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Keywords:

  • Oryza sativa;
  • Zea mays;
  • ammonium;
  • ion fluxes;
  • nitrate;
  • root;
  • structure;
  • uptake

Abstract

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. References

Net fluxes of NH4+ and NO3 along adventitious roots of rice (Oryza sativa L.) and the primary seminal root of maize (Zea mays L.) were investigated under nonperturbing conditions using ion-selective microelectrodes. The roots of rice contained a layer of sclerenchymatous fibres on the external side of the cortex, whereas this structure was absent in maize. Net uptake of NH4+ was faster than that of NO3 at 1 mm behind the apex of both rice and maize roots when these ions were supplied together, each at 0·1 mol m–3. In rice, NH4+ net uptake declined in the more basal regions, whereas NO3 net uptake increased to a maximum at 21 mm behind the apex and then it also declined. Similar patterns of net uptake were observed when NH4+ or NO3 was the sole nitrogen source, although the rates of NO3 net uptake were faster in the absence of NH4+. In contrast to rice, rates of NH4+ and NO3 net uptake in the more basal regions of maize roots were similar to those near the root apex. Hence, the layer of sclerenchymatous fibres may have limited ion absorption in the older regions of rice roots.


INTRODUCTION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. References

Information on the spatial distribution of ion absorption along roots is essential for understanding the mineral nutrition and growth of plants. The age of root tissues may influence their capacity for ion absorption (Clarkson et al. 1968). Nitrogen (N) demands, for example, are large near the root apex due to rapid protein biosynthesis at this location (Clowes 1958; Silk & Erickson 1980). Amino acids translocated from older tissues (Oaks 1966) and high uptake rates of exogenous NH4+ (Grasmanis & Barley 1969) are two sources of nitrogen for root tips (apical 5–10 mm). In contrast to the relatively fast rates of NH4+ absorption near the root tip, NO3 uptake here occurs at rates slower than those in the basal regions, at least for roots of intact maize (Lazof et al. 1992) and barley (Siebrecht et al. 1995) plants. Differences in the spatial patterns of net fluxes of NH4+ and NO3 along roots may affect interactions between these ions at the whole root level during their absorption.

The present report describes experiments in which ion-selective microelectrodes were used to assess net fluxes (Newman et al. 1987; Henriksen et al. 1992) of NH4+ and NO3 at several positions along roots of rice (Oryza sativa L.) and maize (Zea mays L.). Roots of rice differ from those of nonwetland cereals in that they develop a layer of sclerenchymatous fibres on the external side of the cortex (Clark & Harris 1981). The sclerenchymatous layer may function (i) to prevent the collapse of the cortical aerenchyma (Clark & Harris 1981), and (ii) as a barrier to the radial loss of oxygen from the root to the soil, thus enhancing oxygen diffusion to the root tip and increasing the maximum depth to which roots can grow in anaerobic soils (Armstrong 1971, 1979). This physical barrier to oxygen loss is regarded as being of adaptive value to roots of many wetland plants; however, it may also impede nutrient uptake (Clark & Harris 1981; Koncalova 1990), although its influence on ion absorption by roots of rice has not been documented previously.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. References

Plant culture

Rice

Rice (Oryza sativa L. cv Calrose) seeds were imbibed for 3 h in aerated 1·0 mol m–3 CaSO4 and placed in rolled germination paper soaked with this solution in the dark at 28°C. Four-d-old seedlings were transferred to a controlled-environment chamber and their roots placed in an opaque plastic tub containing 22 dm3 of an aerated nutrient solution. The solution contained (in mol m–3): K+, 0·285; Ca2+, 1·0; Mg2+, 0·1; SO42–, 1·11; HPO42–, 0·065; H2PO4, 0·035; Fe-DTPA, 0·011; and micronutrients of 0·20 strength modified Hoagland solution (Epstein 1972). The pH of the solution was 5·9. Depending on the experiment, either 0·10 mol m–3 NH4+, NO3, or both NH4+ and NO3 were supplied using (NH4)2SO4 or Ca (NO3)2. The Ca2+ concentration in all solutions was maintained at 1·0 mol m–3 by altering the amount of CaSO4 supplied. The plants were grown in a controlled-environment chamber at 28 °C with 12 h light/dark cycles. Photon flux density was about 1200 μmol m–2 s–1 at plant height. Nutrient solutions were renewed every 3d.

