Characterization of silicon uptake by rice roots

Transcription

1 Research Characterization of silicon uptake by rice roots Blackwell Publishing Ltd. Kazunori Tamai and Jian Feng Ma Faculty of Agriculture, Kagawa University, Ikenobe 2393, Miki-cho, Kita-gun, Kagawa , Japan Summary Author for correspondence: Jian Feng Ma Tel: Fax: Received: 8 January 2003 Accepted: 12 March 2003 doi: /j x Rice (Oryza sativa) is a typical Si-accumulating plant and it has been suggested that it has a specific uptake system for silicic acid in the roots. Here, we characterized this specific system in rice roots. The ability of rice roots to take up Si was much higher than that of other gramineous species. A kinetic study indicated that Si uptake was mediated by a type of proteinaceous transporter; the K m value was estimated to be 0.32 mm, suggesting that the transporter had a low affinity for silicic acid. Si uptake increased linearly with time, but pretreatment with Si did not affect the uptake of Si, suggesting that the system for Si uptake was not inducible. Mercuric chloride and phloretin, significantly inhibited Si uptake, but 4,4 -diisothiocyanato-stilbene-2,2 -disulfonic acid (DIDS) did not. Mercuric chloride and phloretin also inhibited water uptake, but to a lesser extent. Si uptake was unaffected by the presence of boric acid. Taken together, the data indicate that the uptake of Si by rice roots is a transportermediated process and this transporter contains Cys residues but not Lys residues. Key words: kinetics, inhibitor, Oryza sativa (rice), Si accumulator, transporter, uptake. New Phytologist (2003) 158: Introduction Silicon (Si) is the most abundant mineral in soil and all plants contain Si in their tissues. However, the Si content of the plant varies greatly with the plant species, ranging from 0.1 to 10.0 Si% in d. wt. The difference in Si content has been ascribed to the ability of the roots to take up Si (Takahashi et al., 1990). By comparing the speed of Si uptake with that of water uptake, three modes of Si uptake, active (higher than water uptake), passive (similar with water uptake), and rejective (slower than water uptake), have been suggested for plant species having different Si content (Takahashi et al., 1990). Rice is a typical Si-accumulating plant and is able to accumulate Si at the level of up to 10% Si of shoot d. wt, depending on the Si concentration in the soil solution (Ma et al., 2001b). The Si content in rice shoot is several-fold higher than the essential macronutrients such as N, P and K (Savant et al., 1997). The high accumulation of Si in the top is required to alleviate multiple stresses including biotic and abiotic stresses (Epstein, 1994; Savant et al., 1997; Ma et al., 2001b; Ma & Takahashi, 2002; Ma, 2002). High Si accumulation in rice is controlled by Si-uptake capacity of root. When the rice roots were cut off, the Si accumulation in the shoot was similar to that in the species with a low Si content such as tomato (Okuda & Takahashi, 1962a). However, the mechanism responsible for the high uptake ability of rice roots is poorly understood. Rice plants take up Si in the form of unchanged silicic acid, the major form in the soil under ecologically or agriculturally important condition since the pk a1 of silicic acid is 9.82 at 25 C (Takahashi & Hino, 1978). The permeability coefficient of silicic acid for the plasma membrane is estimated to be m s 1 (Raven, 2001). This low permeability cannot explain the high Si content of rice shoot, suggesting that silicic acid is transported across the plasma membrane not by passive diffusion, but by active uptake in rice roots. There is evidence that the Si uptake by rice roots is an active process. The Si uptake is significantly inhibited by metabolic inhibitors such as NaCN, 2,4-dinitrophenol (Okuda & Takahashi, 1962b) and a low temperature (Ma et al., 2002). The uptake is unaffected by transpiration (Okuda & Takahashi, 1962a). Takahashi (1982) investigated the effect of nutrient anions and cations on the Si uptake by rice. In the presence of each nutrient at 5 mm (20 times concentration of Si) in a nutrient New Phytologist (2003) 158:

