Disruption of the FATB Gene in Arabidopsis Demonstrates an Essential Role of Saturated Fatty Acids...
95
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
Disruption of the FATB Gene in Arabidopsis Demonstrates an Essential Role of Saturated Fatty Acids in Plant Growth Bonaventure et al., 2003 Presented by:
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