Disruption of the FATB Gene in Arabidopsis Demonstrates an Essential Role of Saturated Fatty Acids in Plant Growth Bonaventure et al., 2003 Presented by: Cassandra Jensen Angie Li Feb 10, 2015

Acyl-ACP Thioesterases

Two functions 1)Terminate FA synthesis by releasing free fatty acids 2)Involved in export of acyl chains to the eukaryotic pathway

●Acyl-ACP thioesterases were first purified from soya bean seeds and oilseed rape in 1990 ●Enzyme characteristics were assessed: found two classes of thioesterases based on differences in amino acid sequence and substrate specificity Acyl-ACP Thioesterases

●FATA: High activity for 18:1 ACP. Lower activity for saturated substrates. ●FATB: Highest activity for saturated acyl-ACPS, some activity for 18:1-ACP ●2 FATA genes and 1 FATB gene in Arabidopsis

Main Questions What is the importance of FATB? Why do plants require two classes of acyl-ACP thioesterases?

Potential Answers ●Two thioesterases are needed to control saturated/unsaturated balance of membrane fatty acids ●Membranes require a mixture of both types of fatty acids to maintain a balance of physical properties (i.e. fluidity) ●Saturated fatty acids are precursors for sphingolipids, surface waxes, and cutin ●Unsaturated fatty acids can be precursors for signal molecules

Previous FATB studies Antisense and overexpression study of FATB shows FATB is involved in the production of saturated fatty acids for flowers and seeds (Doermann et al, 2000)

Previous FATB studies Downregulation of FATB expression in soybean causes reduction in seed content of palmitate (Wilson et al, 2001; Buhr et al, 2002)

Q) What is the next logical step to study the function of FATB?

A) Isolate mutants!

Mutant Isolation

Next step?

Basic genetic analysis of mutants 1)Is it heritable? 2)One or more than one nuclear gene? 3)Co-segregation analysis 4)Complementation test

●Heterozygote FATB T- DNA insertion lines were self-fertilized = BASTA RESISTANCE Basic genetic analysis of mutants: Heritable? Number of genes?

●Heterozygote FATB T-DNA insertion lines were self- fertilized ●280:105 Basta resistant:susceptible ●2.5:1 ratio ●considering 50% homozygotes died: 3:1 mutation in a single nuclear gene Bb B b BBBb bb Basic genetic analysis of mutants: Heritable? Number of genes?

●110 Basta resistant plants were analyzed with PCR and GC-FID to determine genotype and fatty acid composition ●Plants with WT appearance and fatty acid composition were heterozygous for tDNA insertion ●Plants with mutant appearance and composition were homozygous for the insertion (fatb-ko) Basic genetic analysis of mutants: Co-segregation Analysis

Complementation analysis ●Vector containing WT FATB cDNA with CaMV35S promoter transformed into homozygous fatb-ko mutant ●Transformed mutants were exposed to hygromycin B and Basta to select for the transgene and fatb-ko, respectively ●Transformed mutants had WT phenotype FATBHYGROMYCIN R35S FAT B BASTA R FATB-KO

Complementation analysis

How effective is the mutation? Q) How does an insertion within an intron of a gene produce a knockout?

How effective is the mutation? INTRON 2 INTRON 3 T-DNA

How effective is the mutation? INTRON 2 INTRON 3 T-DNA

How effective is the mutation? INTRON 2 INTRON 3 T-DNA STOP mRNA

Q) How can we detect correctly spliced mRNA?

