Proceedings Chapter
Reference
Chloroplast Molecular Genetics of Chlamydomonas : a Tool for Studying the Function, Synthesis and Assembly of the Photosynthetic
Apparatus
ROCHAIX, Jean-David, et al .
Abstract
The green unicellular alga Chlamydomonas reinhardtii offers interesting possibilities for studying the function, synthesis and assembly of the photosynthetic apparatus. Chloroplast and nuclear genes coding for subunits of photosystem II, a major multimolecular membrane-associated photosynthetic complex, have been isolated and characterized.
Examination of these genes and of their expression in wild-type cells and in chloroplast and nuclear photosystem II mutants has revealed a complex regulatory circuitry between chloroplast and nucleocytoplasmic compartments. One of the photosystem II subunits which is involved in herbicide binding has been found to have distinct single amino acid substitutions in several herbicide resistant mutants. Some of these mutations do not appreciably affect photosynthetic yield and may therefore be of agronomic interest.
ROCHAIX, Jean-David, et al . Chloroplast Molecular Genetics of Chlamydomonas : a Tool for Studying the Function, Synthesis and Assembly of the Photosynthetic Apparatus. In: Blažević, Marija ; Hranueli, Daslav & Toman, Zora. Genetics of the industrial microorganisms:
proceedings of the Fifth International Symposium . Zagreb : Pliva, 1987. p. 285-293
Available at:
http://archive-ouverte.unige.ch/unige:149609
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Fifth International Symposium on the Genetics of Industrial Microorganisms, 1986 M. Alacevic, D. Hranueli, Z. Toman (eds.)
Chloroplast Molecular Genetics of Chlamydomonas: a Tool for Studying the Function, Synthesis and Assembly of the
Photosynthetic Apparatus
J.D. Rochaix,
s.
Mayfield, J. Erickson, P. Malnoe and M. KuchkaDepartments . of Molecular and Plant Biology, University of Geneva, Geneva, Switzerland SUMMARY
The green unicellular alga Chlamydomonas reinhardtii offer s interesting possibilities for studying the function, synthesis and assembly of the photosynthetic apparatus.
Chloroplast and nuclear genes coding for subunits of photo- system II, a major multimolecular membrane-associated photo- synthetic complex, have been isolated and characterized.
Examination of these genes and of their expression in wild-type cells and in chloroplast and nuclear photosystem II mutants has revealed a complex regulatory circuitry between chloroplast and nucleocytoplasmic compartments. One of the photosystem II subunits which is involved in
herbicide binding has been found to have distinct single amino acid substitutions in several herbicide resistant mutants. Some of these mutations do not appreciably affect photosynthetic yield and may therefore be of agronomic interest.
INTRODUCTION
The biosynthesis of the photosynthetic apparatus in higher plants and algae is achieved through the cooperation of two distinct genetic systems located in the chloroplast a nd nucleocytoplasrnic compartments. Most photosynthetic complexes consist of subunits some of which are encoded by the c h loroplast genome and .translated on chloroplast
ribosomes while others are encoded by the nuclear genome, t ran::;lated on cytoplasmic ribosomes and imported into the chlo roplast where they assemble with their chloroplast partner polypeptides into functional complexes. Our work in recent years has been aimed at unde.rstanding the molecular basis of the cooperative interplay between these two
cellular compartments (cf. fig. 3).
A brief description of photosynthesis may be helpful for understanding the following (cf. fig. 1). Light energy is captured and converted into chemical energy by photo-- system II and photosystem I which act in series and which are connected by an electron transport chain. Photosystem II (PSI!) generates a strong oxidant capable of splitting water irito molecular oxygen, piotoni and electrons.
Electrons are then channelled along the electron transport chain and they ultimately reduce NADP. Electron transport is
285
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CHLOROPLAST MOLECULAR GENETICS OF CHLAMYDOMONAScoupled with an influx of protons into the thylakoid
vesicles thereby creating a proton gradient which is used to produce ATP. Both NADP and ATP are utiiized to drive the reactions of the Calvin cycle whose net result is the fixation of
co
2 and the production of carbohydrates.It is essential to choose a system which allows for a combined genetic and biochemical-molecular approach. The green unicellular alga Ch l amydomonas reinhardtii appears to be a powerful model system in this respec t .
