Received for publication October 24, 1988
and in revised form January 21, 1989
Plant Physiol. (1989) 90, 512-515
0032-0889/89/90/051 2/04/$01 .00/0
Procedures for the Generation of Mature
Chiamydomonas reinhardtii Zygotes for Molecular and
Biochemical Analyses1
Dorothee Wegener, Ulrike Treier, and Christoph F. Beck*
Institut fur Biologie III, Albert-Ludwigs-Universitat, Freiburg, Schaenzlestr. 1, D-7800 Freiburg,
Federal Republic of Germany
MATERIALS AND METHODS
ABSTRACT
Strains and Culture Conditions
Zygotes represent an important stage in the sexual cycle of
the unicellular green alga Chlamydomonas relnhardtii. To study
zygote germination at a molecular level, a protocol was elaborated for the generation of zygotes in large quanftites and a
method was developed for the extraction from zygotes of RNA
that could be translated in vitro.
Chlamydomonas reinhardtii wild-type strains 137c+ and
1 37c- (obtained from R. Matagne) were used for all experiments. One L cultures were inoculated from plates and grown
in TAP2-medium (4) in 1 L round flasks with air bubbling
and continuous illumination (30 uE m-2 s-') at 23C. Gametogenesis was induced by transferring vegetative cultures
in midlog phase (cell density 1 - 2 x 106 cells/mL) to
nitrogen-free TAP-medium (16). Gametogenesis was performed under the conditions described above for the growth
of vegetative cells.
The sexual cycle of Chlamydomonas reinhardtii involves
the fusion of two gametes of opposite mating type resulting
in the formation of a diploid zygote. The mature zygote can
subsequently be induced to undergo germination. After
meiosis, four spores are released (9). The individual steps
within this life cycle (all at a single cell level) represent defined
stages in cell differentiation. The morphological and physiological characteristics of the individual cell types have been
investigated in some detail (3, 12). Although analyses of the
mating reaction and zygote formation have provided insights
into the mechanisms of cell-cell recognition (1, 18) and signal
transduction ( 14), the cellular stages of zygote maturation and
germination have so far not been analyzed at a molecular or
biochemical level in C. reinhardtii. The reasons were the lack
of procedures to obtain mature zygotes (i.e. zygotes able to
germinate) in sufficient quantities and the lack of methods
for the efficient disruption of these thick walled cells. Two
major problems have previously prohibited the production of
large numbers of zygotes. First, zygotes appear to require
contact to a firm surface (e.g. an agar plate) for maturation.
Zygotes left in liquid medium are different from plate matured
zygotes (3, 5). More important, zygotes from liquid medium
germinate very poorly (3). Second, when newly formed zygotes are plated on agar plates for maturation, the mature
zygotes become embedded within the agar and are difficult to
recover. We describe here a novel method for the production
of large numbers of mature zygotes that overcomes both of
these problems, and we also present a reliable procedure for
extraction of functional zygote RNA.
I
Supported by
(SFB206).
a
Production of Zygotes in Large Quantities
Eighteen to 20 h after induction of gametogenesis by nitrogen removal (cell density 4-8 x 106 cells/mL), equal numbers
of mt+ and mt- gametes were mixed, usually 2 L of both.
Portions (50-75 mL) of the mating mixture were then transferred to 100 mL Erlenmeyer flasks and incubated without
shaking in the light (30 ,uE m-2 s-'). After an incubation of 5
to 6 h the zygotes had aggregated to form zygote pellicles at
the surface of the liquid. These pellicles were poured onto
TAP plates (diameter 85 mm, 4% agar). Pellicles from 4 to 5
flasks were transferred to a single TAP plate. Excess liquid
was removed from the plates with a pipette after transfer of
of each pellicle. The surfaces were then allowed to dry under
a flow of sterile air. For maturation, zygotes were incubated
for 16 to 18 h in the light and then in the dark for 5 d. After
maturation the plates were exposed to chloroform vapors (by
inversion over chloroform containing plates) for 90 s in order
to kill unmated cells. The zygote pellicles were scraped off the
plates with a razor blade and the zygotes from two plates were
suspended in 4 mL TAP-medium. The unmated cells were
then destroyed by continuous sonication for 60 s at a power
of 25 to 30 W using a Branson sonifier (model B12) equipped
with a microtip. Zygotes appeared unaffected by sonication.
For separation from the cell debris the volume was adjusted
to 20 mL with TAP-medium. After centrifugation at 15OOg
for 5 min, the zygote pellet was resuspended in 20 mL TAPmedium and the washing procedure was repeated two times.
This procedure yielded essentially pure zygotes as determined
grant of the Deutsche Forschungsgemeinschaft
2Abbreviation: TAP, Tris-acetate-phosphate.
