Chlamydomonas As A Model Organism

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Annu. Rev. Plant Physiol. Plant Mol. Biol. 2001. 52:363406

c 2001 by Annual Reviews. All rights reserved
Copyright
CHLAMYDOMONAS AS A MODEL ORGANISM

Annu. Rev. Plant. Physiol. Plant. Mol. Biol. 2001.52:363-406. Downloaded from www.annualreviews.org
by University of Sussex on 10/06/12. For personal use only.
Elizabeth H Harris
Developmental, Cell and Molecular Biology Group, Biology Department, Duke
University, Durham, North Carolina 27708-1000; e-mail: chlamy@duke.edu
Key Words Volvocales, flagella, chloroplast, basal body, phototaxis

Abstract The unicellular green alga Chlamydomonas offers a simple life cycle,
easy isolation of mutants, and a growing array of tools and techniques for molecular
genetic studies. Among the principal areas of current investigation using this model
system are flagellar structure and function, genetics of basal bodies (centrioles), chloroplast biogenesis, photosynthesis, light perception, cell-cell recognition, and cell cycle
control. A genome project has begun with compilation of expressed sequence tag data
and gene expression studies and will lead to a complete genome sequence. Resources
available to the research community include wild-type and mutant strains, plasmid
constructs for transformation studies, and a comprehensive on-line database.
CONTENTS
INTRODUCTION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
THE GENUS CHLAMYDOMONAS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Major Taxonomic Features . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Laboratory Strains of C. reinhardtii . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
CELL ARCHITECTURE AND FUNCTIONAL SYSTEMS . . . . . . . . . . . . . . . . . .
The Cell Wall . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
The Nucleus and the Nuclear Genome . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Contractile Vacuoles and Osmotic Regulation . . . . . . . . . . . . . . . . . . . . . . . . . . .
Mitochondria . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
The Chloroplast . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Flagella . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Basal Bodies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
The Eyespot and Phototaxis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
MITOTIC AND MEIOTIC LIFE CYCLES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Vegetative Cell Growth . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
The Sexual Cycle . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Tetrad Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
PHYSIOLOGICAL PROCESSES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Nutrient Update and Metabolic Stress . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Light-Mediated Responses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Circadian Rhythms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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TECHNIQUES AND RESOURCES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 387
The Molecular Tool Kit . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 387
Sources of Strains and Information . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 391
INTRODUCTION
At the Fifth International Chlamydomonas Conference in 1992, the venerable
phycologist Ralph Lewin delivered a keynote address with the title, The Cloaked
One Emerges from Obscurity. Lewins talk referred to the development of research
on the unicellular green alga Chlamydomonas (Greek chlamys, a cloak; monas,
solitary), beginning with nineteenth-century morphological descriptions and the
first genetics studies in the early twentieth century. Pascher (1916, 1918, cited in
86) demonstrated the suitability of Chlamydomonas species for genetic analysis
and pointed out the advantages of a haploid system in which all four products of
meiosis could be recovered and analyzed. His investigations were not continued,
but interest was soon renewed in this alga as a eukaryotic organism whose life
cycle could be controlled in the laboratory. The work of Franz Moewus in the
1930s demonstrated that mutants could be isolated and characterized, but was
clouded by irreproducibility of some of the reported results; it was only with the
work of Lewin himself, Ruth Sager, and others in the 1940s and 1950s that a few
Chlamydomonas species, in particular C. reinhardtii and C. eugametos, began to
be developed as laboratory organisms (see 86 for review). The past 50 years have
indeed seen the emergence of this organism from obscure beginnings into one of
the premier model systems for diverse areas of cell and molecular biology.
Lewins 1992 talk concluded with the arrival of his subject in the new age
of molecular biology. Transformation of the chloroplast, nuclear, and mitochondrial genomes had been achieved, and the research presented at that meeting gave
clear evidence that rapid progress lay ahead in developing new technologies for
isolation and manipulation of genes. We are now embarking on a new era, as
Chlamydomonas enters the age of genomics. Sequencing, gene expression studies, and molecular mapping projects are under way, and prospects are good for a
complete genome sequence of C. reinhardtii. A review of the main features of this
organism and its laboratory manipulation seems most appropriate at this time.
THE GENUS CHLAMYDOMONAS

Major Taxonomic Features
Historically, species of Chlamydomonas have been defined based solely on morphological criteria. The genus comprises unicellular chlorophyte algae with two
anterior flagella, a basal chloroplast surrounding one or more pyrenoids, and a
distinct cell wall (Figure 1). Species within the genus have been distinguished by
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CHLAMYDOMONAS
Figure 1 A semidiagrammatic representation of an interphase Chlamydomonas cell. Cell

length, 10 m; BB, basal bodies; Chl, chloroplast; Cv, contractile vacuole; Cw, cell wall;
Er, endoplasmic reticulum; Es, eyespot; F, flagella; G, Golgi apparatus; L, lipid body;
Mi, mitochondria; N, nucleus; No, nucleolus; P, pyrenoid; r, ribosomes; S, starch grain; v,
vacuole. From (85), originally adapted from a figure by H Ettl, courtesy of John Harper.
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differences in overall size and body shape, shape and position of the chloroplast and
pyrenoids, flagellar length, number and position of contractile vacuoles, and more
subtle structural features visible at the light microscope level. Ettl (1976, cited
in 86) recognized 459 species, which he consigned to nine major morphological
groups. Although many of these species are represented in culture collections, only
a few have found significant roles as laboratory research organisms.
C. reinhardtii has emerged as the predominant laboratory species of Chlamydomonas, primarily owing to its ability to grow nonphotosynthetically with acetate as its sole carbon source, and is discussed at length below. Some research
studies have utilized the interfertile species pair C. eugametos and C. moewusii.
C. eugametos, of European origin, derives ultimately from Moewus and has been
used particularly for investigation of sexuality, where it forms a useful contrast to
C. reinhardtii. Gowans isolated a number of nutritional and resistance mutations
in the C. eugametos background, but these are not under active investigation at
present. C. moewusii was isolated in New York in 1948 by Provasoli and was used
soon afterwards by Lewin for selection of flagellar mutants. One of the most active current topics of research with C. eugametos and C. moewusii is phospholipidmediated signal transduction (see 119, 152 and references cited therein). Sequence
of the nuclear genes encoding 18S ribosomal RNA, the nuclear ribosomal DNA
spacer ITS2, and chloroplast ribosomal RNAs place C. moewusii and C. eugametos
in a group more closely allied to Haematococcus and Chlorogonium than to the
C. reinhardtii cluster (see 35). The predicted evolutionary distance between
C. reinhardtii and C. eugametos/C. moewusii is consistent with the marked differences in chloroplast architecture and overall cell morphology and steps in the
mating reaction, and with the inability of C. eugametos and C. moewusii to use
acetate as their sole carbon source, presumably a very fundamental physiological
difference. One suspects that the characters that place all these species within the
same genus (two flagella, cell wall, presence of a pyrenoid) are not sufficient to define the genus Chlamydomonas as a phylogenetic entity. Volvox carteri, Pandorina
morum, and some other colonial Volvocales used as research organisms appear to
be closely allied with C. reinhardtii based on molecular criteria (21, 35, 131).
C. monoica is a homothallic Chlamydomonas species that has been used to
investigate the processes of mating, chloroplast gene inheritance, and zygospore
formation (228, 230). Some experimental work has also been done in various
laboratories on C. geitleri, C. segnis, C. chlamydogama, and several other species
(see 86).
Except as otherwise indicated, the remainder of this article focuses on
C. reinhardtii.
Laboratory Strains of C. reinhardtii

The principal laboratory strains of C. reinhardtii are thought to derive from isolates made by GM Smith in 1945 from soil collected near Amherst, Massachusetts.
Smith gave cultures to Sager, Lewin, Hartshorne, and perhaps others, and three
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main lineages deriving from Smiths collection have been separate since approximately 1950 (see Harris, Chapter 1 in 168). Although analyses of transposon
insertion sites and chloroplast DNA restriction digests strongly suggest that all
these strains do have a common origin, especially when compared with interfertile isolates from other localities (see 35 for citations), they are distinguished by
inability of some of the strains to assimilate nitrate and by variability in light
requirements for gametogenesis (202). AW Coleman (personal communication)
has found significant differences among several of the Smith-derived strains in sequence of the ribosomal spacer ITS2 and has advanced the hypothesis that Smith
may have distributed more than one original isolate. The situation has been complicated further by later crosses among representatives of these three lineages and
by poorly documented transfers of strains among laboratories.
It is only now, as sequencing of the entire genome begins, that nucleotide variations among descendants of the Smith strains are becoming significant. Sagers
strain 21 gr (Chlamydomonas Genetics Center strain CC-1690) has been chosen
as the primary target for sequencing efforts. Extensive EST (expressed sequence
tag) data are also available from strain C9 (4), equivalent to 21 gr but separate from
it since approximately 1955, and from CC-125, Smiths 137C strain as used by
Levine and Ebersold, which differs from 21 gr and C9 in lacking nitrate reductase
activity.
Strains S1 D2 and S1 C5 are isolates from soil collected in Plymouth,
Minnesota (CH Gross et al, 1988, cited in 35). Although fully interfertile with
21 gr and 137C, these strains are distinguished from the Smith isolates by extensive nucleotide polymorphisms, especially in noncoding regions. The genomes appear to be colinear, however, without major chromosome rearrangements. A cross
between 21 gr and S1 C5 has been used as the foundation of the molecular map
(<http://www.biology.duke.edu/chlamy-genome/maps.html>), and EST data are
also being obtained from this strain. Preliminary analyses indicate less than 1%
sequence divergence between S1 D5 and the Smith strains in coding regions but
as much as 7% in 30 untranslated regions (CR Hauser, personal communication).
Isolates of C. reinhardtii have also been made from Quebec, Pennsylvania,
North Carolina, and Florida, and may provide an additional source of diversity in
future (see 35 for citations). A second Massachusetts isolate made by Smith in
1946, designated C. smithii mating type plus by Hoshaw & Ettl, should also be
considered as part of the C. reinhardtii group based on interfertility (86). However,
the strain identified by Hoshaw and Ettl as C. smithii mating type minus (CC-1372,
from Santa Cruz, California) does not belong with the C. reinhardtii group based
on its lack of full fertility and on DNA sequence criteria that suggest it is more
closely related to C. culleus (35). All authentic C. reinhardtii isolates to date thus
appear to derive from North America east of the Rocky Mountains.
Two additional isolates have been placed with C. reinhardtii (UG Schlosser
1984, cited by 35) based on their susceptibility to the C. reinhardtii vegetative
cell lytic enzyme or autolysin, although they do not appear to be cross-fertile with
the authentic C. reinhardtii strains (58). Originally identified as C. incerta (SAG
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7.73, supposedly from Cuba), and C. globosa (SAG 81.72, supposedly from the
Netherlands), these isolates appear to be identical based on restriction fragment
analysis of chloroplast DNA (EH Harris et al 1991, cited by 35), the ribosomal ITS
sequences (35), HindIII and PstI digests of total DNA probed with the beta-tubulin
gene (59), and intron sequences from the Ypt4 gene (59, 131).
CELL ARCHITECTURE AND FUNCTIONAL SYSTEMS

