Genetic and physiological features of the green alga Chlamydomonas reinhardtiihave provided a useful model for elucidating the function, biogenesis and regulation of the photosynthetic apparatus. Combining these characteristics with newly developed molecular technologies for engineering Chlamydomonas and the promise of global analyses of nuclear and chloroplast gene expression will add a new perspective to views on photosynthetic function and regulation.
Addresses
Department of Plant Biology, The Carnegie Institution of Washington, 260 Panama Street, Stanford, California 94305, USA;
e-mail: [email protected]
Current Opinion in Plant Biology2000, 3:132–137
1369-5266/00/$ — see front matter © 2000 Elsevier Science Ltd. All rights reserved.
Abbreviations
ARG 7 ARGININOSUCCINATE LYASE 7
BAC bacterial artificial chromosome
GFP green fluorescent protein
NIT NITRATE REDUCTASE
Introduction
The sequencing of the Arabidopsis genome will be com-pleted within a year. The rice genome, which is about four times as large as that of Arabidopsis, is likely to be the first genome of a commercially important plant for which we will have a complete sequence. The enormous volume of sequence information that is being gathered will facilitate the elucidation of specific gene functions. Nevertheless, the use of angiosperms for functional analyses of genes has some significant limitations, listed below, that make it important to exploit multiple systems to elucidate gene function in vascular plants.
First, because DNA integrates randomly into the genome of angiosperms, an inactivation of any specific gene can only be identified by screening genomic DNA from pooled populations of potential mutants using PCR-based approaches. These approaches can be both difficult and laborious. As an alternative, homologous recombination occurs in the moss Physcomitrella, making it possible to probe gene function by reverse genetics [1].
Second, although it is often possible to use sequence infor-mation to infer catalytic activity encoded by a gene, the exact process in which the polypeptide product is involved might not be apparent; there are numerous genes encoding regula-tory elements that have been identified in Arabidopsis, but the processes that they control have not yet been defined.
Third, plants often have multiple genes with redundant or overlapping functions. This makes it difficult to determine
the function(s) of specific genes even if ‘knockout’ muta-tions are identified. Simpler systems might not have so much functional redundancy and even if there are genes with compensatory functions, it is often easy to create syn-thetic phenotypes that result from more than one lesion.
Fourth, genomic analysis of a range of carefully chosen organisms will reveal a diversity of processes and will show how the environment has influenced the evolution of these processes. Understanding how a process has been tuned to a particular habitat is the first step in developing strategies to alter the range of environments in which spe-cific organisms can grow.
Finally, there are certain processes that are obligate for some organisms but conditional for others. One such process is photosynthesis. All angiosperms that have been studied so far are obligate photoautotrophs; they grow very poorly and often die in the absence of photosynthesis, even when supplied with a source of fixed carbon. It is therefore difficult to exploit mutant generation for probing photosynthetic function. Traditionally, both cyanobacteria and the unicellular green alga Chlamydomonas reinhardtii have served as valuable resources for using genetic and molecular technologies to elucidate the function, biosyn-thesis and regulation of the photosynthetic apparatus. In this review I shall briefly discuss the value of using Chlamydomonas to study photosynthetic processes in plants and how genetics, molecular technologies and genomics are enhancing its utility.
Advantages of
Chlamydomonas
as a model
organism, especially for the analysis of
photosynthesis
Valuable molecular tools have been tailored for use with Chlamydomonas; the facility with which these tools can be used has led investigators to bestow the title of ‘green yeast’ [2] upon this organism. The most important technological developments have been the establishment of selectable markers for the identification of nuclear and chloroplast transformants and relatively simple procedures to introduce DNA into the cells. Furthermore, cosmid [3], yeast artificial chromosome (YAC) [4] and bacterial artificial chromosome (BAC) (Lefebvre, unpublished data; Genome Systems, St. Louis, Missouri, USA) recombinant libraries are being used for complementation of mutant strains, and both an arylsulfatase gene [5] and, more recently, a sequence-tai-lored green fluorescent protein (GFP) gene [6•] have been developed as reporters for monitoring gene expression and subcellular location. Furthermore, sense/antisense technolo-gy has been successfully used to alter the accumulation of a heat shock protein (HSP70B) mRNA [7••]. Nevertheless, the Chlamydomonaswork still has some technological
short-Chlamydomonas reinhardtii
and photosynthesis:
comings. Introduced DNA appears to integrate randomly into the Chlamydomonas nuclear genome (as it does in Arabidopsis), making it difficult to perform reverse genetics. Furthermore, integration of introduced DNA into the nuclear genome frequently causes large deletions that could complicate the analysis of mutants generated by random insertion of exogenous DNA [8].
