GPI Lab Course 2009
Plant Developmental Biology
TU München
Instructors
Prof Dr Kay Schneitz
Dr Lynette Fulton
Balaji Enugutti
Christine Skornia
Prasad Vaddepalli
Table of contents
General Information3
1. Literature Discussion3
2. Presentation3
3. Lab Protocols3
4. Participants3
5. Lectures3
6. Schedule4
Course Schedule
Lab 1: Plant Anatomy and Development 7
Lab 2: CSLM Analysis of GFP Localisation in Ath. Roots.13
Lab 3: Clonal Analysis and Fate Mapping20
Lab 4: Genetic Analysis of Flower Development26
Lab 5: Activation Tagging and Gene Isolation31
Lab 6: Analysis of Gene, Literature Search, Presentation of Results50
General Information
1. Literature discussion
The teaching assistants (TAs) will assist you in finding the appropriate literature you will present at the end of the course.
2. Presentation
Students will prepare a power point presentation to discuss some of their results at the end of the course. Presentation will be in English.
3. Lab protocols
Written experimental protocols (English) should be delivered to the secretary at the end of the course.
The FINAL DATE is Monday 16th of November 2008!!
4. Participants
5. Lectures
Table 1
Lecture / Date / SpeakerIntroduction / October 13th / Kay Schneitz
Fate mapping/clonal analysis / October 14th / Lynette Fulton
Flowers/homeotic genes / October 15th / Lynette Fulton
Plant transformation/reporter genes / October 15th / Prasad Vaddepalli
Activation tagging / October 20th / Balaji Enugutti
Sequencing/BLAST search / October 21th / Christine Skornia
Demonstration: Literature search / October 27th / Christine/Balaji
6. Schedule
Time
/Tuesday
October 13th
/Wednesday
October 14th
/Thursday
October 15th
9:00 - 10:00 / Lecture: Introduction / Lecture: Clonal analysis, fate mapping / Lecture: Flowers, homeotic genes10:00 - 11:00 / Plant Anatomy and Development / Clonal analysis, fate mapping / Floral homeotics
11:00 - 12:00 / Ac::GUS / Floral homeotics
Lunch
13:00 - 14:00 / CLSM imaging of GFP in Arabidopsis / Lecture: plant transformation, reporter genes
14:00 - 15:00 / HOM::GUS
15:00 - 16:00 / Results and Discussion
16:00 - 17:00 / Results and Discussion
Time
/Tuesday
October 20th / WednesdayOctober 21th
/ ThursdayOctober 22th / Friday January October 23th9:00 - 10:00 / Lecture: Activation Tagging (AT) / AT: Secondary TAIL-PCR / AT: prepare gels / Time to write your protocol
10:00 - 11:00 / AT: look at plants / Lecture: Sequencing/Sequence Comparisons (BLAST) / AT: precipitate the sequencing samples
11:00 - 12:00 / AT: run gel, take picture
Lunch
13:00 - 14:00 / AT: extract DNA / AT: run sequencer over weekend (TAs)
14:00 - 15:00 / AT: measure DNA / AT: Set up Tertiary TAIL-PCR and sequencing reaction
15:00 - 16:00 / AT: Set up Primary TAIL-PCR
16:00 - 17:00
Time
/Tuesday
October 27th / WednesdayOctober 28th
/ ThursdayOctober 29th
9:00 - 10:00 / AT: get back sequences, analysis / Read papers / Preparation of presentations
10:00 - 11:00 / AT: BLAST analysis
11:00 – 12:00
Lunch
13:00 - 14:00 / Demonstration: Literature Search / Read papers / Student Presentations (E)
14:00 - 15:00 / Literature search
15:00 - 16:00 / Preparation of presentations / Cheese and Wine
16:00 - 17:00
TUM Plant Developmental Biology GPI Course January 2008
Lab 1: Plant Anatomy and Development
Introduction
This laboratory will introduce you to the structure of wild-type Arabidopsis plants at both the whole plant morphology and internal anatomy levels. We will emphasize the organization and identification of cell and tissue types within the vegetative part of the plant body, the stems, leaves and roots. We will also discuss the relationship between cell structure and function and the developmental relationships between the various cell and tissue types and their progenitors in the embryo and apical meristems. Our primary goal is to gain experience in the interpretation of the mature structure of wild-type Arabidopsis plants (and the developmental basis for that structure) as a baseline for future comparison with mutant phenotypes.
