1. Bonaventure G., Salas J.S., Pollard M.R., Ohlrogge J.B.
Disruption of the FATB Gene in Arabidopsis Demonstrates an Essential Role of Saturated Fatty Acids in Plant Growth
Bonaventure G., Salas J.S., Pollard M.R., Ohlrogge J.B.
Presented by Yang Liu and Matthew Strelau
February 5th, 2019
BIOL 433

2. Fatty Acid (FA) Synthesis Products
FAS produces 16:0-ACP carbon chain fatty acids

3. Acyl-ACP Thioesterases Export FA from Plastids
Courtesy of Kunst, L., Haughn G., Song L.

4. What is a Thioesterase?
Enzyme (FAT) that catalyzes a hydrolysis reaction on the acyl-ACP attached to recently synthesized FA
60% of 16:0 and 18:1 are exported to the cytosol using acyl-ACP thioesterase
Registry of Standard Biological Parts

5. Why are there two classes of acyl-ACP thioesterases?
FAS

6. Why Are Acyl-ACP Thioesterases Important?
Essential role in the partitioning of de novo-synthesized FA
Determine chain length and saturation of FA exported through substrate specificity
Control the balance of saturated and unsaturated FA in the membrane
Why is a mixture of saturated and unsaturated FA important?
Balance of physical properties (e.g. fluidity), adapt to environment (e.g. temperature)
Unsaturated FA are precursors for signal molecules (e.g. linolenic acid for jasmonate)
Saturated FA are precursors for sphingolipids, surface waxes, cutin, and protein acylation

7. What is Known About FATA?
Acyl-ACP thioesterase specific for unsaturated acyl-ACP (18:1), little activity for saturated acyl-ACP
FATA determines 18:1 export levels
Two genes in Arabidopsis

8. What is Known About FATB?
Higher acyl-ACP thioesterase activity for saturated acyl-ACPs and lower activity unsaturated acyl-ACPs
One gene in Arabidopsis

9. Research Question
To what extent does each class of thioesterases, specifically FATB, contribute to the in vivo production of exportable saturated fatty acids?

10. Knock-out the FATB Gene
FATA
18:1-ACP
fatb
18:0-ACP
fatb
16:0-ACP
FAS
PLASTID

11. Mutant Isolation and Complementation
TDNA in the second intron of the FATB gene
How did the authors know the TDNA was in the FATB gene?
(co-segregation with Basta)

12. Segregation Analysis 280 110 85 normal growth 105
25 slow growth and FA composition change AA
280
110
85 normal growth Aa
(1-0.5)+2 = 2.5  2.5:1 ratio
2:0.5 ratio
A>a
A=TDNA
a=WT AA Aa
A_:
Aa:AA aa
DEAD
105

13. Mutant Isolation and Complementation
What “tool” was used in this research?
How did the authors confirm that the TDNA disrupted FATB?
Transgene complementation
Basta + hygromycin resistance
FATB mRNA expression (qPCR)
FATB gel and blot

14. Quantitative-PCR (qRT-PCR)
Measure the amount of desired mRNA using reverse transcriptase

15. qPCR
Critical threshold
BitsizeBio

16. No FATB mRNA Present in the Mutant

17. FATB Is Essential for Normal Seedling Growth
Overall growth less than wild type
Delayed bolting time
Smaller rosettes
Stems elongated more slowly

18. Growth Curve of Arabidopsis WT and fatb-ko plants

19. Recovering the fatb-ko mutant
1% sucrose plates or liquid
no, photosynthesis is not the reason
Different temperature 16°C, 22°C, and 36°C
fresh weight not altered
Exogenous saturated fatty acids
not sufficient
fatb is the first example of mutant with reduced levels of saturated FA with reduced vegetative growth

20. FATB is Essential for Normal Seed Morphology and Germination
Unclear whether mutant seed defects are a consequence of seed development

21. Fatty Acid Composition of fatb-ko Tissues
FATB determine 16:0 in all tissues
FATB contributes to 18:0 in leaves and seeds
Where is the remaining 16:0 in a plant cell produced?