The day prior to net ion flux measurements, 18–20-d-old plants were transferred to a pretreatment solution with the same nitrogen regime as that experienced during growth. The pretreatment solution contained (in mol m–3): K+, 0·001; Ca2+, 1·0; SO42–, 0·95; H2PO4, 0·001; and either 0·10 mol m–3 NH4+, NO3, or NH4+ and NO3. The relatively low level of K+ was used because the NH4+ liquid ion-exchange resin in the microelectrodes was also somewhat sensitive to K+ (10:1 selectivity of NH4+:K+) (Henriksen et al. 1990).

Maize

Maize (Zea mays L. cv Dekalb) seeds were germinated as described above for rice. Two-d-old seedlings were transferred to a pretreatment solution that contained (in mol m–3): K+, 0·001; Ca2+, 1·0; SO42–, 0·95; H2PO4, 0·001; NH4+, 0·10; and NO3, 0·10. Plants used in the net ion flux measurements were 3 d old.

Microelectrode fabrication

The microelectrode fabrication has been described previously (Henriksen et al. 1990; Frensch et al. 1994). In brief, microelectrodes with three active barrels specific for the same ion were constructed as follows. Seven glass pieces, four solid rods and three thin-walled borosilicate capillaries with filament (A-M Systems, Everett, WA) were assembled and pulled in several stages: first, the barrels were twisted together (120°) and then pulled to a combined diameter of about 2 mm on a Nirishige vertical puller (Type PE-2); lastly, the complex was pulled to a fine tip on a Kopf Instruments pipette puller (Model 750). To fill the interbarrel spaces, the capillaries were pressurized with air while the tip was immersed in methylacrylate glue, after which the glue was cured with UV light and heat. The slurry method of Shackel et al. (1987) was used to bevel the micropipette tips on three sides to produce tips of about 3 μm in diameter with three open barrels. Barrels were silanized at 120 °C with tributylchlorosilane (Fluka Chemika, Switzerland).

Micropipettes were backfilled with liquid ion-exchange resins and filling solutions. The NH4+-selective microelectrodes contained a liquid ion-exchange resin from a stock composed of: 6·9 mg Fluka NH4+ ionophore I, 0·7 mg potassium tetrakis (4-chlorophenyl) borate, and 92·4 mg 2-nitrophenyl octyl ether (Fluka Chemika, Switzerland). The filling solution in the NH4+-selective microelectrodes was 500 mol m–3 NH4Cl. The NO3-selective microelectrodes contained Orion NO3-resin, a tris-substituted 1, 10-phenanthroline nickel (II) nitrate in p-nitrocymene, and a filling solution of 500 mol m–3 KNO3 and 100 mol m–3 KCl. Microelectrodes were stored overnight in dry air with their tips down, and conditioned for 2 h in 1 mol m–3 NH4+ or NO3 before use.

The four solid glass rods separated the back-ends of the three active barrels, facilitating the connection of each barrel to a high-impedance amplifier. One end of a fine-gauge silver wire was chloridized and then placed into the filling solution of a barrel, and the other end of the wire was inserted into a socket of an 8 pin Teflon integrated circuit plug (Augat 8058–1G32). The plug was connected into the head stage of a multichannel electrometer amplifier based on earlier designs (Bloom & Chapin 1981; Bloom 1989). This amplifier simultaneously monitored the potential between each of the three microelectrode barrels and a double-junction macro-reference electrode (Fisher Scientific, 13–639–273) that was in the same solution.

