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Quantication of starch in plant tissues

Alison M Smith
1
& Samuel C Zeeman
2
1
Department of Metabolic Biology, John Innes Centre, Norwich NR4 7UH, United Kingdom,
2
Institute of Plant Sciences, ETH Zurich, CH-8092 Zurich, Switzerland.
Correspondence should be addressed to A.M.S. (alison.smith@bbsrc.ac.uk)
Published online 26 October 2006; doi:10.1038/nprot.2006.232
This protocol describes a simple means of measuring the starch content of plant tissues by solubilizing the starch, converting it
quantitatively to glucose and assaying the glucose. Plant tissue must initially be frozen rapidly to stop metabolism, then extracted
to remove free glucose. Starch is solubilized by heating, then digested to glucose by adding glucan hydrolases. Glucose is assayed
enzymatically. The method is more sensitive and accurate than iodine-based protocols, and is suitable for tissues that have a wide
range of starch contents. Measurements on multiple samples can be completed within a day.
INTRODUCTION
Starch is the main form in which plants store carbohydrate, and is
of importance in the carbon economy of many organs, tissues and
cell types in the plant. It is simple to discover whether or not
signicant amounts of starch are present in a plant tissue, and
relatively straightforward to obtain an accurate measure. Starch
occurs as large (1100 mm), water-insoluble semi-crystalline gran-
ules that can stain brown, blue or black in color with iodine,
depending on the nature of the starch. Treating plant tissues with
iodine solution (Lugols solution or aqueous dilutions thereof)
after pigment removal and dissolution of membranes (usually by
boiling in 80% ethanol) reveals whether signicant amounts of
starch are present
1
. Little quantitative information can be gained
from this approach. The intensity of staining is highly dependent
on tissue structure, starch distribution within the tissue and
individual cells, and starch granule size, shape and composition.
An estimate of starch content can be obtained by solubilizing
starch from the ethanol-treated tissue (for example, by boiling, or
by treatment with 4552% perchloric acid or 90% dimethyl
sulfoxide
2,3
), mixing the soluble extract with iodine solution, and
comparing the OD
620
with those of solutions of known amounts of
starch
4
. This method is only semi-quantitative. It is unsuitable for
tissues with low starch contents, and the reliability of the results
depends on the efciency of starch extraction and solubilization,
the suitability of the standard used and the polymeric composition of
the starch. Given that alternative methods can provide an accurate
measure, we do not recommend iodine-based quantication.
Accurate quantication of starch is necessary to allow rates of
accumulation and degradation to be calculated and compared with
other uxes (for example, rates of carbon dioxide assimilation). It is
relatively straightforward, rapid and inexpensive. Here we describe
a simple protocol that can be applied to most plant tissues. We
discuss the checks and modications necessary to ensure accurate
results and to allow other metabolites to be assayed on the same
tissue samples.
MATERIALS
REAGENTS
.
Liquid nitrogen ! CAUTION Liquid nitrogen can cause severe burns. Wear
protective clothing, gloves and a visor.
.
Dry ice ! CAUTION Dry ice can cause severe burns. Wear protective clothing,
gloves and a visor.
.
80% aq. vol/vol ethanol
.
200 mM Na acetate (pH 4.8)
.
High-quality commercial preparations of a-amyloglucosidase and a-amylase
(Roche; among others)
.
Reagent for enzymatic assay of glucose (Lugols solution (Sigma); see
REAGENT SETUP)
.
Commercial preparations of hexokinase (from yeast) and NAD-glucose
6-phosphate dehydrogenase (from Leuconostoc mesenteroides)
EQUIPMENT
.
Bench-top centrifuge with cooling to 4 1C
.
Microcentrifuge with cooling to 4 1C
.
Water baths at 100 1C and 37 1C
.
Spectrophotometer or microtiter plate reader for OD measurements
at 340 nm
.
Three-digit balance
REAGENT SETUP
Enzymatic assay of glucose Prepare a solution containing 100 mM HEPES
pH 7.5, 0.5 mM ATP, 1 mM NAD and 4 mM MgCl
2
. The solution should be
freshly prepared. Solutions containing mixtures of these compounds should be
freshly prepared. Stock solutions (for example, 100 mM) of ATP, NAD and
MgCl
2
may be stored frozen for several weeks.
