Very-Long Chain Fatty Acid Biosynthesis Objectives: 1. Review information on the isolation of mutants deficient in VLCFA biosynthesis 2. Generate hypotheses to explain the absence of mutants with lesions in the 2 reductase and the dehydrase genes 3. Generate a model for the organization and control of VLCFA biosynthesis in plants 4. Design experiments to test the model Reference: Millar and Kunst, 1997. Very-long-chain fatty acid biosynthesis is controlled through the expression and specificity of the condensing enzyme. Plant J. 12, 121-131.
Fatty Acid Synthesis ACC KASIII TA KAS I KR ER DH Ohlrogge and Browse (1995) Plant Cell 7:795
Fatty Acid Synthesis FAS Chloroplast lipids 16:0 AT FATB 16:0-ACP LACS 16:0-CoA 18:0 18:1 AT AT KASII FATB 18:0-ACP LACS DES FATA 18:1-ACP LACS 18:0-CoA 18:1-CoA FAS = fatty acid synthesis FAT = acyl-acp thioesterase LACS = acyl-coa synthetase AT = acyl transferase DES = stearoyl-acp desaturase Main products of FAS are 16:0 and 18:1
Main products of FAS are 16:0 and 18:1 AT FAT Lipid synthesis in the chloroplast Export to the ER How do we know that? From ( 14 C)-acetate labeling experiments of isolated plastids in vitro C16 and C18 fatty acyl chains get radioactively labeled What happens when we add isolated microsomes to the radioactively labeled C16 and C18 fatty acids in chloroplasts?
Microsomes = small ER derived vesicles 100-200 nm in diameter
Microsomes = small ER derived vesicles What happens when we add isolated microsomes to the labeled C16 and C18 fatty acids in chloroplasts? Radioactively labeled very long chain fatty acids (>C18) get made, such as C20, C22, C24 etc.
Very-Long Chain Fatty Acid Biosynthesis Hypothesis: VLCFA synthesis beyond C 18 occurs in which the ER. cellular compartment? Prediction: Each step of fatty acyl chain elongation during VLCFA synthesis requires at least? reactions.
Fatty Acid Synthesis Ohlrogge and Browse (1995) Plant Cell 7:795
Model of Very-Long Chain Fatty Acid PLASTID Biosynthesis Fatty Acid Synthase (FAS) ER C16-ACP C18-ACP REDUCTION C16-CoA C18-CoA CONDENSATION Fatty Acid Elongase (FAE) DEHYDRATION REDUCTION VLCFAs C18 C20 C22 C24 C26 C28 C30 C32 C34
PLASTID Fatty Acid Synthase (FAS) Model of Very-Long Chain Fatty Acid Biosynthesis C16-ACP C18-ACP REDUCTION C16-CoA C18-CoA Malonyl-CoA KCS CONDENSATION Fatty Acid Elongase (FAE) REDUCTION DEHYDRATION VLCFAs All cells Seeds Root C24 C20, C22 C20 to C32 Sphingolipids Triacylglycerols Suberin Epidermis C24 to C34 Waxes
Testing the Model of Very-Long Chain Fatty Acid Biosynthesis How? Isolation of mutants. What kind of phenotype would you look for? Absence of VLCFA in lipids. Which lipids? Seed lipids. How? By gas chromatograph with a flame ionization detector (FID). http://www.shsu.edu/~chm_tgc/sounds/flashfiles/gc.scrubber.swf
Preparation of Fatty Acid Samples for Gas Chromatography 1. Transfer 10-15 seed from each sample to a glass tube. 2. Add 0.5 ml 1 N methanolic-hcl (Supelco) to each tube and cap tubes TIGHTLY. 3. Place samples at 80 o C for 2-3 hrs. 4. Cool down (5 minutes). 5. Add 0.5 ml NaCl (0.9%) and 300 µl hexane. 6. Recap and vortex vigorously to extract fatty acid methyl esters into hexane. 7. Carefully draw 200-250 µl hexane (top phase) and inject into gas chromatograph
Gas Liquid Chromatography http://193.132.193.215/eman2/images/tc2fig2_gcprinciple.jpg
Fae1 Phenotype Fae1 WT
Fae1 Phenotype Fatty acid Wild type Fae1 16:0 8.5 9.3 18:0 3.1 3.6 18:1 14.8 27.3 18:2 29.6 34.4 18:3 21.4 24.1 20:0 2.8 0.5 20:1 18.2 0.8 22:1 1.6 0.0 Values represent Mol % 18:1-CoA-------- 20:1-CoA-------- 22:1-CoA VLCFA = Chain length > C20
Summary of the Screen for Mutants With Reduced Levels of Seed Lipid VLCFAs ~10,000 mutagenized seed samples (M 3 ) analyzed by GC-FID 11 putative mutants identified What do we need to know/do next? Genetic analysis: 1. Is the mutant phenotype heritable? 2. Is the mutation dominant/recessive? 3. Is the phenotype due to a mutation in a single gene? 4. How many different genes have been identified in the screen?