An
impor tant featu:i;-e is that photosynthetic f unction is dispensable in this organism provided a r educed c arbon sourc e s uch as acetate is added to the growth medium. It is therefore possible to isolate acetate requiring mutants that are unable to grow with CO as the unique carbon source. Indeed numerous nuclear andc~loroplast
mutants of this sort have been isolated which are affected either in the photo- synthetic apparatus or in the chloroplast protein synthesizing system.As higher plants, C. reinhardtii contains three genetic systems located in the nucleocytoplasm, chloroplast and mitochondria whose major features are summarized in Table I.
Although the complexity of the chloroplast DNA is rather modest, 190 kb, this DNA represents 14% of the cellular DNA mass which implies that it exists in 80 copies per cell. It is easy to distinguish between nuclear, chloroplast and mitochondrial mutations based on their distinctive
segregation pattern during meiosis. While nuclear mutations are inherited according to Mendelian rules, chloroplast
mutati~ns are preferentially inherited uniparentally from the mt parent (1). Interestingly, mitochondrial genes appear to be inherited uniparentally from the mt- parent
(2).
Light +--Strama--.
NADP·-1 N?PH I
I
t o,j+2H• ...4--- - -- -- - - Chloroplost m~m b rori~ s ---41
Fig. 1. Primary reactions of photosynthesis. Chl, chlorophyll antenna; PSII, PSI, photosystems II, I; ETC, electron transport chain. (2) are catalyzed by ribulose bisphosphate carboxylase-
co
2 fixation (1) and ATP synthesis oxygenase and ATP synthase, respectively._,:; ~;~~. '(;:L~:~(,;~~'~,.:-:;~~ ~-~ ... ~:;~(A~:··_i,.~·.= \:. .·_: _ _:3::,-:~·,;_:~-'~:/(J ... _ , ... ~~~~ ,~-)> -~
,· •.-·.>-: ... : .. :-d:X~ ... :f'~-:..: ('
ROCHAIX et al.
Table 1. Genetic systems of Chlamydomonas reinhardtii
DNA Complexity (kb) %mass Copy Inheritance number
nuclear 7-9x10 4 (99.7%) 85 1 Mendelian ,- . chloroplast 190 (0. 3%) 14 50-80 uniparental
maternal
*
mitochondrial 16 (0.02%) 1 50-80 uni parental paternal
*
This has only been demonstrated in crosses between C. reinhardtii and~· smithii (2).Here we review our recent work on PSII in which we have taken advantage of several chloroplast and nuclear mutants which specifically affect PSII function and assembly.
Photosystem II consist~ of the core complex embedded in the thylakoid membrane, the oxygen evolving complex and the associated light harvesting system (fig. 2). The exact polypeptide composition of PSII is still unknown. At least 11 distinct polypeptides could be recognized in the PSII core complex of C. re i nhardtii (3). The core also contains the reaction center chlorophyl l P680, several core antenna chlorophylls, pheophytin, iron, cytochrome b559, two
acceptor plasto-quinones QA and QB and a primary electron donor Z (Fig.2). The major core proteins (47K, 43K, Dl, D2, cyt b 559) are all encoded by the chloroplast genome and their genes (psbB, psbC, psbA, psbD, psbE respectively) have been sequenced in several systems (4,5). The oxygen evolving system of PSII includes 3 extrinsic proteins of 33, 24 and 18 kd. They are located on the lumen side of the thylakoids and are all encoded by nuclear genes and synthesized as larger precursors in the cytoplasm (6,7).
RESULTS AND DISCUSSION
We have started our studies on PSII with the two core proteins Dl and D2 and their genes. Increased interest in these two proteins has arisen recently because of the similarity between the reaction centers of PSII and of purple photosynthetic bacteria (8,9). In both cases a
pheophy tin acts as intermediate electr9n acceptor and a very similar electron accepting quinone iron complex exists.