2 432 Research solution, the Si uptake during a 72-h period is slightly inhibited by the nitrate anion, but not by Cl, SO 4 2, and H 2 PO 4. Among cations including K +, NH 4 +, Ca 2+ and Mg 2+, only NH 4 + caused inhibition (by 30%). The Si concentration in the rice xylem sap is several hundred-fold higher than that in the external solution after a short exposure time (Okuda & Takahashi, 1962a), indicating that silicic acid is transported against a concentration gradient from the medium into the symplasm or from the symplasm into the xylem. All these observations suggest that a specific uptake system exist in the rice roots which facilitates the transport of silicic acid across the plasma membrane. In the present study, this uptake system is characterized. Our results show that uptake of silicic acid by rice roots is mediated by a kind of transporter containing Cys residues, but not Lys residues, and having a low affinity for silicic acid. Materials and Methods Comparison of Si-uptake ability between rice and other gramineous plants Silicon uptake was compared among various gramineous plants. Seeds of rice (Oryza sativa L. cv. Oochikara), wheat (Triticum aestivum L. cv. Atlas 66), rye (Secale cereale L. cv. Kings), triticale (Triticosecale Wittmark. cv. ST2), corn (Zea mays L. cv. Honey Bantam), sorghum (Sorghum bicolor Moench cv. Sorgou), and barley (Hordeum vulgare L. cv. Kearney) were surface-sterilized in 0.5% (v/v) NaOCl for 15 min, rinsed and soaked in deionized water overnight at 25 C in the dark. The seeds were then transferred to a net floated on 0.5 mm CaCl 2 solution in a plastic container. On day 6, the seedlings were transferred to a 10.0-l plastic pot containing one-fifth -strength Hoagland solution (ph 5.6) in a growth cabinet with a day/night temperature regime of 25 C (14 h)/20 C (10 h). The solution was renewed every 3 d. After a 17-d preculture, the seedlings were used for the uptake experiment. The uptake experiment was conducted in a 0.5-mM CaCl 2 solution (ph 5.6) containing 0.6 mm Si as silicic acid for 24 h at 25 C. Silicic acid was prepared by passing potassium silicate through a cation-exchange resin (Amberlite IR-120B, H + form) (Organo, Tokyo). Before and after the uptake experiment, a 1-ml aliquot of the solution was taken for determination of Si concentration and transpiration (water loss) was also recorded. At the end of the experiment, the roots and shoots were harvested separately and their f. and d. wts were recorded. Each experiment was performed using three replicates for each species. previous paper (Ma et al., 2001a). On day 11, the seedlings (two seedlings per pot) were allowed to take up Si in the nutrient solution ( 1 / 2 (Kimura B, ph 5.6)) containing silicic acid at various concentrations in a 50-ml plastic bottle. The uptake period was 6 h. The amount of Si uptake was measured as described above. To examine whether the uptake system is induced by silicic acid, we transferred the rice seedlings (17-d-old) precultured in the nutrient solution with or without 1.5 mm silicic acid for 24 h, to the nutrient solution containing 1.5 mm silicic acid. Then at intervals (see Fig. 3), a 1-ml aliquot of the solution was taken for determination of Si concentration. Transpiration (water loss) was also recorded at each sampling time. Effect of transporter inhibitors on Si uptake The effect of transporter inhibitors was compared between a wild-type rice (cv. Oochikara) and a rice mutant defective in Si uptake (GR1) (Ma et al., 2002). Rice seedlings (20-d-old) were planted in the nutrient solution containing 0.75 mm silicic acid in the absence or presence of 50 µm, 100 µm 4,4 -diisothiocyanato-stilbene-2,2 -disulfonic acid (DIDS), or 100 µm phloretin., DIDS and phloretin were first dissolved in methanol and the final concentration of methanol in the solution was adjusted to 0.2%. Methanol was also added to the solution without any inhibitors at a similar concentration. A preliminary experiment showed that this concentration of methanol did not have an effect on Si uptake. After 6 h, the plants were harvested and the Si uptake was measured as described above. We further examined the effect of on Si uptake with time using a wild-type rice and a mutant (GR1). 20-d-old seedlings were exposed to the nutrient solution (ph 5.6) containing 0.5 mm silicic acid in the absence or presence of Kinetics of Si uptake Seedlings of rice (cv. Oochikara) were prepared as described above but grown in a one-half-strength Kimura B solution. The composition of the nutrient solution is reported in our Fig. 1 Silicon uptake by various gramineous plants from a solution containing 0.6 mm Si as silicic acid during a 24-h period. Data shown are means ± SD. New Phytologist (2003) 158:

3 Research µm, and at intervals (see Fig. 5), the uptake of Si and water was monitored as described above. The effect of 2-mercaptoethanol on recovery of - induced inhibition of Si uptake was investigated in the nutrient solution containing 0.5 mm silicic acid and 50 µm in the presence of 2-mercaptoethanol ranging from 0 to 10 mm. 4-wk-old seedlings were used in this experiment and the uptake period was 6 h. Effect of B on Si uptake The effect of B (boric acid) on the Si uptake was examined in the nutrient solution containing 0.5 mm silicic acid in the presence of boric acid ranging from 0 to 5 mm. The seedlings (22-d-old) were allowed to take up Si for 6 h. In all uptake experiments described above, one-half-strength Kimura B solution was used as a basic solution. The solution ph was adjusted to 5.6 with 0.1 M HCl or 0.1 M KOH. The experiments were performed in a 50-ml plastic bottle (two seedlings each) with three replicates. Determination of Si concentration The Si concentration in the solution was determined by the colorimetric molybdenum blue method (Ma et al., 2003). Briefly, to 2.7 ml H 2 O, 0.2 ml sample was added, followed by 1.5 ml of 0.26 N HCl, 0.2 ml of 10% (NH 4 ) 6 Mo 7 O 24, 0.2 ml of 20% tartaric acid and 0.2 ml reducing agent. The reducing agent was prepared by dissolving 1 g Na 2 SO 3, 0.5 g 1-amino-2-naphthol-4-sulfonic acid and 30 g NaHSO 3 in 200 ml water. After 1 h, the absorbance was measured at 600 nm with a spectrophotometer ( Jasco, Japan). Results and Discussion According to the distribution of Si accumulators in the phylogenetic tree of higher plants, only the species in Cyperaceae and Gramineae in monocots are high Si-accumulators (Ma & Takahashi, 2002). In the present study, the Si-uptake ability was compared among gramineous species during a period of 24 h under the same condition. The Si uptake by rice roots was much higher than that by other gramineous species including wheat, triticale, sorghum, rye, maize and barley (Fig. 1). This result confirms that rice roots have a different uptake system for silicic acid from other species. The kinetics of this uptake system were then examined. When the Si concentration in the nutrient solution was low, the Si uptake by rice roots increased with increasing external Si concentration (Fig. 2). However, the Si uptake was saturated at a concentration of 1.28 mm Si (Fig. 2). This indicates that Si uptake is mediated by a kind of proteinaceous transporter. Based on this curve, the V max and K m values were estimated to be 6.2 mg Si g 1 root d. wt h 1 and 0.32 mm, respectively. The value of K m for silicic acid was times higher than that for other minerals. For example, the K m for phosphorus is 1 5 µm (Kochian, 2000). This result indicates that the Si transporter in rice roots has a low affinity for silicic acid. One possible explanation for this low affinity is that unlike other minerals, Si is abundant in soil solution and a transporter with high affinity is unnecessary. The Si concentration in soil solution ranges from 0.1 to 0.6 mm (Epstein, 1994). We also measured the Si concentration in a neutral soil and an acidic soil; the Si concentration was 0.6 mm and 0.35 mm, respectively. These Si concentrations are almost 100 times higher than that of phosphorus (Epstein, 1994). Some minerals have two uptake systems, a low-affinity system and a high-affinity uptake system (Epstein et al., 1963). For example, a high-affinity uptake system for K is seen at a low concentration range (0 0.2 mm), while a low-affinity uptake system at a higher external K concentration range Fig. 2 Silicon uptake by rice roots from a solution with various Si concentrations. The seedlings were cultured for 6 h. Data shown are means ± SD. Fig. 3 Change in the Si uptake by rice roots precultured in the solution with Si (1.5 mm Si) or without Si for 1 day. The uptake experiment was conducted in a nutrient solution containing 1.5 mm Si as silicic acid. Data shown are means ± SD. Inlet shows uptake rate. New Phytologist (2003) 158:

