METHOD FOR DETECTING SHIQUIMIC ACID
FIELD OF THE INVENTION The present invention relates to methods for the detection of shikimic acid and more particularly to methods capable of detecting shikimic acid in plant material.
BACKGROUND OF THE INVENTION Shikimic acid is an important intermediate in the biosynthesis of the aromatic amino acids phenylalanine, tyrosine and tryptophan of D-glucose. The glyphosate herbicide, N- (phosphonomethyl) glycine, inhibits the biosynthesis of aromatic amino acids that ultimately cause the accumulation of shikimic acid in plants. Glyphosate is a potent inhibitor of 5-enolpyruvyl-shikimate-3-phosphate (EPSP) synthase, a key enzyme in the biosynthetic pathway of the aromatic amino acid. Glyphosate initiates the accumulation of shikimate-3-phosphate, the EPSP synthase substrate, which is then hydrolyzed in the plant in shikimate. The detection of plant acid in plants can be used to determine if a plant has been exposed to glyphosate and can also be used to determine if plants are resistant to this herbicide. Gaitonde and Gordon, J. Biol. Chem. Vol, 230, no. 1 p. 1 043-1 050 describes a method for quantifying shiquic acid when a shikimic acid solution is oxidized by periodic acid, sodium hydroxide is used to form a yellow chromophore, and glycine is added to stabilize the color. The optical density of the solution is then measured within ten minutes of addition of sodium hydroxide to the solution. Millican, Methods in Enzymology, vol. 1 7A, p. 352-353, 1 970 discloses a method for detecting shikimic acid when a solution containing shikimic acid is oxidized with periodate, and then treated with arsenite. Thiobarbituric acid is added and the solution turns red. The solution is then extracted with cyclohexanone. The clear upper cyclohexanone phase contains the red color and the optical density of the cyclohexanone phase is then determined. Singh and Shaner, Weed Technolgy, vol. 1 2, p. 527-530, 1 998 discloses a method for detecting shikimic acid in plant tissue when a test sample containing shikimic acid is oxidized with periodic acid. The sample is then mixed with sodium hydroxide, glycine is added and the optical density is read. Siehl, in Herbicide Activitv, Toxicoloqy, Biochemistrv and
Molecular Bioloav. R.M. Roe, et al., Editors, IOS Press, 1 997, p. 37-65, describes a method of analysis for DAHP of enzyme when the enzyme is incubated in the presence of its substrates, phosphoenolpyruvate and erythrose-4-phosphate. The reaction is stopped by the periodic acid addition. Sodium sulfite is then added to reduce the excess periodate. Thiobarbituric acid is then added, followed in ten minutes by dimethyl sulfoxide. The absorbance at 549 nm is then determined.
BRIEF DESCRIPTION OF THE INVENTION The present invention provides a method for detecting shikimic acid in an aqueous solution comprising the steps of a) oxidizing shikimic acid with periodic acid or a reagent comprising periodate or periodic acid (periodic acid and reagent collectively referred to as periodate); b) add a strong base to generate a chromophore; c) add sulfite to stabilize the chromophore; d) detect the presence of the chromophore and optionally e) quantify the amount of the chromophore. The presence of chromophore indicates the presence of shikimic acid in the aqueous solution. The amount of chromophore correlates directly with the amount of shikimic acid in the aqueous solution. In a preferred embodiment of the present invention, steps b and c are carried out at the same time with a mixture comprising sulfite and a strong base so that that reduction of excess periodate and the generation of a chromophore take place at the same time. Applicants have found that the use of periodic acid and a periodate provides a faster reaction time with shikimic acid than with periodic acid alone., especially when the oxidation is carried out at temperatures above room temperature. Applicants have further found that the use of sulfite to reduce periodic acid in excess and periodate greatly improves the stability of the chromophore generated by oxidation of shikimic acid followed by the addition of strong base. The method of the applicants can also be used to detect shikimic acid in tissues, preferably plant tissue. Thus, another aspect of the invention provides a method for the detection of shikimic acid in a test sample comprising the steps of (a) treating the test sample with periodic acid or a reagent comprising periodate and periodic acid; (b) adding a strong base to the test sample to generate a chromophore; (c) stabilizing the chromophore with sulfite; (d) detecting the presence of the chromophore; and optionally (e) quantifying the amount of the chromophore. Preferably, steps b and c are carried out at the same time with a mixture comprising sulfite and a strong base so that the reduction of excess periodate and the generation of a chromophore take place at the same time. Different from some previous methods for detecting shikimic acid, the methods of the applicants can be used directly with tissue extracts. Suitable tissue extracts can be produced without grinding or prolonged reflux of the plant material. In addition, unlike some other prior methods, the applicant's method provides a more stable chromophore so that the detection of the chromophore does not need to occur as soon as possible after the periodic acid oxidation cools. The application of the method of the present invention to direct the use with tissue samples, the ability to delay the detection of the chromophore to at least one hour, and the additional quantification of the chromophore produced make the present invention suitable for high yield selections of tissue samples. These and other aspects of the present invention are set forth in the following detailed description of the invention.
