Journal of Experimental Botany, Vol. 53, No. 367, pp. 361–370, February 2002
In situ staining of activities of enzymes involved in
carbohydrate metabolism in plant tissues
Lidiya I. Sergeeva1,2 and Dick Vreugdenhil1,3
1
Laboratory of Plant Physiology, Graduate School of Experimental Plant Sciences, Wageningen University,
Arboretumlaan 4, 6703 BD Wageningen, The Netherlands
2
Institute of Plant Physiology, Russian Academy of Sciences, Botanicheskaya 35, 127 276, Moscow, Russia
Received 24 September 2001; Accepted 27 September 2001
Abstract
A powerful technique is described to localize the
activities of a range of enzymes in a wide variety of
plant tissues. The method is based on the coupling of
the enzymatic reaction to the reduction of NAD and
subsequent reduction and precipitation of nitroblue
tetrazolium. Enzymes that did not reduce NAD could
be visualized by coupling their activities to glucose6-phosphate dehydrogenase activity via one or more
intermediary ‘coupling’ enzymes. The method is
shown to be applicable for the detection of the activities of hexokinase, fructokinase, sucrose synthase,
uridine 59-diphospho-glucose pyrophosphorylase,
ADP-glucose pyrophosphorylase, phosphoglucomutase, and phosphoglucose isomerase. It could be
used for all tissues tested, including green leaves,
stems, roots, fruits, and seeds. The method is specific, very sensitive, and has a high spatial resolution,
giving information at the cellular and the subcellular
level. The localization of sucrose synthase, invertase,
and uridine 59-diphospho-glucose pyrophosphorylase
in transgenic potato plants, carrying a cytokinin
biosynthesis gene, is studied and compared with
wild-type plants.
Key words: Carbohydrate metabolism, enzymes, in situ
staining, plant tissues.
Introduction
In order to interpret the expression patterns of genes, as
routinely judged from Northern blots, it is becoming
increasingly important to localize the expression of genes.
3
The methods available are in situ hybridization and the
use of reporter genes hooked up to the promoter of the
gene under study. Detection of the mRNA or activity of
the reporter gene does not give unequivocal evidence that
the gene is transcribed into a protein; post-transcriptional
regulation might occur. To detect and localize the protein,
encoded for by the gene, immunocytological methods are
available. However, the presence of a protein does not
prove its activity in situ: it might be activateduinactivated
by proteolysis or by phosphorylationudephosphorylation
or by other regulatory mechanisms. Enzyme activities are
routinely determined in extracts, but by this method no
information on the localization of the enzyme within the
plant is obtained.
Thus, strictly speaking, the demonstration of gene
expression by Northern blots or in situ hybridization,
combined with immunological detection of the protein,
and determination of activity in extracts, does not show
the presence of an active enzyme in a certain tissue or
cell type.
For a range of enzymes, protocols have been described
to assess their activities in situ, for example, the activity
of acid phosphatase can be visualized by the lead salt
method, the azo dye method, or the bromoindoxyl
method, and the activity of alkaline phosphatase can be
localized by the metal salt technique (Gahan, 1984).
Another potent method is the coupling of NAD reduction to the reduction of nitroblue tetrazolium (NBT),
leading to the formation of a blue precipitate. This
method has mostly been applied for various reductases:
tetrazole reductase, glucose-6-phosphate dehydrogenase
(G6PDH), isocitrate dehydrogenase, malate dehydrogenase, succinic acid dehydrogenase, and uridine-59diphospho-glucose dehydrogenase (Altman, 1976; Gahan,
1984, 1991; Gahan et al., 1997; Onyia et al., 1985). In
To whom correspondence should be addressed. Fax q31 317 484740. E-mail: [email protected]
ß Society for Experimental Biology 2002
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Sergeeva and Vreugdenhil
animal tissues it was also applied for the determination of
some other enzymes, for example, hexokinase (HK) and
phosphoglucomutase (PGM) (Meijer, 1967a, b). Recently,
a modification of the NBT-method, involving the coupling of a series of enzyme reactions, was described. In this
way the activity of sucrose synthase (Susy) in developing
maize kernels was visualized, by coupling Susy activity via
a series of ‘helper’ enzymes to the reduction of NBT
(Wittich and Vreugdenhil, 1998). Similar protocols were
described several decades ago, but have never found wide
applications in plant studies, partly because the required
intermediary enzymes were not easily available (Altman,
1976). Tecsi et al. also localized the activities of a
few enzymes using helper enzymes (Tecsi et al., 1996),
but these authors used tissue-prints, which have the
disadvantage of low spatial resolution.
