Download isolation and characterization of a cell wall

J. Phycol. 39, 1261–1267 (2003)
ISOLATION AND CHARACTERIZATION OF A CELL WALL-DEFECTIVE MUTANT
OF CHLAMYDOMONAS MONOICA (CHLOROPHYTA)1
Cesar Fuentes and Karen VanWinkle-Swift2
Department of Biological Sciences, Northern Arizona University, Flagstaff, Arizona 86011-5640, USA
Cell wall–defective strains of Chlamydomonas
have played an important role in the development
of transformation protocols for introducing exogenous DNA (foreign genes or cloned Chlamydomonas
genes) into C. reinhardtii. To promote the development of similar protocols for transformation of the
distantly related homothallic species, C. monoica, we
used UV mutagenesis to obtain a mutant strain with
a defective cell wall. The mutant, cw-1, was first
identified on the basis of irregular colony shape and
was subsequently shown to have reduced plating
efficiency and increased sensitivity to lysis by a nonionic detergent as compared with wild-type cells.
Tetrad analysis of crosses involving the cw-1 mutant
confirmed 2:2 segregation of the cw:cw+ phenotypes, indicating that the wall defect resulted from
mutation of a single nuclear gene. The phenotype
showed incomplete penetrance and variable expressivity. Although some cells had apparently normal
cell walls as viewed by TEM, many cells of the cw-1
strain had broken cell walls and others were protoplasts completely devoid of a cell wall. Several cw-1
isolates obtained from crosses involving the original
mutant strain showed a marked enhancement of the
mutant phenotype and may prove especially useful
for future work involving somatic cell fusions or
development of transformation protocols.
mutants of C. reinhardtii. Alternatively, the gametic
autolysin responsible for protoplast formation during
gametogenesis and mating in C. reinhardtii can be used
to strip the walls from vegetative cells and promote
uptake of exogenous transforming DNA (Kindle
1998). Although reports of successful transformation
of walled Chlamydomonas cells are available (Brown
et al. 1991, Dunahay 1993, Stevens and Purton 1997,
Kindle 1998), transformation efficiencies are typically
much higher when protoplasts are used.
Although C. reinhardtii is by far the most wellestablished model, other Chlamydomonas species, including C. moewusii, C. eugametos, and C. monoica, have
provided insights into various aspects of sexual
reproduction and organelle genome structure and
inheritance (Harris 1989). However, further development of these models has been hampered by the absence
of cell wall mutants (or easily purified gametic autolysins)
for the development of transformation protocols.
Our work has focused on the homothallic species
C. monoica because of the species’ unique advantages for the study of sexual development, especially
zygospore formation. Among the large collection of
sexual cycle mutants obtained in C. monoica are those
showing altered mating (VanWinkle-Swift and Bauer
1982, VanWinkle-Swift and Hahn 1986, VanWinkleSwift and Thuerauf 1991), gamete fusion (VanWinkleSwift et al. 1987, Shi 1995), or zygospore formation
(VanWinkle-Swift et al. 1998) as well as those carrying
mutations that affect the uniparental inheritance of
chloroplast genes (VanWinkle-Swift and Salinger 1988,
VanWinkle-Swift et al. 1994). These unique developmental mutants could be used to understand the processes contributing to zygospore morphogenesis and
organelle inheritance, if cloning of the genes by transformation and complementation of mutant strains were
possible. This may depend on the successful isolation of
a wall-deficient C. monoica strain. (The sexual life cycle
of C. monoica does not involve the formation of naked
gametes; thus, autolytic enzymes cannot be recovered
from the culture medium after mating.)
Until now, cell wall–defective mutants have been
described only for C. reinhardtii (Davies and Plaskitt
1971, Hyams and Davies 1972) and the separate natural
isolate C. smithii (Matagne and Beckers 1987). With
the long-term goal of demonstrating transformation of
C. monoica and cloning developmental genes by
complementation, we have attempted to recover a
wall-defective mutant after mutagenesis with UV
radiation. We report here the isolation and characterization of a mutant strain, cw-1, that shows increased
Key index words: cell wall; Chlamydomonas monoica;
mutant; protoplast; transformation
Chlamydomonas, and in particular C. reinhardtii, has
become established as a model system for studying
many basic biological phenomena, including flagellar
structure and function, organelle biogenesis, photosynthesis, and fundamental aspects of Mendelian and
non-Mendelian inheritance (Harris 1989). Although
earlier studies used classical genetic, biochemical, and
cell biological tools, molecular genetic approaches have
now become routine, including the transformation of
mutant strains obtained from earlier classic genetic
searches. The integration of molecular genetics continues to enhance the value of the Chlamydomonas model
(Harris 2001).