Genetic Analysis Fatty acid Wild type Fae1 WTxFae1 (F 1 ) 16:0 8.5 9.3 9.3 18:0 3.1 3.6 3.5 18:1 14.8 27.3 18.9 18:2 29.6 34.4 33.1 18:3 21.4 24.1 23.7 20:0 2.8 0.5 1.1 20:1 18.2 0.8 9.7 22:1 1.6 0.0 0.7 Values represent (Mol %) F 1 progeny has intermediate levels of VLCFAs between WT and Fae1 (co-dominant mutation) F 2 progeny segregated 1:2:1 (co-dominant mutation in a nuclear gene)
Complementation Test All mutants are co-dominant with the wild type. All segregate as mutations in single nuclear genes. How many genes have been identified? Possibilities: 1, 2, 3, 4? Test Deduction Mutant1 x Mutant2 Mutant1 x Mutant 2 Fae Fae f ae1-1/ fae1-1 fae1-2/ fae1-2 Result F1 F1 Fae fae-1-1/ fae1-2 Conclusion: Mutants 1 and 2 fail to complement and must be homozygous for mutations in the same gene Pair-wise crosses between all 11 mutants revealed that all 11 mutations are present in the same gene named FAE1
Generating hypotheses Why were only mutants with mutations in FAE1 recovered? Hypotheses: 1. All fatty acid elongation pathway activities reside on a single multifunctional polypeptide. 2. Other activities may be encoded by genes of a gene family. 3. Mutations in genes encoding other enzymes may be lethal. Can we test these hypotheses? How? FAE1 gene was cloned encodes a polypeptide of 57 kda which functions as a condensing enzyme in seed VLCFA biosynthesis
PLASTID Fatty Acid Synthase (FAS) What are VLCFAs used for in the plant? C16-ACP C18-ACP REDUCTION C16-CoA C18-CoA Malonyl-CoA CONDENSATION Fatty Acid Elongase (FAE) REDUCTION DEHYDRATION VLCFAs All cells Seeds Root C24 C20, C22 C20 to C32 Sphingolipids Triacylglycerols Suberin Epidermis C24 to C34 Waxes
Which of the VLCFA functions may be essential to the organism? SPHINGOLIPID biosynthesis these lipids are important components of plasma membranes in all eukaryotic cells KR VLCFA biosynthesis occurs in all cells KCS FAE DH ER Are the same enzymes used for VLCFA production in all cells?
Which of the VLCFA functions may be essential to the organism? SPHINGOLIPID biosynthesis these lipids are important components of plasma membranes in all eukaryotic cells KR VLCFA biosynthesis occurs in all cells KCS FAE DH ER How can we explain loss of function mutation in the seed KCS (FAE1) that does not affect VLCFA production for sphingolipid biosynthesis?
Mutations in CER6 condensing enzyme gene affect cuticular wax accumulation WT Cer6 Sphingolipid, seed and suberin VLCFA levels are wild type!!!
Model for the Organization of VLCFA Synthesis SEED SHOOT ROOT ALL CELLS KR KR KR KR FAE1 FAE DH CER6 FAE DH KCS2 FAE DH KCS3 FAE DH ER ER ER ER TAG C20,C22 Cuticular Wax C24 to C34 Suberin C20 to C32 Sphingolipids C24 Hypotheses: 1. Condensing enzyme is unique for fatty acid elongation pathway in each tissue 2. Condensing enzyme determines the chain length of fatty acid elongation products
Prediction: Testing the Model of Very-Long Chain Fatty Acid Organization If the 2 reductases (KR and ER) and the dehydrase (DH) are present in all cells, then the expression of the seed specific condensing enzyme FAE1 throughout the plant will result in seed specific VLCFAs (C20 and C22) in all cells Design experiments: Ectopic expression: 1. 35S::FAE1 Transform Arabidopsis thaliana 2. Napin::FAE1 Transform tobacco 3. Gal1::FAE1 Transform Saccharomyces cerevisiae
Expression of 35S::FAE1 transgene in A. thaliana
Expression of Napin::FAE1 Transgene in Tobacco Seeds WT WT + Napin::FAE1
Expression of Gal1::FAE1 Transgene in S. cerevisiae Control 16:0 16:1 18:0 18:1 20:1 22:1 (Empty plasmid) 10.1 55.0 3.1 28.2 0 0 Gal1::FAE1 8.5 60.4 2.1 19.5 4.3 0.5 Values are weight % of total fatty acids
Does the condensing enzyme also control the amounts of VLCFAs in the cell? Fatty acid Wild type Fae1 WTxFae1 (F 1 ) 16:0 8.5 9.3 9.3 18:0 3.1 3.6 3.5 18:1 14.8 27.3 18.9 18:2 29.6 34.4 33.1 18:3 21.4 24.1 23.7 20:0 2.8 0.5 1.1 20:1 18.2 0.8 9.7 22:1 1.6 0.0 0.7 Values represent (Mol %)
Expression of Napin::FAE1 Transgene in Arabidopsis Seeds
Conclusions 1. The expression of FAE1 encoded condensing enzyme is sufficient for the synthesis of VLCFAs, implying that the other three activities of the FAE pathway (KR, DH, ER) are found ubiquitously throughout the plant. 2. The condensing enzyme is the activity of the FAE pathway that determines the acyl chain length of VLCFAs produced and VLCFA amounts. 3. The ability of yeast containing FAE1condensing enzyme to make seedspecific VLCFA suggests that the expression and the specificity of the condensing enzyme may be a universal eukaryotic mechanism for regulating the types and amounts of VLCFAs synthesized in a cell.