FEMS Microbiology Reviews 46 (1987) 13-34
Published by Elsevier
13
FER 00052
Molecular genetics of chloroplasts and mitochondria
in the unicellular green alga Chlamydomonas
J.-D. R o c h a i x
Departments of Molecular Biology' and Plant Biology', Uni~ersi(v of Gener,a, Genes,a, Switzerland
Receivcd 7 July 1986
Accepted 16 September 1986
Key words: Photosynthetic mutants; DNA, chloroplast; DNA, mitochondrial; Chlamydomonas
1. I N T R O D U C T I O N
The green unicellular biflagellate alga Chlamydomonas reinhardtii provides interesting possibilities for a combined genetic and molecular study of
the biosynthesis and function of cellular organelles.
This is due to the fact that numerous mutations
have been identified in Chlamydomonas that affect
photosynthesis, chloroplast protein synthesis,
flagellar structure, motility and mating. This review will focus mostly on the major organelle of
Chlamydomonas, its unique chloroplast, which occupies more than 40% of the cell volume.
It is well established that chloroplasts contain
their own genetic system which comprises DNA,
RNA, DNA- and RNA-polymerases, ribosomes
and translation factors. This system co-operates
closely with its homologue in the nucleocytoplasmic compartment in the biosynthesis of chloroplast components. Most prominent among these
are the photosynthetic apparatus and the chloroplast protein synthesizing system. They consist of
a large number of polypeptides, some of which are
('orrespondence to: J.-D. Rochaix, Depts. of MolecularBiology
and Plant Biology, University of Geneva, 1211 Geneva,
Switzerland.
encoded by the chloroplast genome while others
are coded for by the nuclear genome, translated as
precursors on cytoplasmic ribosomes and imported into the chloroplast where they assemble
with their partner polypeptides into functional
complexes. Little is known about the regulation of
this complex interplay between chloroplast and
nucleocytoplasmic compartments which results in
the establishment of an active photosynthetic system. Among photosynthetic eukaryotes, Chlamydomonas is uniquely suited for studying this problem, since it can be manipulated with ease at the
genetic, biochemical and molecular levels.
The aim of this article is to provide an overview
of the molecular genetics of Chlamydomonas with
special emphasis on photosynthesis. Only recent
developments are covered, which include the
organization of the chloroplast genome in
Chlarnydomonas, the isolation and characterization
of both chloroplast and nuclear photosynthetic
mutants, the use of these mutations for studying
the function and assembly of photosynthetic complexes, the improved correlation between the
genetic and physical chloroplast DNA maps, and
the m o l e c u l a r and g e n e t i c analysis of
mitochondrial DNA. Several recent reviews provide more detailed coverage of various aspects of
chloroplast molecular biology [1-5].
0168-6445/87/$07.70 © 1987 Federation of European MicrobiologicalSocieties
14
2. G E N E R A L P R O P E R T I E S
D O M O N A S RE1NHA R D TII
OF
CHLAMY-
Cells of opposite mating-type (mt) of this heterothallic alga can be propagated vegetatively by
mitosis (Fig. 1). U p o n transfer into a medium
deprived of a reduced nitrogen source, the vegetative cells differentiate into gametes and cells of
opposite mating-type fuse to form a zygote.
Nuclear fusion is followed by chloroplast fusion,
thus allowing the chloroplast genomes of both
parents to mix and recombine. At the end of the
meiotic cycle, haploid daughter cells are released
which can initiate a new cycle. It is also possible
to produce vegetative zygotes which do not enter
meiosis and which divide mitotically as diploid
cells [15].
As in higher plants, there are three genetic
systems in Chlamydomonas, located in the
nucleocytoplasm, chloroplast and mitochondria,
respectively. Table 1 summarizes the major features of these systems. It is noteworthy that in
contrast to higher plants, the size of the
mitochondrial genome is only 16 kb, similar to
animal mitochondrial D N A [12] (see section 7).
Although the complexity of the chloroplast D N A
(190 kb) represents only 0.3% of the total cell
D N A complexity, the amount of chloroplast D N A
constitutes 14% of the cellular D N A mass [7,11].
gamete(-~
-NH~ .'"''"
/
Vegetative ~
gamete~
..... ~
...... ""..
~
Fusion
~
This implies that the chloroplast D N A is present
in more than 50 copies per cell. Similarly, the
mitochondrial D N A also exists in numerous copies
per cell. While nuclear genes are inherited according to mendelian rules, the chloroplast D N A is
inherited in most cases from the mt + parent (see
[15]). In rare cases, biparental zygotes occur in
which the chloroplast genes of both parents are
transmitted to the meiotic progeny, allowing the
study of chloroplast D N A recombination. Surprisingly, in crosses between C. reinhardtii and
Chlamydomonas smithii the mitochondrial genome
of the mt ~ parent is transmitted uniparentally [14]
(see section 7).
Cells can be grown under phototrophic
(minimal medium with light), heterotrophic
(acetate medium in the dark) or mixotrophic
conditions (acetate medium with light). Photosynthetic function is therefore dispensable when the
cells are grown in the presence of acetate. This
property allows one to isolate and maintain
photosynthetic mutants that are unable to grow in
the absence of a reduced carbon source such as
acetate [15]. Conversely, mitochondrial respiratory
function appears to be dispensable when the cells
are grown in the light, but not when they are
grown heterotrophically. Several obligate photoautotrophic mutants which die in the dark have been
characterized (see [15]).
""~'o~~
÷
-NH~
y
Vegetative \
Fig. 1. Life cycle of C. remhardtM rot, Mating type: n, nucleus. Zygote can undergo meiosis or remain diploid and divide mitotically.
15
Table 1
Genetic systems in Chlamydomonas reinhardtii
Nuclear DNA
Chloroplast DNA
Mitochondrial DNA
Complexity (kb)
% genetic
information
% Mass
Copy number
Inheritance
7-9 x 104 [7-9]
190 [10,11]
16 [12]
99.7
0.3
0.02
85
14
1
1
50 80
50-80
Mendelian
Uniparental maternal [13]
Uniparental paternal a [14]
a This has only been demonstrated in crosses between C. reinhardtii and C. smithii [14].
QA and QB, respectively, and channeled along the
electron t r a n s p o r t chain t h r o u g h the plastoquinone pool, the cytochrome b 6 / f complex,
plastocyanin and finally to the PSI reaction center.
This complex generates a strong reductant capable
of reducing ferredoxin and N A D P , the terminal
electron acceptor. The electron flow is coupled to
an influx of protons into the thylakoids, creating a
p r o t o n gradient which drives A T P synthesis
through the A T P synthase complex. Both A T P
and N A D P H are fed into the Calvin cycle which
results in the fixation of CO 2, catalyzed by ribulose 1 , 5 - b i s p h o s p h a t e c a r b o x y l a s e - o x y g e n a s e
(RuBisCO), and in the synthesis of carbohydrates.