A structural homology between Dl, D2 and the L, M subunits of the bacterial reaction center has been noted (10). Michel and coworkers have recently determined by X-ray
cristallographic analysis the molecular structure at 3A resolution of the reaction center of Rhodopseudomona s
viridis (8). Based on the structural and functiDnal homology
287
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CHLOROPLAST MOLECULAR GENETICS OF CHLAMYDOMONASoxygen evolution
,
11
~ ~
,' Z--+ P680--+Pheophytin --+ QA--+0 6Fig. 2. Photosystem protein complex. QA acceptors. Z primary explanations.
II. LHCP, Light harvesting chlorophy ll
Q ,
primary and secondary electron e~ectron donor. See text for furtherbetween the two bacterial and PSII subunits, it has been proposed that 02 and 01 are the a poproteins of the stable primary and secondary electron acceptors of PSII and that they can be folded with 5 transmembrane domains to form a core with chlorophyll, pheophytin a nd quinone which is very simi lar to the bacterial reaction center (8). This model does not however agree completely with protease digestion data on thylakoid membranes (11).
The genes of 01 and 02, psbA and psbO, respectively, have been localized on the chloroplast genome of · C. reinhardtii (cf. fig. 3) and their nucleotide sequences have been determined (12, 13). Since the psbA gene is
located in the inverted repeat of the chloroplast genome, it is present in two copies.
It is well established that 01 is the herbicide binding protein (14). Analysis of psbA from mutants with different levels of resistance and cross-resistance to the herbicides atrazine, diuron and bromacil (15) has allowed us to
identify four residues on the 352 amino acid 01 polypeptide which are changed in these mutants (val 219~ser; phe 255+
tyr, ser 264•ala, leu 275~phe (16, 17, J. Erickson, M.
Rahire and J.D. Rochaix, unpublished results). These results have added new insights into the structure-function
relationship of the 01 protein. They reveal that mutations affecting four different ~mino acid residues of the Dl protein are correlated with different . types and levels -of herbicide resistance. Interestingly the mutations affecting V219, F255 and L275 do not alter electron transport and
ROCHAIX et al.
hence photosynthetic yield, (15) while the mut ation
affecting 8264 does (16). Thus, the first three changes may be of use in genetic engineering of herbicide resistance .
In collaboration with P , Bennoun's group in Paris we have examined several uniparental mutants blocked in PSII function. Among 78 chloroplast PSII mutants examined, 68 were unable 3;o synthesize Dl 4 (as measured by puls€ labelling cells with s-sulfate or C - acetate) (18 ) . Analysis of the chloroplast genome o f 22 of these mutants revealed that they all have deleted both copies of psbA . Although these mutants are able to synthesize and to integrate the other PSII
po l ypeptides in the thylakoid membranes , they are unable t o assemble a stable functional PSII complex (18) . Among the few other PSII mutants that do have an intact psbA gene , one is unable t o produce 02 . We have found recent l y that in this mutant a 46 bp stretch of the 02 gene, p s bD , has b e en
duplicated . Of special interest is the fact that this muta.nt is also unable to synthesize Dl, suggesting that D2 may be involved in the contro l o f translation o f 01 (13) .
By probing a cDNA library from C. reinhardtii in the expression ve.ctor J..gt ll with antibodies (obtained from N. H.
Chua) against the three nuclear e ncoded p o lypeptides from the oxygen evolving complex , cDNA clones corresponding to these three polypeptides were isolated . The gene s o f the 33
(OEEl) and 24kd (OEE2 ) proteins have been sequenced and shown to code for proteins of 2 90 and 245 amino a c ids, respectively (20, unpublished results ). Comparison of the protein sequences derived f r om the DNA sequences with the amino terminal ends of the proteins has revealed that the two transit peptides of the two proteins comprise 52 and 57 amino acids, respectively.
An important advantage of
c.
reinhardtii is that it allows one to easily isolate chloroplast and nuclear mutants affected in PSII function based on their alteredfluorescence properties (19). We have examined in some detail several chloroplast and nuclear PSII mutants by determining the steady state levels of mRNA and proteins of most of the PSII components. In some cases the rates of
prot e~g s ynthesis we l~ established by pulse labelling cells with S-sulfate or C-acetate. The picture which emerges from these studies (13, 18, 20, unpublished results) can be summarized as follows.
