Silicon reduces sodium uptake in rice (Oryza sativa L.) in saline conditions and this is accounted for by a reduction in the transpirational bypass flow

Abstract

Rice is relatively sensitive to salinity and is classified as a silicon accumulator. There have been reports that silicon can reduce sodium uptake in crop grasses in saline conditions, but the mechanism by which silicon might alleviate salinity damage is unclear. We report on the effects of silicon on growth, gas exchange and sodium uptake in rice genotypes differing in salt tolerance. In non-saline media there were no effects of supplementary silicate upon shoot fresh or dry weight or upon root dry weight, indicating that the standard culture solution was not formally deficient with respect to silicon. Plants grown with supplementary silicate had slightly, but significantly, shorter leaves than plants grown in a standard culture solution. Salinity reduced growth and photosynthetic gas exchange. Silicate supplementation partly overcame the reduction in growth and net photosynthesis caused by salt. This amelioration was correlated with a reduction in sodium uptake. Silicate supplementation increased the stomatal conductance of salt-treated plants, showing that silicate was not acting to reduce sodium uptake via a reduction in the transpiration rate. Silicate reduced both sodium transport and the transport of the apoplastic tracer trisodium-8-hydroxy-1,3,6-pyrenetrisulphonic acid (PTS). This implies that the mode of action of silicate was by partial blockage of the transpirational bypass flow, the pathway by which a large proportion of the uptake of sodium in rice occurs. Mechanisms by which silicate might reduce the transpirational bypass flow directly are discussed.

INTRODUCTION

In a recent review Epstein (1994) drew attention to the facts that silicon is abundant in the soil, is a major constituent of many plants, but is not generally classified as ‘essential’. Although silicon has been regarded as an essential element in a number of species of the Poaceae and Cyperaceae it has not been possible to demonstrate, unequivocally, that concentrations of silicon above the level of contamination are essential (Epstein 1994). Added silicon does increase the erectness of the leaves of rice (Yoshida, Navasero & Ramirez 1969) and there have been reports of interactions between silicon supply and the responses of members of the Poaceae to biotic and to abiotic stresses (Epstein 1994) including salinity (Matoh, Kairusmee & Takahashi 1986; Ahmad, Zaheer & Ismail 1992; Liang et al. 1996).

Salinity is a stress to which rice is relatively sensitive (Maas & Hoffman 1977) and rice is a silicon accumulator (Jones & Handreck 1967). Little is known, however, of the role of silicon in the plant other than as a structural component of the cell walls. The reports of interactions between salinity and silicon referred to above do not provide any mechanistic explanation for the alleviation of salinity damage by silicon, although there have been suggestions that silica deposition in the leaf limits transpiration [and hence salt accumulation, see Matoh et al. (1986)] and that silicon complexes sodium in the root [again reducing salt transport to the shoot, see Ahmad et al. (1992)].

Salt damage to rice seedlings occurs as the result of excessive transport of those ions causing the salinity (commonly, but not exclusively, sodium and chloride) to the leaves. This excessive transport is largely the consequence of leakage along an apoplastic pathway to the xylem (Yeo, Yeo & Flowers 1987; Yadav, Flowers & Yeo 1996; Garcia et al. 1997). Calculations (Yeo et al. 1987; Garcia et al. 1997) show that this bypass flow is sufficient to account for the development of toxic ion concentrations within the leaves of rice. If salinity damage in rice is reduced by silicon, it could, therefore, act directly by reducing the bypass flow. Indirect effects are also possible; any reduction in transpiration could also reduce sodium uptake.

We report that silicon does reduce salinity damage in rice and that this is not mediated by a reduction in transpirational volume flow. However, silicon does reduce the bypass flow (the percentage of the transpirational volume flow that crosses the root entirely in an apoplastic pathway). The mechanisms by which this may occur are discussed.

MATERIALS AND METHODS

Plant material

Seeds of rice (Oryza sativa L.) were obtained from the Central Soil Salinity Research Institute (CSSRI, Karnal, India), CSSRI Regional Research Station (Anand, India) and the International Rice Research Institute (IRRI, Philippines). Following preliminary screening, we concentrated on using the genotypes CSR 10 (salt tolerant), GR 4 (salt sensitive) and IR 36 (intermediate tolerance, grown throughout Asia). Seeds were imbibed for 24 h in aerated water and sown on Nylon mesh supported on Perspex grids floated on culture solution. Seedlings were transplanted at 7 d into boxes (3 dm³) or individual tubes (50 cm³), both painted black, containing culture solution and grown in a growth cabinet (Model E15, Conenviron, Winnepeg, Canada). The growing conditions were a 12 h photoperiod of 400–500μmol m^–2 s^–1 photosynthetically active radiation (HQ1 metal halide, Newry & Eyre, Brighton, UK) at 27 °C and 1·0–1·5 kPa saturation vapour pressure deficit (SVPD). The temperature during the dark period was 25 °C and the SVPD was 0·6 kPa.

