JXB Advance Access originally published online on March 29, 2005
Journal of Experimental Botany 2005 56(415):1351-1357; doi:10.1093/jxb/eri136
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RESEARCH PAPER |
Localization of aluminium in the maize root apex: can morin detect cell wall-bound aluminium?
Institute of Plant Nutrition, University of Hannover, Herrenhaeuser Str. 2, D-30419 Hannover, Germany
* To whom correspondence should be addressed. Fax: +49 511 7623611. E-mail: horst{at}pflern.uni-hannover.de
Received 28 October 2004; Accepted 14 February 2005
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Abstract |
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Morin is a fluorochrome which forms a fluorescent complex with aluminium (Al) and is thus used to localize Al in plant tissues. However, reports about the cellular distribution of Al—apoplastic versus symplastic—based on morin staining are often conflicting. The objective of this work was to investigate whether Al localization with morin staining can show the proper cellular distribution of Al. Fresh root cross-sections were made from root apices of maize (cv. Lixis) treated with 25 µM Al for 6 h and stained with morin. Fluorescence microscopic investigation showed Al–morin fluorescence in the cytosol, but not in the cell wall. This is in contrast to the growing evidence which shows that Al mainly accumulates in the cell wall, especially bound to the pectin matrix. Therefore, in vitro analyses were carried out to study whether morin can form a fluorescent complex with Al, which is bound to pectin, cell wall, and other Al-binding ligands such as phosphate, galacturonate, DNA, and ATP. Compared with the control treatment without Al-binding ligands, fluorescence intensity was reduced by about 10-fold in the presence of pectin and isolated cell walls, but fairly unaffected in the presence of phosphate and galacturonate. Al associated with DNA and ATP also formed a fluorescent complex with morin. This implies that, although Al is mainly accumulated in the cell wall, it cannot be detected with morin as it is tightly bound to cell-wall pectin. Thus, morin staining should not be used to study the distribution of Al between cell compartments.
Key words: Al localization, cell wall, cytosol, fresh root cross-section, morin
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Introduction |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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Aluminium (Al) phytotoxicity is a major threat to plant growth on acid soils (Taylor, 1988
). The most commonly observable symptom of Al injury is inhibition of root elongation, which can be recognized within 1 h of exposure to Al (Llugany et al., 1995). There is wide genetic diversity between plant species for Al resistance. Al-sensitive genotypes usually accumulate more Al in the root tissue than Al-resistant ones. For example, Al-sensitive maize cultivars had higher Al contents in the root tip than Al-resistant cultivars (Collet et al., 2002). Similar observations were obtained for wheat (Tice et al., 1992), soybean (Silva et al., 2000), and Arabidopsis (Larsen et al., 1998).
Several methods can be used to assess the uptake and accumulation of Al in root tissue. One of these involves the use of Al-specific dyes. Staining techniques are relatively simple and rapid tools for examining Al accumulation in plant roots. Cancado et al. (1999) used haematoxylin staining as a phenotypic index of selection for Al resistance in maize. Aniol (1983) used eriochrome cyanine R to assess Al uptake of winter wheat varieties. Other chromophores such as aluminon and solochrome azurin were also used to detect Al distribution in biological samples (Denton et al., 1984). However, the low sensitivity and poor spatial resolution of these staining techniques did not allow them to be used as tools for studying the cellular distribution of Al. Fluorophores such as morin and lumogallion are highly sensitive and can detect very low concentrations of Al (Eggert, 1970; Kataoka et al., 1997).
Morin is a pentaprotic acid that forms a highly fluorescent complex with Al. The Al–morin complex has excitation and emission wavelengths of 420 nm and 515 nm, respectively (Browne et al., 1990). Its fluorescence detection limit is as low as 2x10–9 M (Lian et al., 2003b) and thus morin is used along with fluorescence microscopy to sensitively localize Al in plant cells.
Several authors used morin to study the cellular distribution of Al. However, results are conflicting with regard to the major cellular site of Al accumulation. Ahn et al. (2002) observed Al–morin fluorescence in the cell wall of squash root apices after 3 h of Al treatment, whereas Vitorello and Haug (1996) did not see any fluorescence in the cell wall of cultured tobacco cells. They observed Al–morin fluorescence in the cytoplasm in a discrete zone of the cell periphery. Similarly, Tice et al. (1992) observed Al–morin fluorescence, particularly in the cytoplasm and the nucleus and less in the cell wall of wheat root tips. They concluded that the symplastic Al fraction accounted for 60–70% of total cellular Al while the remaining 30–40% represented apoplastic Al.
