A demonstration and hands-on activity to teach plant water transport
By Juliana Medeiros, Holden Arboretum
Contents
Concept and purpose
Background and significance
Procedure
Tips on tailoring for your audience
Suggested companion activities
Resources on plant water transport and plant physiology
Conducting experiments
Concept and purpose
The Drip Races is a simplified version of a device called a hydraulics manifold, which is used in real scientific studies to measure the rate of water transport in plants. The device has been modified for ease of set up and visual demonstration but can still be used to collect real data if desired.
The purpose is to teach the properties of the plant vascular system and how those properties translate into plant growth and drought survival, and to show how different plant species have different water transport properties depending on their leaf traits and the environmental conditions where they live.
The Drip Races can be set up as a demonstration or be conducted as a hands-on activity. The demonstration consists of woody stem sections attached to a hydraulics manifold, creating water flow through the stems, and collecting the water in graduated beakers over time to determine which type of wood transports the most water. You can use wood samples cut from any type of shrub or tree. For hands-on active learning, the device set up and experiments can be conducted by students and/or be combined with other hands-on activities (see Suggested companion activities section below).
This can be tailored to fit any age or skill level (see Tips on tailoring for your audience section below). The time needed to deliver the lesson is very flexible and can range from five minutes to two hours or more, depending on: 1) whether the hydraulic manifold is set up as a demonstration or created during an activity, 2) the number and type of companion activities you include.
Background and significance
In the process of teaching and conducting outreach on plant physiology over the past 15 years, I have had the opportunity to ask many random folks the question “What do you know about plants?”. Invariably, they tell me that plants grow by doing photosynthesis. In fact, children as young as 5 or 6 years old will tell me that plants do photosynthesis. More advanced students and adults may also relay that photosynthesis uses carbon dioxide and light to make sugars, and that oxygen is released in the process. Being that photosynthesis is one of the most prevalent and far-reaching chemical reactions on Earth, and a key component of food production and the global carbon cycle, it is comforting to know that most people have a general appreciation for it.
In addition to general knowledge that plants grow by doing photosynthesis, people are also well-aware that plants need water to grow. But when I ask the follow-up question “Do you know what plants do with their water?”, the answer is almost always “no”. Occasionally someone will relay to me that water is involved in the photosynthetic process, but they are typically under the impression that the water plants use is converted to oxygen in the photosynthetic chemical reaction. In fact, the role of water in photosynthesis is even more impactful and interesting than simply as a chemical reactant: most of the water plants use is evaporated from cell walls inside the leaf, which are exposed to the dry atmosphere when the plant opens its stomata to gain access to carbon dioxide. The plant water transport system, or vascular system, is critical to photosynthesis because it replaces the water that is lost through evaporation, keeping cells inside the leaves hydrated and functioning. Water transport in the vascular system takes place in specialized cells called xylem, which can be visualized a bundle of straws inside the wood. The leaves, wood, roots and flowers all contain xylem tissue, inside which there is an unbroken chain of water connecting all of the cells inside the plant to their water source in the soil.
Without the plant vascular system, leaves would quickly dehydrate and die, so the xylem is critical to plant growth and survival. And, it turns out that plants need to spend a lot of water to keep hydrated: approximately six molecules of water evaporate from the leaf for every single molecule of carbon dioxide that is captured. This trade-off between growth and water loss is an important factor in determining plant heat tolerance and drought resistance. When the air is cool and water is unlimited, plants can transport plenty of water to their leaves and keep their stomata, and thus grow rapidly. But when the air becomes very hot or when water is in short supply, water evaporates rapidly from the leaf surface and plants face a choice: 1) they can keep their stomata closed to prevent leaf dehydration, but this stops the flow of carbon dioxide to photosynthesis, effectively shutting down growth and eventually leading to starvation, or 2) they can keep their stomata open to continue photosynthesis, but as the soil and the atmosphere dry out this will eventually cause the vascular system to fail, cutting off the water supply to the entire leaf canopy and allowing the all of the leaves to dehydrate.