The culture solution used for growing the rice was based on that described by Yoshida et al. (1976) with modifications made over the years (e.g. Yeo & Flowers 1984; Khatun & Flowers 1995) to reduce phosphate toxicity (which had been observed in some experiments) and the sodium concentration in the basic solution (to almost zero, to allow us to distinguish between sodium uptake following salinization and uptake from the non-salinized culture solution). The concentrations of the nutrients added to the solution and their effective concentrations in the solution, as modelled by the program MINTEQA2 (MINTEQA2 1991), are shown in Table 1.

Table 1.  . The composition of the culture solution according to the formulation of Khatun & Flowers (1995), both theoretical (calculated from the added amounts) and as modelled by the program MINTEQA2 ( MINTEQA2 1991). The modelled concentrations of the major and minor nutrients were unaltered by the addition of sodium silicate. The addition of sodium chloride (50 mM) caused a small rise in the concentration of Mn (to 0·33 μM) and a fall in that of silicate (to 96·9 μM)

Treatment with NaCl and silicon

The salinity treatment was NaCl (50 mM) and the silicon treatment was sodium silicate (BDH, 25·5–28·5% SiO₂) solution (added in sufficient quantity to produce a final concentration of 3 mM, slightly in excess of the solubility of silicic acid in water which was reported to be ≈ 2 mM at 25 °C; Raven 1983). MINTEQA2 predicted that the addition of silicate to the culture solution resulted in a precipitation of quartz and an effective silicate concentration of ≈ 0·1 mM (Table 1). We were concerned that during the addition of our stock solution of sodium silicate, when the pH could rise to a value of around 9·5, there would be precipitation of other nutrients. Under such circumstances, haematite, hydrapatite, quartz, tremolite and solid manganese phosphate are predicted by the model to precipitate. However, on adjustment of the pH to 4·5, any solid tremolite and hydrapatite redissolve. Table 1 shows the concentrations of nutrients in the solution after the addition of sodium silicate. The addition of sodium chloride (50 mM) caused a small rise in the concentration of Mn (to 0·33 μM) and a fall in that of silicate (to 96·9 μM).

Seven or 14 d after the treatments, the plants were harvested and weighed. For sodium determination, shoots were extracted in acetic acid (100 mM) for 2 h at 90 °C and the extract analysed by atomic absorption spectroscopy (Unicam 919, Cambridge, UK).

Bypass flow

Bypass flow was determined using a fluorescent, membrane-impermeant dye, the trisodium salt of 8-hydroxy- 1,3,6-pyrenetrisulphonic acid (PTS); former nomenclature 3-hydroxy-5,8,10-pyrenetrisulphonic acid and supplied as Pyranin (Bayer UK). Bypass flow was estimated following methods described by Yeo et al. (1987) and Yadav et al. (1996). Briefly, the seedlings were transplanted into boxes in which the solution could be drained and replaced with the minimum root disturbance. At 14 d, the plants (either treated at 7 d with silicon or left untreated) were exposed to NaCl (50 mM) and PTS (30 mg dm^–3). After 24 h, the shoots were extracted in 10 cm³ of distilled water for 2 h at 90 °C and the fluorescence measured at λ_excitation = 403 nm and λ_emission = 510 nm (Perkin Elmer, LS 3). Sodium content was also measured in the extracts by atomic absorption spectroscopy. Control plants, which had not been exposed to PTS, were extracted to check for natural fluorescence, which was always negligible at this combination of excitation and emission wavelengths. Transpiration was estimated gravimetrically over a period of 48 h for plants in individual Pyrex tubes.

