Use of Arabidopsis eceriferum Mutants to Explore Plant Cuticle Biosynthesis

The plant cuticle is a waxy outer covering on plants that has a primary role in water conservation, but is also an important barrier against the entry of pathogenic microorganisms. The cuticle is made up of a tough crosslink polymer called cutin, and a protective wax layer that seals the plant's surface. The waxy layer of the cuticle is obvious on many plants appearing as a shiny film on the ivy leaf or as a dusty outer covering on the surface of a grape or a cabbage leaf.

Thanks to light scattering crystals present in the wax because the cuticle is an essential adaptation of plants to a terrestrial environment. Understanding that genes involved in plant cuticle formation has applications in both agriculture and forestry. Today we'll show the analysis of plant cuticle MUS identified by forward and reverse genetics approaches.

Hi, my name is Patricia Lam from the laboratory of Lyrica Kunz at the Department of Bot at UVC. Today, myself and others are gonna show you how these plants are AB do can be studied to understand how plants make particular wax. I'm Meen from Brain Heart Jet's Lab at UVC, I'll be showing you how we analyze chemical composition of cuticular wax using gas chromatography.

And I'm Alan Dub Bono from the laboratory of Lacey Samuels, also at the UBC, and I'll be showing you how we analyze the crystal structure on the surface of opsis using cryo scanning electron microscopy. Our overall goal is to understand how plants take ordinary fatty acids of 16 or 18 carbons, extend them to very long chains of 26 to 34 carbons. Then modify them into protective lipids that we find at the plant surface.

So let's get started and I'll show you how we identify particular wax means using forward and reverse genetic approaches. Hi again. So here we have a Cut mutant of rapid opis, which we call sro, which means not bearing wax.

Visual screens were used to OCI isolate these mutants, meaning that they were identified by sight. When you look at the mutants, you can see that the inflow and stems of well type plants is whiteish while the mutants have dark green shiny stems. One of the mutants that was isolated in this type of visual screen done by Martin K and his coworkers is this one called is from four or SIR four.

Another way of discovering cuco mutants is with a reverse genetic approach choosing candidate genes and then studying PLA lines with that genes disrupted in these mutants, TDNA was used to disrupt the gene of interest. TD NA is transferred DNAA segment of DNA inserted into the plant by agrobacterium tumor feces in nature. This bacterium infects TNA as part of its infection process, but scientists have disarms bacterium so it does not pathogenic and uses as a tool to transfer DNA into plants.

In this case of our study of the cuticle, we used the TDNA to disrupt the gene for a cytochrome P four 50 enzyme, which we now call M1.In order to confirm that our TDA insertion has disrupted the M1 gene, we perform PCR. The PCR gel shown here confirms that the TDA insertion is in the gene of interest. When two gene specific primers are used to amplify well type DNA, we see a product shown by the band in this lane, but the TD interrupts it as in the mutant.

There is no product conversely using primer specific for the TDNA and gene. A product is only seen in the mutant. We typically look for homozygous mutant lines where both the maternal and paternal copies of the gene are disrupted.

However, when one copy of the gene is disrupted by the TDNA and the other copy is still well type, the PCR R results are mixed like this. Now that I've shown you how to identify the cuticle mutant surf four ml one me, I will show you how to identify chemical composition using gas chromatography. Thanks Patricia.

Alright, let's go analyze the cuticle I Before performing Gas chromatography. The soluble wax compounds must be removed from the plant surface by deeping it in chloroform. Chloroform completely removes the soluble wax, but also cutin from the plant.

After solubilizing the wax components, the chloroform wax mixture is injected into the gas chromatography column where there will be heated and sensor through on a stream of gas. Different compounds from the wax mixture stick more or less to the walls of the column separating the compounds, and they come out one after the other at the other end of the column. Then as the compounds pass over the flame ionization detector, we see the peaks.

