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Chlamydomonas reinhardtii and photosynthesis:
genetics to genomics
Arthur R Grossman
Genetic and physiological features of the green alga
Chlamydomonas reinhardtii have provided a useful model for
elucidating the function, biogenesis and regulation of the
photosynthetic apparatus. Combining these characteristics
with newly developed molecular technologies for engineering
Chlamydomonas and the promise of global analyses of nuclear
and chloroplast gene expression will add a new perspective to
views on photosynthetic function and regulation.
Addresses
Department of Plant Biology, The Carnegie Institution of Washington,
260 Panama Street, Stanford, California 94305, USA;
e-mail: [email protected]
the function(s) of specific genes even if ‘knockout’ mutations are identified. Simpler systems might not have so
much functional redundancy and even if there are genes
with compensatory functions, it is often easy to create synthetic phenotypes that result from more than one lesion.
Fourth, genomic analysis of a range of carefully chosen
organisms will reveal a diversity of processes and will show
how the environment has influenced the evolution of
these processes. Understanding how a process has been
tuned to a particular habitat is the first step in developing
strategies to alter the range of environments in which specific organisms can grow.
Current Opinion in Plant Biology 2000, 3:132–137
1369-5266/00/$ — see front matter © 2000 Elsevier Science Ltd.
All rights reserved.
Abbreviations
ARG 7 ARGININOSUCCINATE LYASE 7
BAC
bacterial artificial chromosome
GFP
green fluorescent protein
NIT
NITRATE REDUCTASE
Introduction
The sequencing of the Arabidopsis genome will be completed within a year. The rice genome, which is about four
times as large as that of Arabidopsis, is likely to be the first
genome of a commercially important plant for which we
will have a complete sequence. The enormous volume of
sequence information that is being gathered will facilitate
the elucidation of specific gene functions. Nevertheless,
the use of angiosperms for functional analyses of genes has
some significant limitations, listed below, that make it
important to exploit multiple systems to elucidate gene
function in vascular plants.
First, because DNA integrates randomly into the genome
of angiosperms, an inactivation of any specific gene can
only be identified by screening genomic DNA from pooled
populations of potential mutants using PCR-based
approaches. These approaches can be both difficult and
laborious. As an alternative, homologous recombination
occurs in the moss Physcomitrella, making it possible to
probe gene function by reverse genetics [1].
Second, although it is often possible to use sequence information to infer catalytic activity encoded by a gene, the exact
process in which the polypeptide product is involved might
not be apparent; there are numerous genes encoding regulatory elements that have been identified in Arabidopsis, but
the processes that they control have not yet been defined.
Third, plants often have multiple genes with redundant or
overlapping functions. This makes it difficult to determine
Finally, there are certain processes that are obligate for
some organisms but conditional for others. One such
process is photosynthesis. All angiosperms that have been
studied so far are obligate photoautotrophs; they grow very
poorly and often die in the absence of photosynthesis,
even when supplied with a source of fixed carbon. It is
therefore difficult to exploit mutant generation for probing
photosynthetic function. Traditionally, both cyanobacteria
and the unicellular green alga Chlamydomonas reinhardtii
have served as valuable resources for using genetic and
molecular technologies to elucidate the function, biosynthesis and regulation of the photosynthetic apparatus. In
this review I shall briefly discuss the value of using
Chlamydomonas to study photosynthetic processes in plants
and how genetics, molecular technologies and genomics
are enhancing its utility.
Advantages of Chlamydomonas as a model
organism, especially for the analysis of
photosynthesis
Valuable molecular tools have been tailored for use with
Chlamydomonas; the facility with which these tools can be
used has led investigators to bestow the title of ‘green yeast’
[2] upon this organism. The most important technological
developments have been the establishment of selectable
markers for the identification of nuclear and chloroplast
transformants and relatively simple procedures to introduce
DNA into the cells. Furthermore, cosmid [3], yeast artificial
chromosome (YAC) [4] and bacterial artificial chromosome
(BAC) (Lefebvre, unpublished data; Genome Systems,
St. Louis, Missouri, USA) recombinant libraries are being
used for complementation of mutant strains, and both an
arylsulfatase gene [5] and, more recently, a sequence-tailored green fluorescent protein (GFP) gene [6•] have been
developed as reporters for monitoring gene expression and
subcellular location. Furthermore, sense/antisense technology has been successfully used to alter the accumulation of a
heat shock protein (HSP70B) mRNA [7••]. Nevertheless,
the Chlamydomonas work still has some technological short-
Chlamydomonas reinhardtii and photosynthesis Grossman
comings. Introduced DNA appears to integrate randomly
into the Chlamydomonas nuclear genome (as it does in
Arabidopsis), making it difficult to perform reverse genetics.
Furthermore, integration of introduced DNA into the
nuclear genome frequently causes large deletions that could
complicate the analysis of mutants generated by random
insertion of exogenous DNA [8].
When selecting transformed Chlamydomonas strains,
the ARGININOSUCCINATE LYASE 7 (ARG7) [9] and
NITRATE REDUCTASE 1 (NIT1) [10] genes are routinely
used as marker genes to rescue the recessive arg7 (unable to
grow without exogenous arginine) and nit1 (unable to grow
using nitrate as a sole nitrogen source) mutants. A gene for
which there is no selectable phenotype can be co-transformed into cells with the marker genes at relatively high
frequencies. The Chlamydomonas emetine resistance gene
CRY1 [11] and the Streptoalloteichus hindustanus phleomycin
resistance gene ble [12•] have been used as dominant markers for nuclear transformation. The aadA gene confers
spectinomycin/streptomycin resistance on Chlamydomonas
cells, providing a strong selectable marker for introducting
DNA into the chloroplast genome [13]. This gene has
recently been also used for nuclear transformation [14].
There are a number of ways of introducing exogenous
DNA into Chlamydomonas cells including biolistic procedures (i.e. bombarding the cells with microprojectiles
coated with DNA) [15–17], agitation with glass beads [18]
and electroporation [19•]. The simplest method of introducing DNA into the nuclear genome is the procedure
involving glass beads, which requires no special equipment but results in relatively low transformation
efficiencies. Transformation frequencies of more than
1 × 105 transformants per µg of DNA have been achieved
by electroporation [19•]. Such high transformation frequencies provide a strong potential for the ‘shotgun
cloning’ of genes by complementing mutant strains (i.e.
identifying genes that complement a specific mutant phenotype by introduction of a recombinant library).
