Download The Critical Requirement for Linolenic Acid Is Pollen

The Critical Requirement for Linolenic Acid
Is Pollen Development, Not Photosynthesis,
in an Arabidopsis Mutant
Michele McConn and John Browse
Presentation by Rowan van Wersch and Linghui Wang
Trienoic Fatty Acids
alpha-Linolenic acid
Fatty acids with three double bonds (ex. 18:3 and
16:3 fatty acid groups): highly unsaturated
Fatty acid
composition of
Arabidopsis leaves
● Trienoic fatty acids are very common in thylakoid
membrane lipids throughout the higher plants
● Free radicals present in photosynthetic membranes are
expected to lead to fatty acids that are more saturated
Question ?
What does the near-universal prevalence of
trienoic fatty acids suggest?
Trienoic fatty acids are expected to play some
key role in plant biology throughout the plant
kingdom
Due to their overabundant presence in the thylakoid
membranes...
Trienoic acids as crucial for photosynthesis
● At the time this paper was written (1996), trienoic acids were
generally assumed to play some key role in maintaining
photosynthesis
Testing the hypothesis
If it is true that trienoic fatty acids are crucial for
photosynthesis, then plants without trienoic fatty
acids should not photosynthesize.
What is the simplest way to test this?
Using Mutants
The researchers had
access to Arabidopsis
lines with mutations in
each of the known
genes that produced
fatty acids capable of
adding a third double
bond to fatty acid chains
Creating an experimental line
● Using previously discovered mutant lines, researchers generated a triple
mutant
● fad3-2 fad7-2 mutants were crossed to fad7-2 fad8 mutants
● We expect to see fad3-2 fad7-2 fad8 triple mutants in the F2
● The researchers also generated a triple mutant line with fad7-1, a leaky
allele
What proportion of the F2 do we expect to be triple mutants?
1/16th, though if the triple mutant phenotype is lethal we
simply wouldn’t see them
If trienoic fatty acids are absolutely necessary for
photosynthesis, what might we expect to see in these fad3-2
An extremely unhappy or dead plant
fad7-2 fad8 triple mutants?
What they saw
● No visible dwarf plants
● After analyzing fatty acid composition they found a
number of plants (17 out of 240, very close to the
expected number of triple mutants) that contained no
detectable trienoic fatty acids
Suggesting…
● Triple mutants are not deficient in vegetative growth
Which is the triple mutant?
Wild type
Mutant
61%
40-58%
20%
5%
2%
MGDG
DGDG
PG
SQD
PC
PE
● No photosynthesis should lead to no growth
But…
● What if differences in photosynthesis in the
triple mutants are not limiting to growth?
Fluorescence analysis
● Leaves subjected to differing intensities of light
● Chlorophyll fluorescence measured at minimum,
maximum, steady state and saturated levels
● Photosynthetic activity estimated by calculating
potential and steady state quantum efficiency
Wild type and triple mutant plants
do not show significantly different
fluorescent activity
What does this mean?
● There appears to be no difference in photosynthetic
activity between fad3-2 fad7-2 fad8 triple mutants
that contain no detectable trienoic fatty acids and
wild type plants
Are trienoic fatty acids necessary for photosynthesis
in plants?
No
What now?
Why are they there?
● The researchers noticed that homozygous
fad3-2 fad7-2 fad8 mutants did not produce
seeds and lost their flowers late
● This suggested sterility
How do you test whether sterility is in the male,
female, or both organs?
Test outcrossing to the wild type!
Testing sterility
Taking pollen from the
male organs of a wild type
plant let emasculated
mutants produce viable
seed
Taking pollen from the
male organs of triple
mutants did not lead to
successful seed production
in wild type plants
Where is the problem? Are the mutants
male or female sterile?
Nature of the sterility
● Pollen is not properly released from the anther in
mutants
● Even when manually applied to the stigma,
successful fertilization did not occur
● Pollen grains in the mutant appeared outwardly
normal
Question ?
Is the genotype of the parent, or the
genotype of the pollen important for
determining sterility?
Nature of the sterility
● Through staining for DNA, the researchers determined
that both mutant and wild type pollen developed to a
mature, tricellular stage
Nature of the sterility
● Only ~11% of mature pollen was alive and viable in triple
mutants
● Less than 0.6% of these pollen grains germinated
● When they did, they produced much shorter pollen tubes
What does this suggest about the
role of trienoic fatty acids?
Further testing
What can we do to further test whether trienoic
fatty acids are necessary for male fertility?
● Complementation: instead of complementing with a
wild type allele, we complement with exogenous
trienoic acids
● By examining other mutant combinations with
different trienoic acid concentrations we can
estimate what threshold is necessary
Complementation
●
•
•
•
●
●
Three treatments:
Alpha-linolenate (the 18:3 fatty acid typically found in plants)
Omega-linolenate
18:2 fatty acids
The latter two treatments failed to complement, but…
Treatment with the “normal” trienoic acid led to successful pollen
production and self fertilization
● Offspring produced this way exhibit the triple mutant phenotype
Fertility threshold
● Triple mutant lines that incorporated the leaky fad71 allele contain very low (<5%) 18:3 composition
● However, they are fully fertile
● During the complementation test the exogenous fatty
acids only constituted ~5% of fatty acid composition
Other roles
If very low levels of alpha-Linolenate are sufficient
to provide male fertility, why are trienoic acid levels
so high so consistently?
● Low or high temperature stress tolerance
o Photosynthesis may be affected more in triple mutants
that lack trienoic acid at higher or lower temperatures
o Experimental evidence suggests that this is the case,
though mutants can still grow in a wide range of
temperatures
Other roles
● Salt and drought tolerance
o Overexpression of either FAD3 or FAD8 increases
tolerance to drought in tobacco plants and to osmotic
stress in cultured cells.
o Suggest that a drought-induced decreased level of
linolenic acid reflects damage.
Zhang, M., Barg, R., Yin, M., Gueta-Dahan, Y., Leikin-Frenkel, A., Salts, Y., Shabtai, S., and
Ben-Hayyim, G. (2005). Modulated fatty acid desaturation via overexpression of two distinct
ω-3 desaturases differentially alters tolerance to various abiotic stresses in transgenic tobacco
cells and plants. The Plant Journal 44, 361-371
Other roles
● Disease resistance
o Trienoic acids in chloroplast membrane lipids are
involved in defense responses against avirulent bacterial
pathogens.
Yaeno, T., Matsuda, O., and Iba, K. (2004). Role of chloroplast trienoic fatty acids in plant
disease defense responses. The Plant Journal 40, 931-941.