In the present paper it is demonstrated that this histochemical method is widely applicable to localize enzyme
activities in tissue sections from a variety of plants. The
method has been successfully tested for a series of
enzymes, among which are uridine-59-diphosphoglucose
pyrophosphorylase (UGPase), adenosine-59-diphosphoglucose pyrophosphorylase (AGPase), PGM, and phosphoglucose isomerase (PGI), which, as far as is known,
have not been localized in plant tissues before.
Materials and methods
Plant material and growth conditions
The experiments were performed on Gasteria verrucosa (Mill.)
H. Duval leaves, maize (Zea mays L.) leaves, banana (Musa sp.)
leaves, petunia (Petunia hybrida) stem, potato (Solanum
tuberosum L. cv ‘Bintje’) stems, petioles, roots, and tubers,
strawberry (Fragaria 3 ananassa) fruits, arabidopsis (Arabidopsis
thaliana L. Heynh) seedlings, tomato (Lycopersicon esculentum
Mill. cv. Moneymaker) developing fruits and seeds and
germinating seeds.
Plants were grown in a phytotron or in a greenhouse at
standard conditions for the specific plants.
Transgenic potato plants
Two contrasting clones of transgenic potato plants (Solanum
tuberosum cv. Miranda) harbouring the ipt gene from
Agrobacterium tumefaciens, coding for isopentenyltransferase,
the key enzyme of cytokinin biosynthesis, and wild-type plants
were cultivated in vitro as already described in detail
(Macháčková et al., 1997). Leaves, stems, and roots of
23-d-old plants were taken for analysis.
In vitro tuber-induction system
The in vitro tuber-induction system has been described in detail
(Appeldoorn et al., 1997; Hendriks et al., 1991; Vreugdenhil
et al., 1998). In brief: potato (Solanum tuberosum L. cv ‘Bintje’)
plants (maintained in vitro for several years) were grown in vitro,
transferred to soil, and then grown in a phytotron for 3 weeks
under short-day conditions. Each plant delivered 5–6 singlenode cuttings from the main stem containing an axillary bud.
The cuttings were placed on MS medium (Murashige and
Skoog, 1962), containing 1u10 part of standard amount of
KNO3 and NH4NO3, supplemented with 5 mM BAP, 8% (wuv)
sucrose, and 0.8% (wuv) agar. The pH of the medium was
adjusted to 5.8. The explants were incubated in the dark at
20 8C. Developing axillary buds were harvested at 5, 7, 8, and
12 d and fixed for histochemical analysis. After 1 month, tubers
were harvested, the remaining stem piece was removed and the
tubers were kept at 15 8C in darkness for 4–7 months until the
release of dormancy. Dormant and sprouting tubers were used
for histochemical staining as well.
Sectioning and fixation
Sections of 120 or 200 mm thickness were cut with a sledge
microtome. The sections were immediately fixed (first drops of
fixation mixture were applied to the object and to the knife
during sectioning) in 2% paraformaldehyde with 2% polyvinylpyrrolidone 40 and 0.001 M DTT, pH 7.0, at 4 8C for 1 h.
After fixation, sections were rinsed overnight in water at 4 8C
and refreshed at least five times to remove soluble carbohydrates. Sections could be stored at 4 8C up to 5 d without
significant loss of activity.
Staining
General protocol for enzymes tested: To detect the activity of
enzymes, sections were incubated in 1 ml of incubation medium
in 7 ml vials in a water bath at 30 8C for 30 min. In some
experiments the period of incubation was prolonged for 2–3 h.
The duration of incubation depended on the tissues under study
and the stage of plant development. When activities between
tissues or between treatments had to be compared, sections were
always incubated for the same period.
After the incubation period all enzyme reactions were
terminated by rinsing the sections in distilled water. The sections
were stored in distilled water at 4 8C. The sections could be
stored at 4 8C in water for at least 1 month without loss of
staining.
Specific protocols for enzymes tested: Uridine-59-diphosphoglucose pyrophosphorylase (UGPase) activity was visualized
by incubating the tissues in a reaction medium as used for
assaying activity in crude extracts (modified from Appeldoorn
et al., 1997). The reaction mixture contained 100 mM HEPESNaOH buffer (pH 7.5), 1 mM EDTA, 2 mM Mg-acetate,
1 mM NAD, 1 U PGM from rabbit muscle (Boehringer,
Mannheim, Germany), 1 U G6PDH from Leuconostoc
(Boehringer, Mannheim, Germany), 20 mM glucose-1,6bisphosphate, 0.9 mM pyrophosphate (PPi), and 0.03% NBT,
and the reaction was started by adding 5 mM UDPGlc. In
the control reactions UDPGlc, or UDPGlc and PPi, or PPi,
or NAD were omitted.