Chloroplast mutations which prevent the synthesis of either of the PSII core components ( 43K , Ol , 02) lead to the destabilization and degradation of the core comple.x
proteins . Interestingly the PSII core proteins whose genes are not affected are still synthe~ized at wild-type levels and are integrated i n to the thylakoid membrane but they do not accumulate stably (18 ) . It there£o re appears that the stoichiometri c accumulation of the PSII core polypeptides is achieved at the post-translational level. An exc eption is the mutant which has a lesion in its psbD gene . Here both the 02 and Dl polypeptides are no ;Longer synthesi zed (13) . The proteins from the oxygen evolving complex accumulate in these mutants to variable amounts ranging b e tween 10 and 100 % of wild-type levels . It is not yet known whether these
289
290
CHLOROPLAST
~~
CHLOROPLAST MOLECULAR GENETICS OF CHLAMYDOMONAS
NUCLEO-CVTOPLASM
NUCLEUS
Fig. 3. Synthesis and assembly of PSI! . ct DNA, chloroplast DNA with the genes of the core PSI! polypeptides 47K (PsbB), Dl (psbA) 02 (psb'D) and 43K (psbC) in the thylakoid membrane
(darkened) . The nuclear genes of the proteins from the oxygen evolving complex OEEl (psbI), OEE2 (psbII) and OEE3
(psbIII) are indicated. These proteins are synthesized as precursors on cytoplasmic ribosomes. Rl, R2 and R3 refer to nuclear genes whose products regulate the translation of chloroplast proteins. T , translation on chloroplast (left) and cytoplasmic ribosomes (right).
·proteins are still bound to the thylakoid membrane or whether they are free in the thylakoid lumen. In contrast, mutations in the nuclear gene of the OEE2 protein of the oxyge.n evolving complex which abolish the expression of this gene, do not affect the synthesis and accumulation of the other polypeptides of the oxygen evolving complex and of the
PSII
core polypeptides (20). Mutations affecting the OEEl protein gene have a more complex phenotype. While the other polypeptides of the oxygen evolving complex are present in normal amounts, the acc.umulation of the core polypeptides is reduced to about 25 % of wild-type suggesting that the OEEl protein plays some role in stabilizing the PSII complex.,.
ROCHAIX et al.
Nuclear mutations of considerable interest are those which prevent specifically the synthesis of defined
chloroplast encoded PSII core polypeptides. Mutants of this type include F34 which is unable to produce the 43 kd
polypeptide (21) and UVl-8 and XRl-1 which do not synthesize the Dl and D2 proteins. The phenotype of these mutants has been shown to be due to single mutations (21, M. Kuchka, unpublished results). Since the steady state levels of the mRNAs of the missing polypeptides in these mutants are as in wild-type, the mutations either interfere with the
translation of these proteins or render them highly
unstable. The genes affected in these mutants appear to play a regulatory role in PSII synthesis. The results of this PSII analysis are summarized in fig. 3 where several regulatory steps are indicated. From this preliminary
analysis it appears that the nucleus exerts a major control on the synthesis of the chloroplast encoded PSII
polypeptides principally at the translational and/or post-translational level.
PROSPECTS
The photosynthetic apparatus of
c.
reinhardtiiresembles closely that of higher plants. It is especially noteworthy tpat the nucleotide sequences of the PSII core polypeptide genes have been highly conserved. It appears therefore legitimate to extrapolate the results obtained with herbicide-resistant mutants from C. reinhardtii to plants. Because it has now been possible to merg e the biophysical, biochemical, molecular and genetic approaches in this alga, it is an excellent model system for studying the complex regulatory circuitry between chloroplast and nucleocytoplasmic compartments which is involved in the synthesis of PSII.
A transformation system has been established for
c.
reinhardtii that allows one to introduce foreign DNA stably into the cells and autonomously replicating plasmids have been constructed (22, 23, 24). It is hoped thatimprovements in the transformation efficiency will soon allow us to use this system for isolating regulatory genes of the sort described above by complementing, the appropriate mutations with wild type DNA libraries.
ACKNOWLEDGEMENTS
We thank
o.
Jenni for drawings and photography. This work was supported by Swiss National Fund grant 3.587.084.REFERENCES
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