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O DI O SONKIE CHEM 261 – Plant Biochemistry

Ccm1 , a regulatory gene controlling the induction of a carbon-concentrating mechanism in Chlamydomonas reinhardtii by sensing CO 2 availability Fukuzawa et al. 2001. PNAS. 98: 5347-5352. O DI O SONKIE CHEM 261 – Plant Biochemistry. INTRODUCTION. Light. Photosynthesis

cheryl-vazquez
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O DI O SONKIE CHEM 261 – Plant Biochemistry

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  1. Ccm1, a regulatory gene controlling the induction of a carbon-concentrating mechanism in Chlamydomonas reinhardtiiby sensing CO2 availabilityFukuzawa et al. 2001. PNAS. 98: 5347-5352 ODIOSONKIE CHEM 261 – Plant Biochemistry

  2. INTRODUCTION Light Photosynthesis 6 H2O + 6 CO2C6H12O6 + 6 O2 • High CO2 levels are necessary for efficient photosynthesis. • Under CO2-limiting conditions, photosynthetic organisms must adjust, in order to sustain growth and survival. • Aquatic photosynthetic organisms induce a set of genes required for a carbon-concentrating mechanism (CCM) to acclimate to CO2-poor conditions. • These organisms concentrate dissolved inorganic carbon (DIC) intracellularly to optimize the fixation of CO2 by Rubisco. • Chlamydomonas reinhardtti (green algae) is one of such aquatic photosynthetic organisms.

  3. INTRODUCTION • In C. reinhardtti , several genes are regulated in response to low CO2 availability, including: • Periplasmic Carbonic Anhydrase • Mitochondrial Carbonic Anhydrase • Chloroplast Envelope Protein, LIP-36 • Sensory mechanisms enable cells to sense CO2 shortage, and induce the expression of specific genes, in order to achieve optimal photosynthesis. Low CO2 H+ + HCO3- CO2 + H2O Carbonic Anhydrase Cah1 gene Cah1 mRNA Calvin Cycle

  4. MAIN IDEA • A C. reinhardtti mutant (C16) is deficient in CCM induction: Unable to induce CCM in response to low CO2 availability. • A regulatory gene, Ccm1, complements the C16 mutation and restores: • Active transport of dissolved inorganic carbon. • Development of the pyrenoid structure in the chloroplast. • Induction of CCM-related genes. HYPOTHESIS • CCM1 (the protein product of Ccm1) controls the expression of several genes, which are necessary for the induction of CCM. • CCM1 contains a putative zinc finger motif and a His54 residue that is essential to its regulatory function.

  5. RESULTS Figure 1: Organization of the Ccm1 Gene Figure Legend Protein-Coding Regions P-1, P-3, P-5: Ccm1-specific Probes (Hybridization) C16-5 and C16-3: Oligonucleotide Probes (Isolation of Ccm1 gene) Restriction Enzymes: B (BamHI), X (XhoI), A (Apa1), S (SacI) Approach • C16 mutant (CCM-deficient) contains Nia1 gene. • Test Cross: C16 x Nia-  CCM-deficiency co-segregates with Nia+  CCM mutation was tagged by Nia1 coding sequence • Determination of Ccm1 sequence: flanking regions of Nia1 coding region • Isolation of Ccm1 gene (4 genomic clones) from WT using C16-5 and C16-3 probes. • Ccm1 Genomic Clones: pK14, pK14Xh, pK14XA, pK14Sc

  6. RESULTS Figure 2: Complementation of the C16 Mutation by Transformation Purpose To observe the effect of Ccm1 on the phenotype of transformed C16 mutants. Approach Transformation of pK14 Ccm1 genomic clones into C16 mutants Results • 2A (Relative Positions of Ccm1 clones): Restoration of growth under low CO2. • 2B (Northern Analysis of Cah1 mRNA): Induction of Cah1 under low CO2. • 2C (Southern Analysis of BamHI-digested genomic DNA): C16::pK14 contains 4.8-kb fragment (WT) and 9.4-kb fragment (Nia1 insertion). Conclusion 2A: Complementation region is a 5.1-kb fragment in pK14XA

  7. RESULTS Figure 3: Photosynthetic Characteristics of C16::pK14XA transformant Purpose: To observe the effect of the Ccm1 gene on CCM induction and Photosynthesis Approach: Growth of WT, C16, C16::pK14XA strain under low CO2. Measurement of photosynthesis rate, DIC accumulation. Results • 3A: C16::pK14XA has a high affinity for DIC (similar to WT). • 3B: C16::pK14XA accumulates DIC similar to WT. • 3C: C16::pK14XA performs carbon fixation similar to WT. Conclusion The Ccm1 gene induces a CCM and leads to efficient photosynthesis under low CO2 levels.

