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40 Hermand, D. et al. (2001) Specificity of cdk
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41 Yamaguchi, M. et al. (1998) A rice homolog of
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42 Yamaguchi, M. et al. (2000) Activation of CDKactivating kinase is dependent on interaction with
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Biotechnol. 11, 138–145
Functional genomics of plant
photosynthesis in the fast lane using
Chlamydomonas reinhardtii
Rachel M. Dent, Miehie Han, and Krishna K. Niyogi
Oxygenic photosynthesis by algae and plants supports much of life on Earth.
Several model organisms are used to study this vital process, but the unicellular
green alga Chlamydomonas reinhardtii offers significant advantages for the
genetic dissection of photosynthesis. Recent experiments with
Chlamydomonas have substantially advanced our understanding of several
aspects of photosynthesis, including chloroplast biogenesis, structure–function
relationships in photosynthetic complexes, and environmental regulation.
Chlamydomonas is therefore the organism of choice for elucidating detailed
functions of the hundreds of genes involved in plant photosynthesis.
Rachel M. Dent
Miehie Han
Krishna K. Niyogi*
Dept of Plant and
Microbial Biology,
University of California,
Berkeley, CA 94720-3102,
USA.
*e-mail: niyogi@
nature.berkeley.edu
With the genome sequence of the first photosynthetic
eukaryote fully characterized, the path appears set
for Arabidopsis to dominate the field of plant biology
in the next decade. But Arabidopsis is not the ideal
organism for all fields of plant research, and
photosynthesis is one area where other models have
major advantages (Table 1). Chlamydomonas
reinhardtii (Fig. 1), for example, has been used as a
model organism in photosynthesis research for
>40 years, and the use of this unicellular green alga
in biochemical, biophysical and genomic approaches,
to the study of photosynthesis and photoprotection
has been reviewed by several authors recently1–7.
This review describes several examples of how
molecular genetic studies of Chlamydomonas have
http://plants.trends.com
provided new insights into photosynthesis. We will
highlight the comparative merits of Chlamydomonas
as a model photosynthetic organism and discuss how
it can make future contributions to the functional
genomics of photosynthesis.
Advantages of Chlamydomonas for studying
photosynthesis
Chlamydomonas has several attributes that make it
an excellent organism for basic genetic studies of
plant photosynthesis (Table 1). Its photosynthetic
apparatus is closely related to that of vascular
plants, and it is also a eukaryote, with
photosynthesis genes encoded by both the nuclear
and chloroplast genomes. As a unicellular organism,
Chlamydomonas has the advantages of a microbial
lifestyle without the complications of
multicellularity. Synchronous or asynchronous
cultures of Chlamydomonas grow quickly with a
doubling time of less than ten hours, and the cells
behave homogeneously in terms of physiological and
biochemical characteristics. Because
Chlamydomonas is haploid and has a controlled
sexual cycle with the possibility of tetrad analysis
(Fig. 2), it is an excellent genetic model.