Furthermore, biolistic procedures appear to be the most
efficient way of introducing DNA into the chloroplast
genome [17], probably because the chloroplast occupies
over half of the volume of the cell providing a large target
for a microprojectile.
Many of the characteristics of Chlamydomonas, a eukaryote
with haploid genetics, make it an ideal organism for the
analysis of photosynthesis. The considerable power of a
model system with haploid genetics is exemplified by the
use of the yeast Saccharomyces cerevisiae to augment our
understanding of basic cellular processes such as nutrient
utilization, cell cycle control and signal transduction.
Furthermore, Chlamydomonas can be grown heterotrophically on acetate as a sole source of carbon; when growing
heterotrophically dark-grown cells exhibit normal photosynthetic capability and chloroplast development. This
capacity for heterotrophic growth has permitted the devel-
133
opment of a number of different genetic screens to isolate
mutants that have impaired photosynthesis (reviewed in
[20]). Most of these screens, which are not readily applied
to vascular plants, allow the rapid analysis of thousands of
colonies on solid medium. Furthermore, because
Chlamydomonas grows well heterotrophically in the dark, it
is relatively easy to isolate and maintain photosynthetic
mutants that are light sensitive [21].
Many procedures in vivo can be incorporated into studies of
Chlamydomonas; these procedures might be difficult or
impossible to perform with more complex systems.
Spectrophotometric studies in vivo have provided an abundance of information on photosynthetic exciton and
electron transfer (reviewed in [22]). The analysis of various
aspects of photosynthetic activity and chloroplast biogenesis in vivo is facilitated by the uptake of certain electron
donors and acceptors, inhibitors of photosynthetic electron
transport and chloroplast and cytoplasmic protein synthesis,
and 35SO42– to label proteins, by intact Chlamydomonas
cells. The use of Chlamydomonas for the characterization of
chloroplast biogenesis in vivo is exemplified by the work of
Howe and Merchant [23]. Pulse labeling of proteins in vivo
was used to define discrete steps in the biosynthesis of the
nucleus-encoded chloroplast polypeptides cytochrome c6,
plastocyanin and the 33 kDa oxygen-evolving complex protein. In contrast, pulse-labeling of proteins is much more
difficult in vascular plants. There might be a considerable
lag between the time at which the label is administered (to
the roots or to a cut edge of the stem) and the time at which
it is incorporated into chloroplast proteins.
It is relatively easy to generate Chlamydomonas ‘tagged’
mutants by transformation with the ARG7 or NIT1 genes.
This strategy facilitates the isolation of genes that are
altered in any of a number of different processes, including
photosynthesis. This approach, however, generally causes
the production of null mutations that, in some cases, will
be lethal under the conditions used for screening. It will be
advantageous to generate point mutants that have defective photosynthesis (many of which are already available)
and to rapidly identify BAC clones that rescue the mutant
phenotypes. This approach can be made routine by generation of a physical map of the Chlamydomonas genome that
is linked to the genetic map and the creation of a set of
overlapping BAC clones that cover the entire genetic map.
Because Chlamydomonas doubles its cell numbers in 5–7 h
when grown on acetate-containing medium and completes its sexual cycle in less than two weeks, genetic
analysis is rapid. As genomic analyses generate more
mapped physical markers, mutations will be readily
placed on the genetic map. In addition, efficient transformation methods make it possible to test a candidate BAC
clone for rescue of a mutant phenotype within one or two
weeks. In the near future, it should be feasible to map an
interesting mutation and to assay overlapping BAC clones
from the neighbouring regions of the genome for rescue
134
Genome studies and molecular genetics
of the mutant phenotype within 6–8 weeks. In
Arabidopsis this process often takes more than a year.
DNA is readily introduced into Chlamydomonas chloroplasts using particle bombardment [17] and transformants can
be easily selected because of their antibiotic (spectinomycin) resistant growth. Because integration into the
Chlamydomonas chloroplast genome occurs by homologous
recombination, any gene in the genome can be inactivated
and the resultant phenotype evaluated rapidly with regard
to photosynthetic function, assembly of the photosynthetic apparatus and regulation of transcription/translation of
chloroplast-encoded genes. The inactivation of some
chloroplast genes results in lethality, but the function(s) of
these genes can be studied in heteroplasmic transformants
[24]. Furthermore, the Chlamydomonas chloroplast transformation system has allowed investigators to use
site-directed mutagenesis to perform structure–function
studies of both electron-transport and reaction-center components [25–27,28•]. Although chloroplast transformation
can be performed with tobacco cell protoplasts [29], it is
much more problematic and the results are inherently
more difficult to interpret.
Global regulation of gene expression under different environmental conditions is more readily studied in a
single-celled organism such as Chlamydomonas than in vascular plants. First, alterations in growth conditions can be
rapidly imposed on Chlamydomonas cells by changing the
growth medium and/or moving the cells to different environmental conditions. Second, Chlamydomonas cultures
represent a relatively uniform population of cells and the
analysis is therefore not complicated by the presence of different organs and tissue types that might exhibit radically
different responses to the stimulus. Nevertheless, complex
higher-order processes that involve communication between
different tissue types cannot be studied in Chlamydomonas.
Recent progress using Chlamydomonas in
understanding photosynthetic function
Historically, Chlamydomonas has been important in elucidating photosynthetic mechanisms. Early characteriztion
of Chlamydomonas mutants identified components of
photosynthetic electron transport, photosystem I, photophosphorylation and the Calvin cycle (summarized in
[30]). The first physically defined mutation of the chloroplast genome was recovered from Chlamydomonas and was
shown to be in the rbcL gene (large subunit of ribulose-1,5bisphosphate carboxylase) [31]. Analysis of the labile
32 kDa D1 and D2 polypeptides of photosystem II helped
researchers to recognize that these polypeptides were
functionally analogous to the integral membrane L and M
polypeptides of the reaction centers in the purple non-sulfur bacteria (summarized in [32]). In addition, freeze-etch
electron microscopy of thylakoid membranes from mutant
and wild-type Chlamydomonas strains has led to the association of defined particles of the thylakoid membranes with
specific complexes involved in photosynthesis [33].
Visualization of these particles suggested that complexes
of the photosynthetic apparatus are in a fluid state within
the membranes, as shown by the lateral movement of
phosphorylated light-harvesting complexes to nonappressed regions of the thylakoid membranes (this
movement is associated with a state transition) [34].
