Center, Tokyo (in Japanese).
Ikeuchi, M. and S. Murakami (1982a) Behavior of the 36,000-dalton protein in the internal membranes of squash
etioplasts during greening. Plant & Cell Physiol. 23: 575-583.
Ikeuchi, M. and S. Murakami (1982b) Measurement and identification of NADPH :protochlorophyllide oxidoreductase solubilized with Triton X-100 from etioplast membranes of squash cotyledons. Plant & Cell Physiol.
23: 1089-1099.
Ikeuchi, M. and S. Murakami (1983) Separation and characterization of prolamellar bodies and prothylakoids
from squash etioplasts. Plant & Cell Physiol. 24: 71-80.
Kesselmeier, J. and H. Budzikiewicz (1979) Identification of saponins as structural building units in isolated
prolamellar bodies from etioplasts of Avena saliva L. Z. Pflanzenphysiol. 91: 333-344.
Kesselmeier, J. and H. G. Ruppel (1979) Relation between saponin concentration and prolamellar body structure
in etioplasts of Avena sativa during greening and re-etiolation and in etioplasts of Hordeum vulgare and Pisum sativum.
Z. Pflanzenphysiol. 93: 171-184.
Downloaded from http://pcp.oxfordjournals.org/ at Penn State University (Paterno Lib) on May 12, 2016
In the purified intact etioplast fraction which was prepared from the P-2 fraction by Percoll
density gradient centrifugation, avenacosides A and B were markedly reduced (Fig. 2, lane 6).
The saponin content in this fraction was not much compared with those of galactolipids which
were exclusively contained in the etioplast membranes.
Steroidal saponins were scarcely detected in the prolamellar bodies with attached
prothylakoids (Fig. 2, lane 7), which had been isolated from etiolated Cucurbita cotyledons
(Fig. 3A). High resolution scanning electron microscopy showed that these prolamellar bodies
have paracrystalline structures (Fig. 3B). This is contradictory to the view that good crystallinity
of tubular elements in the prolamellar body is related to its higher content of saponins (Liitz
1981b).
The results presented in this work have revealed that steroidal saponins are contained in the
crude fractions of the etioplast and the prolamellar body, but they are markedly reduced to
negligible amounts after purification of the fractions. Therefore we do not support the concept
that steroidal saponins are essential constituents of the prolamellar body in etioplasts. Our
previous study demonstrated that the 36,000-dalton NADPH :protochlorophyllide oxidoreductase is a main constitutive protein of the paracrystalline prolamellar body in etioplasts
(Ikeuchi and Murakami 1983).
Localization of steroidal saponins in Avena leaf cells is not yet known. Distribution of
saponins in the cell fractions was affected by the amount of salts added to the isolation medium.
In this work, 1 nw MgCl2 was included in the medium and saponins were found to be most
abundant in the lightest fraction P-30. If more MgCl2 was included, more aggregates of the
various cellular components were formed and saponins were found in the heavier fractions
P-l and P-2, which contained most of the etioplasts. The Avena intact etioplast fraction which
had been prepared by sucrose density gradient centrifugation contained a large amount of other
cell structures and saponins (data not shown). We were able to separate saponins from the
etioplasts when the fraction was further purified by Percoll density gradient centrifugation.
Evidently salt concentrations in the isolation medium affect the separation of the intact etioplasts
from other cellular components. Saponins are present in the etioplasts fraction because of insufficient fractionation of the intact etioplasts.
586
S. Murakami, M. Ikeuchi and M. Miyao
Liitz, C. (1978) Separation and comparison of prolamellar bodies and prothylakoids of etioplasts from Avena sativa
L. In Chloroplast Development. Edited by G. Akoyunoglou and J. H. Argyroudi-Akoyunoglou. p. 481-488.
Elsevier, Amsterdam.
Liitz, C. (1981a) New concepts for the formation of photosynthetic membranes in etioplasts. In Photosynthesis V.
Chloroplast Development. Edited by G. Akoyunoglou. p. 619-630. Balaban International Science Services,
Philadelphia.
Liitz, C. (1981b) On the significance of prolamellar bodies in membrane development of etioplasts. Protoplasma
108: 99-115.
Liitz, C. and S. Klein (1979) Biochemical and cytological observations on chloroplast development. VI Chlorophyll
and saponins in prolamellar bodies and prothylakoids separated from etioplasts of etiolated Avena sativa L. leaves.
(Received August 5, 1982; Accepted February 15, 1983)
Downloaded from http://pcp.oxfordjournals.org/ at Penn State University (Paterno Lib) on May 12, 2016
Z. Pflanzenphysiol. 9 5 : 227-237.
Liitz, C. and U. Manning (1980) Determination of chlorophylls, saponins and proteins in separated prolamellar
bodies and prothylakoids of etiolated Avena saliva. In Photoreceptor and Plant Growth. Edited by J. De Greef.
p. 229-236. Antwerpen Univ. Press, Antwerpen.
Liitz, C , U. Roper, N. S. Beer and T. Griffiths (1981) Sub-etioplast localization of the enzyme NADPH:protochlorophyllide oxidoreductase. Eur. J. Biochem. 118: 347-353.
Murakami, S. and M. Ikeuchi (1982) Biochemical characterization and localization of the 36,000-dalton
NADPH :protochlorophyllide oxidoreductase in squash etioplasts. In Cell Function and Differentiation, Part B.
Edited by G. Akoyunoglou et al. p. 13-23. Alan R. Liss, Inc., New York.
Nagano, M., K. Kodama, N. Baba, K. Kanaya and M. Osumi (1982) Filamentous structure in yeast mitochondria
by freeze-etching replica combined with rapid freezing. J. Electron Microscopy 31: 268-272.
Touchstone, J. C. and M. F. Dobbins (1978) Practice of thin layer chromatography. p. 205. John Wiley & Sons, Inc.,
New York.
Yamada, N., M. Osumi, M. Nagano, S. Murakami and T. Saito (1982) Methodology of the observation on the
ultrastructure of biological specimens by high resolution SEM. J. Electron Microscopy 31: 302.