DETAILED DESCRIPTION OF THE INVENTION The shikimic acid is oxidized by periodic acid to give trans-aconitic acid and a dialdehyde. When the excess periodic acid is removed by the addition of sufficient sodium hydroxide to raise the pH above 10, an intense yellow color develops over the duration. Only shichic acid, quinic acid and tryptophan have been found to produce a chromophore that has a yellow color under alkaline conditions, after periodic acid treatment. The formation of the yellow color of shikimic acid, which may be characteristic of the formed dialdehyde, is the basis of several methods for detecting shikimic acid. The chromophore that produces the yellow color is not stable and some methods employ a compound such as glycine to stabilize the color. Applicants have found that sulfite stabilizes the chromophore much better than known methods and provides a method that can be used directly with tissue samples. The present invention provides a method for detecting shikimic acid in an aqueous solution comprising the steps of a) oxidizing shikimic acid with periodic acid or a reagent comprising periodate or periodic acid (collectively referred to as periodate); b) add a strong base to generate a chromophore; c) add sulfite to stabilize the chromophore; d) detect the presence of the chromophore and optionally e) quantify the amount of the chromophore. The presence of chromophore indicates the presence of shikimic acid in the aqueous solution. The amount of chromophore correlates directly with the amount of shikimic acid in the aqueous solution. The aqueous solution may be known to contain shikimic acid, such as when a known amount is added to an aqueous solution to create a standard, or is suspected of containing shikimic acid, such as a test sample. Detection of shikimic acid in plants can be used to determine if a plant has been exposed to glyphosate herbicide and can also be used to determine if plants are resistant to this herbicide. In this manner, the methods of the present invention are useful in situations where the detection and / or quantification of shikimic acid is desired. For example, the methods of the present invention can be used to select plants or plant tissues or cells to determine glyphosate herbicide resistance or to test crop plants to determine whether they have been exposed to glyphosate. The methods of the present invention are conveniently carried out in aqueous solution. The aqueous solution can be water or a mixture of water and organic solvent miscible with water. The methods of the invention are suitable for use with a variety of tissue types including microorganism, animal and plant tissue. Preferably, the tissue is a plant tissue. When the tissue samples are used, it is first necessary to extract the shikimic acid in a suitable solvent, such as a water solution and a water miscible organic solvent and then, if necessary, clarify the resulting solution. Shikimic acid can be extracted from tissues using any suitable method. Applicants have found that shikimic acid can be extracted easily and quantitatively from plant tissue at room temperature by a solution comprised of water and organic solvent miscible with water. Preferably, the plant tissue has been previously frozen below about -4 ° C. The solution used to extract shikimic acid from the tissue may have an acidic or basic pH. The water-miscible organic solvent can be any solvent in which the shikimic acid is soluble, preferably a low molecular weight alcohol, more preferably isopropanol. The tissue is incubated in the extraction solution until all or a sufficient amount of shikimic acid has been extracted. The extraction is usually completed after approximately 24 hours, but it is usually completed after 16 to 24 hours, or even sooner. A preferred solution for extracting shikimic acid is a 2: 1 (volume: volume) mixture of 0.05 M alkali metal hydroxide (in water) and isopropanol. Preferably, the alkali metal hydroxide is sodium hydroxide or potassium hydroxide. In a preferred method for extracting shikimic acid from plant tissue, the frozen tissue below about -4 ° C is cut into small pieces and immersed in a sufficient quantity of a 2: 1 (v: v) mixture of 0.05 M Sodium hydroxide or potassium hydroxide (in water) and isopropanol. Generally, for convenience, 1 mL of extraction mixture is used for each 0.1 gm of plant tissue, but other extraction: tissue mixing ratios are also suitable. The extraction is allowed to proceed at room temperature until the additional shiquímico acid is extracted in solution. Shikimic acid in many plant tissues is completely extracted by this procedure in 1 5-22 hours. The aliquots of the extraction mixture can then be analyzed for the presence of shikimic acid according to the method of the invention without further manipulation, making this process convenient for the analysis of large numbers of samples. For example, tissue extractions can be conducted in small amounts of plant tissue in plastic plates, or the like, of 95 cavities from which the aliquots can be removed with automatic sample handling equipment for the determination of absorbency and thus the amount of shikimic acid in plant samples. The oxidation of shikimic acid can be done with periodic acid, or