When selecting transformed Chlamydomonas strains, the ARGININOSUCCINATE LYASE 7 (ARG7) [9] and NITRATE REDUCTASE 1 (NIT1) [10] genes are routinely used as marker genes to rescue the recessive arg7(unable to grow without exogenous arginine) and nit1(unable to grow using nitrate as a sole nitrogen source) mutants. A gene for which there is no selectable phenotype can be co-trans-formed into cells with the marker genes at relatively high frequencies. The Chlamydomonas emetine resistance gene CRY1[11] and the Streptoalloteichus hindustanusphleomycin resistance gene ble[12•] have been used as dominant mark-ers for nuclear transformation. The aadA gene confers spectinomycin/streptomycin resistance on Chlamydomonas cells, providing a strong selectable marker for introducting DNA into the chloroplast genome [13]. This gene has recently been also used for nuclear transformation [14].
There are a number of ways of introducing exogenous DNA into Chlamydomonas cells including biolistic proce-dures (i.e. bombarding the cells with microprojectiles coated with DNA) [15–17], agitation with glass beads [18] and electroporation [19•]. The simplest method of intro-ducing DNA into the nuclear genome is the procedure involving glass beads, which requires no special equip-ment but results in relatively low transformation efficiencies. Transformation frequencies of more than 1×105transformants per µg of DNA have been achieved by electroporation [19•]. Such high transformation fre-quencies provide a strong potential for the ‘shotgun cloning’ of genes by complementing mutant strains (i.e. identifying genes that complement a specific mutant phe-notype by introduction of a recombinant library). Furthermore, biolistic procedures appear to be the most efficient way of introducing DNA into the chloroplast genome [17], probably because the chloroplast occupies over half of the volume of the cell providing a large target for a microprojectile.
Many of the characteristics of Chlamydomonas, a eukaryote with haploid genetics, make it an ideal organism for the analysis of photosynthesis. The considerable power of a model system with haploid genetics is exemplified by the use of the yeast Saccharomyces cerevisiae to augment our understanding of basic cellular processes such as nutrient utilization, cell cycle control and signal transduction. Furthermore, Chlamydomonas can be grown heterotrophi-cally on acetate as a sole source of carbon; when growing heterotrophically dark-grown cells exhibit normal photo-synthetic capability and chloroplast development. This capacity for heterotrophic growth has permitted the
devel-opment of a number of different genetic screens to isolate mutants that have impaired photosynthesis (reviewed in [20]). Most of these screens, which are not readily applied to vascular plants, allow the rapid analysis of thousands of colonies on solid medium. Furthermore, because Chlamydomonas grows well heterotrophically in the dark, it is relatively easy to isolate and maintain photosynthetic mutants that are light sensitive [21].
Many procedures in vivo can be incorporated into studies of Chlamydomonas; these procedures might be difficult or impossible to perform with more complex systems. Spectrophotometric studies in vivo have provided an abun-dance of information on photosynthetic exciton and electron transfer (reviewed in [22]). The analysis of various aspects of photosynthetic activity and chloroplast biogene-sis in vivo is facilitated by the uptake of certain electron donors and acceptors, inhibitors of photosynthetic electron transport and chloroplast and cytoplasmic protein synthesis, and 35SO
42– to label proteins, by intact Chlamydomonas cells. The use of Chlamydomonas for the characterization of chloroplast biogenesis in vivo is exemplified by the work of Howe and Merchant [23]. Pulse labeling of proteins in vivo was used to define discrete steps in the biosynthesis of the nucleus-encoded chloroplast polypeptides cytochrome c6, plastocyanin and the 33 kDa oxygen-evolving complex pro-tein. In contrast, pulse-labeling of proteins is much more difficult in vascular plants. There might be a considerable lag between the time at which the label is administered (to the roots or to a cut edge of the stem) and the time at which it is incorporated into chloroplast proteins.