Measurements of net ion fluxes

About 4 h prior to measurements of net ion fluxes, the root system of an intact plant was inserted into a temperature-controlled root cuvette (Henriksen et al. 1990, 1992) at 28 °C, with 50 ml min–1 flow of the relevant aerated pretreatment solution. Roots were exposed to the same concentration and form of nitrogen throughout their growth, pretreatment, and measurements of net ion fluxes. For rice adventitious roots, NH4+ net fluxes were measured with 0·10 mol m–3 NH4+ as the sole nitrogen source, or with both NH4+ and NO3 at 0·10 mol m–3. Likewise, NO3 net fluxes were measured with 0·10 mol m–3 NO3 as the sole nitrogen source, or with both NH4+ and NO3 at 0·10 mol m–3. For maize primary seminal roots, NH4+ and NO3 net fluxes were measured for roots supplied with both NH4+ and NO3 at 0·10 mol m–3; net fluxes of sole nitrogen sources were not examined due to time constraints in this study. Shoots were illuminated at a photosynthetic photon flux density of 700 μmol m–2 s–1 during the root ion flux measurements.

Microelectrodes were calibrated before and after each experiment. Calibrations were done in solutions at 28 °C that contained (in mol m–3): K+, 0·001; Ca2+, 1·0; SO42–, 0·95; H2PO4, 0·001; and 0·040, 0·080, or 0·120 mol m–3 of NH4+ or NO3. The responses (in mV decade–1) to the ion of interest were: –57·5 ± 0·8 and 46·7 ± 0·8 (mean ± SE, n = 24) for NH4+- and NO3-selective microelectrodes, respectively.

Following calibration, microelectrodes were positioned in the bulk solution and the flow through the chamber was stopped. After 15 min, ion concentrations at 5000 (bulk solution), 200 and 100 μm radially from the root surface and once again at 5000 μm were determined. This cycle of measurements was then repeated. These measurements were taken at 1, 3·5, 6, 11, 16, 21, 31, 41, and 61 mm behind the root apex, which was discernible through the translucent root cap. The root cuvette was positioned with respect to the microscope using a rack and pinion slide, and microelectrode tips were positioned using a motor-driven micromanipulator (Sutter, Model MP-185) and a calibrated eyepiece reticle. The large number of lateral roots at positions further back than 61 mm along the main axes prevented measurements being made at more basal locations. After measurements were taken at each location, the solution flow was restarted for 30 min to flush the cuvette and fill it with fresh experimental solution; the flow was then stopped again for a second set of measurements. This second set of measurements was taken to check whether the net flux rates at each position were relatively constant over time. Rates at each position were found to be similar at both times, so these estimates of net ion flux at each position along individual roots were then used to calculate the average net flux rate at each position along the root. Such measurements were taken along individual roots of four different plants grown in each solution, to provide four replicates.

Net ion fluxes at the root surface were calculated using eqn 17 of Henriksen et al. (1992):

Jroot = KD*°× (c2 c1)/ rroot× ln (r2/r1)

where, Jroot is the net flux (nmol m–2 s–1) at the root surface, rroot is the root radius (μm), D*° is the self diffusion coefficient for the ion of interest (m2 s–1), c1 and c2 are the concentrations of the ion of interest (nmol cm–3) at radial distances r1 and r2 (μm) from the centre of the root, and K is a units conversion coefficient. The self diffusion coefficients (D*°) of NH4+ and NO3 in aqueous solution at 28 °C are 2·089 × 10–9 and 2·044 × 10–9 m2 s–1, respectively, as calculated using the Nernst–Einstein relation (see eqns 5 and 6 of Henriksen et al. 1992).

Staining and microscopy of root cross-sections

Free-hand sections were taken across roots at various positions behind the apex using a hand-held razor blade, and stained for lipids with Fluoral yellow, as described by Brundrett et al. (1991). In brief, fresh sections were stained for 1 h in an aliquot from a stock containing 1 ml Fluoral yellow in 499·5 ml polyethylene glycol 400, 399·5 ml glycerol, and 100 ml deionized water. After staining, sections were rinsed for 10 s in deionized water and then mounted in an aliquot from a stock containing 75 ml glycerol and 25 ml deionized water. Sections were examined under ultraviolet illumination using an Olympus Vanox-T microscope and photographed on Ektachrome 160 slide film.