In this lab we will use several methods that allow rapid microscopic examination of plant cells and tissues with minimum distortion: clearings, surface impressions, epidermal peels and toluidine blue O-stained hand sections of living tissue. Much can be learned about the plant phenotype from these methods, and these are a good place to start before embarking on the more time-consuming protocols for preparation of tissues for light, electron and confocal microscopy.
Plant morphology
1.Obtain a plate of wild-type Arabidopsis seedlings. Gently pull the seedlings from the agar and mount on a microscope slide in lactophenol. Coverslip and observe using a compound microscope.
Identify cotyledons, hypocotyl, and primary root bearing emerging lateral roots. Locate the shoot apical meristem region surrounded by developing leaf primordia and root apical meristem protected by a root cap. Observe vascular tissue, noting the continuity of the vasculature throughout the plant.
2.Examine the shoot of older Arabidopsis seedlings growing on soil. Note the helical phyllotaxis and leaf heteroblasty. Starting with the cotyledons, remove leaves one at a time in the order that they were formed, and mount on double stick tape. How do successively formed leaves differ in size, shape and trichome distribution? How many juvenile leaves and how many adult leaves are produced?
3.Examine Arabidopsis plants that are flowering. Once the stem has elongated, it is possible to recognize the reiterated basic units of shoot construction, the metamers (each consisting of leaf, axillary bud and internode). Compare type 1, type 2, and type 3 metamers (Schultz and Haughn, 1991).
Surface micromorphology
1.Pattern, shape and size of surface cells can be determined quickly by isolating the epidermal layer or by making a surface impression. First, practice removing the abaxial epidermal layer from a Kalenchoe leaf by making two shallow cuts in the margin, grasping the tab of leaf tissue with forceps, and pulling toward the midvein. Mount the epidermal strip in water on a microscope slide, coverslip, and observe. Identify epidermal cells, guard cells and trichomes.
2.Making an epidermal peel is not as easy with Arabidopsis! Two other approaches that can be used are: (1) Make a surface impression using cellulose nitrate (a.k.a. clear nail polish). Brush on a thin layer, let dry two minutes, use forceps to pull off, and mount dry on a microscope slide. Coverslip and examine. (2) Isolate a living epidermal layer using medical adhesive spray. Spray medical adhesive spray on a glass microscope slide and spread using a Kimwipe. Let the adhesive dry for 5 min and then press the upper surface of an Arabidopsis leaf gently onto the spray. Flood the upper surface of the leaf with water and then gently scrape away the lower epidermis, mesophyll and veins, using a single edge razor blade. Mount coverslip in a drop of water and examine.
3.Compare your observations with scanning electron micrographs of surface replicas of intact Arabidopsis leaves.
Internal anatomy
1.Start with the large stems and easy-to-identify phloem of a squash (Cucurbita) stem. Remove a 1-2 centimeter section of stem and cut cross sections using a double edge razor blade. Stain for 30 sec. in 0.05% aqueous toluidine blue O (TBO) in a watch glass (or petri dish), rinse in water in a second watch glass, and transfer to a drop of water on a microscope slide. Coverslip and observe. TBO is a metachromatic dye that stains lignified secondary cells walls blue/green and pectin-rich cell walls purple/pink.
Locate the three tissue systems and their component tissues. Note that the vascular bundles of squash stem are unusual in having both internal and external phloem.
Cut longitudinal sections, stain with 0.05% TBO and examine. Identify xylem tracheary elements, phloem sieve tubes, parenchyma, collenchyma, sclerenchyma, and epidermal cells.
