Disruption of FATB Gene in Arabidopsis Demonstrates an Essential Role - - PowerPoint PPT Presentation

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
• f 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

fab2 fad2 fad6 fad6 fad6 fad6

FATB

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

(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 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

• 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

• Range of seed shapes

→ 20% of mutant seeds severely deformed (fig. F) → 16% germination of highly deformed seeds ~50% germination for mutant seeds

• Sucrose did not rescue germination rate

→Reduced photosynthesis is NOT the cause

• FATB products essential for seed development
• Seed coat still present in mutants

W/T fatb fatb fatb 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

Oleoyl-CoA (18:1-CoA) 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

• 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 FATB act

• FATB gene is necessary for regular saturated fatty acid production
• The fatty acids produced in FATB pathway are crucial for growth, seed

morphology + germination, wax production

Why are the fatty acids produced by FATB crucial?

• NOT likely to be important for photosynthesis

→ Sucrose did not rescue mutant phenotypes

• NOT likely to be important for maintaining membrane fluidity

→ Mutant growth reduced in all temperatures → May be due to more subtle membrane changes

• May be due to impaired biosynthesis of important compounds

→ eg. slower sphingolipid production FUTURE WORK WILL TELL!