Effective transformation protocols in Chlamydomonas
often take advantage of the availability of wall-defective
1
Received 9 May 2003. Accepted 5 August 2003.
Author for correspondence: e-mail karen.vanwinkle-swift@
nau.edu.
2
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CESAR FUENTES AND KAREN VANWINKLE-SWIFT
cell lysis during routine manipulation or when exposed
to non-ionic detergent, reduced plating efficiency,
and altered cell wall integrity when viewed by
TEM. The mutant phenotype, which is enhanced at
lower temperatures and in certain genetic backgrounds, is the consequence of a single Mendelian
gene mutation.
MATERIALS AND METHODS
Strains and culture conditions. The wild-type strain of
C. monoica Strehlow used in this study was obtained from
the University of Texas Culture Collection of Algae (UTEX
220). A wall-defective mutant clone 10A3-1 (referred to
hereafter as cw-1) was obtained from UTEX 220 after mutagenesis with UV light. For genetic analysis the cw-1 mutant
was crossed to each of three zygote maturation mutants, zym6, zym-13, and zym-27. Several tetrad products (designated 1a,
2b, and 1c) carrying the cw-1 mutant allele were obtained
from these crosses and maintained for further phenotypic
characterization. Tetrad product 1c also carries the zym-27
mutant allele. All strains were maintained as vegetative stocks
on solid HS medium under continuous cool white fluorescent
illumination (80 mmol photons m 2 s 1) at 201 C.
Mutagenesis. Vegetative cells of UTEX 220 were suspended in 1 mL sterile HS medium (Sueoka 1960) to a final
cell density of approximately 5 106 cells mL 1. Several 30mL aliquots of the suspension were plated onto solid BM
medium (Bischoff and Bold 1963) supplemented with 1%
(w/v) soluble potato starch (as a potential osmotic protectant
for wall-defective mutants). The plated samples were irradiated for 10, 20, 30, and 40 s using a 8-W germicidal UV
lamp at a distance of 15 cm from the agar surface. Irradiated
plates were placed in darkness overnight to prevent photoreactivation. The plates were then returned to continuous
illumination for 5–7 days at 201 C. Postmutagenesis populations were then scraped from the irradiated plates and were
suspended in HS medium, diluted appropriately, and plated
onto solid BM medium (supplemented with soluble potato
starch) to obtain isolated postmutagenesis clones. Approximately 10,000 colonies were screened for abnormal colony
morphology after allowing at least 14 days of vegetative
growth. Colony morphology was determined by dissection
microscope. Twenty-two colonies showing unusual irregular
outlines were selected for further analysis. One colony (10A31; cw-1) derived from a 10-s UV light exposure showed an
amoeboid colony shape with extensive cell lysis along the
colony periphery.
Genetic analysis. The cw-1 mutant was crossed to each of
three zygote maturation mutants, zym-6, zym-13, and zym-27,
following procedures described by VanWinkle-Swift and
Burrascano (1983). Cells were mated in LPN medium for 7
days. To reduce the frequency of cw-1 self-mating, mating
induction was initiated using a 1:2 ratio of cw-1:zym cells.
Although this increases the frequency of zym self-matings,
these matings do not produce viable zygotes. Thus, the
frequency of heterozygotes among the viable zygotes recovered is increased. Each tetrad product recovered from the
crosses was self-mated and tested for the presence of the zym
markers as described by VanWinkle-Swift and Burrascano
(1983). The 2:2 segregation of zym:zym þ alleles in tetrads
verified that the tetrad was derived from crossing rather than
cw-1 selfing. The cw-1 phenotype was scored on the basis of
colony morphology and peripheral cell lysis. Tetrads were
classified as parental ditype, nonparental ditype, or tetratype with regard to the cw-1 and zym markers. Linkage
(% recombination) was determined using the formula: 1/2
tetratype þ nonparental ditype/parental ditype þ nonparental
ditype þ tetratype 100. The zym-6 and zym-13 alleles are
centromere-linked markers. Accordingly, the distance of cw-1
from its centromere could be calculated as 1/2% tetratype for
cw-1/zym-6 or cw-1/zym-13.