  8. RESULTS Figure 4: Formation of Pyrenoid Structures in the Transformant Purpose: To observe the effect of Ccm1 on pyrenoid structure formation in the transformant. Approach: Growth of WT, C16, C16::pK14XA strain under high and low CO2. Results: • Under low CO2, WT and C16::pK14XA (but not C16) developed the pyrenoid structure. Conclusion • The Ccm1 gene in pK14XA reverses multiple defects in the CCM. • A single mutation in Ccm1 (C16 mutation) results in multiple defects in the CCM that are needed to acclimate to low CO2 conditions.

  9. RESULTS Figure 5: Activation of CCM-related Genes in C16::pK14XA transformant Purpose To observe the effect of Ccm1 on the induction of CCM- related genes in the transformant. Approach Northern Blot Analysis of CCM- related genes (mRNA) Results • Under low CO2, activation of 5 CCM-related genes in WT and C16::pK14XA. • Cah3 and Cyp1 detected in WT and C16::pK14XA. Conclusion • The 5.1-kb DNA fragment in pK14XA encodes a gene (Ccm1) which controls the expression of at least 7 genes needed for CCM.

  10. RESULTS Figure 6A: Expression of the Ccm1 mRNA at High and Low CO2 Purpose: To observe the expression of the Ccm1 mRNA in WT and C16 at high and low CO2. Approach • Agarose Gel Electrophoresis of RNA from WT and C16 • Hybridization with 32P-labeled Ccm1-specific probes • Northern Blot Analysis of Ccm1 gene (mRNA) Results: Ccm1 mRNA detected in WT at high and low CO2, but NOT in the C16 mutant. Conclusion: Ccm1 gene is constitutively expressed in WT but NOT in the C16 mutant (Nia1 insertion). Probes: P5 P1 P3 Cblp DNA

  11. RESULTS Figure 6B: Expression of the CCM1 protein at High and Low CO2 Purpose: To observe the expression of CCM1 protein in WT and C16 at high and low CO2. Approach • Labeling of WT and C16 cells with 35S-Met and 35S-Cys. • Immunoprecipitation of CCM1 protein with anti-CCM1 antibody. • SDS-PAGE of proteins from WT and C16. Results: 76 kDa protein in WT at high and low CO2 (CCM1 protein) 80 kDa protein in C16 at low CO2 (from 2.2-kb fusion mRNA: 5’ Ccm1 + 5’ Nia1) Conclusion • CCM1 protein is constitutively expressed in WT but not in C16 mutant (Nia1 insertion). 80 kDa 76 kDa

  12. RESULTS Figure 7: Predicted Amino Acid Sequence of CCM1 Protein Important AA: Possible role in regulation of CCM A Putative Zinc Finger Motif Glutamine Repeat Glycine-rich Domain CCM1 AA Sequence Conservation B Green Algae Humans Frog Fruit Fly Fruit Fly Flowering Plant Wheat Highly Conserved

  13. CONCLUSION • Aquatic photosynthetic organisms induce a carbon-concentrating mechanism (CCM) to acclimate to CO2-poor conditions. • In Chlamydomonas, the Ccm1 gene encodes a CCM1 protein which regulates the activation of CCM by controlling : • The expression of CCM-related genes. • The induction of DIC accumulation and active DIC transport. • Development of the pyrenoid structure in chloroplasts. • The CCM1 protein contains a putative zinc finger motif, and a conserved His54 reside, which is important for CCM activation. High CO2 Low CO2 CCM-related genes ChloroplastCarbonic Anhydrase Cah1 Mca Ccp2 Lci1 Att1 Cyclophilin Cah3 Cyp1 Cah3 Cyp1 Basal Level Induction Ccm1 CCM1 protein Ccm1 CCM1 protein

  14. QUESTIONS • Why do aquatic photosynthetic organisms accumulate dissolved inorganic carbon rather than using gaseous CO2 like most terrestrials? • CO2 is readily available to terrestrial photosynthetic organisms (air). • CO2 is not readily available to aquatic organisms. They must make CO2 from dissolved HCO3-. • What problem(s) do photosynthetic organisms that concentrate CO2 face? • CO2 can easily diffuse through biological membranes. • Aquatic photosynthetic algae overcome the problem of CO2 diffusion by accumulating HCO3-. • Being a charged species, HCO3- diffuses through membranes much more slowly than CO2.

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