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Keywords:

  • active uptake;
  • dicots;
  • monocots;
  • passive uptake;
  • silicon

Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  • • 
    Here, we characterized silicon (Si) uptake and xylem loading in Oryza sativa, Zea mays, Helianthus annuus and Benincase hispida in a series of hydroponic experiments. Both active and passive Si-uptake components co-exist in all the plants tested. The active component is the major mechanism responsible for Si uptake in O. sativa and Z. mays.
  • • 
    By contrast, passive uptake prevails in H. annuus and B. hispida at a higher external Si concentration (0.85 mm), while the active component constantly exists and contributes to the total Si uptake, especially at a lower external Si concentration (0.085 mm).
  • • 
    Short experiments showed that Si uptake was significantly suppressed in O. sativa and Z. mays by metabolic inhibitors or low temperature, regardless of external Si concentrations. By contrast, Si uptake in H. annuus and B. hispida was inhibited more significantly by metabolic inhibitors or low temperature at lower (for example, 0.085 mm) than at higher (for example, 1.70 mm) external Si concentrations.
  • • 
    It can be concluded that both active and passive Si-uptake components co-exist in O. sativa, Z. mays, H. annuus and B. hispida, with their relative contribution being dependent much upon both plant species and external Si concentrations.

Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Silicon (Si) is the second most abundant element both on the Earth's crust and in the soil. It is accumulated in plants at a level equivalent to that of macronutrient elements such as calcium, magnesium and phosphorous (Epstein, 1999). Despite its higher accumulation in plants, Si has not been listed among the essential elements for higher plants because the strictly defined essentiality of Si in the Arnon & Stout (1939) sense has not been proven (Lewin & Reimann, 1969; Epstein, 1994, 1999). The beneficial effects of Si on stimulating plant growth, however, have recently received increasing attention, particularly in plants subject to both abiotic (e.g. aluminium, salt and heavy metal toxicity) and biotic (e.g. plant diseases and pests) (Savant et al., 1997; Epstein, 1999; Liang, 1999; Ma et al., 2001a; Liang et al., 2003, 2005a,b) stresses.

All plants growing in soils take up Si, but Si content in plant tissues varies greatly among plants, ranging from c. 0.1 to 10% (w/w) Si on a dry weight basis (Epstein, 1994, 1999). Based on the Si content of the plant tops, plants are classified into Si accumulator, intermediate-type and excluder species (Jones & Handreck, 1967; Takahashi et al., 1990). The difference in Si content in plants has been attributed to the different mechanisms involved in Si uptake by the roots. Three modes of Si uptake (active, passive and rejective) have been suggested for the corresponding Si accumulator, intermediate-type and excluder plants, respectively (Takahashi et al., 1990). Oryza sativa, a typical Si accumulator, takes up Si actively (Takahashi et al., 1990; Ma et al., 2001a). Apart from O. sativa, some graminaceous plants, such as Triticum aestivum (van der Vorm, 1980; Jarvis, 1987; Rafi & Epstein, 1999; Casey et al., 2003), Lolium perenne (Jarvis, 1987) and Hordeum vulgare (Barber & Shone, 1966) take up Si actively, while some others, such as Avena sativa, take it up passively (Jones & Handreck, 1965). By contrast, some dicots, such as Cucumis sativus, C. melo, Fragaria vesca and Glycine max Merr. (Takahashi et al., 1990; Ma et al., 2001b; Mitani & Ma, 2005) take up Si passively, and Lycopersicon esculentum (Takahashi et al., 1990), Phaseolus vulgaris (Jones & Handreck, 1967) and Vicia faba (Liang et al., 2005c) exclude Si from uptake.