Adenosine-59-diphosphoglucose pyrophosphorylase (AGPase)
activity was visualized by incubating the tissues in a reaction
medium as used for assaying the activity in crude extracts
(modified from Appeldoorn et al., 1999). The reaction mixture
contained 75 mM HEPES-NaOH (pH 8.0), 0.44 mM EDTA,
5 mM MgCl2, 0.1% BSA, 1 mM NAD, 20 mM glucose-1,6bisphosphate, 2 U PGM, 6 U G6PDH, 2 mM 3-phosphoglycerate, 10 mM NaF, 1.4 mM PPi, and 0.03% NBT, and
the reaction was started by adding 2 mM ADPGlc. In the
control reactions ADPGlc, or ADPGlc and PPi, or PPi, or NAD
were omitted.
Sucrose synthase (Susy) activity was visualized by incubating the tissues in a reaction medium as described earlier
In situ staining of enzymes
(Wittich and Vreugdenhil, 1998) with some modifications. The
incubation medium contained 50 mM HEPES-NaOH buffer
(pH 7.4), 5 mM MgCl2, 1 mM EDTA, 0.1% BSA, 1 mM
EGTA, 1 mM NAD, 1 U PGM, 1 U G6PDH, 20 mM glucose1,6-bisphosphate, 1 U UDPase from beef liver (Boehringer,
Mannheim, Germany), and 0.03% NBT, and the reaction was
started by adding substrate solution (containing sucrose, UDP
and PPi) resulting in final concentration of substrates in the incubation medium of 3.6 mM, 71 mM, and 71 mM, respectively.
Control sections were incubated without sucrose, or without
PGM, glucose-1,6-bisphosphate and PPi, or without NAD.
Invertase activity was determined according the protocol of
Doehlert and Felker (Doehlert and Felker, 1987). The incubation medium contained 38 mM sodium phosphate buffer
(pH 6.0), 25 U glucose oxidase (GOD) (Boehringer, Mannheim,
Germany), 0.024% NBT, 0.014% phenazine methosulphate,
and 1% sucrose. In the control reactions sucrose, or GOD, or
phenazine methosulphate were omitted.
Hexokinase (HK) and fructokinase (FK) activities were
visualized by incubating the tissues in a reaction medium as
used for assaying the activity in crude extracts (modified from
Appeldoorn et al., 1997). The incubation medium contained
50 mM 2-(bis(2-hydroxyethyl)amino)-2-(hydroxymethyl)-1,3propanediol (Bistris) buffer (pH 8.0), 6.25 mM MgCl2, 2.5 mM
ATP, 1 mM NAD, 1 U G6PDH, 1 U PGI (Boehringer,
Mannheim, Germany) (only for FK determination), 12.5 mM
HEPES-KOH (pH 7.4), 0.25 mM EGTA, 0.25 mM EDTA,
0.025% BSA, 0.03% NBT, and 0.5 mM glucose (or 0.5 mM
fructose in the case of FK determination) to start the assay. In
the control reactions glucose (or fructose for FK assay), or ATP
or G6PDH or NAD were omitted. The HEPES-KOH buffer
may be omitted without affecting the staining.
Phosphoglucose isomerase (PGI) and phosphoglucomutase
(PGM) activities were visualized by incubating the tissues in
a reaction medium as used for assaying the activity in crude
extracts (modified from Appeldoorn et al., 1999). The reaction
medium contained 42 mM HEPES-NaOH buffer (pH 7.4),
4.2 mM MgCl2, 0.84 mM EDTA, 0.84 mM EGTA, 0.084%
BSA, 1.4 mM NAD, 1 U G6PDH, 0.03% NBT, and 4.35 mM
Fig. 1. Scheme of reactions for histochemical assay of enzymes.
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fructose-6-phosphate (Fru6P) (or 4.35 mM glucose-1-phosphate
(Glc1P) for PGM determination). In control reactions Fru6P
(or Glc1P for PGM assay), or NAD were omitted.
Microscopy and photographs
The sections were studied with a Leica binocular or a Nikon
Optiphot microscope in bright field mode. Photographs were
taken with a digital Panasonic Colour Video Camera or a Sony
CCD Camera DKR 700.