Finally, detailed biochemical approaches, targeted disruptions and site-directed mutagenesis are all helping to
define interactions between the different polypeptides of
the photosystems.
A number of recent reports describe research in which
molecular tools have been used to elucidate photosynthetic processes. Mutants in chlorophyll b biosynthesis were
isolated by transformation tagging with the ARG7 gene
[35••]. The lesions were found to be in a gene encoding a
putative protein with sequence similarity to a methylmonooxygenase. This work has helped to elucidate both
mechanistic and evolutionary aspects of chlorophyll
biosynthesis [36••].
Wollman, Merchant and collaborators [37,38,39•] have isolated several nuclear mutants that define elements
involved in the biogenesis of the cytochrome complex.
The nuclear loci CCS1,2,3,4 are involved in the attachment
of c-heme to cytochrome f, and the nuclear loci CCB1,2,3,4
are involved in b-heme binding to cytochrome b6. Other
nuclear loci are important for the maturation and/or stabilization and translation of transcripts encoding the
individual components of the cytochrome b6f complex
[40•]. The work on the cytochrome complexes has opened
new horizons that would have been very difficult to
explore in vascular plants. This work also highlights the
fact that much of the control of chloroplast biogenesis is at
the level of post-transcriptional and translational events,
which we are only just beginning to understand
[41,42••,43,44••,45•,46].
Recently, an elegant selection was performed that yielded
several mutants with an altered ability to transport proteins
across the thylakoid membranes [47••]; analysis of these
mutants, designated TIP (for thylakoid insertion proteins),
should add greatly to our knowledge of protein targeting
and chloroplast biogenesis.
Another topic of considerable interest is the acclimation of
the photosynthetic apparatus to changing environmental
conditions. Chlamydomonas and vascular plants must tune
their photosynthetic activity to their environmental nutrient status (which determines their growth potential);
these alterations include the accumulation of photoinhibited reaction centers and the formation of centers that
exhibit a much lower rate of QB (i.e. the secondary electron acceptor of photosystem II) reduction [48•].
Chlamydomonas also modulates photosynthetic function as
a direct response to light levels. Recently, mutants resistant to very high light [49] or defective in the dissipation
of excess excitation energy [50] have been isolated. A
Chlamydomonas reinhardtii and photosynthesis Grossman
video-imaging
screen
identified
mutants
of
Chlamydomonas that were defective in the xanthophyll
cycle and also exhibited a reduced ability to eliminate
excess absorbed light energy [50]; this was the first genetic evidence linking energy dissipation to the production of
specific xanthophylls. Similar findings were recently
obtained with Arabidopsis [51••], although the xanthophyll
cycle does seem to have a more dominant role in energy
dissipation in vascular plants than in Chlamydmonas.
Recently, the analysis of another mutant unable to dissipate excess absorbed light energy was shown to be
missing a minor component of photosystem II designated
PsbS, which is related to the light harvesting chlorophylla,b-binding proteins [52••]. This finding has important
implications with respect to both light harvesting function
and evolution. A video-imaging screen was also used to
isolate mutants that are unable to undergo state transition
(or to perform phosphorylation of the light-harvesting
antennae proteins) [53•,54•]. Furthermore, recent work of
Schroda et al. [7••] has demonstrated that the chloroplastlocalized chaperone HSP70B is involved in both the
protection and repair of photosystem II in high light.
Applications of genomics, and conclusions
The physiological, genetic and molecular manipulations
that have become routine for Chlamydomonas make this
organism ripe for genome-wide analyses. The global
analysis of the genome will add considerably to our
understanding of photosynthetic function and the way in
which photosynthesis is modulated as environmental
conditions change [55]. Sequence analyses of cDNAs
derived from RNA isolated from Chlamydomonas cells
that are exposed to a number of different environmental
conditions will inundate the research community with
interesting sequences that can be used as substrates for
functional genomics. cDNA microarrays will provide a
global view of gene regulation under a number of different environmental conditions. Analysis of high-density
DNA microarrays with cDNA probes generated from
putative ‘regulatory mutants’ will enable investigators to
identify the targets of specific regulators and the
processes that are affected in these mutant strains;
Chlamydomonas has a number of putative regulatory
mutants that can be used immediately for such studies.
Finally, establishing a high-density physical map that is
linked to the genetic map [56] will make the positional
cloning of genes that are altered (e.g. point mutations) in
specific mutants at least an order of magnitude more
rapid than in vascular plants.
Acknowledgements
I thank all of the co-investigators, including Pete Lefebvre, John Davies,
Carolyn Silflow, Elizabeth Harris and David Stern, who worked with me to
create a Chlamydomonas genome project and acknowledge the National
Science Foundation for funding this project. There are many excellent
articles in which genetics and molecular strategies were used to dissect
photosynthetic processes in Chlamydomonas that have not been discussed in
this review because of severe length restrictions. I wish to apologize to those
whose work has not received appropriate attention.
135
References and recommended reading
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cytochrome f) were identified. Whereas the chloroplast ccsA gene appears to
be transcribed in the ccs1-4 mutant, the CcsA protein is absent in the ccs1
mutant and more susceptible to degradation in the ccs3 and ccs4 mutants.
These results suggest that a multi-subunit complex involved in holocytochrome c assembly might contain the proteins encoded by the CCS loci.
27.
Redding K, MacMillan F, Leibl W, Brettel K, Hanley J, Rutherford AW,
Breton J, Rochaix J-D: A systematic survey of conserved histidines
in the core subunits of photosystem I by site-directed
mutagenesis reveals the likely axial ligands of P-700. EMBO J
1998, 17:50-60.
28. Zito F, Finazzi G, Delosme R, Nitschke W, Picot D, Wollman FA: The
•
Qo site of cytochrome b6f complex controls the activation of the
LHCII kinase. EMBO J 1999, 18:2961-2969.
The petD gene, which encodes subunit IV of the cytochrome b6f complex,
was mutated in a way that disrupted the Qo pocket of the complex. The
mutation eliminated both electron transport through the complex and plastoquinol binding at Qo, although it did not prevent assembly of the polypeptides of the complex. The mutant cells remained locked in state I (the
light-harvesting-center II [LHCII] antennae complex associated with photosystem II) and the polypeptides of the LHCII were only poorly phosphorylated. These results suggest that plastoquinol binding at the Qo site is required
for the activation of the LHCII kinase and the transition to state II (LHCII
antennae associated with photosystem I).
