Note: Descriptions are shown in the official language in which they were submitted.
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METHOD OF DETECTING SHIIQIVIIC ACID
FIELD OF THE INVENTION
The present invention relates to methods for 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 from D-glucose.
The herbicide glyphosate, N-(phosphonomethyl) glycine, inhibits the
biosynthesis of
aromatic amino acids which ultimately causes accumulation of shikimic acid in
plants.
Glyphosate is a potent inhibitor of 5-enolpyruvyl-shikimate-3-phosphate (EPSP)
synthase, a
key enzyme in the aromatic amino acid biosynthetic pathway. Glyphosate
initially causes
accumulation of shikimate-3-phosphate, the substrate of EPSP synthase, which
is then
hydrolyzed in the plant to shikimate. Detection of shikimic acid in plants can
be used to
determine whether a plant has been exposed to glyphosate and can also be used
to determine
whether plants are resistant to this herbicide.
Gaitonde and Gordon, J. Biol. Chem., vol. 230, no. 1, p. 1043-1050 discloses a
method for quantifying shikimic acid wherein a solution of shikimic acid is
oxidized by
periodic acid, sodium hydroxide is added 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 adding sodium hydroxide to the solution.
Millican, Methods in Enzymology, vol. 17A, p. 352-353, 1970 discloses a method
of
detecting shikimic acid wherein a solution containing shikimic acid is
oxidized with
periodate, and then is treated with arsenite. Thiobarbituric acid is added and
the solution
takes on a red color. 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 Technology, vol. 12, p. 527-530, 1998 discloses a
method of
detecting shikimic acid in plant tissue wherein a test sample containing
shikimic acid was
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oxidized with periodic acid. The sample was then mixed with sodium hydroxide,
glycine
was added and the optical density was read.
Siehl, in Herbicide Activity: Toxicology, Biochemistry and Molecular Biology,
R.M.
35 Roc et al., editors, IOS Press, 1997, p. 37-65, discloses an assay method
for the enzyme
DAHP wherein the enzyme is incubated in the presence of its substrates
phosphoenolpyruvate, and erythrose-4-phosphate. The reaction is stopped by
addition of
periodic acid. Sodium sulfite is later added to reduce excess periodate.
Thiobarbituric acid
is then added, followed in ten minutes by dimethylsulfoxide. Absorbance at 549
nm is then
40 determined.
SUMMARY OF THE INVENTION
The present invention provides a method of detecting shikimic acid in an
aqueous
solution comprising the steps of a) oxidizing shikimic acid with periodic acid
or a reagent
comprising periodate and periodic acid (periodic acid and the reagent are
referred to
45 collectively herein as periodate); b) adding a strong base to generate a
chromophore; c)
adding sulfite to stabilize the chromophore; d) detecting the presence of the
chromophore;
and optionally e) quantifying the amount of the chromophore. The presence of
the
chromophore signals 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
50 a preferred embodiment of the present invention, steps b and c are
performed at the same
time with a mixture comprising sulfite and a strong base so that reduction of
excess periodate
and generation of a chromophore take place at the same time.
Applicants have discovered that the use of periodic acid and periodate
provides a
more rapid reaction time with shikimic acid than periodic acid alone,
especially when the
55 oxidation is carried out at temperatures above ambient room temperature.
Applicants have additionally found that use of sulfite to reduce excess
periodic acid
and periodate greatly improves the stability of the chromophore generated by
oxidation of
shikimic acid followed by addition of strong base.
Applicants' method can also be used to detect shikimic acid in tissues,
preferably
60 plant tissue. Thus, another aspect of the invention provides a method for
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)
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detecting the presence of the chromophore; and optionally (e) quantifying the
amount of the
65 chromophore. Preferably, steps b and c are performed at the same time with
a mixture
comprising sulfite and a strong base so that reduction of excess periodate and
generation of a
chromophore take place at the same time.
Unlike some prior methods of detecting shikimic acid, Applicant's methods can
be
used directly with tissue extracts. Suitable tissue extracts can be produced
without grinding
70 or prolonged reflux of the plant material. Further, unlike some other prior
methods,
Applicants' method provides a more stable chromophore such that the detection
of the
chromophore need not occur as soon as possible after quenching the periodic
acid oxidation.