Phenotypic characterization of the cw-1 mutant. The original
cw-1 mutant clone, several cw-1 tetrad products (see genetic
analysis above), and UTEX 220 wild-type cells were assayed
for growth rates in liquid HS medium at 201 C and in HS
medium supplemented with sucrose (50 mM). Cultures were
inoculated at a starting cell density of 1 105 cells mL 1. To
minimize damage to wall-deficient cells, cultures were neither
shaken nor aerated. Hemacytometer counts were taken daily
for 10 days, and doubling times were calculated during the
exponential phase of growth (between days 2 and 6). The
number of doublings occurring over a period of 3–4 days was
calculated according to the formula d 5 log10 ending cell
density – log10 starting cell density/0.301.
The original cw-1 mutant clone, cw-1 tetrad products, and
UTEX 220 wild-type cells were tested for plating efficiency.
Strains were suspended in 1 mL of HS liquid medium and were
cultured under standard conditions at 201 C for 1–5 days. Four
10-fold dilutions were prepared, and a 10-mL aliquot of each
dilution was dropped onto modified HS solid medium (see
below). Cells in the undiluted cultures were counted by
hemocytometer to estimate the number of colonies expected
after each dilution. In a separate experiment designed to test
for temperature effects on cell viability, stock plates of each
strain were prepared by streaking cells onto HS solid medium.
The strains were grown at 15, 20, and 251 C for 2–3 days before
testing. Cells were then suspended in 1 mL HS liquid, and cell
densities were determined by direct hemocytometer count.
Serial dilutions were prepared and plated at 15, 20, and 251 C.
Recently, we observed reduced plating efficiencies on agarsolidified medium. Therefore, our assays of plating efficiency
used HS medium supplemented with additional calcium
chloride (25 mg L 1) and magnesium sulfate (75 mg L 1)
and solidified with Gel-Gros (ICN Biochemicals, Cleveland,
OH, USA). The higher divalent cation concentration is needed
to improve the gelation of HS medium. Supplementation of
media with soluble potato starch was discontinued because no
consistent protective effect was observed. To test for potential
temperature sensitivity of the cw-1 phenotype, all strains were
tested for colony formation at all three test temperatures,
regardless of the growth temperature maintained before
testing. Plating efficiency was determined by dividing the
visible colony count by the predicted number based on direct
hemocytometer count (adjusted by the dilution factor).
Vegetative cells of the original cw-1 clone and derived tetrad
products were tested for sensitivity to lysis by the non-ionic
detergent NP-40. Results were compared with UTEX 220 wildtype cells. Cells grown in liquid HS culture medium were
sampled and counted by hemocytometer. A second sample of
the same volume (200 mL) was diluted 1:1 with 1% (w/v) NP-40
and counted after a 10-min incubation period. The percent
lysis was calculated according to the formula %L 5 [1–2 (cell
count of detergent treated sample)/control cell count] 100.
TEM. TEM of wild-type UTEX 220 cells and cells of the
original cw-1 mutant was performed as described by
VanWinkle-Swift and Rickoll (1997), with the exception that
the cells were immobilized after glutaraldehyde fixation in 3%
sodium alginate (w/v) rather than in agarose. The alginate
beads were then solidified by incubation in cold 30 mM CaCl2
for 30 min. Secondary fixation was in 1% osmium tetroxide,
dehydration in a graded ethanol series, and embedment in
Spurr’s low viscosity resin, as described by VanWinkle-Swift
and Rickoll (1997). Grids were double-stained in ethanolic
uranyl acetate and aqueous lead citrate before viewing with a
transmission electron microscope (model 1200 EX, JOEL,
Peabody, MA, USA) operated at 60 kV.
