Lab 4: Photosynthesis & Respiration II

Transcription

1 BIOL 153L General Biology II Lab Black Hills State University Lab 4: Photosynthesis & Respiration II Lab exercises in BIOL 153 have to this point emphasized photosynthetic organisms that are small (single cells or small aggregations of cells) and reside in aquatic or marine habitats. Today and in most subsequent labs we will focus on multicellular terrestrial plants. Life on land presents many physical challenges to plants, and as outlined below, the process of photosynthesis is intricately related to the movement of water in soil, through the plant body, and into the atmosphere. Transpiration is the process by which water moves through a plant, generally entering roots and exiting leaves through stomata. (Some plants have stomata on their stems or lack roots, but these are exceptional and will not be discussed here.) Stomata are located on the leaf epidermis and consist of paired guard cells that inflate to open pores and deflate to close pores, thus regulating gas exchange. Open pores are the means by which plants obtain carbon dioxide for photosynthesis. However, when the pores are open, water evaporates from the stomata and this is the driving force behind movement of water in the plant body! Evaporation of water at the leaf surface pulls water molecules through the vascular system, up from the roots and through the stem. Transpiration is responsible for carrying nutrients through the plant body and also provides evaporative cooling in an analogous manner to sweat in humans and some other animals. The down side of transpiration, however, is that water loss from stomata may prove to be excessive for plants, especially for species residing in dry environments. In such circumstances, plants may close their stomatal pores, slowing growth but also reducing loss of essential water molecules to the atmosphere (Chapter 30, pp ). Plants have other tricks for reducing loss of water during photosynthesis. For example, some plants will develop small or sparsely distributed stomata structures, while others will produce stomata only on the underside of their leaves. Plants may reduce overall water loss by having smaller (or fewer) leaves, by maintaining vertical leaf orientations, or production of leaf hairs (pubescence). The latter reduce water loss by making the leaf surface reflective to the warming rays of sunlight and by expanding the boundary layer (thin area of still air immediately surrounding the leaf). Watch the short video linked below and then proceed to page 2 of this lab exercise. 1

2 Introduction to Hedera In today s lab, you will be studying photosynthesis-related characteristics of the flowering plant Hedera also known as ivy which is in the Ginseng family (Araliaceae). Hedera species are native to Eurasia and North Africa and grow in relatively moist and cool forest habitats. They also have a peculiar life cycle. "Juvenile" plants have lobed leaves and herbaceous growth, and spend their time growing at ground level in the forest. In contrast, "adult" plants have un-lobed leaves and woody stems that climb trees, walls, and other vertical structures. Adult ivies reproduce in the forest canopy, producing fleshy fruits that are consumed by birds. In this lab you'll be working with juvenile ivy plants that were grown by the Ramsey Lab you are doing real science, while also learning about photosynthesis, anatomy and morphology of land plants, and genetics. The Ramseys study ivies because they are pernicious weeds of forest habitats in the U.S. While relatively well-behaved in the extreme climate of South Dakota, in forests of the Pacific Northwest and Atlantic Seaboard, ivy often covers the understory with thick growth (smothering native herbs) and entombing trees in evergreen foliage (increasing their susceptibility to windfall and ice damage). Despite these problems, ivies remain a lucrative crop of the horticultural industry, with ~10 million units sold each year in the U.S. Cultivated ivies often escape and colonize natural areas, furthering the invasion of urban forest habitats. The following video discusses the problems of invasive ivy in U.S. forests (first 1:30 is most informative). AIM field class project: invasive ivy video: Some progress has been made in unraveling the invasion biology of Hedera. First, it is known that two closely-related species Hedera helix and H. hibernica from inland and coastal areas of Europe, respectively are invasive in the U.S. The other thirteen species of Hedera occur naturally in the Mediterranean Basin, on islands of the Atlantic Ocean, in the Middle East, and in East Asia but are not invasive here in North America. Second, the two invasive species differ in ploidy level. Like most animals, H. helix is diploid and has two sets of chromosomes. Hedera hibernica, in contrast, is tetraploid and originated via chromosome mutation in populations of the diploid H. helix. Polyploid plants have larger cells and slower cell division compared to diploid plants, and it is hypothesized that polyploidy may affect whole organism appearance, such as structural size. (You can learn more polyploidy and its biological significance in your textbook, pp ) Third, the occurrence of diploid H. helix and tetraploid H. hibernica differ markedly across the U.S. Thus, ivies in east coast forests are mostly diploid while ivies in west coast forests are mostly tetraploid, thus mirroring the longitudinal distribution of the two species in their native European range. The aforementioned results suggest an approach for reducing ivy invasion. If the horticultural industry cannot be convinced to remove Hedera entirely from the market it is a lucrative specialty crop, and growers have so far resisted bans on these plants then perhaps less invasive ivy varieties and species can be selected for use in particular geographic areas. The Ramseys are evaluating the physiology and growth performance of Hedera spp., and today, you'll be using these materials too. Invasive ivy: juveniles in the forest understory and adults climbing trees (Seattle, WA, USA). 2

