A Mutant of Arabidopsis Lacking a Chloroplast-

Specific Lipid

Presented by Christine Phillips and Elsha Noah

J. Browse, P. McCourt, and C.R. Somerville

Science, 15 February 1985: Vol. 227 no. 4688 pp. 763-765

INTRODUCTIONWhat is the objective of this research?

– To investigate the functional significance of membrane lipid acyl unsaturation.

How did they do this?

– Isolate a series of Arabidopsis thaliana mutants that are deficient in a particular membrane fatty acid.

What did they find?

– One mutant lacks trans-C16:1, but shows no physiological differences.

What is their conclusion?

– This mutant could help with future investigations of membrane structure and function.

∆3-trans-hexadecenoate or 16:1∆3t or trans-C16:1

or trans-3-16:1 or trans-hexadecenoic acid

C C CC C CCCC CC CC C CC

O

HO

H

H H

H HH H

H HH

H H

H

H

HH

H

H

H

H

H

H H H

H

HHH

H

- -- -- - --- - -- - - -==

-

-

-

--

---

- -- -

-- -

-- -

- --

-- -

--

--

-It is a fatty acyl group

LipidsLipids can be organized into classes based on a shared chemical structure.

In eukaryotes, each lipid class has a particular fatty acyl composition.But what is the functional significance of this?

Present Hypotheses? (1985)

• Lipid unsaturation in membranes causes chilling sensitivity.

• Chloroplast membranes have unusual fatty acid composition, so this may be critical to photosynthetic function.

• Trans-C16:1 might be important to the structure and/or function of thylakoid membranes. Now

What?

Make the Mutants

20,000 wild-type

16 hours

0.3% aqueous ethyl methanesulfonate

Grow and self M1

M2

What do we do now?

Why do we screen the M2

plants?

Gas-Liquid Chromatography (1985)

Nitrogen

1ml of 1.0M methanolic

HCl

Reflux for 1 hour at 80°C

Cool for 5minutes

1ml of 0.9%NaCl

0.3ml of hexane

vortex

Carefully draw out 0.2-0.25ml of

hexane (top phase) 170°C on a 2m

column

Table 1

Each value is the mean of independent

measurements made for 10 leaves of each line.

• 89 putative mutants were identified.• What is our next step?

Is It Heritable?

Putative mutants were selfed to get M3.

4-10 individuals from each line were analyzed for inheritance of the anomaly.

M3 putative mutants were advanced by single seed descent.After several generations, at least 7 of

the original lines stably inherited the anomaly.

JB60

Besides this, the putative mutants were indistinguishable from the wild type in every other way

There are 7 putative mutants, what else do we need to know about them?

Perform a genetic complementation test. There are 5 genes involved in

producing normal leaf membrane fatty acyl composition.

Genetic Analysis!!!FATTY ACID WILD TYPE WT X MUTANT

(F1)

trans-C16:1 1.74 ± 0.21 0.89 ± 0.25

Tells us there is a simple inheritance of the nuclear mutation

JB60 (Mutant) X Wild Type = F1

F2

self

13 out of 57 plants lacked trans-C16:1

3:1 = There is a single nuclear mutation at a locus Authors name this mutant fadA (fatty acid desaturase)

Trans-C16:1

We know that it only esterifies to position 2 of phosphatidyl glycerol (PG)–IT IS NOT present in any other lipids of the thylakoid membrane!!

So let us see how the fatty acid composition of PG differs in the mutant and wild type.

Preparative Thin-Layer Chromatography

Figure 1

solvent

C16:0 (34%)Trans-C16:1 (20%)C18:1 (4%)

C18:2 (10%)

C18:3 (32%)

What does this tell us?

Mutant must lack a desaturase that converts C16:0 to trans-C16:1 at position 2 of PG– Then the intermediate levels of trans-

C16:1 in the heterozygote suggests that the fatty acid is regulated by the amount of enzyme activity.

– Unfortunately this hypothesis could not be evaluated at the time because the enzyme had not been discovered yet

Recent research• The mutant fadA is now called fad4-1

• FAD4 is now believed to be the enzyme that converts C16:0 to position 2 of PG to trans-C16:1

• Proving this is difficult because membrane associated fatty acid desaturases are very difficult to assay in vitro

• Evidence: FAD4 protein is limiting for the biosynthesis of trans-C16:1, it contains features similar to known membrane-bound desaturases, and no other mutants were recovered that caused a loss of trans-C16:1

Source:Gao, J., Ajjawi, I., Manoli, A., Sawin, A., Xu, C., Froehlich, J. E., Last, R. L. and Benning, C. (2009), FATTY ACID DESATURASE4 of Arabidopsis encodes a protein distinct from characterized fatty acid desaturases. The Plant Journal, 60: 832–839. 

