PLANT DEVELOPMENT - EXTERNAL FACTORS Name: Class ID: Introduction: This lab focuses on an evaluation of the effects of environmental factors on plant growth and development. Nearly all aspects of the plant life cycle are at least partly regulated by external cues. Most seeds, for example, require highly specific light or temperature treatments for germination. Similarly, environmental changes indicative of seasonal progression such as changes in photoperiod or diurnal temperature range frequently control the timing of flowering and/or the onset of dormancy in temperate plant species. The vegetative growth of plants is also affected by external factors. The direction of plant organ growth, for example, is typically influenced by environmental stimuli; i.e., the bending growth of shoots towards the light is a familiar phenomenon to botanists and non-botanists alike. The growth orientation of plant organs is also typically affected by gravity. In addition, some plants also exhibit “preprogrammed” non-growth movements, termed nastic movements, such as the closure of the trap-leaves of Venus flycatcher plant, or the daily cycles of flower or leaf opening and closing, that are regulated by external stimuli. I) Light and Plant Development Since plants are photoautotophic organisms, it is hardly surprising that they are highly responsive to their light environment, in terms of both the quantity and quality of illumination. The perception of light is mediated by multiple classes of pigments (i.e., phytochrome, cryptochrome, phototropin, xeoxanthin, etc.) that can be distinguished based on their absorption spectra and, in some instances, kinetic analyses (lag time, escape time, reversibility, etc.) of the growth and development responses they mediate. The following exercises will involve an evaluation of the contribution of different classes of photoreceptors to plant morphogenetic responses. In addition, the site of light perception in seedling shoot tips will be assessed and the effects of light absence (dark growth) on plant morphogenesis will be evaluated. A) Phytochrome and Seed Germination The seeds of most plant species are not initially competent for germination. The requirements for breaking seed dormancy are diverse, and are generally related to the climate and ecological niche of the species. However, for species that produce small seeds, as is typical of many weed and grass species, light exposure is frequently required. The detection of light by seeds is mediated by phytochrome, a blue to blue-green protein pigment. Phytochrome alternates between two conformationally distinct forms, termed the red (Pr) and far red (Pfr) forms of phytochrome, depending upon the wavelength of red light absorbed. The Pr form of phytochrome absorbs red light, with an absorption maximum at 660 nm, while the Pfr form absorbs far red light, with an absorption maximum at 730 nm. The absorption of red light by the Pr form results in its conversion to Pfr and visa versa (Fig. 1). The Pfr form of phytochrome, induced by red light, is physiologically active. However phytochrome is synthesized in the Pr form. In terms of seed germination, the effects of red light and far red light exposure are typically reversible, when applied in direct sequence, with the ultimate response depending on which wavelengths were last absorbed. Biol 219, Lab 2 - 2 - In this experiment, we will evaluate whether lettuce seeds (cv. Grand Rapids) have a light requirement for germination and whether phytochrome is involved in the light detection process. Figure 1. Diagram of alternative phytochrome conformations and the effects of red and far red light on their interconversion. Experimental Protocol: 1) Lettuce seeds that have been imbibed overnite (12-16 hr) are provided in the biology darkroom. 2) Working in pairs, obtain 7 petri plates. Add two sheets of filter paper to each plate and wet the filter paper with distilled water. 3) Working under green safelights add 10-15 seeds to each plate. Seal each plate twice with parafilm and assign the plates randomly to experimental treatments (next page), being sure to write you name and the experimental treatment on each plate. Wrap each plate in a foil envelope and use a Sharpie marker to write the treatment number on the envelope NOTE: The envelopes must be able to be opened and closed to apply light treatments. 4) After all of the petri plates have been sealed, turn on the flood lights in the darkroom. Three boxes are provided below the floodlights. The lids of the boxes have been replaced with either red acetate filters, far red filters (combined yellow and blue filters), or gray filters (for the white light treatment). Each box should be positioned below a floodlight. 5) To apply the light treatments place the petri plates into the appropriate box and open the foil envelop on the plate to allow for light exposure. Close the foil envelopes prior to removing the petri plates. After the treatments have been employed, verify that the seals on the plates are light-tight and return the plates to the lab instructor. 