Effect of fixation on enzyme activities
Tissue sections were fixed and rinsed according to the standard
protocol. Control sections were incubated for 1 h in buffer
(similar to extraction buffer, Appeldoorn et al., 1997), instead
of fixative. Soluble proteins were subsequently extracted from
the tissue and enzyme activities were quantified according to
Appeldoorn et al. (Appeldoorn, 1997, 1999). Enzyme activities
were determined in sections of growing potato tubers (AGPase,
UGPase, PGM, PGI, HK, FK) or in vitro-grown potato
plantlets (Susy, soluble and cell-wall bound acid invertases).
Results
The principle of the visualization of the activities of
enzymes, as used in the present research, is the coupling
of the reduction of NAD to the reduction of NBT,
resulting in precipitation of the blue tetrazolium salt. For
all enzymes described here, this method is indirect; the
product of the reaction to be visualized is used as a
substrate for a series of subsequent steps, eventually
resulting in NAD reduction. The scheme of the reactions
used is shown in Fig. 1.
Before staining and visualization of enzyme activities,
the tissue had to be fixed, since all enzymes (except cell-wall
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bound invertase) are soluble. The effect of fixation on
total enzyme activity was tested for all enzymes by
quantifying activities in extracts from fixed tissues and
control tissues. Table 1 shows that fixation and subsequent rinsing of the tissue resulted in a loss of activity,
ranging from 31% to 69% for the different enzymes,
the average loss being 54%.
The enzymes included in the scheme in Fig. 1 are
involved in carbohydrate metabolism. Therefore, as an
object for initial testing, developing potato tubers were
chosen because they are metabolically very active and
because the activities of various enzymes has been studied
using conventional extraction procedures, followed by
in vitro assays (Appeldoorn et al., 1997, 1999; Ross et al.,
1994; Vreugdenhil et al., 1998).
All enzymes tested could be visualized in growing
potato tubers or potato stems. Figure 2 shows an
overview of the staining patterns in potato tubers for
FK, HK, UGPase, AGPase, PGM, and PGI. Staining for
Susy activities is shown in potato stems in Fig. 3 (E2, E4).
Susy activity could also be detected in swelling stolons
and in tubers (NJG Appeldoorn and LI Sergeeva,
unpublished results). The control incubations, in which
the immediate substrate for the enzymes under investigation was omitted, never showed blue staining for any of
the enzymes. Other controls, in which NAD or one of the
‘helper’ enzymes was omitted, did not show any staining
either (data not shown). Only representative controls for
AGPase and PGM (Fig. 2A2, D3) are shown.
In general, the vascular tissue showed heavier staining
than the other tissues, which in potato tubers mainly
consist of storage parenchyma cells. This pattern cannot
be explained simply by overall higher metabolic activity in
the cells in or surrounding the vascular tissue, since this
Table 1. Effect of fixation on inhibition of enzyme activities,
assayed in sections of growing potato tubers (AGPase, UGPase,
PGM, PGI, HK, FK) or in vitro-grown potato plantlets (Susy,
soluble and cell-wall bound acid invertases)
Tissue sections were fixed and rinsed according to the standard protocol.
Enzymes were extracted, their activities quantified, and compared with
the activities in extracts from similar sections, that had been treated with
buffer instead of fixative. Data are averages of two independent
determinations.
Enzyme
Inhibition
(% of control)
AGPase
UGPase
PGM
PGI
HK
FK
Susy
Soluble acid invertase
Cell-wall bound acid invertase
Average
31
56
64
56
59
69
52
54
42
54
pattern was not observed to the same degree for all the
enzymes (compare AGPase and FK activities, Fig. 2A1,
B1). The method appeared to be sensitive enough to give
information at the level of activity in individual cells as,
for instance, shown for PGM activity: the parenchyma
cells immediately bordering the vascular strands and
lacking starch, showed a much higher activity than the
neighbouring starch-storing parenchyma cells (Fig. 2D2).
Figure 2F2 suggests that even subcellular localization
might be possible, at least for HK activity. The NBT
precipitate accumulated around the nuclei. This pattern is
unlikely to be an artefact, since it was observed when
staining for HK, Susy and AGPase, but not for FK, PGI
or PGM (data not shown).
Having shown that activities of all enzymes tested
could be visualized in potato tuber tissue, whether the
method could also be successfully applied to other tissues
was tested. A wide variety of organs from various plant
species were analysed: roots, stems, hypocotyls, leaves,
fruits, and petioles. Only a limited number of enzymes
and tissues will be shown.