22. Fatty Acid Composition of Individual Leaf Glycerolipids
Extraplastid
plastid

23. Fatty Acid Composition of Individual Leaf Glycerolipids
16:0 reduction mainly occurred in extraplastidial lipids
PE, PA, and PI had ~50% reduction in 16:0
PC had ~80% reduction in 16:0
All had reduced level of 18:0 except PI
16:0 was less affected in plastid lipids
Only SL (from DAG) had 40% reduction in 16:0
PA had a 50% reduction in 16:0
Increased level of 18:1 18:2 but reduced level of 18:3 in phospholipids and SL
Similar fatty acid accumulation per fresh weight
Could FATA compensated for FATB?
No major changes in relative proportions between WT and fatb-ko

25. Acyl-ACP Thioesterase Activity
FATA acyl specificity: 18:1 >> 18:0 >> 16:0
FATB acyl specificity: 16:0 >> 18:1 >> 18:0
WT and fatb-ko had similar 18:1-ACP hydrolytic activity
Both 16:0 -ACP hydrolytic activity were close to the background levels
The FATA hydrolytic activity did not upregulate due to the similar hydrolytic activity between WT and fatb-ko (no compensatory effect)

26. Total Palmitate Content in Arabidopsis Leaf Tissue
Strong alkaline hydrolysis:
Total leaf tissue, chloroform extracted lipids and solvent-extracted residue
Almost all 16:0 and 18:0 were co-extractable
Solvent-extracted residue had similar reduction in all of the fraction analyzed WT
fatb-ko
Reduction
16:0
1.87±0.02 umol/g
1.14±0.03 umol/g
39% (42%)
18:0
0.16±0.01 umol/g
0.075±0.005 umol/g
50% (50%)
FATB reduced saturated fatty acids levels in both organic soluble and insoluble components.

27. Leaf Surface Wax Analysis
FAS
FATB
LACS
Why wax analysis?
VLCFA are made from 16:0

28. Leaf Surface Wax Analysis
Leaf and stem epicuticular waxes
20% reduction leaves; 50% reduction stems
No novel component or significant changes in wax composition distribution
-FATB has a greater effect in stems than leaves

29. Sphingoid Base Analysis
Sphingoid bases
backbone of sphingolipid
Amino alcohol long chain base (LCB)
Predominantly by 18 carbon atoms
Why?
Lynch and Fairfieldl., 1993; Tessema et al., 2017

30. Pata et al., 2009

31. Sphingoid Base Analysis
-No major difference in sphingoid base composition between WT and fatb-ko

32. fatb-ko act1 Double Mutant
fatb was only ~50% saturated fatty acid levels compared to WT
act1 reduced level of G3P: acyl-ACP AT activity
How to make the fatb-ko act1 double mutant?
Cross fatb x act1 and identify homozygous double mutant in the F2
Why was the fatb-ko act1 double mutant was generated?

33. fatb-ko act1 Double Mutant

35. fatb-ko act1 Double Mutant
Further reduced in saturated FA to ~30% (70% reduction vs. WT)
Smaller size
act1 had minor effect on 16:0 accumulation in plastidial glycerolipids
act1/fatb-ko
act1
Saturated FA are essential in maintaining normal plant growth
fatb WT

36. Simplified Model of C16 and C18 Fluxes

37. Conclusions The fatb-ko shows:
A reduction in saturated FA exported to the cytosol
Altered seed morphology and germination
Reduced wax lipids but minor effect on sphingolipids
First Arabidopsis mutant with reduced saturated FA and reduced growth in standard conditions
A lack in change reflects the importance of the respective role (sphingolipids)

38. Supplementary references
Alberts, B., Bray, D., Hopkin, K., Johnson, A. D., Lewis, J., Raff, M., ... & Walter, P. (2015). Essential cell biology. Garland Science.
Browse, J., & Somerville, C. (1991). Glycerolipid synthesis: biochemistry and regulation. Annual review of plant biology, 42(1),
Dörmann, P., Voelker, T. A., & Ohlrogge, J. B. (2000). Accumulation of palmitate in Arabidopsis mediated by the acyl-acyl carrier protein thioesterase FATB1. Plant Physiology, 123(2),
Kunst, L., & Somerville, C. (1988). Altered regulation of lipid biosynthesis in a mutant of Arabidopsis deficient in chloroplast glycerol-3-phosphate acyltransferase activity. Proceedings of the National Academy of Sciences, 85(12),
Lynch, D. V., & Fairfield, S. R. (1993). Sphingolipid long-chain base synthesis in plants (characterization of serine palmitoyltransferase activity in squash fruit microsomes). Plant physiology, 103(4),
Pata, M. O., Hannun, Y. A., & Ng, C. K. Y. (2010). Plant sphingolipids: decoding the enigma of the Sphinx. New Phytologist, 185(3),
Tessema, E. N., Gebre-Mariam, T., Lange, S., Dobner, B., & Neubert, R. H. (2017). Potential application of oat-derived ceramides in improving skin barrier function: Part 1. Isolation and structural characterization. Journal of Chromatography B, 1065,