3 Step 1: Visualize leaf anatomy under light microscope In the first part of this exercise, you will inspect leaves and view slides prepared by your instructors to learn more about leaves and anatomical features of Hedera. 1. Select two specimens from the array of Hedera species that your instructor has set out for the lab take both a pressed leaf and prepared slide for each specimen. Note that there are materials for >10 species available, including species with unusual appearances and geographic distributions. Take one specimen that is diploid (2x) and one with a different ploidy (4x, 6x, or 8x). 2. Compare and contrast the leaves of the two species you've selected. How are they different in size and shape? Sketch the leaves in the space below. 3. Inspect images reprinted below to orient yourself to microscopic features of ivy leaves. (You may also want to review your textbook; see Fig , p. 597 & Fig. 30-2, p. 710). 4. Now for one of your selected specimens, use a light microscope to examine the peels from the leaf top and bottom. Sketch what you see at medium (100x) and high (400x) powers for one field of view. Be diligent with your drawings! Use fine focus to move slightly up and down note that some stomata aren t visible until you do this, because the depth (thickness) of the nail polish impression is too great to see all layers at once. For each drawing, indicate species, ploidy, and top or bottom; also, label stomata, trichomes, and epidermal cells. 3

4 5. Do you see any differences in abundance or size between stomata on the two peels (i.e., from the top vs. bottom of the leaf)? If so, why do you think these differences exist? 6. Now examine your second species. Use a light microscope to examine the peels from the leaf top and bottom. Sketch what you see at 400x. Draw to scale and use field of view to show scale. For each drawing, indicate species, ploidy, and top/bottom draw each stomata and trichome you see. 7. Do you see difference between stomata of the two species? What could explain these? 8. Now sketch a close-up view of one stomate, labeling the guard cells and pore use 400x. (Note there may be some accessory cells surrounding the stomata that look a bit like guard cells.) Step 2. Make your own leaf peels and stomatal impressions: You will paint nail polish onto a leaf surface, and then use clear Scotch tape to remove polish and attach it to a glass slide. The polish will make an impression of the Hedera leaf surface including stomata, trichomes, and epidermal cells that can be viewed under the microscope. You will then count (i.e., determine density) and measure (i.e., determine size) of these structures. Please read the entire procedure and talk through steps with your partner(s) before you start! 1. To ensure that leaves are divided properly across five lab sections, work in groups of two. If your lab section has an odd number, there may be one group of three. 2. Do NOT mix up leaves. Keep track of the names assigned to leaves, and label slides properly. Do not discard your finished slides or leaves you will submit them at the end of lab. 3. Each group will need the following items to perform lab activities: 2 Hedera leaves (distributed by instructor) 2 self-adhesive labels for each assigned leaf (distributed by instructor) 1 container of clear nail polish (shared among groups) 1 roll of clear Scotch tape (shared among groups) 1 glass slide (each slide will be used for two leaf impressions and turned in at the end of lab) 4

5 4. Reminder! Before starting the leaf peels, read all the directions and talk through each step with partner. Ask the instructor if something in these directions seems unclear to you! 5. Before receiving your leaves, make space on the lab table. Instructor will distribute leaves and labels at the back bench. Each group will be given 2 leaves (one is diploid and one is tetraploid). Keep each leaf and label in its own Petri dish so leaves aren t mixed up. 6. The bottom of the leaf is generally lighter and less shiny than the top and it has a more prominent midrib. Flip so that the leaf underside is facing up. 7. Paint a thin layer of nail polish onto the bottom of the leaf. Flip a coin to determine if you will put the polish on the right or left side of the leaf (heads = right; tails = left). Put the polish on the area shown in the diagram below. The polished area should be a square that has sides that are the width of a piece of Scotch tape. Polish should completely cover this area (it will appear shiny) but don t glop it on as a puddle. 8. Repeat above steps for both leaves. Each group member should apply polish to one leaf. 9. While waiting for polish to dry, attach labels to slide (pull paper backing off the label to expose the sticky back). There should be labels on both ends of the slide, with your group s two peels placed on the same slide (see below). You will still have one label in each dish, set this aside for later! 10. Allow nail polish to dry on leaf surfaces (5-10 minutes). The polish will become less shiny as it dries, and if you carefully touch an edge it won't feel sticky. 11. Tear off a piece of Scotch tape that is a bit longer than the width of your glass slides. (It is okay if a small amount of tape wraps around and sticks to the bottom of slide.) 12. Press tape onto polished area of leaf, rubbing your finger on the tape to make sure it is firmly stuck to the polish. The tape will become slightly cloudy when the polish transfers. 5