40. Drager RG, Girard-Bascou J, Choquet Y, Kindle KL, Stern DB: In vivo
•
evidence for 5′′→3′′ exoribonuclease degradation of an unstable
chloroplast mRNA. Plant J 1998, 13:85-96.
A mutant (mcd1-1) was isolated in which the petD mRNA (encoding subunit
IV of the cytochrome b6f complex) was unstable. The instability of the petD
mRNA in the mutant background was a consequence of an element located
in the 5′ untranslated region of the mRNA. This element seems to promote
5′ to 3′ exoribonuclease degradation of the mRNA in the absence of a binding protein, possibly the MCD1 gene product.
41. Kim J, Mayfield SP: Protein disulfide isomerase as a regulator of
chloroplast translational activation. Science 1997, 278:1954-1957.
Chlamydomonas reinhardtii and photosynthesis Grossman
42. Yohn CB, Cohen A, Danon A, Mayfield SP: A poly(A) binding protein
•• functions in the chloroplast as a message-specific translation
factor. Proc Natl Acad Sci USA 1998, 95:2238-2243.
A 47 kDa polypeptide was identified that binds to the 5′ untranslated region
of the chloroplast psbA mRNA (encoding the D1 protein of photosystem II).
This protein, which might be involved in controlling the light-regulated translation of the psbA mRNA, is a member of the polyA-binding protein family.
PolyA-binding proteins are thought to bind the 3′ polyA tail of cytoplasmic
mRNAs in eukaryotes and to have a role in translational control. The data in
this paper suggest that members of the polyA-binding protein family can also
interact with chloroplast messages and possibly modulate their translation.
43. Yohn CB, Cohen A, Rosch C, Kuchka MR, Mayfield SP: Translation
of the chloroplast psbA mRNA requires the nuclear-encoded
poly(A)-binding protein, RB47. J Cell Biol 1998, 142:435-442.
44. Choquet Y, Stern DB, Wostrikoff K, Kuras R, Girard-Bascou J,
•• Wollman F-A: Translation of cytochrome f is autoregulated through
the 5′′ untranslated region of petA mRNA in Chlamydomonas
chloroplasts. Proc Natl Acad Sci USA 1998, 95:4380-4385.
The synthesis of the chloroplast-encoded cytochrome f subunit of the
cytochrome b6f complex is diminished when other subunits of the complex
are not synthesized. This process, termed CES (control of epistasy of synthesis), involves autoregulation at the level of translation. It appears to be
mediated by either direct or indirect interaction of the 5′ untranslated region
of the cytochrome f mRNA (petA mRNA) with the C-terminal domain of
unassembled cytochrome f. The CES mechanism might also be important for
controlling the synthesis of other multi-protein complexes in the chloroplast.
45. Nickelsen J, Fleischmann M, Boudreau E, Rahire M, Rochaix J-D:
•
Identification of cis-acting RNA leader elements required for
chloroplast psbD gene expression in Chlamydomonas. Plant Cell
1999, 11:957-970.
Site-directed mutagenesis was used to identify RNA stability determinants
in the psbD transcript (which encodes the D2 polypeptide of the reaction
center of photosystem II). Other elements in the mRNA were shown to alter
translation but not RNA stability. These results suggest that the post-transcriptional regulation of psbD expression might involve both translational
control and mRNA turnover.
46. Fargo DC, Boynton JE, Gillham NW: Mutations altering the
predicted secondary structure of a chloroplast 5′′ untranslated
region affect its physical and biochemical properties as well as its
ability to promote translation of reporter mRNAs both in the
Chlamydomonas reinhardtii chloroplast and in Esherichia coli. Mol
Cell Biol 1999, 19:6980-6990.
47.
••
Bernd KK, Kohorn BD: Tip loci: six Chlamydomonas nuclear
suppressors that permit the translocation of proteins with mutant
thylakoid signal sequences. Genetics 1998, 149:1293-1301.
Mutations in the leader sequence of the cytochrome f protein that inhibit its
translocation into the photosynthetic membranes and prevent photoautotrophic growth are defined. Suppressors of the mutant phenotype were
generated that define six nuclear loci. Both biochemical analysis of the
mutants and sequence characterization of the genes altered in the suppressor strains will elucidate the apparatus of the photosynthetic membranes
that is involved in protein translocation.
48. Wykoff DD, Davies JP, Grossman AR: The regulation of
•
photosynthetic electron transport during nutrient deprivation in
Chlamydomonas reinhardtii. Plant Physiol 1998, 117:129-139.
Nutrient limitation markedly influences photosynthetic electron flow; this
modulation of photosynthetic activity is critical for cell survival. Starving
137
Chlamydomonas of either sulfur or phosphorus leads to a downregulation of
photosystem II activity that involves the generation of QB-nonreducing centers, a decline in the maximum quantum efficiency of photosystem II and a
decreased efficiency of excitation energy transfer to the photosystem II reaction centers.
49. Forster B, Osmond CB, Boynton JE, Gillham NW: Mutants of
Chlamydomonas reinhardtii resistant to very high light. J Photochem
Photobiol 1999, 48:127-135.
50. Niyogi K, Björkman O, Grossman AR: Chlamydomonas xanthophyll
cycle mutants identified by video imaging of chlorophyll
fluorescence quenching. Plant Cell 1997, 9:1369-1380.
51. Niyogi KK, Grossman AR, Björkman O: Arabidopsis mutants define
•• a central role for the xanthophyll cycle in the regulation of
photosynthetic energy conversion. Plant Cell 1998, 10:1121-1134.
A video-imaging system developed for use with Chlamydomonas was used
to screen Arabidopsis plants for alterations in non-photochemical quenching.
This screen resulted in the isolation of mutants that are defective in the xanthophyll cycle. The npq1 mutant, which was unable to convert violaxanthin to
zeaxanthin in intense light, was more severely limited in its ability to dissipate
excess excitation than the analogous mutant in Chlamydomonas. Hence,
although both Chlamydomonas and Arabidopsis use the xanthophyll cycle for
the dissipation of excess excitation, the proportions of excess excitation energy that is dissipated in this way seems to differ in the two organisms.
52. Li X-P, Bjôrkman O, Shih C, Grossman AR, Rosenquist M, Jansson S,
•• Niyogi KK: A pigment binding protein essential for regulation of
photosynthetic light harvesting. Nature 2000, 403:391-395.
