Download A Mutant of Arabidopsis Lacking a Chloroplast

A Mutant of Arabidopsis Lacking a
Chloroplast – Specific Lipid
By Browse J
McCourt P
Somerville CR
What are lipids ?
(Bile Salt)
Lipid classification
Fatty acids
eg. Palmitic acid
Trans C16:1
Storage
lipids
Triacylglycerols
Waxes
Glycerol phospholipids
lipids
Membrane
lipids
eg. Phosphatidyl glycerol
Sphingolipids
Cholesterol
Other
functions
Nutrition: Vitamins
Signaling: Steroid hormones
Eicosanoids
Emulsification: Bile salt
Lipid classification
Fatty acids
eg. Palmitic acid
Trans C16:1
Storage
lipids
Triacylglycerols
Waxes
Hydrophobic
Fatty acyl chain
lipids
Palmitic acid ( C16:0)
Hydrophilic
Carboxylate
3
(Trans- C16:1 Δ3 t)
Lipid classification
Glycerol phospholipids
eg. Phosphatidyl glycerol
lipids
Membrane
lipids
Sphingolipids
Cholesterol
Head group
Phosphatidyl glycerol (PG)
R1 = saturated FA
R2 = saturated/unsaturated FA
Phosphatidyl glycerol (PG)
CH2 OH
CHOH
CH2
Lipid classification
Fatty acids
eg. Palmitic acid
Trans C16:1
Storage
lipids
Triacylglycerols
Waxes
Glycerol phospholipids
lipids
Membrane
lipids
eg. Phosphatidyl glycerol
Sphingolipids
Cholesterol
Other
functions
Nutrition: Vitamins
Signaling: Steroid hormones
Eicosanoids
Emulsification: Bile salt
Fatty acids
Chain
Length
Fatty acids
Degree of
unsaturation
Short chain =< 6C
Medium chain 8 – 12C
Long chain 14 - 18C
Very long chain >= 20C
Saturated - Straight chain
Unsaturated – double bond (s)
ie. Cis vs. Trans
H
H
H
H
Fatty acids
Packed acyl chains,
Semicrystalline
-High energy
required to separate
-Less fluidity
Less packed,
Liquid crystalline
-Less energy
required to separate
-More fluidity
Fluid Mosaic Model of Plasma Membrane
Unsaturation on plasma membrane
Packed acyl chains,
Semicrystalline
-High energy
required to separate
-Less fluidity
/ trans
-Less packed,
Liquid crystalline
-Less energy
required to separate
-More fluidity
Cis
Major hypotheses about compositions of membrane lipids before this
paper:
1. Lipid unsaturation increases chilling resistance
2. Lipid unsaturation  differences in thermal tolerance between species
3. Trans unsaturation  photosynthetic functions
Chilling (~ 0-12 C) causes:
-Reduced growth and
development
-Phase transition from the
liquid crystalline state to the
semicrystalline
So, elevated level of cisunsaturation to maintain
membrane fluidity in some
species, such as Arabidopsis.
Chromatography background
 Thin-layer chromatography (TLC)
 Gas-liquid chromatography (GC)
TLC
 Mobile phase:
solvent
 Stationary phase: a
sheet of glass
coated with silica
gel
Silica gel
Less polar
More polar
Thin-layer chromatography
 Rf value
Silica gel
Thin-layer chromatography
 Recycle the
component we
want
degreaser
Silica gel
Gas-liquid chromatography
 Mobile phase: gas
 Stationary phase: a high
boiling point liquid
absorbed onto a solid
Gas-liquid chromatography
 retention time
TB
Solubility
TLC
GC
Advantages:
 More efficient (time
and money)
 Recycle the sample
Advantages:
 Easy to use
 get the amount of
the sample
Disadvantage
 Detector is limited
Disadvantage
 Destroy the sample
Mutant
 What type of mutant?
 Alterations in leaf membrane fatty acyl
composition.
Material
Arabidopsis
thaliana (L.)