Recently, rapid progress has been made in characterizing Si uptake and transport in O. sativa (Ma et al., 2001b, 2002, 2004, 2006; Tamai & Ma, 2003). These authors agree that Si uptake and transport in O. sativa is an active process mediated by a specific transporter having a low affinity (Km = 0.32 mm) compared with phosphorous or potassium. The low-silicon rice 1 (Lsi1) gene, which is responsible for xylem loading of Si, has recently been mapped to chromosome 2 of O. sativa (Ma et al., 2004). The Lsi1 gene, localized on the plasma membrane of the distal side of both exodermis and endodermis cells, belongs to the aquaporin family and is constitutively expressed in roots (Ma et al., 2006). Suppression of Lsi1 expression resulted in reduced silicon uptake, and expression of Lsi1 in Xenopus oocytes resulted in transport activity for silicon only (Ma et al., 2006). This work at the molecular level clearly shows that Si uptake and transport is mainly an active process in rice. However, even in the Lsi1, defective in active Si uptake, shoot Si content still reached up to 1.43% when the rice mutant was grown hydroponically at 1.5 mm Si for 4 wk (Ma et al., 2002). In the Lsi1 mutant used for gene mapping, cloning and characterization, shoot Si content was ≈ 0.5% when the mutant was grown in the paddy field (Ma et al., 2006), suggesting that the Si transporter-mediated system is not the only mechanism responsible for Si uptake in rice and that a passive uptake component, mediated by transpiration, may co-exist. In contrast to O. sativa, information is still scant on Si uptake and transport in the other monocotyledonous and dicotyledonous plants. More recently, Mitani & Ma (2005) have reported that the Si concentration was lower in the xylem sap of C. sativus than in the external solution, suggesting that xylem loading of Si was mediated by a passive diffusion mechanism in C. sativus, although the concentrations of Si were higher in the root–cell symplast than in the external solution. However, in a series of hydroponic experiments with C. sativus, we have demonstrated that Si uptake and transport is an active process (Liang et al., 2005c). Such distinct discrepancies reported between investigators regarding Si uptake and xylem loading in C. sativus might be caused by the different methods used for collecting xylem sap and the preculture of the plants, and/or by different cultivars used. In the experiment of Mitani & Ma, seedlings of C. sativus precultivated in an Si-deprived solution for 20 d were transferred to a nutrient solution containing 0.5 mm Si, and the stem was severed, after the plants were grown for 0.5, 1, 2, 3, 4, 6 and 8 h, respectively, for collecting xylem sap (Mitani & Ma, 2005). In our previous study, seedlings of C. sativus grown in an Si-containing solution for 18 d were decapitated, and xylem sap was then collected at different time-points (Liang et al., 2005c). Thus, it appears that further investigation is needed to characterize Si uptake and transport in C. sativus under the same experimental conditions.

Based on the current knowledge in the literature, we hypothesize that except for the excluder plant species there are two general mechanisms for Si uptake and transport (i.e. active and passive components) co-existing in a plant species, with their relative contribution being dependent much upon the plant species and external Si concentration. Because it has been well documented that the excluder plant species, such as tomato and faba bean (Liang et al., 2005c; Mitani & Ma, 2005) exclude Si from uptake, regardless of the external Si concentration, in this study we focused on characterizing Si uptake and xylem loading in O. sativa, Zea mays, Helianthus annuus and Benincase hispida. Choosing O. sativa, Z. mays, H. annuus and B. hispida as case studies is based on such considerations that O. sativa and Z. mays, the representatives of monocots, are Si accumulators, and that H. annuus and B. hispida, the representatives of dicots, are intermediate types but not excluders. We also investigated Si concentration in xylem sap with time in this study because information in the literature is scarce on the time-course changes of Si concentrations present in the form of monosilicic acid in xylem, although it has been reported that the composition of xylem sap changed with time and the concentration of solutes in xylem sap increased with time (Canny & McCully, 1988). The objectives of this study were to determine whether the two general mechanisms for Si uptake and transport co-exist in the same Si accumulator or intermediate-type species, and to demonstrate that active Si uptake and transport is involved not only in O. sativa, but also in Z. mays and some dicots, such as H. annuus and B. hispida.

Materials and Methods

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Growth conditions and plant materials

Four plant species were selected: rice (O. sativa L. cv. Shanyou 63), maize (Z. mays L. cv. Nongda 108), sunflower (H. annuus L. cv. Hungary 4) and wax gourd (B. hispida L. cv. Maqun 1). Seeds of the tested plants were surface sterilized with 10% H2O2 (v/v) for 10 min, rinsed thoroughly with distilled water, and germinated on moist filter paper for 3 d in an incubator at 25°C. After germination, the seeds were then transferred to a net floated on 0.5 mm CaCl2 in a plastic container. At 6 d, the seedlings were transferred to a 10-l plastic pot in a growth chamber with a day/night temperature regime of 25°C (14 h) : 18°C (12 h) and a light intensity of 400 µmol m−2 s−1. The nutrient solution for O. sativa was half-strength Kimura B solution containing the following macronutrients: 0.18 mm (NH4)2SO4, 0.27 mm MgSO4·7H2O, 0.09 mm KNO3, 0.18 mm Ca(NO3)2·4H2O, and 0.09 mm KH2PO4 and the micronutrients 20 µm NaEDTAFe·3H2O, 6.7 µm MnCl2·4H2O, 9.4 µm H3BO3, 0.015 µm (NH4)6Mo7O24·4H2O, 0.15 µm ZnSO4·7H2O and 0.16 µm CuSO4·5H2O. The nutrient solution for the other three plant species had the following compositions: 2.0 mm Ca(NO3)2·4H2O, 0.7 mm K2SO4, 0.1 mm KH2PO4, 100 µm NaEDTAFe·3H2O, 10 µm H3BO3, 0.5 µm MnSO4·5H2O, 0.5 µm ZnSO4·7H2O, 0.2 µm CuSO4·5H2O and 0.01 µm (NH4)6Mo7O24. In the nutrient solutions, silicon was supplied at a rate of 1.70 mm Si as sodium metasilicate (Na2SiO3·9H2O), which had been neutralized with 300 ml of 0.1 m HCl per 10 l of nutrient solution before use. The nutrient solution prepared with purified water (Milli-Q, Billerica, MA, USA) was aerated daily and renewed every 3 d. The pH of the solution was adjusted to 5.6, daily, with dilute HCl and/or NaOH. All the experiments were repeated at least three times.