The oxygenase activity of RuBisCO initiates the
first step of photorespiration, a competing reaction of photosynthesis which ultimately results in
the loss of C O 2 [16]. The existence of a respiratory
chain in the thylakoid membranes of C. reinhardtii
has been recently demonstrated [17]. This chloro-
3. P H O T O S Y N T H E T I C A P P A R A T U S
Since the following sections will deal with the
genes and proteins involved in photosynthesis, the
main features of the photosynthetic apparatus will
be briefly described. The primary reactions of
photosynthesis occur in the thylakoid membranes,
in which five major macromolecular complexes
can be recognized: photosystem II (PSII), photosystem I (PSI), the c y t o c h r o m e b 6 / f complex, the
A T P synthase complex and the light-harvesting
system (Fig. 2). Light energy is harvested by the
pigment antenna and directed to the PSII and PSI
reaction centers where the primary p h o t o c h e m istry results in a charge separation across the
membrane. The PSII reaction center generates a
strong oxidant capable of splitting water into
molecular oxygen, protons and electrons. The latter
are transferred on the reducing side of PSII to the
primary and secondary quinone electron acceptors
CF1
stroma
Nt
DP (~
!FO~
PSII
Ithy[ak°id
intrathylakoid space
Fig. 2. Tentative structural model of the photosynthetic complexes in the thylakoid membrane. Numbers indicate the Mr ( × 10 3) of
the polypeptides of PSII. LHCP, light-harvesting chlorophyll binding proteins: PC, plastocyanin: CPI, chlorophyll-protein complex
of PSI. Rieske, Rieske Fe-S protein. CF1 refers to the coupling factor (ATP synthase) and CFo to its membrane anchor.
16
plast respiratory chain oxidizes N A D ( P ) H at the
expense of oxygen and shares the plastoquinone
pool with the photosynthetic chain.
4. O R G A N I Z A T I O N OF T H E C H L O R O P L A S T
G E N O M E OF CHLAMYDOMONAS
4.1. Organization, structure and function of chloroplast genes of C. reinhardtii
The chloroplast genome of C. reinhardtii consists of 190-kb D N A circles [10,11]. Its physical
map is shown in Fig. 3. As in m a n y higher plants,
the two ribosomal regions are located within two
repeats that are oriented in opposite directions.
The chloroplast genes which have been identified
fall into three groups. The first set encodes
polypeptides of the photosynthetic apparatus, the
second set codes for components of the chloroplast protein-synthesizing apparatus which include
ribosomal RNA, tRNAs, R N A polymerase subunits, transcription and translation factors, and
ribosomal proteins, and there may be a third set
which encodes components involved in D N A replication and recombination (table 2; and Fig. 3).
While over 20 protein genes have been localized
on the chloroplast genorne of C. reinhardtii (Fig.
3), the existence of at least 40 chloroplast genes of
known function can be inferred based on the
assumption that all the proteins synthesized within
the organelle are encoded by the chloroplast DNA.
Indeed, there is no convincing evidence for m R N A
transport across the chloroplast envelope. By using
inhibitors specific for cytoplasmic translation, it
has been shown that close to 20 ribosomal proteins are synthesized in the chloroplast of C. reinhardtii [18]. A list of R N A s and proteins known to
be encoded or synthesized by the chloroplast is
given in Table 2. Sequencing of several chloroplast
genes has revealed a high degree of sequence
conservation between the algal and higher plant
genes [3]. As an example, the rbcL and psbA
genes are 77 and 80% homologous, respectively,
between C. reinhardtii and spinach [19,20].
Four chloroplast genes of C. reinhardtii (out of
a total of ten examined at the nucleotide level)
have been shown to contain introns: the 23S r R N A
gene [21,54] psbA, coding for the D1 protein of
PSII [20]; psaA1 coding for the P700 apoprotein
A1 of PSI (U. Kiick, unpublished results); and
rpoC, coding for the/3' subunit of an R N A polymerase (S. Surzycki, unpublished results). The
ribosomal intron consists of 888 bp and can be
folded with a secondary structure that is typical of
group I introns of fungal mitochondrial genes
[54-56]. It also has sequences homologous to the
box 9 and box 2 consensus sequences in which
cis-dominant mutations leading to splicing deficiency have been found in yeast mitochondrial
introns [57]. The intron contains a 489-bp open
reading frame coding for a potential polypeptide
that is related to mitochondrial maturases (Fig. 4)
[54,55]. The four introns of psbA range from
1.1-1.8 kb and appear to belong to the group I
introns based on the presence of box 9 and box 2
consensus sequences [20]. Intron 3 of psbA is
completely absent from C. smithii and therefore
represents an optional intron [36]. Introns 2 and 3
have been entirely sequenced except for a small
gap in intron 3 (J. Erickson, M. Rahire and J.D.
Rochaix, unpublished results). It is interesting to
note that open reading frames of 69 and 160
codons in introns 2 and 3, respectively, prolong
the upstream exons (Fig. 4). The presence of similar open reading frames in several yeast
mitochondrial genes is well documented and has
led to the 'maturase' concept [58]. The open reading frame of intron 2 is followed, after a frameshift,
by a second open reading frame of 298 codons
(Fig. 4). Recently, a 2.2-kb intron has been found
in psaA1, which appears to belong to the group lI
introns (U. KiJck, unpublished results).
4.2. Comparison of the chloroplast genome organization of C. eugametos, C. moewusii, C. reinhardtii
and C. smithii
The genus Chlamydomonas is one of the largest
genera of the green algae. The physical map of the
chloroplast genomes of the two interfertile species
Chlarnydomonas eugametos and Chlamydomonas
moewusii have been determined and shown to
consist of 242 and 292-kb circles, respectively
[59,60]. The two genomes display the same
arrangement of common sequences, except for two
major insertions in the chloroplast D N A of C.
moewusii: a 21-kb insertion in the inverted repeat
17
0 0
Q'O.
...........
~.~
e o_
~'o
.-'"
.-
...--
--.~,aoc ~
"" "-.9,>
~
__
• boo 4
i
3o,~o ,,"
t
2~.
L.) I1~
16
,~Qt~8
~ .~.
07s,6 ' c
O
F'ig. 3. Physical map of the chloroplast genome of C. reinhardtii. The inner circles represent the BamHI (Ba) and Ec, RI (R)
restriction fragments [11]. The two segments of the inverted rcpcat containing the ribosomal RNA genes [21] and pshA [20] are drawn
on the outside and bounded by' arrows. Introns are drawn in thinner lines relative to the coding sequences. Dark ~edgcs indicate 12
identified tRNA genes of which three are located within the inverted rcpcat [53] (scc Table 2). Characterized protein genes in the
single copy region are marked: psaA1 (U. Ki~ck, unpublished results), psaA2 (M. Schneider et al., unpubli,,,hed rcsults): l~shB, p~h("
(C. Kovacic, P. Malnoe and J.D. Rochaix, unpublished results) p~hD [22], atpA [23], atpB [24,25] atpE. atpF, atptt (J. Woessncr, J.
Robertson, J. Boynton and N. Gillham, unpublished rcsuhs), rhcL [19], tufa [26], rpoA [27], q~oB, q~oC, dnuA [28] (S. Surzvcki,
unpublished results) q~s-12, rps-7, rpl-2 [50]. Recently, Surzycki and collaborators have found chloroplast DNA ~,equcnccs that
cross-hybridize to the E. coh genes ut,rC (Rll), re<A (RI3, R22) and the gene of rho factor (RI1. marked with *). The eight
identified chloroplast ARS sequences are indicated by 01 to 08 [29 31]. The four chloroplast DNA sequences promoting autonomous
replication in C. reinhardtii are marked by ARC1, ARC2, ARC3a, ARC3b [32]. Two authentic origins of replication are indicated by
oriA and oriB [33]. Abbreviations for genes are explained in Table 2.
a n d a 5.9-kb insert in the single c o p y r e g i o n close
to the 16S r R N A g e n e s [61,62]. I n t e r e s t i n g l y , this
5.9-kb D N A also exists as a free l i n e a r p l a s m i d in
t h e s e cells a n d it has b e e n s h o w n to b e i n h e r i t e d
uniparentally
[63,64]. It h a s n o t yet b e e n
d e t e r m i n e d w h e t h e r the p l a s m i d r e p l i c a t e s a u t o n o m o u s l y o r w h e t h e r it is a m p l i f i e d f r o m its c o p y in
the c h l o r o p l a s t g e n o m e . S i n c e the p l a s m i d is abs e n t f r o m C. eugametos, the a n a l y s i s o f the t r a n s m i s s i o n p a t t e r n of the free a n d i n t e g r a t e d f o r m s in
i n t e r s p e c i f i c h y b r i d s b e t w e e n C. eugametos a n d C.
moewusii s h o u l d p r o v i d e n e w i n s i g h t s i n t o the
m e c h a n i s m o f r e p l i c a t i o n o f this p l a s m i d .