However, this is in clear contrast to the growing evidence which shows that symplastic Al is many-fold lower than apoplastic Al. Marienfeld et al. (2000) measured a higher concentration of Al in the cell wall of maize and bean root tips using laser microprobe mass analysis. They attributed the differences in cellular localization and tissue distribution of Al to differences in cell-wall pectin content of the plant species. In agreement with this, an increase in cell-wall pectin content resulted in a higher accumulation of Al in maize suspension cells (Schmohl and Horst, 2000). Furthermore, a decrease in the degree of esterification of cell-wall pectin enhanced Al accumulation (Schmohl et al., 2000). Using a fractionated extraction, Wang et al. (2004) measured about 85% of the total Al in the cell wall of maize root tips. Taylor et al. (2000) determined even much higher (>99%) accumulation of Al in the cell wall of the giant alga Chara corallina after physically separating the cell wall from the protoplast.
The objectives of the present study were to investigate the cellular localization of Al in maize root apex using morin staining, and to determine whether morin can form a fluorescent complex with pectin-bound Al.
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Materials and methods |
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Root sectioning, staining, and microscopy
Maize (cv. Lixis) seeds were germinated in rolls of wet filter paper. Several rolls, each having about 10 seeds, were placed in a glass beaker containing a small amount of tap water and placed in a dark chamber at a temperature of 30 °C. After germination, the seedlings were exposed to light for 1 d before the treatment. Either the whole root system or only root cross-sections were treated with Al. To treat the intact root system, the seedlings were transplanted into a continuously aerated solution containing 0.5 mM CaCl2 with or without 25 µM Al as AlCl3 at pH 4.3 for 6 h. Root tips were excised, inserted in wet styrofoam and thin cross-sections (a few cell layers) were made from the 1–3 mm zone of the root apex. Free-hand sectioning, without fixation and embedding, was employed in order to reduce artefacts related to cellular redistribution of Al. The sections were made with sharp razor blades (Wilkinson Sword GmbH, Solingen, Germany). The blade was dipped in water before cutting so that the sections would remain on the blade after sectioning. The sections were carefully removed with a paintbrush and collected in Petri dishes containing 0.5 mM CaCl2 solution. After collecting sufficient root sections, they were transferred to staining tubes which had a nylon mesh at the base to facilitate washing. The sections were rinsed with double-deionized water and stained with a 100 µM aqueous solution of morin (C15H10O7) for 30 min. Then they were washed twice with double-deionized water for 5 min each. Slides were prepared, mounting the sections in distilled water, and examined with an Axioscope microscope (Zeiss, Axioscope, Jena Germany) equipped with epifluorescence illumination (Mercury lamp, HBO 50 W). The filter set used to observe Al–morin fluorescence consisted of a band pass filter BP 395–440 nm (exciter), a beam splitter FT 510 nm, and a long-wave pass filter LP 515 nm (emitter) since Al–morin has excitation and emission wavelengths of 420 nm and 515 nm, respectively (Browne et al., 1990
). Pictures were taken with a digital camera (Sony Cyber-Shot, DSC-S85, Japan) mounted on the microscope.
Treating root cross-sections with Al
Free-hand sections of maize cv. Lixis root tips were made as described above but from plants not treated with Al. The sections were treated with 0 nM, 10 nM, 100 nM, and 37 µM Al for 5 min in order to study the sensitivity of morin staining. In another experiment, the effect of membrane damage on Al-associated but unspecific fluorescence was studied. For this purpose, root sections were treated with either Al (0 nM, 10 nM 100 nM, and 10 µM) or digitonin (10 and 100 µM) for 30 min. Staining and microscopy was carried out as explained above.