Si-uptake experiments

The experiment for short-term Si uptake was continued for 12 h. For this, the 14-d-old seedlings were placed in a 100-ml plastic bottle containing half-strength nutrition solution (described above under Growth conditions and plant materials) with either 0.085 or 0.85 mm Si. Silicon was supplied as sodium metasilicate (Na2SiO3·9H2O), which had been neutralized with 0.1 m HCl before use. A 1.0-ml aliquot of uptake solution was taken at 2, 4, 6, 8, 10 and 12 h after the experiment had started. Water loss, resulting from transpiration, was also recorded by weight at each sampling time. At the end of the uptake experiment, the plants were harvested and the dry weights of roots recorded after being oven-dried at 60°C for 72 h.

Inhibitors and low-temperature experiments

To investigate the effect of metabolic inhibitor on Si uptake, 14-d-old seedlings were exposed to half-strength nutrition solution (described above under Growth conditions and plant materials) containing 0.085, 0.85 or 1.70 mm Si, with or without 1.0 mm NaF, 1.0 mm NaCN or 0.1 mm 2,4-dinitrophenol (2,4-DNP). Silicon was supplied as sodium metasilicate (Na2SiO3·9H2O), which had been neutralized with 0.1 m HCl before use. Stock solution of 2,4-DNP was dissolved in ethanol and the final concentration of the ethanol in the uptake solution was less than 0.4% (v/v). Ethanol was also added to the solution, without inhibitors, at the same concentration. A preliminary experiment showed that this concentration of ethanol had no effect on Si uptake. After 6 h, a 1.0-ml aliquot of uptake solution was taken for determination of Si, and water loss, via transpiration streams, was also recorded by weight. The plants were harvested and the dry weights of roots recorded after being oven dried at 60°C for 72 h.

For the treatment with low temperature, 14-d-old seedlings were exposed to half-strength nutrient solution containing 0.085, 0.85 or 1.70 mm Si. The solutions had been precooled to 4°C (O. sativa, H. annuus and B. hispida) or 10°C (Z. mays) before the onset of the experiments, and the low temperature was maintained for 6 h using an ice-bath. Si concentration in the treatment solution was then determined, and water loss via transpiration streams was also recorded by weighing. In order to examine the effect of a short-term (6 h) pretreatment of low temperature on the subsequent Si uptake under normal conditions (25°C), the ice-bath was removed. The solution temperature rose to 25°C progressively at 10 h after the removal of the ice-bath. The experiment for Si uptake at the normal temperature (25°C) was continued for a further 6 h. The Si concentration in the treatment solution was then determined. The plants were harvested and the dry weights of the roots were recorded after being oven-dried at 60°C for 72 h.

Xylem-loading experiments

For xylem-loading experiments, 14-d-old plants were decapitated 1 h after the nutrient solutions containing 0.085 and 0.85 mm Si were renewed. The plants were decapitated 3–4 cm above the roots and the detopped stalks were sealed with a rubber tube for collecting xylem exudates with a micropipette at the time-points indicated in Tables 1–4. The Si concentration in the xylem sap was determined immediately in order to avoid analytical errors caused by Si polymerization.

Table 1.  Time-course changes of silicon (Si) concentration in xylem exudates and in external solutions, and the ratios of Si in xylem exudates to Si in external solutions in Oryza sativa (rice) grown hydroponically with varying concentrations of Si
External Si concentration Time (h)
24681012
  1. Plants were decapitated 3–4 cm above the roots for collection of xylem exudates (at the time-points indicated in the table) using a micropipette.