C o m p a r i s o n o f the c h l o r o p l a s t g e n o m e s o f C.
eugametos a n d C. reinhardtii, w h i c h are n o t i n t e r fertile, reveals a g r e a t d e a l of d i v e r g e n c e in the
p r i m a r y s e q u e n c e s a n d in g e n e o r g a n i z a t i o n [65].
F o r e x a m p l e , the o r i e n t a t i o n of psbA r e l a t i v e to
the r i b o s o m a l R N A g e n e s is d i f f e r e n t , a n d rbcL is
i n c l u d e d in the i n v e r t e d r e p e a t o f C. eugametos. In
Table 2
Chloroplast genes and characterized mutations in
Chlamydomonas
reinhardtii
The location of the genes on the chloroplast genome is shown in Fig. 3. - , Not determined; P, point mutation; A, deletion; v,
insertion. In addition 9 distinct tRNA genes have identified: t r n T , t r n P , t r n F (2 genes) t r n N , t r n L , t r n L t r n A , t r n H , of which the last
three are located within the inverted repeat [53].
Complex
Component
(,4) Photosynthetic
ATP synthase
a subunit
/3 subunit
c subunit
CI subunit
Photosystem II "
Photosystem I
RuBisCO
(B) Components
Gene
Introns
atpA
-
-
atpB
0
FuD50
atpE
0
-
atpF
-
-
CIII subunit
atpH
-
-
D1
psbA
4
FUD7, 8-36C
DCMU4, Dr2,
Ar207, Br202
47-50-kDa protein
43-47-kDa protein
psbB
-
psbC
-
FUD34, MA16?
D2
psbD
0
FUD47
A1 P700 apoprotein
A2 P700 apoprotein
psaA1
1
psaA 2
0
Large subunit
rbcL
0
i n v o l v e d in c h l o r o p l a s t t r a n s c r i p t i o n
Ribosome
R N A polymerase
Mutant
Nature of mutation
Reference
components
23S rRNA
16S rRNA
Ribosomal protein
Elongation factor Tu
a suburtit
fl subunit
13' subunit
Termination factor Rho factor
( C ) P r o t e i n s i n v o l v e d in D N A
1241
10-6C
18-7G
18-5B
and translation
[23,24]
[24,25,34]
(J.D. Woessner, J. Robertson,
J. Boynton and N. Gillham,
unpublished results)
a n d in D N A
rnaL
1
CapR,EryR b
rnaS
0
Spc R, Str R b
,x (8-10 kb)
P
P
x7 (46 bp)
[35,36]
[37-40] (J. Erickson, L. Mets
and J.-D. Rochaix,
unpublished results)
(M. Kuchka, P. Bennoun
and J.-D. Rochaix,
unpublished results)
[41]
a (4 bp)
(U. Kiick, unpublished results)
(M. Schneider et al.,
unpublished results)
P
P
P
[42,43]
[44,45]
[44,451
replication and recombination
p?
p?
[46-48]
[48,49]
rpl-2
-
-
rps-7
-
-
[501
[501
rps-12
-
-
[50]
tufa
-
-
rpoA
-
-
-
rpoB
-
-
-
[26]
[27]
[281
rpoC
1
-
-
[281
rhoC c
_
_
_
(S. Surzycki,
unpublished results)
replication and recombination
dnaA
~
-
uvrC c
_
recA c
(S. Surzycki,
unpublished results)
(S. Surzycki,
unpublished results)
(S. Surzycki,
unpublished results)
" The PSII core contains in addition apocytochrome b559, whose gene has not yet been located on the chloroplast genome of C.
reinhardtii.
b The assignment of uniparental chlorampheniol (CapR), erythromycin (Eryg), spectinomycin (Spc g) and streptomycin (Str R)
resistance mutations to the 23S and 16S rRNA genes is based on the location of similar mutations in the yeast mitochondrial rRNA
gene [51], in the chloroplast 16S rRNA gene of E u g l e n a g r a c i l i s [52] and on recent correlations between restriction site alterations
and resistance phenotypes (see section 6 for details).
c These gene assignments are based on cross-hybridizations detected between E . coil gene probes and chloroplast restriction
fragments of C. r e i n h a r d t i i (S. Surzycki and S. Hong, unpublished results).
19
23 S rDNA
PI
,00 bc
psbA
a~
oa
aa
c o m p l e x e s have been identified a n d c h a r a c t e r i z e d
in Chlamydomonas. M e t h o d s of m u t a n t isolation
have been covered in recent articles a n d will not
be discussed here [67,68].
5.1. Ribulose
oxvgenase
P TGA
ATG
P
Fig. 4. Chloroplast introns in the 23S rDNA and pshA of C.
reinhardtii • exon sequences, [] intron sequences (not determined), [] intron regions with blocked reading frames, []
intron open reading frames that prolong the upstream exons, []
open reading frame within introns. The box 9 (zx) and box 2
(O) consensus sequences [57] characteristic of group I introns,
are indicated. P1 marks a conserved dodecapeptide found in
mitochondrial intron open reading frames [55]. Sizes of polypeptides encoded by the open reading frames are indicated in
amino acids (aa). Numbers above psbA correspond to the
amino acids contained in each of the five exons [20]. The
boundary between the first and second open reading frame in
the second intron of pshA is enlarged in the lower part of the
figure (J. Erickson, Rahire and Rochaix, unpublished results).
Abbreviations for gcnes are explained in Table 2.
a d d i t i o n , transfer of c h l o r o p l a s t D N A sequences
have occurred from one single c o p y region to the
o t h e r d u r i n g the evolution of these two species.
T h e v a r i a t i o n in c h l o r o p l a s t D N A between them
greatly exceeds that o b s e r v e d in the vast m a j o r i t y
of higher p l a n t s [65,66]. W h e t h e r this v a r i a t i o n
results from a longer p e r i o d of evolution s e p a r a t ing these two algae as c o m p a r e d to that of higher
plants, or w h e t h e r it reflects a high rate of D N A
r e a r r a n g e m e n t in algae, r e m a i n s an o p e n question.
T h e latter p o s s i b i l i t y has to be c o n s i d e r e d since
p h o t o s y n t h e t i c m u t a n t s i n d u c e d b y 5-fluorodeo x y u r i d i n e have suffered extensive sequence rea r r a n g e m e n t s p r i n c i p a l l y in the region of the inverted repeat [36]. In two of these m u t a n t s the
inverted r e p e a t has been e x p a n d e d from 20 kb to
63 kb [36].
5. P H O T O S Y N T H E T I C M U T A T I O N S : T O O L S
FOR UNDERSTANDING
THE FUNCTION
AND ASSEMBLY OF PHOTOSYNTHETIC
COMPLEXES
A large n u m b e r of c h l o r o p l a s t a n d nuclear mutations affecting p h o t o s y n t h e t i c enzymes a n d
1,5
bisphosphate
carboxvlase /
A m o n g the p h o t o s y n t h e t i c complexes, R u B i s C O
has the most simple structure. T h e h o l o e n z y m e
consists of eight identical large subunits (LS) and
eight identical small subunits (SS), which are enc o d e d b y the c h l o r o p l a s t and nuclear genomes,
respectively. The large subunit c o n t a i n s the catalytic sites for two c o m p e t i n g reactions: c a r b o x y l a tion by C O 2 a n d o x y g e n a t i o n by 02 of ribulose
b i s p h o s p h a t e [16]. W h i l e there are between 5 0 - 1 0 0
LS genes (rbcL) p e r Chlamydomonas cell [69],
there are o n l y two closely linked genes of the
small subunit (rbcS) in the nuclear g e n o m e [70].