A) Reverse transcriptase PCR

Reverse Transcription PCR (RT-PCR)

Q) Why is this a problem? ➢ If there is a substantial amount of correctly spliced mRNA producing WT FATB protein, this line is not an efficient knockout

Quantification of mRNA Transcripts

●WT Ct = ~6 cycles earlier than fatb-ko (hom) ●Mutant transcript levels were ~150-fold lower than WT

Quantification of mRNA Transcripts

FATB Essential for Seedling Growth WT fatb-ko WTfatb-ko

Bolting Time

Decreased Growth Rate

●During these growing experiments, morphology between WT and fatb-ko plants remained similar. ●Reduced growth rate not caused by carbon limitation

Effect of Temperature ●Growth rate could be affected by temperature due to membrane properties ●Plants were grown at 22°C for 2 weeks and then transferred to 16, 22, and 36°C ●fatb-ko plants showed the same percentage of reduction (~50%) in fresh weight per seedling compared to WT at each temperature

FATB Essential for Seed Development

Wild-type Wild-type-like Intermediate deformed Very deformed

Causes of Irregular Seed Phenotype ●Alterations during seed developmental phases? ●Deficiencies in nutrient supply from maternal tissues?

Triacylglycerol: an O-linked glycerolipid Sphingolipid: an N-linked lipid Fatty Acid Composition of fatb-ko Tissues

●Reduction of palmitate (16:0) in leaves (42%), flowers (56%), roots (48%), and seeds (56%) Fatty Acid Composition of fatb-ko Tissues

●Reduction of stearate (18:0) in leaves (50%) and seeds (30%) ●No change to flowers and roots Fatty Acid Composition of fatb-ko Tissues

● % Increase in oleate (18:1) and % increase in linoleate (18:2) in leaves, flowers, and roots Fatty Acid Composition of fatb-ko Tissues

●(18:3) decreased % in leaves, flowers, and roots Fatty Acid Composition of fatb-ko Tissues

●Unsaturated fatty acids in seed tissues were less affected Fatty Acid Composition of fatb-ko Tissues

Summary 1.FATB has a major role in determining 16:0 levels in all tissues analyzed 2.FATB influences the level of 18:0 in leaves and seeds

Triacylglycerol: an O-linked glycerolipid Sphingolipid: an N-linked lipid Total Palmitate Content in Leaves

●39% reduction in total 16:0 in fatb-ko mutants ●Similar to 42% reduction of 16:0 in glycerolipids ●18:0 was reduced by 50%

Fatty Acid Composition of Individual Leaf Glycerolipids Individual leaf glycerolipids were separated and isolated by class using thin layer chromatography, then analyzed by GC-FID

Fatty Acid Composition of Individual Leaf Glycerolipids Extraplastidial Plastidial PC PE PG SQD DGDG MGDG

●16:0 reductions occurred mainly in extraplastidial lipids Fatty Acid Composition of Individual Leaf Glycerolipids PC PE

●16:0 reductions in plastidial lipids were less affected Fatty Acid Composition of Individual Leaf Glycerolipids PG SQD DGDG MGDG

●18:0 reductions occurred mainly in extraplastidial lipids Fatty Acid Composition of Individual Leaf Glycerolipids PC PE

Q)Saturated fatty acid reductions mainly occurred in extraplastidal membranes. Is this surprising?

Q) Is this surprising? A) No

Fatty Acid Composition of Individual Leaf Glycerolipids PC PE PG SQD DGDG MGDG ●No major difference in % total of each leaf glycerolipid between WT and fatb-ko ●fatb-ko does not affect net fatty acid accumulation

Is the lack of FAT-B activity compensated for by an increase in activity of FAT-A? ●18:1 ACP hydrolytic activity in leaves are similar in WT and mutants ●FATA activity is not upregulated in the mutant Acyl-ACP Thioesterase Activity

Leaf Surface Wax Analysis

●20% reduction of total wax load in fatb-ko mutant

Leaf Surface Wax Analysis ●20% reduction of total wax load in fatb-ko mutant ●No changes in distribution of wax components

●Consistent 20% decrease in leaf wax at different developmental stages ●Primary stems showed 50% decrease in wax load Greater effect on stems because they accumulate more epicuticular waxes ●Wax biosynthesis is limited by the supply of saturated fatty acids by FATB Leaf Surface Wax Analysis

Sphingoid Base Analysis

●N-linked fatty acids (sphingolipids) are more difficult to remove from lipids compared to O-linked fatty acids (glycerolipids) ●Strong alkaline hydrolysis was used to prepare the lipids for fatty acid analysis

Sphingoid Base Analysis

Sphingolipid synthesis begins with palmitoyl-CoA and serine Export Saturated fatty acids in glycerolipids Sphingoid bases Why do you think we see this?