  2. Data are expressed as mean ± standard deviation (n = 3). Data followed by different letters in the same row denote significant difference at P < 0.05.

0.085 mmSi concentration (mm) 3.64 ± 0.74b 3.64 ± 0.17b 3.95 ± 0.12a 4.91 ± 0.21a 4.24 ± 0.22a 4.61 ± 0.42a
0.85 mmin xylem exudates12.10 ± 0.88c14.28 ± 0.22b15.43 ± 0.52a16.15 ± 1.12a15.95 ± 1.10a16.43 ± 1.32a
0.085 mmSi concentration (mm)0.073 ± 0.002a0.072 ± 0.002a0.071 ± 0.003a0.070 ± 0.006ab0.069 ± 0.006ab0.062 ± 0.004b
0.85 mmin external solutions 0.70 ± 0.02a 0.65 ± 0.02b 0.62 ± 0.02bc 0.60 ± 0.01c 0.55 ± 0.02d 0.50 ± 0.02e
0.085 mmRatios of Si in xylem exudates49.86 ± 1.35e49.19 ± 1.64e55.63 ± 2.05d70.13 ± 1.65c61.45 ± 1.40b74.35 ± 2.41a
0.85 mmto Si in external solutions17.54 ± 0.18d21.91 ± 1.30c28.89 ± 1.13b26.91 ± 1.42b29.01 ± 1.51b32.83 ± 1.40a
Table 2.  Time-course changes of silicon (Si) concentration in xylem exudates and in external solutions, and the ratios of Si in xylem exudates to Si in external solutions in Zea mays (maize) grown hydroponically with varying concentrations of Si
External Si concentration Time (h)
24681012
  1. Plants were decapitated 3–4 cm above the roots for collection of xylem exudates (at the time-points indicated in the table) using a micropipette.

  2. Data are expressed as mean ± standard deviation (n = 3). Data followed by different letters in the same row denote significant difference at P < 0.05.

0.085 mmSi concentration (mm) 0.88 ± 0.01f 0.97 ± 0.03e 1.02 ± 0.01d 1.07 ± 0.01c 1.17 ± 0.01b 1.24 ± 0.00a
0.85 mmin xylem exudates 4.41 ± 0.02f 5.02 ± 0.02e 5.31 ± 0.04d 5.47 ± 0.01c 5.76 ± 0.05b 6.28 ± 0.04a
0.085 mmSi concentration (mm)0.084 ± 0.00a0.080 ± 0.005ab0.079 ± 0.004ab0.078 ± 0.004ab0.074 ± 0.004ab0.071 ± 0.003b
0.85 mmin external solutions 0.83 ± 0.00a 0.83 ± 0.01b 0.80 ± 0.00c 0.79 ± 0.01d 0.79 ± 0.00d 0.77 ± 0.00d
0.085 mmRatios of Si in xylem exudates10.46 ± 0.31f12.06 ± 0.02e12.99 ± 0.51d13.76 ± 0.43c15.85 ± 0.54b17.42 ± 0.72a
0.85 mmto Si in external solutions 5.29 ± 0.03f 6.09 ± 0.02e 6.66 ± 0.03d 6.94 ± 0.05c 7.33 ± 0.28b 8.14 ± 0.078a
Table 3.  Time-course changes of silicon (Si) concentration in xylem exudates and in external solutions, and the ratios of Si in xylem exudates to Si in external solutions in Helianthus annuus (sunflower) grown hydroponically with varying concentrations of Si
External Si concentration Time (h)
24681012
  1. Plants were decapitated 3–4 cm above the roots for collection of xylem exudates (at the time-points indicated in the table) using a micropipette.

  2. Data are expressed as mean ± standard deviation (n = 3). Data followed by different letters in the same row denote significant difference at P < 0.05.