Statistical analyses

Net flux data at 1, 11, 21, 41, and 61 mm behind the apex of rice roots supplied with NH4+ and NO3 as sole or combined nitrogen sources (Fig. 1 and appropriate data from Fig. 2) were analysed using a three-way analysis of variance to determine the main effects and interactions between ions (NH4+ or NO3), sources (sole or combined), and position along the root. Net flux data for rice and maize roots supplied with both NH4+ and NO3 (Figs 2 and 3) were analysed using a two-way analysis of variance to determine the main effects and interactions between ions and position along the root, so that differences between the species were examined.

image

Figure 1. . Net fluxes of NH4+ (closed symbols) and NO3 (open symbols) along intact rice adventitious roots, with either form of nitrogen as the sole source. Net fluxes were calculated from ion concentration gradients between 200 and 100 μm from the root surface (Henriksen et al. 1992). Plant growth and net flux measurements were in nutrient solutions with either 0·1 mol m–3 NH4+ or NO3. Each data point represents the mean ± standard error of four replicates, with each replicate being a separate experiment on an individual plant.

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image

Figure 2. . Net fluxes of NH4+ (closed symbols) and NO3 (open symbols) along rice adventitious roots, when both forms of nitrogen were present. Net fluxes were calculated from ion concentration gradients between 200 and 100 μm from the root surface (Henriksen et al. 1992). Plant growth and net flux measurements were in nutrient solutions with 0·1 mol m–3 NH4NO3. Each data point represents the mean ± standard error of four replicates, with each replicate being a separate experiment on an individual plant.

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image

Figure 3. . Net fluxes of NH4+ (closed symbols) and NO3 (open symbols) along maize primary seminal roots, when both forms of nitrogen were present. Net fluxes were calculated from ion concentration gradients between 200 and 100 μm from the root surface (Henriksen et al. 1992). Plant growth and net flux measurements were in nutrient solutions with 0·1 mol m–3 NH4NO3. Each data point represents the mean ± standard error of four replicates, with each replicate being a separate experiment on an individual plant.

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RESULTS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. References

NH4+ and NO3 net fluxes along adventitious roots of rice

Rates of NH4+ and NO3 net uptake varied significantly along the adventitious roots of rice (Tables 1 & 2). Net uptake of NH4+ was most rapid at 1 mm behind the apex of roots supplied with either NH4+ alone, or both NH4+ and NO3, and it tended to decline with distance behind the apex (Figs 1 & 2; Tables 1 & 2). By comparison, NO3 net uptake first increased with distance behind the apex and then it declined at distances more basal than 21 mm from the apex (Figs 1 & 2). In the statistical analysis these differences were confirmed as a significant interaction between ion and position (Tables 1 & 2).

Table 1.  . Three-way analysis of variance of NH4+ and NO3 net flux data along intact adventitious roots of rice, when either form of nitrogen was the sole source and when both forms were present. Data from Fig. 1 and the corresponding locations from Figs 2 (1, 11, 21, 41 and 61 mm behind the apex) Thumbnail image of
Table 2.  . Two-way analysis of variance of NH4+ and NO3 net fluxes along intact adventitious roots of rice and the primary seminal root of maize, when both forms of nitrogen were present. Data from Figs 2 and 3Thumbnail image of

Rates of NH4+ net uptake were, on average, equal for rice roots supplied with NH4+ alone, or with both NH4+ and NO3 (cf. Figs 1 & 2). In contrast, NO3 net uptake was slower when both NH4+ and NO3 were present, than when NO3 was the sole nitrogen source (cf. Figs 1 & 2; Table 1). For example, when NO3 was the sole nitrogen source its net uptake rate at 1 mm behind the root apex was not statistically different from that of NH4+ (Fig. 1); however, when both forms of nitrogen were offered, NO3 net uptake was only 40% of the sole source rate (Fig. 2). An inhibitory effect of NH4+ on net uptake of NO3 was evident at all positions along the root axis (cf. Figs 1 & 2), and this was expressed as a significant interaction between ion and source in the statistical analysis (Table 1).

