Fatty Acid Desaturation Objectives: 1. Isolation of desaturase mutants 2. Substrates for fatty acid desaturation 3. ellular localization of desaturases References: Buchanan et al. 2000. Biochemistry and Molecular Biology of Plants. American Society of Plant Physiologists, Rockville MD. hapter 10. Wallis and Browse (2002) Progress in Lipid Research 41, 254278..
Glycerolipid Synthesis 16:3 plants Arabidopsis, spinach ER PA 16:0 18:2 18:2 18:2 18:2 16:0 18:2 Plastid Where does come from?
Fatty Acid Synthesis FAS hloroplast lipids 16:0 AT AT 16:0AP 18:0AP DES AP FATB FATA LAS LAS 16:0oA oa FAS = fatty acid synthesis FAT = acylap thioesterase LAS = acyloa synthetase AT = acyl transferase DES = stearoylap desaturase Main products of FAS are 16:0 and
Fatty Acid Desaturation Where does come from? It is made in the plastid stroma by the only known soluble desaturase enzyme in eukaryotes Fig. 1018
Plant Membrane Lipids are ighly Unsaturated The fatty acid compositions of the major leaf glycerolipids from wild type Arabidopsis Fatty acid Lipid P PE PI SL DGD PG MGD
Plant Membrane Lipids are ighly Unsaturated In all plant tissues the first synthesized glycerolipids PA and DAG contain only 16:0 and fatty acyl groups. is made in the plastid stroma by the stearoylap desaturase Where does desaturation beyond occur? Where does desaturation of 16:0 to 16:1, 16:2 and 16:3 occur? Prediction: Desaturase enzymes must exist in both the plastid and the ER to carry out desaturation of to 18:2 and 18:3 Unique desaturase enzymes must exist in the plastid to carry out desaturation of 16:0 to 16:1, 16:2 and 16:3
Approaches to Identification and haracterization of Membrane Bound Plant Desaturase Enzymes 1. Traditional biochemical approach: Detergent solubilization results in loss of enzyme activity > isolation and characterization were unsuccessful! 2. Genetic approach: Requires isolation of mutants an we isolate mutants deficient in fatty acid unsaturation? What kind of phenotype would such mutants have?
Glycerolipid Synthesis 16:3 plants Arabidopsis, spinach ER PA 16:0 18:2 18:2 18:2 18:2 16:0 18:2 Plastid Marker for prokaryotic pathway
Identification of Plant Fatty Acid Unsaturation Mutants ow many mutants would you expect to find? 16:0 t16:1 16:0 c16:1 16:1 16:2 16:2 16:3 18:0 Plastid 18:2 18:2 18:3 ER 18:2 18:2 18:3
Identification of Plant Fatty Acid Unsaturation Mutants What types of mutants have we isolated? Fatty acid WT JB60 JB27 JB67 LK3 JB1 LK9 JB12 JB25 LK8 16:0 15 18 17 24 15 15 13 15 13 12 16:1 cis 0 0 0 0 11 2 2 0 0 0 16:1 trans 2 0 0 2 2 2 3 4 2 2 16:2 0 0 0 0 0 12 12 0 0 0 16:3 12 12 13 0 0 3 4 10 0 0 18:0 1 1 1 1 1 1 2 1 1 1 3 3 3 2 21 3 3 27 8 9 18:2 18 19 19 14 14 26 32 4 23 23 18:3 48 47 47 56 36 36 29 36 53 53
Identification of Plant Fatty Acid Unsaturation Mutants What types of mutants have we isolated? SUBSTRATE MUTANT GENE 16:0 t16:1 on sn2 of PG JB60 and JB27 FAD4 16:0 c16:1 on sn2 of MGD JB67 FAD5 16:1 16:2 and 18:2 LK3 FAD6 16:2 16:3 and 18:2 18:3 JB1 and LK9 FAD7 PLASTID Mutations in FAD4, FAD5, FAD6 and FAD7 affect plastid lipids 18:2 on sn1 and sn2 of P JB12 FAD2 ER Mutations in FAD2 affect extraplastidial lipids and plastid lipids