Sphingoid Base Analysis Explanations: ●Sphingolipids are essential for cell growth. o Sphingoid base synthesis is maintained at the expense of acyl composition changes in other glycerolipids ●Slow growth rate in mutants could be due to slower supply of 16:0 for sphingolipid synthesis

fatb-ko act1 Double Mutant

Q) Where are the remaining saturated fatty acids coming from?

fatb-ko act1 Double Mutant

fatb-ko act1 fatb-ko act1 Wild-type

fatb-ko act1 Double Mutant ●fatb-ko act1 double mutant had 70% decreased 16:0 compared to wild type

fatb-ko act1 Double Mutant ●18:1 fatty acid levels are higher in the double mutant than the fatb-ko mutant

fatb-ko act1 Double Mutant ●18:0, 18:2, and 18:3 levels are the same in both fatb-ko and double mutants

●Analysis of extraplastidial lipid classes showed similar C16 composition and abundance between fatb-ko act1 and fatb-ko mutants ●act1 mainly affects 16:0 in plastidial glycerolipids while fatb-ko affects extraplastidial lipids o Size Growth rate Saturated fatty acid o Essential role in maintaining growth rate fatb-ko act1 Double Mutant

Q) In the double mutant, saturated fatty acid content was reduced to 30% of the wild type content. If both FATB and ACT-1 pathways are blocked, where do the remaining portion of saturated fatty acids come from?

Other sources of saturates ●Plastidial phosphatidylglycerol from unknown prokaryotic pathway ●FATA activity ●Mitochondrial pathway

Comparisons with previous studies ●Doermann et al. (2000): 35S FATB antisense study resulted in reduced 16:0 only in flowers and seeds, not other tissues. No visual phenotype ●Contrasts this study: 16:0 decreased in all tissues, slow growth phenotype ●Shows that the FATB enzyme or mRNA may be in excess and difficult to reduce to levels that would result in a growth phenotype

Comparisons with previous studies ●Most mutants with fatty acid composition changes could not be differentiated from wild-type ●Exception: fab2

fab2 mutants: ●high 18:0 (increased saturated fatty acids) ●rigid membranes ●mutant phenotype partially rescued by increasing growth temperature Comparisons with previous studies fatb-ko mutants: ●reduced saturated fatty acids ●fluid membranes ●slow growth phenotype not allieviated by low temperature, neither exacerbated by high temperature

●Effects other than membrane property changes limit fatb-ko growth ●Reduction of saturates may alter the biosynthesis and function of critical cell components Comparisons with previous studies

How does information from this study add to previous knowledge ?

Comparisons with previous studies

●16:0 ACP elongation is regulated primarily by substrate availability ●FATB and acyltransferase effects on 16:0 exhibit additional regulation Summary

Conclusion ●fatb-ko line shows a reduction in saturated fatty acids exported to the cytosol ●17% reduction in growth rate ●Altered seed morphology and germination

Specific functions of saturated fatty acids in sustaining normal growth remain unknown. Is growth rate linked to: o biosynthesis of critical cell components? o variations in membrane properties? o changes in fatty acid synthase? o lipid turnover rates? o all of the above? Potential Future Studies

Subsequent Studies ●Isotope labelling experiment (Bonaventure et al., 2004) o Fatty acid synthesis increased by 40% in fatb-ko o Fatty acid degradation also increased o Increased fatty acid turnover rate as a response to decreased saturated fatty acid production

Questions?