NH4+ and NO3 net fluxes along the primary seminal root of maize

Net fluxes of NH4+ and NO3 were measured along the primary seminal root of maize supplied with NH4+ and NO3 in the same solution (Fig. 3). Net uptake of NH4+ was most rapid at 1 mm behind the apex, while NO3 net uptake was slowest at 1 mm behind the apex (Fig. 3), although overall the statistical analysis indicated that position along the root did not have a significant effect on net flux rates of these ions in maize (Table 2).

Root anatomy

Root cross-sections stained with Fluoral yellow (Brundrett et al. 1991) showed rice (Fig. 4), but not maize (data not shown), to have a layer of sclerenchymatous fibres on the outermost side of the cortex, three cell layers from the exterior of the root. The sclerenchymatous layer in rice was absent at 6 mm behind the apex, but was well developed at 21 mm, and even more prominent at 41 and 61 mm behind the apex (data not shown).

image

Figure 4. . Cross-section of a rice adventitious root taken 21 mm behind the apex and stained for lipids with Fluoral yellow (Brundrett et al. 1991). Fresh sections were stained for 1 h with Fluoral yellow as described in the Materials and Methods, and examined under ultraviolet illumination with a Olympus Vanox-T microscope. A layer of sclerenchymatous fibre cells was absent in sections taken at 6 mm, but was evident at 21, 41, and 61 mm behind the apex. (Magnification ×25).

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DISCUSSION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. References

The form of inorganic nitrogen absorbed by root tips may play a role in the regulation of root growth and development (cf. Bloom et al. 1993). At 1 mm behind the apex, NH4+ net uptake by roots of rice and maize was faster than that of NO3 when both forms of nitrogen were present (Figs 2 and 3). These findings are in agreement with earlier studies which found that NH4+ net uptake tended to be most rapid near the apices of roots of barley (Henriksen et al. 1992) and pea (Grasmanis & Barley 1969), but that NO3 uptake near root tips was slower than uptake by the more basal regions in maize (Lazof et al. 1992) and barley (Siebrecht et al. 1995). NH4+ assimilation requires less energy than that of NO3 (Bloom et al. 1992); therefore, utilization of NH4+ by the root apical meristem may supply nitrogen for protein synthesis with the least expenditure of energy. Moreover, assimilation of NH4+ into amino acids generates a proton that is usually released into the rhizosphere (Allen 1988), so that NH4+ utilization should acidify the root tip and enhance root elongation, because cell-wall extensibility is controlled, at least partially, by pH (Taiz & Zeiger 1991). Thus, although high concentrations of NH4+ impede root growth, at low concentrations of NH4+(which may be of more relevance to nature) root growth is equal to, or greater than, that under NO3 (Bloom et al. 1993). Nevertheless, above-ground biomass is generally highest when both NH4+ and NO3 are supplied (Marschner 1995).

For the maize roots studied, a net NH4+ or NO3 uptake of 300 nmol m–2 s–1 (per unit root-surface area) corresponds to a rate based on a unit of root length of about 3·1 nmol mm–1 h–1, and a per unit dry weight rate of 1·0 μmol g–1 dry weight min–1. These uptake rates are very similar to other values reported for NH4+ (Vale et al. 1988) and NO3 (Pan et al. 1985) absorption by whole root systems of maize seedlings, and to rates of 15NO3 accumulation in the apical 35 mm of maize seminal roots (Lazof et al. 1992). Sharp et al. (1990) calculated that rates of net K+ deposition ranged from 5 to 33 nmol mm–1 h–1 over the apical 9 mm of maize root tips, and K+ made a large contribution to the osmotic potential of the expanding cells. When absorbed, NH4+ is usually assimilated relatively quickly, whereas NO3 may not be, so that the NO3 absorbed at 3·1 nmol mm–1 h–1 may have also made a substantial contribution to the osmotic potential of the root cells.

