Very-Long Chain Fatty Acid Biosynthesis

Transcription

1 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, Very-long-chain fatty acid biosynthesis is controlled through the expression and specificity of the condensing enzyme. Plant J. 12,

2 Organization of FAS Enzymes TYPE I (Animals and Fungi) Location: Cytosol Organization: A complex of 2 large subunits (250 kda each) catalyzing all the reactions of FAS KAS ER KR DH TYPE II (Plants and Bacteria) Location: Plastid stroma Organization: Enzyme activities reside on individual proteins this reflects prokaryotic origin of plastids KAS ER KR DH

3 Fatty Acid Synthesis ACC KASIII TA KAS I KR ER DH Ohlrogge and Browse (1995) Plant Cell 7:795

4 Fatty Acid Synthesis FAS Chloroplast lipids 16:0 AT 16:0-ACP FATB LACS KASII 18:0-ACP 16:0-CoA FAS = fatty acid synthesis FAT = acyl-acp thioesterase LACS = acyl-coa synthetase AT = acyl transferase DES = stearoyl-acp desaturase

5 Fatty Acid Synthesis FAS Chloroplast lipids 16:0 AT FATB 16:0-ACP LACS 16:0-CoA 18:0 AT KASII FATB 18:0-ACP LACS 18:0-CoA DES 18:1-ACP FAS = fatty acid synthesis FAT = acyl-acp thioesterase LACS = acyl-coa synthetase AT = acyl transferase DES = stearoyl-acp desaturase

6 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

7 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?

8 Microsomes = small ER derived vesicles nm in diameter

9

10 SER RER

11 Microsomes = small ER derived vesicles What happens when we add isolated microsomes to the labeled C16 and C18 fatty acids in chloroplasts?

12 TLC analysis of fatty acid elongation products in leek microsomes Radioactively labeled very long chain fatty acids (>C18) get made, such as C20, C22 Evenson and Post-Beitenmiller,1995

13 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 4 reactions.

14 Fatty Acid Synthesis Ohlrogge and Browse (1995) Plant Cell 7:795

15 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

16 PLASTID Fatty Acid Synthase (FAS) C16-ACP C18-ACP Model of Very-Long Chain Fatty Acid Biosynthesis 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

17 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?

18 PLASTID Fatty Acid Synthase (FAS) C16-ACP C18-ACP Model of Very-Long Chain Fatty Acid Biosynthesis 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

19 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 or waxes. How? By gas chromatograph with a flame ionization detector (FID).

20 Preparation of Fatty Acid Samples for Gas Chromatography 1. Transfer 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 µl hexane (top phase) and inject into gas chromatograph

21 Gas Liquid Chromatography The mixture to be analyzed is injected into the stream of carrier gas. As it passes along the it separates into the different substances. Substances with a greater affinity for the mobile (gas) phase reach the detector at the end of the column more quickly. Substances with a greater affinity for the stationary (liquid) phase move more slowly through the column.

22 Analyzing Gas Chromatograms Three main pieces of information can be obtained from a gas chromatogram: the number of compounds in the mixture - represented by the number of peaks how much of each compound is present - represented by the height of the peak (higher = more) the retention time - indicated by the position of the peak (can be compared to reference standard)

23 Analyzing Gas Chromatograms This gas chromatogram shows that: substance A was present in the smallest quantity (it has the smallest peak) substance A had the shortest retention time substances B and C were present in equal amounts substance F had the longest retention time substance F was present in the greatest quantity (it has the largest peak) substance F had the greatest affinity for the stationary phase

24 Fae1 Phenotype Fae1 WT

25 Fae1 Phenotype Fatty acid Wild type Fae1 16: : : : : : : : Values represent Mol % 18:1-CoA à 20:1-CoA à 22:1-CoA VLCFA = Chain length > C20

26 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? 1. Is the mutant phenotype heritable? 2. Is the mutant phenotype due to a recessive/dominant/co-dominant mutant allele? 3. Is the phenotype due to a mutation in a single or more nuclear genes? 4. Do mutants with the same phenotype carry mutations in the same gene or different genes?

27 Fatty acid Wild type Fae1 WTxFae1 (F 1 ) 16: : : : : : : : Values represent (Mol %) Genetic Analysis 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)

28 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 fae1-1/ 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

29 Generating hypotheses Why were only mutants with mutations in FAE1 recovered? Hypotheses: 1. Functional redundancy 2. Lethality 3. FAE1 encodes a single multifunctional protein How can we proceed from here?