1360-1385/01/$ – see front matter © 2001 Elsevier Science Ltd. All rights reserved. PII: S1360-1385(01)02018-0
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365
Table 1. Comparison of model photosynthetic organisms
Synechocystis PCC6803
Chlamydomonas
Arabidopsis
Maize
Unicellular prokaryote
Unicellular eukaryote
Multicellular eukaryote
Multicellular eukaryote
Rapid growth rate (doubling
time <10 h)
Rapid growth rate (doubling
time <10 h) and life cycle
(<2 weeks)
Complete life cycle takes
6 weeks
Complete life cycle takes at
least 4 months
Oxygenic photosynthesis;
Oxygenic photosynthesis;
heterotrophic growth possible heterotrophic growth
possible with normal
assembly of photosynthetic
machinery in the dark
Oxygenic photosynthesis;
heterotrophic growth
possible at seedling stage
Oxygenic photosynthesis;
heterotrophic growth
possible at seedling stage
Transformation by
homologous recombination
Transformation of nuclear
genome; transformation of
chloroplast genome has
low success rate
Transformation of nuclear
genome only
Transformation of nuclear,
mitochondrial and chloroplast
genomes; homologous
recombination in chloroplast
and mitochondrial genomes
Segregation of wild-type
Haploid, therefore immediate Diploid (possible ancestral
Ancestral tetraploid, therefore
copies of the circular genome expression of nuclear mutant tetraploid), therefore
recessive mutations not
required for expression of
phenotype
recessive mutations not
expressed in heterozygotes
loss-of-function mutant
expressed in heterozygotes
phenotypes
Low level of functional overlap Degree of functional overlap
not known
High degree of functional
overlap with many genes
belonging to gene families
Replica plating for large-scale
screening
Replica plating for large-scale Field space required for largescreening not possible
scale screening
Replica plating for large-scale
screening
High degree of functional
overlap
Tetrad analysis not applicable Tetrad analysis possible
because no sexual
reproduction
Tetrad analysis possible, but
only in specific genetic
backgrounds (e.g. the qrt
mutant)
Tetrad analysis not possible
Genome size = 3.5 Mbp
Genome size = 100–160 Mbp
Genome size = 120 Mbp
Genome size = 2500 Mbp
Genome sequenced
Limited sequence available,
Genome sequenced
but sequencing projects have
been initiated
However, the most valuable characteristic that
Chlamydomonas possesses with respect to the study
of photosynthesis is its ability to grow
heterotrophically using acetate as a sole carbon
source. This allows the isolation of viable mutants
that are unable to perform photosynthesis, and lightsensitive mutants can be maintained in complete
darkness (Fig. 2). Plants such as Arabidopsis or maize
with mutations that affect photosynthesis are often
inviable or at least susceptible to light-induced
photo-oxidation, which can complicate biochemical
analyses. Unlike angiosperms, Chlamydomonas can
synthesize chlorophyll and assemble a complete
photosynthetic apparatus in the dark. It is therefore
possible to perform detailed biochemical or
biophysical studies of dark-grown nonphotosynthetic
and/or light-sensitive mutants. Nonphotosynthetic
mutants of other green algae, such as Chlorella and
Scenedesmus, and the cyanobacterium Synechocystis
PCC6803, can also be isolated and maintained in the
dark, but genetic analysis is not possible with these
organisms because of the lack of a known sexual cycle.
Synechocystis has the advantage of a completely
sequenced genome8 and photosynthetic reaction
centers that are highly similar to those of plants,
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Limited sequence available,
but sequencing projects have
been initiated
making it an especially useful organism for studying
photosystem II and I (PSII and PSI). The limitations
of Synechocystis as a plant model include differences
in its light-harvesting system and oxygen-evolving
complex, and, because the cytochrome b6 f complex is
part of both the photosynthetic and respiratory
electron transport chains in cyanobacteria, mutants
severely affected in this complex cannot be recovered.
Chlamydomonas is the only known eukaryote in
which the nuclear, chloroplast and mitochondrial
genomes can all be transformed, and studies of
photosynthesis have made extensive use of this.
Chloroplast transformation is achieved using particle
bombardment and occurs by homologous
recombination, simplifying chloroplast gene
knockouts and site-directed mutagenesis1. Chloroplast
transformation is not currently practical in
Arabidopsis9 or maize: it is possible in tobacco10, but
the nuclear genetics are not as easy and rapid to
perform in tobacco as they are in Chlamydomonas or
Arabidopsis. Because Chlamydomonas contains a
single chloroplast, the generation of homoplasmic
strains is simplified compared with plants: a single
plant mesophyll cell can contain hundreds of
chloroplasts. The bacterial aadA gene encoding
366
Fig. 1. The morphology of
Chlamydomonas
reinhardtii.