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C. MONOICA CELL WALL MUTANT
RESULTS
Isolation and preliminary phenotypic characterization of
the cw-1 mutant. From more than 10,000 postmutagenesis clones obtained after UV mutagenesis of the
wild-type UTEX 220 strain, only one clone, derived
from a 10-s UV, dose showed a consistent phenotype
indicative of a cell wall defect. Wild-type C. monoica
cells produce spherical colonies with well-defined
edges (Fig. 1a). Cells of the mutant clone produced
flattened amoeboid colonies with irregular outlines, similar to that described for cw mutants of
C. reinhardtii (Hyams and Davies 1972). Lysis of cells
at the periphery of the colony was also evident (Fig.
1b), and manipulation of cells on the agar surface
resulted in further extensive lysis.
Growth rates for the cw-1 strains (the original isolate
and tetrad products derived from the crosses described
below) were variable with some strains showing shorter
doubling times than wild-type cells, whereas others
grew more slowly. The doubling time for standing
cultures of wild-type cells was 41 h. The original cw-1
mutant and tetrad product 1a showed doubling times
of 47 h, whereas tetrad products 2b and 1c showed
doubling times of 32 h. Addition of sucrose (50 mM) to
the growth medium did not improve growth rates; in
fact, doubling times were increased by 1–10 h depending on strain (data not shown).
Cell morphology also varied greatly among cw-1
strains (Fig. 2). cw-1 strains showing more rapid growth
rates and higher stationary cell densities were smaller
and more spherical than wild-type cells or the original
cw-1 clone.
Detergent sensitivity, a characteristic of C. reinhardtii
cw mutants (Harris 1989), was also characteristic of the
C. monoica cw-1 mutant strain, although considerable
strain-to-strain variation in the percentage of cells
undergoing detergent-induced lysis was detected
(Table 1). Repeated testing also revealed considerable
within-strain variation in the percentage of lysis for
some strains (note the large SDs in Table 1). Withinstrain variation showed no consistent correlation with
stage in the growth cycle, and subcloning did not
reduce the variability (data not shown). This suggests
that the absence of complete detergent induced-lysis
within a culture reflects variable expressivity of the
mutant allele rather than an accumulation of wild-type
revertant cells within the original mutant clone.
In general, cw-1 strains showing increased detergent
lysis also showed reduced plating efficiency (Table 1).
An effect of temperature on plating efficiency was also
observed (Fig. 3). Both strains tested (the original
isolate and tetrad product 2b) showed improved
plating efficiency when pregrown and plated at higher
temperature (251 C; Fig. 3). These strains were also
more resistant to detergent lysis when grown at 251 C
rather than 201 C (data not shown).
Genetic analysis. Tetrads were dissected and analyzed from crosses between the original cw-1 isolate
and each of three marked strains carrying Mendelian
recessive zygotic lethal mutations (zym-6, zym-13, and
zym-27). In each of these crosses, some tetrads were
derived from self-mating within the cw-1 strain and
showed 4:0 segregation for the cw-1 allele. Thus, the
cw-1 mutation appears to have no effect on the
zygospore wall because homozygosity for cw-1 did not
affect zygospore viability. For all tetrads in which the
zym markers showed 2:2 segregation, the cw-1
phenotype (abnormal colony morphology) segregated 2:2 and showed no linkage to the zym-6 or
TABLE 1. Detergent and plating sensitivity of cw-1 strains.
Strain
FIG. 1. Chlamydomonas monoica colony morphology. (a) Wildtype cells produce round colonies with well-defined edges.
(b) Cells of the cw-1 mutant strain produce smaller flattened
colonies with irregular edges resulting from spontaneous lysis of
cells along the periphery (arrow). Colonies were photographed
on thin agar at 400 using a standard Zeiss ( Jena, Germany)
phase-contrast microscope. Scale bar, 30 mm.
Wildtype
UTEX220
cw-1
10A3-1
1a
2b
1c
Detergent-induced cell lysis (%)
Plating efficiency (%)
070
94711
42715
7879
72728
9272
26722
2279
1179
271
Data are based on 3–7 independent tests on each strain.
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CESAR FUENTES AND KAREN VANWINKLE-SWIFT
FIG. 2. Variation in cell size and shape among cw-1 isolates. (a) Wild-type strain UTEX 220. (b) Original cw-1 mutant isolate 10A3-1.