Thus, plants have evolved a wide array of xylem adaptations to keep the water flowing under different environmental conditions, so they can keep growing. This process is taking place every day, in all the plants on Earth, and the amount of water that evaporates from large plant canopies can be enormous. Approximately eight trillion metric tons of water evaporates from the Amazon annually, so plant water transport is a critical component of the global water cycle. In addition, because water transport determines how much the stomata can be opened, and thus how much carbon the plant can obtain through photosynthesis, it represents an important aspect of global food production and the global carbon cycle. In fact, plant water transport is critical for all life on earth. Because plants are the primary producers, their ability to grow determines the resources available for humans and other living things. The plant vascular system, in turn, is a key component determining where, and how fast, plants can grow. Given this grand importance, it seems as though the role of water should be more commonly understood. The Drip Races seeks to fill this gap by showing how wood transports water and how the water transport properties of wood relate to the leaf structure and environmental conditions where a plant grows.
Procedure
Step 1. Build the hydraulics manifold. The manifold consists of tubing, flow control valves, a water source, graduated beakers, a drain bucket and plastic hose clamps (Fig. 1). The woody stems are attached to the manifold by inserting into tubing. You will use small tubing for the backbone of the tubing system, and larger tubing to hook up your wood samples. The water source must be placed about 1 meter above the wood samples. The drain is used to initially fill the tubing, by opening the drain the water will flow from the source and fill the tubing, then close the drain. At the end of the day, the drain can be used to flush all water out of the system. The other thing you will need is a framework to hold all of the tubing and items in place. I use ring stands and clamps but note that the assembled manifold can take many forms, essentially, it’s just a tube hooked to a water source at one end and a stem at the other end. You can add as many or as few stem slots as you like. You may need to obtain tubing adapters to get the different tubing and source container connected. An example of how my manifold is set up is shown in Fig. 2.
Step 2. Collect woody stems. You can use any woody stem, but typically it’s good to choose plants that will be familiar to your students as this helps people understand what the wood is doing. Choose woody stems of a diameter that will fit snuggly inside your tubing (to prevent leaks). For the tubing described in the supplies section I use stems with diameter approximately 0.8-1.2 cm but you should check the fit with your tubing to be sure. Cut the stems with pruning shears, the sharper the better. Cut a large branch (1 m) and then when you get back to the lab trim the sample back from both ends to about 4-10 cm (trimming from both ends removes damage caused by pruning). For your final sample, you’ll want a straight section that has no major side branches. You can also trim the sample in the field, but this may damage the vascular system and taking the whole branch has the advantage of letting you look at the leaf type and total leaf area that was supplied by your stem.
Step 3. Prepare wood samples. This step is optional and designed to remove air bubbles from your stem. Air bubbles inside the stem are a type of damage experienced by wood called an embolism, which blocks water flow to the leaves. These air bubbles will inevitably be introduced into your stem sample when you cut it off the tree, and they can be created during drought and frost events, or by a small insect piercing the surface of the vascular tissue. Embolism can be a more difficult concept, so I don’t recommend this as an activity for elementary school students, and please see the reference section at the end of this document for literature about embolism.
Use a razor blade to carefully trim any wood that was damaged by the pruning shears. Fill a vacuum flask about ¼ full of water and place your stem samples in there (trimmed to fit). Turn on the vacuum, and the air will be pulled out of the stem immediately (you’ll have to keep your eye on it to see it). Some stems will produce bubbles for a while. Leave them on there as long as you like, but I usually do it until bubbles are no longer visibly pouring out of the stem. There are more technical ways to remove embolisms, but the vacuum is a fun way that allows people to see the air being sucked out of the stem.
Note that I often take two samples from each tree and remove the air from only one. When you place the two stems on the manifold the one with the air removed with have a faster rate of flow than the one where air has not been removed.
Step 4. Place samples on manifold and measure water flow. Place stems on manifold with water flowing so bubbles are not present, as bubbles can block the water flow. Open the valve leading to each stem and allow the water to drip into the graduated beaker. You can measure the flow rate per time (for example, mL per minute), which is the hydraulic conductivity of the stem. For stems with low flow rates, you may need to let it drip for a while before you have a measurable flow.
Step 5. Talk about what you observed. After setting up the demonstration I initiate a discussion of what students already know about the different types of wood, and then relating this to what they know about how fast and where that plant typically grows. For this reason, it is good to obtain plant species that will be familiar to a general audience, for example Oaks and Maples are well-known to the general public in our region of Northeastern United States, and people have a general idea that Oaks grow more slowly but are more drought tolerant than Maples. Another great topic of discussion is the physics behind the system. The force driving water flow on the hydraulics manifold is gravity (flowing down from the source into and out of the stem). In the living plant the force for water flow is the evaporation of water from the leaf surface, while the cohesive and adhesive forces of water cause all of the water contained in the plant to cling together such that they are pulled up as a continuous chain as water molecules evaporate from the leaf surface.