(a) Ultrastructure of the
Chlamydomonas cell,
showing the central
nucleus (N) with
nucleolus (Nu),
surrounded by the cupshaped chloroplast (C)
containing thylakoid
membranes (T), starch
grains (S) and pyrenoid
(P), within the stroma (St).
An eye-spot (ES) is
positioned against the
inner envelope
membrane of the
chloroplast. Two flagella
(F) project from the apical
region of the cell, and
vacuoles (V) might also be
visible in the cytoplasm.
(b) Chlamydomonas cells
growing in liquid culture,
viewed under 500×
magnification using
Smith DIC optics. A single
Chlamydomonas cell is
~10 µm in diameter.
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TRENDS in Plant Science Vol.6 No.8 August 2001
Transformation of the Chlamydomonas nuclear
genome can be accomplished using either endogenous
or foreign selectable marker genes. The expression of
foreign genes in Chlamydomonas has been a
longstanding problem, but this has been overcome by
the use of strong endogenous promoters, such as that
of RBCS2, and by the introduction of
Chlamydomonas introns within the recombinant
sequence14. In contrast with chloroplast
transformation, nuclear transformation results in
random insertion of recombinant DNA. Insertional
mutagenesis has often been used in forward genetic
studies of photosynthetic processes. Highly efficient
nuclear transformation has also been used to clone
genes by the complementation of existing mutations.
Recent successes in identifying, cloning and
analyzing novel genes involved in photosynthesis
using these techniques are apparent in the studies
described below. Although these examples focus on
the light reactions of photosynthesis, it should be
noted that Chlamydomonas has also proved useful in
studies of other aspects of photosynthesis, such as
starch metabolism15.
(a)
F
C
ES
V
M
S
Nu
St
N
T
P
Biogenesis of the photosynthetic apparatus
(b)
F
ES
S
C
TRENDS in Plant Science
spectinomycin resistance has been the most widely
used selectable marker for chloroplast
transformation11, but recently a new series of vectors
have been developed12 for the targeted insertion and
expression of foreign genes that are based on the
bacterial gene aphA-6. As with Chlamydomonas
chloroplasts, Synechocystis PCC6803 can also be
transformed easily by homologous recombination.
Site-directed mutagenesis has therefore been used
extensively in the study of structure–function
relationships in the reaction centers of this organism13.
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The ability to transform both the nuclear and
chloroplast genomes of Chlamydomonas has
revolutionized the study of chloroplast biogenesis and
the role of nuclear genes in chloroplast gene
expression. Protein subunits of the photosynthetic
complexes within the thylakoid membrane are
encoded by both the nuclear and chloroplast genomes
(Fig. 3). A complex crosstalk between the two separate
genetic compartments is therefore required to ensure
the correct subunit and photosystem stoichiometry.
Whereas induction of nuclear gene expression is often
controlled at the transcriptional level, mounting
evidence suggests that chloroplast gene expression is
primarily regulated by post-transcriptional events
that are under the control of nucleus-encoded
factors16. Mutants with defects in these processes are
generally nonphotosynthetic and light sensitive, and
exhibit high chlorophyll fluorescence, enabling easy
screening of mutant populations (Fig. 2). Pulselabelling experiments can also be performed routinely
with Chlamydomonas to test whether a mutation
affects RNA or protein synthesis or stability in vivo.