The Cell Wall
The wild-type C. reinhardtii cell (Figure 1) averages about 10 m in diameter
(with significant variation through the cell cycle) and is enclosed within a wall
consisting primarily of hydroxyproline-rich glycoproteins that resemble plant extensins. Contrary to a few erroneous early reports that have unfortunately been
perpetuated in some textbooks, the C. reinhardtii cell wall does not contain cellulose. The wild-type wall comprises seven principal layers (241). Genes for some
wall components have been cloned and sequenced, and many mutants with defects
in cell wall biogenesis have been isolated. Most of these mutants seem to make the
precursor proteins of the wall in normal amounts but fail to assemble them into
complete walls (232). Cell wall mutants have found widespread use as recipients
for transformation with exogenous DNA, a process that is much more efficient
with wall-less cells.
The Nucleus and the Nuclear Genome

The cell nucleus and nucleolus are prominent in cross-sections of Chlamydomonas
cells. The nuclear membrane is continuous with the endoplasmic reticulum, and
one to four Golgi bodies are situated nearby. Chromosome cytology is poor, with
only eight discrete chromosomes being consistently visible by light microscopy in
metaphase cells. Electron microscopy of synaptenemal complexes suggested 16
or more chromosomes, a number that is consistent with the 17 linkage groups now
defined by genetic analysis. Attempts to separate chromosomes electrophoretically have not been fully successful (82). Vegetative cells are normally haploid,
but stable diploids can be selected using auxotrophic markers. The nuclear genome
size is estimated at approximately 1 108 base pairs (86, 194). It is GC-rich, approximately 62% overall in denaturation studies; sequence analysis gives a similar
figure. This high GC content may produce difficulties in cloning genes. Amplification is improved by selection of primer sequences with 45% to 50% G-C content,
and by including c7dGTP in the PCR reaction mixture (see 189 for methods).
Contractile Vacuoles and Osmotic Regulation

Two contractile vacuoles are located at the anterior end of the C. reinhardtii cell.
Mutants requiring hyperosmotic media for survival (136) may provide a starting
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point for a study of genetic control of vacuole structure and function. Salt-sensitive
mutants have also been isolated but are thought to affect ion transport across the
plasma membrane and have not been implicated directly in vacuole function (179).
Mitochondria
Mitochondria are dispersed throughout the cytosol and are sometimes seen in
electron micrographs as elongated or branching structures. The difficulty of purifying mitochondria free of chloroplast contamination has limited biochemical
research on Chlamydomonas mitochondria, and methods for purification of active
mitochondria have been developed only recently (14, 53). The 15.8-kb mitochondrial genome is linear and contains only a few genes: cob, cox1, five subunits of
mitochondrial NADH dehydrogenase, the mitochondrial rRNAs (which are fragmented in the DNA sequence); three tRNAs, and an opening reading frame that
resembles a reverse transcriptase (see GenBank Accession number U03843 for
complete sequence and citations). Mutants that delete the cob gene are unable to
grow on acetate in the dark but are viable when grown phototrophically. Point mutations in the cob gene can confer myxothiazol resistance. Nuclear mutants with
respiratory deficiencies and a dark-dier phenotype have also been obtained (see 45
for citations).
The Chloroplast
A single cup-shaped chloroplast occupies the basal two thirds of the cell and partially surrounds the nucleus. Thylakoid membranes are arranged in well-defined
appressed and non-appressed domains whose composition and functional organization have been extensively investigated in wild-type and mutant strains of
Chlamydomonas (Olive & Wollman, in 196). A distinctive body within the chloroplast, the pyrenoid, is the site of CO2 fixation and the dark reactions of photosynthesis. Starch bodies surround the pyrenoid and are also seen dispersed
throughout the chloroplast under some conditions of growth. Presence or absence
of a pyrenoid distinguishes Chlamydomonas from the genus Chloromonas, and
within the genus Chlamydomonas, the number and arrangement of the pyrenoids
is an important species character in traditional taxonomy.
Sequencing of the 195-kb chloroplast genome is nearly complete (DB Stern and
colleagues, personal communication). The chloroplast genomes of all Chlamydomonas species examined have an inverted repeat structure reminiscent of that
of most land plants, but gene order differs markedly from the plant model and
cannot be accounted for by any simple scheme of rearrangements or inversions.
The gene content of the chloroplast genome largely does resemble that of land
plants, however, with only a few significant differences (19).
Chlamydomonas became known early on as an excellent model system in which
to study both photosynthesis and biogenesis of the chloroplast. The y1 mutant, originally isolated by Sager, loses chlorophyll and forms only a rudimentary proplastid
when cultured on acetate-containing medium in the dark. On exposure to light,
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y1 cells become green and form a complete chloroplast structure over the course
of approximately 8 h. Although not precisely analogous to greening of an etiolated plant seedling in terms of how this process is regulated, y1 re-greening has
nevertheless proved to be a powerful and accessible system for studying synthesis
and assembly of the photosynthetic machinery, and for investigation of the relative
roles of nuclear and chloroplast genomes in this process (237 and references cited
therein).
One avenue of research arising from these early studies has been investigation of the genetic control of chlorophyll synthesis (see 12 for review of chlorophyll biosynthesis in general). Besides y1, there are several additional, nonallelic,
mutants that show a yellow-in-the-dark phenotype, including some temperaturesensitive alleles (see 23). C. reinhardtii has two pathways for conversion of
protochlorophyllide to chlorophyllide. The y mutants are blocked in the lightindependent pathway, homologous to the protochlorophyllide reductase seen in
organisms ranging from purple bacteria through gymnosperms (23, 126). The core
enzyme in Chlamydomonas consists of three subunits coded by the chloroplast
genes chlB, chlL, and chlN. The nuclear loci Y1 and Y5 through Y10 in C. reinhardtii
all appear to be involved in expression of these chloroplast genes and/or assembly of
their products (23). The pc1 mutant is blocked in light-mediated protochlorophyllide conversion because of a deletion in the gene encoding NADPH:protochlorophyllide oxidoreductase (130), which is equivalent to the light-dependent enzyme
found in angiosperms (126). Other mutants in the chlorophyll biosynthetic pathway
in Chlamydomonas include strains in which formation of Mg protoporphyrin from
protoporphyrin IX is blocked and mutants specifically deficient in chlorophyll b.
A signal transduction pathway for light-induced expression of glutamate 1-semialdehyde aminotransferase, an early enzyme in synthesis of both chlorophyll and
heme, has been analyzed by Im & Beale (101).
In the 1960s, Levines laboratory at Harvard produced a series of papers demonstrating that the photosynthetic electron transfer chain was amenable to genetic
dissection. Nonphotosynthetic mutants of C. reinhardtii were isolated, and identified by the prefix ac, for acetate-requiring. Many of these mutants were assigned
to specific processeswater oxidation, photosynthetic electron transport, ATP
synthesis, CO2 fixationbut identification of lesions in specific proteins was not
possible with technology available at that time. Investigation in the early years was
limited to mutations in nuclear genes. A breakthrough was made in 1979, however, with development of methods to select nonphotosynthetic mutations by using
5-fluorodeoxyuridine to reduce the number of copies of the chloroplast genome
prior to mutagenesis (HS Shepherd et al 1979, cited in 86). The list of photosynthetic genes cloned and marked by mutations is now impressive (41) (Table 1).
Chloroplast transformation with exogenous DNA occurs by homologous replacement (see below), thereby potentially permitting analysis by site-directed mutagenesis of every chloroplast gene. More than 50 nuclear gene loci affecting chloroplast
biogenesis and photosynthetic functions are marked by mutations, and nearly all
the structural genes for chloroplast components known in land plants have also
been identified in Chlamydomonas either by complete sequencing or as ESTs.
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TABLE 1 Representative mutations affecting photosynthesis and the chloroplast in