Fluorometry
The fluorescence of Al bound to pectin, plant cell walls, and other Al-binding ligands was investigated using morin reagent according to Browne et al. (1990). Two citrus pectins differing in degree of methyl esterification were purchased from Sigma, Steinheim, Germany. The degrees of esterification were 92% and 28.5% while galacturonic acid contents were 82% and 65%, respectively, for the two pectins. A solution resulting in 100 mg l–1 galacturonic acid was prepared from both pectins. In addition, solutions of pure galacturonic acid (100 mg l–1), KH2PO4 (30 µM) and control (only double-deionized water) were prepared. To each of the above solutions, Al was added to a final concentration of 1 µM in the assay. The pH of the solutions was adjusted to 4.8 using 0.1 N HCl/NaOH and left to equilibrate for 1 h at room temperature. Samples were taken from the solutions and filtered through 0.025 µm membrane filters (Schleicher & Schuell, Dassel, Germany) on a Millipore filtration unit (Millipore GmbH, Germany). Samples of 25 ml were taken from both the filtered and the unfiltered solutions. Then 7.5 µl of 33.3 mM morin dissolved in dimethyl sulphoxide (DMSO) was added to make up 10 µM morin in the assay. The samples were vortexed and kept in the dark for 15 min. Finally, a 2 ml sample was transferred to a microcuvette and Al–morin fluorescence was measured with a fluorescence spectrophotometer (F 2000; Hitachi Ltd, Tokyo, Japan) at the optimized (compared with Browne et al., 1990) excitation and emission wavelengths of 418 and 502 nm, respectively.
The formation of Al–morin fluorescence in the presence of the symplastic ligands DNA and ATP was tested at pH 7.5 typical for the cytosol. Al (200 nM) was added to 0.1% DNA (herring sperm DNA; Sigma) solution. Similarly, 1 µM Al was added to 0.5 mM ATP (Sigma) solution. After 1 h of equilibration at room temperature, morin reagent was applied and the fluorescence was measured as explained above.
A similar assay was performed using cell-wall material extracted from the maize root apex. Root tips (1 cm) were excised from maize (cv. Lixis) seedlings treated with or without 25 µM Al for 12 h and cell-wall material was prepared according to Schmohl and Horst (2000). Dried cell-wall material (4 mg) was suspended in 2 ml of 1 µM Al solution and was shaken on a rotary shaker (Landgraf Laborsysteme, Germany) for 1 h. The pH was adjusted to 4.8, and 4 µl of 5 mM morin in DMSO solution was added to the suspension to make up 10 µM morin in the assay. Next, the samples were shaken for 15 min, centrifuged at 23 000 g, the supernatant was collected and the fluorescence was determined.
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Results |
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There was bright green morin fluorescence in the root sections of Al-treated plants but there was hardly any fluorescence in the control (–Al) (Fig. 1). During the 6 h treatment, Al reached the endodermis but was detected neither in living nor in non-living cells in the stele, which could indicate that the radial transport of Al was restricted by the endodermis. Epidermal, cortical, and endodermal cells were heavily stained (Fig. 1B). The absence of fluorescence in the stele showed that cross-contamination during root sectioning and staining operations was minimal.
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In thin root sections, more intense fluorescence was observed in the cytosol than in the cell wall (Fig. 1). The brightest fluorescence in the cytosol appeared in the nucleus. In cells where the cytoplasmic contents were lost through cutting, there was apparently no fluorescence (see the arrows in Fig. 1C and compare with the fluorescent image in Fig. 1B). This can be visualized from an ultra-thin longitudinal section of the epidermis (Fig. 2). From the bright light image, the cell wall was clearly seen; however, there is virtually no fluorescence in the cell wall. Fluorescence can be observed only in the cells with cytoplasm.
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In order to test the sensitivity of morin for Al staining, root cross-sections from plants not treated with Al were exposed to Al from nanomolar to micromolar concentrations for 5 min only and then stained with morin. Fluorescence was observed again only in the cytosol of the root cells exposed to Al for a short time (Fig. 3D) similar to Fig. 1, where the whole root was treated with Al for a much longer time. There was low but distinct fluorescence in sections exposed to nanomolar concentrations of Al (Fig. 3B, C) versus the control (Fig. 3A).
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There is hardly any doubt that fluorescence was associated with Al treatment (Figs 1–3
), but there is uncertainty whether the fluorescence in the cytosol originated from Al–morin or other cation–morin complexes since some metallic cations such as Br, Mg, and Zn may also form fluorescent complexes with morin (Lian et al., 2003a). This led to doubts that the fluorescence in the cytosol might come not from Al–morin but from complexes of morin with other cations, and that Al may merely disrupt the plasma membrane and open a gateway for high morin permeation into the cytosol. In order to clarify this, thin cross-sections of the maize root apex were treated with digitonin, which is reported to permeabilize the plasma membrane (Tsay et al., 1999), and then stained with morin to compare sections treated with or without Al.
