Very-Long Chain Fatty Acid Biosynthesis - Home | UBC...
Transcript of Very-Long Chain Fatty Acid Biosynthesis - Home | UBC...
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
Very-Long Chain Fatty Acid Biosynthesis
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.
PLASTIDFatty AcidSynthase (FAS)
C16-ACPC18-ACP
C16-CoAC18-CoA
Malonyl-CoA
DEHYDRATION
REDUCTION
REDUCTION
Fatty AcidElongase
(FAE)CONDENSATION
C24 C24 to C34C20, C22Sphingolipids WaxesTriacylglycerols
All cells Seeds RootC20 to C32
Suberin
Epidermis
VLCFAs
Model of Very-Long Chain Fatty Acid Biosynthesis
Testing the Model of Very-Long Chain Fatty Acid Biosynthesis
How?Isolation of mutants.
How?By gas chromatograph with a flame ionization detector (FID).
What kind of phenotype would you look for?Absence of VLCFA in lipids.
Which lipids?Seed lipids.
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)
Fatty acid Wild type Fae1
16:0 8.5 9.318:0 3.1 3.618:1 14.8 27.318:2 29.6 34.418:3 21.4 24.120:0 2.8 0.520:1 18.2 0.822:1 1.6 0.0
18:1-CoA--------à20:1-CoA--------à22:1-CoA
VLCFA = Chain length > C20
Values represent Mol %
Fae1 Phenotype
Summary of the Screen for Mutants With Reduced Levels of Seed Lipid VLCFAs
~10,000 mutagenized seed samples (M3) analyzed by GC-FID
Why were M3 seeds used for the screen?
M0 seeds
M2 seedsM1 seedsM1 plants M2 plants
mutagen
Harvest from individual M2 plants
M3 seed families
Use 10-15 seeds
from eachM3 seed
family for mutant
isolation by GC-FID analysis
Modified from: http://file.scirp.org/Html/8-3000817_48085.htm
Summary of the Screen for Mutants With Reduced Levels of Seed Lipid VLCFAs
~10,000 mutagenized seed samples (M3) analyzed by GC-FID
11 putative mutants identified
What do we need to know/do next?
1.
2.
3.
4.
Fatty acid Wild type Fae1 WTxFae1 (F1)
16:0 8.5 9.3 9.318:0 3.1 3.6 3.518:1 14.8 27.3 18.918:2 29.6 34.4 33.118:3 21.4 24.1 23.720:0 2.8 0.5 1.120:1 18.2 0.8 9.722:1 1.6 0.0 0.7
Values represent (Mol %)
Genetic Analysis
F1 progeny has intermediate levels of VLCFAs between WT and Fae1 (co-dominant mutation)
F2 progeny segregated 1:2:1 (co-dominant mutation in a nuclear gene)
Complementation Test
Test DeductionMutant1 x Mutant2 Mutant1 x Mutant 2
Fae Fae fae1-1/ fae1-1 fae1-2/ fae1-2
ResultF1 F1Fae fae-1-1/ fae1-2
All mutants are co-dominant with the wild type. All segregate as mutations in single nuclear genes.
How many genes have been identified?
Pair-wise crosses between all 11 mutants revealed that all 11 mutations are present in the same gene that was named FAE1
Conclusion: Mutants 1 and 2 fail to complement and must be homozygousfor mutations in the same gene
Possibilities: 1, 2, 3, 4?
Generating hypothesesWhy were only mutants with mutations in FAE1 recovered?
Hypotheses:
1. Functional redundancy of other fatty acid elongation enzymes
2. Lethality
3. FAE1 gene encodes a single multifunctional protein with KCS, KR, DH and ER activities
How can we proceed from here?
Generating hypothesesWhy were only mutants with mutations in FAE1 recovered?
Hypotheses:
1. Functional redundancy of other fatty acid elongation enzymes
2. Lethality
3. FAE1 gene encodes a single multifunctional protein with KCS, KR, DH and ER activities
How can we proceed from here?FAE1 gene was cloned – encodes a polypeptide of 57 kDa which functions as a condensing enzyme (KCS) exclusively in seed VLCFA biosynthesis
FAE DH
KR
ER
FAE1
How can we explain loss of function mutation in the seed KCS (FAE1) that does not affect VLCFA production for
sphingolipid, suberin or wax biosynthesis?