The NAC2 gene is a good example of a nuclear gene
controlling chloroplast gene expression. It is involved
specifically in the control of the half-life of the
chloroplast psbD mRNA encoding the D2 reaction
center polypeptide of PSII (Ref. 17). By chloroplast
transformation and mutagenesis using chimeric
psbD mRNAs, two cis-elements have been identified
within the 5′UTR that are required for psbD RNA
stabilization18. There is also evidence to suggest a
close connection between the processes of RNA
stabilization, 5′-end maturation and translation
initiation. Additional support for the interconnection
of these processes has been provided by recent
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(b)
(c)
Chlorophyll fluorescence
(a)
TRENDS in Plant Science Vol.6 No.8 August 2001
Tetrads
Parental
strains
WT
lts5
WT
lts5
WT
lts5
TRENDS in Plant Science
Fig. 2. Genetic analysis of a nonphotosynthetic Chlamydomonas mutant. The mutant (lts5) was
isolated in a screen for mutants that do not grow in the presence of light, and crossed with the wildtype strain. The phenotypes of the parental strains and two tetrads when grown (a) in the dark, or (b)
in low light conditions. (c) False-color digital image of chlorophyll fluorescence showing that darkgrown lts5 also has a phenotype of high chlorophyll fluorescence compared with the wild type. The
analysis shows that the light sensitivity and the chlorophyll fluorescence phenotypes are the result of
a mutation at a single nuclear locus.
cross-linking experiments showing that a 40 kDa
protein interacts specifically with a U-rich translation
element in a NAC2-dependent manner19.
The NAC2 gene was cloned by complementation of
the nac2 mutation with a cosmid library20, and found
to encode a hydrophilic polypeptide with nine
tetratricopeptide-like repeats (TPRs) in its C-terminal
domain. TPR motifs might coordinate the assembly of
proteins into multisubunit complexes, and indeed the
Nac2 protein was shown to be part of a high molecular
weight complex associated with non-polysomal RNA.
RNA binding by Nac2 has not been shown, therefore
Nac2 might bind RNA via an additional unidentified
subunit. Candidates for this role include three
independent nuclear products that have been
genetically identified as suppressors of psbD 5′UTR
mutations that destabilize psbD mRNA21. This
suppressor analysis of nonphotosynthetic mutants, a
powerful approach for identifying interacting genes, is
easily applied using Chlamydomonas. Although
nonphotosynthetic mutants can be isolated using
plant models such as Arabidopsis or maize, their
mutants can only be propagated as heterozygotes,
making suppressor analysis much more difficult.
The mechanism of control of psbD RNA stability is
shared perhaps by other chloroplast mRNAs. The
MBB1 gene, which encodes a Nac2 homolog, has
recently been isolated22 and shown to be required for
psbB mRNA accumulation23. Mbb1 also functions as
part of a high molecular weight complex, and although
Mbb1 and Nac2 are different sizes, it is possible that
these two specific complexes might recruit common
http://plants.trends.com
367
factors via their TPR domains. This process might not
be limited to Chlamydomonas because the maize
Crp1 protein, which is needed for chloroplast RNA
processing, is also part of a high molecular weight
complex and contains pentatricopeptide repeats
related to the TPR repeats24.
Structure–function analysis of photosystem I
Nuclear and chloroplast transformation of
Chlamydomonas can be coupled with sophisticated
spectroscopic measurements in the study of
structure–function relationships within
photosynthetic complexes such as PSI. PSI functions
as a light-driven oxidoreductase that transfers
electrons from plastocyanin or cytochrome c6 (in the
thylakoid lumen) to ferredoxin (in the stroma), which
in turn generates NADPH. The Chlamydomonas PSI
consists of eight nucleus-encoded polypeptides (PsaD,
PsaE, PsaF, PsaG, PsaH, PsaK, PsaL and PsaN) and
at least six chloroplast-encoded polypeptides (PsaA,
PsaB, PsaC, PsaI, PsaJ and PsaM) (Fig. 3). PsaA and
PsaB are related proteins that form the heterodimeric
reaction center core of PSI. Structural studies of PSI
and PSII have revealed striking similarities in the
arrangement of transmembrane helices that bind
symmetrical cofactor branches25.