Glycerolipid Synthesis 16:3 plants Arabidopsis, spinach ER fad2 fad3 PA act1 fad4 fad5 16:0 fab2 fab1 fad6 fad6 fad6 fad6 fad7 fad8 fad7 fad8 fad7 fad8 18:2 18:2 18:2 18:2 16:0 18:2 fad7 fad8 Plastid Marker for prokaryotic pathway
Identification of Plant Fatty Acid Unsaturation Mutants What types of mutants have we isolated? SUBSTRATE MUTANT GENE 16:0 t16:1 on sn2 of PG JB60 and JB27 FAD4 16:0 c16:1 on sn2 of MGD JB67 FAD5 16:1 16:2 and 18:2 LK3 FAD6 16:2 16:3 and 18:2 18:3 JB1 and LK9 FAD7 SUBSTRATE SPEIFI SUBSTRATE NONSPEIFI PLASTID Mutations in FAD4, FAD5, FAD6 and FAD7 affect plastid lipids 18:2 on sn1 and sn2 of P JB12 FAD2 18:2 18:3 on sn1 and sn2 of P FAD3 ER Mutations in FAD2 and FAD3 affect extraplastidial lipids and plastid lipids
Glycerolipid Synthesis 16:3 plants Arabidopsis, spinach ER fad2 fad3 PA act1 fad4 fad5 16:0 fab2 fab1 fad6 fad6 fad6 fad6 fad7 fad8 fad7 fad8 fad7 fad8 18:2 18:2 18:2 18:2 16:0 18:2 fad7 fad8 Plastid
JB1 (Fad7) Story Fatty acid WT JB60 fad4 JB27 fad4 JB67 fad5 LK3 fad6 JB1 fad7 LK9 fad7 JB12 fad2 JB25 act1 16:0 15 18 17 24 15 15 13 15 13 12 16:1 cis 0 0 0 0 11 2 2 0 0 0 16:1 trans 2 0 0 2 2 2 3 4 2 2 16:2 0 0 0 0 0 12 12 0 0 0 16:3 12 12 13 0 0 3 4 10 0 0 18:0 1 1 1 1 1 1 2 1 1 1 3 3 3 2 21 3 3 27 8 9 18:2 18 19 19 14 14 26 32 4 23 23 18:3 48 47 47 56 36 36 29 36 53 53 LK8 act1
JB1 (Fad7) Phenotype The Fad7 phenotype is apparent only when plants are grown at temperatures greater than 18 o
JB1 (Fad7) Phenotype Effect of temperature on the proportion of trienoic fatty acids (16:3 + 18:3) in leaves WT JB1 ypothesis: This effect is due to a temperature sensitive mutation
JB1 (Fad7) Phenotype ypothesis: This effect is due to a temperature sensitive mutation Several additional alleles of fad7 were isolated. They were all temperature sensitive!!! What does that suggest about our hypothesis? It is likely not correct. Why? Temperature sensitive mutations are very rare! Alternate hypothesis: There must be a second plastidial 16:2, 18:2 desaturase in addition to FAD7 that functions at low temperature
Isolation of the Low Temperature ow? Desaturase Mutant (Fad8) 1. Mutagenize the Fad7 mutant 2. Grow M 2 population at low temperature 3. Screen for alterations in leaf fatty acid composition by gas chromatography Which phenotype would you look for? 4. Identify mutants with lower 16:3 and 18:3 content than that found in the Fad 7 mutant
Isolation of the FAD8 gene FAD8 gene was cloned by heterologous hybridization using FAD3 ER Δ15 desaturase gene as a probe. FAD8 gene is not linked to the FAD7 gene. FAD7 and FAD8 genes share about 75% nucleotide identity. The FAD8 gene functionally complements the fad7 mutation when expressed using the FAD7 promoter. This demonstrates that FAD7 and FAD8 gene products are functionally equivalent. fad81 mutation created a premature stop codon 149 amino acids from the aminoterminal end of the 435 amino acid long predicted polypeptide, suggesting that this mutation results in a complete loss of FAD8 activity.