Gas exchange

Instantaneous measurements of the exchange of carbon dioxide and water between leaves and the surrounding air were made in a temperature-controlled glasshouse under natural daylight supplemented with high-pressure sodium illumination (SON/T, Camplex Plantcare Ltd). The plants, which had been raised in a growth chamber, were acclimated to the glasshouse for 48 h prior to measurements. Measurements of carbon dioxide and water vapour exchange were made on the fourth leaf by infrared gas analysis in an open system at ambient partial pressures of carbon dioxide and water vapour (CIRAS-1, PP Systems, UK). At the time of measurement, the leaf temperature was 22–28°C and the light flux density was 600–800μmol m^–2 s^–1. Net assimilation and stomatal conductance were calculated by the on-board software. Subsequently, measured leaves were excised, dried, weighed, extracted in acetic acid, and the sodium concentration determined.

RESULTS AND DISCUSSION

Effects of silicon on growth with and without NaCl

The effect of added silicon on growth was investigated in the three selected genotypes in non-salinized culture solution (Table 2). Without added silicon, the concentration of silicon in the complete culture solution was below the quoted sensitivity (1 part per million) of the Unicam SP 919 atomic absorption spectrometer and so was less than 35 μM. There was no significant effect of added sodium silicate on shoot fresh or dry weight, nor on root dry weight, implying that the plants were not nutrient deficient with respect to silicon in the modified Yoshida culture solution over a treatment period of 7 d. Therefore, we assumed in our studies that the silicon available from the water and the chemicals used to prepare the culture solution satisfied any specific micronutrient requirement for silicon. Matoh et al. (1986) also found that silicon did not significantly improve growth without salt, even though the silicon content of the shoots was increased 50-fold. If precautions are taken to reduce the silicon supply in ‘minus-silicon’ treatments, a positive response of growth to silicon fertilization can be demonstrated (Ma & Takahashi 1993). Calcium silicate, in the form of a slag, is used in Southeast Asia as a fertilizer for paddy fields (Ma & Takahashi 1993).

Table 2.  . The effect of silicon on the growth of three genotypes of Oryza sativa L. Plants were grown in culture solution without NaCl or added silicate for 14 d and then treatments [with and without sodium silicate and NaCl (50 mM)] were applied for the times indicated. Mean values (n not less than 10) and analysis of variance are given

Although there was no effect upon weight, there was a reduction of ≈10% (significant at P < 0·001) in plant height (i.e. length of the longest leaf) with the addition of sodium silicate (Table 2). This result contrasts with that of Yoshida et al. (1969) who reported a small (c. 5%) increase in leaf length using pooled data across three levels of nitrogen supply and five varieties. Further investigation would be needed to account for the effect of silicon on leaf length, but high levels of deposition of silicon in the cell walls may lead to wall hardening at an earlier stage of cell elongation than in the absence of added silicon.

The growth experiment was repeated for shoot dry weight over a longer period of 14 d (Table 2), but again there were no significant effects of added silicon. When plants were grown in salinized (50 mM NaCl) solution for 14 d, then the addition of silicate did significantly (P = 0·001) counteract the growth reduction caused by salt (Table 2: an average of 70% in the absence of and 64% in the presence of silicon).

Effect of silicon on sodium uptake from saline culture medium

Silicon reduced sodium uptake from saline solution into the shoots of all genotypes (Table 3). The least responsive to silicon was the tolerant CSR 10 and the most responsive was the sensitive GR 4: IR 36 was intermediate in both respects.

Table 3.  . Effect of silicon (sodium silicate) on shoot sodium content of three cultivars of Oryza sativa L. grown for 7 d in culture solution salinized with NaCl (50 mM). Treatments were applied at 14 d. Means and standard deviations (n = 10) are given. The effect of silicon is significant at P < 0·05 in all cases

Gas exchange

Photosynthetic gas exchange in rice is known to be highly sensitive to salt accumulation in the leaves (Yeo, Caporn & Flowers 1985) and consequently the amelioration of sodium uptake by silicon might be expected to enhance photosynthesis. This was investigated with the three selected genotypes and the results are shown in Table 4. The addition of silicon significantly (P < 0·001) reduced leaf sodium concentration and increased both the assimilation rate and stomatal conductance (Table 4). The relative responsiveness of the shoot sodium concentration of the three genotypes, CSR 10, GR 4 and IR 36, to silicon seen in Table 3 (reductions of 14·5%, 63·7% and 22·5%, respectively) was confirmed (Table 4: 14·2%, 65·8% and 24·3% for CSR 10, GR 4 and IR 36, respectively). The increased assimilation rate of salt-grown plants in the presence of silicon could broadly be accounted for by an increase in stomatal conductance: conductance and assimilation rate were increased proportionately with the addition of silicon (Fig. 1). Our results do not suggest a mechanism for this. One hypothesis had been that silica bodies act as ‘windows’ that enhance light transfer to the mesophyll. However, Agarie et al. (1996) could find no evidence that silica bodies affected the optical properties of rice leaves.