The authors show that the gene encoding PSBS, which is a chlorophyllbinding protein that is intrinsic to photosystem II, is necessary for the dissipation of excess absorbed light energy, but is not required for photosynthetic
oxygen evolution. A four membrane-helix PSBS-like protein was probably the
evolutionary precursor of the three membrane-helix LHC proteins, the major
components of the light harvesting antennae. These findings suggest that
the function of energy dissipation may have evolved prior to the current-day
light-harvesting function found in vascular plants.
53. Kruse O, Nixon PJ, Schmid GH, Mullineaux CW: Isolation of state
•
transition mutants of Chlamydomonas reinhardtii by fluorescence
video imaging. Photosynth Res 1999, 61:43-61.
This paper, like [54•], describes how video-imaging techniques can be used
to isolate state transition mutants. Characterizations of these mutants are
helping to elucidate the dynamics of the photosynthetic apparatus and the
environmental factors that modulate photosynthetic activities.
54. Fleishmann MM, Ravanel S, Delosme R, Olive J, Zito R, Wollman FA,
•
Rochaix J-D: Isolation and characterization of photoautotrophic
mutants of Chlamydomonas reinhardtii deficient in state
transition. J Biol Chem 1999, 274:30987-30994.
The work in this paper and [53•] demonstrates that video-imaging techniques
can be used to isolate state transition mutants. Characterizations of these
mutants are helping to elucidate the dynamics of the photosynthetic apparatus and the environmental factors that modulate photosynthetic activities.
55. Davies JP, Grossman AR: The use of Chlamydomonas
(Chlorophyta: Volvocales) as a model algal system for genome
studies and the elucidation of photosynthetic processes. J Phycol
1998, 34:907-917.
56. Lefebvre PA, Silflow CD: Chlamydomonas: the cell and its genome.
Genetics 1998, 151:9-14.
Chlamydomonas reinhardtii and photosynthesis:
genetics to genomics
Arthur R Grossman
Genetic and physiological features of the green alga
Chlamydomonas reinhardtii have provided a useful model for
elucidating the function, biogenesis and regulation of the
photosynthetic apparatus. Combining these characteristics
with newly developed molecular technologies for engineering
Chlamydomonas and the promise of global analyses of nuclear
and chloroplast gene expression will add a new perspective to
views on photosynthetic function and regulation.
Addresses
Department of Plant Biology, The Carnegie Institution of Washington,
260 Panama Street, Stanford, California 94305, USA;
e-mail: [email protected]
the function(s) of specific genes even if ‘knockout’ mutations are identified. Simpler systems might not have so
much functional redundancy and even if there are genes
with compensatory functions, it is often easy to create synthetic phenotypes that result from more than one lesion.
Fourth, genomic analysis of a range of carefully chosen
organisms will reveal a diversity of processes and will show
how the environment has influenced the evolution of
these processes. Understanding how a process has been
tuned to a particular habitat is the first step in developing
strategies to alter the range of environments in which specific organisms can grow.
Current Opinion in Plant Biology 2000, 3:132–137
1369-5266/00/$ — see front matter © 2000 Elsevier Science Ltd.
All rights reserved.
Abbreviations
ARG 7 ARGININOSUCCINATE LYASE 7
BAC
bacterial artificial chromosome
GFP
green fluorescent protein
NIT
NITRATE REDUCTASE
Introduction
The sequencing of the Arabidopsis genome will be completed within a year. The rice genome, which is about four
times as large as that of Arabidopsis, is likely to be the first
genome of a commercially important plant for which we
will have a complete sequence. The enormous volume of
sequence information that is being gathered will facilitate
the elucidation of specific gene functions. Nevertheless,
the use of angiosperms for functional analyses of genes has
some significant limitations, listed below, that make it
important to exploit multiple systems to elucidate gene
function in vascular plants.
First, because DNA integrates randomly into the genome
of angiosperms, an inactivation of any specific gene can
only be identified by screening genomic DNA from pooled
populations of potential mutants using PCR-based
approaches. These approaches can be both difficult and
laborious. As an alternative, homologous recombination
occurs in the moss Physcomitrella, making it possible to
probe gene function by reverse genetics [1].
Second, although it is often possible to use sequence information to infer catalytic activity encoded by a gene, the exact
process in which the polypeptide product is involved might
not be apparent; there are numerous genes encoding regulatory elements that have been identified in Arabidopsis, but
the processes that they control have not yet been defined.
Third, plants often have multiple genes with redundant or
overlapping functions. This makes it difficult to determine
Finally, there are certain processes that are obligate for
some organisms but conditional for others. One such
process is photosynthesis. All angiosperms that have been
studied so far are obligate photoautotrophs; they grow very
poorly and often die in the absence of photosynthesis,
even when supplied with a source of fixed carbon. It is
therefore difficult to exploit mutant generation for probing
photosynthetic function. Traditionally, both cyanobacteria
and the unicellular green alga Chlamydomonas reinhardtii
have served as valuable resources for using genetic and
molecular technologies to elucidate the function, biosynthesis and regulation of the photosynthetic apparatus. In
this review I shall briefly discuss the value of using
Chlamydomonas to study photosynthetic processes in plants
and how genetics, molecular technologies and genomics
are enhancing its utility.
Advantages of Chlamydomonas as a model
organism, especially for the analysis of
photosynthesis
Valuable molecular tools have been tailored for use with
Chlamydomonas; the facility with which these tools can be
used has led investigators to bestow the title of ‘green yeast’
[2] upon this organism. The most important technological
developments have been the establishment of selectable
markers for the identification of nuclear and chloroplast
transformants and relatively simple procedures to introduce
DNA into the cells. Furthermore, cosmid [3], yeast artificial
chromosome (YAC) [4] and bacterial artificial chromosome
(BAC) (Lefebvre, unpublished data; Genome Systems,
St. Louis, Missouri, USA) recombinant libraries are being
used for complementation of mutant strains, and both an
arylsulfatase gene [5] and, more recently, a sequence-tailored green fluorescent protein (GFP) gene [6•] have been
developed as reporters for monitoring gene expression and
subcellular location. Furthermore, sense/antisense technology has been successfully used to alter the accumulation of a
heat shock protein (HSP70B) mRNA [7••]. Nevertheless,
the Chlamydomonas work still has some technological short-
Chlamydomonas reinhardtii and photosynthesis Grossman
comings. Introduced DNA appears to integrate randomly
into the Chlamydomonas nuclear genome (as it does in
Arabidopsis), making it difficult to perform reverse genetics.