Summary of Plant Fatty Acid Unsaturation Mutants SUBSTRATE MUTANT GENE 16:0 t16:1 on sn2 of PG JB60 and JB27 FAD4 16:0 c16:1 on sn2 of MGD JB67 FAD5 SUBSTRATE SPEIFI 16:1 16:2 and 18:2 LK3 FAD6 16:2 16:3 and 18:2 18:3 JB1 and LK9 FAD7 16:2 16:3 and 18:2 18:3 FAD8 (Low temperature inducible) PLASTID SUBSTRATE NONSPEIFI Mutations in FAD4, FAD5, FAD6, FAD7 and FAD8 affect plastid lipids 18:2 on sn1 and sn2 of P JB12 FAD2 18:2 18:3 on sn1 and sn2 of P FAD3 ER Mutations in FAD2 and FAD3 affect extraplastidial lipids and plastid lipids
Isolation of the other FAD genes All seven genes identified by mutation in the seven classes of fad mutants have been cloned and all encode desaturase enzymes: FAD2 cloned by two research teams by TDNA tagging and mapbased cloning FAD3 cloned by mapbased cloning FAD4 cloned by mapbased cloning FAD5 cloned by mapbased cloning FAD6 cloned by heterologous hybridization using FAD2 ER Δ12 desaturase gene as a probe FAD7,8 cloned by heterologous hybridization using FAD3 ER Δ15 desaturase gene as a probe
ontributions of Plant FAD Mutants to Understanding of the Fatty Acid Unsaturation Process 1. Substrates of all membranebound FAD enzymes are lipidbound fatty acids 2. Fatty acid unsaturation is a sequential process insertion of the first double bond is required (16:1 or ) before the next desaturase can use this fatty acid chain as a substrate for insertion of the second double bond
Fatty Acid Unsaturation Where does come from? It is made in the plastid stroma by the only known soluble desaturase enzyme in eukaryotes which acts on a soluble 18:0AP substrate Fig. 1018
Glycerolipid Synthesis 16:3 plants Arabidopsis, spinach ER fad2 fad3 PA act1 fad4 fad5 16:0 fab2 fab1 fad6 fad6 fad6 fad6 fad7 fad8 fad7 fad8 fad7 fad8 18:2 18:2 18:2 18:2 16:0 18:2 fad7 fad8 Plastid
ontributions of Plant FAD Mutants to Understanding of the Fatty Acid Unsaturation Process 1. Substrates of all membranebound FAD enzymes are lipidbound fatty acids 2. Fatty acid unsaturation is a sequential process insertion of the first double bond is required (16:1 or ) before the next desaturase can use this fatty acid chain as a substrate for insertion of the second double bond 3. Because some desaturases can use both 16 and 18 fatty acid substrates, they must determine the site of double bond insertion relative to an existing double bond or relative to the methyl end of the fatty acyl chain
Δ9 > 18:2Δ9, 12 > 18:3 Δ9, 12, 15 >16:3 Δ7, 10, 13 > 16:2Δ7, 10 arboxyl end Methyl end O O O O O O O O 16:1Δ7 O O O O O O
ontributions of Plant FAD Mutants to Understanding of the Fatty Acid Unsaturation Process 1. Substrates of all membranebound FAD enzymes are lipidbound fatty acids 2. Fatty acid unsaturation is a sequential process insertion of the first double bond is required (16:1 or ) before the next desaturase can use this fatty acid chain as a substrate for insertion of the second double bond 3. Because some desaturases can use both 16 and 18 fatty acid substrates, they must determine the site of double bond insertion relative to an existing double bond or relative to the methyl end of the fatty acyl chain 4. fad mutants made it possible to clone the FAD genes and characterize FAD enzymes biochemically
Biological Roles of Fatty Acid Unsaturation Objectives: 1. Analysis of the role of Δ3 trans16:1 (Tutorial) 2. Determining the role of trienoic fatty acids (16:3 and 18:3) using the Fad3, Fad7, Fad8 triple mutant (Tutorial) 3. The importance of polyunsaturated fatty acids for photosynthesis References: Lightner et al. (1994) Altered body morphology is caused by increased stearate levels in a mutant of Arabidopsis. Plant J. 6, 401412. Mconn and Browse (1998) Polyunsaturated membranes are required for photosynthetic competence in a mutant of Arabidopsis. Plant J. 15, 521530..