6 13. Slowly peel the tape off the leaf. It generally works best if you peel the tape from the bottom left (if you are right handed) or bottom right (if you are left handed) toward the tip of the leaf. 14. Confirm that you see a polish impression on the tape. If you do not see an impression, consult your instructor! Each partner should participate in the peeling process. 15. Stick the tape to slide. Be sure the leaf peel is placed directly under the correct label and leave room for the second peel! Center the impression on the slide, and wrap any extra tape around the bottom of slide. Pull the tape taut as you are placing it so that there aren t bubbles. Be obsessive about matching each leaf to the correct label. If you aren t sure how to do this, see examples at back table or ask instructor. 16. Keep the leaf intact and safe in the Petri plate! You'll be gluing and pressing it later in the lab. Step 3: Investigate and measure stomatal traits from your leaf peel. Now you can inspect your leaf and the leaf peel! You'll need a microscope and dissecting scope. 1. Use a dissecting scope to examine your leaves wait until after you have made peels, and don t mix up leaves. Sketch what you see. (Note, our dissecting scopes have magnification from 10-30x). 2. Can you see stomata or trichomes at this magnification on the dissecting scope? Would it work to place the leaf blade under the compound light microscope? Explain your response. 3. Use a compound light microscope at your table to examine both of your leaf peels. Confirm that you can see stomata at medium and high powers; if not, please consult your instructor. 4. Use high power (400x) on a scope to count stomata. For each leaf peel, count the number of stomata in 10 different fields of view at 400x magnification. Use fine focus to move up and down to make sure you find all stomata. Select the fields of view haphazardly (i.e., don t target areas that seem especially dense or sparse in the occurrence of stomata). Move the slide left-to-right (or up-to-down) in a manner that ensures you don t double sample the same leaf area with your fields of view. Record your findings in the data sheet below. Each partner should count the stomata on one leaf; while one of you counts, the partner can record the data. 6

7 Use the brand microscope for stomata counts! Data table for one leaf peel: count the number of stomata in 10 fields of view at 400x (your partner will enter stomata counts for your group s second leaf in their lab handout). Population Ploidy Field o' view no. No. stomata Microscope brand Example: Weston 4x FoV1 45 FoV 1 FoV 2 FoV 3 FoV 4 FoV 5 FoV 6 FoV 7 FoV 8 FoV 9 FoV Use high power (400x) on a scope with an ocular micrometer to measure stomata. Note, there are only a few of these scopes so you will need to share if they are all in use, skip ahead until one is available. For each leaf peel, use the ocular micrometer to measure the length of four stomata. Make measurements from the outside of the guard cells, as shown in the illustration below! Haphazardly select one stomate from four different fields of view. You will record the number of segments (aka tick marks ) from the ocular micrometer (e.g., 10, 12, 15, etc.). You can move the micrometer to line up with the stomata just twist the ocular lens. Each partner should count the stomata on one leaf; while one of you counts, the partner can record the data. Use the scopes at the Ocular Micrometer stations for stomata lengths! Data table for one leaf peel: measure length of four stomata, in different fields of view, at 400x (your partner will enter stomata lengths for your group s second leaf in their lab handout). Population Ploidy Stomate number Length (micrometer units) Scope ID (see note on scope) Example, Weston 4x Stomate 1 13 A Stomate 1 Stomate 2 Stomate 3 Stomate 4 7