Furthermore, integration of introduced DNA into the
nuclear genome frequently causes large deletions that could
complicate the analysis of mutants generated by random
insertion of exogenous DNA [8].
When selecting transformed Chlamydomonas strains,
the ARGININOSUCCINATE LYASE 7 (ARG7) [9] and
NITRATE REDUCTASE 1 (NIT1) [10] genes are routinely
used as marker genes to rescue the recessive arg7 (unable to
grow without exogenous arginine) and nit1 (unable to grow
using nitrate as a sole nitrogen source) mutants. A gene for
which there is no selectable phenotype can be co-transformed into cells with the marker genes at relatively high
frequencies. The Chlamydomonas emetine resistance gene
CRY1 [11] and the Streptoalloteichus hindustanus phleomycin
resistance gene ble [12•] have been used as dominant markers for nuclear transformation. The aadA gene confers
spectinomycin/streptomycin resistance on Chlamydomonas
cells, providing a strong selectable marker for introducting
DNA into the chloroplast genome [13]. This gene has
recently been also used for nuclear transformation [14].
There are a number of ways of introducing exogenous
DNA into Chlamydomonas cells including biolistic procedures (i.e. bombarding the cells with microprojectiles
coated with DNA) [15–17], agitation with glass beads [18]
and electroporation [19•]. The simplest method of introducing DNA into the nuclear genome is the procedure
involving glass beads, which requires no special equipment but results in relatively low transformation
efficiencies. Transformation frequencies of more than
1 × 105 transformants per µg of DNA have been achieved
by electroporation [19•]. Such high transformation frequencies provide a strong potential for the ‘shotgun
cloning’ of genes by complementing mutant strains (i.e.
identifying genes that complement a specific mutant phenotype by introduction of a recombinant library).
Furthermore, biolistic procedures appear to be the most
efficient way of introducing DNA into the chloroplast
genome [17], probably because the chloroplast occupies
over half of the volume of the cell providing a large target
for a microprojectile.
Many of the characteristics of Chlamydomonas, a eukaryote
with haploid genetics, make it an ideal organism for the
analysis of photosynthesis. The considerable power of a
model system with haploid genetics is exemplified by the
use of the yeast Saccharomyces cerevisiae to augment our
understanding of basic cellular processes such as nutrient
utilization, cell cycle control and signal transduction.
Furthermore, Chlamydomonas can be grown heterotrophically on acetate as a sole source of carbon; when growing
heterotrophically dark-grown cells exhibit normal photosynthetic capability and chloroplast development. This
capacity for heterotrophic growth has permitted the devel-
133
opment of a number of different genetic screens to isolate
mutants that have impaired photosynthesis (reviewed in
[20]). Most of these screens, which are not readily applied
to vascular plants, allow the rapid analysis of thousands of
colonies on solid medium. Furthermore, because
Chlamydomonas grows well heterotrophically in the dark, it
is relatively easy to isolate and maintain photosynthetic
mutants that are light sensitive [21].
Many procedures in vivo can be incorporated into studies of
Chlamydomonas; these procedures might be difficult or
impossible to perform with more complex systems.
Spectrophotometric studies in vivo have provided an abundance of information on photosynthetic exciton and
electron transfer (reviewed in [22]). The analysis of various
aspects of photosynthetic activity and chloroplast biogenesis in vivo is facilitated by the uptake of certain electron
donors and acceptors, inhibitors of photosynthetic electron
transport and chloroplast and cytoplasmic protein synthesis,
and 35SO42– to label proteins, by intact Chlamydomonas
cells. The use of Chlamydomonas for the characterization of
chloroplast biogenesis in vivo is exemplified by the work of
Howe and Merchant [23]. Pulse labeling of proteins in vivo
was used to define discrete steps in the biosynthesis of the
nucleus-encoded chloroplast polypeptides cytochrome c6,
plastocyanin and the 33 kDa oxygen-evolving complex protein. In contrast, pulse-labeling of proteins is much more
difficult in vascular plants. There might be a considerable
lag between the time at which the label is administered (to
the roots or to a cut edge of the stem) and the time at which
it is incorporated into chloroplast proteins.
It is relatively easy to generate Chlamydomonas ‘tagged’
mutants by transformation with the ARG7 or NIT1 genes.
This strategy facilitates the isolation of genes that are
altered in any of a number of different processes, including
photosynthesis. This approach, however, generally causes
the production of null mutations that, in some cases, will
be lethal under the conditions used for screening. It will be
advantageous to generate point mutants that have defective photosynthesis (many of which are already available)
and to rapidly identify BAC clones that rescue the mutant
phenotypes. This approach can be made routine by generation of a physical map of the Chlamydomonas genome that
is linked to the genetic map and the creation of a set of
overlapping BAC clones that cover the entire genetic map.
Because Chlamydomonas doubles its cell numbers in 5–7 h
when grown on acetate-containing medium and completes its sexual cycle in less than two weeks, genetic
analysis is rapid. As genomic analyses generate more
mapped physical markers, mutations will be readily
placed on the genetic map. In addition, efficient transformation methods make it possible to test a candidate BAC
clone for rescue of a mutant phenotype within one or two
weeks. In the near future, it should be feasible to map an
interesting mutation and to assay overlapping BAC clones
from the neighbouring regions of the genome for rescue
134
Genome studies and molecular genetics
of the mutant phenotype within 6–8 weeks. In
Arabidopsis this process often takes more than a year.
DNA is readily introduced into Chlamydomonas chloroplasts using particle bombardment [17] and transformants can
be easily selected because of their antibiotic (spectinomycin) resistant growth. Because integration into the
Chlamydomonas chloroplast genome occurs by homologous
recombination, any gene in the genome can be inactivated
and the resultant phenotype evaluated rapidly with regard
to photosynthetic function, assembly of the photosynthetic apparatus and regulation of transcription/translation of
chloroplast-encoded genes. The inactivation of some
chloroplast genes results in lethality, but the function(s) of
these genes can be studied in heteroplasmic transformants
[24]. Furthermore, the Chlamydomonas chloroplast transformation system has allowed investigators to use
site-directed mutagenesis to perform structure–function
studies of both electron-transport and reaction-center components [25–27,28•]. Although chloroplast transformation
can be performed with tobacco cell protoplasts [29], it is
much more problematic and the results are inherently
more difficult to interpret.