Chlamydomonas reinhardtii
Component
Photosystem II reaction center and water-splitting complex
Deletions of chloroplast-encoded psbA gene
Site-directed mutations in psbA affecting photosynthesis
Herbicide-resistance mutations in psbA
Nuclear mutations that cause accumulation of excess D1 protein or
make D1 unstable at high light intensity
Nuclear mutations that destabilize psbB mRNA
Mutation in chloroplast-encoded psbC gene
At least four nuclear mutations affecting translation of psbC
Induced and site-directed mutations in chloroplast-encoded psbD gene
At least three nonallelic nuclear mutations affecting psbD translation
Chloroplast psbD mutation producing unstable mRNA;
three nuclear loci have been identified that suppress this mutation
psbE null mutant
Disruption of chloroplast-encoded psbH gene and site-directed
mutations in this gene
Disruption of chloroplast psbI gene; can grow photosynthetically
but is light sensitive
Disruption of chloroplast psbK gene
Disruption of chloroplast ycf 8 gene; impairs PS II function under
stress conditions
Transposon insertion in PsbO gene
Two allelic nuclear mutants deficient in OEE2 protein
State-transition mutations affecting LHCII phosphorylation
Cytochrome b6 /f complex
Site-directed point and deletion mutations in chloroplast-encoded
petA gene
Five allelic nuclear mutations affecting petA mRNA stability
and/or maturation
Point and deletion mutations in chloroplast-encoded petB gene
A nuclear mutation affecting petB mRNA stability and/or
maturation
Induced, null and site-directed mutations in the nuclear-encoded
PetC gene
Numerous induced and site-directed mutations in chloroplast-encoded
petD gene
Two nuclear mutations affecting petD mRNA stability and/or
maturation
Several allelic mutations in nuclear-encodedPetE gene
Deletion of chloroplast-encoded petG gene
A nuclear mutation affecting petG mRNA stability and/or maturation
References
See (86)
(87, 137, 220, 249)
See (86)
(254)
(225)
(195)
(195, 253)
(107)
(18, 155)
(155)
(150)
(163, 219)
See (219)
See (219)
See (194)
See (194)
(200)
(118)
(11, 31, 32, 36)
(41)
(41, 257)
(41)
(41)
(41, 92, 256)
(41)
(129)
See (41)
(41)
(Continued )
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TABLE 1 (Continued )
Component
Five nuclear gene loci involved in synthesis of chloroplast c-type
cytochromes
Chloroplast mutations in the ccsA ( ycf5) gene, encoding a protein
required for heme attachment of cytochrome c
At least four nuclear mutations affecting heme attachment to
cytochrome b6
Disruptions of chloroplast ycf 7 ( petL) gene; distabilization of
the cytochrome b6/f complex
Deletion of petO gene
Photosystem I reaction center
Deletion or disruption of chloroplast-encoded psaA gene
Site-directed point mutations in psaA
Disruption of tscA, a 430-nt RNA involved in psaA trans-splicing
At least 5 nuclear loci affecting psaA exon 23 splicing
At least 2 nuclear loci affecting both trans-splicing steps
At least 7 nuclear loci affecting exon 12 trans-splicing
Frame-shift and site-directed mutations in chloroplast psaB gene
Mutation in a nuclear gene that blocks a post-transcriptional step in
psaB expression
Site-directed mutations in chloroplast psaC gene
Mutations in nuclear PsaF gene
Disruptions of chloroplast ycf3 and ycf4 genes; produce PS I
deficiency
Site-directed mutations in ycf3 and ycf4
Insertional mutations in nuclear Crd1 gene; blocked in response to
copper deficiency, fail to accumulate PS I and LHC I
Xanthophyll cycle and photoprotection
At least three nonallelic nuclear mutants blocked in xanthophyll cycle
Several nonallelic nuclear mutants resistant to very high light intensities
Photophosphorylation
Site-directed mutations in chloroplast atpA gene
Nuclear mutation affecting translation of chloroplast atpA gene
Nuclear mutation that destabilizes atpA mRNA
Nuclear mutation that destabilizes atpB mRNA
Many point and deletion mutations in chloroplast atpB gene
Site-drected mutations in atpB
Site-directed mutations in nuclear AtpC gene
Induced mutations in chloroplast atpE, atpF and atpI genes
Nuclear mutation affecting expression of the chloroplast-encoded
atpH and atpI genes
CO2 uptake
Mutation in nuclear CAH3 gene encoding intracellular carbonic
anhydrase
Additional mutants that require high levels of CO2 for growth
References
(102, 246)
(246, 247)
(120)
(222)
(222)
(83)
(36)
(55)
(195)
(173, 195)
(63)
(195, 215)
(63)
(62, 93)
(20, 41)
(20)
(151)
(161)
(65)
(44)
See (41)
See (86)
e.g. (29, 30, 99)
(114, 199)
See (44)
(71, 218)
(110, 166)
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TABLE 1 (Continued )
Component
Disruption of chloroplast ycf10 gene; produces inefficient carbon

uptake into chloroplast
nit1-tagged mutants affecting CO2 uptake
Carbon fixation
Many mutations in chloroplast rbcL gene
Site-directed mutation in rbcL that alters its specificity for
Rubisco activase
Nuclear mutation that inhibits rbcL expression; second-site
suppressors of this mutation
Point mutation in structural gene for phosphoribulokinase
Chlorophyll biosynthesis
Deletion mutation in gene encoding NADPH:protochlorophyllide
oxidoreductase
Nuclear mutations in at least six loci affecting expression of the
chloroplast-encoded chlB, chlL, and chlN genes and/or assembly
of the protochlorophyllide reductase complex
Two allelic nuclear mutants in the nuclear gene encoding Mg
chelatase
Chloroplast protein synthesis and protein translocation
Many antibiotic resistance mutations in 16S and 23S ribosomal
RNA genes
Nuclear mutation that blocks processing of chloroplast rRNA
Additional nuclear mutations that result in deficiency in
chloroplast ribosomes
Nuclear mutations that suppress site-directed alterations
in thylakoid signal sequences
Disruption of chloroplast-encoded clpP protease
Nuclear mutation affecting LHC assembly, probably at the
level of a chaperone protein
References
(197)
(69)
See (191)
(125)
(98)
(7)
(130)
(23)
(E Chekunova,
personal
communication)
See (86)
(96)
See (86)
(15)
See (194)
(H Naver,
personal
communication)
Among the greatest strengths of Chlamydomonas as a model organism in which