0.085 mmSi concentration (mm) 0.21 ± 0.01f 0.38 ± 0.00e 0.71 ± 0.01d 0.88 ± 0.02c 0.94 ± 0.01b 1.02 ± 0.01a
0.85 mmin xylem exudates 1.49 ± 0.02f 1.58 ± 0.01e 2.29 ± 0.05d 2.44 ± 0.04c 2.71 ± 0.02b 2.77 ± 0.02a
0.085 mmSi concentration (mm)0.084 ± 0.001a0.080 ± 0.003b0.073 ± 0.002c0.070 ± 0.002c0.068 ± 0.002c0.067 ± 0.003c
0.85 mmin external solutions 0.85 ± 0.01a 0.85 ± 0.00a 0.83 ± 0.01ab 0.83 ± 0.00b 0.82 ± 0.00b 0.82 ± 0.00b
0.085 mmRatios of Si in xylem exudates 2.51 ± 0.31f 4.73 ± 0.02e 9.73 ± 0.51d12.55 ± 0.43c13.85 ± 0.54b15.21 ± 0.72a
0.85 mmto Si in external solutions 1.76 ± 0.03c 1.86 ± 0.02c 3.48 ± 0.03b 2.94 ± 0.15a 3.33 ± 0.28a 3.37 ± 0.28a
Table 4.  Time-course changes of silicon (Si) concentration in xylem exudates and in external solutions, and the ratios of Si in xylem exudates to Si in external solutions in Benincase hispida (wax gourd) grown hydroponically with varying concentrations of Si
External Si concentration Time (h)
24681012
  1. Plants were decapitated 3–4 cm above the roots for collection of xylem exudates (at the time-points indicated in the table) using a micropipette.

  2. Data are expressed as mean ± standard deviation (n = 3). Data followed by different small letters in the same row denote significant difference at P < 0.05.

0.085 mmSi concentration (mm)0.180 ± 0.003f0.370 ± 0.008e0.560 ± 0.017d0.717 ± 0.01c0.803 ± 0.008b0.883 ± 0.02a
0.85 mmin xylem exudates 0.92 ± 0.03f 1.23 ± 0.02e 1.81 ± 0.02d 2.12 ± 0.01c 2.33 ± 0.02b 2.39 ± 0.01a
0.085 mmSi concentration (mm)0.084 ± 0.002a 0.08 ± 0.002b0.076 ± 0.001c0.073 ± 0.002d0.071 ± 0.002d0.070 ± 0.003d
0.85 mmin external solutions 0.85 ± 0.01a 0.84 ± 0.00a 0.84 ± 0.01a 0.84 ± 0.00ab 0.83 ± 0.00b 0.83 ± 0.00b
0.085 mmRatios of Si in xylem exudates 2.14 ± 0.01a 4.63 ± 0.16e 7.37 ± 0.22d 9.81 ± 0.39c11.28 ± 0.17b12.58 ± 0.82a
0.85 mmto Si in external solutions 1.08 ± 0.03a 1.46 ± 0.02e 2.15 ± 0.02d 2.54 ± 0.01c 2.80 ± 0.08b 2.88 ± 0.00a

Determination of Si concentration in solutions

The Si concentration in the uptake and xylem exudate solutions was determined by the colorimetric molybdenum blue method at 700 nm. For O. sativa, Z. mays and H. annuus, 0.1 ml, and for B. hispida, 0.2 ml of xylem sap solution was sampled and diluted with 2.0 ml of water, followed by the addition of 1.0 ml of 0.25 M H2SO4 and 1.0 ml of 5% (NH4)6Mo7O24. The reaction solution was mixed well and allowed to stand for 10 min at 25°C. Then, 1.0 ml of 5% oxalic acid was added, followed by the addition of 1.0 ml of 5% ferrus ammonia sulphate [(NH4)2SO4·FeSO4·6H2O] for color development. The reducing agent was prepared by dissolving 5 g of (NH4)2SO4·FeSO4·6H2O into 100 ml of 3 M (3 mol l−1) H2SO4 before use. After 20 min, the absorbance was measured at 700 nm with a spectrophotometer (722; Shanghai Analytical Instruments Factory, Shanghai, China). A standard curve was prepared from Si standard solution.

Statistical analysis

All experimental data reported were the means ± standard deviation (SD) of at least three independent assays with four replicates each and examined statistically by analysis of variance (anova). Statistical significances of the means were determined by Duncan's new multiple range test at a 0.05 probability level, using spss 12.0 for windows (SPSS, Chicago, IL, USA).