Is trans-C16:1 synthesis encoded by the chloroplast?

• In favour: barley mutants deficient in chloroplast ribosomes were also deficient in trans-C16:1

• In favour: chloroplast protein synthesis inhibitors also inhibited trans-C16:1 synthesis

• Not in favour: Mendelian segregation of the fadA mutant does not follow predicted pattern

• Inconclusive: certain proteins encoded by the chloroplasts may also be required for trans-C16:1 synthesis but this hypothesis was not tested

Trans-C16:1 is present in the thylakoid membranes of most plants and algae

Potentially involved in membrane function?

Potentially involved in membrane structure?

Light-harvesting reaction

Is trans-C16:1 associated with light harvesting chlorophyll a/b proteins (LHCP) that in turn are thought to affect grana membrane formation?

Experiment used Fourier analysis of high-resolution electron micrographs

Predictions?

A is the wild-type and B is the mutant.

The paper’s authors suggest no significant difference was found.

However, while the grana in micrograph B appear to be the same length and thickness as those in micrograph A, in micrograph B they are much more compressed.

What do you think?

Electron Micrograph Procedure

Whole leaves fixed in 4% glutaraldehyde solution

washed in 0.1M phosphate (pH 7.2)

stained with OsO4

infiltrated with a graded series of ethanol (25% to 100%)

placed in epoxy resin

Electron Micrograph Procedure

Porter-Blum MT-2 microtome

to make 80nm-100nm sections

2% aqueous uranyl acetate Reynold lead citrate solution

for post-staining

Philips 201 transmission electron micrograph

x22500 magnification

LHCP: Light-harvested chlorophyll protein

• There is one bound molecule of trans-C16:1 per LHCP monomer

• LHCP exists as a trimer

• LHCP enhances light energy capture in photosystem II

Does trans-C16:1 affect LHCP?

• Experiment to determine if fadA mutants have a difference in irradiance response curves

• If trans-C16:1 played a large role in LHCP then the mutant would have reduced rate of electron transport at low irradiance

Irradiance Curves

The white circles represent the mutant JB60 and the black circles represent the wild-type.

Graph a shows the results of both mutant and WT in photosystem II, while graph b shows both mutant and WT in photosystem I.

Researchers found no significant differences in irradiance between mutant and wild-type.

Irradiance Experimental Procedure

Thylakoids were ground up in a solvent of:

• 50mM Tricine (pH 7.8),• 10mM NaCl,• 10mM EDTA• 400mM sorbitol

then filtered through cheesecloth, centrifuged and washed with more solvent

Irradiance Experimental Procedure

Photosystem II

O2 evolution with ferricyanide as an electron acceptor

Photosystem I

inhibiting photosystem II and using an electron donor

How was irradiance measured?

Energy Transfer Experiment

• Energy transfer from LHCP to the photosystem II in mutants and wild-type was also tested using a wide variety of temperatures and salinities

• No functional differences between the mutant and the wild type were found

Conclusions of the Paper

• In order to be present in 100% of eukaryotic photosynthetic membranes, trans-C16:1-PG must be selected for in some way

• Trans-C16:1 -PG does not appear to play any functional or structural role under normal environmental conditions

More recent research• FAD4 encodes a predicted integral membrane protein • it appears to be unrelated to classic membrane bound fatty

acid desaturases based on overall sequence conservation. • FAD4 is targeted to the plastid. • Overexpression of the cDNA in transgenic Arabidopsis led to

increased accumulation of trans-C16:1 -PG relative to wild type. • FAD4 is the founding member of a novel class of fatty acid

desaturases.

Source: Plant J. 2009 Dec;60(5):832-9. doi: 10.1111/j.1365-313X.2009.04001.x. Epub 2009 Aug 13.FATTY ACID DESATURASE4 of Arabidopsis encodes a protein distinct from characterized fatty acid desaturases.Gao J, Ajjawi I, Manoli A, Sawin A, Xu C, Froehlich JE, Last RL, Benning C

Thank you!

Questions?