Both nuclear genes c o n t a i n three introns which are
l o c a t e d at different p o s i t i o n s within the c o d i n g
sequence than in higher p l a n t s [71]. The genes
e n c o d e two slightly distinct p r o t e i n s which differ
b y four a m i n o acids, and their expression is differentially regulated by the growth c o n d i t i o n s [71].
T h e first u n i p a r e n t a l R u B i s C O mutant, 10-6C,
was isolated as a light-sensitive a c e t a t e - r e q u i r i n g
m u t a n t by Spreitzer a n d Mets [42]. T h e m u t a t i o n
alters the isoelectric p o i n t of the large subunit and
it greatly reduces c a r b o x y l a s e a n d oxygenase activity. T w o a d d i t i o n a l m u t a n t s 18-5B and 18-7G
were isolated by screening for u n i p a r e n t a l mutations that did not r e c o m b i n e with the first m u t a tion in 10-6C [44]. These two m u t a n t s d o not
a c c u m u l a t e either large or small subunit. The rbcL
genes of the three m u t a n t s were isolated, p a r t i a l l y
sequenced a n d f o u n d to c o n t a i n single base pair
changes. M u t a n t 10-6C has a missense m u t a t i o n
that changes a gly (residue 171) to asp near one of
the active sites of the large s u b u n i t (Fig. 5) [43].
T h e significance of this base change was conf i r m e d by isolating a revertant in which the wildt y p e gly 171 was f o u n d to be restored [72]. The
o t h e r two m u t a n t s c o n t a i n nonsense m u t a t i o n s
n e a r the 3' and 5' ends of rbcL (Fig. 5) [45].
Interestingly, the 18-5B m u t a n t which p r o d u c e s a
t r u n c a t e d large s u b u n i t only 25 a m i n o acids shorter
20
i~
~
.~
HA
I
~0 6C
G IAT °~:
+
--
R/. 7
OiGy;g ,l
"t"
"1"
18-5B
18-7G
TGI A ' ¢'c
TAOlcmee
Fig. 5. Mutations in the gene of the large subunit of ribulose
bisphosphate carboxylase. The rhcL coding sequence is shown
with its 5'- and 3' untranslated regions. Regions I, III and IV
correspond to the active sites of the large subunit of ribulose
bisphosphate carboxylase and region II corresponds to the
CO 2 activator region [16]. Base and corresponding amino acid
changes are shown for 10-6C [43], R4-7, a revertant of 10-6C
[72], 18-5B and 18-7 G [45]. + / refer to the presence or
absence of holoenzyme (H) and of enzymatic activity (A).
than its wild-type counterpart, is unable to form a
stable holoenzyme [44].
Since the intact large subunit is no longer
synthesized in these mutants, they allow one to
study whether synthesis of the two subunits of
RuBisCO is tightly coordinated. In both nonsense
mutants examined, the steady-state levels of the
large and small subunit m R N A s are not appreciably changed relative to the wild type. Pulse-labeling of cells reveals that the small subunit is
synthesized and processed to its mature size, thus
strongly suggesting that it is imported into the
chloroplasts. However, pulse-chase experiments
reveal that the small subunit is rapidly degraded
in these mutants [45]. Similar results have been
obtained by Schmidt and Mishkind who inhibited
chloroplast protein synthesis with chloramphenicol or used a chloroplast ribosome deficient strain
[73]. It can be concluded that there is no tight
coordination of synthesis of the two subunits and
that the stoichiometric accumulation of the two is
achieved at the post-translational level by specific
degradation of the subunits present in excess. It
will be of interest to examine the fate of the large
subunit in mutants unable to produce intact small
subunit. However, mutants of this type have not
yet been reported.
One of the nonsense mutants, 18-5B, reverts at
a high spontaneous frequency, 6 x 10 6 [74]. These
revertants are unstable: they segregate both wildtype and acetate-requiring phenotypes in crosses
or under permissive growth conditions for
acetate-requiting mutants [74]. Spreitzer et al. [74]
presented a model based on the observation that
the acetate-requiring segregants were always unstable. The model postulates a heteroplasmic
population of chloroplast D N A molecules in which
all of them carry the rbcL mutation of 18-5B and
where some fraction of these D N A molecules contains in addition a suppressor mutation capable of
restoring the wild-type large subunit. The wild-type
allele of the suppressor gene would be required for
performing its normal function. While homoplasmicity of the wild-type allele would confer a
selective advantage for cells grown under heterotrophic conditions (where photosynthetic function
is not required), homoplasmicity of either allele
would be lethal under phototrophic conditions. In
this model, heteroplasmicity would be maintained
by constant selection for photosynthetic function.
The molecular basis of this suppression has not
yet been elucidated and it could involve tRNAs,
ribosomal proteins, ribosomal RNAs or other factors involved in chloroplast translation.
5.2. Photosystem H (PSII)
The PSII core, which is embedded in the
thylakoid membrane consists of at least five chloroplast-encoded proteins (Table 2) [76]. The genes
of four of these polypeptides have been located on
the chloroplast genome of C. reinhardtii [20,22]
(C. Kovacic, P. Malnoe and J.-D. Rochaix, unpublished results), psbB and psbC encode two proteins of 47-50 and 43-47 kDa, respectively, which
have been shown to be chlorophyll a-binding proteins [75]. The polypeptides D1 and D2 are encoded by psbA and psbD, respectively [20,22]. The
gene of the apoprotein of cytochrome b559, psbE,
has not yet been located on the chloroplast D N A
map of C. reinhardtii. Three nuclear-encoded polypeptides of 33, 24 and 18 kDa are involved in
oxygen evolution (Table 3). Several low-molecular-weight proteins appear to be associated with
the PSII core [76].
Mutants deficient in PSII activity can usually
be recognized by their high fluorescence yield [68].
A striking feature of these mutants is that they all
lack the core PSII polypeptides, indicating that
they are unable to assemble a stable PSII complex
21
Table 3
Properties of photosystem II m u t a n t s of
Chlamydomonas reinhardtii
psB, pshC, psbA and psbD are the chloroplast genes of the 50-kDa, 47-kDa, D1 and D2 proteins of the PSII core. The 33-kDa,
24-kDa and 18-kDa proteins of the oxygen evolving complex (OEC) of PSII are encoded by nuclear genes. I - [
gene, + stable accumulation of protein, ( + ) reduced a m o u n t of protein,
Mutant
PSII core
p r i m a w lesion in the
no accumulation of protein.
OEC of PSII
psbB
psbC
pshA
psbD
(50 kDa)
(47 kDa)
D1
D2
FuD7
-
~l
-
(+ )
FuD47
-
FuD34
~?
-
-
33 k D a
24 k D a
18 k D a
+
+
+
+
+
-
+
-t-
+
Chloroplast
MA16
-
~?
BF25
+
+
+
+
+
FuD39
+
+
+
+
+
FuD44
(+)
(+)
(+)
(+)
Nuclear
[35,41]. Pulse-labeling of cells with [14C]acetate in
the presence of an inhibitor of cytoplasmic ribosome translation allows one to easily detect the
most abundant polypeptides synthesized in the
chloroplast: the large subunit of RuBisCO and
several thylakoid polypeptides which include the
PSII core proteins (Fig. 6). It is therefore possible
to measure the synthesis of thylakoid polypeptides
of mutants that are unable to stably insert these
proteins into the membranes. Analysis of several
PSII mutants by this method has revealed that
more than 85% lack the D1 polypeptide (Fig. 6).
