COMPILED AND CIRCULATED BY BANGAMOTI HANSDA, ASSISTANT PROFESSOR, DEPARTMENT OF BOTANY, NARAJOLE RAJ COLLEGE GUS 1. What are the benefits and limitations of the GUS Gene Reporter System in Plants? Gene reporters enable valuable insight into gene expression. The GUS gene reporter system is one of the popular and common plant reporter systems. GUS is short for glucuronidase, an enzyme in the bacterium E. coli. GUS is a good reporter for plants, as it does not occur naturally, and thus, has a low background. With some simple genetic techniques, one can attach the promoter of the gene you want to investigate to the GUS coding region. You can then transform your reporter construct into your plant species of choice to monitor its expression. Transformation can be accomplished in plants via methods like Agrobaterium-mediated gene transfer. The GUS assay does not require the presence of any cofactors or ions for function. Beta- glucuronidase can function through a wide range of pH values, and is fairly resistant to thermal inactivation. However, GUS is susceptible to inhibition from certain heavy metal ions, such as Cu2+ and Zn2+. Additionally, the interpretation of the assay is limited by the movement of diX-indigo throughout the cell. DiX-indigo, can associate with lipids to diffuse far from the site of enzyme activity, which shows a lack of cytosolic localization and irregularity of substrate penetration. This can potentially lead to an incorrect interpretation of GUS protein localization. Despite a lack of cellular localization, nuclear localization of GUS has been well observed. GUS assays can be carried out in the presence of potassium ferricyanide to prevent the stain from diffusing. BOATNY: SEM-VI, PAPER-CC14T: PLANT BIOTECHNOLOGY, UNIT 4: METHODS OF GENE TRANSFER COMPILED AND CIRCULATED BY BANGAMOTI HANSDA, ASSISTANT PROFESSOR, DEPARTMENT OF BOTANY, NARAJOLE RAJ COLLEGE 2. What is GUS? The GUS reporter system (GUS: β glucuronidase) is a reporter gene system, particularly useful in plant molecular biology and microbiology. Several kinds of GUS reporter gene assay are available, depending on the substrate used. The term GUS staining refers to the most common of these, a histochemical technique. 3. What are substrates used for the β-glucuronidase? There are different possible glucuronides that can be used as substrates for the β- glucuronidase, depending on the type of detection needed (histochemical, fluorimetrical, spectrophotometrical). The most common substrate for GUS histochemical staining is 5- bromo-4-chloro-3-indolyl glucuronide (X-Gluc). X-Gluc is hydrolyzed by GUS into the product 5,5'-dibromo-4,4'-dichloro-indigo (diX-indigo). DiX-indigo will appear blue, and can be seen using light microscopy. This process is analogous to hydrolysis of X- gal by Beta-galactosidase to produce blue cells as is commonly practiced in bacterial reporter gene assays. For other types of detection, common substrates are p-nitrophenyl β-D-glucuronide for the spectrophotometric assay and 4-methylumbelliferyl-beta-D-glucuronide (MUG) for the fluorimetric assay. 4. What is the GUS gene assay in transformed tissue? In order to identify transformed cells or plants that have been growing on a selective medium, it is necessary to have an easily assayable reporter gene. The most useful reporter genes encode an enzyme activity not found in the organism being studied. A number of genes currently are being used, however one of the most popular is the E. coli glucuronidase. The protein has a molecular weight of 68,200 and appears to function as a BOATNY: SEM-VI, PAPER-CC14T: PLANT BIOTECHNOLOGY, UNIT 4: METHODS OF GENE TRANSFER COMPILED AND CIRCULATED BY BANGAMOTI HANSDA, ASSISTANT PROFESSOR, DEPARTMENT OF BOTANY, NARAJOLE RAJ COLLEGE tetramer. It is very stable, and will tolerate many detergents, widely varying ionic conditions, and general abuse. It is most active in the presence of thiol reducing agents such as mercaptoethanol or DTT. It may be assayed at any physiological pH, with an optimum between 5.2 and 8.0. The GUS gene is usually used in a gene fusion. This means that the GUS coding sequence is under the direction of the controlling sequence of another gene. For this exercise the GUS gene is under the control of the Cauliflower Mosaic Virus 35S promotor. The GUS gene was developed initially as a gene fusion marker in E. coli and in the nematode C. elegans, but has more recently been used extensively to monitor chimeric gene expression in plants. There is little or no detectable b-glucuronidase activity of yeast, Drosophila, C. elegans, Dictyostelium, or in almost any higher plant. Agrobacterium containing some of the GUS plasmids show significant GUS activity. This seems to be due to in part read-through transcription from the gene into which the GUS coding region might be located. Agrobacterium without these constructs shows little if any detectable GUS activity. In order to solve this problem, one laboratory has constructed GUS genes carrying an intron, which much be processed before expression takes place. This totally eliminates