to study chloroplast biogenesis has been its use to identify nuclear genes that
regulate the expression of genes encoded in the chloroplast. Many of the mutants
isolated in Levines laboratory have turned out to be involved in processing of
chloroplast mRNAs or other regulatory steps. For example, the ac115 mutant,
isolated by Gillham in 1960 and initially described as lacking several proteins
of photosystem II (PS II), has finally revealed its true nature (190). The Ac115
gene product, a small basic protein with a potential membrane-spanning domain
at the carboxyl terminus, is required for translation of mRNA for the chloroplastencoded psbD gene, encoding the D2 protein of the photosystem II reaction center.
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The original ac115 allele is a nonsense mutation near the 50 end of this gene.
The nuclear NAC1 and NAC2 loci, unlinked to AC115, are also involved in psbD
translation, and a mutation that suppresses both ac115 and nac1 has been found
(HY Wu & MR Kuchka, cited in 190). In nac2 mutants, the psbD mRNA is unstable,
leading to failure of assembly of the PS II reaction center. The Nac2 gene product
is a 140-kDa hydrophilic polypeptide containing nine tetratricopeptide repeats
(18). Additional nuclear mutants have been isolated that suppress a site-directed
mutation within the 50 untranslated region of the psbD gene (155).
The chloroplast psaA gene presents an even more complex example of interactions between nuclear and chloroplast genes in Chlamydomonas. This gene is split
into three separately transcribed exons in C. reinhardtii, and the mRNA is assembled by trans-splicing. At least 14 different nuclear genes affect the trans-splicing
process, and a small chloroplast-encoded RNA molecule (tscA) is also required
(81, 155, 173).
Synthesis and assembly of components of the cytochrome b6/f complex have
been investigated in several laboratories (e.g. 11, 32, 120, 257). As is also true for
the reaction centers of photosystems I and II, this complex contains both chloroplast
and nuclear gene products, and additional nuclear genes may be required for control
of chloroplast gene expression. For example, one chloroplast locus and at least four
nuclear loci are required for heme attachment to the cytochrome c apoprotein (246).
Wollman et al (cited in 32) have proposed a general model for stoichiometric
accumulation of chloroplast-encoded proteins based on the concept of control of
epistasy of synthesis or CES. Synthesis of some chloroplast-encoded subunits
of the cytochrome b6/f complex (CES subunits) is strongly attenuated if other
subunits of the complex (dominant subunits) are absent. For example, mutants
deficient in either cytochrome b6 or subunit IV show a greatly reduced rate of
translation of cytochrome f, encoded by the chloroplast petA gene (32). However,
in mutants lacking cytochrome f, cytochrome b6 and subunit IV are synthesized at
normal rates and then degraded. Choquet et al (32) showed that the 50 untranslated
region of the petA mRNA regulates its own translation by interaction, either directly
or through an intermediary protein, with the C-terminal domain of the unassembled
cytochrome f protein.
Merchant and colleagues have studied regulation of the copper-containing protein plastocyanin (129), and the c-type cytochromes (144, 248). Copper deficiency
in Chlamydomonas results in degradation of plastocyanin and induction of cytochrome c6 and coproporphyrinogen oxidase, and copper-responsive sequences
have been identified within the promoters of the Cyc6 and Cpx1 genes (185, 186).
Mutants at the newly identified CRD1 locus are chlorophyll deficient in the absence of copper and have defects in photosystem I (151). Restoration of copper
rescues both phenotypes. The Crd1 gene product is a 47-kDa hydrophilic protein
with a carboxylate-bridged di-iron binding site, and it appears to be required for
adaptation to either copper or oxygen deficiency.
These are only a few examples of the ways in which Chlamydomonas is currently
being used for investigation of the chloroplast and photosynthesis. For a much
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more comprehensive treatment, the recent book edited by Rochaix, GoldschmidtClermont & Merchant (196) is highly recommended. Rochaix (194) and Davies
& Grossman (38) have reviewed the use of Chlamydomonas for elucidation of
photosynthetic processes. Niyogi (159) has reviewed photoprotection and photoinhibition, a process for which Chlamydomonas is proving to be a very useful model (9, 65, 90, 104, 139, 160, 161, 210). Xiong et al (250) have published
a three-dimensional model for the photosystem II reaction center of Chlamydomonas in a paper that thoroughly reviews the literature on components of this
complex. For an assessment of photosystem I function and its indispensability, see Redding et al (192). There is also a substantial literature on transcriptional and translational control of chloroplast gene expression in Chlamydomonas
(44, 57, 73, 88, 116, 141, 252) and on processing of introns in chloroplast rRNA
and psbA genes (95). Goldschmidt-Clermont (76) has reviewed coordinated expression of nuclear and chloroplast genes in plant cells, including Chlamydomonas,
and Nickelsen & Kuck (156) have reviewed the use of C. reinhardtii as a model
system for study of chloroplast RNA metabolism. Chloroplast DNA replication
has also been studied in several laboratories (28 and references cited therein).
Flagella
Two anterior flagella, 10 to 12 m in length, protrude through specialized collar
regions in the cell wall. The structure of the flagellar axoneme has been described
thoroughly (147), and more than 250 component proteins have been resolved
by two-dimensional electrophoresis. From the very beginning, Chlamydomonas
has been among the very best organisms for research on flagellar function and
assembly. Mutants with defects in motility were among the first to be isolated both
in C. moewusii and C. reinhardtii. Many of these early mutants are extant and are
finally revealing their precise defects.
Mutations have been identified that affect nearly all of the principal components of the flagellar axoneme. More than 75 genetic loci have been identified in
C. reinhardtii that affect flagellar assembly and/or function, and more than 40
genes for flagellar components have been cloned and sequenced. Some of these
mutants have parallels in mutations affecting animal cilia and sperm cells (33,
167169). Complementation in the transient dikaryons formed after mating can
be used to identify specific proteins of the radial spokes, central pair, and other
complexes. Fusion of gametes of opposite mating type in C. reinhardtii produces a
cell with two nuclei and four flagella. This quadriflagellate cell remains motile for
about two hours before the flagella are resorbed and formation of the zygospore
wall begins. During this motile period, flagellar assembly continues, and polypeptides contributed by both parental gametes are incorporated into all four flagella.
Thus mating of two nonallelic paralyzed mutants usually results in restoration
of full motility in this transient dikaryon, as each partner supplies a wild-type
copy of the defective flagellar protein produced by the other gamete. The simple observation of restoration of motility makes a very nice laboratory exercise
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for students. Luck and colleagues (cited in 46, 86) used radioactive labeling and
two-dimensional gel electrophoresis to identify the specific proteins that were restored by mating labeled mutant cells to unlabeled wild-type cells. The reviews by
Dutcher (46) and Mitchell (147) of the genetics of flagellar assembly and structure
are highly recommended as an introduction to this field of research for the nonspecialist. Johnson (106) has reviewed flagellar beating motility and its structural
correlates. Detailed reviews have also been published on dyneins (176), radial
spokes (37), the central apparatus (213), kinesins (16), and intraflagellar transport
(198).
In addition to mutants with primary defects in flagellar components, many
second-site suppressor mutations have been found that restore partial or complete
motility, and these mutations have permitted identification of additional structural
components. Mutants with unusually long or short flagella are also known (5), and
the regulation of flagellar length through the cell cycle in wild-type cells is being
elucidated (224).
Gliding motility, by means of movement of the flagellar membrane, has long
been known, and nongliding mutants have been isolated (RA Bloodgood in 43).
This gliding motion can also be visualized by transport of adherent particles or
polystyrene beads along the rigid, extended flagella of certain mutants with defects
in the central pair microtubules (e.g. pf18; 17).
Flagellar assembly depends on yet another type of motility, intraflagellar
transport (IFT), first identified in Chlamydomonas by Rosenbaum and colleagues
(34, 117, 198). This process involves bi-directional movement of protein complexes (rafts) along the flagella. Transport toward the flagellar tip is mediated by
a kinesin, first identified as the site of the flagellar assembly mutation fla10 (117).
The return transport of rafts back toward the cell body is dependent on cytoplasmic dynein 1b and dynein light chain LC8 (34, 175). Mutants in which anterograde
IFT is disrupted may have short or stumpy flagella and may accumulate flagellar
proteins in the cell body, whereas mutants in which retrograde IFT is defective
display a bulge in the flagellar membrane (34, 175). One of the components of
IFT complex B shows homology to a mammalian protein implicated in a form of
polycystic kidney disease, known from a mouse mutant in which renal cilia are
abnormal. The IFT system is also implicated in retinitis pigmentosa, where retinal
photoreceptor cells, whose outer sector is a modified cilium, are progressively lost
(33, 34, 198).
Basal Bodies
The flagella arise from a pair of basal bodies located just beneath the apical end
of the cell, surmounting the cell nucleus. The basal bodies are connected to one
another by a distal striated fiber (Figure 2) and are attached at their proximal ends
to four sets of microtubules that extend around the anterior portion of the cell.
Proximal fibers connect the basal bodies to the nucleus, and a cruciate fibrous root
is located directly beneath the basal bodies.
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Figure 2 Flagellar root system of Chlamydomonas. B, basal bodies; P, pro-basal bodies; DSF,
distal striated fiber; PSF, proximal striated fiber; SMAF, striated microtubule-associated fiber;
2MTR and 4MTR, 2- and 4-membered rootlet microtubules. Figure by Andrea Preble, courtesy
of Susan Dutcher.
In cross-section, the basal bodies show a progression from a ring structure

through a series of cartwheel configurations also seen in centrioles of animal cells,
to the final 9 + 2 microtubule structure characteristic of eukaryotic flagella and
cilia (Figure 3). Chlamydomonas has proved an especially favorable system in
which to investigate the formation and function of basal bodies and flagella. Centrin, the 20-kDa contractile protein of the distal striated fiber and the nucleus-basal
body connector, was discovered in Chlamydomonas (see 204), as was delta-tubulin,
required for assembly of triplet microtubules in the basal body (49). Three additional centriole-associated proteins, BAp90, BAp95, and striated fiber assemblin,
were also first identified in green algae (127).
Prior to mitosis, the basal bodies assume their alternative role as centrioles,
components of the microtubule organizing center as in animal cells (138). The
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Figure 3 Cross-sectional views of a Chlamydomonas basal body, showing progression

through triplet microtubule and stellate morphologies to the axonemal 9 + 2 microtubule
structure (from 178; courtesy of S Dutcher).
connections from the flagella to the basal bodies are lost early in mitosis, and the
flagella are resorbed. The basal bodies (centrioles) duplicate in late G1 phase by
forming a new partner next to each pre-existing one, and by prophase the cell has
a pair of centrioles at each spindle pole, each pair consisting of an old and a new
centriole. After cytokinesis, the basal bodies return to the cell anterior, and new
flagella are formed. Recent studies indicate that centrioles are able to form de novo
(49, 138).
When Chlamydomonas cells are treated with weak organic acids, mastoparan,
calmodulin antagonists, detergent, or various other stimulants, the flagella are
severed at the level of the transition region between the basal body and the flagellar
axoneme (see Figure 3). The process is thought to be similar to katanin-mediated
severing of cytoplasmic microtubules in mitotic Xenopus and starfish oocytes (see
132, 133, 182). Investigation of this phenomenon in Chlamydomonas has revealed
a complex signal transduction pathway that is proving to be amenable to genetic
analysis. Mutants at two genetic loci fail to sever their flagella in response to
any stimulus and are thought to be defective in the calcium-activated severing
process. The product of one of these loci is a 171-kDa protein with an N-terminal
coiled-coil domain and three Ca+2/calmodulin-binding domains (60). Mutants at
a third locus are blocked in acid-stimulated deflagellation but respond to nonionic
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detergent plus Ca+2 and are proposed to affect protein-stimulated Ca+2 influx
(61).
Deflagellation is followed by active transcription of the genes encoding tubulin
and other flagellar components, and regeneration of the flagella over a 3-h period.
This has proved an excellent system both for purification of axonemes and for
study of the assembly process [see articles by SM King and PA Lefebvre in (43) for
methods]. Deflagellation induces both transcription of genes for flagellar proteins
and changes in mRNA half-life (79, 109, 172). In the presence of inhibitors of
protein synthesis, assembly of half-length flagella still occurs, suggesting that
there is a cytoplasmic pool of flagellar precursors (66). Pre-assembled complexes
move from the cytoplasm to the flagellar compartment, where they are attached to
microtubule-associated docking sites (162).
The Eyespot and Phototaxis

The eyespot, or stigma, appears bright orange at the light microscope level, owing
to a high concentration of carotenoid pigments. Electron microscopy reveals it as a
region of electron-dense granules located just inside the chloroplast membrane at
the cell equator. The carotenoid-containing granules of the eyespot are thought to
act as a quarter-wave plate to direct light to the true photoreceptor (KW Foster &
RD Smyth, cited in 206), located in the overlying plasma membrane and now
identified as a retinal-binding rhodopsin homologue (chlamyopsin; 42, 89). The
complex functions as a directional antenna that enables swimming cells to orient themselves with respect to unidirectional light. Mutants lacking the eyespot
structure show reduced efficiency of phototaxis but may still be able to perceive
light through the photoreceptor. Mutants with defects in eyespot assembly have
been grouped into six complementation groups, several of which map to a closely
linked cluster of loci (123; DGW Roberts, MR Lamb & CL Dieckmann, submitted).
Mutants deficient in the signal transduction pathway essential to the phototactic
response have also been isolated (115, 170, 201). Methods for assaying phototaxis
have been described by Moss et al (contained in 43).
The position of the eyespot gives the Chlamydomonas cell an inherent asymmetry, because it is associated with the distal end of one of the flagellar roots, specifically the four-membered rootlet emanating from newly formed basal body from
the previous mitosis. This basal body is referred to as cis (relative to the eyespot),
whereas the parent basal body is trans. The flagellar beat in normal forward swimming is a breast-stroke action that propels the cell in a helical path, with constant orientation of the eyespot relative to the helical axis. However, the cis and trans flagella
respond differently to phototactic signals, thus effecting a turning response (205).
The position of the eyespot is established during mitosis, when the old eyespot disappears and a new one is formed, invariably opposite the site of the cleavage plane.
Two distinct photoresponses have been observed in Chlamydomonas, the oriented movements of phototaxis in response to a constant unidirectional light and
the photophobic or stop response to sudden light flashes. The same photoreceptor
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appears to mediate both responses (P Kroger & P Hegemann 1994, cited in 205).
Analysis of mutants that show phototactic but not photophobic responses suggests
that the photophobic response requires a calcium-dependent all-or-none electric
current induced by photoreceptor-mediated depolarization of the flagellar membrane (140). Phototaxis also depends on calcium-induced changes in flagella beating, but at a lower molarity of calcium that shifts the balance in beating strength
of the two flagella.
MITOTIC AND MEIOTIC LIFE CYCLES

