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)

WTFae1

Fae1 Phenotype

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:

Expression of 35S::FAE1 transgene in A. thaliana

Expression of Napin::FAE1 Transgene in Tobacco Seeds

WT WT + Napin::FAE1

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?

Expression of Napin::FAE1 Transgene in Arabidopsis Seeds

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

Chloroplasts from 35S–FAE1 Expressing Plants Have Altered Thylakoid Membrane Shape

Millar AA, Wrischer M, Kunst, L. (1998) Plant Cell. 1998 11:1889-902