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

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

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

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

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

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

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

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

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

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

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)

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

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

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

qPCR Critical threshold BitsizeBio

No FATB mRNA Present in the Mutant

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

Growth Curve of Arabidopsis WT and fatb-ko plants

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

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

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?

Fatty Acid Composition of Individual Leaf Glycerolipids Extraplastid plastid

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

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)

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.

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

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

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

Pata et al., 2009

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

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?

fatb-ko act1 Double Mutant

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

Simplified Model of C16 and C18 Fluxes

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)

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), 467-506. 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), 637-644. 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), 4143-4147. 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), 1421-1429. Pata, M. O., Hannun, Y. A., & Ng, C. K. Y. (2010). Plant sphingolipids: decoding the enigma of the Sphinx. New Phytologist, 185(3), 611-630. 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, 87-95.