To study the oxidizing side of PSI, insertional
mutagenesis was used to generate a mutant in the
nuclear psaF gene26. Analysis of the mutant showed
that PsaF is important for the docking of plastocyanin
to PSI. The positively charged N-terminal domain of
PsaF in Chlamydomonas and plants is not present in
cyanobacteria, and it has been hypothesized that the
evolution of this domain allowed the formation of a
stable complex between plastocyanin and PSI, and
thus fast electron transfer6. Site-directed
mutagenesis of PsaF has illustrated that a single
amino acid (K23) might represent a specific
recognition site for the interaction of plastocyanin
with PSI (Ref. 27). The interaction of PsaF with
plastocyanin might also involve the small
hydrophobic subunit PsaJ. Although PsaJ could be
cross-linked to PsaF, targeted mutation of the
chloroplast psaJ gene does not affect the level of PsaF
in isolated PSI particles28. However, flash-absorption
spectroscopy has revealed that only 30% of PSI
particles isolated from the PsaJ-deficient strain
oxidized plastocyanin with the same kinetics as the
wild type. This suggests that PsaJ is involved in the
maintenance of PsaF in the correct orientation to
enable fast electron transfer from soluble donor
proteins to the PSI reaction center28.
On light-induced excitation of P700, the chlorophyll
dimer in the PSI reaction center, an electron is first
transferred to the primary acceptor A0 and then
rapidly to the secondary acceptor A1 (a phylloquinone).
From there, the electrons are transferred to the
(4Fe–4S) cluster FX, and finally to the terminal
iron–sulfur acceptors FA and FB, which are bound by
the extrinsic polypeptide PsaC. Several site-directed
368
Fig. 3. Photosynthetic
complexes of the
thylakoid membrane. The
composition of the
complexes is drawn
according to structural
data where available, but
position of subunits
within the complexes is
largely arbitrary. Nucleusencoded subunits are
shaded blue, chloroplastencoded subunits are
white. An arrow indicates
the direction of
movement of the rotor
upon ATP synthesis.
Based on Ref. 46.
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TRENDS in Plant Science Vol.6 No.8 August 2001
ATP synthase
b
b
d
Stroma
Photosystem I
LHCII
II
E
C
D
Lhcb
Lhcb
IV
D2
D1
XM J KW S I
EFL HN
O
P
I
g
e
CP43 CP47
Lhcb
a
b
Fd
Cyt b6f
complex
Photosystem II
a
MG
IV-Cyt b6
LO
HGJ
A-B
MK I L
IIIIIIIIIIII
R
QT
Thylakoid lumen
Rieske
F
N
PC
Cyt f
TRENDS in Plant Science
mutations in the two PSI reaction center subunits,
PsaA and PsaB, have been used to identify the
residues that function as ligands to the redox cofactors
P700, A0, A1 and FX. For example, mutagenesis studies
in Chlamydomonas showed that only changes in the
histidines of helix 10 of both PsaA and PsaB result in
significant changes in the spectroscopic properties of
P700, indicating that these residues are the most
probable ligands of the chlorophyll dimer29.
Helix 10 of PsaA and PsaB is also involved in quinone
binding, and mutation of the tryptophans PsaA-W693
and PsaB-W673 has been used recently to investigate
the kinetics of electron transfer from the quinone (A1) to
FX (Ref. 30). Previous evidence suggested that electron
transport through PSI is unidirectional along a single
cofactor pathway, as is the case in PSII. However, it has
been shown that electron transfer from the quinone has
two kinetic components and that these components can
be independently modulated by mutating W693 in PsaA
and the corresponding W673 in PsaB (Ref. 30). The
fast component of electron transfer can be assigned to
the PsaB side, whereas the slow component occurs on
the PsaA side. Thus, in spite of the structural
similarity of PSII and PSI, electron transfer in PSI
appears to use both cofactor branches30, unlike PSII.
Regulation of photosynthesis in response to light
and nutrients
Light and nutrients are important environmental
factors that affect photosynthesis in algae and plants.