PROCEDURE
1| Harvest plant material (0.20.5 g) into capped tubes and freeze immediately in liquid nitrogen. Once tissue is frozen,
tubes can be transferred to dry ice. If starch content is to be expressed on a fresh-weight basis, sample weight can be
determined either by weighing very rapidly prior to freezing, by re-weighing the sample-containing collection tubes, or by
weighing out part of the frozen material for analysis. If starch content is to be expressed on a dry-weight basis, the sample can
be freeze-dried and then weighed. To obtain a robust estimate of the starch content of a particular tissue, it will normally be
necessary to collect and analyze several biological replicates.
PAUSE POINT Samples can be stored at 80 1C for several months at this stage. For measurements on a fresh-weight basis,
sample weight must be determined before long-term storage.


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1342 | VOL.1 NO.3 | 2006 | NATURE PROTOCOLS
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2| Transfer tissue to a tube containing 5 ml 80% aq. vol/vol ethanol and incubate in a boiling water bath for 3 min. Spin
samples in a benchtop centrifuge (Z 3,000 g for 510 min at room temperature (2025 1C)) to pellet solids (note: starch is in
the pellet) and discard supernatant. Repeat this ethanol extraction twice more, discarding the supernatants. Allow ethanol to
evaporate from the nal pellet.
m CRITICAL STEP It is important that no particulate matter is discarded with the supernatant.
3| Transfer pellet to a mortar with a minimum volume of water and homogenize thoroughly to a smooth consistency. Transfer
to a volumetric container and make up to 5 ml with water washings from the mortar. Where large numbers of samples are to be
processed, ethanol-extracted pellets can be homogenized in small volumes of water in a multi-tube ball mill (for example, the
Sample Prep Geno Grinder; SPEX, Metuchen, NJ).
4| Add 0.5 ml of the homogenate to each of four tightly sealing screw-capped microcentrifuge tubes. After sealing, heat to
100 1C for 10 min to gelatinize starch granules. Alternatively, samples can be autoclaved at 120 1C (ref. 5).
m CRITICAL STEP Ensure complete mixing of the homogenate before sampling.
m CRITICAL STEP For each new type of tissue assayed, rst check that the heat treatment completely gelatinizes the starch.
Use light microscopy to examine material stained with Lugols solution before and after heating: no intact starch granules
should remain within tissue fragments after heating.
5| Allow to cool. Add 0.5 ml 200 mM Na acetate (pH 5.5) to each tube. To two of the tubes add B6 units of
a-amyloglucosidase and at least 0.5 units of a-amylase. Add a solution of equivalent volume and composition without the
enzymes to the other two tubes (control samples). Incubate tubes at 37 1C for 4 h.
PAUSE POINT After incubation, samples can be frozen at 20 1C for a few days, or at 80 1C for several months, prior to
glucose measurements.
m CRITICAL STEP Preparations of a-amyloglucosidase and a-amylase must be free of enzymes that degrade glucose-containing
polymers other than starch (e.g., cellulose).
? TROUBLESHOOTING
6| Spin tubes in a microcentrifuge (Z 10,000 g) for 5 min at room temperature to remove particulate material.
7| Assay samples of the supernatant for glucose. Several assays for glucose are available. We recommend an enzymatic
assay in which hexokinase and glucose 6-phosphate dehydrogenase are used to convert glucose to 6-phosphogluconate with
concomitant reduction of NAD to NADH
6
. It is simple, rapid and sensitive, and if NADH production is monitored at 340 nm
on a spectrophotometer it requires no standard curve. If many samples are to be processed, the assay can be adapted for a
microtiter plate reader. In this case we recommend the assay of at least four separate samples from each incubation tube,
and use of a standard curve of glucose (as the light path length is not 1 cm, the extinction coefcient for NADH should
not be used). For spectrophotometric assay, sample volumes of between 0.02 and 0.2 ml are mixed with the assay reagents
and water in a 1-cm-wide cuvette to give a nal volume of 1 ml containing the reagents at the concentrations specied
in MATERIALS.
