Disruption of FATB Gene in Arabidopsis Demonstrates an Essential Role of Saturated Fatty Acids in Plant Growth Abby Holden & Nik Wilson
acyl-ACP Thioesterase (FAT) - Involved in the release of free fatty acids - Free fatty acids are re-esterified to CoA after export from the plastid, which are then used glycerolipid biosynthesis in the ER Two major roles of thioesterase: 1. Partitioning de novo fatty acid synthesis between the prokaryotic and eukaryotic pathways 2. Substrate specificity. - Which determines chain length and saturation of fatty acids exported from the plastid
What are the two classes of thioesterases? ● FATA ○ Highest in vitro activity, specifically 18:1, and lower activity for saturated fatty acids ○ Two genes ● FATB ○ Higher activity for saturated acyl groups ○ In vivo production of saturates in seeds and flowers ○ One gene What is known about FATB? ● When is it downregulated, it shows a reduction in palmitic acid ○ Which is a major product of fatty acid synthesis What is not known about FATB? ● The extent to which this class contributes to the in vivo production of exportable fatty acid by different tissues
-FATA/FATB may control balance of saturated/unsaturated fatty acids in membrane → Benefits: -Maintain specific fluidity in varying temp. -Prevent phase transition -Arabidopsis can grow with wide range of FA mixtures → Mutants with no 16:1 trans- △ 3, 16:3, 18:3, or much less 18:2 grow normally - Certain fatty acids required in mixture for proper growth → fab2 mutant increased 18:0, decreased growth → fad2/fad6 mutant lost photosynthetic ability → saturated FAs are precursors for waxes, sphingolipids, etc. - What is the role of the FATB thioesterase and the fatty acids it helps produce? → Knockout of FATB created using T-DNA insertion
fad2 FATB fad6 fab2 fad6 fad6 fad6
What did they do next? Mutation Isolation - T-DNA tagged Arabidopsis plants screening using PCR approach -T-DNA insertion in the second intron Complementation Analysis -Segregation analysis was done to observe if the frequencies agreed with a single gene insertion - They found the expected frequency of 3:1, half of the fatb-ko plants were lost during germination so the frequency was 2.5:1, 110 plants had normal growth and 25 plants had a slow-growing phenotype out of 280 -Mutants were homozygous for the insertion
How did they determine the extent of the gene disruption? -Expression analysis was done using PCR and protein gel blot -Reverse-transcriptase mediated PCR was performed to ensure that the insertion was not spliced out and mature mRNA was produced -Protein gel blot analysis confirmed that FATB protein was absent in the mutant plants -Indicating the T-DNA insertion generated a full knockout mutation
Phenotype of fatb mutant
Growth Reduced -Rosettes were ~50% diameter in mutant seedlings → Adult plants more similar in size -Exogenous sucrose did not rescue slow growth phenotype → Reduced photosynthesis is NOT the cause of phenotype -Exogenous saturated fatty acids did not rescue phenotype → FATB specific to saturated FAs → May need FATB pathway to produce specific fatty acid -Relative growth same at 3 different temperatures → Change in membrane fluidity not likely cause of growth reduction (A) Four-week-old wild-type (left) and fatb-ko (right) plants. (B) Two-week-old wild-type (left) and fatb-ko (right) plants.
Growth Reduced -Rosettes were ~50% diameter in mutant seedlings → Adult plants more similar in size -Exogenous sucrose did not rescue slow growth phenotype → Reduced photosynthesis is NOT likely cause of phenotype -Exogenous saturated fatty acids did not rescue phenotype → FATB specific to saturated FAs → May need FATB pathway to produce specific fatty acid -Relative growth same at 3 different temperatures → Change in membrane fluidity NOT likely cause of phenotype (A) Four-week-old wild-type (left) and fatb-ko (right) plants. (B) Two-week-old wild-type (left) and fatb-ko (right) plants.