TAGC20,C22
SEED
What about other tissues?
Model for the Organization of VLCFA Synthesis
SEED
FAE DH
KR
ER
FAE1
TAGC20,C22
FAE DH
KR
ER
KCS1
SHOOT
Cuticular WaxC24 to C34
FAE DH
KR
ER
KCS2
ROOT
SuberinC20 to C32
FAE DH
KR
ER
KCS3
ALL CELLS
SphingolipidsC24
1. Condensing enzyme is unique for fatty acid elongation pathway in each tissue
Hypotheses:
2. Condensing enzyme determines the chain length of fatty acid elongation products
KCS1 is CER6 mutations in CER6 condensing enzyme gene
uniquely affect cuticular wax accumulation
WT cer6
Seed TAG, suberin and sphingolipid VLCFA levels are wild type!!!
Model for the Organization of VLCFA Synthesis
SEED
FAE DH
KR
ER
FAE1
TAGC20,C22
FAE DH
KR
ER
CER6
SHOOT
Cuticular WaxC24 to C34
FAE DH
KR
ER
KCS2
ROOT
SuberinC20 to C32
FAE DH
KR
ER
KCS3
ALL CELLS
SphingolipidsC24
What about the other 3 predicted VLCFA biosynthetic enzymes?
Model for the Organization of VLCFA Synthesis
SEED
FAE DH
KR
ER
FAE1
TAGC20,C22
FAE DH
KR
ER
CER6
SHOOT
Cuticular WaxC24 to C34
FAE DH
KR
ER
KCS2
ROOT
SuberinC20 to C32
FAE DH
KR
ER
KCS3
ALL CELLS
SphingolipidsC24
1. Condensing enzyme is unique for fatty acid elongation pathway in each tissue
Hypotheses:
2. Condensing enzyme determines the chain length of fatty acid elongation products3. Two reductases (KR and ER) and the dehydratase (DH) are expressed in all cells and have broad substrate specificities
Testing the Model of Very-Long Chain Fatty Acid Organization
If the 2 reductases (KR and ER) and the dehydratase (DH) are expressed in all cells, then the expression of the seed specific condensing enzyme FAE1throughout the plant will result in…
Prediction:
Testing the Model of Very-Long Chain Fatty Acid Organization
If the 2 reductases (KR and ER) and the dehydratase (DH) are expressed in all cells, then the expression of the seed specific condensing enzyme FAE1throughout the plant will result in production of 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
Prediction:
16:0 16:1 18:0 18:1 20:1 22:1Control(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
Expression of Gal1::FAE1 Transgene inS. cerevisiae
Fatty acid Wild type Fae1 WTxFae1 (F1)
0.79.71.123.733.118.93.59.3
0.01.622:10.818.2 20:1 0.52.820:0
24.121.418:3 34.429.618:227.314.818:13.63.118:09.38.516:0
Values represent (Mol %)
Does the condensing enzyme also control the amounts of VLCFAs in the cell?
1. The expression of FAE1 encoded condensing enzyme is sufficient for the synthesis of VLCFAs, implying that the other three predicted activities of the FAE pathway (KR, DH, ER) are found ubiquitously throughout the plant.
Conclusions
2. The condensing enzyme is the activity of the fatty acid elongation pathway that determines the acyl chain length of VLCFAs producedand VLCFA amounts.
3. The ability of yeast containing FAE1condensing enzyme to make seed-specific 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.
Initial hypothesesWhy were only mutants with mutations in FAE1 recovered?
Hypotheses:
1. Functional redundancy of other fatty acid elongation enzymesNO KR (2 genes, but only one is expressed)NO DH (single gene)NO ER (single gene)
2. Lethality:YES KR (2 genes, but only one is expressed)YES DH (single gene)NO- Surprise !!! ER (single gene)
3. FAE1 gene encodes a single multifunctional protein with KCS, KR, DH and ER activities
NO
Transgenic 35S–FAE1 Arabidopsis Plants That Accumulate High Levels of VLCFAs
Millar AA, Wrischer M, Kunst, L. (1998) Plant Cell. 1998 11:1889-902
High Levels of VLCFAs Co-segregate with the Unusual Morphological Phenotype
Millar AA, Wrischer M, Kunst, L. (1998) Plant Cell. 1998 11:1889-902