Surprisingly, all the mutants of this class which
were examined have both copies of psbA deleted
from their chloroplast genome [35]. It is known
that the chloroplast inverted repeat of C. reinhardtii contains numerous repetitive elements, interspersed throughout the chloroplast genome
[6,21,77]. Since psbA is known to be surrounded
by these repeats, it is possible that the deletions
arise through recombination between the repeats
[35,361.
I I
4-
+
Another class of nuclear and chloroplast PSII
mutants unable to synthesize both the D1 and D2
polypeptides is of special interest. Genetic and
molecular analysis of one of these chloroplast
mutants, FUD47, has revealed that its psbA is
intact and transcribed at wild-type levels [41]. The
mutation was found to be a 46-bp direct D N A
duplication within psbD, the gene of the D2 protein, thus causing a frameshift which results in a
truncated D2 polypeptide that is highly unstable.
With the exception of D1 and D2, all the other
PSII core polypeptides are synthesized and integrated into the membrane in this mutant, but
never accumulate. It appears therefore that D2 is
not only involved, directly or indirectly, in the
stable assembly of the PSII core complex, but also
regulates D1 synthesis or stability at either the
translational or the post-translational level.
Two other chloroplast PSII mutants, F U D 3 4
and MA16, also lack the PSII core (J. GirardBascou and P. Bennoun, unpublished results).
Analysis of the newly synthesized chloroplast pro-
22
--
0'}
n
LL
LL
LL
r-~
D
LL
LL
i i % 1 ¸ ~........
CFI<
CFI<
6
....
Cyt.
D2
D2
E
19
L2
L3
L4
.... *
.........
- ~
~~
~
........
34
36
C F I,(::{,,p -~__
5 -6
--
~
.........
Fig. 6. Gel electrophoretic fractionation of thylakoid membrane polypeptides from wild-type cells and photosystem II
mutants missing psbA (FUD7, F U D l l 2 and FUD13: see
Table 2 for details) of C. reinhardtii labelled for 45 rain with
[14C]acetate in the presence of anisomycin. Stained gel and
corresponding autoradiogram are shown in the left and right
panels, respectively. Upper part: 12-18% SDS polyacrylamide
gel with 8 M urea. Lower part: 7.5-15% SDS polyac~lamide
gel: only the region of polypeptide 5 is shown because this
band comigrates with the a subunit of ATP synthase in the
SDS urea gel shown above. PSII polypeptides are marked with
dots (from [35] with permission).
teins in these mutants reveals that F U D 3 4 specifically lacks the 43-47 k D a polypeptide, and that
this protein is altered in MA16. The other PSII
core polypeptides, including D1 and D2, are
synthesized normally, but do not accumulate. It is
likely that the primary lesion in these two mutants
is located in psbC, although no molecular evidence of this has yet been found. These results
suggest that any of the PSII core polypeptides
may play a role in the stable assembly of the core
complex. It is interesting to note that nuclear
mutations have been isolated which have the same
phenotype as FUD34, i.e., these mutants are unable to synthesize the 4 3 - 4 7 - k D a polypeptide.
Recently, a nuclear mutant has been isolated from
F U D 3 4 in which the chloroplast mutation of the
latter is specifically suppressed (J. Girard-Bascou,
unpublished results).
The oxygen-evolving complex of PSI! consists
of at least three nuclear-encoded polypeptides of
33, 24 and 18 kDa (see [79] for review). By screening a c D N A library of C. reinhardtii (M.
Goldschmidt-Clermont, A. Shaw, P. Malnoe, S.
Mayfield, unpublished results) in the expression
vector X g t l l [80] with monospecific antibodies
against these proteins, it has been possible to
isolate the three respective c D N A and genomic
clones (S. Mayfield and J.-D. Rochaix, unpublished results). Southern hybridizations of genomic
D N A with the three c D N A clones reveals that the
corresponding genes are present in single copies.
Three low-fluorescent nuclear mutants deficient in
oxygen evolution have been isolated [78] (P. Bennoun, unpublished results). Two of these, FUD39
and BF25, lack the 24-kDa polypeptide, but not
the 33 and 18 kDa polypeptides, while FUD44
lacks the 33 kDa polypeptide but not the 24 and
18 kDa polypeptides (S. Mayfield and J.-D.
Rochaix, unpublished results). The core PSII complex is still present in FUD39 and BF25, indicating that it can assemble independently from the
oxygen evolving complex. This also holds true for
FUD44, except that the amount of core complex
proteins is reduced. No m R N A for the missing
polypeptide is detectable in any of these mutants.
Genomic rearrangements have occurred at or near
the 24-kDa and 33-kDa protein genes in FUD39
and FUD44, respectively. Revertants of FUD44,
which occur at a frequency of 10-7, all display a
novel, identical genomic arrangement that is different from both wild type and mutant. The
molecular basis of this phenomenon, which may
involve transposable elements, is under study.
A great deal of similarity exists between the
23
reaction centers of PSII and of purple photosynthetic bacteria. In both cases a pheophytin acts as
an intermediate electron acceptor and a very similar electron accepting quinone iron complex exists
[81,82]. A structural homology between D1, D2
and the L and M subunits of the bacterial reaction
center has been noted [83,84,123]. Recently the
molecular structure at 0.3 nm resolution of the
reaction center of Rhodopseudomonas viridis has
been determined by X-ray diffraction studies on
crystallized reaction centers [85]. Based on the
structural and functional homology between the
two bacterial and PSII subunits, it has been proposed that D2 and D1 are the apoproteins of the
stable primary and secondary electron acceptors
of PSII, respectively, and that they can be folded
with 5 transmembrane domains to form a core
with chlorophyll, pheophytin and quinone which
is very similar to the bacterial reaction center
[82,85]. While this model of D1 (Fig. 7) fits nicely
with data obtained from herbicide-resistant
mutants (see below), it does not completely agree
NH2
250~
F#~~
. --
'
5C
G
I
1
~ G
~
H
COOH
Fig. 7. Membrane-spanning model of the photosystem II polypeptide DI of C. remhardtii. This model proposed by Trebst [82] is
based on the homology between D1 and the L subunit of the bacterial photosynthetic reaction center [83,123] and on the X-ray
structure of the latter [85]. Amino acids are represented in the single letter code. The five transmembrane domains are framed.
Stroma and intrathylakoid space are on the upper and lower side, respectively. Residues V219, F255, $264 and L275 which are
individually changed in four different herbicide resistant mutants [39,40], (J. Erickson et al., unpublished results) are indicated. The
region which binds azidoatrazine (residues 244-224) is marked [118]. The trypsin cleavage sites at R225 and R238 are indicated [86].
The two H215 and H272 residues together with the corresponding residues of the D2 polypeptide may be involved in iron and QB
binding [82,85].
24
with results obtained with protease digestion of
thylakoid membranes which indicate that the
carboxy-terminal end of D1 is on the stromal side
[86].
Several uniparental mutants with different levels
of resistance and cross-resistance to the herbicides
atrazine, diuron and bromacil have been isolated
in C. reinhardtii [37-39,87]. Analysis of psbA from
some of these mutants has revealed four distinct
residues on the D1 polypeptide which can be
changed (Fig. 7) [39~40] (J. Erickson, L. Mets and
J.-D. Rochaix, unpublished results). It is noteworthy that although the four possible mutation
sites identified to date in D1 (Val 219 ~ Ser; Phe
255 ---, Tyr; Ser 264 ~ Ala; Leu 275 --* Phe) are
scattered over a stretch of 56 amino acids, they are
located close to each other in the above model in a
region of D1 that is oriented towards the stromal
side of the thylakoid membrane, close to the presumed acceptor quinone-binding niche (Fig. 7).