Table 4.  . Effect of added silicon (sodium silicate) on leaf sodium concentration and photosynthetic gas exchange (A; net assimilation rate, G_s; stomatal conductance) in three genotypes of Oryza sativa L. grown in culture solution salinized with NaCl (50 mM). Means and standard deviations (n = 20) are given

Figure 1. . Effect of added silicon (sodium silicate) on assimilation rate and stomatal conductance of leaves of Oryza sativa L. growing in culture solution salinized with NaCl (50 mol m^–3). Pooled data of three genotypes (n = 60 for plus and minus added silicon).

The addition of silicon increased stomatal conductance (instantaneous single-leaf measurement) in the presence of salt, the effect differing with variety in the order GR 4 > IR 36 > CSR 10. Whole-plant transpiration (gravimetric measurement) was significantly higher in the silicon-supplemented plants of IR 36 and GR 4 than in those without silicon, although there was not a significant effect with the more salt-tolerant CSR 10 (Table 5). In no case did silicon supplementation reduce transpiration in salinized plants. Therefore, silicon did not reduce sodium transport as a consequence of reduced transpiration. In non-saline media, silica has been reported to reduce transpiration (Jones & Handreck 1967; Ma & Takahashi 1993), but in saline conditions any such effect must have been subordinate to effects mediated by reduced leaf sodium concentration.

Table 5.  . Effects of silicon on transpiration by salt-treated plants over a period of 48 h following the imposition of treatments at 14 d. Seedlings were grown in individual tubes and transpiration was monitored gravimetrically. Means and standard deviations (n = 12 or 24) are given

The increase in stomatal conductance and net assimilation due to silicon under saline conditions (Table 4) was associated with a positive effect of silicon on growth under these saline (although not under non-saline) conditions (Table 2). Because silicon decreased sodium uptake despite an increase in transpiration, this implies that silicon had a direct effect upon sodium transport across the root.

It has been suggested, but without direct evidence, that silicates can complex sodium and prevent its transport into the plant (Ahmad et al. 1992). However, complexing of sodium within the root could not account for the reduction in sodium transport observed. The concentration of sodium in the shoot of GR 4 was reduced by 500 μmol g^–1 dry weight (Table 4). If this quantity of salt were stored uniformly throughout the cells of the root it would produce a concentration of over 2000 mM. If the salt was complexed in the cell walls alone then this would be equivalent to a concentration at least 10 times greater and many times the aqueous solubility of NaCl.

Bypass flow and sodium uptake

The effect of silicon on the transport of sodium and PTS (as a tracer for the bypass flow) was investigated to provide some insight into the mechanism of action of silicon (Table 6). In the three selected genotypes, silicon significantly (P = 0·001) reduced the quantities of both PTS and sodium in the shoots. This implies that silicon blocked both the movement of sodium and of water at sites of apoplastic leakage. The reduction in PTS transport by silicon suggests that it is partially blocking the bypass flow across the root in rice. The bypass flow is at most a few per cent of the transpirational volume flow (Yeo et al. 1987; Garcia et al. 1997). It is important to emphasize that this is the proportion of water leaking to the xylem without ever crossing a membrane – we do not imply that this is a quantification of water movement in the apoplast [see Steudle & Peterson (1998)]. Even a complete blockage of the bypass flow would not offset the increase in total transpirational volume flow that resulted from the increase in stomatal conductance brought about by the reduced leaf sodium concentration. It is essential to distinguish between the total flux of water through the root and the small proportion of uncontrolled flux in the apoplastic pathway that carries a high proportion of the sodium.

Table 6.  . Effect of added silicon (sodium silicate) on sodium and trisodium-8-hydroxy-1,3,6-pyrenetrisulphonic acid (PTS) transport by three genotypes of Oryza sativa L. in culture solution salinized with NaCl (50 mM). Means and standard deviations (n = 20) are given

The physical basis of the bypass pathway has not yet been identified in rice, but the literature suggests that it is likely to be in root apices before the completion of rhizodermal and endodermal differentiation and at sites where lateral root development breaches both endodermal and rhizodermal barriers: see, for example Bell & McCully (1970) and Dumbroff & Pierson (1971). Although the endodermis rather than the rhizodermis was the barrier to PTS movement in aeroponically grown maize (Steudle & Peterson 1998), our observations show that the mature, intact rhizodermis is a potent barrier to apoplastic movement in rice (Plant, Cell and Environment20(9) front cover).