Hints for getting the water to flow: if you set everything up, open the valve and water is not flowing out the stems, then you most likely have air blocking water flow somewhere in your system. Check your tubing for air, and re-flush using the drain, check the tubing leading to each stem. If you are sure that the tubing has no blockages, it’s probably your stem sample that is blocked by air. To alleviate this, you can raise the water source higher, try flushing with the vacuum as described in Step 3, or try a different plant species. Plants that grow fast and have large vessels are your best bet for producing good water flow.
Figure 1. The basic set up of the hydraulics manifold.
Supply list
Flow control valves – I use Nalgene 2-way and 3-way stopcocks (Fisher Catalog No.14-630-2C and 14-630-1A). The Nalgene valves are very water tight and long-lasting but are very expensive. For the short term, you can use any kind of 2-way or 3-way valves that attach to plastic tubing.
Graduated beakers – any will do, I use plastic Nalgene 150 mL (Fisher Catalog No. 02-591-15D)
Plastic hose clamps – should be the right size to fit over your larger tubing and snuggly secure your stem (Fisher Catalog No. 05-815)
Source container with bottom spout – I currently use a Kimax Reservoir Bottle (Fisher Catalog No. 12-141-303), but this can be a drink dispenser, or any reservoir with a bottom spout.
Tubing connectors – plastic, range of sizes for connecting the odd tubing bits (Fisher Catalog 13-717-23)
Plastic tubing – I use clear pvc or vinyl. Note that the size of the tubing depends on the size of the flow control valves and stems that you will attach. I am currently using for smaller backbone tubing 0.18 in. ID and 0.31 in. OD (Fisher Catalog 14-174-1A). For the larger tubing to hook up stems I use 3/8 in. ID and ½ in. OD (Fisher Catalog 14-174-1G)
Figure 2. An example of an assembled hydraulics manifold. In this build have attached the tubing to a metal bar using zip ties, then attached the bar to ring stands using clamps. The water source was a large drinking jug (left), and I used a small bucket for my drain (middle, behind the stems). I used laminated labels to show the species names. My manifold has 10 slots, but you can build it with as few or as many stem slots as you like The colored light was added to create a little drama.
Tips on tailoring for your audience
This activity can be tailored for any audience from 1-99. For beginner audiences, the idea that wood supplies water to leaves, and that different plants have different wood and leaves is typically enough to get through. For more advanced audiences, the Drip Races can be a jumping off point to address concepts in physics, environmental science, ecology, evolution and cell physiology. For the most advanced audience, the Drip Races can be used as an experimental device, for conducting real experiments on plant water transport (see Conducting experiments section below).
Event table: For this I do steps 1-4 ahead of time and conduct it as a demonstration. When conducting outreach at an event table you will encounter mixed ages and skill levels, and your audience will have a short attention span of about 2-5 minutes. At my table I have the Drip Races set up with familiar wood types, and I laminate a few leaves of each species. When a guest comes up to the table I simply ask, “Do you know what plants do with their water?”, or “Do you know what wood does for plants?” Then I describe how the manifold works, show how the water is flowing through the wood, point out that different woods have different flow rates, talk about the fact that the wood supplies water to the leaves, then point out the differences in the laminated leaves that go with the different wood samples. That is about enough for most event-goers, some people will then stick around and ask questions about the types of wood or relate what you have just told them to what they know about wood from their day-to-day experiences. For this group, I’ve found that it’s nice to have one valve that has no woody stem attached. Keep it turned off, but when a guest arrives you can turn that valve on briefly to show that the water flows faster without a stick attached, allowing guests to quickly understand that the wood regulates how much water is supplied to the leaves.
Elementary school classroom: For this I do steps 1-4 ahead of time and for step 5 I do leaf sorting and leaf matching games (see below Suggested companion activities section). K-5 students will be interested in the idea that wood is like a bundle of drinking straws, and they like the idea that leaves drink water from their wooden straws. They can also have a bit of fun turning the water valves of the manifold off and on. As when working an event table, for elementary students, I’ve found that it’s nice to have one valve that has no woody stem attached. Keep it turned off, then turn that valve on briefly to show that the water flows faster without a stick attached.
