BiolB239 Saul Purton Lecture 4: Chlamydomonas - a model for plant cells What is Chlamydomonas reinhardtii? • • • • • Unicellular eukaryote Green alga (Chlorophyte) [note: plants have evolved from within the chlorophyte lineage, so green algae can be considered as simple plants]. Chlamydomonas species found in freshwater and damp soils around world (over 400 reported species). C. reinhardtii appears to be restricted to Eastern side of North America. Major C. reinhardtii strain used in lab (137c) dates from 1945 - isolated from field in Massachusetts by G.M. Smith. Cell architecture: • ovule cell 10-20 um in length • Single cup-shaped chloroplast (204 kb genome - completely sequenced) partially surrounding the nucleus (~100 Mb haploid genome – completely sequenced) • Two anterior flagella • Eyespot (stigma) • several small mitochondria (15.8 kb genome - completely sequenced) • cell wall made of glycoproteins Chlamy life cycle Vegetative • Vegetative cells are haploid • Will grow by binary fission to form colonies, or in liquid culture to ~2 x 10^7 cells/ml. • Can be synchronised by 12h light : 12 hour dark cycle. Sexual cycle (response to adverse conditions) • • • • • • Two stable mating types mt+ and mt–. Vegetative cells will undergo transformation to form sexually-competent gametes if deprived of nitrogen source. Flagellar pairing initiates a cascade of events. Ultimately leading fusion of the two cells and formation of a diploid zygote. Thick cell wall forms around zygote (allows long-term survival in dormant state). Restoration of nutrients and light -> germination of zygote. Meiosis results in the formation of 4 haploid daughter cells (=Tetrad). Inheritance patterns/genetic mapping • Nuclear markers inherited according to Mendelian rules (ie 2 : 2 in tetrad). 1 • Chloroplast and mitochondrial mutations inherited uniparently (4:0) from mt+ and mt– parents, respectively. Hence, it is easy to discriminate between the three possible sites of mutation. • • For nuclear mutations, can do simple genetic analysis to look for linkage of two markers. Can also do complementation studies and analyse dominant vrs recessive mutations by creating vegetative diploid lines. Nuclear genetic map comprises17 linkage groups (probably equates to 17 chromosomes in haploid cell). Several hundred genetic markers now placed on maps. Mutagenesis • • • Chlamy will grow in liquid culture or as clonal colonies on solid medium. Can therefore add mutagen (eg EMS) to culture or zap with UV, X-rays, etc., and then plate millions of cells. Because, nuclear genome haploid then mutants will have a phenotype (lethal mutations will obviously not be recovered, but can use stable diploid strains to isolate these). Classes of mutant • Photosynthetic (require acetate for survival) • Respiratory (require light) • Flagella • Pigment and starch metabolism • Phototaxis, chemotaxis • Various metabolic mutations (e.g. arginine biosynthesis) Molecular-genetics An important reason for using Chlamy as a model system is that all three genomes can be transformed. Nuclear transformation: Methods for DNA delivery: • Gun • Glass beads (preferred) • Electroporation Selectable markers: • Auxotrophic markers eg ARG7 (rescues arg7 mutant to growth without exogenous arginine) • Dominant markers (eg BLE – resistance to the drug bleomycin) 2 Integration pattern: • Random (illegitimate recombination) • Copy no. high with gun, low with beads/electroporation (can set conditions to give ~1 integration event per transformant cell) Applications: • Relating gene to mutation by showing can complement mutation by transformation with wild-type gene (eg arg7 with ARG7), and fine mapping the ends of gene. • Isolation of genes by genomic complementation (pools of cosmid clones) • Positional cloning (testing for complementation by transformation using BACs, cosmids, etc). • Using GFP constructs as reporters of promoter activity/promoter traps. • Expression of foreign genes. • Insertional mutagenesis (to create tagged genes). • Specific gene knockouts (saturation approach). • Gene knockdowns using RNAi constructs. Organellar Transformation Transformation of both the chloroplast and mitochondrial genomes has been reported. So far only chloroplast transformation has been exploited, but recent work indicates that genetic manipulation of mitochondrial genes is now feasible. • • • • Method for DNA delivery - biolistics Mode of integration - homologous recombination Markers - bacterial antibiotic resistance genes (fused to chloroplast promoter) • aadA (-> Spec resistance) Can use marker to: • Disrupt/delete specific chloroplast genes • Introduce site-directed changes to specific chloroplast genes • Introduce other foreign genes Chlamy - a powerful model for studying photosynthesis • • • • • • • • • Photosynthetic apparatus very similar to that of plants Quick and easy to grow Single cell type Easy classical genetics Can dispense with photosynthesis (when supplied with acetate as C source) Makes chlorophyll (and hence complexes) in dark (unlike flowering plants) Can genetically engineer key photosynthetic genes in chloroplast Can down regulate nuclear photosynthetic genes by RNAi (or obtain null mutants using insertional mutagenesis screens) Nuclear genome has been sequence (extensive EST collection already), chloroplast genome also done. 3 • Large culture collection of photosynthetic mutants available. Example of using Chlamy to study photosynthesis Understanding e-flow in PSI • By engineering codon changes within the chloroplast-encoded proteins (PsaA, PsaB or PsaC) that bind the redox centres (chlorophylls, quinone and iron-sulphur clusters) it is possible to alter their redox properties and therefore disrupt electron flow. Analysis of PSI in these mutants using spectroscopic techniques then provides insights into how electron transfer processes work. One such experiment involved replacement of the key tryptophans that modulate the redox properties of the phylloquinone, A1 (see figs on handout). References • • • • • • Gumpel NJ & Purton S (1994). Playing tag with Chlamydomonas. Trends Cell Biol. 4, 29930 Rochaix J-D (1997). Chloroplast reverse genetics: new insights into the function of plastid genes. Trends in Plant Sciences 2: 419-425 Davies JP & Grossman AR (1998). The use of Chlamydomonas as a model algal system for genome studies and the elucidation of photosynthetic processes. J. Phycol. 34: 907-917 Harris EH (2001). Chlamydomonas as a model organism. Annu. Rev. Plant Physiol. Plant Mol. Biol. 52: 363-406 Dent RM, Han M & Niyogi KK (2001) Functional genomics of plant photosynthesis in the fast lane using Chlamydomonas reinhardtii. Trends Plant Sci 6: 364-371 Xiong L, Sayre RT. Engineering the chloroplast encoded proteins of Chlamydomonas. Photosynth Res 2004; 80:411-419 4