Vegetative Cell Growth
Wild-type C. reinhardtii is easily grown in defined liquid or agar media at neutral
pH, and has no requirements for supplementary vitamins or other co-factors (see
86). Strains in the Ebersold/Levine 137C background cannot assimilate nitrate and
therefore require a reduced nitrogen source (usually NH4Cl). Acetate can be used
as a carbon source by wild-type strains, with the consequence that growth can occur
in the dark, and mutants blocked in photosynthesis are viable if acetate is provided.
Other intermediates in the citric acid cycle do not support growth in the dark, nor do
various pentose or hexose sugars, ethanol, glycerol, or other organic compounds.
For wild-type strains, growth in light either with or without acetate is faster
than dark growth and is therefore recommended. Optimal growth temperature
is from 20 to 25 . At 25 , in minimal medium and with adequate light (200
400 Einsteins/m2sec photosynthetically active radiation), an average doubling
time of 6 to 8 h should be achieved.
When grown on a 12:12, 14:10, or 16:8 light-dark cycle, cells remain in G1
throughout the light phase and divide during the dark phase, usually with two or
sometimes three mitotic divisions taking place in rapid succession. Four daughter
cells are retained within a common mother cell wall and released simultaneously
on secretion of a specific lytic enzyme. Commitment to divide appears to be determined at a specific point in G1 phase, thought to be analogous to the START
event in the yeast cell cycle (see 85 for review). Beyond this point, division will
still occur even if light and nutrients are removed from the culture. The number
of successive divisions that take place in a given cycle depends on the cell size
reached during G1. A circadian oscillator may also be involved in determining
timing of division (78).
The progress of mitosis in C. reinhardtii was described in 1968 by Johnson and
Porter in a classic paper (cited in 86). Since that time, computer-assisted analysis
of serial sections and immunofluorescence techniques have added to our understanding of the changes in cytoplasmic microtubules, actin, and the chloroplast
during mitosis (51, 85).
Conditional mutants blocked at specific points in the cell division cycle at
restrictive temperature were described more than 25 years ago by Howell and
colleagues (reviewed in 85), but the state of knowledge of the cell cycle at that
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time did not permit full characterization of their defects. New conditional cell
cycle mutants have been isolated by John and colleagues (203, 242), and the old
mutants have been subjected to further study as well. The results now emerging
indicate that Chlamydomonas has great potential as a system for genetic analysis
of cell cycle control.
A nonconditional mutant defective in size control has been found to have a
deletion in a gene encoding a homologue of the retinoblastoma family of tumor
suppressors (3; J Umen, unpublished). This protein appears to function at two
points in the cell cycle, in G1 and again in S phase. The result of its deletion in
Chlamydomonas is impairment both in timing of commitment to divide and in
control of the number of divisions that eventually occur.
Mutants of C. moewusii described as twins and monsters were reported
by Lewin in 1952 (reviewed in 85). The twinning mutant is now thought to be
defective in formation of the cleavage furrow. Cultures of monster mutants have
a significant proportion of cells that are blocked in division but continue to grow.
The cyt1 mutant of C. reinhardtii resembles the monster mutants in its failure to
complete cytokinesis in many cells of a culture, with the consequence that cells
may be multinucleate, large, and multilobed (JR Warr, cited in 85). Ehler & Dutcher
(51) found that cyt1 cultures often produce cells with incomplete cleavage furrows.
They also isolated an insertional allele of cyt1 and a mutant at a second locus, cyt2,
which makes additional, misplaced, cleavage furrows. The number of flagella on a
given cell is correlated with the number of nuclei. Similar mutants (oca1 and oca2,
for occasional cytokinesis arrest) have been described by Hirono & Yoda (94).
Mutants of C. reinhardtii with abnormalities in the basal body cycle have proved
to be particularly amenable to study. Several classes of mutants were first identified
by their variable number of flagella. Vf l1 cells have lost control of the timing and
placement of basal body and flagellar formation and show abnormalities in the
direction of the flagellar beat, as well as structural abnormalities in several of the
doublet microtubules of the flagellar axoneme (223). New basal bodies can appear
at any point during G1 phase, and the flagellar insertions can be anywhere on
the cell surface. Vf l2 mutants have alterations in the structural gene for centrin
(221) and show random segregation of the basal bodies. Pedigree analysis of
mitotic progeny from a vf l2 mutant has been used to demonstrate de novo centriole
assembly (138). Vf l3 mutants are defective in placement of the probasal bodies
and have been shown to have defects in striated fibers (HJ Hoops, RL Wright, JW
Jarvik & GB Witman 1984, and RL Wright, Chojnacki & JW Jarvik, 1983, cited in
86). Uni mutants were so named because the majority of cells in a culture have
only a single flagellum. The uni1 and uni2 mutants both show alterations in the
transition zone (Figure 3, level 7) but are distinguishable at the ultrastructural level
(49). The uni3 mutant may have zero, one, or two flagella and exhibit aberrant
cytokinesis. In this mutant, the C tubule is missing in the triplet microtubule of the
basal body (Figure 3, level 3). The defect in uni3 has been traced to delta-tubulin,
a protein first identified when the Uni3 gene was cloned from Chlamydomonas
(49).
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Bld (bald) mutants lack flagella altogether. One such mutant, bld2, lacks
functional basal bodies and has defects in cytokinesis consistent with loss of
centrioles (52). The cleavage furrow loses its precise orientation with respect to the
mitotic spindle. Another mutation affecting cytokinesis is f la10, originally isolated
as a temperature-conditional flagellar assembly mutant, but subsequently shown
to have a defect in a kinesin-homologous protein that functions in basal bodies
both in flagellar assembly and in organization of the mitotic spindle (117, 231).
The Sexual Cycle

C. reinhardtii cells are normally haploid and are of one of two genetically fixed
mating types, designated plus (mt+) and minus (mt). The mating-type locus is a
complex region of recombinational suppression on linkage group VI, comprising
approximately 1 megabase and containing genes involved in cell recognition and
fusion, zygospore maturation, and in the mating-type controlled inheritance of
organelle genes, as well as some additional closely linked loci that have no apparent
role in the sexual cycle (58, 59). Additional genes that map elsewhere in the genome
but whose expression is sex-limited have also been identified (25, 77, 122, 226).
When deprived of nitrogen, cells of both mating types differentiate into sexually
competent gametes (Figure 4). Some strains have an additional requirement for blue
light to progress from a pregamete state to mating competence (13, 74, 165, 202).
Plus and minus gametes pair initially along the lengths of their flagella in a reaction
mediated by sex-specific agglutinin proteins. Flagellar pairing initiates a cAMPmediated signal transduction cascade, which has been investigated extensively
(181, 239). Pairing is followed by a morphological change (activation) in the
flagellar tips and by dissolution of the cell walls of the mating partners by a gametespecific lytic enzyme. Flagellar agglutination, activation of the flagellar tips, and
wall lysis can all be by-passed by supplying exogenous dibutryl cAMP and isobutyl
methylxanthine (SM Pasquale & UW Goodenough, cited in 86; this observation
has been exploited as a means of genetic analysis of mutants lacking flagella).
Fusion of the mating partners begins at sex-specific structures at the anterior ends
of the cells (238, 240), and continues laterally from anterior to posterior. The
newly formed diploid zygote remains motile for several hours as a quadriflagellate
cell.
In C. moewusii, C. eugametos, C. monoica, and some other Chlamydomonas
species, flagellar contact is followed initially by fusion only at the extreme anterior
ends of the cells, and the partner cells swim about for several hours as a visa` -vis pair before full cell fusion occurs (229). This distinction in the mating
process is probably a very fundamental character separating different ancestral
lineages within the green algae. Sexual cycles have been described in relatively
few Chlamydomonas species, so this has not been used as a character in traditional
taxonomy. The overall process of recognition, signal transduction, and cell fusion
in C. eugametos resembles that of C. reinhardtii in many ways, however, and a
substantial body of literature exists on the sexual cycle in this species (see 229).
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Figure 4 The sexual cycle of Chlamydomonas reinhardtii. Courtesy of William Snell.
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Zygote-specific transcripts appear within minutes of gamete fusion (121 and