a reagent comprising periodic acid or periodate (periodate reagent). For ease of reference herein, the periodic acid and reagent comprising periodic acid and periodate will generally be referred to as periodate. Preferably, a reagent comprising periodic acid and periodate in a ratio of 1: 1 is used for oxidation. The reagent can be prepared in water to be 0.5% (weight / volume) of periodic acid and 0.5% (weight / volume) of sodium meta-periodate. The periodate is added to an aqueous solution that contains, or is suspected to contain, shikimic acid. Sufficient periodate is added to the aqueous solution so that there is a substantial molar excess of periodate over known or suspected amounts of shikimic acid in the aqueous solution. The oxidation of shikimic acid can be carried out at room temperature or at elevated temperature. Preferably, the oxidation has to bind at a temperature between 25 and 65 ° C, more preferably between 35 and 50 ° C. Applicants have found that the oxidation of shikimic acid is complete after approximately thirty to forty-five minutes at 37 ° C. When the oxidation takes place at room temperature, the oxidation takes approximately three hours. The oxidation of shikimic acid is stopped or cooled by the addition of a strong base to the aqueous solution that generates a chromophore, and sulfite is added to reduce the excess periodate that stabilizes the chromophore. Preferably, a mixture comprising strong base and sulfite is added to the aqueous solution to cool the reaction and stabilize the chromophore at the same time. However, it is possible to perform this step by adding the freshly prepared sulfite base and solution separately. Preferably, the base is a strong base such as hydroxide, more preferably the base is an alkali metal hydroxide, such as sodium hydroxide or potassium hydroxide, more preferably sodium hydroxide. Sufficient base is added to make the alkaline aqueous solution at a pH above 1 0, preferably above 1 1. Preferably, the base is mixed with water or another solvent, more preferably a mixture of the base and water is used in the methods of the invention. The addition of base and sulfite to the aqueous solution produces a stabilized chromophore, the presence of which can be detected in the aqueous solution until approximately one hour after the addition of base and sulfite. The sulfite is added to the aqueous solution, separately or in a mixture with a strong base, in an amount sufficient to stabilize the chromophore generated by the addition of strong base to the completed periodate oxidation reaction. Sulfite is added to the aqueous solution so that there is at least a lot of sulfite on a molar basis as periodate. Excess sulfite does not interfere with the method of the invention.
Any soluble form of sulfite can be used in the method of the invention, such as the potassium or sodium salt, more preferably the sulfite is sodium sulfite. Preferably, the sulfite is mixed with water or another solvent, more preferably a mixture of sulfite and water is used in the methods of the invention. The presence of chromophore in the aqueous solution, which signals the presence of shikimic acid, can be detected by any suitable method such as measuring the optical density at 382 nm using a spectrometer, or visual inspection of the aqueous solution for a yellow color. Optionally, the amount of chromophore in the test sample can be quantified, thus providing the amount of shikimic acid in the aqueous solution. The quantification can be done by any suitable method, such as comparison with a standard shikimic acid prepared using the method of the present invention with known amounts of shikimic acid. In a preferred embodiment of the invention, the optical density of the aqueous solution is measured at 382 nm using a spectrometer, and the amount of chromophore is determined by comparison with a standard shikimic acid generated using known amounts of shikimic acid and measured at 382 nm using a spectrometer. In carrying out the methods of the invention, for the detection of the chromophore, it is preferable to have an absorbency of at least 0.05 absorbance units (AU) for the untreated control plant test sample and an absorbance of less than 2.0 AU for the test sample with greater amounts of shikimic acid / shikimate in order to obtain optimal results of spectrophotometric measurement of the chromophore. Applicants have found that most plants treated with a lethal amount of glyphosate accumulated shikimic acid / shikimate at a level such that 5-10 μL of a prepared aqueous solution of about 50 mg of base sheet tissue in 8 times ( w / v) of 0.25 N hydrochloric acid could be added to 250 μL of periodate reagent and water to give a volume of 0.5 mL. After the reaction with periodate reagent and cooling by 0.5 ml of a mixture comprising sulfite and sodium hydroxide, there was 1 ml of solution with an absorbance of approximately 1.8 AU, while an untreated control plant gave an absorbance of approximately 0.1 AU. In a preferred embodiment of the invention, the method of the invention is used to determine the presence and / or levels of shikimic acid in an aqueous acidic extract of plant tissue. In this embodiment of the invention a test sample of a tissue, preferably