6) After 72 hrs, collect germination frequency data using the table provided (next page) Experimental Treatments (Phytochrome Experiment) Biol 219, Lab 2 - 3 - Treatment 1: Continuous darkness (wrap in foil immediately) Treatment 2: Continuous light Treatment 3: White light for 10 minutes, then darkness (foil) Treatment 4: Red light for 10 minutes, then darkness (foil) Treatment 5: Far red light for 10 minutes, then darkness (foil) Treatment 6: Red light for 10 minutes, followed by far red light for 10 minutes,then darkness (foil) Treatment 7: A) Red light for 10 minutes, far red light for 10 minutes, red light for 10 minutes, then darkness (foil) B) Red light for 10 minutes, 120 minutes of dark incubation, far red light for 10 minutes, then darkness (foil) Table 1. Lettuce seed germination Treatment Proportion Germinated Percentage Germination Continuous Dark Continuous Light White Dark Red Dark Far Red Dark Red Far Red Dark Red Far Red Red Dark Red 120 min Far Red Dark B) Light and Seedling Morphology Plants with large seeds (indicative of the presence of large food reserves) frequently do not have a light requirement for germination. However, when germination occurs in the dark the seedlings exhibit an alternative pattern of growth and development. These seedlings, which are long and spindly with small underdeveloped leaves and a white to pale yellow color (lacking chlorophyll) are said to be etiolated. In this exercise, we will formally compare the morphological traits of etiolated and light grown bean seedlings. The seedlings provided (approximately 14 days old) were sown on the same day but in the light vs. the dark. Working in pairs collect the data indicated in the following table. Collect data for two seedlings of each type (light grown vs. etiolated) Table 2. Etiolated bean seedling data Biol 219, Lab 2 - 4 - Response Variable Seedling Type Light Grown Etiolated Total height (cm) Number of leaves Leaf sizes; L x W [cm] (Leaf A), (Leaf B), etc. top down C) Phototropism in Oat Coleoptiles The directional growth of plant shoots towards the light is one of the most familiar examples of a plant tropic response. Tropisms are growth movements in which the direction of the growth response is affected by the position of the stimulus, with growth (curving) being either towards or away from the stimulus. Movements towards a stimulus are said to be positive while movements away from a stimulus are ‘negative’. Since plant growth is indeterminate and tropic responses modify the relationship between plants and their environment, plant tropisms have been compared with the “behavior” of animals. According to the authors of one of the most popular introductory botany textbooks (Evert, R.F. et al., 2003), “the sequence of growth stages in plants corresponds to a whole series of motor acts in animals, especially those associated with obtaining food and water” The scientific study of plant “behavior” as manifested by their tropic movements has long been a subject of fascination to botanists. Some of the first controlled studies of plant phototropism were carried out by Charles Darwin and his son Francis, with the results being published in 1881 in a book entitled ‘The Power of Movement in Plants’. Working with oat and grass seedlings the Darwins demonstrated that the sites of light perception and differential growth, resulting in plant curving, were distinct. Using a combination of caps and collars they were able to demonstrate that the perception of light occurred in the shoot tip while shoot bending occurred at a more proximal (lower) position, suggesting that an influence, possibly a hormone, that caused the bending was transmitted from the tip of the seedlings to the area below where bending normally occurs In this experiment we will repeat some of the experimentation of Charles Darwin and his son, but will also evaluate the effects of different colors of light (red, green, blue, and white) on the phototropic response with the goal of making inferences about the specific pigments involved in light detection. Experimental Protocol: 1) Working in groups of three to four, under reduced light intensity, obtain two petri plates of oat seedlings (5 day old) that have been cultured in the dark. Each plate should have 10-15 seedlings with 2-5 cm tall coleoptiles. 2) One of the petri plates will be assigned to the white light treatment (gray acetate filters) and the second will be assigned to a colored light treatment. The selection of the colored light treatments will be coordinated with the laboratory instructor to ensure that all of the light treatments are tested to an equivalent degree at the class level. After the treatments have been selected label the front edge of each petri plate, which will face the light source, with your names and the treatment name. Biol 219, Lab 2 - 5 - 3) For the white light treatment, cover the coleoptile tips of four seedlings with foil caps. To prepare the caps squares of foil approximately 8 mm in diameter are provided. Place each square over the tip of a blunt pencil and mold it to create a cap. 4) After the treatments have been applied place both sets of petri plates in the appropriate light box and record the start time. 5) After 2-2.5 hrs the seedlings will be removed from the light boxes and evaluated for phototropism. For each treatment, collect data on the frequency of phototropism and the degree of curvature, as measured separately for each seedling using protractors. You may also want to compare the seedlings for differences in the degree of green coloration (chlorophyll induction). Table 3. Oat phototropism data Treatment Primary (Color) Secondary N Proportion with Curvature (˚, degrees) phototropism White None Foil Cap Red None Green None Blue None II) Gravity and Plant Development Gravity is an important organizer of plant growth with roots typically showing positive gravitropic responses (downward growth into the soil) and shoots showing negative gravitropic responses. According to the Cholodny-Went model of plant tropisms (1937), tropic