8| Record OD
340
and leave the cuvette in the spectrophotometer.
9| Place a minimal volume of a mix of 1 unit (i.e., 1 mmol min
1
) hexokinase and 1 unit of glucose 6-phosphate dehydrogen-
ase onto a plastic microspatula and mix rapidly with the contents of the cuvette without removing it from the spectrophot-
ometer. The OD
340
is recorded again after a constant value is reached. This should take no more than 10 min.
m CRITICAL STEP Optical density should be constant with respect to time when the initial and nal ODs are recorded.
m CRITICAL STEP Addition of the enzymes should be in a minimal volume (around 5 ml), and should be done without moving the
cuvette relative to the light beam. These precautions are to minimize OD changes that are due to factors other than NADH
production.
mCRITICAL STEP Changes in OD during the assay must be within the linear range of the assay. If the OD change is large (e.g., Z 1),
check that a further assay with half the volume of the same sample gives half the OD change. If the changes are very small and close
to the limit of detection (e.g., r0.01), increase the sample volume assayed to obtain an accurate reading.
10| Calculate the starch content of the tissue as follows. The amount of glucose in the cuvette (A), in mmol, is calculated as:
Change in OD
6:22
A
where 6.22 is the millimolar extinction coefcient of NADH at 340 nm. Note that this equation is valid only if the lights path
length (i.e., cuvette width) is 1 cm. If the volume in the cuvette is different from 1 ml, this factor must also be multiplied by
the volume in ml.
The mean value of A for control samples (A
c
) is subtracted from the mean value of A for samples incubated with enzymes
(A
s
), to give a net value of A (A
e
).


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NATURE PROTOCOLS | VOL.1 NO.3 | 2006 | 1343
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From this value, the starch content of the tissue in mmol glucose equivalents g
1
fresh weight can be calculated:
A
e
vol of incubation assayed between 0:02 and 0:2 ml
2
5
wt of tissue g
To convert this value to mg starch g
1
(fresh or dry) weight, multiply by 162 (the mass of anhydroglucose). To obtain a robust
estimate of the starch content of a particular organ or tissue, we recommend that measurements are made on at least ve
independent biological replicates.
m CRITICAL STEP The glucose content of control samples is expected to be very low. If it is not, see TROUBLESHOOTING below.
? TROUBLESHOOTING
Troubleshooting advice can be found in Table 1.
ANTICIPATED RESULTS
Starch contents of plant tissues vary enormously with organ or tissue type, species, developmental stage, environmental
conditions and time of day. In a mature leaf of a non-owering Arabidopsis plant, starch content is typically between 5 and
15 mg g
1
fresh weight at the end of the day, and between 0.5 and 1 mg g
1
fresh weight at the end of the night. Starch
content in maturing potato tubers is typically in the range 80 to 150 mg g
1
fresh weight. Between 50 and 80% of the weight
of mature, dry seeds of cereals (wheat, rice, maize, etc.) and pulses (peas and beans) consist of starch.
The protocol is generally suitable for tissue containing 0.1 to 4100 mg starch per g fresh weight. At the top end of this
range, it is advisable to reduce the fraction of the homogenate used in Step 4, and to check rigorously that digestion of starch
to glucose (Step 5) is complete.


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TABLE 1 | Troubleshooting table.
Problem Possible reason Solution
Step 5: Control samples have high glucose
contents.
Failure to remove free glucose from the tissue
through ethanol extraction (Step 2).
Increase the number of ethanol extractions,
and/or the volume of ethanol per extraction.
Fragment the frozen tissue before ethanol
extraction.
Step 10: Results are highly variable between
replicate samples from a single homogenate.
Failure to take representative samples of the
homogenate (Step 4).
Ensure that tissue is completely disrupted
during homogenization.
Mix homogenate thoroughly before taking
samples for incubation.
Step 10: Values for starch content are lower
than expected from published values for the
tissue and growth conditions.
Starch-containing material has been
discarded with the supernatant at Step 2.
Increase time or speed of centrifugations at
Step 2. Ensure that no particulate material is
discarded.
Digestion of starch is not complete. Complete the checks suggested in
Further comments below.
