Slide 1

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

Slide 2

Acyl-ACP Thioesterases

Slide 3

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

Slide 4

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

Slide 5

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

Slide 6

Slide 7

Slide 8

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

Slide 9

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

Slide 10

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)

Slide 11

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

Slide 12

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

Slide 13

A) Isolate mutants!

Slide 14

Mutant Isolation

Slide 15

Next step?

Slide 16

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

Slide 17

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

Slide 18

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?

Slide 19

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

Slide 20

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

Slide 21

Complementation analysis

Slide 22

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

Slide 23

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

Slide 24

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

Slide 25

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

Slide 26

Q) How can we detect correctly spliced mRNA?

Slide 27

A) Reverse transcriptase PCR

Slide 28

Reverse Transcription PCR (RT-PCR)

Slide 29

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

Slide 30

Quantification of mRNA Transcripts

Slide 31

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

Slide 32

Quantification of mRNA Transcripts

Slide 33

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

Slide 34

Bolting Time

Slide 35

Decreased Growth Rate

Slide 36

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

Slide 37

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

Slide 38

FATB Essential for Seed Development

Slide 39

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

Slide 40

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

Slide 41

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

Slide 42

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

Slide 43

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

Slide 44

150-200% Increase in oleate (18:1) and 40- 60% increase in linoleate (18:2) in leaves, flowers, and roots Fatty Acid Composition of fatb-ko Tissues

Slide 45

(18:3) decreased 15- 20% in leaves, flowers, and roots Fatty Acid Composition of fatb-ko Tissues

Slide 46

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

Slide 47

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

Slide 48

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

Slide 49

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%

Slide 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

Slide 51

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

Slide 52

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

Slide 53

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

Slide 54

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

Slide 55

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

Slide 56

Q) Is this surprising? A) No

Slide 57

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

Slide 58

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

Slide 59

Leaf Surface Wax Analysis

Slide 60

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

Slide 61

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

Slide 62

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

Slide 63

Sphingoid Base Analysis

Slide 64

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

Slide 65

Sphingoid Base Analysis

Slide 66

Slide 67

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

Slide 68

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

Slide 69

fatb-ko act1 Double Mutant

Slide 70

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

Slide 71

fatb-ko act1 Double Mutant

Slide 72

fatb-ko act1 fatb-ko act1 Wild-type

Slide 73

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

Slide 74

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

Slide 75

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

Slide 76

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

Slide 77

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?

Slide 78

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

Slide 79

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

Slide 80

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

Slide 81

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

Slide 82

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

Slide 83

How does information from this study add to previous knowledge ?

Slide 84

Comparisons with previous studies

Slide 85

Slide 86

Slide 87

Slide 88

Slide 89

Slide 90

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

Slide 91

Slide 92

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

Slide 93

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

Slide 94

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

Slide 95

Questions?