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Gus’ unique selections seeds

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The T-DNA promoter/gus fusion. This represents the T-DNA region of an Agrobacterium tumefaciens vector to make transgenic plants containing promoter-gusA fusions. The T-DNA borders are processed out of the vector and delimit the DNA that will be transferred to the plant cell. The selectable marker (kanamycin-resistance, in this case) is used to select plant cells that contain this T-DNA region. The fusion gene is made of the promoter of interest added to create a translational fusion to the gusA gene and a polyadenylation signal sequence. This fusion gene will express GUS activity in appropriate cell types under the appropriate environmental conditions.

Gus’ unique selections seeds

Susan J. Karcher and Stanton B. Gelvin
Department of Biological Sciences
Purdue University
West Lafayette, IN 47907-1392

We present a series of investigative laboratories using transgenic plants for the introductory biology curriculum. The transgenic plants to be used in these laboratories contain an uidA (gus A) reporter gene under the control of various promoters that respond to different environmental or developmental signals. Following induction by these environmental or developmental signals, the gusA gene will respond by producing the enzyme �-glucuronidase (GUS). When plant tissue is stained with the chromogenic compound X-gluc, those tissues that produce GUS will turn blue. Using investigative experiments, students will monitor both the physiological response of plants to these signals, as well as the induction of gene activity as reflected by GUS activity. Thus, students will be able to see that physiological and developmental responses correlate with specific gene activity. The GUS assay is highly visible, safe for the undergraduate laboratory, easy to conduct, and relatively inexpensive.

A specific system that we have tested in classes at Purdue uses transgenic Arabidopsis plants containing the GUS reporter gene fused to the cor15a gene promoter, which responds to cold stress.

Students can design their own investigations of plant responses to environmental stimuli using transgenic Arabidopsis plants containing the chimeric reporter fusion gene cor15a/gusA. This gene is regulated by a promoter that shows interesting environmental responses which can be assayed by simple color changes.

Plant biology is frequently an area that is neglected in the teaching of biology, yet plants display unique characteristics in their developmental and physiological processes that set them apart from their animal and microbial counterparts. In addition, plants can be easily cultured, manipulated, and stored (as seeds)–which make them ideally suited for undergraduate teaching laboratories.

Plants have developed unique and interesting mechanisms to deal with problems of predation, environmental stresses (such as heat and cold, salinity, heavy metal toxicity, lack of light) and biotic stresses (such as pathogen attack). Some of these mechanisms are very rapid (i.e., the response occurs within seconds or a few minutes of the stimulus) while some responses occur only after many minutes or hours. In some instances, (i.e., pathogen attack) secondary responses can occur in remote portions of the plant not under attack a day or two after the initial inducing insult. These latter responses generally result from changes in gene activity, and lead to the production of proteins not normally made by the plant, or normally made only in small quantities in specific organs. In addition, plants take many of their developmental cues form their environment.

If one has isolated a gene that is induced in response to a specific stimulus; if one has developed an assay for the activity of an induced enzyme, or if one has raised an antibody to an induced protein, one can follow the induction of that specific gene by using RNA or Western blot analysis, in situ RNA or protein visualization (tissue printing), or enzymatic assays for the activity or the specific gene. However, because these techniques and assays are cumbersome and time-consuming, researchers often turn to following “reporter gene” activity to monitor the response of genes to specific stimuli.

A reporter gene encodes an enzyme with an easily assayable activity that is used to report on the transcriptional activity of a gene of interest. Using recombinant DNA methods, the original promoter of the reporter gene is removed and replaced by the promoter of the gene to be studied. The new chimeric gene (Fig. 1) is introduced into an organism and the expression of the gene of interest is monitored by assaying for the reporter gene product. A reporter gene allows the study of expression of a gene for which the gene product is not known or is not easy to identify. To determine the patterns of expression of environmentally or developmentally regulated genes, reporter genes are placed under the transcriptional regulation of promoters that show interesting developmental and/or stress responses. In bacteria, the lacZ gene from E. coli, that encodes ß-galactosidase, as a reporter of gene activity. lacZ can be used as a reporter in bacteria that are naturally lac-, or that are lac- due to a mutation. This gene can also be used in many animal systems that lack endogenous ß-galactosidase activity. Other reporter genes often used in bacteria and animals include cat (encoding the enzyme chloramphenical acetyl transferase), fus (encoding the jellyfish green fluorescent protein), and lux (encoding the enzyme firefly luciferase). Plants contain endogenous ß-galactosidase activity, so lacZ is not generally a useful reporter gene for plants. A widely used reporter gene in plants is the uidA, or gusA, gene that encodes the enzyme ß-glucuronidase (GUS). This enzyme can cleave the chromogenic (color-generating) substrate X-gluc (5-bromo-4-chloro-3-indolyl ß-D-glucuronic acid; Fig. 2), resulting in the production of an insoluble blue color in those plant cells displaying GUS activity. Plant cells themselves do not contain any GUS activity, so the production of a blue color when stained with X-gluc in particular cells indicates the activity of the promoter that drives the transcription of the gusA-chimeric gene in that particular cell. The GUS assay is easy to perform, sensitive, relatively inexpensive, highly reliable, safe, requires no specialized equipment, and is highly visual (Jefferson, 1987; Jefferson and Wilson, 1991; Jefferson et al., 1987).