Glucose assay has not gone to completion. Decrease the volume of the sample used
in the assay; check that OD change is
proportional to amount of sample added
over the working range.
Check that the pH of the glucose assay
is optimal (pH 7.9). Addition of large sample
volumes can reduce the pH. Solve by
decreasing sample volume and/or increasing
buffer (HEPES) concentration in the assay.
Published values are incorrect or not
applicable.
Step 10: Values for starch content are higher
than expected from published values for the
tissue and growth conditions.
Preparations of a-amyloglucosidase and/or
a-amylase are contaminated with enzymes
that release glucose from non-starch polymers.
Try alternative commercial sources of
enzymes.
Published values are incorrect or not
applicable.
1344 | VOL.1 NO.3 | 2006 | NATURE PROTOCOLS
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Further comments
This method is appropriate where starch is the only metabolite to be assayed. Soluble metabolites from the tissue sample are
extracted in the ethanolic supernatants produced at Step 2, and can potentially be recovered for assay by pooling the
supernatants, drying them down in vacuo, and redissolving the dry material in a small volume of water. However, this method is
not generally recommended if soluble metabolites are to be assayed. Boiling in ethanol does not inactivate some enzymes
sufciently rapidly to prevent changes in concentrations of soluble metabolites during the killing process (for example,
non-specic phosphatases
7
). Heating can also accelerate the chemical breakdown of some metabolites.
If both starch and soluble metabolites are to be assayed on the same tissue sample, we recommend the use of an extraction
protocol that is specically suitable for soluble metabolites. Starch can readily be assayed when such protocols are used. It
remains in the insoluble fraction when the soluble extract for metabolite analysis is prepared, and it can be recovered and
assayed from a pellet after centrifugation. Typical quenching agents in such protocols are dilute perchloric acid
8
(0.71 M) and
trichloroacetic acid
9
. In both cases, tissue is harvested into liquid nitrogen, powdered while frozen in a mortar or ball mill, then
added to the killing agent at 0 1C. After a short period of incubation, the extract is centrifuged and the supernatant is further
treated before the assay of soluble metabolites. The starch-containing pellet should be washed to remove the quenching agent
by repeated re-suspension in water and centrifugation. It can then be treated as from Step 3 in the PROCEDURE above.
Different types of tissue might require different treatments to ensure that starch is completely digested to glucose. We
recommend that the following test is carried out for each type of tissue to be analysed. Using replicated samples, check
whether doubling any of the following increases the estimated starch content of the tissue: (i) length of homogenization time,
(ii) length of boiling or autoclave time, (iii) length of incubation with a-amylase and a-amyloglucosidase and amounts of
these enzymes. If the estimated starch content is affected by any of the above treatments, the method should be optimized
to ensure that it does not underestimate the starch content.
Several companies offer kits for the determination of starch content. Some of these use the principles of starch hydrolysis
and glucose measurement through hexokinase and glucose 6-phosphate dehydrogenase outlined above. Others use a glucose
assay that is based on glucose oxidase (e.g., from Megazyme). Glucose is oxidized by glucose oxidase with the generation of
hydrogen peroxide. Peroxidase uses the hydrogen peroxide to convert a colorless substance to a colored dye. This method has an
advantage in that it does not require a spectrophotometer with UV capability. However, it does require the preparation of a
standard curve from glucose solutions of known concentration. The Further comments and TROUBLESHOOTING sections of this
protocol should still be considered together with the manufacturers instructions when using kits for starch determination.
ACKNOWLEDGMENTS We thank the many members of our laboratories, past and
present, who have contributed to troubleshooting and streamlining this protocol.
The John Innes Centre is supported by a core strategic grant from the
Biotechnology and Biological Sciences Research Council, UK. S.C.Z. is partly
supported by an EMBO Young Investigators Award.
COMPETING INTERESTS STATEMENT The authors declare that they have no
competing nancial interests.
Published online at http://www.natureprotocols.com
Reprints and permissions information is available online at http://npg.nature.com/
reprintsandpermissions
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(1978).
6. Kunst, A., Draeger, B. & Ziegenhorn, J. in Methods in Enzymatic Analysis 3rd edn.
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