The applicability of the method of the present invention to direct use with
tissue samples, the
ability to delay detection of the chromophore for up to at least one hour, and
optional
75 quantification of the chromophore produced make the present invention well
suited for high
throughput screening of tissue samples.
These and other aspects of the present invention are set out in the following
detailed
description of the invention.
DETAILED DESCRIPTION OF THE INVENTION
80 Shikimic acid is oxidized by periodic acid to give trans-aconitic acid and
a
dialdehyde. When excess periodic acid is removed by addition of sufficient
sodium
hydroxide to raise the pH above 10, upon standing an intense yellow color
develops. Only
shikimic acid, quinic acid and tryptophan have been found to produce a
chromophore having
a yellow color under alkaline conditions, after treatment with periodic acid.
The formation
85 of the yellow color from shikimic acid, which may be characteristic of the
dialdehyde
formed, 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
90 samples.
The present invention provides a method of detecting shikimic acid in an
aqueous
solution comprising the steps of a) oxidizing shikimic acid with periodic acid
or a reagent
comprising periodate and periodic acid (referred to collectively herein as
periodate); b)
adding a strong base to generate a chromophore; c) adding sulfite to stabilize
the
95 chromophore; d) detecting the presence of the chromophore; and optionally
e) quantifying
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the amount of the chromophore. The presence of the chromophore signals the
presence of
shikimic acid in the aqueous solution. The amount of chromophore directly
correlates with
the amount of shikimic acid in the aqueous solution. The aqueous solution can
be known to
contain shikimic acid, such as when a known amount is added to an aqueous
solution to
100 create a standard, or suspected to contain shikimic acid, such as a test
sample.
Detection of shikimic acid in plants can be used to determine whether a plant
has
been exposed to the herbicide glyphosate and can also be used to determine
whether plants
are resistant to this herbicide. Thus, the methods of the present invention
are useful in
situations where detection and/or quantification of shikimic acid is desired.
For example,
105 the methods of the present invention can be used for screening plants or
plant tissues or cells
to determine resistance to the herbicide glyphosate, or for testing crop
plants to determine if
they have been exposed to glyphosate.
The methods of the present invention are conveniently performed in aqueous
solution.
The aqueous solution can be water or a mixture of water and a water miscible
organic
110 solvent. The methods of the invention are suitable for use with a variety
of types of tissue,
including plant, animal, and microorganism tissue. Preferably, the tissue is a
plant tissue.
When tissue samples are used, it is first necessary to extract the shikimic
acid into a suitable
solvent, such as a solution of water and a water miscible organic solvent and
then, if
necessary, to clarify the resulting solution.
115 Shikimic acid can be extracted from tissues using any suitable method.
Applicants
have found that shikimic acid can be easily and quantitatively extracted from
plant tissue at
room temperature by a solution comprised of water and a water miscible organic
solvent.
Preferably, the plant tissue has been previously frozen below about -4 C. The
solution used
to extract shikimic acid from the tissue can have a basic or acidic pH. The
water miscible
120 organic solvent can be any solvent in which 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.
Extraction is generally complete after about 24 hours, but is usually complete
after 16 to 24
hours, or even sooner.
125 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.
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In a preferred method for extracting shikimic acid from plant tissue, the
tissue frozen
below about -4 C is cut into small pieces and immersed in a sufficient amount
of a 2:1 (v:v)
130 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 mixture:tissue ratios are also suitable. The
extraction is allowed
to proceed at room temperature until no additional shikimic acid is extracted
into solution.
The shikimic acid in many plant tissues is completely extracted by this
procedure in 16-22
135 hours.
Aliquots of the extraction mixture can then be analyzed for the presence of
shikimic
acid in accordance with the method of the invention without additional
manipulation, making
this process convenient for the analysis of large numbers of samples. For
example, the tissue
extractions can be conducted on small amounts of plant tissue in 96-well, or
similar, plastic
140 plates from which aliquots can be removed with automated sample handling
equipment for
determination of the absorbance and thereby the amount of shikimic acid in the
plant
samples.