(c) cw-1 tetrad product 1a. (d) cw-1 tetrad product 2b. (e) cw-1 zym–27 tetrad product 1c. Scale bar, 10 mm.
zym-13 markers (Table 2). Thus, the cw-1 mutation
marks a single Mendelian gene. Because these zym
alleles are also centromere-linked markers, the
percentage of tetratype tetrads in crosses between
cw-1 and zym-6 or zym-13 gives a measure of the
distance of cw-1 from its centromere. The data indicate that cw-1 is not closely linked to its centromere.
The cw-1 tetrad products 2b and 1a from the cross to
zym-13 showed an enhanced cw-1 phenotype and were
saved for further analysis (see earlier sections). Most
tetrads dissected from the cross between cw-1 and zym27 were derived from cw-1 self-matings. However in
seven tetrads showing 2:2 segregation for the zym-27
and zym-27 þ alleles, the cw-1 and cw-1 þ alleles also
segregated 2:2. Tetrad product 1c, with the genotype
cw-1 zym-27, was retained for further analysis (see
earlier sections).
Ultrastructural analysis. The wild-type UTEX 220
strain and the original cw-1 isolate were analyzed by
TEM. Wild-type cells were surrounded by an intact
FIG. 3. Temperature effects on plating efficiencies for wildtype and cw-1 strains. (a) Between-strain temperature effects.
(b) Within-strain temperature effects. Strains suspended and
plated at a particular temperature were maintained on stock
plates at the same temperature for 3 days before testing.
well-defined cell wall (Fig. 4a), including a surface
layer with a highly ordered crystalline structure (Fig.
4b). In cw-1 mutant cells, wall ultrastructure varied
from cell to cell with some cells having apparently
normal intact walls (Fig. 5a), whereas others had
numerous breaks and discontinuities in the wall (Fig.
5b). A small proportion of the cells examined were
naked protoplasts completely devoid of the vegetative
cell wall (Fig. 5, c and d). Thus, in classic genetic
terms, the cw-1 allele shows incomplete penetrance
(i.e. some cells carrying the cw-1 allele show no
apparent defect) and variable expressivity (i.e. among
FIG. 4. The wild-type UTEX220 cell wall. (a) An intact wall
surrounds the entire cell. Scale bar, 2 mm. (b) The highly ordered
crystalline lattice of the normal cell wall is apparent at higher
magnification. Scale bar, 500 nm.
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C. MONOICA CELL WALL MUTANT
TABLE 2. Tetrad analysis of crosses involving the cw-1
mutant.
Tetrad class
Marker pair
PD
NPD
TT
% Recombination
% TT
cw-1/zym-6
cw-1/zym13
5
7
2
5
13
11
42.5
45.6
65
48
PD, parental ditype; NPD, nonparental ditype; TT, tetratype; 12% tetratype estimates the cw-1—centromere distance in
map units.
those cells showing wall defects, the extent of the
defect varies). This incomplete penetrance and variable
expressivity is also apparent from the data on plating
efficiency and detergent sensitivity.
DISCUSSION
We describe here the isolation of a cell wall–defective
strain of C. monoica that shows altered cell wall ultrastructure, reduced plating efficiency and enhanced
sensitivity to lysis by non-ionic detergent. The severity
of the phenotype is affected by temperature and by
genetic background. When the original mutant clone,
derived from the UTEX 220 wild-type strain, is grown
at 251 C, the mutant phenotype is barely visible. The
cw-1 tetrad products 1a and 2b used in this study were
derived from crosses to zygote maturation mutants that
had been isolated in a different wild-type background
(wt15c derived from the C. monoica strain maintained in
the Cambridge Culture collection; see VanWinkle-Swift
and Burrascano 1983). Although these cw-1 products
do not carry the zym mutant alleles, other unidentified
FIG. 5. Variable wall ultrastructure in individual cells of the cw-1 mutant. (a) A cw-1 cell with an intact cell wall. The plasma membrane
is not closely affixed to the wall, and amorphous material and vesicles accumulate in the periplasmic space. Scale bar, 2 mm. (b) A cw-1 cell
with a broken outer cell wall layer. Fibrous material as well as amorphous material accumulates in the enlarged periplasmic space. Scale
bar, 1 mm. (c) A cw-1 protoplast devoid of cell wall. Amorphous material and vesicles appear in the region separating the protoplast from
the alginate embedding medium. Scale bar, 500 nm. (d) A cw-1 protoplast showing release of vesicles from the naked plasma membrane.