Results

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

The time-dependent Si uptake by the four plant species was examined in a short-term solution experiment with both low (0.085 mm Si) and high (0.85 mm Si) external Si concentrations (Fig. 1). The measured Si uptake and Si uptake calculated via transpiration streams increased linearly with time in all the plant species tested at both external Si concentrations. However, measured Si uptake was significantly higher than Si uptake calculated via transpiration streams at both external Si concentrations. The greatest difference was observed between measured Si uptake and Si uptake calculated in O. sativa at both Si concentrations, followed by Z. mays (Fig. 1). Compared with O. sativa or Z. mays, measured Si uptake was not greatly higher than Si uptake calculated via transpiration streams in H. annuus, and especially in B. hispida (Fig. 1), although the difference was still statistically significant (P < 0.01). This is more significant, especially at higher external Si concentrations (0.85 mm Si).

image

Figure 1. Measured silicon (Si) uptake (closed circles) and Si uptake calculated via transpiration streams (open circles) by Oryza sativa (a, b), Zea mays (c, d), Helianthus annuus (e, f) and Benincase hispida (g, h) grown hydroponically in a nutrient solution containing 0.085 or 0.85 mm Si during a 12-h uptake period. At the onset and times indicated in the panels, a 1-ml aliquot of nutrient solution was taken for assay of Si concentration, and meanwhile water loss from transpiration was recorded by weight. At the end of the experiment, the roots were harvested and dry weights were recorded after they had been oven-dried at 60°C for 72 h. Si uptake was measured by both analysis and calculation from transpiration rate on a root dry weight basis within a given time-period based on the data of both Si concentration in solutions and water losses via transpiration from the pots. Significant differences between measured Si uptake and Si uptake calculated via transpiration streams: *, P < 0.05; **, P < 0.01.

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Treatment with metabolic inhibitors (NaF, NaCN and 2,4-DNP) and low temperature inhibited Si uptake in the four plant species tested at all the three external Si concentrations (0.085, 0.85 and 1.7 mm) (Fig. 2). However, the degree of inhibition varied with external Si concentrations and the plant species. At the same external Si concentration, the inhibitory effect on Si uptake was the greatest in O. sativa and Z. mays, followed by H. annuus and B. hispida. On the other hand, the inhibitory effect decreased with increasing external Si concentrations in all plant species, especially in H. annuus and B. hispida. For example, treatment with metabolic inhibitors or low temperature exhibited little inhibitory effects on Si uptake in H. annuus or B. hispida at 1.7 mm Si (Fig. 2), and it is interesting to note that Si uptake was not fully inhibited in O. sativa or Z. mays under these experimental conditions.

image

Figure 2. Silicon (Si) uptake by the different plant species grown hydroponically in a nutrient solution containing 0.085, 0.85 and 1.70 mm Si, as influenced by metabolic inhibitors (NaF, NaCN and 2,4-dinitrophenol) and low temperature [4°C for Oryza sativa (rice), Helianthus annuus (sunflower) and Benincase hispida (wax gourd), and 10°C for Zea mays (maize)] in a 6-h uptake experiment. At the onset and end of the experiment, a 1-ml aliquot of nutrient solution was taken for assay of Si concentration. At the end of the experiment, the roots were harvested and dry weights were recorded after they had been oven-dried at 60°C for 72 h. Si uptake was measured by analysis of the Si in the nutrient solution on a root dry weight basis. Different small letters above the bars denote a significant difference in Si uptake between any two of the treatments at P < 0.05.

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In order to examine the effect of a short-term (6 h) pretreatment of low temperature on the subsequent Si uptake under normal conditions (25°C), we performed the Si-uptake experiments at normal temperature by removing the ice-bath following the experiments at low temperature (4°C for O. sativa, H. annuus and B. hispida, and 10°C for Z. mays). As shown in Fig. 3, the inhibitory effect of low temperature on Si uptake was significantly and greatly eliminated in O. sativa and Z. mays respectively, 16 h after removal of the ice-bath, irrespective of external Si concentrations. In H. annuus and B. hispida, this inhibitory effect of low temperature on Si uptake was also eliminated, especially at low external Si concentrations (0.085 mm Si) (Fig. 3).

image

Figure 3. Silicon (Si) uptake by Oryza sativa (rice), Zea mays (maize), Helianthus annuus (sunflower) and Benincase hispida (wax gourd) in a nutrient solution containing 0.085, 0.85 or 1.7 mm Si at low temperature (4°C for rice, sunflower and wax gourd and 10°C for maize) for 6 h compared with Si uptake at the normal temperature (25°C), which was created by removing the ice-bath. After the ice-bath was removed, the solution temperature rose to 25°C progressively. Si uptake at normal temperature (25°C) was then continued for a further 6 h and assayed. Different letters above the bars in the same plant species denote a significant difference in Si uptake at 25°C and at low temperature at P < 0.05.