The anticipated membrane disruption through digitonin treatment did not result in fluorescence different from the control (data not shown). Distinct fluorescence was observed only in Al-treated sections. This indicates that the fluorescence in the cytoplasm resulted specifically from the Al–morin complex. Similar to the above observations (Figs 1–3), bright Al–morin fluorescence was mainly localized in the symplast. This observation leads to the formulation of two possible hypotheses: (i) Al is mainly accumulated in the symplast but not in the cell wall; (ii) Al may accumulate in the cell wall but cannot be detected with morin. Further elucidation of these hypotheses is presented below.
In the cell wall, and particularly in the symplast, the activity of free Al3+ is rather low. It can be assumed that most Al is bound to ligands. Thus the formation of the Al–morin complex was studied in vitro in the presence of Al-binding ligands such as galacturonate, phosphate, and pectin, likely Al-binding compounds in the cell apoplast, and DNA and ATP, likely Al-binding compounds in the cell symplast (Crawford et al., 1998; Chang et al., 1999; Schmohl and Horst, 2000; Zhang et al., 2002). Phosphate and galacturonate did not have a significant influence on Al–morin fluorescence in non-filtered samples (Fig. 4). Filtered samples generally had lower fluorescence compared with non-filtered samples, indicating that a larger proportion of the added Al was precipitated even in the control (deionized distilled water) samples at pH 4.8 used in this experiment. It was evident that morin could form complexes with freshly precipitated Al [Al(OH)3, AlPO4, Al-galacturonate] but not with pectin-bound Al. It appeared that Al–pectin is more stable than the Al–morin complex. The formation of the fluorescent Al–morin complex was greatly reduced in the presence of pectin, particularly of the pectin with a low degree of esterification (DE 30%) compared with the pectin with a high degree of esterification (DE 90%) regardless of the filtration of the samples.
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Al–morin fluorescence was detected in DNA and ATP solutions even without adding Al, showing that the commercial DNA and ATP contained trace levels of Al. Addition of Al to the DNA and ATP samples increased the fluorescence intensity (Fig. 5) indicating that Al bound to DNA and ATP can be detected with morin.
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The sorption of Al to cell-wall materials derived from maize root tips was investigated by applying 1 µM Al followed by testing with morin. Al was strongly sorbed to the cell-wall material, causing a great reduction in the fluorescence intensity of Al–morin (Fig. 6). The origin of the cell-wall material had significant influence on the amount of Al sorbed, as reflected by a decrease in fluorescence intensity. Al was more strongly bound to cell-wall material derived from control plants (which were not treated with Al, i.e. Al-0), possibly due to the availability of more free Al-binding sites. Morin did not desorb Al from the cell-wall material showing that Al has a higher affinity to the cell wall than to morin.
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Discussion |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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The cellular distribution of Al between apoplast and symplast is still a matter of debate. Several methods have been employed by different authors to investigate Al uptake and distribution. Zhang and Taylor (1989
, 1990, 1991) used a kinetic approach and concluded that a rapid apoplastic binding was followed by slow uptake across the plasma membrane. Electron diffraction X-ray microanalysis showed that Al was predominantly localized in the cell wall of Al-treated Avena sativa roots (Marienfeld and Stelzer, 1993). In addition, laser microprobe mass analysis indicated a higher accumulation of Al in the cell wall of maize roots after a short-term (1–3 h) Al treatment (Marienfeld et al., 2000). On the other hand, using the morin Al staining technique, Tice et al. (1992) operationally allocated 30–40% of the Al to the apoplastic and the remainder to the symplastic fraction. Lazof et al. (1994) used secondary ion mass spectrometry and, after removing cell-wall Al by washing with citrate, detected symplastic accumulation of Al in intact soybean root tips after only 30 min of Al exposure.
All of the above approaches face specific methodological limitations unequivocally to give the precise cellular distribution of Al. The first unambiguous and direct measurement of Al uptake and distribution was achieved by Taylor et al. (2000), who used the rare 26Al isotope, accelerator mass spectroscopy, and a surgical technique to physically separate the cell wall from the cytosol in single cells of the giant alga Chara corallina. They observed that Al accumulation in the cell wall dominated total uptake (up to 99.99%), but transport across the plasma membrane was also detected within 30 min of exposure to Al. Chara showed a growth response to Al similar to that of wheat (Reid et al., 1995). Moreover, the electrical properties of Chara and wheat root cell walls were similar (Reid et al., 1996). In agreement with the observation in Chara, Chang et al. (1999) found that the cell wall isolated from Al-treated tobacco cells contained as much Al as the intact cells. Thus, there is little doubt that the majority of the cellular Al is located in the cell wall. Accordingly, genuine Al-localization methods have to reflect similar observation. However, reports are usually conflicting in this regard.