Scale bar, 500 nm.
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CESAR FUENTES AND KAREN VANWINKLE-SWIFT
allele(s) introduced by the cross appear to enhance the
cw-1 phenotype. Thus, the recognition of a cw
phenotype (i.e. the successful isolation of a walldefective mutant) may be greatly influenced by the
choice of genetic background as well as the growth
conditions used after mutagenesis.
The cell wall–defective mutants of C. reinhardtii have
been divided into three phenotypic classes: those
producing little if any wall material, those producing
wall material detached from the plasma membrane,
and those producing wall material attached to the
plasma membrane (Davies and Plaskitt 1971). Our
ultrastructural studies on the cw-1 C. monoica mutant
indicate that all three of these phenotypes can be
produced within a single mutant strain.
Davies and Plaskitt (1971) also suggested that cell
wall biogenesis involves at least two levels of control:
synthesis of the wall precursors as dictated by nuclear
gene expression and the three-dimensional assembly
of the wall precursors after secretion. Furthermore, the
composition of the culture medium can affect the
assembly of the cell wall in cw mutants of C. reinhardtii
(Davies and Lyall 1973). For our C. monoica cw-1
isolates, the proportion of cells exhibiting a particular
phenotype within a given strain appears to be controlled both by genetic background and by environmental conditions. These influences may reflect the
distinct levels of control suggested by Davies and
Plaskitt (1971).
Cell wall composition and ultrastructural details,
although not known in C. monoica, are well characterized in C. reinhardtii. In this species, the wall includes a
salt-soluble outer layer made up of at least four
glycoproteins (three of which are hydroxyproline-rich)
that assemble into a crystalline lattice (Goodenough
et al. 1986). The glycoproteins of the inner wall layers
are covalently cross-linked, making these layers insoluble and more difficult to analyze (Woessner and
Goodenough 1994). As viewed by standard TEM, the
surface layer(s) of the C. monoica cell wall also displays a
regular crystalline pattern reminiscent of the C. reinhardtii cell wall. Although not as well characterized, the
cell wall of C. moewusii (a species more closely related to
C. monoica) is also a multilayered structure (Horne et al.
1971).
The cold sensitivity of the C. monoica cw-1 mutant
suggests that the defect may involve wall assembly
rather than an enzymatic process. Although not
reported in the literature, we have found that the
cw-15 mutant of C. reinhardtii—like cw-1 of C. monoica—
is cold sensitive, showing greatly reduced plating
efficiency at lower temperatures (data not shown).
Loss or alteration of a particular structural protein
component would be expected to affect assembly of
one or more wall layers. However, studies on several
classes of cw mutants of C. reinhardtii that differ in the
amount of cell wall produced or in the extent of
attachment between the cell wall and the plasma
membrane revealed no differences in the amounts
or patterns of intracellular synthesis of cell wall
precursors (Voigt et al. 1997). These observations
suggest that the C. reinhardtii cw mutants are also
assembly mutants.
The availability of the cw-1 mutant and the derived
cw-1 strains showing enhanced mutant phenotypes
opens the door to a variety of experiments that were
not heretofore possible with C. monoica. In particular,
the strains will be useful for the production of vegetative diploids by somatic cell fusion as in C. reinhardtii
(Matagne et al. 1979) and for the development of
transformation protocols. Vegetative diploids can be
used to evaluate dominance relationships between
alleles of genes of interest and to extend our studies
on chloroplast gene inheritance. The development
of transformation protocols, involving both the expression of foreign genes and the complementation
of interesting mutant phenotypes by the introduction of cloned C. monoica gene sequences, will be a
central part of our continuing work with this unique
species.
This research was supported by grant R25-GM56931 from the
National Institutes of Health and by grant MCB-9728461 from
the National Science Foundation. We thank Marilee Sellers for
providing excellent training in TEM techniques.
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