curvatures result from the unequal distribution of the plant hormone auxin (IAA) between the two sides of the curving organ with the auxin concentration gradient resulting from auxin redistribution across the organ. In the case of gravitropism, the auxin redistributes to the lower side of horizontal oriented plant organs; however, the response to the elevated auxin content on the lower surface apparently differs between organ types with growth being stimulated in shoots, resulting in upward curvatures, and inhibited in roots, resulting in downward growth. A) Corn Seedling Gravitropism (Demonstration) Examine the beakers of germinated corn seeds. The seeds were sown in multiple orientations and cultured in the dark to eliminate and possible light effects of their growth response. Did the roots and shoots of the seedlings both exhibit gravitropic responses and if so in what directions? B) Arabidopsis Gravitropism Mutants Biol 219, Lab 2 - 6 - The current model for root gravitropism in Arabidopsis is outlined in pp. 446-448 of your textbook. Gravity perception is correlated with the sedimentation of starch grains located in the root cap with the differential growth response being mediated by changes in auxin distribution across the growth zone of the root. Most auxins in the root tip are apparently imported from the shoot via the phloem. These auxins pass into the root cap and are laterally redistributed into the epidermis and outermost cortex where they inhibit root elongation. In vertically oriented roots the auxin that arrives at the root cap is redistributed equally to all sides of the root. However, in horizontally positioned roots most of the auxin is redistributed to the lower side of the root. The degree of root growth inhibition is greater on the lower side of the root resulting in a downward curvature. This model has been both developed and confirmed via the analysis of Arabidopsis mutants. In this exercise you will compare the gravitropic responses of wild type Arabidopsis seedlings and mutants defective for components involved in the normal gravitropic response. For each Arabidopsis genotype (mutant or wild type), seeds have been sterilized, stratified (cold treated), and germinated on petri plates of tissue culture media. The seeds were germinated two days prior to lab with the plates being cultured vertically to provide a gravity stimulus oriented parallel to the media surface. For wild type seedlings the initial growth direction of the roots should have been synchronized as a result of the vertical orientation of the plates. After an additional 2-3 days, the plates will be rotated 90 degrees. The plates will be marked with arrows to indicate the position of the gravity stimulus both before (black ink) and after reorientation (blue ink). Today you will examine the plates to assess whether the primary roots have exhibited positive gravitropic responses. In next week’s lab the plates will be re-examined, with the primary and secondary roots both be evaluated for gravitropic responses. In addition, for each root type the degree of gravitropism will be quantified in the form of growth angle measurements. The mutants may exhibit either no root gravitropism or an attenuated gravitropic response. In addition, the gravitropic responses observed may change with seedling age (i.e., before/after plate rotation) and may differ among root types (primary vs. secondary). Background information on the mutants is provided below. Gravitropism Mutants: phosphoglucomutase-1 (pgm-1, CS210): A mutant that is homozygous recessive for a loss of function allele of the phosphoglucomutase gene, conferring a starchless phenotype. The roots of the pgm-1 mutant have been reported to exhibit positive gravitropism but to be less sensitive to gravity, requiring higher threshold g-values) than wild type. auxin-resistant1-7 (aux1-7, CS3074): A homozygous recessive mutant lacking a functional AUX1 auxin uptake carrier (permease). The AUX 1 permease is asymmetrically localized on the plasma membrane at the upper end of root protophloem cells (i.e. away from the tip), a distribution pattern that has been proposed to promote the net movement of auxin from the phloem to the root apex. AUX1 is also strongly expressed in a cluster of cells in the columella of the root cap as well in the lateral root cap cells that overlay the cells of the distal elongation zone of the root (Fig. 19.19 in your text), but is not thought to be involved in the lateral redistribution of auxin in the root cap. agravitropic1 (agr1, CS268): A homozygous recessive mutant lacking an auxin efflux carrier that is normally localized at the basal (distal) end of cortical cells near the root tip and is thought to be responsible for the flow of auxin away from the root cap following its lateral redistribution. Table 4. Arabidopsis gravitropism: Initial data Biol 219, Lab 2 - 7 - Genotype Proportion of seedlings with positively gravitropic primary roots1 Wild Type pgm-1 aux1-7 agr1 1 Consider any roots growing downward at an angle of at least 45 degrees positively gravitropic. Table 5. Arabidopsis gravitropism: Following reorientation Genotype Growth angle1 Other observations Primary Roots Secondary Roots Wild Type pgm-1 aux1-7 agr1 1 Compare the direction of root growth, as assessed at the root tip, with the direction of the gravity stimulus with growth angles of 0 degrees indicating growth parallel with and towards the gravity vector, growth angles of 90 degrees indicating horizontal root growth, and angles of 180 degrees indicating growth