The iron which connects the two quinone acceptors in the bacterial reaction center would also be
located in the same region and held by four His
residues (His 215 and His 275 from D1; Fig. 7;
and two from D2 at the same positions). It is
noteworthy that the mutations affecting Val 219,
Phe 255 and Leu 275 do not markedly alter electron transport and thus photosynthetic yield, in
contrast to the Ser 264 mutation, which significantly retards electron transport [38,39]. Atrazine
resistance in higher plants has also been shown to
be due to a change at the same Ser 264 residue,
but in contrast to C. reinhardtiL this serine is
replaced by glycine [88,89].
5.3. Photosystem I
This photosynthetic complex consists of a large
number of polypeptides: two chloroplast encoded
polypeptides of apparent M r 60000-70000 (subunit I) both of which appear to be the apoproteins
of the CPI chlorophyll-protein complex which
comprises the reaction center chlorophyll P700
[90,91], and several smaller subunits with sizes
ranging between 25 and 8 kDa [92]. Recently, the
two genes of subunit I, psA1 and psaA2, of C.
reinhardtii have been sequenced (U. Khck, M.
Schneider, M. Dron, Y. Choquet, unpublished results). Whilst psaA2 is continuous, psaA1 con-
tains a large intron near its 5' end. The function of
the other subunits of PSI has not yet been clearly
established. Some of the smaller subunits have
been tentatively identified as the apoproteins of
the stable Fe-S electron acceptors of PSI [93].
Comparisons of PSI defective chloroplast and
nuclear mutants of C. reinhardtii with wild-type
cells have shown that all the mutants lack subunits
I and the same set of low-molecular-weight
thylakoid polypeptides [94]. Genetic analysis of 25
nuclear PSI mutants has revealed that they belong
to 13 c o m p l e m e n t a t i o n g r o u p s scattered
throughout the nuclear genome [94]. Eight chloroplast PSI mutants fall into four distinct genetic
loci as defined by the inability of mutants of the
same group to recombine with each other (J.
Girard-Bascou, unpublished results; see section
6). One chloroplast PSI mutant, FUD26, produces a truncated form of subunit I. Partial sequence analysis of psaA2 from this mutant has
revealed that it has suffered a 4-bp deletion which
results in a frameshift (M. Schneider, Y. Choquet,
J. Girard, F. Galangau, M. Dron, P. Bennoun,
unpublished results).
5.4. Cytochrome b 6 / f complex
As in the other photosynthetic protein complexes, the cytochrome b 6 / f complex consists of
subunits of nuclear and chloroplast origin. It is
well documented that cytochrome f , cytochrome
b6 and subunit IV are encoded by the chloroplast
genome whereas the Rieske protein is coded for
by the nuclear genome [95]. A fifth subunit of this
complex has recently been identified in C. reinhardtii by comparing the polypeptide profiles of
the purified complex with that of thylakoids from
mutants lacking this complex [96]. Translation of
this subunit V appears to occur on cytoplasmic
ribosomes [96]. Analysis of thylakoid polypeptides
of pulse labelled cells from several chloroplast and
nuclear mutants affected in the function of the
cytochrome b 6 / f complex has revealed that it is
assembled in two steps. The three chloroplast-encoded subunits are inserted independently in the
thylakoid membrane and assembled into a subcomplex in the absence of the nuclear-encoded
Rieske protein and subunit V. These two proteins
are unable to insert and assemble in the mem-
25
brane without the presence of the chloroplast-encoded sub-complex. The assembly of the cytochrome b 6 / f complex resembles to some degree
that of PSII, where the chloroplast-encoded subunits can also assemble in a core complex in the
absence of the nuclear-encoded subunits. However, while the latter are peripheral membrane
proteins, the Rieske protein of the cytochrome
b 6 / f complex is a transmembrane polypeptide.
5.5. A TP synthase complex
This complex consists of a peripheral component which included 5 subunits a, /3, y, 6, ~ and a
membrane-embedded component which contains
at least three subunits (CI, CII, C I I I proteolipid)
[97]. The genes of the chloroplast-encoded subunits, atpA (cO, atpB (/3), atpE (~), atpF (CI)
and atpH (CIII) have been identified on the chloroplast genome of C. reinhardtii (see Fig. 3). Sixteen chloroplast mutations affecting the ATP synthase complex have been characterized [24]. Complementation assays in young zygotes and recombination analysis has revealed five complementation groups. Although this number agrees with
the number of identified ATP synthase chloroplast
genes, this correlation still needs to be proven
rigorously at the molecular level. In one case it has
been possible to assign mutants of one complementation group to the atpB locus based on the
fact that some of the mutants have chloroplast
D N A deletions in this region [34].
6. R E C O M B I N A T I O N A N A L Y S I S OF C H L O ROPLAST GENES: CORRELATION
BETWEEN PHYSICAL AND GENETIC CHLOR O P L A S T D N A MAPS
The localization and characterization of several
mutations on the chloroplast genome of C. reinhardtii has allowed one to attempt to correlate
physical distances with recombination frequencies
between markers. Genetic mapping of chloroplast
genes in Chlamydomonas has been performed in
several ways including recombination and cosegregation analysis in pedigrees [98] and segregation and recombination analysis in the progeny of
biparental zygotes [15,98]. Since these methods
have been extensively discussed (see [15]), only the
zygote clone analysis, which is commonly used,
will be described. One first selects for biparental
zygotes and scores the frequencies of different
chloroplast genotypes among the progeny of each
biparental zygote, usually 64 or more randomly
selected cells. Because thousands of cells need to
be examined in order to obtain reproducible maps
[48], the standard zygote clone analysis has been
modified in order to obtain reliable mapping data
with a smaller number of cells [99]. Cells are
selected that carry at least one marker from the
paternal mt parent, and only one cell from each
biparental clone is examined, thus ensuring that
each cell scored has a genotype that is established
independently from the others [99].
Chloroplast gene mutations conferring resistance to erythromycin, neamine, spectinomycin
and streptomycin were the first to be mapped by
recombination analysis [15,48,98]. These mutations were shown to be genetically linked and
their order and relative position were determined.
A first indication that these mutations may affect
the chloroplast r R N A genes was provided by interspecific hybrids of C. eugametos and C.
moewusii whose chloroplast D N A restriction patterns differ. Linkage between a streptomycin
sensitivity locus and a chloroplast restriction fragment which hybridizes to the 16S r R N A gene
region of C. reinhardtii was shown by an analysis
of recombinant chloroplast restriction patterns
from the hybrid progeny [100]. Similarly, a correlation was found in other hybrids between the
inheritance of chloroplast mutations conferring
resistance to streptomycin and erythromycin and
the small specific deletion/addition differences at
the 5'-end of the 16S r R N A gene and the 3'-end of
the 23S r R N A gene, respectively [101]. Streptomycin resistant mutants from Euglena gracilis
have been shown to contain a single base change
in an invariant position of their chloroplast 16S
r R N A gene [52].
Recently, four independent mutations conferring resistance to spectinomycin have been mapped
to the 16S r R N A gene of C. reinhardtii based on
alterations of a restriction site in a highly conserved region of this gene (J. Boynton, E. Harris
and N. Gillham, unpublished results). The same
26
restriction site change has also been observed for
similar mutants in E. coli [102] and tobacco [103].
A uniparental chloramphenicol resistant mutant
has been isolated but its mutation has not yet
been mapped relative to the other antibiotic resistance markers [46]. Based on the sequence homology between chloroplast and mitochondrial
r R N A s [6] and on the localization of mutations
conferring resistance to chloramphenicol and
erythromycin in the yeast mitochondrial large
r R N A gene [51], it is tempting to think that the
homologous chloroplast mutations will be found
in the 23S r R N A gene of C. reinhardtii.