Silicon is a major structural component of cell walls in those species that readily accumulate it. Although taken up as monosilicic acid, the fate of most silicon in the plant is to be deposited as insoluble silica which has a role in the cell wall as a compression-resisting element analogous to lignin (Raven 1983). More recently, Inanaga & Okasaka (1996) provided evidence of the formation of silicon–organic complexes in the shoots of rice. As a structural element, silicon is found mostly in the shoot and predominantly at the termini of the transpirational pathway. However, silicon has been found in the endodermis of roots of some species where it is confined principally to the inner tangential wall (Sangster 1978): suggested roles in Sorghum were in mechanical strengthening and resistance of water loss during severe soil water stress (Sangster 1978). Silica has also been found to be deposited in the inner tangential and radial walls of the endodermis in rice roots (Parry & Soni 1972). Further studies are needed to determine whether silicon is used to enhance endodermal integrity or whether silica deposition is only a consequence of permeability changes during endodermal development and of localized saturation of the solubility of silicic acid. The accumulation of silica in the endodermis of Sorghum bicolor was greater in the proximal regions and negligible in the distal regions, implying that endodermal development reduced its ability to allow the passage of water containing silicic acid (Sangster & Parry 1976). This mirrors the longitudinal distribution of calcium transport (Robards et al. 1973) suggesting that silicon transport, like calcium transport, is predominantly apoplastic.

A possible explanation with respect to the bypass flow is the production of colloidal precipitates of silica in the apoplast. Yoshida, Ohnishi & Kitagishi (1962) concluded from infrared absorption spectroscopy and hot-water solubility that silicon in rice was mostly in the form of silica gel or polysilicic acids. Because the supply to the plant was the silicate ion, polymerization must, therefore, have occurred within the plant. Polymerization of silicate is caused by an increased concentration and proceeds via colloidal silica (Yoshida et al. 1962). As well as the deposition of amorphous silica, it is also probable that colloidal silica is formed in the apoplast of roots and this could help to block leakage paths such as around emerging laterals. Yeo & Flowers (1984) observed non-osmotic effects of polyethylene glycols (PEG) on sodium uptake in rice: concentrations too small to affect transpiration reduced sodium uptake. At the time, the bypass flow was dismissed as an explanation of the PEG effect because: (1) the quantitative importance of the bypass pathway was not appreciated; and (2) PEG bound to membranes and could, therefore, have a stabilizing effect analogous to that of calcium (Yeo & Flowers 1984). Subsequent measurements (Yeo et al. 1987; Yadav et al. 1996) permitted the additional explanation that PEG molecules also acted by blocking the bypass flow as the size of the molecule is comparable to the pore size of plant cell walls (Carpita et al. 1979).

The differences in response of the genotypes used to silicon would then imply that these genotypes differ in the size of the bypass flow and/or in wall properties that affect the polymerization of silicate.

Genetically determined differences in the bypass flow in rice have been demonstrated. Yadav et al. (1996) showed that pure line selections differing in sodium transport differed proportionately in PTS transport and concluded that selection for differences in sodium transport was due to selection of heritable differences in bypass flow.

The deposition of silica in the endodermis and rhizodermis, and the polymerization of silicate via colloidal silica to silica gel or polysilicic acid throughout the root apoplast, offer possible mechanisms by which silicon could physically block the transpirational bypass flow across the roots of rice. Silica gel is formed on acidification of aqueous solutions of silicic acids, a process that might be facilitated by activity of the root P-ATPase. This could account for the reduction in sodium uptake caused by silicon which is not accountable by the effects on transpiration.

Further studies will seek to correlate the sites and nature of silicon accumulation in the roots of rice with sites of sodium uptake and bypass leakage.

Acknowledgements

This work was supported by collaborative projects funded by the Department for International Development (DfID, formerly the ODA, London, UK), International Institute for Land Reclamation and Improvement (IILRI, Wageningen, The Netherlands) and the Indian Council of Agricultural Research (ICAR, New Delhi, India). Gururajah Rao is grateful to IILRI and to ICAR for a fellowship to the University of Sussex. The award of a Royal Society Fellowship to N. Senanayake is gratefully acknowledged. We thank Dyestuffs Division, Bayer UK for the gift of PTS.