512
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513
C. REINHARDTII ZYGOTES FOR MOLECULAR AND BIOCHEMICAL ANALYSES
by microscopic observation. The zygote pellets were stored at
-80°C.
Determination of Zygote Germination
Sonication of the zygotes provided the additional advantage
of disrupting the zygote pellicles into individual zygotes and
clusters of up to 30 zygotes. The purified zygotes were suspended in TAP-medium at a density of 1 X I07 cells/mL and
exposed to light to induce germination. Samples of this suspension were plated on TAP plates (4% agar) at 0, 24, 48,
and 72 h after the onset of illumination. The vegetative cells
(products of already germinated zygotes) were killed after
plating by exposure of the plates to chloroform vapors. Zygotes which had not germinated withstood the chloroform
treatment. The number of colonies observed on the plates
after a 6 d incubation was taken as the number of zygotes
which had not germinated prior to plating. The number of
colonies from the 0 h sample was set as 100%. The reciprocal
values give the percentage of zygotes germinated.
Disruption of Zygotes
For zygote disruption, a Mikro-Dismembrator (Braun-Melsungen, model II, D-3508 Melsungen, F.R.G.) was used. The
pellets of the frozen zygotes and a tungsten carbide ball were
dispensed into Teflon vessels that had been precooked in liquid
nitrogen. Approximately 1 x I09 zygotes in a volume of about
2 mL were used for each 20 mL vessel. The vessel was shaken
vigorously for 30 s with a frequency of 50 Hz and maximal
amplitude. After this, the vessel was again immersed in liquid
nitrogen in order to avoid thawing of the zygotes. This procedure was repeated 20 times.
F.
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Figure 1. Zygote pellicles on agar plate.
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RNA Isolation
.
60
m
After disruption, the zygotes were suspended in 5 mL lysis
buffer (l 00 mM Tris-HCl [pH 8.0], 0.6 M NaCl, 10 mM EDTA,
4% SDS). Several standard methods were tested for the
extraction of RNA from zygote homogenates. Whereas all
other methods failed, the following method routinely yielded
RNA that could be translated in vitro. In this method, the
zygote homogenate was extracted with buffer-saturated
phenol:chloroform:isoamylalcohol (1: 1:0.04, v/v) until an
interphase was no longer visible (usually 5 times). After
two additional extractions with chloroform:isoamylalcohol
(1:0.04, v/v), the aqueous phase was mixed with 0.33 volumes
of 8 M LiCl and stored overnight at 40C. This procedure
resulted in a selective precipitation of RNA (10). This RNA
was collected by centrifugation, washed twice with 2 M LiCl,
dried under vacuum, and dissolved in distilled water at the
desired concentration, assuming that one absorbance unit at
260 nm corresponds to 40 ,gg RNA/mL (1 1). The purity of
the RNA obtained was determined by measuring the absorption at 230, 260, 280, and 320 nm, as well as by agarose gel
electrophoresis.
In Vitro Translation
The isolated RNA was tested by in vitro translation using a
rabbit reticulocyte lysate (6). Total RNA of vegetative cells
.E
40
02
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01
20 iF
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20
40
60
80
time (h)
Figure 2. Kinetics of zygote germination.
and zygotes was translated in nuclease-treated rabbit reticulocyte lysates (kindly provided by K. Hilse, Univ. of Freiburg)
using [35S]methionine (specific activity >30 TBq/mmol) as
label. The amount of radioactivity incorporated into protein
was determined by TCA precipitation (15), and these values
were used as a measure of the translational efficiency of the
RNA.
SDS-PAGE
Translation products were separated on 7.5 to 20% (w/v)
polyacrylamide (acrylamide: N'N'-methylene bisacrylamide
30:0.8 w/w) gradient gels with 5% (w/v) polyacrylamide stacking gels using the discontinuous buffer system of Laemmli
(8). Gels were prepared for fluorography by the method of
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WEGENER ET AL.
514
Kinetics of Zygote Germination
V
M.2M.. -IA-4
Germination of zygotes was induced by exposing zygote
suspensions in TAP-medium to light (30 gE m-2 s-'). The
kinetics of zygote germination in liquid are shown in Figure
2. Twenty-four h after induction of germination about 50%
of the zygotes had germinated and 80% by 80 h. The number
of zygotes germinated after 24 h was lower than that usually
observed when zygotes are generated in small quantities according to the procedure described by Levine and Ebersold
(9). The reduced germination observed can be accounted for
by the presence of zygote clusters (with up to 30 cells) among
the mature zygotes resuspended in liquid medium. Since
germination of individual members of a zygote cluster would
not be detected, our results are an underestimation of the
degree of germination.
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Disruption of Zygotes
143.
Figure 3. Translation of total cellular RNAs in a rabbit reticulocyte
lysate system. The RNA concentration in the assay mixes was 150
sg/mL. The translation products were separated by SDS-PAGE.
