Prepare to be be fully submerged into root physiology as we enter the deep-end of our waterlogging project. For months now, you have read update after update on the tissue harvests and photosynthesis measurements we have been taking. The suspense is building and I have received many emails from anxious readers demanding some results. Just kidding, I have received zero emails. If I do, I will surely cherish them, print them out, and stick them to the Root Lab fridge for motivation. In the mean time, I'm motivated purely by the groundbreaking science we're doing here. By "groundbreaking," I mean we literally had to break artificial ground to get to these samples. Keep reading to learn about how we prepped the tissue samples for next week's tests and some background info on assessing stress in plants.

Grinding tissue samples

How do you get over 300 samples containing fine roots, chunky coarse roots, and stems, down to a fine powder? Lots of liquid nitrogen and human-sourced grinding power. Decades down the road, when the Root Lab is automated like the Amazon Fresh grocery stores, there will surely be robots zipping around with mortars and pestles, left with the responsibilities of the monotonous tasks some researchers despise. Personally, I enjoyed the monotony, even though Marvin did most of the grinding (my hands got cold, okay?!).

Let me back up and explain why we needed to grind these samples in the first place. The stress and nonstructural carbohydrate (NSC) tests we will be running on the waterlogging tree samples requires the tissues to be ground to a fine powder in order to homogenize the sample. I will explain the specific chemical markers we're looking at for each stress test later on in this post. For now, let's continue with our tissue grinding protocol.

The samples harvested for this project were immediately frozen in liquid nitrogen, a common procedure taken by researchers collecting any type of live tissue. This helps maintain cell structures and prevents further degradation and/or metabolic processes from occurring in the tissues. Now we have a snapshot of what was going on inside the giraffe, human, or tree, at the time of sampling. When grinding, liquid nitrogen saves the day again by making it easier to pulverize the sample into a powdery substance easily mistaken for different spices and flours. In fact, crushed foodstuffs were the original end products of mortar and pestle use.

The fine root samples, containing the least amount of mass, were ground in full. The roots were dumped from their falcon tubes into a mortar, covered with liquid nitrogen, and then smashed and ground with a pestle. Samples were then transferred back into a falcon tube, weighed, and stored in liquid nitrogen. The same procedure was followed with the coarse roots and stem, except these samples were larger, so we clipped off small portions with pruners prior to transferring them to a mortar.

Lexikon Blogion

Fine roots: the smallest, most distal roots in a plant's root system. We focus on these roots specifically because they are responsible for a plant's uptake capacity. Functions such as water uptake, nutrient uptake, mycorhizzal association, and root exudation, are all mediated through fine roots.

Liquid nitrogen: the most commonly used cryogenic liquid. At a chilly temp of − 320 °F, it is used by scientists for a number of purposes. We use it to preserve and grind samples.

Mortar and pestle: traced back to the stone age, a set of simple tools including a bowl (mortar) and a club (pestle) that are used to grind hard substances into powders and pastes. Tough seeds and grains were probably the first materials ground with the mortar and pestle, but pretty soon the tools' uses and cultural impacts diversified. You can now find a mortar in pestle in the hands of a cosmetologist, artist, chef, pharmacologist, chemist, or anyone else attempting to make guacamole.

Nonstructural carbohydrate (NSC): carbohydrates in plants that are available as a source of energy for growth and other metabolic processes.

Homogenize: to equally distribute or mix. Each of our samples are composed of different tissue layers. We want the chemical analyses we perform to assess the entire sample, not just the bark of the stem or the cortex of the root.

Above you can see our grinding station, equipped with dewars holding liquid nitrogen, a scale, mortars, a pestle, and the mini spatulas/scoopulas that we used to scrape the samples in and out of the falcon tubes.

A fine root sample during and post grinding.

A coarse root sample followed by a stem sample. You can see these samples took an extra stage of chopping before grinding. The fine roots were easier to get down to a powder, but these woodier fellows weren't too difficult to work with - eventually we ground them down.

Stress tests - a final reveal

By now, the stress tests that I've mentioned many times in previous posts may seem like a perpetual mystery that will never be explained. It was all a ploy to keep you coming back for more! Hopefully the pay off is as thrilling as Bruce Willis's character discovering he's been a ghost THE WHOLE TIME. For those of you who haven't seen The Sixth Sense, Willis, assuming the role of a psychologist, tries to help a young patient cope with his visions of the dead. I won't spoil the ending for you here (if you want spoilers, shift your eyes up about three lines of text). I know, I know, "what does this have to do with roots?!" Everything, actually. This research was funded by Mr. Willis himself.

To transition away from my poor attempts at sarcasm that are probably difficult to read over the internet, let's move on to the tests we're running on our fine root samples to assess their stress levels and the cellular damage they accrued over the course of the experiment:

Peroxidase activity
Peroxidase is a ubiquitous
enzyme found throughout the bacterial, animal, fungal, and plant kingdoms. In plants, these proteins are involved in many processes of growth and development. Our leafy friends have multiple types of peroxidases (just like we do) that are up- and down-regulated in response to various stimuli. Previous research shows that introduction of some stressors (e.g. herbivory, heavy metal pollution, excessive heat), impacts the activities of these peroxidases differently. These proteins are known as stress enzymes because they serve the essential function of degrading reactive oxygen species (ROS). ROS are natural byproducts of the chemical reactions that occur within cells to create energy, otherwise known as cellular respiration. The production of ROS increases under stressful conditions, such as waterlogging, and their build up leads to cellular damage and inhibition of many cellular functions. Enough ROS induced damage will eventually cause tissue death, as seen in the root die-back of our intolerant tree species. Check out this in-depth review on plant peroxidases if you want to learn more about their functions and applications.