Photosynthetic light harvesting is regulated by
changes in both light quantity and quality. In
response to high light intensities that exceed a
photosynthetic organism’s capacity for CO2 fixation,
nonphotochemical quenching (NPQ) mechanisms are
induced that dissipate excess absorbed light energy
harmlessly as heat. Chlamydomonas mutants that
are defective in NPQ have been isolated by digital
video imaging of chlorophyll fluorescence31. The lack
of the xanthophyll cycle in the npq1 mutant was the
http://plants.trends.com
first genetic evidence for the importance of
zeaxanthin synthesis in NPQ. Characterization of the
lor1 mutant has revealed a possible role for lutein in
NPQ (Ref. 32), and other mutants appear to identify
specific proteins that are involved in the process.
In response to changes in light quality, a
mechanism known as state transition regulates the
relative excitation of PSII and PSI. When PSII is
preferentially excited by illumination with red light,
plastoquinone, the lipid-soluble electron carrier
between PSII and PSI, becomes reduced. A protein
kinase that phosphorylates the PSII light-harvesting
complex (LHCII) proteins is then activated, resulting
in a decrease in excitation energy reaching PSII
(state 2). Far-red light excites PSI preferentially,
resulting in oxidation of the plastoquinone pool,
dephosphorylation of LHCII, and restoration of
energy transfer from LHCII to PSII (state 1). A
crucial role for the plastoquinol-binding site of the
Chlamydomonas cytochrome b6 f complex in redox
sensing and kinase activation has been demonstrated
by site-directed mutagenesis33. Nuclear mutants
defective in state transition have been identified
using chlorophyll fluorescence video imaging under
conditions that induce either reduction or oxidation
of the plastoquinone pool34,35. The stt7 and stm1
mutants are blocked in state 1 and are deficient in
LHCII phosphorylation. These mutants are still
capable of photoautotrophic growth, although at a
slower rate than the wild type34,35, and further
analysis should answer longstanding questions
about the physiological role of state transitions.
Acclimation of the photosynthetic apparatus also
occurs in response to a deficiency of various essential
nutrients. In a screen to identify genes involved in the
response to copper deficiency36, crd1 mutants were
isolated that failed to accumulate PSI, LHCI and to a
lesser extent LHCII under copper-deficient
conditions. Because the mutants were produced and
tagged by insertional mutagenesis, it was possible to
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Insertional
mutagenesis
TRENDS in Plant Science Vol.6 No.8 August 2001
Genome
sequencing
EST
sequencing
Isolation of
Isolation of
nonphotosynthetic nonphotosynthetic
mutants
mutants
Generation of
genetic map
Genome
annotation
Isolation and
sequencing
of flanking DNA
Chemical
mutagenesis
Reverse genetics
Map-based
cloning
Insertional
mutants
RNAi EMS/dHPLC
Identification of all genes necessary for
photosynthesis and photoprotection
Pulse-labeling
Proteomics
Metabolite
profiling
Biochemical,
Site-directed
biophysical, and
mutagenesis
spectroscopic
analyses
Identification of specific functions and interactions
of each photosynthetic gene
TRENDS in Plant Science
Fig. 4. Functional genomics of photosynthesis using Chlamydomonas reinhardtii.
recover the genomic sequence flanking the insertion
by inverse PCR. The CRD1 gene encodes a 47 kDa
hydrophilic protein with a consensus carboxylatebridged di-iron binding site. CRD1 homologs are
found exclusively in photosynthetic organisms, with
the highest levels of conservation found in plants such
as rice and Arabidopsis. Although the specific
function of the Crd1 protein is not clear, these results
have uncovered a previously unrecognized role for
copper in photosynthetic metabolism.
A bright future (with a few shadows to dispel)
Although Chlamydomonas has many unique genetic
and biochemical attributes for the study of
photosynthesis, there are problems that need to be
solved to realize the full potential of the organism.
Storage of Chlamydomonas strains is difficult, with
viable cells only being recovered from lines stored in
liquid nitrogen37. Large mutant collections therefore
require specialized storage facilities. Alternatively,
diploid zygospores can be stored at room temperature
for many years, but this requires the mating of each
individual mutant line and subsequent recovery of
the mutant after sporulation.