In order to use GUS as a reporter of promoter activity, a transgenic plant containing the promoter-gusA chimeric gene is made. (A transgenic organism has added foreign DNA.) There are several ways to generate transgenic plants. For many plant species, the easiest method is Agrobacterium-mediated gene transfer. When Agrobacterium infects a host plant, a part of the Ti (tumor-inducing) plasmid of the bacterium is transferred from the bacterium to the plant. The transferred DNA (called the T-DNA) is integrated into the plant nuclear DNA (Binns and Thomashow, 1988; Watson, 1992). The normal T-DNA contains genes that encode plant growth hormones and cause the production of a plant tumor called a crown gall. None of the T-DNA genes are necessary for T-DNA transfer from the bacterium to the plant cell. Other genes (the virulence, or vir genes) on the Ti-plasmid are necessary for transfer. Vir genes are not normally transferred to the plant, but encode proteins needed for the processing of the T-DNA from the Ti-plasmid, proteins that form the channels in bacterial walls through which T-DNA exits to the plant, and proteins that accompany the T-DNA to the plant cell, target it to the nucleus, protect it from nuclease digestion, and perhaps aid in the integration of the T-DNA into the plant chromosome. Any DNA within the T-DNA will be transferred to the plant and integrated into the plant nuclear DNA. Using recombinant DNA methods, the tumor causing genes were deleted from the T-DNA and any DNA of interest can be inserted. In addition to the gene of interest, the gene for a selectable marker is put between the T-DNA borders. This allows the selection of plant cells that have been genetically transformed. Selectable markers used include antibiotic resistance genes and herbicide resistance genes. The modified T-DNA is put back into Agrobacterium and is transferred to the plant by the normal infection process. Intact plants are produced (Fig. 3). Plants are regenerated, self-pollinated, and seed from these plants is germinated on the selective media. Only plants that have been shown to be homozygous for the inserted T-DNA (containing the gusA gene) are used for these laboratory experiments.

Many plants respond to low, nonfreezing temperatures by changing the intracellular concentrations of carbohydrates and free amino acids, their isozyme patterns, and their membrane composition. In addition, there is an alteration in the activity of many genes in response to low temperatures. Dr. Michael Thomashow of Michigan State University studies how plants respond to cold temperatures. He has examined cold-induced genes in the plant Arabidopsis and cloned cor (cold related) genes. The response of one of these genes, cor15a will be used in the laboratory experiments presented in this paper. The cor15a gene is induced by cold temperatures, drought, and the hormone abscisic acid. cor15a is a nuclear gene that encodes a 15 kDa protein (Baker, et al., 1994; Hajela, et al., 1990). The function of this protein is not known. The protein is translocated to the chloroplasts and may have a role in protecting plants from cold. Baker et al. (1994) isolated a DNA fragment containing the promoter region and the first few amino acids from the cor15a gene and joined it, in the correct translational reading frame, to the gusA gene, generating a cor15a-gusA translational fusion that was introduced into Arabidopsis plants using Agrobacterium. This chimeric reporter gene can be used to monitor the response of the cor15a gene in various plant tissues.

In this laboratory investigation, transgenic Arabidopsis plants that contain the cor15a/ GUS fusion gene are used. Students can investigate the expression of the cor gene under different conditions by assaying for GUS activity. Procedures to examine the cold response of the cor gene are given in detail.

The expected results for cold response are that plants from the cold will show blue indicating GUS activity in the leaves, stems, and roots while the control plants will not be blue. If the plants are incubated in X-gluc for more than 16 hours, some light blue staining of the control plants may also occur because the cor15a gene is transcriptionally active to a low level at room temperature.

1. Seeds of transgenic Arabidopsis thaliana plants harboring a cor15a-gusA chimeric gene are germinated in sterile soil at room temperature (approximately 14 hours light-10 hours dark) until the plants are approximately 1 inch tall (2-3 weeks). [Seeds may be obtained from the authors.] Seeds should be covered with just a small amount of soil. Pots should be covered with plastic wrap to keep the soil moist until germination occurs.

Note: The soil should not be allowed to dry out because desiccation stress can induce the expression of the cor15a gene.

2. Response to cold stress. Plants, still in soil, are placed in a plastic bag (so they do not dry out) are refrigerated for 24 to 48 hours.

3. Control plants are kept at room temperature.

4. Plants are placed in X-gluc solution. Plants are gently removed from soil and put in a microfuge tube containing a small volume of X-gluc solution. (Use the smallest volume that will cover the plants.) Plants are left in the X-gluc solution for 2 hours at 37oC or overnight at room temperature.

5. To aid in visualizing the blue staining of the plant tissues, incubate the tissues in several changes of 70% ethanol. Ethanol extracts the chlorophyll.