Thylakoid Membranes are the Most ighly Unsaturated Membranes in Eukaryotes Thylakoid membrane glycerolipids from wild type Arabidopsis hloroplast Fatty acid Lipid Lipid SL DGD Lipid PG MGD Thylakoid Thylakoid membranes are 7580% polyunsaturated Are such high levels of thylakoid membrane unsaturation critical for photosynthesis?
None of the Isolated Unsaturation Mutants Displays a Visible Phenotype SUBSTRATE MUTANT GENE 16:0 t16:1 on sn2 of PG JB60 and JB27 FAD4 16:0 c16:1 on sn2 of MGD JB67 FAD5 SUBSTRATE SPEIFI 16:1 16:2 and 18:2 LK3 FAD6 16:2 16:3 and 18:2 18:3 JB1 and LK9 FAD7 16:2 16:3 and 18:2 18:3 FAD8 (Low temperature inducible) PLASTID SUBSTRATE NONSPEIFI Mutations in FAD4, FAD5, FAD6, FAD7 and FAD8 affect plastid lipids 18:2 on sn1 and sn2 of P JB12 FAD2 18:2 18:3 on sn1 and sn2 of P FAD3 ER Mutations in FAD2 and FAD3 affect extraplastidial lipids and plastid lipids
an we conclude that unsaturation is irrelevant to membrane functions? Why or why not? ypothesis: More substantial changes in membrane unsaturation are probably required to affect membrane functions. Tutorial: Fad3 Fad7 Fad8 triple mutant is not impaired in photosynthesis Under normal growth conditions indicating that 16:3 and 18:3 are not essential for this process Which double/triple mutant would you try to make?
The Fad2Fad6 Story ER fad2 fad3 PA act1 fad4 fad5 16:0 fab2 fab1 fad6 fad6 fad6 fad6 fad7 fad8 fad7 fad8 fad7 fad8 18:2 18:2 18:2 18:2 16:0 18:2 fad7 fad8 Plastid
Generation of the Fad2Fad6 Double Mutant Fad2 x Fad6 F 1 x F 1 (selfing) F 2 No Fad2Fad6 double mutant was found ypotheses: 1. Double mutant failed to germinate 2. Double mutant could not get established autotrophically
Seed Germination and Seedling Establishment
Fad2Fad6 Double Mutant Grows on Sucrose F 2 population Fad2Fad6 double mutants are chlorotic and contain only 10% of WT chlorophyll levels WT Fad2Fad6 When maintained on sucrose medium Fad2Fad6 double mutants develop relatively normal shoots and roots, but no flowers When transplanted into soil, Fad2Fad6 double mutants die within 7 days onclusion: Fad2Fad6 double mutant is not capable of autotrophic growth (photosynthesis)
Fad2Fad6 Phenotype 77% polyunsaturated fatty acids 6% polyunsaturated fatty acids The observation that growth and organ development of Fad2Fad6 plants is almost normal on sucrose medium indicates that: 1. The majority of membrane functions required for these processes is not compromised; 2. Photosynthesis is the only function that requires high levels of membrane polyunsaturation.