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369
Insertional mutagenesis has been a valuable
technique in the generation of banks of photosynthetic
mutants. A complicating factor is that not all mutants
are actually tagged by the DNA insertion. Genetic
crosses have shown that ~50% of mutants are
tagged31,34,36, a frequency similar to that observed
using comparable T-DNA tagging approaches in
plants. Insertional mutagenesis in Chlamydomonas
also often results in the deletion of large fragments of
genomic DNA (up to 20 kb). This makes identification
of the gene responsible for a mutant phenotype more
difficult, but does have the advantage that fewer
transformants are needed to saturate the nuclear
genome. As an alternative to insertional mutagenesis
by transformation, the development of a transposon
tagging system should provide a more precise
mechanism for the insertion of foreign DNA into the
genome. Several active transposons have already
been identified in Chlamydomonas38.
An additional tool that would aid in the isolation of
photosynthetic genes is an autonomously replicating
shuttle vector for cloning by complementation.
Current methods require plasmid rescue to recover
an integrated cosmid after complementation of the
mutant phenotype20. An alternative strategy involves
transformation with indexed libraries that consist of
pools of specific cosmids, with subsequent
identification of an individual complementing cosmid
clone39. It can therefore be seen that although there
are technical challenges that remain to be met in
Chlamydomonas research, progress is being made in
finding solutions to each of these issues.
The ultimate goal of photosynthesis research is to
identify all the genes that are involved in
photosynthesis and to understand how their proteins
function and interact with one another (Fig. 4). A
central resource to this goal is a catalog of all the genes
in Chlamydomonas, and progress towards achieving
this aim is being made through a Chlamydomonas
genome project. Current efforts4 include sequencing
of full-length cDNAs that are expressed under various
environmental conditions. These will be used to
construct DNA microarrays to investigate global
patterns of gene expression. However, the sequencing
of cDNAs is unlikely to find all the genes, so
complete DNA sequencing of the ∼100–160 Mbp
Chlamydomonas nuclear genome will be necessary.
In the meantime, cDNA sequencing from
polymorphic strains of Chlamydomonas can be used
to identify single nucleotide polymorphisms and DNA
fragment length polymorphisms (insertions or
deletions) that will enable construction of a detailed
genetic map. The current map of 240 markers on
17 linkage groups is based on progeny from a cross
between the standard laboratory strain of
Chlamydomonas and a highly polymorphic field
isolate from Minnesota, USA (strain S1D2)40. The
markers used to generate the map include random
cDNAs, random genomic fragments, and cloned genes
already in the database.
370
Fig. 5. Comparison of
map-based cloning in
Chlamydomonas and
Arabidopsis, showing the
approximate minimum
time to obtain a rough
map position for a
nonphotosynthetic
mutation. A collection of
markers for
Chlamydomonas
mapping is currently
being developed. The
Arabidopsis timeline
assumes that a collection
of M3 families (seeds
from individual M2
plants) is available for
screening, thereby
enabling the identification
of heterozygous siblings
of mutants that are lethal
as homozygotes.
Review
TRENDS in Plant Science Vol.6 No.8 August 2001
Chlamydomonas
Mutant
picked
Streak mutant to retest phenotype
Total = 6 weeks
to map position
Do cross and dissect tetrads
Confirmation of gene
by complementation
takes a further 2 weeks
Grow up tetrads
Restreak colonies, score phenotype and extract DNA
Score PCR-based markers
Arabidopsis
Mutant
picked
in M3
family
Confirmation of gene
by complementation
takes a further 6 weeks
Total = 16 weeks
to map position
Cross heterozygous sibling
and get F1 seeds
Grow F1 and get F2 seeds
Grow F2, score phenotype
and extract DNA.