Aniline blue fluorescence can be used to identify phloem sieve tubes. Mount unstained sections in 0.1% aniline blue and observe using a fluorescence microscope.
2.Section, stain and observe a wild-type Arabidopsis inflorescence stem. Note dermal tissue with epidermal cells, stomates and trichomes, vascular tissue with xylem and phloem, and ground tissue with chlorenchyma, sclerenchyma and parenchyma cells. Small or thin plant parts can be more readily sectioned if supported between pieces of carrot (make a slit about halfway down a 3 cm carrot stick; insert the plant material to be sectioned, hold firmly, wet razor blade and plant material in a petri dish of water, and hold horizontally to section.
3.Use the carrot method to make cross sections of Arabidopsis leaves. Identify epidermal layers, palisade and spongy mesophyll, and veins.
4.Compare your freehand sections with prepared slides of Arabidopsis stem, leaf and root cross sections (shown by the TAs). The tissue on these slides has been chemically fixed, dehydrated, and embedded in Spurr's resin. The sections reveal detailed structure because they are thin (2m thick), but tissue also has been altered by the fixation, dehydration, and sectioning processes.
Locate the Arabidopsis root cross sections on the prepared slides (next to black line). Identify dermal, cortex, and vascular tissue (stele). Locate the endodermal layer and xylem and phloem of the stele. Compare the appearance of these layers in cross section with the GUS-stained cleared seedling roots.
Analysis of locations of cell cycling using a GUS reporter construct
- Obtain cyc1At::GUS Arabidopsis seedlings. They have been stained previously by the TAs.
- Mount the seedlings in lactophenol on a microscope slide, and coverslip. First observe the seedlings under a dissecting microscope and then under a compound microscope. Identify GUS-stained cells. Determine the relative frequency of cells with GUS activity in the root apical meristem, sites of initiation of lateral roots, the shoot apex region, and expanding leaves.
- Repeat with older plant material, i.e. rosette leaf stage and after bolting. Focus on above-ground tissue. Compare main SAM and lateral meristems. Look at IM, flower primordia and floral organ primordia. Compare floral organ primordia of various stages.
TUM Plant Developmental Biology GPI Course January 2008
Lab 2:
CLSM Analysis of GFP Localisation
In Arabidopsis Roots
Visualisation of the Arabidopsis root apex and of GFP-labelled subcellular structures in Arabidopsis cells using confocal laser scanning microscope (CLSM)
Introduction
Confocal laser scanning microscopy (CLSM) represents one of the most significant advances in optical microscopy ever developed. This technique enables visualization deep within both living and fixed cells and tissues and affords the ability to collect sharply defined images of cellular components or of cells as a whole.
A fundamental aspect of confocal microscopy is the use of fluorescent molecules. Fluorescent dyes and fluorescent protein tags, such as GFP, are used to highlight known structures within cells. When excited with light, these molecules emit light at a lower wavelength that can be detected as an image. As a result, the labeled cellular components are visualized. The microscope itself scans precise focal planes to obtain optical sections of a specimen, that is, a 2-dimensional image of that specimen at a particular plane. When a series of these sections (a Z-stack) is obtained, it can be rendered into a 3-dimensional image of that specimen. Thus, confocal microscopy, in conjunction with fluorescent labels, can provide insights into three-dimensional cell and tissue morphology in organisms, as well as subcellular structures within cells. Importantly, confocal microscopy enables live cell imaging, where dynamic processes can be observed such as cell division, chromosome replication and organelle dynamics, or the activities of a particular protein of interest.
In this lab we will demonstrate two common applications of CLSM using Arabidopsis as a model: the three-dimensional visualization of cellular organization in the root apex and the imaging of GFP-tagged subcellular structures within cells. Our imaging instrument is the Olympus FluoView 1000 with an IX81 inverted microscope stand. The primary aim is to gain an appreciation of how cellular structures can be imaged through use of confocal microscopy.