It is relatively easy to generate Chlamydomonas ‘tagged’ mutants by transformation with the ARG7or NIT1genes. This strategy facilitates the isolation of genes that are altered in any of a number of different processes, including photosynthesis. This approach, however, generally causes the production of null mutations that, in some cases, will be lethal under the conditions used for screening. It will be advantageous to generate point mutants that have defec-tive photosynthesis (many of which are already available) and to rapidly identify BAC clones that rescue the mutant phenotypes. This approach can be made routine by gener-ation of a physical map of the Chlamydomonas genome that is linked to the genetic map and the creation of a set of overlapping BAC clones that cover the entire genetic map.
of the mutant phenotype within 6–8 weeks. In Arabidopsis this process often takes more than a year.
DNA is readily introduced into Chlamydomonas chloroplas-ts using particle bombardment [17] and transformanchloroplas-ts can be easily selected because of their antibiotic (spectino-mycin) resistant growth. Because integration into the Chlamydomonaschloroplast genome occurs by homologous recombination, any gene in the genome can be inactivated and the resultant phenotype evaluated rapidly with regard to photosynthetic function, assembly of the photosynthet-ic apparatus and regulation of transcription/translation of chloroplast-encoded genes. The inactivation of some chloroplast genes results in lethality, but the function(s) of these genes can be studied in heteroplasmic transformants [24]. Furthermore, the Chlamydomonas chloroplast transfor-mation system has allowed investigators to use site-directed mutagenesis to perform structure–function studies of both electron-transport and reaction-center com-ponents [25–27,28•]. Although chloroplast transformation can be performed with tobacco cell protoplasts [29], it is much more problematic and the results are inherently more difficult to interpret.
Global regulation of gene expression under different envi-ronmental conditions is more readily studied in a single-celled organism such as Chlamydomonas than in vascu-lar plants. First, alterations in growth conditions can be rapidly imposed on Chlamydomonas cells by changing the growth medium and/or moving the cells to different environ-mental conditions. Second, Chlamydomonas cultures represent a relatively uniform population of cells and the analysis is therefore not complicated by the presence of dif-ferent organs and tissue types that might exhibit radically different responses to the stimulus. Nevertheless, complex higher-order processes that involve communication between different tissue types cannot be studied in Chlamydomonas.
Recent progress using
Chlamydomonas
in
understanding photosynthetic function
Historically, Chlamydomonas has been important in eluci-dating photosynthetic mechanisms. Early characteriztion of Chlamydomonas mutants identified components of photosynthetic electron transport, photosystem I, pho-tophosphorylation and the Calvin cycle (summarized in [30]). The first physically defined mutation of the chloro-plast genome was recovered from Chlamydomonas and was shown to be in the rbcLgene (large subunit of ribulose-1,5-bisphosphate carboxylase) [31]. Analysis of the labile 32 kDa D1 and D2 polypeptides of photosystem II helped researchers to recognize that these polypeptides were functionally analogous to the integral membrane L and M polypeptides of the reaction centers in the purple non-sul-fur bacteria (summarized in [32]). In addition, freeze-etch electron microscopy of thylakoid membranes from mutant and wild-type Chlamydomonas strains has led to the associ-ation of defined particles of the thylakoid membranes with specific complexes involved in photosynthesis [33].
Visualization of these particles suggested that complexes of the photosynthetic apparatus are in a fluid state within the membranes, as shown by the lateral movement of phosphorylated light-harvesting complexes to non-appressed regions of the thylakoid membranes (this movement is associated with a state transition) [34]. Finally, detailed biochemical approaches, targeted disrup-tions and site-directed mutagenesis are all helping to define interactions between the different polypeptides of the photosystems.
A number of recent reports describe research in which molecular tools have been used to elucidate photosynthet-ic processes. Mutants in chlorophyll b biosynthesis were isolated by transformation tagging with the ARG7 gene [35••]. The lesions were found to be in a gene encoding a putative protein with sequence similarity to a methyl-monooxygenase. This work has helped to elucidate both mechanistic and evolutionary aspects of chlorophyll biosynthesis [36••].