Score PCR-based markers
0
Acknowledgements
We thank Irene Baroli,
Heidi Ledford, and Patricia
Müller for their critical
reading of this article and
Jai Shin for isolation of the
lts5 mutant. Microscope
work was carried out by
Denise Schichnes of the
CNR Biological Imaging
Facility, UC Berkeley. This
work was supported by
grants from the National
Institutes of Health
(GM58799-01), the US
Dept of Energy, the Torrey
Mesa Research
Institute/Syngenta
Research and Technology,
and the Searle Scholars
Program/the Chicago
Community Trust to K.K.N.
2
4
6
8
Time (weeks)
The genome project has also initiated the
development of a physical map linked to the genetic
map of the nuclear genome. The physical map will
consist of overlapping BAC clones that are anchored
on the genetic map using the set of 240 unique
markers as probes. To date, ~2000 different BACs
have been aligned on the genetic map, representing
~25% of the genome. Work is currently being carried
out to fill in gaps between BAC contigs, and an
updated map will soon be placed in the
Chlamydomonas database (C. Silflow and
P. Lefebvre, pers. commun.).
These genetic and physical maps will facilitate the
positional cloning of genes identified in classical
forward genetics screens for nonphotosynthetic
mutants. Use of mutagens such as EMS or UV light
should enable the recovery of a variety of partial
loss-of-function and gain-of-function mutations, in
addition to complete null mutations. Because of the
rapid life cycle of Chlamydomonas, map-based cloning
can be completed much faster than the positional
cloning of genes in Arabidopsis (Fig. 5), an organism
for which this procedure has become routine.
Collections of insertional mutants can be used in
both forward and reverse genetics strategies to
identify genes involved in photosynthesis. Sequencing
of flanking DNAs from nonphotosynthetic insertional
mutants should enable the rapid identification of
novel photosynthetic genes, especially once the
Chlamydomonas genome sequence is available.
Although forward genetics screens can potentially
identify all the individual genes that are necessary for
http://plants.trends.com
10
12
14
16
TRENDS in Plant Science
photosynthesis, reverse genetics approaches will also
be necessary where, for example, members of gene
families have overlapping function. To obtain
mutations in specific genes, collections of random
insertion mutants can be screened using PCR-based
strategies similar to those used for Arabidopsis and
maize41. As an additional reverse genetics approach,
we are currently generating an EMS-induced
Chlamydomonas mutant population that can be
screened by denaturing HPLC for point mutations in
any gene of interest42. Because EMS usually causes
C-to-T transition mutations, this approach is
particularly suited to Chlamydomonas, given the
GC-rich nature of its genome. The technique has the
advantage that it can be used to find nonsense (null)
alleles of a gene, as well as missense mutations that
might have more subtle phenotypes.
Recently developed strong promoters should enable
silencing of specific genes by antisense technology or
RNA interference (RNAi). For example, the
HSP70A–RBCS2 expression system43 has been used
to silence the COP gene encoding the photoreceptor for
phototaxis (Chlamyrhodopsin; M. Fuhrmann and
P. Hegemann, pers. commun.). After transformation of
wild-type cells with an inverted repeat construct,
COP gene expression was reduced by almost 90%. An
advantage of this approach is the feasibility of
silencing duplicated genes in a single experiment. The
development of this technology should be aided by the
recent use of Chlamydomonas as a model system for
the study of the mechanisms of post-transcriptional
gene silencing44.
Review
TRENDS in Plant Science Vol.6 No.8 August 2001
Using a combination of these genetic approaches, it
should thus be possible to obtain mutations in every
Chlamydomonas gene involved in photosynthesis.
Many photosynthetic mutants have already been
generated in Synechocystis, and a complete collection
of knockout mutants will soon be available for
Arabidopsis. These resources should enable the
annotation of many new genes that are necessary for
photosynthesis45. However, because of the inviability
of nonphotosynthetic mutants of Arabidopsis, it will be
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