a test sample comprised of an aqueous solution of a plant tissue prepared as described herein, is treated with periodic acid or a reagent comprising periodate and periodic acid to oxidize shiquímico acid. A strong base is added to the test sample to generate a chromophore, and the chromophore is stabilized with sulfite. The presence of the chromophore is then detected, which signals the presence of shikimic acid in the test sample. Optionally, the amount of chromophore in the test sample is quantified to provide the amount of shikimic acid in the test sample. The ability to delay the detection and / or quantification of the chromophore to at least one hour and the ability to use the tissue extracts without further manipulation make the method of the invention very suitable for use in high performance selection and other occasions where large Sample numbers are about to be tested. For example, tissue extractions can be conducted in small amounts of plant tissue in 96-well plastic plates, or the like, whose aliquots can be removed with automatic sample handling equipment for the determination of absorbency and the amount of shikimic acid in The plant samples can be determined by comparing the absorbance with a standard shikimic acid. EXAMPLES Example 1 - Absorbency Background of Reagents 250 μl of water, 250 μl of periodate reagent, 0.3 ml of 1 N NaOH and 0.2 ml of 0.056 M Na2SO3 were mixed together and the absorbance was measured at 382 μm. The absorbance was 0.047 and does not pile up significantly for five minutes. The concentration of 0.056 M sodium sulfite was chosen to be equimolar with the added periodate. The periodate reagent was prepared in water to be 0.5% by weight of periodic acid and 0.5% by weight of sodium mepha periodate. Example 2 - Detection of shikimic acid Ten μl of 2 mM samples of shikimic acid were added to 250 μl of water. Then 250 μl of periodate reagent were added. After 30 minutes at 37 ° C, the reactions were cooled with a) 0.3 ml of 1 N NaOH followed by 0.2 ml of 0.056 M sodium sulfite, or b) 0.5 ml of a combined solution prepared by mixing 0.3 ml of 1 N NaOH and 0.2 ml of 0.056 M sodium sulfite (referred to as the combined solution). The absorbance (A) of the solution at 382 nm was read immediately, and then after 5, 30 and 60 minutes of holding at room temperature. The averaged results of 3 replicates shown in Table 1 demonstrate an increase in absorbance of 382 nm in the presence of added shikimic acid.
Table 1 Addition to Solution A (382) to A (382) to A (382) to A (382) a Analysis T = 0 min T = 5 min T = 30 min T = 60min
NaOH then Na2So3 0.949 0.949 0.941 0.924 combined solution 0.959 0.957 0.953 0.930 The addition of base followed by Na2SO3 provides a chromophore with excellent stability for 30 min and only about 3% loss in absorbency after 60 min while the addition of the combined reagent provides slightly increased sensitivity and equivalent stability. The combined solution is stable at room temperature for approximately 12 hours, but additional testing may show that it is stable for longer periods of time. Preparation of the Calibration Curve - The amount of 2 mM of shikimic acid indicated for each entry in Table 2 was placed in 1.5 ml of micro polypropylene test tubes with water to make 250 μl. Then, 250 μl of periodate reagent are added, and the tubes are maintained at 37 ° C for 30 min, during which time 0.5 ml of the NaOH / sulfite combo reagent were added. The absorbance at 382 nm of each of the resulting solutions was determined within 5 min, and the average of the two replications are shown in column two of Table 2.
Table 2 μl 2 mM of A (382) with A (382) with shikimate water only plant extract 0 0.066 0.095 2 0.21 8 0.266 4 0.409 0.479 6 0.575 0.664 1 0 0.944 1 .028 14 1 .243 1 .320 Lolium perennial It grew on the ground in a greenhouse for 2-3 weeks until the plants reached stage 3-4 of the leaf. The aerial parts of the plants were harvested, and were immediately placed in a freezer maintained at -80 ° C. For the preparation of plant extracts, samples of approximately 1 gm of tissue were tanned and weighed. The tissue was frozen in liquid N2 and ground into a fine powder with a mortar and pestle. Then, a volume of 0.25 N HCl equal to 4 times the weight of the tissue was added, and the pulp was milled for 5 min. plus. This material was centrifuged at 25,000 x g for 1 5 min, and the supernatant was removed by pipette and kept in an amber glass vial. The method used to prepare the calibration curve was repeated, except that 5 μl of the untreated Lolium extract was added to each tube. The results are shown in the third column of Table 2. If these data are plotted as absorbance vs. Shiquimato aggregate, two parallel straight lines are obtained. The data for the shikimate only shows that this analysis procedure gives a direct correlation between the present shikimate and the absorbance at 382 nm, at least up to an absorbance of approximately 1.25. The data for the study, in which the plant extract is added together with the shiquimato, demonstrate both that the untreated perennial Lolium extracts contain a small amount of shiquimato and that the addition of a plant extract does not interfere with this chemical analysis for shiquimato