Using the paternal marker selection mapping
method, Mets and Geist [99] have demonstrated
linkage between three photosynthetic mutations
and the uniparental linkage group comprising the
antibiotic resistance mutations. One of the photosynthetic mutants~ 10-6 C, was shown to have a
single base-pair change in rbcL (see section 5.1)
and therefore provided the first correlation site
between the physical and genetic chloroplast D N A
maps [43,99]. No recombinants could be recovered
between the other two mutants (Dr2, which is
herbicide-resistant, and 8-36C which lacks photosystem II), suggesting that the same gene is affected in both mutants [99]. Indeed, a molecular
analysis of these mutants has revealed that the
first carries a single base-pair change in psbA (see
section 5.2) and the second has a double deletion
in its inverted repeat that includes all of psbA
[35,40]. Surprisingly, the results of crosses in which
the same mt parent carried the herbicide and
antibiotic resistance markers, were quite different,
depending on whether the marker of the mt +
parent was in rbcL or psbA. In the first case, the
parental class accounted for two-thirds of the
progeny and the data obtained with four markers
could not be resolved into a single linear map. In
the second case, the frequency of recombination
was higher: the parental class accounted for only
1 / 4 of the progeny and the frequency of recombination between the same two antibiotic resistance
markers was nearly twice as high as in the first
cross [99]. It was suggested that the organization
of the chloroplast D N A into nucleoids could lead
to incomplete mixing of the parental chloroplast
D N A and that the extent of mixing may depend
on the organization of the thylakoids, which is
different in PSII mutants compared to wild-type
and rbcL mutants [99]. It is also conceivable that
the deletion in 8-36C may stimulate chloroplast
D N A recombination in crosses with strains lacking the deletion. Effects of this sort are well-documented in yeast: in heteropolar crosses of yeast
strains that differ by the presence and absence of
the omega insertion in the mitochondrial large
r R N A gene, striking perturbations occur in the
recombination frequencies of nearby markers
[104].
Another complication in the study of genetic
linkage of chloroplast markers arises from the
structure of the chloroplast genome. Since intramolecular recombination is known to occur
within the inverted repeat [36,105], one single copy
region may be inverted relative to the other. If this
recombination occurs frequently, markers in each
of the single copy regions would appear to be
unlinked with respect to each other. On the other
hand, genetic linkage would be expected between
markers in the single copy region and in the
inverted repeat, as has been found between
markers of rbcL and psbA [99]. This can be
attributed to an efficient gene conversion which
appears to operate between the two segments of
the inverted repeat. In at least one case this has
been demonstrated for a single base pair mutation
conferring herbicide resistance [40] and in several
cases double symmetrical deletions have been
found in the inverted repeat [34,36]. Because of
this mismatch correction mechanism, intramolecular recombination in this region would not disturb
recombination frequencies between markers outside and inside of the inverted repeat.
The observed genetic" linkage between markers
in rbcL and psbA or in the ribosomal region
which are more than 10 kb distant from each
other on the chloroplast genome, indicates that
chloroplast markers recombine at a lower rate
than yeast mitochondrial markers. In the latter
case, genetic linkage is only apparent between
mitochondrial markers separated from each other
by less than 1 kb [104]. An alternate gene linkage
analysis (J. Girard-Bascou, unpublished results)
uses vegetative zygotes obtained from gametes of
opposite mating-type each carrying a photosyn-
27
thetic mutation and either the arg-2 or arg-7
nuclear mutation which are closely linked [106].
Vegetative zygotes remain diploid, divide mitotically and can be selected on medium lacking
arginine. The rate of recombination is estimated
from three parameters: the number of biparentals,
usually higher than 40%, the frequency of cells
able to grow on minimal medium and the differential growth rate of wild-type and mutant cells.
Using this method, Girard-Bascou (unpublished
results) has grouped eight different chloroplast
PSI mutants into four distinct genetic loci. Mutants
of the same locus recombine with a frequency of
less than 5 x 10 s while recombination between
mutations of presumably unlinked loci is greater
than 10 1. The same method yields a recombination frequency of 2 x 10 2 between mutations in
rbcL and psaA2, which are 1.9 kb apart (M.
Schneider, Y. Choquet, M. Dron, J. Girard-Bascon
and P. Bennoun, unpublished results).
In conclusion, chloroplast D N A recombination
has been clearly demonstrated in Chlamydomonas.
Six distinct loci on the chloroplast genome have
been correlated with defined photosynthetic mutations in C. reinhardtii. The correct interpretation
of recombination frequencies between chloroplast
markers is not an easy task, however, since it
depends on several parameters, such as the input
of the parental chloroplast genomes in the cross
[107,108], the extent of mixing of these two genomes in the zygotic cell, and possible alterations
in recombination frequencies due to somatic mutations. Before recombination frequencies can be
safely correlated with physical distances on the
chloroplast genome, it is clearly important to examine more markers with different and independently expressed phenotypes and to compare carefully the recombination frequencies between the
same markers obtained with the different mapping
methods. It may then be possible to use genetic
mapping to locate interesting mutations on the
chloroplast genome, such as those involved in the
regulation of the assembly and function of photosynthetic complexes.
It is noteworthy that a complementation test
has been developed for photosynthetic mutations
which examines the fluorescence induction kinetics or the luminescence properties of young zygotes
[109]. This test works for all nuclear photosynthetic mutations examined and for the chloroplast
mutations affecting D1, D2 and the ATP synthase
subunits, but apparently not for chloroplast
mutants blocked in the electron transport chain
downstream from the plastoquinone pool (J.
Girard-Bascou and P. Bennoun, unpublished results). This observation may be related to the
specific deficiency of PSI activity in gametes as
compared to wild-type cells (P. Bennoun, unpublished results).
7. G E N E T I C F U N C T I O N A N D
T A N C E OF M I T O C H O N D R I A L
C H L A M YD O M O N A S
INHERID N A IN
It is well documented that the mitochondrial
D N A of higher plants ranges between 400 and
2000 kb [110]. In C. reinhardtii, this D N A appears
to consist only of 16-kb linear molecules with
unique ends and a homogenous sequence [12].
W h e t h e r this D N A represents the entire
mitochondrial D N A of C. reinhardtii has not yet
been proven rigorously because of the difficulty of
obtaining pure mitochondria from this organism.
The genes of apo-cytochrome b, of subunit I of
cytochrome oxidase and of the mitochondrial
r R N A s have been mapped on the 16-kb linear
D N A molecule [111]. This D N A has also been
shown to contain open reading frames which are
homologous to U R F 2 and U R F 5 of the m a m malian mitochondrial genome [112-114]. The
products of these two open reading frames appear
to be components of the N A D H : u b i q u i n o n e reductase of the inner mitochondrial membrane
[115]. From the available partial sequence of the
C. reinhardtii mitochondrial genome it can be
concluded that tryptophan is specified by T G G
rather than by TGA, as in the mitochondrial
D N A s of fungal and animal cells [104,116]. Although the mitochondrial genomes of C. reinhardtii and animal cells have similar sizes, their
genome organization is different [111]. N o discrete
D N A species around 16 kb are detectable in C.
eugametos and C. moewusii, but sequences homologous to the 16-kb D N A from C. reinhardtii
have been found [111]. The size of mitochondrial
28
D N A s in these two species is not yet known.
The mitochondrial D N A s of C. reinhardtii and
C. smithii can be distinguished based on small
differences in size and restriction patterns [14].