Oxidation of the shikimic acid can be done with periodic acid, or a reagent
comprising periodic acid and periodate (periodate reagent). For ease of
reference herein,
145 periodic acid and the reagent comprising periodic acid and periodate will
be referred to
generally as periodate. Preferably, a reagent comprising periodic acid and
periodate in a 1:1
ratio is used for oxidation. The reagent can prepared in water to be 0.5%
(weight/volume)
periodic acid and 0.5% (weight/volume) sodium meta-periodate. The periodate is
added to
an aqueous solution containing, or suspected of containing, shikimic acid.
Sufficient
150 periodate is added to the aqueous solution so there is a substantial molar
excess of periodate
over known or suspected amounts of shikimic acid in the aqueous solution.
Oxidation of the shikimic acid can be performed at ambient room temperature or
at
an elevated temperature. Preferably, the oxidation takes place at a
temperature between 25
and 65 C, more preferably between 35 and 50 C. Applicants have found that
oxidation of
155 the shikimic acid is complete after about thirty to forty-five minutes at
37 C. When the
oxidation takes place at ambient room temperature, the oxidation takes about
three hours.
Oxidation of the shikimic acid is stopped or quenched by addition of a strong
base to
the aqueous solution which generates a chromophore, and sulfite is added to
reduce excess
periodate which stabilizes the chromophore. Preferably, a mixture comprising
the strong
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160 base and sulfite is added to the aqueous solution to quench the reaction
and stabilize the
chromophore at the same time. However, it is possible to accomplish this step
by adding the
base and freshly prepared sulfite solution separately.
Preferably the base is a strong base such as a hydroxide, more preferably the
base is
an alkali metal hydroxide, such as sodium hydroxide or potassium hydroxide,
most
165 preferably sodium hydroxide. Sufficient base is added to make the aqueous
solution alkaline
to a pH above 10, preferably above 11. Preferably, the base is mixed with
water or other
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 for
up to about
170 an hour after the addition of base and sulfite.
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 addition of
strong base to
the completed periodate oxidation reaction. Sulfite is added to the aqueous
solution so there
is at least at much sulfite on a molar basis as periodate. Excess sulfite does
not interfere with
175 the method of the invention. Any soluble form of sulfite can be used in
the method of the
invention, such as the sodium or potassium salt, more preferably the sulfite
is sodium sulfite.
Preferably, the sulfite is mixed with water or other solvent, more preferably
a mixture of
sulfite and water is used in the methods of the invention.
The presence of the chromophore in the aqueous solution, which signals the
presence
180 of shikimic acid, can be detected by any suitable method such as measuring
optical density at
382 nm using a spectrophotometer, or visual inspection of the aqueous solution
for a yellow
color. Optionally, the amount of chromophore in the test sample can be
quantified, thereby
providing the amount of shikimic acid in the aqueous solution. Quantification
can be done
by any suitable method, such as comparison with a shikimic acid standard
prepared using the
185 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 spectrophotometer, and the amount of chromophore determined by
comparison
with a shikimic acid standard generated using known amounts of shikimic acid
and measured
at 382 nm using a spectrophotometer.
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190 In the performance of the methods of the invention, for detection of the
chromophore,
it is preferable to have an absorbance 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 the greatest amount of shikimic acid/shikimate in order to obtain optimal
results from
spectrophotometric measurement of the chromophore. Applicants have found that
most
195 plants treated with a lethal amount of glyphosate accumulated shikimic
acid/shikimate to a
level such that 5-10 L of an aqueous solution prepared from about 150 mg of
leaf tissue
ground in 8-fold (w/v) of 0.25N hydrochloric acid could be added to 250 L of
periodate
reagent and water to give a volume of 0.5 ml. After reaction with the
periodate reagent and
quenching by 0.5 ml of a mixture comprising sulfite and sodium hydroxide,
there was I ml
200 of solution with an absorbance of about 1.8 AU, while an untreated control
plant gave an
absorbance of about 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 acidic aqueous
extract of plant
tissue. In this embodiment of the invention a test sample of a tissue,
preferably a test sample
205 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 shikimic
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
210 chromophore in the test sample is quantified to provide the amount of
shikimic acid in the
test sample.
The ability to delay detection and/or quantification of the chromophore for up
to one
at least one hour and the ability to use tissue extracts without additional
manipulation make
the method of the invention well-suited for use in high throughput screening
and other
215 occasions where large numbers of samples are to be tested.