Heynh
0.3% EMS
(Ethyl
methanesulfonate)
16h
X
200 μ E/m2/sec
23℃
70%humidity
GC
Gas-liquid chromatography
 Column:
2m
170℃
10% diethylene glycol
succinate
 Detector:
flame ionization
detector
recessive?
heritable?
0.3% EMS
16h
X
X
X
genetic
complementation
X
200 μ E/m2/sec
23℃
70%humidity
One gene?
X
GC
Result
 No difference of Morphology
 No difference of growth rate
 Fatty acid composition changes
 JB60: no trans-C16:1
A chloroplast-specific mutant
Biochemical Phenotypes:
- Lacks trans-C16:1
- Elevated level of C16:0
A single nuclear mutation ?
A chloroplast-specific mutant
P:
F1:
JB60
No trans-C16:1
X
Wild type
Trans-C16:1
½ amount of trans-C16:1
-codominant
-suggesting single nuclear gene
A chloroplast-specific mutant
P:
F1:
F2:
JB60
No trans-C16:1
X
Wild type
Trans-C16:1
½ amount of trans-C16:1
trans-C16:1
3
:
So, Single nuclear mutant
-Mendelian ratio for one gene
no trans-C16:1
1
A chloroplast-specific mutant
What is unusual about trans-C16:1?
1. Trans-configuration
2. Delta-3 double bond
3. It only attaches to position two of PG !!
=> compare WT-PG vs. Mut-PG
Fig. 1 FA composition of PG
Wild type
Mutant
C16:0
< 34%
C16:0
34%
Trans
-C16:1
20%
TransC16:1
0%
http://www.wfu.edu/chem/courses/organic/GC/index.html
Table 1. FA composition of leaves
A chloroplast-specific mutant
Proposal: a desaturase specifically converts
C16:0 at position two of PG to trans-C16:1 is
involved
However…no gene was cloned by that time…
Name the locus: fadA (=FAD4)
Thylakoid membrane
Stroma
S
PS II
Appressed
membrane of grana
LHC II trimer
Is fadA a chloroplast-encoded gene ?
 Exp 1. mutant in chloroplast ribosome
No trans-C16:1
 Exp 2. add chloroplast protein synthesis
inhibitor
No trans-C16:1
 Exp 3. Mendelian segregation
 => Not supportive evidence
Other evidence
Trans-C16:1
 Location
 Etiolated tissue
 Light-induced chloroplast development
LHCP
 Why are we focusing on the association of
LHCP (Light-harvesting chlorophyll a/b
protein complex) with trans-C16:1 ?
 light-induced chloroplast development
 The structure (trimer): each subunit
associates with trans-C16:1-PG
The main function of LHCP
 Contribute to formation of the grana
 Enhance the capture of light energy
the thylakoid ultrastructure
Wild type
mutant
The main function of LHCP
 Contribute to formation of the grana
 Enhance the capture of light energy
The effects of light intensity
PS ΙΙ
PS Ι
Sensitive fluorescence
techniques
 Aim:
Extensive analysis of energy transfer from LHCP to
photosystem ΙΙ
 Result:
NO functional difference
Since 1985…
Specials about trans-C16:1 of PG
-only found in thylakoid membranes
-always esterified specifically to the sn-2 position of the glycerol backbone
of PG
-found exclusively in eukaryotic, Chl a/b-containing photoautotrophs
Functions of trans-C16:1 of PG
-phosphatidylglycerol (PG) and its fatty acid composition play a crucial role
in stabilizing the oligomeric state of LHCII (1983).
-not involved in energy transfer (1985)
-the apparent stability of oligomeric LHCII is dependent not on PG content,
but rather on the molecular species composition of PG such that
oligomeric LHCII was stabilized when thylakoids exhibit high levels of PG
16:0/trans-Δ3-hexadecenoic acid (trans-16:1) relative to PG 16:0/16:0 (1998)
Formation of trans-C16:1 of PG
-by a trans-desaturase located within the inner recesses of the thylakoid
compartment. (1991)
Gene cloned
-FAD4 (2009)