8 6. Use a ruler and measure the length and width of your leaf in centimeters. Width = distance between widest points; length = distance from petiole attachment point to tip (see diagram). Population Ploidy Length (cm) Width (cm) Example, Weston 4x Having completed data tables for the two leaves assigned to your group one leaf per student please enter the findings into the Excel spreadsheet on the computer at the front of class. Be sure to enter data for all cells. Read instructions next to computer regarding data entry! (We will analyze the data from all sections and present a summary at the beginning of lab next week.) 8. Bring your leaf and remaining label to rear of room. Use one piece of Scotch tape to attach the petiole of your leaf to a rectangle of herbarium paper top side of leaf should face up. Place the label on the bottom right side (see example). Step 4: Stomatal conductance (and photosynthesis) Stomatal conductance is a measure of water loss (i.e., transpiration) and uptake of carbon dioxide through stomata it is thus an index of the instantaneous photosynthetic rate in a plant! Conductance is influenced by a number of factors (size, density and aperture of stomata; temperature and humidity levels surrounding the leaf; health and phenological state of the plant; etc.) and requires specialized devices that can measure movement of small volumes of gas through a leaf. High values of stomatal conductance (which are typically measured in units of mmol H 2 O per mm 2 of leaf surface area per second) indicate that a plant is experiencing high transpiration rates and thus loosing a lot of water to the atmosphere through its stomata but at the same time, gaining CO 2 from the atmosphere that may be used in the dark reactions of photosynthesis. Watch this video that illustrates use of a LI-COR for physiological research. The LiCor Field Portable - Infrared Gas Analyzer video: Measurement of stomatal conductance has many potential uses in plant biology from analysis of the effects of environmental factors on plant stress to comparison of photosynthetic capacity between varieties or species of plants. Today we will use measurements of stomatal conductance made by the Ramseys to look at relationships between air temperature, humidity, and transpiration. Physiological experiments with Hedera In summer 2013, the Ramseys measured stomatal conductance on 100s of ivies that had recently been sampled from Europe including many of the same plants on which you measured stomatal sizes and densities today! These plans had been clonally propagated and were grown in a common garden in upstate New York. Measurements were made over five days between 9AM and 12:30PM, which are times of highest transpiration rates for Hedera; we also measured environmental conditions (temperature, humidity, cloud cover, etc.) through this time period. 8

9 The images below show relationships for time-of-day vs. air temperature (upper graph) and time-ofday vs. humidity (lower graph) for two days on which stomata conductance was evaluated. Review these graphs and answer the questions below. Humidity is reported as relative humidity, which is the amount of moisture in the air compared to the total amount the air can hold at that temperature. Temperatures are reported here in F. 1. Why do you think temperature increases with time-of-day? Why does relative humidity decrease? 2. What do you think the effect of increased temperature will be on leaf transpiration? Explain. 3. What do you think the effect of increased relative humidity will be on leaf transpiration? Explain. 9

10 The table below shows the relationship between air temperature and relative humidity (%) for two days of Hedera experiments, based on measurements every 30 minutes through the 3.5 hour sampling period (9:00AM 12:30PM). Also shown is the average stomatal conductance value for ivy plants in these time periods (in units of mmol H 2 O per mm 2 of leaf area per second). 4. Using data from the table above, sketch two scatter plots in the graph template below to show the relationship between air temperature and relative humidity separately for day 1 and day 2 using. Next to the "dots" in each scatter plot, write the mean stomatal conductance values observed. 5. Is there a positive or negative correlation between air temperature and stomatal conductance and does this finding match your expectation in #2 above? Explain. 6. Is there a positive or negative correlation between relative humidity and stomatal conductance and does this finding match your expectation in #2 above? Explain. 10

11 7. In the afternoons of summer 2013, temperatures continued to increase and relative humidity dropped (warmest, driest part of the day was late afternoon) but stomatal conductance of ivy plants dropped in this time (sharply decreased around noon). How do you explain this finding? Step 5: Synthesis about photosynthesis and water relations in plants Watch the video linked below about adaptations to avoiding water loss. Based on info presented in the video, as well as knowledge gained from this exercise, answer the following questions Explain mechanistically how the following traits may help reduce water loss in terrestrial plants. smaller (or fewer numbers) of leaves vertical leaf orientation leaf pubescence stomata mostly on the leaf bottoms 2. What are potential disadvantages to closing stomata to reduce water loss? 3. Do the single-celled aquatic organisms we viewed in Labs 1 & 2 have stomata? If so, explain. If not, explain how carbon dioxide and oxygen exchange takes place. 4. For the stomatal data set collected in your lab section, do we see differences in density and size between diploid and tetraploid ivy? Explain your response. 11