references cited therein). Formation of a hard, impermeable zygospore wall begins, chloroplasts appear to disintegrate, with loss of chlorophyll, and lipid bodies
accumulate over the ensuing 4 to 6 days. The zygospore wall in sexual species
of Chlamydomonas affords protection against adverse environmental conditions.
Like the vegetative cell wall, the zygospore wall of C. reinhardtii contains hydroxyproline-rich glycoproteins, some of which are marked by (SerPro)x repeats. Zygospores can remain viable in soil for many years. However, under laboratory
conditions only a few days are required for zygospore maturation before germination can be induced by restoration of nitrogen in the presence of light. Meiosis
occurs, with the subsequent release of the four haploid meiotic products. Under
some conditions, a mitotic division follows meiosis prior to opening of the zygospore wall, with the resulting release of eight rather than four progeny cells;
predilection for release of eight rather than four cells may be strain dependent
(86).
A small percentage of mated pairs fail to initiate the zygospore maturation
pathway and begin instead to divide mitotically as stable vegetative diploids (see
86). These can be deliberately selected using complementing auxotrophic markers,
and can be recognized 3 or 4 days after mated cells have been plated on agar, as
bright green hemispherical colonies visible using a dissected microscope, on a
lawn of unmated gametes and immature zygospores.
Tetrad Analysis
Separation of all meiotic products from a single zygospore is the foundation of
traditional Chlamydomonas genetics by tetrad analysis (see SK Dutcher in 43 and
86 for methods). Nuclear genes show 2:2 inheritance in crosses and are scored
by their segregation in parental ditype, nonparental ditype, or tetratype tetrads.
Chloroplast and mitochondrial genes show primarily uniparental inheritance, from
the plus and minus mating types, respectively. Elucidating the mechanism by
which uniparental inheritance is achieved was one of the earliest challenges in
Chlamydomonas research. Although the mysteries are still not entirely solved,
the tools of molecular biology are now being brought to bear on this problem
(146, 158).
Mutations are readily induced in the nuclear genome of haploid Chlamydomonas cells by UV or chemical mutagenesis (86), or by insertional mutagenesis
in which transformation with exogenous DNA results in disruption of nuclear
genes (LW Tam & PA Lefebvre in 43). This technique is becoming increasingly
common as a preliminary step in cloning genes for which a mutated phenotype is
known or predictable and is discussed further below.
The spectrum of mutants available in C. reinhardtii includes nonphotosynthetic,
nonmotile, and nonphototactic strains, auxotrophs, mutants resistant to antibiotics
or herbicides, and other phenotypes (see 86, 128). As of this writing, nearly 200
nuclear gene loci have been identified by mapped mutations.
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PHYSIOLOGICAL PROCESSES
Nutrient Update and Metabolic Stress

Most wild-type strains of C. reinhardtii can assimilate nitrogen as nitrate, nitrite,
ammonium, or other small molecules such as urea, acetamide, etc. Strains in the
137C background carry two mutations blocking nitrate reductase activity. The Nia1
gene (NIT1 locus) is the structural gene for nitrate reductase (108); the Nia2 gene
(NIT2) encodes a regulatory protein in the nitrate assimilation pathway (207). The
nitrate transport system has been characterized in some detail and several component genes have been cloned (153, 184, 255). Nitrite is transported by a separate
system, regulated by blue light (187). The structural gene for nitrite reductase
(Nii1) maps to a cluster that also includes nitrate assimilation genes and a lightregulated gene (183). Genes for some additional enzymes involved in nitrogen
metabolism have also been cloned and are under investigation.
Arginine is the only amino acid for which auxotrophic mutations are readily
isolated in Chlamydomonas. Mutations at six loci affect the arginine biosynthetic
pathway (see 86). Only the argininosuccinate lyase gene has been completely
sequenced (6), but ESTs have been found corresponding to ornithine transcarbamoylase (the step blocked by the arg4 mutation) and argininosuccinate synthase (arg8). Efforts to obtain a broader spectrum of amino acid auxotrophs have
not been successful, possibly owing to lack of active transport systems for these
compounds. Some mutants resistant to amino acid analogs have been isolated,
however. Auxotrophic mutants are also known for thiamine, nicotinamide, and
para-aminobenzoic acid.
Carbon assimilation involves a complicated pathway with multiple forms of
carbonic anhydrase (218), the genes for several of which have been cloned from
C. reinhardtii. Expression of the periplasmic enzyme is regulated by acetate and
pH (227). Carbonic anhydrase activity associated with the chloroplast (2, 71)
is required for photosynthesis at ambient concentrations of CO2. A novel 29.5kDa alpha-type carbonic anhydrase associated with the thylakoid membrane has
been cloned from Chlamydomonas by Karlsson et al (110). Mitochondrial carbonic anhydrase is induced at low CO2 concentrations (54). Mutants blocked
in carbonic anhydrase activity are dependent on high CO2 levels for growth
(69, 112, 214).
Limitation for CO2 in the light or for acetate in the dark results in accumulation
of large amounts of starch (amylopectin) by Chlamydomonas cells (22). Mutants
deficient in amylose biosynthesis resemble waxy mutants of maize and other plants
in accumulating an aberrant amylopectin. A mutant blocked in ADP-glucose pyrophosphorylase has also been isolated.
Sulfur deprivation induces expression of a high-affinity transport system (251)
and a periplasmic arylsulfatase that has found utility as a reporter gene (164).
Mutants at three loci show aberrant responses to sulfur deprivation; two of these
loci have been shown to correspond to genes for regulatory proteins (39, 40).
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Phosphorous starvation induces expression of several phosphatases (1, 8, 188) and

of the Psr1 gene, encoding a regulatory protein (245).
When grown under anaerobic conditions in the light, Chlamydomonas cells are
capable of producing molecular hydrogen. Sulfur deprivation enhances hydrogen
production by repressing photosynthetic oxygen evolution, and under laboratory
conditions, cultures can be maintained that cycle between photosynthesis and
hydrogen production as sulfur is alternately removed and resupplied (142). The
wild-type hydrogenase enzyme is very sensitive to inhibition by oxygen, but efforts
are under way to select mutants with a more oxygen-tolerant enzyme (T Flynn &
M Ghirardi, in preparation). The process is potentially of enormous value as a
source of renewable fuel.
Another potential commercial application of Chlamydomonas is in removal
of heavy metal wastes from the environment. Tolerance for cadmium and other
metals by Chlamydomonas cells can be altered by growth conditions, by mutation
(177, 234), by treatment with phytochelatin inhibitors (24), or by genetic engineering (R Sayre, personal communication).
As our knowledge increases of genes involved in individual metabolic pathways, studies of the integration of these pathways is becoming feasible. Huppe
& Turpin (100) provide a good summary of the relationships between carbon and
nitrogen metabolism. Wykoff et al (244) have assessed effects of nutrient deprivation on photosynthesis. Rochaix (194) has discussed state transitions, chlororespiration and interactions between the chloroplast and mitochondria. The study
of stress responses in Chlamydomonas is in fact emerging as an integrated discipline, encompassing nutrient limitation, excess light, heavy metal contamination,
osmotic stress, and heat responses. Bell and colleagues have published a series of
papers dealing with genetic fitness and response of Chlamydomonas cells to experimentally controlled environmental fluctuations (75, 111 and references cited
therein).
Light-Mediated Responses
As mentioned in connection with the sexual cycle, blue light photoreceptors
may be involved in regulation of gametogenesis in Chlamydomonas (74, 165).
The blue-light-responsive LRG5 locus encodes a protein rich in arginine, lysine,
and alanine with a putative nuclear-localization signal at its C-terminal end. No
significant sequence homology was found to proteins from other organisms, but
DNA hybridization experiments suggest some conservation of related sequences
in other Volvocales and in land plants. The blue-light signaling pathway in gametogenesis appears to involve consecutive action of a phosphatase and a kinase
resembling protein kinase C (165). Disruption of the LRG6 gene, encoding a protein
with significant homology to the yeast membrane transport facilitator YJR124p,
eliminates the requirement for blue light in gametogenesis (G Dame, G Glockner
& CF Beck, personal communication). Blue light has also been implicated in
cell cycle regulation in Chlamydomonas (233), in chlorophyll biosynthesis (91),
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and in repair of UV-induced DNA damage (174). An early report that antibodies to plant phytochrome reacted with a Chlamydomonas protein was misleading;
Bonenberger et al (1994, cited in 74) have shown that a monoclonal antibody
to pea phytochrome reacts with a totally unrelated protein in C. reinhardtii, and
there is no evidence to date for any phytochrome-mediated responses. The absence of red/far-red responses increases the utility of Chlamydomonas for the
study of other light-regulated responses. A cryptochrome photoreceptor, CPH1,
has recently been cloned from C. reinhardtii (NA Reisdorph & GD Small, in
preparation). Although cryptochrome proteins have been implicated in molecular clock mechanisms in some organisms, the CPH1 gene of C. reinhardtii
does not seem to show circadian regulation. Promoter regions of light-responsive
genes in Chlamydomonas do not have the conserved control elements seen in
higher plants but do appear to have characteristic light-regulated cis sequences
(80).
Circadian Rhythms
Like many other algae, Chlamydomonas does have a circadian clock system, however, and its potential for genetic analysis increases its utility as a model for this
area of research. Circadian rhythms of phototactic aggregation were observed by
Bruce and coworkers in the early 1970s, and mutants with altered rhythms were
obtained (see 86). Mergenhagen isolated additional mutants and explored the nature of the timer, or zeitgeber, under various environmental conditions, including
zero gravity. Circadian rhythms have also been found in abundance of mRNAs
for a number of genes involved in nitrogen metabolism and in photosynthesis
(68, 103), and in UV sensitivity (157). Circadian rhythm phases can be shifted by
brief pulses of light given during the dark period. The action spectrum for this
response shows peaks at 520 and 660 nm, but is not far-red reversible (T Kondo
et al 1991, cited in 103). Mittag (1994, cited in 148) discovered a regulatory factor
in the dinoflagellate Gonyaulax polyedra that binds to a cis element, a UG repeat
in the 30 untranslated region of the gene encoding the luciferin-binding protein in
that alga. Further investigation revealed that Chlamydomonas has an analogous
clock-controlled RNA-binding protein (148, 149) whose target sequence is also a
UG repeat. This sequence appears in the 30 untranslated region of many Chlamydomonas genes, including several involved in nitrogen metabolism and previously
reported to exhibit temporal expression (H Waltenberger, C Schneid, J Grosch, A
Bariess & M Mittag, submitted).
TECHNIQUES AND RESOURCES