Global regulation of gene expression under different environmental conditions is more readily studied in a
single-celled organism such as Chlamydomonas than in vascular plants. First, alterations in growth conditions can be
rapidly imposed on Chlamydomonas cells by changing the
growth medium and/or moving the cells to different environmental conditions. Second, Chlamydomonas cultures
represent a relatively uniform population of cells and the
analysis is therefore not complicated by the presence of different organs and tissue types that might exhibit radically
different responses to the stimulus. Nevertheless, complex
higher-order processes that involve communication between
different tissue types cannot be studied in Chlamydomonas.
Recent progress using Chlamydomonas in
understanding photosynthetic function
Historically, Chlamydomonas has been important in elucidating photosynthetic mechanisms. Early characteriztion
of Chlamydomonas mutants identified components of
photosynthetic electron transport, photosystem I, photophosphorylation and the Calvin cycle (summarized in
[30]). The first physically defined mutation of the chloroplast genome was recovered from Chlamydomonas and was
shown to be in the rbcL gene (large subunit of ribulose-1,5bisphosphate carboxylase) [31]. Analysis of the labile
32 kDa D1 and D2 polypeptides of photosystem II helped
researchers to recognize that these polypeptides were
functionally analogous to the integral membrane L and M
polypeptides of the reaction centers in the purple non-sulfur bacteria (summarized in [32]). In addition, freeze-etch
electron microscopy of thylakoid membranes from mutant
and wild-type Chlamydomonas strains has led to the association of defined particles of the thylakoid membranes with
specific complexes involved in photosynthesis [33].
Visualization of these particles suggested that complexes
of the photosynthetic apparatus are in a fluid state within
the membranes, as shown by the lateral movement of
phosphorylated light-harvesting complexes to nonappressed regions of the thylakoid membranes (this
movement is associated with a state transition) [34].
Finally, detailed biochemical approaches, targeted disruptions and site-directed mutagenesis are all helping to
define interactions between the different polypeptides of
the photosystems.
A number of recent reports describe research in which
molecular tools have been used to elucidate photosynthetic processes. Mutants in chlorophyll b biosynthesis were
isolated by transformation tagging with the ARG7 gene
[35••]. The lesions were found to be in a gene encoding a
putative protein with sequence similarity to a methylmonooxygenase. This work has helped to elucidate both
mechanistic and evolutionary aspects of chlorophyll
biosynthesis [36••].
Wollman, Merchant and collaborators [37,38,39•] have isolated several nuclear mutants that define elements
involved in the biogenesis of the cytochrome complex.
The nuclear loci CCS1,2,3,4 are involved in the attachment
of c-heme to cytochrome f, and the nuclear loci CCB1,2,3,4
are involved in b-heme binding to cytochrome b6. Other
nuclear loci are important for the maturation and/or stabilization and translation of transcripts encoding the
individual components of the cytochrome b6f complex
[40•]. The work on the cytochrome complexes has opened
new horizons that would have been very difficult to
explore in vascular plants. This work also highlights the
fact that much of the control of chloroplast biogenesis is at
the level of post-transcriptional and translational events,
which we are only just beginning to understand
[41,42••,43,44••,45•,46].
Recently, an elegant selection was performed that yielded
several mutants with an altered ability to transport proteins
across the thylakoid membranes [47••]; analysis of these
mutants, designated TIP (for thylakoid insertion proteins),
should add greatly to our knowledge of protein targeting
and chloroplast biogenesis.
Another topic of considerable interest is the acclimation of
the photosynthetic apparatus to changing environmental
conditions. Chlamydomonas and vascular plants must tune
their photosynthetic activity to their environmental nutrient status (which determines their growth potential);
these alterations include the accumulation of photoinhibited reaction centers and the formation of centers that
exhibit a much lower rate of QB (i.e. the secondary electron acceptor of photosystem II) reduction [48•].
Chlamydomonas also modulates photosynthetic function as
a direct response to light levels. Recently, mutants resistant to very high light [49] or defective in the dissipation
of excess excitation energy [50] have been isolated. A
Chlamydomonas reinhardtii and photosynthesis Grossman
video-imaging
screen
identified
mutants
of
Chlamydomonas that were defective in the xanthophyll
cycle and also exhibited a reduced ability to eliminate
excess absorbed light energy [50]; this was the first genetic evidence linking energy dissipation to the production of
specific xanthophylls. Similar findings were recently
obtained with Arabidopsis [51••], although the xanthophyll
cycle does seem to have a more dominant role in energy
dissipation in vascular plants than in Chlamydmonas.
Recently, the analysis of another mutant unable to dissipate excess absorbed light energy was shown to be
missing a minor component of photosystem II designated
PsbS, which is related to the light harvesting chlorophylla,b-binding proteins [52••]. This finding has important
implications with respect to both light harvesting function
and evolution. A video-imaging screen was also used to
isolate mutants that are unable to undergo state transition
(or to perform phosphorylation of the light-harvesting
antennae proteins) [53•,54•]. Furthermore, recent work of
Schroda et al. [7••] has demonstrated that the chloroplastlocalized chaperone HSP70B is involved in both the
protection and repair of photosystem II in high light.
Applications of genomics, and conclusions
The physiological, genetic and molecular manipulations
that have become routine for Chlamydomonas make this
organism ripe for genome-wide analyses. The global
analysis of the genome will add considerably to our
understanding of photosynthetic function and the way in
which photosynthesis is modulated as environmental
conditions change [55]. Sequence analyses of cDNAs
derived from RNA isolated from Chlamydomonas cells
that are exposed to a number of different environmental
conditions will inundate the research community with
interesting sequences that can be used as substrates for
functional genomics. cDNA microarrays will provide a
global view of gene regulation under a number of different environmental conditions. Analysis of high-density
DNA microarrays with cDNA probes generated from
putative ‘regulatory mutants’ will enable investigators to
identify the targets of specific regulators and the
processes that are affected in these mutant strains;
Chlamydomonas has a number of putative regulatory
mutants that can be used immediately for such studies.
Finally, establishing a high-density physical map that is
linked to the genetic map [56] will make the positional
cloning of genes that are altered (e.g. point mutations) in
specific mutants at least an order of magnitude more
rapid than in vascular plants.