Wollman, Merchant and collaborators [37,38,39•] have iso-lated several nuclear mutants that define elements involved in the biogenesis of the cytochrome complex. The nuclear loci CCS1,2,3,4are involved in the attachment of c-heme to cytochrome f, and the nuclear loci CCB1,2,3,4 are involved in b-heme binding to cytochrome b6. Other nuclear loci are important for the maturation and/or stabi-lization and translation of transcripts encoding the individual components of the cytochrome b6f complex [40•]. The work on the cytochrome complexes has opened new horizons that would have been very difficult to explore in vascular plants. This work also highlights the fact that much of the control of chloroplast biogenesis is at the level of post-transcriptional and translational events, which we are only just beginning to understand [41,42••,43,44••,45•,46].
Recently, an elegant selection was performed that yielded several mutants with an altered ability to transport proteins across the thylakoid membranes [47••]; analysis of these mutants, designated TIP(for thylakoid insertion proteins), should add greatly to our knowledge of protein targeting and chloroplast biogenesis.
video-imaging screen identified mutants of Chlamydomonas that were defective in the xanthophyll cycle and also exhibited a reduced ability to eliminate excess absorbed light energy [50]; this was the first genet-ic evidence linking energy dissipation to the production of specific xanthophylls. Similar findings were recently obtained with Arabidopsis [51••], although the xanthophyll cycle does seem to have a more dominant role in energy dissipation in vascular plants than in Chlamydmonas. Recently, the analysis of another mutant unable to dissi-pate excess absorbed light energy was shown to be missing a minor component of photosystem II designated PsbS, which is related to the light harvesting chlorophyll-a,b-binding proteins [52••]. This finding has important implications with respect to both light harvesting function and evolution. A video-imaging screen was also used to isolate mutants that are unable to undergo state transition (or to perform phosphorylation of the light-harvesting antennae proteins) [53•,54•]. Furthermore, recent work of Schroda et al. [7••] has demonstrated that the chloroplast-localized chaperone HSP70B is involved in both the protection and repair of photosystem II in high light.
Applications of genomics, and conclusions
The physiological, genetic and molecular manipulations that have become routine for Chlamydomonas make this organism ripe for genome-wide analyses. The global analysis of the genome will add considerably to our understanding of photosynthetic function and the way in which photosynthesis is modulated as environmental conditions change [55]. Sequence analyses of cDNAs derived from RNA isolated from Chlamydomonas cells that are exposed to a number of different environmental conditions will inundate the research community with interesting sequences that can be used as substrates for functional genomics. cDNA microarrays will provide a global view of gene regulation under a number of differ-ent environmdiffer-ental conditions. Analysis of high-density DNA microarrays with cDNA probes generated from putative ‘regulatory mutants’ will enable investigators to identify the targets of specific regulators and the processes that are affected in these mutant strains; Chlamydomonas has a number of putative regulatory mutants that can be used immediately for such studies. Finally, establishing a high-density physical map that is linked to the genetic map [56] will make the positional cloning of genes that are altered (e.g. point mutations) in specific mutants at least an order of magnitude more rapid than in vascular plants.Acknowledgements
I thank all of the co-investigators, including Pete Lefebvre, John Davies, Carolyn Silflow, Elizabeth Harris and David Stern, who worked with me to create a Chlamydomonas genome project and acknowledge the National Science Foundation for funding this project. There are many excellent articles in which genetics and molecular strategies were used to dissect photosynthetic processes in Chlamydomonas that have not been discussed in this review because of severe length restrictions. I wish to apologize to those whose work has not received appropriate attention.
References and recommended reading
Papers of particular interest, published within the annual period of review, have been highlighted as:
• of special interest
••of outstanding interest
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• Identification of cis-acting RNA leader elements required for chloroplast psbDgene expression in Chlamydomonas.Plant Cell
1999, 11:957-970.
Site-directed mutagenesis was used to identify RNA stability determinants in the psbDtranscript (which encodes the D2 polypeptide of the reaction center of photosystem II). Other elements in the mRNA were shown to alter translation but not RNA stability. These results suggest that the post-tran-scriptional regulation of psbDexpression might involve both translational control and mRNA turnover.
46. Fargo DC, Boynton JE, Gillham NW: Mutations altering the predicted secondary structure of a chloroplast 5′′untranslated region affect its physical and biochemical properties as well as its ability to promote translation of reporter mRNAs both in the
Chlamydomonas reinhardtiichloroplast and in Esherichia coli.Mol Cell Biol1999, 19:6980-6990.