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The data presented in Tables 1–4 show the time-course changes of Si concentrations in both xylem exudates and external solutions, and the ratios of Si in xylem exudates to Si in external solutions. The results clearly showed that Si concentration was much higher in xylem exudates than in the external solution throughout the duration of the experiment, regardless of the plant species or of the external Si concentrations used (Tables 1–4). The Si concentration in xylem exudates also increased with increasing duration after decapitation, and the ratios of Si concentrations in xylem exudates to Si in external solutions were higher at the lower (0.085 mm Si) than at the higher (0.85 mm Si) Si concentration. Furthermore, the ratios were much higher in O. sativa and Z. mays than in H. annuus and B. hispida, especially at higher external Si concentrations (Tables 1–4).

Discussion

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

The results from the present study convincingly suggest that the active Si-uptake process prevails in both O. sativa and Z. mays, and the passive component also exists at both lower and higher external Si concentrations with its relative contribution being greater at higher external Si concentrations (Fig. 1). However, for H. annuus and B. hispida, an active component contributes to the uptake and translocation of Si, especially at the lower external Si concentration (i.e. 0.085 mm Si, Fig. 1) despite much greater contribution of the passive component, especially at the higher external Si concentration (i.e. 0.85 mm Si, Fig. 1). More importantly, Si uptake by O. sativa and Z. mays (Si accumulators) and H. annuus and B. hispida (intermediate-type species) tested in the present study was significantly inhibited by treatment with either metabolic inhibitors (NaF, NaCN and 2,4-DNP) or low temperature, particularly at the lower external Si concentration (0.085 mm) (Fig. 2). The reduced Si uptake caused by these metabolic inhibitors and low temperature implies that Si uptake is an ‘uphill’ process against the electrochemical potential gradient, which is driven by the H+-ATP pump (Marschner, 1995) or ATP-binding cassette transporters (Jasinski et al., 2003) and characterized by its high selectivity in uptake and energy-consuming mechanism. As is well established, a metabolic inhibitor, such as 2,4-DNP, acts primarily as an uncoupler of oxidative phosphorylation (Kennedy et al., 1983; Nie & Hill, 1997; Liang et al., 2005c), whereas NaF primarily inhibits glycolysis (Kennedy et al., 1983), and cyanide primarily inhibits cytochrome oxidase (Hewitt & Nicholas, 1963; Kennedy et al., 1983; Nie & Hill, 1997). Evidence supporting the active-uptake mechanism in O. sativa, Z. mays, H. annuus and B. hispida is illustrated by the data given in Fig. 3, which show that reduced Si uptake by low-temperature treatment fully recovered at 16 h after removal of the ice-bath when the solution temperature was elevated to 25°C. On the other hand, the inhibitory effect of metabolic inhibitors and low temperature on Si uptake was still significant at a high external Si concentration (1.70 mm) in both O. sativa and Z. mays, but not in H. annuus or B. hispida (Fig. 2), suggesting that Si-uptake mechanisms are dependent upon both plant species and external Si concentration. However, it is worth noting that Si uptake was not fully inhibited by the treatment with either metabolic inhibitors or low temperature, even in O. sativa, a typical Si accumulator (Fig. 2). These results unequivocally demonstrate that both active and passive mechanisms are operating in Si uptake and transport in the same Si accumulator and intermediate-type species, with their contribution being dependent upon plant species and external Si concentrations (see the Introduction). Therefore, it appears that an active mechanism for Si uptake and transport is involved not only in some members of the Cyperaceae, and wetland and upland species of Gramineae, such as O. sativa and Z. mays (Figs 1–3), but also in some dicots, such as C. sativus (Liang et al., 2005c), H. annuus (Figs 1–3 in this study; van der Vorm, 1980) and B. hispidas (Figs 1–3). Evidence supporting the existence of active uptake and transport mechanisms in all the plant species tested is the data given in Tables 1–4, which show that Si concentrations were constantly higher in xylem exudates than in external solutions.

Time-course changes of Si concentrations in xylem sap (Tables 1–4) were in good agreement with the previous report that the concentration of solutes in xylem sap increased with time (Canny & McCully, 1988). It was also reported that the organic and inorganic composition of xylem sap changed with time (e.g. amino acids and nitrate; see Canny & McCully, 1988). This suggests that the collection of xylem sap for the investigation on composition and concentration of solutes, including Si, should be completed within a shorter time (2 h), and these data for Si concentrations in xylem sap collected after a prolonged time period (after 4 h) (Tables 1–4) should be interpreted with caution.