In developing tomato fruits, a clear pattern of activity
of UGPase and AGPase was observed (Fig. 3A1, A2).
Both enzymes exhibited high activities in the young
seeds, the vascular tissue and in the subepidermal layers,
and lower activities in the pericarp parenchyma. The
specificity of the staining method is clear from the different
pattern observed within the seeds: UGPase showed high
activity in different parts of developing seeds whereas
AGPase activity was confined to one part of the
seed, probably the embryo sack. PGM was also active
in tomato fruits, preferentially labelling the vascular
bundles, with much lower activity in the seeds
(Fig. 3A3). The apparent staining in the epidermal layers
was mainly due to endogenous pigments as concluded
from the control (Fig. 3A4).
In cross-sections of petunia stems UGPase activity was
observed in most tissues (Fig. 3B1). The highest activity
was present in the internal and external phloem areas of
the vascular ring, and in the chlorophyll-containing outer
layers of the cortex, as judged from the comparison with
the control (Fig. 3B2). The hairs on the stem also showed
a high activity of UGPase, especially in the apical cells
(Fig. 2B3). This activity was specific for UGPase, since no
activity was observed when stained for the closely related
AGPase activity, even after prolonged incubation
(Fig. 2B4). Other tissues of petunia stems showed activity
in the AGPase assay (data not shown).
In green leaves it was also possible to localize activities,
despite the interference with chlorophyll. The succulent
leaves of Gasteria verrucosa showed high UGPase activity
in the palisade and spongy parenchyma, whereas the
activity was nearly absent in the epidermal cells and
in the central non-green parenchyma (Fig. 3C1, C2). In
maize leaves high Susy activity was localized in the
In situ staining of enzymes
365
Fig. 2. Staining of activity of a variety of enzymes in developing potato tubers. (A) AGPase activity in 12-d-old tubers; (A1) overview of a longitudinal
section showing AGPase activity in most tissues (bar ¼ 2 mm); (A2) no staining in the control reaction without ADPGlc (bar ¼ 2 mm); (A3) detail of
(A1), showing intense staining in starch-storing parenchyma cells (bar ¼ 30 mm). (B) FK activity in developing potato tubers; (B1) overview of a
longitudinal section from 12-d-old tuber showing high activities in most tissues, especially in the vascular bundles (bar ¼ 2 mm); (B2) detail of (B1),
showing the pattern of activity in the apical bud of the tuber (bar ¼ 150 mm); (B3) high FK activity in cells bordering the vascular strand of 7-d-old
potato tuber (bar ¼ 30 mm). (C) UGPase activity in developing potato tubers; (C1) apical bud of 8-d-old potato tuber (bar ¼ 100 mm); (C2) activity in
lenticel of 12-d-old potato tuber (bar ¼ 150 mm); (C3) staining in parenchyma cells of 12-d-old tuber (bar ¼ 30 mm). (D) PGM activity in dormant
potato tuber; (D1) transverse section of 4-month-old, dormant tuber (bar ¼ 3 mm); (D2) detail of (D1), showing PGM activity in parenchyma cells
bordering the vascular strand and lacking starch (bar ¼ 30 mm); (D3) absence of staining in the control reaction without Glc1P (bar ¼ 30 mm). (E) PGI
activity in sprouting 5-month-old tuber. High staining around the sprout and in the vascular strands (bar ¼ 500 mm). (F) HK activity in developing
tubers; (F1) apical bud of 5-d-old tuber. High staining in leaf primordia, central and lateral zones of apex (bar ¼ 100 mm); (F2) HK activity around
nuclei in cells of subapical zone of 5-d-old tuber (bar ¼ 30 mm).
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Sergeeva and Vreugdenhil
Fig. 3. Staining of enzyme activities in various plant organs and tissues. (A) Developing tomato fruit; (A1) high activity of UGPase in most tissues
(bar ¼ 2 mm); (A2) AGPase activity, note lower activity in seed than for UGPase (bar ¼ 2 mm); (A3) PGM activity (bar ¼ 2 mm); (A4) control for
UGPase staining, lacking UDPGlc; note staining by endogenous pigments in epidermis (bar ¼ 2 mm). (B) Petunia stem; (B1) high UGPase staining in
internal and external phloem and in the chlorophyll-containing outer layers of the cortex in cross-section (bar ¼ 500 mm); (B2) no staining in control
reaction without UGPglucose (bar ¼ 500 mm); (B3) UGPase and (B4) AGPase activities in hairs of petunia stems (bars ¼ 30 mm). (C) Gasteria leaf;
(C1), UGPase activity (bar ¼ 500 mm); (C2) control without UGPGlc (bar ¼ 500 mm); (C3) PGM activity in stomatal guard cells of Gasteria leaf
(bar ¼ 30 mm); (C4) control without Glc1P (bar ¼ 30 mm). (D) Maize leaf; (D1) Susy activity (bar ¼ 100 mm); (D2) control without sucrose
(bar ¼ 100 mm). (E) Potato stem; HK staining in stomata of stem (bar ¼ 30 mm).
