Journal of Experimental Botany, Vol. 53, No. 367, pp. 361-370,
February 1, 2002
© 2002 Oxford University Press
In situ staining of activities of enzymes involved in carbohydrate metabolism in plant tissues
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
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Abstract |
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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 glucose-6-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 5'-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 5'-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.
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Introduction |
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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. 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 activated/inactivated by proteolysis or by phosphorylation/dephosphorylation 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-5'-diphospho-glucose dehydrogenase (Altman, 1976; Gahan, 1984, 1991; Gahan et al., 1997; Onyia et al., 1985). In 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-5'-diphosphoglucose pyrophosphorylase (UGPase), adenosine-5'-diphosphoglucose pyrophosphorylase (AGPase), PGM, and phosphoglucose isomerase (PGI), which, as far as is known, have not been localized in plant tissues before.
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Materials and methods |
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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 (Fragariaxananassa) 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 (Machá
ková 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 single-node cuttings from the main stem containing an axillary bud. The cuttings were placed on MS medium (Murashige and Skoog, 1962), containing 1/10 part of standard amount of KNO3 and NH4NO3, supplemented with 5 µM BAP, 8% (w/v) sucrose, and 0.8% (w/v) agar. The pH of the medium was adjusted to 5.8. The explants were incubated in the dark at 20 °C. 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 °C 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 µm 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 °C for 1 h. After fixation, sections were rinsed overnight in water at 4 °C and refreshed at least five times to remove soluble carbohydrates. Sections could be stored at 4 °C 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 °C 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 °C. The sections could be stored at 4 °C in water for at least 1 month without loss of staining.
Specific protocols for enzymes tested:
Uridine-5'-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 HEPES-NaOH 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 µM glucose-1,6-bisphosphate, 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-5'-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 µM glucose-1,6-bisphosphate, 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 (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 µM glucose-1,6-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 µM, and 71 µM, 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)-1,3-propanediol (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 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).
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Results |
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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.
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Before staining and visualization of enzyme activities, the tissue had to be fixed, since all enzymes (except cell-wall 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%.
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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.
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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 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 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 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 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). 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).
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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).
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Discussion |
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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 organ/tissue, 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 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 and/or 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 5'-diphosphoglucose (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 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 mg-1 FW min-1 (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 various plants and organs, yielding information not available via other localization methods, for example, in situ hybridization or immunolocalization.
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Acknowledgments |
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The authors are grateful to Professor I Machá
ková (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. |
Notes |
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3 To whom correspondence should be addressed. Fax +31 317 484740. E-mail: dick.vreugdenhil{at}pph.dpw.wau.nl
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