One may argue that silicic acid collected in the xylem sap may be polymerized at higher Si concentrations, thus affecting the determination of true Si concentrations, because in natural water and soil solutions at pH < 9, Si existing primarily as orthosilicic or monosilicic acid can reach concentrations of only ≈ 0.1–2.0 mm in equilibrium with simple silicon oxides such as quartz, cristobalite or amorphous silica (Gunnarsson & Arnórsson, 2000). It is interesting to note that Si at a concentration of up to 6–8 mm in the xylem exudates is still present in the form of monosilicic acid [Si(OH)4] and not polymerized (Tables 1–4, Casey et al., 2003; Ma et al., 2004), However, obvious precipitation of Si arising from polymerization occurred both in the xylem exudates in a matter of minutes after collection and in the xylem exudates subject to freezing and thawing, with a consequence of considerable reduction of Si concentration (data not shown), implying that both in vitro storage and freezing of xylem exudates have removed silica from the solution as a result of silica precipitation. This is why an immediate assay of Si concentration in the xylem exudates is necessary to avoid analytical errors (see the Materials and Methods).

As shown in Figs 1 and 2, an active component was clearly involved in Si uptake and transport in H. annuus at 0.085 or 0.85 mm Si. However, Si uptake was mainly passive in H. annuus at 1.7 mm Si because it was not significantly inhibited by the treatment with either metabolic inhibitors or low temperature (Fig. 2). It was reported by van der Vorm (1980) that the measured Si uptake was less than Si uptake calculated via transpiration streams in H. annuus treated with higher external Si concentrations (0.5 or 2.7 mm Si), but was several-fold higher than Si uptake calculated at a lower external Si concentration (0.013 mm Si), suggesting the occurrence of an active-uptake process in H. annuus at a lower external Si concentration. It thus appears that the Si-uptake mechanism in the representative intermediate-type species, H. annuus, is dependent upon the external Si concentration.

We have recently reported that Si uptake and transport is active in C. sativus, but rejective in V. faba (Liang et al., 2005c). Measured Si uptake in C. sativus was more than twice as high as calculated from the rate of transpiration. Measured Si uptake in V. faba, however, was significantly lower than the calculated uptake (Liang et al., 2005c), suggesting that Si uptake by V. faba is rejective. Si concentrations in xylem exudates were several-fold higher in C. sativus, but significantly lower in V. faba compared with the Si concentrations in external solutions, regardless of external Si concentration (Liang et al., 2005c). The characterization of Si uptake and transport in B. hispidas (Figs 1–3, Tables 1–4) was similar to that in C. sativus (Liang et al., 2005c).

Clearly, the mechanisms for Si uptake and transport in higher plants are complex, depending upon not only plant species but also external conditions, such as Si concentration. The three modes (accumulator, intermediate and excluder) proposed previously to distinguish Si-uptake patterns (Jones & Handreck, 1967; Takahashi et al., 1990) are unreliable for determining Si-uptake mechanisms, thus needing re-evaluation with caution because these modes do not refer or correlate to a molecular uptake mechanism, such as a channel, pump or carrier, but are based upon measurements of silicon concentration and transpiration rates relative to a preset baseline (Richmond & Sussman, 2003). The Si-uptake mechanism in a specific plant species can be judged by considering both the ratio of measured Si uptake to that calculated from transpiration streams and the responses of plants to metabolic inhibitors and/or low temperature. The ratio of Si in xylem exudates to Si in external solution is of particular importance in elucidating the mechanisms for xylem loading of Si (Liang et al., 2005c).

The first identification of the gene family encoding a Si transporter using a rice mutant, Lsi1, has recently been reported in higher plants (Ma et al., 2006). Further investigations should focus on characterizing Si uptake and xylem loading in the Si-accumulating dicots, such as cucumber and sunflower, at the molecular level.

In conclusion, Si uptake and transport in the four plant species tested is dependent much upon plant species and external Si concentrations. Both active and passive Si-uptake components co-exist in O. sativa, Z. mays, H. annuus and B. hispida, with their relative contribution being dependent upon plant species and external Si concentrations. The contribution of passive component cannot be overlooked in O. sativa and Z. mays, especially at higher external Si concentration; while the active component is also important in H. annuus and B. hispida, especially at a lower external Si concentration. Furthermore, xylem loading of Si is an active process in O. sativa, Z. mays, H. annuus and B. hispida, regardless of external Si concentrations.

Acknowledgements

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

This research was supported by the Distinguished Talent Program of the Chinese Academy of Agricultural Sciences granted to Y. Liang. It was also partly supported by Alexander von Humboldt Foundation Research Fellowship granted to Y. Liang. We are very grateful to the three anonymous referees and the editor who gave constructive comments on this manuscript. We also thank Professor Andrew Smith, The University of Adelaide, Australia for his critical reading of the manuscript and his useful comments.

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  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
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