phloem areas of the vascular bundles (Fig. 3D1, D2), but
was absent in surrounding tissues.
The high resolution of the method, yielding information at the cellular level, is also shown in Fig. 3E:
stomatal guard cells of potato show relatively high
staining of HK, this activity being much lower in the
rest of the epidermis and in parenchyma cells. Note that
small granules of tetrazolium salt are present in the
epidermal cells, suggesting localization of HK in a specific
type of organelle, presumably mitochondria. PGM
In situ staining of enzymes
activity was also high in guard cells, as illustrated in
Fig. 3C3 and C4 for Gasteria leaves.
In stems and roots of plants, or developing seedlings,
enzyme activities could also be visualized, as shown in
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Fig. 4 for potato stems and tomato hypocotyls and roots.
Some activity of FK was observed in the apical region
of axillary bud of potato stems (Fig. 4A1). The activity
of HK in the same tissue was much higher (Fig. 4A2).
Fig. 4. Staining of enzyme activities in potato and tomato tissues. (A) axillary bud of potato cutting before culturing; (A1) weak activity (pink areas) of
FK in central and lateral zones zones of apex (bar ¼ 150 mm); (A2) high activity of HK in same region (bar ¼ 150 mm). (B) HK in vascular bundles and
parenchyma cells of potato stem (bar ¼ 30 mm). (C) Rows of cells with high HK activity in hypocotyl of tomato seedling (bar ¼ 100 mm). (D) Roots of
tomato seedling; (D1) HK staining in root hairs (bar ¼ 150 mm); (D2) no staining in control reaction without glucose (bar ¼ 150 mm); (D3) root tip
with high HK activity (bar ¼ 100 mm). (E) Invertase and Susy activities in wild type and ipt-transgenic potato plants; (E1) invertase activity in wild-type
stem (bar ¼ 150 mm); (E2) invertase activity in stem of ipt-transgenic plant (bar ¼ 150 mm); (E3) Susy activity in wild-type stem (bar ¼ 150 mm); (E4)
Susy activity in stem of ipt-transgenic plant (bar ¼ 150 mm). (F) Enzyme activities in roots of wild type and transgenic potato plants; (F1) UGPase
activity in root of wild type (bar ¼ 150 mm); (F2) UGPase activity in root of ipt-transgenic plant (bar ¼ 150 mm); (F3) Susy activity in root of
ipt-transgenic plant (bar ¼ 150 mm); (F4) Susy activity in root of wild type (bar ¼ 150 mm).
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In potato stems a clear staining of HK was observed in
the companion cells, and some activity was associated
with organelles (presumably mitochondria) in the bordering parenchyma cells (Fig. 4B). In hypocotyls of germinating tomato seeds, a remarkable pattern of HK staining
was observed; some longitudinal files of cells showed
high activity, whereas neighbouring files had a much lower
activity (Fig. 4C). This pattern is unlikely to be a fixation
artefact since it was only observed for HK, and not for
FK, or other enzymes tested. In roots of tomato seedlings
high activity of HK was seen in the root tip (Fig. 4D3),
and in root hairs (Fig. 4D1, D2).
In a previous study on carbohydrate metabolism in
transgenic potato plants, it was found that the presence
of the Agrobacterium tumefaciens ipt gene, influencing
cytokinin synthesis in plants, had a marked effect on the
activities of sucrose hydrolysing enzymes: in stolons of
wild-type plants, sucrose breakdown is dominated by
invertases, except when induced to tuberize. Under these
conditions Susy takes over. One of the ipt-transgenic lines
(clone 1), which never formed tubers, always showed
a high activity of Susy, thus biochemically resembling
tubers, whereas not showing tuber morphology (Sergeeva
et al., 2000). The present localization method was used to
study if Susy activity in the ipt-transgenic lines was
localized in the same tissues as invertase activity in the
wild type. The activity staining in Fig. 4 confirms the
high invertase and low Susy activities in wild-type stems,
whereas the opposite is observed in an ipt-transgenic line
(Fig. 4E1–E4). Invertase activity in wild-type stems was
present in the outer layers of the cortex and in the
vascular tissue, with much lower activities in the inner
cortex and the pith. Susy activity in the ipt-plants was
mostly confined to the vascular tissue, and the pith, and
not in the outer layers of the cortex. In wild-type plants
weak activity of Susy was seen in companion cells (data
not shown).