Equal amounts of radioactivity per slot were applied. The mixture of
14C-labeled mol wt standards used contained myosin (Mr 200,000),
phosphorylase b (Mr 92,500), bovine serum albumin (Mr 69,000),
ovalbumin (Mr 46,000), carbonic anhydrase (Mr 30,000), and lysozyme (Mr 14,300). Lane V shows the translation products from total
cellular RNA of vegetative cells. In lane Z, the translation products of
total cellular RNA from zygotes 3 h after induction of germination are
shown. The arrows indicate the major differences in proteins translated from the two RNA preparations.
Bonner and Laskey (2) and dried under
posure to Fuji x-ray film RX at -80'C.
vacuum
before
ex-
RESULTS AND DISCUSSION
Production of Mature Zygotes in Large Quantities
With the method described, we were able to reproducibly
2 to 3 x I09 zygotes from a mixture of about 8 x I09
gametes. With a mating efficiency of 80 to 90%, the yield of
mature zygotes was about 70% that expected. This reduction
in yield probably was due to losses during transfer to and
recovery from the plates. Pellicle transfer to plates was difficult
for two reasons: (a) The pellicles tended to stick to the walls
of the Erlenmeyer flasks prohibiting total transfer, and (b)
they had a tendency to form clumps when they were manipulated. Zygotes that stuck together in clumps remained green
and did not germinate (our unpublished observations and ref.
3). To obtain mature zygotes competent for germination, it
was essential to transfer the pellicles as a layer. Figure 1 shows
an agar plate onto which three pellicles had been transferred.
During maturation, the color of the zygotes changed from
green to orange-brown.
prepare
Plant Physiol. Vol. 90, 1989
Several methods were tested to achieve quantitative disruption of zygotes. Only two methods produced significant breakage of zygotes. Grinding with quartz sand in liquid nitrogen
for 3 h resulted in the disruption of about 30% of the zygotes.
The method of choice was the use of a Mikro-Dismembrator.
By the procedures outlined in "Materials and Methods,"
about 80% of the zygotes could routinely be disrupted.
RNA Isolation and in Vitro Translation
We tested several methods for RNA isolation in order to
obtain high quantities ofintact mRNA from Chlamydomonas
zygotes. In addition to absorbance measurements and agarose
gel electrophoresis, in vitro translation of the RNA in a rabbit
reticulocyte lysate was used as a crucial parameter for RNA
purity. The main problem of RNA extraction from zygotes
(but not from vegetative cells) was the elimination of contaminants that inhibited in vitro translation. Candidates for such
contaminants are acidic polysaccharides, which Jackson and
Hunt (6) reported to be strong inhibitors of translation. We
presume these polysaccharides to be components of the thick
zygote wall that were set free during cell disruption. The
inhibitory contaminants could not be removed by washing
with 3 M sodium acetat (7, 13) of phenol extracted and ethanol
precipitated nucleic acids. Such RNA was essentially untranslatable. The contaminants could partially be eliminated
by salt-ethanol-precipitations using 0.1 M sodium acetate (pH
6.0) and ethanol at a final concentration of 10 to 20% (17).
The pellets obtained by this method of precipitation were free
of nucleic acids. Zygote RNA purified in this way could be
translated in vitro, but only at an efficiency of about 10% of
that obtained with RNA from vegetative cells. The best results
were obtained when nucleic acids from zygote homogenates
were precipitated with LiCl after phenol extraction. Translatable RNA was routinely obtained by this method and the
translational efficiency of such zygote RNA preparations was
about 50% of that obtained with control RNA (using equal
RNA concentrations) from vegetative cells. By this extraction
procedure 1 x I09 zygotes yielded approximately 300 gg of
RNA and the preparations were essentially free of DNA. In
vitro translation of total RNA isolated from zygotes and
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C. REINHARDTII ZYGOTES FOR MOLECULAR AND BIOCHEMICAL ANALYSES
vegetative cells gave the protein patterns shown in Figure 3.
Although a few proteins may be common to both, most of
the proteins encoded by RNA from vegetative cells and zygotes were unique to each cell type. These results suggest that
the differences in mRNA populations between vegetative cells
and zygotes may reflect the differences in gene expression
from cells representing different stages of the Chlamydomonas
sexual cycle. Zygotes, which represent a resting form and
eventually will undergo germination and meiosis, can be
expected to express sets of genes not expressed in vegetative
cells. With the ability to obtain RNA from zygotes and
subsequent germination stages, an analysis of gene expression
during Chlamydomonas zygote maturation, germination and
meiosis, is now possible.
ACKNOWLEDGMENT
We thank F. Muller, T. Quayle, and W. Wakarchuk for critical
reading of this manuscript.
1.
2.
3.
4.
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