-Fresh weight used to measure growth -Mutant seedlings grew slower/accumulated fresh weight slower → At 4 weeks, mutant was ~50% fresh weight -Mutants had much slower growth initially -After 8 weeks, relatively similar size -FATB products important for early growth A) Fresh weight of aerial parts of seedlings B) Log scale of fresh weight
Seed Morphology altered W/T fatb -Range of seed shapes → 20% of mutant seeds severely deformed (fig. F) → 16% germination of highly deformed seeds ~50% germination for mutant seeds fatb -Sucrose did not rescue germination rate →Reduced photosynthesis is NOT the cause fatb -FATB products essential for seed development -Seed coat still present in mutants fatb
-Germination severely reduced in mutant seeds → Deformed seeds
-Germination severely reduced in mutant seeds → Deformed seeds -Bolting time (increased flower growth) delayed in mutants → Likely due to decreased growth → Similar morphology in mutants, >90% eventually bolted
Fatty Acid Composition in fatb-ko tissues -16:0 (Palmitic Acid) was reduced in all tissue types -in vivo role of FATB is a major determinant for 16:0 -FATB contributes to 18:0 levels in both leaves and seeds -When a fatb-ko plant is transformed with WT cDNA with the 35S promoter, we see similar results of fatty acid levels with WT -FATB cDNA complemented the biochemical phenotype of the mutant
Fatty acid composition of individual leaf glycerolipids
Palmitic and Stearic acid in leaves -Palmitic acid = 16:0 fatty acid Stearic acid = 18:0 fatty acid -Needed to use strong akaline hydrolysis instead of typical method to test for N-linked fatty acids → Present on sphingolipids, etc. → Other method too slow (for O-linked) -61% palmitic acid content in mutant 50% stearic acid content in mutant -Decrease in N-linked fatty acids similar to O-linked (glycerolipids) Where does the remaining saturated fatty acid come from ? → ACP bound saturated fatty acid may be used in prokaryotic lipid synthesis pathway → Mitochondria may help produce and export saturated fatty acid → FATA has weak binding to saturated fatty acids
Leaf surface wax analysis -Total wax load was analyzed to determine if the reduction observed in saturated fatty acids has any influence -No significant changes were observed in leaf wax components between WT and fatb-ko -FATB supply of saturated fatty acids is one factor that limits wax biosynthesis -However, reduction did not result in 18:1 replacing 16:0
Sphingolipids -Essential for proper growth + development -Make up part of lipid bilayers -Contribute to cell signalling, stress response, senescence, apoptosis, etc.. -Sphingolipids have fatty acids attached to bases → Can be saturated or unsaturated - Was sphingolipid fatty acid base composition altered in mutants? → May not have as flexible of a balance as other lipids.. → Gas chromatography used to determine composition
-Only major difference between mutant and wild-type was in t18:0 fatty acid bases → Mutant had much more t18:0 -More abundant fatty acids had similar composition in mutant and wild-type -Mutant may keep mostly consistent sphingolipid abundance at expense of other lipids → Sphingolipid composition may be too important to alter heavily → Slow growth may be due to slow sphingolipid synthesis
Why create a fatb-ko and act1 double mutant? - Mutants still seemed to have 50% of saturated fatty acids -despite FATB being absent - To determine if some fatty acids were derived from the prokaryotic lipid synthesis - As it would disrupt the first step of the plastid pathway - The Act1 mutant had reduced plastidial glycerol-3-phosphate: acyl-ACP transferase activity - Act1 mutant had reduced 16:3, but showed a normal growth phenotype
ACT1
What did the double mutant show? -Severe growth impairment -18:1 levels were increased in double mutant compared to fatb-ko -Double mutant had even further reduced saturated fatty acid content -Showed that more severe growth phenotypes are associated with a greater reduction in saturated fatty acids -Saturated fatty acids play a role in maintaining normal growth rate
Is FATA upregulated? -FATA has high specificity for 18:1-ACP in the plastid → 18:1-ACP hydrolyzed by FATA thioesterase, converted to 18:1-CoA in cytosol Oleoyl-CoA (18:1-CoA) -Researchers wanted to know if FATA activity was upregulated to compensate for FATB knockout -Enzyme assay was performed to determine if 18:1-ACP hydrolysis increased in mutants → 18:1 hydrolysis not significantly different vs wild-type Conclusion: FATA is NOT upregulated in mutants
FATA FATB
Conclusions Predicted flux of fatty acids through different pathways in W/T and mutants -Saturated fatty acids in mutant likely mostly from prokaryotic pathway + from FATA (weak binding) -Double mutant still has some saturated fatty acid → Likely from FATA or unknown pathway to produce PG
FATA act FATB
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