parallel with but away from the gravity vector (negative gravitropism). Collect gravitropism data for the second gravity vector; i.e., following plate orientation, see the blue arrow. For the secondary roots data should be collected only for the subset of roots that had developed from the upper surface of the primary roots (relative to the gravity vector). If the responses of the seedlings are variable, indicate the range of growth angles. See Figure 2 (below) for an example. = initial gravity vector = current (2º) gravity vector Figure 2. Using root growth angles to score gravitropism. The growth angle of the primary root is approximately 0 (parallel with the gravity vector), while that of the secondary root is 45 degrees. C) The Role of Auxin Transport in Shoot Gravitropism Biol 219, Lab 2 - 8 - This experiment will involve an evaluation of the role of auxin in shoot gravitropism via the use of traditional plant physiology techniques (organ removal, inhibitor studies, etc.). As described for roots, the placement of shoots in a horizontal orientation results in auxin accumulation on the lower side of the organ. However, in contrast, with the growth inhibition observed in roots, the rate of growth (elongation) on the lower side of the shoot is increased, resulting in an upward growth curvature. The processes of shoot and root gravitropism also differ in terms of the sites of gravity perception and auxin redistribution, and the degree of tip-localization of the region of growth (curving) (textbook, p 444-445) In this experiment we will apply indole acetic acid (IAA) and triiodobenzoic acid (TIBA) in the form of lanolin pastes. IAA is the most important of naturally produced auxins in plants. IAA is thought to be synthesized primarily in shoot tips and young leaves and subsequently transported polarly towards the roots, with the polar transport of auxin occurring in the vicinity of the shoot vasculature. TIBA is an auxin transport inhibitor. TIBA applications would be expected to result in auxin (IAA) accumulation distal to the size of application. Experimental Protocol: 1) Working in pairs obtain 6 sunflower seedlings (approximately 14 days old) sown in plastic 50 ml centrifuge tubes. Place the tubes containing the seedlings in a plastic rack and place the rack on its side so the seedlings are horizontally oriented. Use duct tape to secure the tubes of seedlings to the rack. Additional soil may also be added to the tubes to secure any seedlings which are exceptionally ‘wobbly’. 2) Add a piece of labeling tape on the top of each test tube and assign the seedlings randomly to the following treatments (1 seedling per treatment). Treatment 1: Control (no treatment) Treatment 2: Seedling left intact, IAA (1000 ppm) applied on upper side (tip back 5-6 cm) Treatment 3: Seedling left intact, 1% TIBA applied as a ring 1-2 cm below the apex Treatment 4: Seedling decapitated Treatment 5: Seedling decapitated, then 1000 ppm IAA applied to the cut surface Treatment 6: Seedling decapitated, 1000 ppm IAA applied to the cut surface and 1% TIBA as a ring 1 cm below the cut surface. NOTE: Decapitation will involve removal of the terminal 5-10 mm of the sunflower seedlings using a razor blade, being sure to leave at least one node of leaves intact below the point of decapitation. You may want to use only the tallest seedlings for the decapitation treatment. 3) Place your rack of seedlings into a cardboard box along with those of other student groups in the lab section, making sure that the orientation of the tubes is unchanged (i.e., with the tape labels facing up) 4) After 72 hours examine your seedlings and collect data on their growth responses using the table provided (next page). Be sure to indicate the auxin concentration used for Treatment 2. In addition, please be sure to shut the box again after you have collected your data to maintain dark conditions. Table 6. Shoot gravitropism data Biol 219, Lab 2 - 9 - Treatment Gravitropism (Type)1 Relative Degree (+ - +++)2 Treatment 1 Treatment 2 Treatment 3 Treatment 4 Treatment 5 Treatment 6 Treatment 7 1 Negative or positive gravitropism, positive = downards, negative = upwards 2 Rank the degree of gravitropic curvature on a relative scale from 1 to 3 (+ to +++) with higher numbers indicating a relatively greater degree of curvature Questions and Review Problems: 1) Can you think of any natural fitness advantages that may be conferred by having a light requirement for germination in the case of plants with small seeds? 2) For each etiolated phenotype demonstrated in the lab (i.e., more rapid rates of elongation growth, smaller/underdeveloped leaves, and a lack of chlorophyll synthesis) suggest a possible adaptive value. Biol 219, Lab 2 - 10 - 3) To produce a far-red (730 nm) light environment, blue and orange acetate filters were overlapped. The transmission spectrum for the orange filter used is shown below. Using a different color of ink (blue or red) please draw the approximate transmission spectrum of the blue filter. 4) Using your data or the demonstration date (provided on the class website), answer the following questions. Indicate which treatments you compared to make your decisions. Use the back side of this sheet if necessary. a) On what side of a horizontally positioned sunflower seedling does auxin normally accumulate? b) Is an intact shoot tip required for shoot gravitropism? c) Does the shoot tip affect the degree of gravitropic curvature? d) Does TIBA application substitute for tip removal?
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