The retention time of each peak is characteristic for different compounds and using mass spectrometry. We have identified each of the components of ADOS wax. Looking at the chromatogram for a wild type plant, we can see the major peaks the correspond to a 29 carbon stray chain LK keone in secondary alcohol.

The minor peaks are the primary alcohols out heights, and as the area under the peaks relative to standard tells us how much of each compound is present. Here are the chromatograms for the plant lines that Patricia just showed us. Notice how lacks the primary alcohols and esters while ma one lacks secondary alcohols and ketones.

These phenotypes can tell us a lot about the function of the gene of interest in the plant. For example, before we knew which gene was mutated in the pho line, the phenotype told us that there was a problem in the primary alcohol branch of the biosynthetic pathway. After the molecular identity of that gene was studied in the consta lab, it was discovered that it is part of gene family of fatty acid reductases, which fits well with this chemical phenotype.

We can now place this genes on the pathway showing how the plant makes this component of the wax. Okay, that's how we analyze the chemical composition of our cuticle mutants. Now Alan will show us how we examine the structure of cuticle using scanning electro microscopy.Thanks.

Ow.Does anyone wanna see some pretty images of the cuticle? Let's get to work. When we study the Rapid opsys sine mutants by eye, we can see that they have a dark green glossy phenotype.

When we study wild type plants, we can see a whiteish appearance on the stem to determine the basis of these phenotypes that we can see with the naked eye. The scanning electron microscope is used to look at the cuticle structure on the surface of the plant. Since we know the chemical composition of the wax from the gas chromatography work, we can see how the chemistry is related to the structure of the wax crystals.

These crystals will be the first thing encountered by an insect or a pathogen landing on the stem surface. So they are an important part of cuticle algae. When performing cryo SEM plants must be frozen.

In liquid nitrogen plants are kept between minus 150 and 100 degrees Celsius during viewing. Under the scope, extremely low temperatures are essential for keeping cells intact because the SEM requires a vacuum to operate and if they were not frozen samples would dry out and collapse. Now we are ready to dissect surfer four and mah one samples along with the wild type controls.

I first removed the flowers and CLICs from these plants and then cut out developmentally matched segments of stem from the wild type and mutants. Then I mount them onto the SEM step. When the samples are transferred to the cold stage of the microscope and visualized, you can see that the surface of the wild hyper avid opsis stem cuticle is covered with crystals.

The light from those crystals gives the stem its whitish appearance. Now remember how the stem looked shiny on the mutants. You can see in the SEM images that it lacks the crystals on the surface.

So what are these crystals made from and what gives them their funny shapes? The answer is different. Wax chemical components give different shapes of crystals.

In experiments in which a single pure wax compound from the cuticle of a plant was isolated and re crystallized, the crystals form the same shapes as those found on the plants. In rabbit opsis, we believe the crystals are made up of a mixture of compounds and the situation is much more complicated. For example, the chemical analysis done by MEO shows that in surf four only the minor component of primary alcohols and their derivative esters are missing.

Yet the crystals on the surf four stem are gone. In the case of M1, the effect of the mutation in the cytochrome P four 50 enzyme is that the mutant plants lack two major components of the wax, secondary alcohols and ketones. Yet the crystals on the MAH one plants form normally.

So you see the absence of light scattering crystals, which gives the SRA mutants. Their glossy phenotype is always positively correlated with a change in cuticle composition. However, a mutant like mah one would never have been found using this approach because its crystals are normal despite the changes in the chemical phenotype as revealed by gas chromatography.

So reverse genetics approaches are also required to identify cuticle genes from known gene families. We've just shown that both forward and reverse genetics allow us to identify rub dous cuticle mutants. The chemical analysis of mutants phenotype using gas chromatography has helped us to understand how the plants make the protective cuticle lipids Cryo SEM has shown us how the crystals on the surface of the plant change when the chemistry of the cuticle changes.

So that's it. Thanks for watching and good luck with your experiments.