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 Fatty acids with three double bonds (ex. 18:3 and 16:3 fatty acid groups): highly unsaturated alpha-Linolenic acid

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? Due to their overabundant presence in the thylakoid membranes... Trienoic fatty acids are expected to play some key role in plant biology throughout the plant kingdom

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 fad7-2 fad8 triple mutants? An extremely unhappy or dead plant

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

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●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

Is the genotype of the parent, or the genotype of the pollen important for determining sterility? Question ?

Nature of the sterility ●Through staining for DNA, the researchers determined that both mutant and wild type pollen developed to a mature, tricellular stage

● 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 Nature of the sterility What does this suggest about the role of trienoic fatty acids?

Further testing ● 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 What can we do to further test whether trienoic fatty acids are necessary for male fertility?

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 fad7- 1 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 ● 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 If very low levels of alpha-Linolenate are sufficient to provide male fertility, why are trienoic acid levels so high so consistently?

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,

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,