Root cross-sections stained for lipids with Fluoral yellow (Brundrett et al. 1991) showed that older regions of adventitious roots of rice (Fig. 4), but not the primary seminal root of maize (data not shown), had a layer of sclerenchymatous fibre cells on the outermost side of the cortex (see also Clark & Harris 1981). The sclerenchymatous layer is distinct from hypodermal casparian bands which have been shown to develop in the roots of many species (Peterson 1988; Perumalla et al. 1990), including maize (Clarkson et al. 1987) and rice (Clark & Harris 1981). The hypodermal casparian bands block the apoplastic movement of solutes, so that nutrient ions are absorbed by epidermal cells and transported symplastically to the xylem (Clarkson et al. 1968; Ferguson & Clarkson 1975; Kochian & Lucas 1988). In contrast to the hypodermis, the sclerenchymatous layer may pose a barrier to nutrient uptake by rice roots (as suggested by Clark & Harris 1981). Our findings show that net uptake rates of NH4+ and NO3 were fastest in the younger regions and slowest in the older regions of the roots of rice, whereas in maize the net uptake rates of these ions were similar at the youngest and oldest locations measured along the root (Figs 2 and 3; Table 2). The decline in NH4+ and NO3 net uptake in the older regions of roots of rice, but not maize, is consistent with the sclerenchymatous layer limiting the capacity for ion absorption by the roots of rice.

The sclerenchymatous cells differentiate immediately inside the hypodermis as short fibres, which subsequently elongate and form thick secondary walls which are lignified with relatively little pitting (Clark & Harris 1981). The degree of lignification may determine the ion absorption capacity at locations along roots of rice. Sclerenchymatous cells were evident 21 mm behind the apex, but the wall thickening was not as prominent at this position as the thickening in the more basal regions, and the rates of NH4+ and NO3 net uptake at 21 mm behind the apex were higher than those in more basal regions (Figs 1 & 2). Perhaps the symplastic connections became restricted in the older regions of rice roots as the degree of lignification of the fibres increased. It should also be noted that in the rice roots used in the studies reported here, aerenchyma had not developed at 21 mm behind the apex, but it had at 41 and 61 mm. Clark & Harris (1981) suggested the formation of aerenchyma may reduce the capacity for ion transport across the root, which in turn may also slow the rate of ion absorption. However, studies of maize roots with and without aerenchyma showed no difference in the capacity of these roots to transport ions, probably due to the radial files of cells which span the cortex in roots with lysigenous aerenchyma (Drew & Saker 1986). The cross-sections taken at 41 and 61 mm behind the apex showed radial files of cortical cells in the rice roots used in our study.

The issue of whether NH4+ influences NO3 net uptake at the whole root level has received considerable experimental attention, with conflicting results. The discrepancies may be due to different responses among genotypes, different experimental time frames, and different pretreatments (Ullrich et al. 1984; Bloom & Finazzo 1986; Aslam et al. 1994). Nevertheless, short-term studies generally support the view that additions of NH4+ inhibit NO3 net uptake (Lee & Drew 1989), whereas there are few longer-term studies. In our study of adventitious roots of rice under nonperturbing conditions, NO3 net uptake was decreased by the presence of NH4+. In contrast, NH4+ net uptake was not affected by the presence of NO3 (cf. Figs 1 and 2; Table 1). Ullrich et al. (1984) suggested that NH4+-induced inhibition of net NO3 uptake may result from high rates of NH4+ influx decreasing the proton driving force for anion transport. This mechanism may explain the short term inhibition of net NO3 uptake, but probably not that under the longer-term nonperturbing conditions used in the studies described here.

Acknowledgements

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. References

We thank S. Baum for advice on the Fluoral yellow staining technique, and Prof. T. Rost for access to the microscope and photography equipment. This research was supported by NSF IBN-93–06521 and USDA 93–37305–9143.

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  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. References
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