One of the easiest and most commonly used Al localization methods is the use of the fluorophore morin (Eggert, 1970; Tice et al., 1992; Larsen et al., 1996, 1998; Vitorello and Haug, 1996, 1997; Ezaki et al., 2000; Ahn et al., 2001, 2002). Morin (2,3,4,5,7-pentahydroxy flavone) makes a highly fluorescent complex with Al. It is specific to Al, especially at low pH (Browne et al., 1990), and highly sensitive, with an in vitro detection limit of 2 nM (Lian et al., 2003b). This makes it very attractive for Al studies. However, Tice et al. (1992) and Vitorello and Haug (1996), who used morin staining to localize Al, appeared to have greatly underestimated the proportion of Al found in the cell wall. Therefore, Archambault et al. (1996) questioned whether morin can detect cell-wall-bound Al.
In an attempt to clarify the prospects and limitations of morin as a stain for in vivo cellular distribution of Al, thin hand-sections (one to three cell layers) of maize root tips were used in the present study. The advantage of thin cross-sections is that free apoplastic Al and Al from the symplast of damaged cells could easily be removed by simple washing with double-deionized water. Moreover, the desorption of Al from the cell wall during staining and washing procedures was reduced by using an aqueous solution of morin. In the conventional method of morin staining, acetate or MES buffers, which readily complex Al, were used as a solvent for morin and also as a washing solution before and after staining (Tice et al., 1992; Larsen et al., 1996). These buffering chemicals may enhance desorption of Al from the cell wall.
Al localization using morin staining detected the presence of Al in the cytosol but not in the cell wall. The result was consistent throughout the experiments (Figs 1–3) and also similar to the observations of Tice et al. (1992) and Vitorello and Haug (1996). This does not necessarily show that Al is more abundant in the cytosol than in the cell wall. It may indicate that morin cannot detect cell wall-bound Al.
Chang et al. (1999) reported that about 71–82% of the total cellular Al was found associated with pectin of the cell wall. Hence the interaction between morin and pectin-bound Al was tested. The results clearly indicated that morin could not form a fluorescent complex with pectin-bound Al (Fig. 4). Experiments with isolated cell-wall material also reflected similar phenomena (Fig. 6). Even using the common and strong metal-chelating agent, EDTA (Al–EDTA binding affinity constant log K=16.5; Orvig, 1993), Chang et al. (1999) were able to desorb only 17% of the cell wall-bound Al. Therefore, it can be speculated that Al has a higher affinity to the cell wall than to EDTA. Thus, morin (Al–morin binding affinity constant log K=6.5; Katyal and Prakash, 1977) could not form a fluorescent complex with cell-wall-bound Al.
Intracellular Al may exist in association with cytosolic ligands with lower binding affinity and thus can form fluorescent complexes with morin. Intense fluorescence was observed in the nucleus as was previously reported (Tice et al., 1992; Vitorello and Haug, 1996). Al binds to DNA in the nucleus. The binding site of Al on DNA was shown to be the phosphate backbone but not the bases (Zhang et al., 2002). Similarly, Crawford et al. (1998) reported that Al appeared to be co-localized with P in the nuclei of root cap and meristematic cells. Al associated with phosphates (KH2PO4, DNA, ATP) can be detected with morin (Figs 4, 5) that is why Al in the nuclei gives a bright fluorescence when stained with morin.
In conclusion, the results clearly show that morin is not able to detect Al tightly bound to the cell-wall pectin and as such should not be used to determine the relative distribution of Al in the different parts of the cell.
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Acknowledgements |
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This study was financially supported by the EU, Science-Research-Development, International Cooperation Project (ICA4 CT 2000 0017).
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References |
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B. Ezaki, K. Sasaki, H. Matsumoto, and S. Nakashima Functions of two genes in aluminium (Al) stress resistance: repression of oxidative damage by the AtBCB gene and promotion of efflux of Al ions by the NtGDI1gene J. Exp. Bot., October 1, 2005; 56(420): 2661 - 2671. [Abstract] [Full Text] [PDF]
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