The Molecular Tool Kit
Over the past 12 years transformation of the nuclear and chloroplast genomes with
exogenous DNA has become routine (see 113 for review). The first successful
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transformations were accomplished using biolisticTM bombardment with tungsten or gold particles. Vortex-mixing with DNA-coated glass beads or
silicon carbide whiskers is also effective, especially for nuclear gene transformation, as is electroporation (211). The glass bead method is especially recommended, as it requires no specialized equipment or expensive supplies. Highest
frequencies are obtained with glass bead transformation and electroporation when
cell walls are removed prior to transformation, and when steps are take to improve plating efficiency of cells after transformation (180). Wall-deficient mutant strains can be used, or the walls of wild-type cells can be removed prior
to transformation with a preparation of the gamete lytic enzyme (113). With
all methods, cotransformation with two different plasmids occurs at a high frequency (216). When selection is made for one of the introduced genes, most
transformants are found to carry the unselected DNA as well. This observation suggests that the critical event in transformation is in sustaining a nonlethal hit by the particles, glass beads, fibers, etc, and that cells that survive
such a hit are likely to assimilate whatever DNA was present at the moment of
impact.
Chloroplast transformation usually occurs by homologous replacement and has
been extensively used to study proteins of the photosynthetic complexes by sitedirected mutagenesis (e.g. 63, 99, 124, 137, 193, 249, 257). Selectable markers for
cotransformation include antibiotic resistance mutations in the chloroplast ribosomal RNA genes, the bacterial antibiotic resistance genes aadA (56, 57, 64) and
aphA-6 (10), uidA (GUS; 57), and Renilla luciferase (145). Transformation of the
mitochondrial genome has also been reported (B Randolph-Anderson et al, 1993,
cited in 45).
In contrast to the organelles, nuclear gene transformation generally occurs by
nonhomologous insertion into the genome of one or (usually) more copies of the
transforming DNA. Transformation with glass beads usually results in integration
of fewer copies of the transforming DNA than does particle bombardment (79).
Insertion usually results in deletion of 10 to 20 kb or more of DNA at the integration
site (128). When this event disrupts a nonessential gene, the insertion can be used
as a probe for hybridization to clone the gene (LW Tam & PA Lefebvre, in 43).
More than 50 genes have already been cloned using this technique. Because the
insertion nearly always disrupts the gene function, creating a null mutation, this
method is not useful for cloning essential genes. However, insertion frequencies
are sufficiently high that the method is a very efficient means of obtaining populations of mutant cells to screen for specific phenotypes, e.g. loss of motility or
photosynthetic function (128).
Insertional mutagenesis has usually been accomplished by transformation of a
mutant deficient in either nitrate reductase (nit1) or argininosuccinate lyase (arg7 )
with the corresponding wild-type gene. Transformants are selected by restoration
of ability to grow on nitrate as sole N source (NIT1), or on minimal medium
(ARG7 ), and can then be screened for other phenotypes resulting from nonhomologous insertion of the transforming DNA.
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Transposon tagging of Chlamydomonas genes has also been used as a cloning

strategy, although less extensively to date than ARG7 or NIT1 insertions. Transposable elements characterized in C. reinhardtii include a class I retrotransposon
(TOC), three class II elements (Gulliver; Pioneer, and TOC2); and three elements,
Tcr1 through Tcr3, which are characterized by inverted terminal repeat sequences
[for review, see (236)].
Other constructs that have been used for nuclear transformation include several selectable markers that can confer inhibitor resistance on wild-type cells.
The CRY1 and ALS markers are endogenous Chlamydomonas genes from mutant
cells resistant to emetine and sulfmeturon methyl, respectively (JAE Nelson &
PA Lefebvre, in 43; 70). Several bacterial antibiotic resistance genes have also
been expressed successfully in Chlamydomonas (27, 212, 217).
The nuclear transforming constructs used to date generally include a native
C. reinhardtii promoter and 30 untranslated region, most commonly from the RbcS2
gene encoding the small subunit of ribulose bisphosphate carboxylase. Improved
expression of nuclear genes has been achieved by including the first intron of
RbcS2 (135). Further enhancement has been obtained with a construct that fuses
the promoter of the HSP70A (heat shock protein) gene upstream of the promoter
and first intron from RbcS2 (208).
Failure to achieve satisfactory expression of some introduced genes has been
attributed to posttranscriptional gene silencing, codon bias, and incomplete promoters, enhancers, or other regulatory sequences. Finally, these problems seem to
be yielding to creativity, hard work, and persistence. Cerutti and colleagues (26)
have investigated silencing of the bacterial aadA gene after transformation into
Chlamydomonas in a construct where it is flanked by the RbcS2 50 and 30 noncoding regions. Expression of aadA was found to be unstable in approximately
half the transformants recovered. Inactivation of the gene was epigenetic, i.e. no
alterations were observed in the presence or sequence of the transforming DNA,
and the changes were reversible in selected clones transferred to and from selective
conditions (spectinomycin). Spectinomycin resistance always cosegregated with
the integrated construct in crosses. Direct analysis of aadA expression showed that
the gene was inactivated at the transcriptional level and that this inactivation was
not correlated with cytosine methylation or with accessibility of the integrated construct to restriction enzymes (which would have suggested silencing by chromatin
condensation). Insertional mutagenesis has been used to recover suppressors of
this epigenetic silencing (243). Although methylation was not implicated in the
aadA silencing, work with Volvox suggests that CpG methylation does have a role
in expression of introduced genes in this alga, and by extension may be expected
to have significance in Chlamydomonas as well (P Babinger, I Kobl, W Mages &
R Schmitt, submitted).
C. reinhardtii nuclear genes show a pronounced codon bias, a consequence
of the GC-rich genome. The search for foreign genes that can be used as selectable markers has led to bacterial genes whose codon usage is similar to that of
Chlamydomonas. The ble gene from Streptoalloteichus hindustanus, conferring
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phleomycin resistance, and the aphVIII gene of Streptomyces rimosus, conferring

resistance to aminoglycoside antibiotics, were chosen for this reason (212, 217).
Expression of the green fluorescent protein gene was improved by resynthesizing
it with a codon set more typical of native C. reinhardtii genes (67).
The molecular tool kit for Chlamydomonas includes several additional constructs useful for analyzing gene expression (also see 128, 134, 216). The arylsulfatase gene (Ars1), whose synthesis is induced when cells are starved for sulfur,
can be used as a reporter for promoter function (164). The enzyme is assayed using a chromogenic sulfate substrate. Haring & Beck (84) have adapted insertional
tagging to create a promoter trap system. Cells of a double mutant, arg7 pf14, are
transformed with the Arg7 gene and a promoter-less Rsp3 gene (radial spoke protein 3, complementing the pf14 mutation). Transformants are selected for ability to
grow on minimal medium, and then screened for restoration of motility resulting
from integration of the Rsp3 gene downstream of an endogenous promoter. The
gene whose promoter is now tagged with Rsp3 can thus be cloned. Auchincloss
et al (6) have reported development of a shuttle vector based on the cDNA for the
Arg7 gene that is able to complement both the C. reinhardtii arg7 mutant and the
E. coli argH mutant. Constructs have also been made to permit epitope tagging of
cloned genes (e.g. 105).
Although targeted disruption of Chlamydomonas nuclear genes by transformation has succeeded only rarely owing to the rarity of homologous insertion (154),
RNAi is showing promise as an alternative means of targeting and inactivating
expression of specific genes (50, 209). For example, expression of chlamyopsin
(the photoreceptor protein) has been studied in constructs driven by the Hsp70A
and RbcS2 promoters (M Fuhrmann, S Rank, E Govorunova & P Hegemann, in
preparation). When the complete Cop gene encoding this protein, including introns, is expressed from these promoters, chlamyopsin levels are 5 to 10 times
higher than in untransformed cells. Replacing the Cop gene with an inverted gene
sequence capable of forming a double-stranded RNA structure reduced expression
to as little as 10% of the wild-type level.
Fortunately, lack of directed homologous integration has not prevented rescue
of mutants by transformation with cosmid or BAC clones derived from C. reinhardtii (71, 74, 189), and this technique is now becoming routine. In a variation of
this technique suitable for genes with no selectable phenotype, Purton & Rochaix
(180) prepared a cosmid library in a vector carrying the wild-type Arg7 DNA.
Arginine-independent transformants were selected and then screened for complementation of the original mutation. Rescue of mutant phenotypes by genes from
other organisms may become feasible as better expression of heterologous genes
is achieved.
A BAC library (http://www.biology.duke.edu/chlamy/ChlamyGen/libraries.
html) with approximately 10- to 12-fold coverage of the Chlamydomonas genome
is commercially available from Incyte Genomics and is being used to generate a
molecular map (P Kathir, PA Lefebvre & CD Silflow, in preparation). Positional
cloning using individual BAC clones together with this map has already been
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successful (CD Silflow & PA Lefebvre, personal communication) and will undoubtedly become more practical as the density of markers on the molecular map
increases. As BAC clones are identified that hybridize to molecular markers, they
can be used to assemble overlapping contigs that will eventually cover the entire
genome.
The current molecular map contains 240 markers, with an average physical
spacing of 400 to 500 kb. All molecular markers analyzed to date have been placed
on 17 linkage groups, suggesting that this is indeed the definitive number of linkage
groups for C. reinhardtii1 . For nearly all of the linkage groups, the molecular and
genetic maps can be anchored and oriented to each other by markers in common.
Sources of Strains and Information