Middle and High school classroom: For this I do step 1 ahead of time and depending on how long we have I may also do steps 2-4 ahead. This age-group can be the most difficult to engage, so I find that doing the sample collection and preparation with the students adds to their enjoyment, increases their attention span and helps them better connect to the information. For interpretation at this level, I often stick to linking the role of water in plant growth and stress resistance to ecological patterns.
College/Adult workshop: For this I don’t have a typical set-up, just depends on the amount of time I have and the goals of the class, but in general people get the most fun out of steps 2-4. Although not typical, I have built a new manifold with students and this offers a nice challenge along with more opportunities to talk about the physics involved. For the interpretation at this level, I generally take a more in-depth look at cellular processes of water transport taking place in the wood and leaves.
Suggested companion activities
Wood anatomy:The anatomical properties of wood are largely responsible for how water flows in the wood. Specifically, the size of the xylem cells (vessels and tracheids) and the way they are arranged in the stem have a big impact. Investigating the different cell types of wood, and how this differs across species that have different rates of water transport is a fantastic companion activity for the Drip Races. Excellent online resources for this activity include Mauseth’s Plant Anatomy (http://www.sbs.utexas.edu/mauseth/weblab/) and the InsideWood Database (https://insidewood.lib.ncsu.edu/welcome).
Leaf diversity: Activities that invite discussion on the properties of leaves that determine their growth rate and drought tolerance are a good complement to The Drip Races. For example, Oak leaves are thick, waxy and small compared to Maple leaves, so it is easy for people to see that Oak leaves should use less water than Maple leaves. A nice primer on leaf diversity (https://www.ck12.org/biology/leaf-types/lesson/Leaf-Types-Advanced-BIO-ADV/)
Plant identification: Activities that involve observing plant parts to make an identification of species go well with the Drip Races, because in the process students will observe many features that relate to plant function and environmental conditions. An online plant ID key can be found here (https://www.colby.edu/info.tech/BI211/PlantFamilyID.html)
Photosynthesis and leaf anatomy: The Drip Races makes a fantastic complement to lessons on photosynthesis and leaf anatomy because of direct physiological link with water transport. There are many online primers concerning photosynthesis, one of which can be found here (https://opentextbc.ca/biology2eopenstax/chapter/overview-of-photosynthesis/). Mauseth’s Plant Anatomy (http://www.sbs.utexas.edu/mauseth/weblab/) is also good for leaves.
Physics of water: The action in the Drip Races is all driven by the physics of water, which has unique properties. Water physics is summarized here (https://www.encyclopedia.com/environment/encyclopedias-almanacs-transcripts-and-maps/physics-water).
And so much more: The role of plant water transport in plant biology is a rich subject area. For more ideas about how to expand the Drip Races to connect with other concepts, please visit the Resources section below, and the section on Conducting experiments at the end of this document.
Resources on plant water transport and plant physiology
On-line primers on plant water transport
https://jplanthydro.org/article/view/25
Catalogs of plant hydraulic methods
Sperry Lab http://sperry.biology.utah.edu/methods.html
Jacobsen Lab https://www.csub.edu/~ajacobsen/Research_Methods.htm
Books about plant physiology and water transport
Nobel, PS. 2009. Physicochemical and Environmental Plant Physiology. 4th edition. Amsterdam, Academic Press.
Tyree, M.T. and M.H. Zimmermann. 2002. Xylem Structure and the Ascent of Sap. Berlin, Springer-Verlag.
Carlquist, S. 2001. Comparative Wood Anatomy: Systematic, Ecological, and Evolutionary Aspects of Dicotyledon Wood. Berlin, Springer-Verlag.
Conducting experiments
If you intend to conduct experiments and collect data, you can use the manifold as described in this protocol, but the results will be more accurate if the water is measured by a balance, rather than graduated beakers. The section “Catalogs of plant hydraulic methods” above provides links to detailed protocols for plant hydraulics. The following provides questions that need to be answered (pre-tests that need to be conducted) before taking on a full-scale plant hydraulics experiment. Many of the concepts in this section have not been covered in this document, so consultation with the books and on-line resources listed above, and the broader scientific literature on this subject is strongly suggested for more complete understanding of proper protocols. Basically there are two main concerns: 1) because plants differ widely in their structure, and because the structure determines vascular function, in order to make comparisons across treatments, your samples will need to be standardized (based on the size, age, position, etc.), and 2) the plant vascular system is dynamic, and anything you do can cause a change in the state of the system, so care must be taken in how samples are collected and handled.