In roots of the ipt-transgenic potato line, very high
activity of Susy was also observed, especially in the root
tip (Fig. 4F3). The activity in the wild type was much
lower (Fig. 4F4). To check whether this might be due to
an overall difference in metabolic activity, similar roots
were also stained for UGPase activity. For this enzyme no
clear difference in activity was seen (Fig. 4F1, F2).
Discussion
This paper describes a general protocol for in situ staining
of activities of a variety of enzymes, provided the activity
to be analysed can be coupled to the reduction of NAD,
which in turn is coupled to the reduction and precipitation of nitroblue tetrazolium (NBT). The potential of
the method was illustrated with a selection of enzymes
involved in carbohydrate metabolism for a range of
tissues.
The use of NBT to visualize enzyme activity has been
described before (Altman, 1976; Gahan, 1984, 1991;
Gahan and Kalina, 1968). However, so far this method
has been used to localize enzymes that reduce NAD
themselves, whereas in the present study, it has been
demonstrated that enzymes that do not directly use NAD,
can be visualized using one or more additional enzymatic
steps, eventually leading to the reduction of NAD.
Earlier, a similar protocol was used for the staining of
Susy activity in developing maize kernels (Wittich and
Vreugdenhil, 1998).
For the protocol to be effective, the enzyme under
investigation has to be fixed at the site where it is present
in the organutissue, without destroying its activity. The
combination of a mild fixative and immediate fixation by
sectioning in the presence of the fixative appeared to be
successful.
Only the enzyme to be studied is fixed inside the tissue,
whereas substrates and ‘coupling enzymes’, added to the
incubation media, are fully soluble. Hence, the precipitating NBT will only represent the localization of the
enzyme if the whole chain of reaction, following the initial
step, catalysed by the fixed enzyme, is much faster than
the rate of diffusion of intermediary products. The high
spatial resolution observed, and the consistent patterns
of staining in all tissue investigated, shows this to be
the case.
Possible artefacts
Fixation of the tissue might lead to artefacts. The fixative
used is considered to be a mild one, and results in losses of
enzyme activities of 0–61% (Gahan, 1984). The possible
losses of activities due to fixation were calculated by
extracting fixed and non-fixed tissues and determining the
activities in the extracts: after fixation, the activities in the
buffer-soluble fraction were reduced by 31–69%, with an
average inhibition for all enzymes of 54%. It is known
from the literature that even when up to 75% of the
activity is lost, reliable localization studies can be done
(Shnitka and Seligman, 1971).
These findings do not necessarily imply that the
enzymes were inactivated; the activity could partly be
recovered from the buffer-insoluble fraction by subsequent extraction of the pellets with triton-containing
buffer. It is concluded that fixation results in cross-linking
of proteins (as is to be expected), rendering them less
soluble, but not inactive.
From the enzyme stainings it can further be inferred
that the fixation protocol does not lead to artefacts, since:
(i) in axillary buds of potato, relatively low activities of
FK and high activities of HK were detected (Fig. 4A1,
A2). This is in agreement with activities of the same
enzymes as determined in extracts from these tissues
(Appeldoorn et al., 1997); (ii) the activities of Susy and
In situ staining of enzymes
invertase in stems sections of wild type and ipt-transgenic
potato plants (Fig. 4E1–E4) are in accordance with
activities determined in extracts from similar tissues
(Sergeeva et al., 2000).
It might also be possible that the precipitation of NBT
does not reflect the localization of the enzyme, since NBT
might preferentially precipitate in certain cells or tissues,
for example, in cytoplasm-rich cells. If so, it would be
expected that the patterns of activities of different
enzymes would be identical in the same organ. This was
clearly not the case, as can be seen by comparing AGPase
and UGPase activities in petunia hairs (Fig. 3B3, B4).
Side reactions, due to unwanted or non-specific
activities resulting in the reduction of NAD, might also
result in erroneous staining patterns. To check for such
artefacts, controls were run for all enzymes tested, in
which the substrate of the reaction under investigation
was omitted. These blanks were all negative. Moreover,
further controls, in which one of the coupling enzymes
anduor NAD (or other cofactors) were omitted, also did
not show staining (data not shown).