47. Bernd KK, Kohorn BD: Tip loci: six Chlamydomonas nuclear
•• suppressors that permit the translocation of proteins with mutant thylakoid signal sequences.Genetics1998, 149:1293-1301. Mutations in the leader sequence of the cytochrome fprotein that inhibit its translocation into the photosynthetic membranes and prevent photoau-totrophic growth are defined. Suppressors of the mutant phenotype were generated that define six nuclear loci. Both biochemical analysis of the mutants and sequence characterization of the genes altered in the suppres-sor strains will elucidate the apparatus of the photosynthetic membranes that is involved in protein translocation.
48. Wykoff DD, Davies JP, Grossman AR: The regulation of
• photosynthetic electron transport during nutrient deprivation in
Chlamydomonas reinhardtii.Plant Physiol 1998, 117:129-139. Nutrient limitation markedly influences photosynthetic electron flow; this modulation of photosynthetic activity is critical for cell survival. Starving
Chlamydomonas of either sulfur or phosphorus leads to a downregulation of photosystem II activity that involves the generation of QB-nonreducing cen-ters, a decline in the maximum quantum efficiency of photosystem II and a decreased efficiency of excitation energy transfer to the photosystem II reac-tion centers.
49. Forster B, Osmond CB, Boynton JE, Gillham NW: Mutants of
Chlamydomonas reinhardtiiresistant to very high light.J Photochem Photobiol1999, 48:127-135.
50. Niyogi K, Björkman O, Grossman AR: Chlamydomonas xanthophyll cycle mutants identified by video imaging of chlorophyll fluorescence quenching.Plant Cell1997, 9:1369-1380.
51. Niyogi KK, Grossman AR, Björkman O: Arabidopsis mutants define
•• a central role for the xanthophyll cycle in the regulation of photosynthetic energy conversion.Plant Cell1998, 10:1121-1134. A video-imaging system developed for use with Chlamydomonas was used to screen Arabidopsis plants for alterations in non-photochemical quenching. This screen resulted in the isolation of mutants that are defective in the xan-thophyll cycle. The npq1mutant, which was unable to convert violaxanthin to zeaxanthin in intense light, was more severely limited in its ability to dissipate excess excitation than the analogous mutant in Chlamydomonas. Hence, although both Chlamydomonas and Arabidopsis use the xanthophyll cycle for the dissipation of excess excitation, the proportions of excess excitation ener-gy that is dissipated in this way seems to differ in the two organisms. 52. Li X-P, Bjôrkman O, Shih C, Grossman AR, Rosenquist M, Jansson S,
•• Niyogi KK: A pigment binding protein essential for regulation of photosynthetic light harvesting.Nature2000, 403:391-395. The authors show that the gene encoding PSBS, which is a chlorophyll-binding protein that is intrinsic to photosystem II, is necessary for the dissi-pation of excess absorbed light energy, but is not required for photosynthetic oxygen evolution. A four membrane-helix PSBS-like protein was probably the evolutionary precursor of the three membrane-helix LHC proteins, the major components of the light harvesting antennae. These findings suggest that the function of energy dissipation may have evolved prior to the current-day light-harvesting function found in vascular plants.
53. Kruse O, Nixon PJ, Schmid GH, Mullineaux CW: Isolation of state
• transition mutants of Chlamydomonas reinhardtiiby fluorescence video imaging.Photosynth Res1999, 61:43-61.
This paper, like [54•], describes how video-imaging techniques can be used
to isolate state transition mutants. Characterizations of these mutants are helping to elucidate the dynamics of the photosynthetic apparatus and the environmental factors that modulate photosynthetic activities.
54. Fleishmann MM, Ravanel S, Delosme R, Olive J, Zito R, Wollman FA,
• Rochaix J-D: Isolation and characterization of photoautotrophic mutants of Chlamydomonas reinhardtii deficient in state transition.J Biol Chem1999, 274:30987-30994.
The work in this paper and [53•] demonstrates that video-imaging techniques
can be used to isolate state transition mutants. Characterizations of these mutants are helping to elucidate the dynamics of the photosynthetic appa-ratus and the environmental factors that modulate photosynthetic activities. 55. Davies JP, Grossman AR: The use of Chlamydomonas
(Chlorophyta: Volvocales) as a model algal system for genome studies and the elucidation of photosynthetic processes.J Phycol
1998, 34:907-917.