1 mM X-Gluc (5-bromo-4-choloro-3-indolyl ß-D-glucuronic acid)

[Supplier: Rose Scientific; 4027 97th St.; Edmonton, Alberta; CANADA T6E 5Y5; phone 1-800-661-9288] in 50 mM Na2HPO4, pH 7.0 and 0.1% Triton X-100. Store aliquots of the solution in the dark in a refrigerator or freezer.

Using these transgenic Arabidopsis plants harboring the cor15a/gusA reporter gene, students can carry out investigative studies of their own design. For example, students might devise experiments to determine the shortest exposure to cold that will induce the cor15a gene. They might test the effect of different temperatures, including high temperatures on the expression of the reporter gene. They might examine the effects of drought or other environmental changes or the effects of abscisic acid.

Other GUS reporter gene fusions used in plants are in the scientific literature. A student might study environmental effects on other reporter gene fusions (Gelvin and Karcher, 1996). For example, genes that respond to the phytohormone auxin have been studied by Hagen and Guilfoyle (1985). They identified genes (the SAURs or small auxin up RNAs) that respond very rapidly to auxin (McClure and Guilfoyle, 1987; McClure et al., 1989). Li et al. (1991) and Liu et al. (1994) have used SAUR gene/gusA reporter gene fusions to investigate auxin-stimulated events in transgenic tobacco plants.

We thank Dr. Michael Thomashow for providing the first seeds for the cor15a Arabidopsis transgenic plants and Dr. Burgund Bassuner for maintenance of the seed stocks. This project was funded by National Science Foundation grant #9354721.

Baker, S.S., Wilhelm, K.S., & Thomashow, M.F. (1994). The 5�-region of Arabidopsis thaliana cor15a has cis-acting elements that confer cold-, drought- and ABA- regulated gene expression. Plant Molecular Biology 24: 701-713.

Binns, A.N., & Thomashow, M.R. (1988). Cell biology of Agrobacterium infection and transformation of plants. Annual review of Microbiology 42: 575-606.

Gelvin, S.B., & Karcher, S.J. (1996). Reporter genes and transgenic plants to study reponse to environmental signals. Tested studies for laboratory teaching, Volume 17 (J. C. Glase, Editor). Proceedings of the 17th Workshop/Conference of the Associaton for Biology Laboratory Education (ABLE): 71-83.

Hagen, G., & Guilfoyle, T.J. (1985). Rapid induction of selective transcription by auxin. Molecular and Cellular Biology 5: 1197-1203.

Hajela, R.K., Horvath, D.P., Gilmour, S.J., & Thomashow, M.F. (1990). Molecular cloning and expression of cor (cold-regulated) genes in Arabidopsis thaliana. Plant Physiology 93: 1246-1252.

Jefferson, R.A. (1987). Assaying chimeric genes in plants: The GUS gene fusion system. Plant Molecular Biology Reporter 5: 387-405.

Jefferson, R. A., Kavanagh, T. A., & Bevan, M.W. (1987). GUS fusions: ß-glucuronidase as a sensitive and versatile gene fusion marker in higher plants. EMBO Journal 6: 3901-3907.

Jefferson, R.A., & Wilson, K.J. (1991). The GUS gene fusion system. In S.B. Gelvin and R.A. Schilperoort (eds.) Plant Molecular Biology Manual, Kluwer Academic Publishers (Dordrecht). B14: 1-33.

Li, Y., Hagen, G., & Guilfoyle, T.J. (1991). An auxin-responsive promoter is differentially induced by auxin gradients during tropisms. Plant Cell 3:1167-1175.

Liu, Z.-B., Ulmasov, T., Shi, X., Hagen, G., & Guilfoyle, T.J. (1994). Soybean GH3 promoter contains multiple auxin-inducible elements. Plant Cell 6:645-657.

McClure, B.A., & Guilfoyle, T.J. (1987). Characterization of a class of small auxin-inducible soybean polyadenylated RNAs. Plant Molecular Biology 9:611-623.

McClure, B.A., Hagen, G., Brown, C.S., Gee, M.A., & Guilfoyle, T.J. (1989). Transcription, organization, and sequence of an auxin-regulated gene cluster in soybean. Plant Cell 1:229-239.

Watson, J. D., Gilman, M., Witkowski, J., & Zoller, M. (1992). Recombinant DNA (2nd ed.) New York: W.H. Freeman and Co.

X-Gluc or 5-bromo-4-chloro-3-indolyl-ß-D-glucuronide is cleaved by ß-glucuronidase to produce glucuronic acid and chloro-bromoindigo. The chloro-bromoindigo dimerizes to produce the insoluble blue precipitatie dichloro-dibromoindigo.

The T-DNA promoter/gus fusion. This represents the T-DNA region of an Agrobacterium tumefaciens vector to make transgenic plants containing promoter-gusA fusions. The T-DNA borders are processed out of the vector and delimit the DNA that will be transferred to the plant cell. The selectable marker (kanamycin-resistance, in this case) is used to select plant cells that contain this T-DNA region. The fusion gene is made of the promoter of interest added to create a translational fusion to the gusA gene and a polyadenylation signal sequence. This fusion gene will express GUS activity in appropriate cell types under the appropriate environmental conditions.

Figure 3
T-DNA vector to insert foreign DNA into plants.