The Chlamydomonas Genetics Center at Duke University provides cultures of
wild-type and mutant strains of C. reinhardtii, C. eugametos, and C. moewusii.
The web site is at http://www.biology.duke.edu/chlamy; mailing address c/o
Elizabeth H. Harris, DCMB Box 91000, Duke University, Durham, NC 277081000. Wild-type strains of other genera are available from several major algal
collections worldwide, including Culture Centre of Algae and Protozoa (CCAP;
Freshwater Biology Association, The Ferry House, Ambleside, Cumbria, LA22
0LP, UK; http://wiua.nwi.ac.uk/ccap/ccaphome.html); Institute of Applied Microbiology, Tokyo (IAM; The University of Tokyo, 1-1-1 Yayoi, Bunkyou-ku, Tokyo
113, JAPAN); Sammlung von Algenkulturen (SAG; Pflanzen physiologisches
Institut, Universitat Gottingen, Nikolausberger Weg 18, D-3400 Gottingen,
Germany (http://www.gwdg.de/botanik/phykologia/epsag.html); University of
Texas Algal Collection (UTEX; Department of Botany, Austin, TX 78713-7640,
Section of Molecular Cell and Developmental Biology, University of Texas, Austin,
TX 78713 (http://www.bio.utexas.edu/research/utex/); University of Toronto
Culture Collection (UTCC; Department of Botany, University of Toronto, Toronto,
Ontario M5S 3B2, Canada (http://www.botany.utoronto.ca/utcc/). ChlamyDB is
a comprehensive database for information on Chlamydomonas and other algae
in the Volvocales, including publications, sequence citations, gene descriptions,
and genetic maps. This is available in a web presentation through the USDAARS Center for Bioinformatics and Comparative Genomics at Cornell University (http://arsgenome.cornell.edu/cgi-bin/WebAce/webace?db=chlamydb).
Recommendations for nomenclature of Chlamydomonas genetic loci have been
published by Dutcher (47) in the Trends in Genetics Genetic Nomenclature
1 In
early genetic mapping studies, 19 linkage groups were identified and numbered. Subsequent analysis (48) has shown that linkage groups XII and XIII in fact are colinear, as are
groups XVII and XVIII. The UNI linkage group, or ULG, previously reported by Ramanis
& Luck to be a circular linkage group having a specific association with the basal body (see
235), now appears to be a linear nuclear linkage group like the others and is now designated
as group XIX (97).
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Guide. The Chlamydomonas Genetics Center coordinates availability of names

for loci and mutant alleles; contact Elizabeth Harris (chlamy@duke.edu) for assistance. The bionet.chlamydomonas newsgroup (http://www.bio.net:80/hypermail/
CHLAMYDOMONAS/) provides a moderated forum for discussion of Chlamydomonas and other algae. The 10th International Conference on the Cell and
Molecular Biology of Chlamydomonas will be held at the University of British
Columbia (UBC) Conference Center in Vancouver, BC, Canada, June 1116, 2002.
The Chlamydomonas Genetics Center is sponsored by National Science Foundation Grant 9970022. The initial phase of the Chlamydomonas Genome Project
is sponsored by NSF Grant 9975765 under the direction of Arthur Grossman,
Carnegie Institution of Washington, Stanford, CA.
Visit the Annual Reviews home page at www.AnnualReviews.org
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Annual Review of Plant Physiology and Plant Molecular Biology

Volume 52, 2001
CONTENTS
FIFTY YEARS AS A PLANT PHYSIOLOGIST, James HM Henderson
ALKALOID BIOSYNTHESIS IN PLANTS: Biochemistry, Cell Biology,
Molecular Regulation, and Metabolic Engineering Applications, Peter J
Facchini
HOW GIBBERELLIN REGULATES PLANT GROWTH AND
DEVELOPMENT: A Molecular Genetic Analysis of Gibberellin
Signaling, Donald E Richards, Kathryn E King, Tahar Ait-ali, Nicholas P
Harberd
CYTOKININ METABOLISM AND ACTION, David WS Mok,
Machteld C Mok
ONE-CARBON METABOLISM IN HIGHER PLANTS, Andrew D
Hanson, Sanja Roje
CIRCADIAN RHYTHMS IN PLANTS, C Robertson McClung
MACRONUTRIENT UTILIZATION BY PHOTOSYNTHETIC
EUKARYOTES AND THE FABRIC OF INTERACTIONS, Arthur
Grossman, Hideki Takahashi
PLANT PHOSPHOLIPASES, Xuemin Wang
ENDOSPERM DEVELOPMENT: Cellularization and Cell Fate
Specification, Odd-Arne Olsen
MECHANISTIC FEATURES OF THE MO-CONTAINING
NITROGENASE, Jason Christiansen, Dennis R Dean, Lance C Seefeldt
MOLECULAR ENGINEERING OF C4 PHOTOSYNTHESIS, Makoto
Matsuoka, Robert T Furbank, Hiroshi Fukayama, Mitsue Miyao
THE PLASTID DIVISION MACHINE, Katherine W Osteryoung,
Rosemary S McAndrew
VARIATIONS IN THE BIOSYNTHESIS OF SEED-STORAGE LIPIDS,
Toni Voelker, Anthony J Kinney
CHLAMYDOMONAS AS A MODEL ORGANISM, Elizabeth H Harris
ISOPRENE EMISSION FROM PLANTS, Thomas D Sharkey, Sansun
Yeh
BIOSYNTHESIS OF ASCORBIC ACID IN PLANTS: A Renaissance,
Nicholas Smirnoff, Patricia L Conklin, Frank A Loewus
TONOPLAST TRANSPORTERS: Organization and Function,
Masayoshi Maeshima
PROBING PLANT METABOLISM WITH NMR, R George Ratcliffe,
Yair Shachar-Hill
29
67
89
119
139
163
211
233
269
297
315
335
363
407
437
469
499
FUNCTION AND MECHANISM OF ORGANIC ANION EXUDATION

FROM PLANT ROOTS, PR Ryan, E Delhaize, DL Jones
PLANT MITOCHONDRIA AND OXIDATIVE STRESS: Electron
Transport, NADPH Turnover, and Metabolism of Reactive Oxygen
Species, Ian M Mller
PHOTOSYSTEM I: Function and Physiology, Parag R Chitnis
561
593
GUARD CELL SIGNAL TRANSDUCTION, Julian I Schroeder, Gethyn

J Allen, Veronique Hugouvieux, June M Kwak, David Waner
627
527
TRANSPORTERS RESPONSIBLE FOR THE UPTAKE AND

PARTITIONING OF NITROGENOUS SOLUTES, LE Williams, AJ
Miller
DEFENSIVE RESIN BIOSYNTHESIS IN CONIFERS, Susan Trapp,
Rodney Croteau
MOLECULAR BIOLOGY OF FRUIT MATURATION AND
RIPENING, Jim Giovannoni
CYTOKINESIS AND BUILDING OF THE CELL PLATE IN PLANTS,Desh Pal S Verma
RIBOSOME-INACTIVATING PROTEINS: A Plant Perspective, Kirsten

Nielsen, Rebecca S Boston
PLANT PLASMA MEMBRANE H+-ATPases: Powerhouses for Nutrient
Uptake, Michael G Palmgren
THE COHESION-TENSION MECHANISM AND THE ACQUISITION
OF WATER BY PLANT ROOTS, Ernst Steudle
659
689
725
751
785
817
847

Chlamydomonas As A Model Organism

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Annu. Rev. Plant Physiol. Plant Mol. Biol. 2001. 52:363406

CHLAMYDOMONAS AS A MODEL ORGANISM

Key Words Volvocales, flagella, chloroplast, basal body, phototaxis

THE GENUS CHLAMYDOMONAS

Figure 1 A semidiagrammatic representation of an interphase Chlamydomonas cell. Cell

Laboratory Strains of C. reinhardtii

CELL ARCHITECTURE AND FUNCTIONAL SYSTEMS

The Nucleus and the Nuclear Genome

Contractile Vacuoles and Osmotic Regulation

TABLE 1 Representative mutations affecting photosynthesis and the chloroplast in

Disruption of chloroplast ycf10 gene; produces inefficient carbon

Among the greatest strengths of Chlamydomonas as a model organism in which

In cross-section, the basal bodies show a progression from a ring structure

Figure 3 Cross-sectional views of a Chlamydomonas basal body, showing progression

The Eyespot and Phototaxis

MITOTIC AND MEIOTIC LIFE CYCLES

The Sexual Cycle

Figure 4 The sexual cycle of Chlamydomonas reinhardtii. Courtesy of William Snell.

Zygote-specific transcripts appear within minutes of gamete fusion (121 and

Nutrient Update and Metabolic Stress

Phosphorous starvation induces expression of several phosphatases (1, 8, 188) and

TECHNIQUES AND RESOURCES

Transposon tagging of Chlamydomonas genes has also been used as a cloning

phleomycin resistance, and the aphVIII gene of Streptomyces rimosus, conferring

Sources of Strains and Information

Guide. The Chlamydomonas Genetics Center coordinates availability of names

flagella locus and extragenic suppressor

Phylogeny of the Chlamydomonadales

cal for survival of Chlamydomonas reinhardtii during sulfur deprivation. EMBO J.

quences are not required for translation

1996. Translational regulation of chloroplast genesproteins binding to the

106. Johnson KA. 1995. Keeping the beat:

subunit of chloroplast ATPase. Plant

PG, Sobolev V, et al. 1998. Biophysical,

blet severing during deflagellation. Cell

with non-permeabilizing concentrations

Linck RW. 1996. Structural comparison

duced by phosphorus-deprived Chlamydomonas reinhardtii. Plant Physiol.

gene NIT2 by transposon tagging. Genetics 134:73747

mRNAs of Chlamydomonas reinhardtii.

genes and nucleosome structure. Nucleic

photosynthetic electron transport during

1997. Translation of the chloroplast psbC

two gene products. FEBS Lett. 466:225

Annual Review of Plant Physiology and Plant Molecular Biology

FUNCTION AND MECHANISM OF ORGANIC ANION EXUDATION

GUARD CELL SIGNAL TRANSDUCTION, Julian I Schroeder, Gethyn

TRANSPORTERS RESPONSIBLE FOR THE UPTAKE AND

CYTOKINESIS AND BUILDING OF THE CELL PLATE IN PLANTS,Desh Pal S Verma

RIBOSOME-INACTIVATING PROTEINS: A Plant Perspective, Kirsten

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