For example, the tissue extractions can be conducted on small amounts of plant
tissue
in 96-well, or similar, plastic plates from which aliquots can be removed with
automated
sample handling equipment for determination of the absorbance and the amount
of shikimic
acid in the plant samples can be determined by comparison of the absorbance
with a shikimic
220 acid standard.
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EXAMPLES
Example 1 - Background Absorbance of Reagents
250 1 of water, 250 l of periodate reagent, 0.3 ml 1N NaOH and 0.2 ml of
0.056M
Na.rSO3were mixed together and the absorbance was measured at 382 m.
Absorbance was
225 0.047 and did not drift significantly over five minutes. The 0.056M
concentration of sodium
sulfite was chosen to be equimolar with the periodate added.
The periodate reagent was prepared in water to be 0.5% by weight periodic acid
and
0.5% by weight sodium meta-periodate.
Example 2 - Detection of Shikimic acid
230 Ten l samples of 2 mM shikimic acid were added to 240 l of water. Then
250 l
of periodate reagent was added. After 30 minutes at 37 C, the reactions were
quenched with
a) 0.3 ml of 1N NaOH followed by 0.2 ml of 0.056M sodium sulfite, or b) 0.5m1
of a
combined solution prepared by mixing 0.3 ml of 1N NaOH and 0.2 ml of 0.056M
sodium
sulfite (referred to as the combined solution). Absorbance (A) of the solution
at 382 nm was
235 read immediately, and then after 5, 30, and 60 minutes of standing at room
temperature.
Results averaged from 3 replicates shown in Table 1 demonstrate an increase in
382 nm
absorbance in the presence of added shikimic acid.
Table 1
Addition to A(382) at A(382) at A(382) at A(382) at
240 Assay Solution T= 0 min T= 5 min T= 30 min T= 60 min
NaOH then Na. SO3 0.949 0.949 0.941 0.924
combined solution 0.959 0.957 0.953 0.930
245 The addition of base followed by Na2SO3 provides a chromophore with
excellent
stability over 30 min and only about 3% loss in absorbance after 60 min while
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 it is stable for longer periods of time.
250 Preparation of Calibration Curve-The amount of 2 mM shikimic acid
indicated for each
entry in Table 2 was placed in 1.5 ml polypropylene micro test tubes with
water to make 250
l. Then 250 l of periodate reagent was added, and the tubes were maintained
at 37 C for
30 min, at which time 0.5 ml of the combined NaOH/sulfite reagent was added.
The
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absorbance at 382 nm of each of the resulting solutions was determined within
5 min, and the
255 average of two replications is shown in column two of Table 2.
Table 2
l 2mM A(382) with A(382) with
shikimate water only plant extract
260
0 0.066 0.095
2 0.218 0.266
4 0.409 0.479
6 0.575 0.664
265 10 0.944 1.028
14 1.243 1.320
Lolium perrenne was grown in soil in a greenhouse for 2-3 weeks until the
plants
reached the 3-4 leaf stage. The aerial parts of the plants were harvested, and
were
270 immediately placed in a freezer maintained at -80 C. For preparation of
plant extracts,
samples of about 1 gm of tissue were thawed and weighed. The tissue was frozen
in liquid
NZ and ground to a fine powder with a mortar and pestle. Then a volume of 0.25
N HCI
equal to 4 times the weight of tissue was added, and the slurry was ground for
an additional 5
min. This material was centrifuged at 25,000 x g for 15 min, and the
supernatant was
275 removed by pipette and kept in an amber glass vial.
The method us.ed to prepare the calibration curve was repeated except that 5 l
of the
untreated Lolium extract was added to each tube. Results are shown in the
third column of
Table 2. If these data are plotted as absorbance vs. shikimate added, two
parallel straight
lines are obtained. The data for the shikimate only study show that this assay
procedure
280 gives a direct correlation between shikimate present and the absorbance at
382 nm, at least up
to an absorbance of about 1.25. The data for the study in which plant extract
was added
along with the shikimate demonstrates both that extracts of untreated Lolium
perrenne
contain a small amount of shikimate and that addition of a plant extract does
not interfere
with this chemical assay for shikimate.
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