Crosses between these interfertile algae reveal that
whereas chloroplast genes from the mt parent
are rarely transmitted to the meiotic progeny, the
mitochondrial D N A from the m t - parent is uniparentally transmitted in these crosses [14]. While
C. reinhardtii can grow in the dark on medium
containing acetate, this is not the case for C.
smithii. It is interesting that this trait is also transmitted by the m t - parent to the meiotic progeny
suggesting that the function required for growth in
the dark may be specified by the mitochondrial
genome (N. Gillham, unpublished results).
The 16-kb D N A can be eliminated when C.
reinhardtii is grown in the presence of acriflavin or
ethidium bromide [117,119]. These drugs do not
appear to affect chloroplast and nuclear D N A .
This treatment induces with 100% efficiency a
class of lethal small colony mutants called minutes,
whose cyanide sensitive respiration is eliminated.
Photosynthetic function is not affected under the
same conditions.
8. C O N C L U D I N G R E M A R K S
Photosynthesis in eukaryotes is achieved
through the close cooperation of two genetic systems. Most studies in Chlamydomonas have
Table 4
Codon usage in nuclear and chloroplast genes of Chlamydomonas reinhardtii
The percentage of nuclear codons (Nu) used is based on the nucleotide sequences of the genes of a l , a2, fll and f12 tubulin
[121,122], of the two genes of the small subunit of ribulose 1,5 bisphosphate carboxylase [71] and the genes of the 33 kDa and 24 kDa
proteins from the oxygen-evolving complex (S. Mayfield, M. Rahire and J.D. Rochaix, unpublished results). The percentage of
chloroplast codons (Ct) used is taken from [22].
Nu
Ct
1.6
98.4
21.5
78.5
0
0
1.0
4.0
0.5
94.5
58.5
1.5
27.0
0
13.0
0
16.5
83.5
0
60
36
4
Phe
UUU
UUC
Ile
AUU
AUC
AUA
Met
AUG
100
100
3.8
44.0
0
52.2
45.5
0
54.5
0
Val
GUU
GUC
GUA
GUG
Ct
7.2
43.9
0.6
33.9
0
14.4
36
2
45
2
9
5
Ser
Leu
UUA
UUG
CUU
CUC
CUA
CUG
Nu
UCU
UCC
UCA
UCG
AGU
AGC
Nu
Ct
0
100
18
82
Tyr
UAU
UAC
His
CAU
CAC
Nu
Ct
Cys
4.4
95.6
6
94
UGU
UGC
0
100
Trp
UGG
100
96.5
3.5
100
Arg
Pro
CCU
CCC
CCA
CCG
Thr
ACU
ACC
ACA
ACG
Gin
3.2
87.9
0
8.9
38
0
57.5
4.5
9.5
89.1
0
1.4
59
0
39
2
18.0
76.2
0
5.8
64.5
0.5
19.5
15.5
Ala
GCU
GCC
GCA
GCG
CAA
CAG
Asn
AAU
AAC
0
100
1.9
98.1
87.5
12.5
3.5
96.5
Lys
AAA
AAG
Glu
GAA
GAG
10.2
89.0
0
0.8
0
0
96.0
3.0
0
0
1.0
0
22.0
76.6
0
0.4
95.5
4
0.5
0
Gly
0
100
95.0
5.0
Asp
GAU
GAC
CGU
CGC
CGA
CGG
AGA
AGG
16.1
83.9
0
100
31.0
69.0
87.0
13.0
GGU
GGC
GGA
GGG
Stop
UAA
UAG
UGA
100
0
0
100
0
0
29
focused on the structure, function, expression a n d
i n h e r i t a n c e of c h l o r o p l a s t genes. However, it has
b e c o m e increasingly clear that the nucleus p l a y s a
p r e d o m i n a n t role in this process. A l t h o u g h only
few n u c l e a r genes involved in p h o t o s y n t h e s i s have
been characterized, a striking difference in the
c o d o n usage of n u c l e a r a n d c h l o r o p l a s t genes of
C. reinhardtii is a p p a r e n t ( T a b l e 4). W h i l e chloroplast c o d o n s are highly b i a s e d t o w a r d s A a n d T in
the w o b b l e position, nuclear c o d o n s use m o s t l y C
a n d G at this position. It is n o t e w o r t h y that the
n u c l e a r c o d o n usage is very restricted. In most
cases only one c o d o n is used, e.g., a m o n g s t the 6
p o s s i b l e Leu codons, o n l y one was p r e f e r e n t i a l l y
f o u n d in the eight nuclear genes examined. These
o b s e r v a t i o n s strongly suggest that these two gen o m e s have a different origin a n d / o r that they
have evolved u n d e r very different selective pressures.
A stage has b e e n r e a c h e d where the a p p r o a c h e s
of genetics, b i o c h e m i s t r y , m o l e c u l a r b i o l o g y a n d
b i o p h y s i c s can be c o u p l e d fruitfully for s t u d y i n g
the structure, function a n d r e g u l a t i o n of p h o t o s y n t h e t i c complexes. A n efficient t r a n s f o r m a t i o n
system in Chlamydomonas w o u l d be an a d d i t i o n a l
p o w e r f u l tool for these studies. T r a n s f o r m a t i o n in
C. reinhardtii can be achieved at the n u c l e a r level
with a frequency of a b o u t 10 6 t r a n s f o r m a n t s p e r
t r e a t e d cell (see [120] for review). U n f o r t u n a t e l y ,
this efficiency is t o o low for isolation of n u c l e a r
genes b y c o m p l e m e n t a t i o n of defined p h o t o s y n thetic m u t a t i o n s with a g e n o m i c library. W i t h the
a v a i l a b i l i t y of n u m e r o u s c h l o r o p l a s t m u t a t i o n s
which have been c h a r a c t e r i z e d at the n u c l e o t i d e
level, it is p o s s i b l e to explore selection schemes for
c h l o r o p l a s t t r a n s f o r m a t i o n . T h e psbA d e l e t i o n
m u t a n t s are of special interest, since they m a y
p o s s i b l y b e t r a n s f o r m e d with the c l o n e d psbA
gene from h e r b i c i d e - r e s i s t a n t m u t a n t s . However,
e x p e r i m e n t s a l o n g these lines have n o t yet been
successful in Chlamydomonas. Because of its large
c u p - s h a p e d chloroplast, which occupies over 40%
of the cell volume, Chlamydomonas a p p e a r s to be
a p r o m i s i n g system for a t t e m p t s at c h l o r o p l a s t
t r a n s f o r m a t i o n by microinjection. Even in the a b sence of an efficient t r a n s f o r m a t i o n system,
Chlamydomonas will r e m a i n a p o w e r f u l m o d e l for
s t u d y i n g b i o e n e r g e t i c systems since its d i s p e n s a b l e
p h o t o s y n t h e t i c a n d r e s p i r a t o r y function m a k e this
alga a m e n a b l e to extensive e x p e r i m e n t a l analysis.
ACKNOWLEDGEMENTS
I thank P. Bennoun, J. Boynton, N. G i l l h a m , J.
G i r a r d - B a s c o u , R. Lee, C. L e m i e u x a n d U. Kiick
for c o m m u n i c a t i n g u n p u b l i s h e d results a n d O.
Jenni for d r a w i n g s a n d p h o t o g r a p h y . I a m i n d e b t ed to P. Bennoun, J. G i r a r d - B a s c o u , J. Erickson,
M. G o l d s c h m i d t - C l e r m o n t a n d S. M a y f i e l d for
critical r e a d i n g of the m a n u s c r i p t . The work in the
a u t h o r ' s l a b o r a t o r y was s u p p o r t e d by g r a n t 3.5870.84 from the Swiss N a t i o n a l F o u n d a t i o n .
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