Experimental procedure
1. Obtain Arabidopsis seedlings (supplied by the TAs) that represent wildtype and a selection of transgenic GFP-gene fusion marker lines (Table 1).
Table 1. Transgenic Arabidopsis GFP-fusion marker lines
Plant Line / GFP-fused gene presentLer-0 / none present
pEGAD GFP1 / cytoplasmic GFP
LTi6b1 / SIMIP
ER1 / Q4
GFP::TALIN2 / Mouse TALIN
GFP::MAP43 / MAP4
1. Cutler et al (2000), 2. Kost et al (1998), 3. Marc et al (1998)
2. Using 4-day wild-type seedlings, mount individuals in a drop of FM4-64 dye (4 M solution) on a microscope slide. Stain for 5 minutes, coverslip and image using the confocal microscope, as demonstrated by your TA. You will focus on cells in the root apex and hypocotyls of seedlings.
3. Obtain a Z-stack for a wild-type root specimen. When imaging, take note of parameters such as the laser used, excitation and emission wavelengths of light source, objective (including magnification) and optical zoom. Note the number of optical sections used and the distance at which sections are made. Were any technical difficulties experienced? Using a midsection image obtained, observe the organization of cell types within the root apex. Make an estimate of cell sizes within the meristematic region of the root.
4. With each marker line provided, make identical preparations for GFP imaging. Your TA will help you to visualize cells at higher magnifications. Note down any changes in parameters used for imaging GFP signal, as compared to FM4-64. Obtain images of cells that clearly demonstrate GFP signal for that marker line. Note the appearance of the GFP localization and whether it corresponds to what is expected.
Questions to consider
- What is the purpose of using FM4-64 dye in this experiment? What component of the cell does it stain and how does it help cellular visualization?
- What is GFP and from where is this marker protein derived?
- Describe the individual gene constructs represented in this experiment. What is known about the genes that are fused to the GFP reporter protein? Why are these valuable constructs to have?
- Summarise, with pictures to illustrate, the GFP localization for each transgenic line.
References
General references on plant structure, development and microtechnique
Esau K 1977 Anatomy of Seed Plants. 2nd ed. Wiley
Jurzitza, G 1987 Anatomie der Samenpflanzen. Thieme Verlag
Raven PH, RF Evert, SE Eichhorn 1999 Biology of Plants. 6th ed. Worth.
Ruzin SE 1999 Plant microtechnique and microscopy. Oxford.
Steeves TA, IJ Sussex 1989 Patterns in plant development. 2nd ed. Cambridge.
Strasburger, E. 2002 Lehrbuch der Botanik für Hochschulen. 35nd ed. Heidelberg
Selected references on wildtype Arabidopsis structure and development
Bowman J (ed.) 1994 Arabidopsis: An atlas of morphology and development. Springer-Verlag.
Busse JS and RF Evert 1999 Vascular differentiation and transition in the seedling of Arabidopsis thaliana (Brassicaceae) Int. J. Plant Sci. 160: 241-251.
Dolan LK Janmaat, V Willemsen, P Linstead, S Poethig, K Roberts and B Scheres 1993 Cellular organization of the Arabidopsis thaliana root. Development 119: 71-84.
Pyke KA and RM Leech 1992 Temporal and spatial development of the cells of the expanding first leaf of Arabidopsis thaliana (L.) Heyng. J. Exp. Bot. 42: 1407-1416.
Schultz EA and GW Haughn 1991 LEAFY, a homeotic gene that regulates inflorescence development in Arabidopsis. Plant Cell 3: 771-781.
Zhong, R, Taylor JJ, Y ZH 1997 Disruption of interfasicular fiber differentiation in an Arabidopsis mutant. Plant Cell 9: 2159-2170.
References on cell proliferation and development in Arabidopsis
DeBlock M and D Debrouwer 1992 In situ enzyme histochemistry on plastic-embedded plant material. The development of an artefact-free -glucuronidase assay. Plant J 2: 261-266.