What is the xylem structure? Xylem structure (tracheids versus vessels, diffuse versus ring-porous, conduit diameter and length) will influence the magnitude of flow rates and the likelihood of accidentally introducing embolisms during sample preparation. Short stems with lots of large, open vessels will have the highest conductivity because there is less end wall resistance, but these will also refill more easily.
How closely can the size and developmental stage of samples be constrained? The more closely the samples match the better. In most cases samples cannot be perfectly matched, so it will be very important to standardize flow rates in some way, e.g. by sapwood area or by leaf area.
Will any samples have difficult features? Some features, like strong tapering, will make standardization across samples more difficult. Other features, like branch points and absorbent bark, can alter measured out flow rates substantially. If some plants must be excluded because of these features it is worth considering what the effect might be on the overall results of the study.
Is there evidence for a wounding response to cutting? Sample preparation is a crucial time when artifacts may be introduced. Wounding responses to cutting can reduce or eliminate flow, and they may take 30 mins or an hour to resolve.
What is the native ion concentration of the xylem sap? Perfusion solution impacts the magnitude of flow rates, a perfusion solution of pure DI water will reduce hydraulic conductance. Choosing a perfusion solution that most closely matches the ionic concentration of the sample will reduce the amount of time needed to establish steady-state flow. If treatments are expected to vary in sap ionic concentration then a single measurement solution should be used for all treatments.
What is the expected range of flow rates? Do the flow rates in the samples you have chosen seem reasonable (proper magnitude) given what was expected?
How does the expected magnitude of flow compare to the sensitivity of my measurement device? For very low flow rates more sensitive devices, or more time, will be needed to distinguish among treatments. For very high flow rates a less sensitive device will do the job.
What pressure head is sufficient to establish measurable flow? Perfusion pressure should be kept below the point where refilling is occurring. This will depend on the diameter of the conduits, length of the sample vs. conduit length, etc. Having too small of a pressure difference can be a problem because this really lowers the signal to noise ratio. Recommended pressure head is in the range of 1kPa for plants with really large conduit diameters, below 6kPa for those with smaller diameter conduits.
What is the background flow, i.e. without a pressure head, relative to flow at my chosen pressure head? Background flow should be accounted for during each measurement. If background flow is very high compared to the flow established during measurement this could cause high variability in measurements. With higher background flows it may be necessary to make measurements using a larger pressure head (but not large enough to re-fill if native embolism is of interest). Alternatively, measurements can be made over a longer time period.
Can steady-state flow be established? Once flow is established in a sample does it remain constant for a good period of time (30 mins)? If flow rates drop over time refilling, wounding, clogging or a response to ionic concentration may be occurring.
How long does it take to establish stead-state flow? If flow rates increase over time there may be a response to the ionic concentration of your solution.
Are repeated flow measurements on the same sample consistent? Measurements should be repeatable, that is one should be able to return to a sample after a period of time (30 mins to an hour) and be able to establish the same flow rate under the same pressure head.
What pressure head is required to refill non-functional conduits? A test demonstrating the minimum pressure at which re-filling is observed is useful for setting an upper bound on the measurement pressure head. Also, if re-filling will be part of the experimental design (e.g. for maximum hydraulic conductance) choosing a moderate pressure head that accomplishes refilling will avoid unintended damage to the sample.
How long will elevated pressure need to be applied before refilling is complete? This can be determined by applying pressure, then measuring flow rate, then again applying pressure and measuring flow rate, etc until the measured flow rate does not increase.
How much variation is there among individuals in relation the variation expected between treatments? This will be important for determining the sample size needed to detect significant differences among treatments.
How much time is needed to make a good measurement at the chosen pressure head? The time needed to make a single flow measurement is in the range of 5 to 60 minutes (depending on the technique, pressure head, species, sample characteristics, perfusion solution, etc.). This will determine the total number of samples that can be measured within the time constraints of the study.
Can the temperature in the lab (or field location) be held constant? Flow rates will depend on the viscosity of water, which changes with temperature.
Are there temporal patterns in embolism formation? Flow rates can change over time in intact plants, for example, some plants can have lower flow rates in the afternoon as compared to the morning. If temporal patterns exist it will be better to confine sampling to a particular time period. If this can’t be done then randomizing the time of measurement across treatments can help disentangle time of measurement versus treatment effects.
For more information, or to suggest additions or corrections to this information, please contact:
Dr. Juliana Medeiros at jmedeiros@holdenfg.org