expression in any untransformed system. Histochemical assay The best substrate currently available for histochemical localization of b-glucuronidase activity in tissues and cells is 5-bromo-4-chloro-3-indolyl glucuronide (X-Gluc). The substrate works very well, giving a blue precipitate at the site of enzyme activity. There are numerous variables that affect the quality of the histochemical localization, including all aspects of tissue preparation and fixation as well as the reaction itself. BOATNY: SEM-VI, PAPER-CC14T: PLANT BIOTECHNOLOGY, UNIT 4: METHODS OF GENE TRANSFER COMPILED AND CIRCULATED BY BANGAMOTI HANSDA, ASSISTANT PROFESSOR, DEPARTMENT OF BOTANY, NARAJOLE RAJ COLLEGE It is necessary to understand the nature of the reaction to better eliminate the variables. The product of glucuronidase action on X-Gluc is not colored. Instead, the indoxyl derivative produced must undergo an oxidative dimerization to form the insoluble and highly colored indigo dye. This dimerization is stimulated by atmospheric oxygen, and can be enhanced by using an oxidation catalyst such as a K+ ferricyanide/ferrocyanide mixture. Without a catalyst, the results are often very good, but one must be concerned about the possibility that localized peroxidases may enhance the apparent localization of glucuronidase. Fixation conditions will vary with the tissue, and its permeability to the fixative. Glutaraldehyde which can be used, does not easily penetrate leaf cuticle, but is immediately available to stem cross sections. Formaldehyde seems to be a more gentle fixative than glutaraldehyde, and can be used for longer times. Whole tissues, callus, suspension culture cells and protoplasts or whole plants or plant organs, can be stained, but survival of the stained cells is not by means certain. After staining, clearing the tissue with 70% ethanol seems to improve contrast in many cases. Fluorimetric assay Although spectrophotometric substrates for GUS are available, GUS activity in solution is usually measured with the fluorometric substrate 4-methylumbelliferyl-b-D- glucuronide (MUG). Fluorometry is preferred over spectrophotometry because of its greatly increased sensitivity and wide dynamic range. The assay is highly reliable and simple to use. Occasionally, endogenous compounds will interfere with the assay, either by quenching or by producing high background fluorescence. In these situations, BOATNY: SEM-VI, PAPER-CC14T: PLANT BIOTECHNOLOGY, UNIT 4: METHODS OF GENE TRANSFER COMPILED AND CIRCULATED BY BANGAMOTI HANSDA, ASSISTANT PROFESSOR, DEPARTMENT OF BOTANY, NARAJOLE RAJ COLLEGE fluorometric substrates with differing excitation and emission wavelengths are available (the most popular is resorufin b-D-glucuronic acid). The substrate 4- trifluoromethylumbelliferyl b-D-glucuronic acid (4-TFMUG) allows continuous monitoring of GUS activity because, unlike MUG, it becomes fluorescent upon hydrolysis at the assay pH. In contrast, after hydrolysis of MUG by GUS, the reaction first must be terminated with basic solution. This not only stops the enzyme reaction, but also causes fluoresce. Spectrophotometric assay A continuous spectrophotometric assay has been developed for detecting β-glucuronidase activity. In the assay, Para-nitrophenyl β-D-glucuronide is cleaved to yield a chromophoric product. With the commercial E. coli enzyme, it is demonstrated that the reactions can be continuously monitored by the increase of absorbance at 405 nm. The method is highly sensitive and able to detect less than 1.4 × 10-4 U/mL of the enzyme activity in solution. Such a new assay offers significant advantages over the existing discontinuous methods and should be useful for both routine enzyme assay and accurate kinetic studies. 5. What are the target organisms suitable for GUS gene assay? An organism is suitable for a GUS assay if it lacks naturally occurring β-glucuronidase activity or if the activity is very low (background activity). For this reason, the assay is not useful in most vertebrates and many molluscs. Since there is no detectable GUS activity in higher plants, mosses, algae, ferns, fungi and most bacteria, the assay is ideally suited for gene expression studies in these organisms, and considered the reporter gene of choice for in plant science. BOATNY: SEM-VI, PAPER-CC14T: PLANT BIOTECHNOLOGY, UNIT 4: METHODS OF GENE TRANSFER COMPILED AND CIRCULATED BY BANGAMOTI HANSDA, ASSISTANT PROFESSOR, DEPARTMENT OF BOTANY, NARAJOLE RAJ COLLEGE Left: light grown and right: dark grown tobacco seedlings expressing the GUS gene driven by the PAL1 promotor. Work of Dr. Tomoko Fugisaka Akada References: 1. https://bitesizebio.com/33226/benefits-gus-gene-reporter-system-plants/ 2. https://www.cas.miamioh.edu/~meicenrd/anatomy/Ch2_Ultrastructure/GUS_assay.html 3. https://www.future-science.com/doi/pdf/10.2144/01304rr02 4. https://en.wikipedia.org/wiki/GUS_reporter_system (All the information is collected from above references and will be used only for teaching and learning purposes) BOATNY: SEM-VI, PAPER-CC14T: PLANT BIOTECHNOLOGY, UNIT 4: METHODS OF GENE TRANSFER .