Donnelly PM, D Bonetta, H Tsukaya, RE Dengler and NG Dengler 1999 Cell cycling and cell enlargement in developing leaves of Arabidopsis. Devel. Biol. 215: 407-419.
Doonan J 2001 Social controls on cell proliferation in plants. Curr. Op. Plant Biol. 3:482-487.
Doonan J and P Fobert 1997 Conserved and novel regulators of the plant cell cycle. Curr. Op. in Cell Biol. 9: 824-830.
Ferreira PCG, AS Hemerly, J de Almeida Engler, M Van Montagu, G. Engler and D Inze 1994 Developmental expression of the Arabidopsis cyclin gene cyc1At. Plant Cell 6: 1763-1774.
Hemerly A, J de Almeida Engler, C Bergounioux, M Van Montagu, G Engler, D Inze and P Ferreira 1995 Dominant negative mutants of the Cdc2 kinase gene uncouple cell division from iterative plant development EMBO J 14: 3925-3936.
Hemerly AS, PCG Ferreira M Van Montagu and D Inze 1999 Cell cycle control and plant morphogenesis: is there an essential link? BioEssays 21: 29-37.
References on cell imaging in Arabidopsis
Cutler SR, Ehrhardt DW, Griffitts JS, and Somerville CR. (2000) Random GFP::cDNA fusions enable visualization of subcellular structures in cells of Arabidopsis at a high frequency.
Dolan L, Janmaat K, Willemsen V, Linstead P, Poethig S, Roberts K and Scheres B. (1993) Cellular organization of the Arabidipsis thaliana root. Development. 119: 71-84.
Kost B, Spielhofer, P and Chua NH. (1998) A GFP-mouse talin fusion protein labels plant actin filaments in vivo and visualizes the actin cytoskeleton in growing pollen tubes. Plant J. 16: 393-401.
Marc J, Granger CL, Brincat J, Fisher DD, Kao T, McCubbin, AG and Cyr RJ. (1998) A GFP-MAP4 reporter gene for visualizing cortical microtubule rearrangements in living epidermal cells. Plant Cell 10: 1927-1939.
Prasher, D. C., Eckenrode, V. K., Ward, W. W., Prendergast, F. G. & Cormier, M. J. (1992) Primary structure of the Aequorea victoria green fluorescent protein. Gene 111:229233.
Chalfie, M., Tu, Y., Euskirchen, G., Ward, W. W. & Prasher, D. C. (1994) Green fluorescent protein as a marker for gene expression. Science 263:802805.
Haseloff, J., Siemering, K., Prasher, D. & Hodge, S. (1995) Stable expression of GFP in Arabidopsis. Posting on the bionet.genome.arabidopsis newsgroup, 13th June 1995.
Sheen J, Hwang S, Niwa Y, Kobayashi H and Galbraith DW. (1995) Green-fluorescent protein as a new vital marker in plant cells. Plant J. 8: 777-784.
Websites of interest
Olympus
Ehrhardt Lab
Haseloff Lab
History of GFP
TUM Plant Developmental Biology GPI Course January 2008
Lab 3:
Clonal Analysis and Fate Mapping
Introduction
This laboratory will introduce you to the concept and technique of clonal analysis and fate mapping.
The developmental history of any structure or organ can be traced back through its cell lineages to a founding cell or cell population. It is important to be able to determine the size and identity of the founding cell population in order to understand the influences that shape the development of a structure or organ. To follow the cell lineage, cells needs to be marked. The marker needs to be cell-autonomous and be neutral in a sense that it does not interfer with the behaviour and development of the marked cell(s). To mark single cells at a particular time in development, one can for example irradiate a plant, generating mutations or chromosome breaks at low frequency.The mutations or chromosomes breaks inactivate or remove the marker gene. For example, the loss of both active copies of a pigmentation gene can result in a colorless sector that stands out in the surrounding pigmented tissue. A common approach has been to delete a pigmentation gene, which is present in heterozygous form and located near the tip of a chromosome.