We will test the peroxidase activity in our fine root samples by using a reagent containing the compound guaiacol. Guaiacol is helpful substrate to use when quantifying peroxidase activity because it turns the sample to a brownish color that can then be measured spectrophotometrically. When peroxidase is present in a sample, it catalyzes the following reaction:

2 H2O2 → 2 H2O + O2

For anyone who is not fond of chemical equations, no worries. Basically, peroxidase is speeding up the decomposition of hydrogen peroxide, and producing water and oxygen. That lonely diatomic oxygen molecule you see towards the end of the reaction combines with guaiacol to form a brown color. Higher peroxidase levels lead to quicker production of oxygen. The more oxygen in the sample, the more bonds guaiacol can form, producing darker brown hues.

What can we expect to see in our trees? We're not sure, but we have some ideas. The tolerant trees that showed little root die-back, i.e. the silver maples, could be synthesizing more peroxidase proteins in order to scavenge the ROS produced by the stress of waterlogging. Intolerant trees may show high peroxidase activity as well, and that would tell us that having more scavengers wasn't enough to mitigate tissue damage and save the tree.

Lexikon Blogion

Peroxidase: a large group of enzymes involved in many cellular processes. The peroxidases found in plants are defined as class III peroxidases. Within that class are different forms of peroxidase with unique structures and functions. Genetic research has found that the number of genes coding for these enzymes vary between species of plants. In rice, 42 members of the class III peroxidases have been sequenced. Numbers are closer to a dozen in tobacco plants.

Enzyme: proteins produced in all living organisms that catalyze, or speed up, biochemical reactions. Technically, there are also some non-protein enzymes, such as ribozymes, which are ribonucleic acid enzymes. This is a rabbit ribo-hole topic for another time!

Reactive oxygen species (ROS): an unstable molecule that readily reacts with lipids, proteins, and DNA. ROS, often called 'radicals,' are reactive because they contain one or more unpaired electrons. These sneaky thieves zoom around, stealing electrons through a process called oxidation.

Spectrophotometry: a branch of electromagnetic spectroscopy that measures the number of photons emitted vs. absorbed by a sample.

Lipid: a class of organic compounds composed of fatty acids and their byproducts. These include fats, waxes, oils, and steroids, to name a few.

Oxidative damage: imbalance that occurs within a biological system when the amount of ROS exceeds the ability of the organism to detoxify and/or repair the damage caused by the radicals.

Oxidize: a chemical reaction which causes electrons to be transferred to another molecule.

Lipid oxidative damage

Remember all of that cellular damage I was talking about earlier? The lipids that form a plant's cellular membrane are the unlucky receivers of such harm. To throw in a sports reference for all the sporty readers out there, lipid membranes are like the offensive line on a football team. ROS are the defensive players on the other team, continuously pushing back against the offensive line. When the offensive line is strong and intact, they form a barrier and protect their key players. Tom Brady can serve as the nucleus for this metaphor, and some running-back (apologies, I don't know the names of any current running-backs) can play the role of another organelle, the mitochondria. When the lipid membrane is weakened by too much ROS damage, Tom Brady gets sacked or the anonymous running-back gets tackled behind the line. According to football fans, that's bad (if it's your team).

I'll throw in some microbiological explanations for all the microbiology-y readers out there. Lipid peroxidation, the chain reaction caused by lipid oxidative damage, is a common biomarker used to assess stress and cellular damage in plants, animals, bacteria, and so on. When ROS oxidize the unsaturated fatty acids that form the lipid membrane, this deleteriously impacts the membrane's properties, leading to protein degradation, limitation of ionic transport in and out of the cell, and ultimately cell death. According to cell fans, that's bad.

The cellular membrane destabilizes and becomes permeable as a result of oxidative damage. Image source

To test the levels of lipid oxidative damage in our fine root samples, we will be using thiobarbituric acid-reactive substances (TBARS). When lipid peroxidation occurs, one of the byproducts is a chemical called malondialdehyde (MDA). MDA reacts with the acid in the TBARS test to form a pinkish color. We can then quantify the damage by measuring the sample spectrophotometrically, just like the previous peroxidase activity test. If the root's offensive line really took a beating, there will be a darker color change.

The oxidative damage is expected to be much higher in the waterlogged sugar maples compared to the controlled counterparts. They experienced a fair amount of root die-back, which is assumed to be influenced by high lipid oxidative damage. As for the rest of the species, predictions for both low and high levels seem sensible. The silver maples held onto their roots, but it's possible that a lot of internal damage was endured and the samples could still contain high amounts of MDA. Both magnolias experienced root die-back followed by a new flush of growth during the recovery period. Will some older age classes (pre-waterlogging) express higher damage, while the younger classes (post-waterlogging) show no damage at all? We'll find out soon enough.

Special shout out to Dr. Chad Rigsby and Dr. Carla Rosenfeld who have both been a huge help in deciding on protocols and running these tests!