From the relatively high staining in the vascular
tissue it might be argued that the method merely reflects
protein levels in different tissue, since protein levels are
expected to be high in vascular regions. However, the
comparison of wild type and ipt-transgenic potato plants
(Fig. 4E1–E4) makes this unlikely, since the staining
patterns in the vascular tissue are very different, whereas
the protein levels in these plants were found to be similar
(data not shown). Hence it is concluded that the staining
pattern as observed after fixation adequately reflects the
in vivo situation.
Specificity
The staining protocol for all enzymes is based on the
conversion on glucose-6-phosphate (Glc6P) to 6-phosphogluconate and the concomitant reduction of NAD.
The type of substrate and ‘coupling enzymes’ determines
the specificity of the method for each individual enzyme.
For each enzyme studied a specific and consistent pattern
was observed. For instance, the chains of reactions used
to visualize AGPase and UGPase are similar, except for
the first step, using ADP-glucose (ADPGlc) or uridine 59diphosphoglucose (UDPGlc) as substrates, respectively.
Yet completely different staining patterns were observed,
showing the specificity of the protocol. Furthermore, the
reactions catalysed by FK and HK are highly similar, but
the patterns observed are specific for each enzyme tested,
in various tissues (Fig. 4A1, A2).
Sensitivity
As applied in the present paper the method can not be
used for absolute quantitative determinations of activities. In a series of papers, Gahan and co-workers have
369
used NBT staining of sections in a quantitative way
(Gahan, 1991; Gahan et al., 1983; Gahan et al., 1997).
However, this requires on line determination of NBT
precipitation in each section to be analysed, making the
protocol labour intensive and unsuitable for large series
of sections.
Strictly speaking, it is not possible to quantify the
detection limits of the method. However, comparing the
in situ staining method with quantitative data, obtained
from analyses using extracts, gives information about the
relative sensitivity; it has been reported that the activity of
FK in axillary buds of potato stems before culturing (day
0 of the in vitro tuberization system), was very low, i.e.
less than 0.3 nmol substrate converted mg1 FW min1
(or less than 15% of the activity observed in growing
tubers) (Appeldoorn et al., 1997). Figure 4A1 shows that
the staining method did reveal activity well before
culturing (day 0). Thus, for FK, the present protocol is
at least as sensitive as conventional extraction methods.
In cases where the enzyme is only present in a few cells, it
might be even more sensitive, since no dilution by inactive
cells or tissues occurs.
A high spatial resolution was obtained, and in many
instances staining was observed in individual cells, for
example, companion cells in potato tubers (Fig. 1D2) and
stomatal guard cells (Fig. 3C3, E).
Case study on enzyme activities in ipt-transgenic
potato plants
Using the enzyme localization method, the earlier finding
that ipt-transgenic potato plants have largely reduced
levels of invertase and increased levels of Susy activity
(Sergeeva et al., 2000) was confirmed. In addition, it
shows that the tissue localization of these enzymes is
different: Susy is localized in the vascular tissue and in the
pith, whereas invertase activity was found in the outer
layer of the cortex, in the vascular tissue, but not or
hardly in the pith. From this it is concluded that Susy
activity might take over the function of invertase in the
transgenic plants, but with a different tissue localization.
This might be related to the suggested role of Susy in
symplastic unloading, whereas invertase most likely
functions during apoplastic unloading (Helder and
Vreugdenhil, 1999). High Susy activities were also found
in roots of the ipt-transgenic plants, indicating that the
effect of the ipt gene is not confined to tuber-forming
tissues, but rather that it has an overall effect on
carbohydrate metabolism, consistent with the analysis
of sugar levels in these lines (Sergeeva et al., 2000). The
analysis of the activity of a control enzyme (UGPase)
shows that the ipt gene does not cause a non-specific
overall change in enzyme activities.
In summary, the histochemical method described may
be useful in studies on carbohydrate metabolism in
370
Sergeeva and Vreugdenhil
various plants and organs, yielding information not
available via other localization methods, for example,
in situ hybridization or immunolocalization.
Acknowledgements
The authors are grateful to Professor I Macháčková (Prague)
for critically reading the manuscript. We thank Diaan Jamar
and Margo Claassens for their excellent help with the inhibition
studies. Part of this work was funded by the European Union,
project BI04-CT96-0529, and by the Netherlands Ministry of
Agriculture, Nature Management and Fisheries.
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