Acknowledgements
I thank all of the co-investigators, including Pete Lefebvre, John Davies,
Carolyn Silflow, Elizabeth Harris and David Stern, who worked with me to
create a Chlamydomonas genome project and acknowledge the National
Science Foundation for funding this project. There are many excellent
articles in which genetics and molecular strategies were used to dissect
photosynthetic processes in Chlamydomonas that have not been discussed in
this review because of severe length restrictions. I wish to apologize to those
whose work has not received appropriate attention.
135
References and recommended reading
Papers of particular interest, published within the annual period of review,
have been highlighted as:
• of special interest
•• of outstanding interest
1.
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reverse genetics. Trends Plant Sci 1998, 3:209-210.
2.
Goodenough UW: Green yeast. Cell 1992, 70:533-538.
3.
Zhang H, Herman PL, Weeks DP: Gene isolation through genomic
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Vashishtha M, Segil G, Hall JL: Direct complementation of
Chlamydomonas mutants with amplified YAC DNA. Genomics
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Davies JP, Weeks DP, Grossman AR: Expression of the arylsulfatase
gene from the β2-tubulin promoter in Chlamydomonas reinhardtii.
Nucleic Acids Res 1992, 20:2959-2965.
6.
•
Fuhrmann M, Oertel W, Hegemann P: A synthetic gene coding for
the green fluorescent protein (GFP) is a versatile reporter in
Chlamydomonas reinhardtii. Plant J 1999, 19:353-361.
A gene encoding the GFP was synthesized on the basis of the codon usage
of Chlamydomonas and fused to the RBCS2 promoter (whose chimeric
gene designation is cgfp). When this synthetic gene is fused to either the
phleomycin resistance gene ble (the gene product is localized in the nucleus) or the chlamyopsin gene cop (the gene product is localized in the eyespot) and introduced into Chlamydomonas, GFP fluorescence is visualized
in the correct subcellular compartment. The cgfp gene will be a very useful
tool for studying both gene regulation and the localization of gene products
in vivo.
7.
••
Schroda M, Vallon O, Wollman F-A, Beck C: A chloroplast targeted
heat shock protein 70 (HSP70) contributes to the photoprotection
and repair of photosystem II during and after photoinhibition.
Plant Cell 1999, 11:1165-1178.
Both sense and antisense constructs of the Chlamydomonas HSP70B gene
(encoding heat shock protein of 70 kDa, a chloroplast-localized chaperone)
were transformed back into Chlamydomonas to alter the expression level of
HSP70B in the cells. Chlamydomonas cells synthesizing high levels of
HSP70B were more resistant to photoinhibition and recovered from photoinhibition more rapidly than cells expressing normal levels of HSP70B.
Furthermore, cells with lowered expression of the HSP70B gene exhibited
increased light sensitivity. These results suggest that the HSP70B chaperone
functions in both protecting photosystem II reaction centers from damage in
intense light and the process by which damaged reaction centers are repaired.
8.
Tam LW, Lefebvre PA: Cloning of flagellar genes in
Chlamydomonas reinhardtii by DNA insertional mutagenesis.
Genetics 1993, 135:375-384.
9.
Debuchy R, Purton S, Rochaix J-D: The argininosuccinate lyase
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•
Identification of cis-acting RNA leader elements required for
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••
Bernd KK, Kohorn BD: Tip loci: six Chlamydomonas nuclear
suppressors that permit the translocation of proteins with mutant
thylakoid signal sequences. Genetics 1998, 149:1293-1301.
Mutations in the leader sequence of the cytochrome f protein that inhibit its
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mutants and sequence characterization of the genes altered in the suppressor strains will elucidate the apparatus of the photosynthetic membranes
that is involved in protein translocation.
48. Wykoff DD, Davies JP, Grossman AR: The regulation of
•
photosynthetic electron transport during nutrient deprivation in
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137
Chlamydomonas of either sulfur or phosphorus leads to a downregulation of
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•• a central role for the xanthophyll cycle in the regulation of
photosynthetic energy conversion. Plant Cell 1998, 10:1121-1134.
A video-imaging system developed for use with Chlamydomonas was used
to screen Arabidopsis plants for alterations in non-photochemical quenching.
This screen resulted in the isolation of mutants that are defective in the xanthophyll cycle. The npq1 mutant, which was unable to convert violaxanthin to
zeaxanthin in intense light, was more severely limited in its ability to dissipate
excess excitation than the analogous mutant in Chlamydomonas. Hence,
although both Chlamydomonas and Arabidopsis use the xanthophyll cycle for
the dissipation of excess excitation, the proportions of excess excitation energy that is dissipated in this way seems to differ in the two organisms.
52. Li X-P, Bjôrkman O, Shih C, Grossman AR, Rosenquist M, Jansson S,
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photosynthetic light harvesting. Nature 2000, 403:391-395.
The authors show that the gene encoding PSBS, which is a chlorophyllbinding protein that is intrinsic to photosystem II, is necessary for the dissipation of excess absorbed light energy, but is not required for photosynthetic
oxygen evolution. A four membrane-helix PSBS-like protein was probably the
evolutionary precursor of the three membrane-helix LHC proteins, the major
components of the light harvesting antennae. These findings suggest that
the function of energy dissipation may have evolved prior to the current-day
light-harvesting function found in vascular plants.
53. Kruse O, Nixon PJ, Schmid GH, Mullineaux CW: Isolation of state
•
transition mutants of Chlamydomonas reinhardtii by fluorescence
video imaging. Photosynth Res 1999, 61:43-61.
This paper, like [54•], describes how video-imaging techniques can be used
to isolate state transition mutants. Characterizations of these mutants are
helping to elucidate the dynamics of the photosynthetic apparatus and the
environmental factors that modulate photosynthetic activities.
54. Fleishmann MM, Ravanel S, Delosme R, Olive J, Zito R, Wollman FA,
•
Rochaix J-D: Isolation and characterization of photoautotrophic
mutants of Chlamydomonas reinhardtii deficient in state
transition. J Biol Chem 1999, 274:30987-30994.
The work in this paper and [53•] demonstrates that video-imaging techniques
can be used to isolate state transition mutants. Characterizations of these
mutants are helping to elucidate the dynamics of the photosynthetic apparatus and the environmental factors that modulate photosynthetic activities.
55. Davies JP, Grossman AR: The use of Chlamydomonas
(Chlorophyta: Volvocales) as a model algal system for genome
studies and the elucidation of photosynthetic processes. J Phycol
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