4 434 Research Fig. 4 Effect of transporter inhibitors, mercuric chloride, phloretin and DIDS on Si (a) and water uptake (b) by a wild-type rice and a rice mutant defective in Si uptake (GR1). The uptake experiment was conducted in a nutrient solution containing 0.5 mm Si as silicic acid for 6 h in the presence of 50 µm, 100 µm phloretin or 100 µm DIDS. Values are means ± SD. ( 50 mm). However, it is impossible for Si to perform the uptake experiment at higher Si concentrations. This is because unlike most minerals, Si (silicic acid) starts to polymerize when the concentration is higher than 2.0 mm at 25 C. It is well known that the uptake of some minerals is inducible. A typical example is nitrate (Siddiqi et al., 1989). We examined whether the Si uptake is inducible by comparing the Si uptake between rice plants precultured with and those without silicic acid. The uptake of Si increased linearly with time (Fig. 3), but pretreatment with silicic acid did not cause any change in Si uptake rate (Fig. 3). No difference was observed in the Si uptake between the seedlings precultured with and without Si at a low external Si concentration (0.15 mm) (data not shown). These results suggest that the uptake of Si by rice roots is not inducible. The Si uptake system was further characterized by examining the effect of three transporter inhibitors for Si uptake in a wild-type rice and a mutant (GR1). GR1 is a mutant in which the active uptake system for silicic acid was disrupted (Ma Fig. 5 Time course of Si (a) and water (b) uptake in the presence and absence of mercuric chloride in a wild-type rice and a rice mutant defective in Si uptake (GR1). Seedlings of the wild-type and the mutant were exposed to a nutrient solution containing 0.5 mm Si as silicic acid in the presence and absence of 50 µm. Data shown are means ± SD. et al., 2002). In the wild-type rice, mercuric chloride and phloretin inhibited the Si uptake by 91.0% and 70%, respectively (Fig. 4a), but DIDS hardly affected the Si uptake (Fig. 4a). The Si uptake was hardly inhibited by either inhibitor in the mutant. The uptake of water was inhibited by and phloretin in both the wild-type and the mutant (Fig. 4b), and the inhibition was similar (approx. 50% inhibition) in the two lines (Fig. 4b). Mercuric chloride is a channel inhibitor which has been extensively used in the water uptake study (Maurel, 1997). Recently, it was reported that also inhibited the uptake of boric acid (Dordas & Brown, 2001). It is suggested that blocks water channels via oxidation of cystein residue(s) proximal to the aqueous pore and subsequent occlusion of the aqueous pore by the large mercury ion. On the other hand, phloretin is a nonspecific membrane inhibitor that has been used extensively in animal studies to inhibit water, urea, and glycerol transport through aquaporins and other channels (Macey, 1984). It has been suggested that phloretin binds with one of the sites on the transporter and inhibits its function although the exact mode of action of phloretin remains unclear (Krupka, 1985). DIDS is also a New Phytologist (2003) 158:

5 Research 435 channel inhibitor which inhibits both water and nonelectrolyte movement as does (Macey, 1984). DIDS has two isothiocyanate groups, that can react with lysine residues, annulling the transport properties of the protein (Sitsapesan, 1999). A strong inhibition of Si uptake by and absence of inhibition by DIDS in rice roots suggests that the Si transporter contains cystein residue(s), but not lysine residue(s) (Fig. 4). Less inhibition of water uptake by compared with Si uptake suggests that the -induced inhibition of Si uptake was not caused by the inhibition of water uptake. This is supported by the result of the experiment with the mutant (Fig. 4). Although water uptake was inhibited by both in the mutant and in the wild type, the Si uptake was hardly affected by in the mutant (Fig. 4). The time-course experiment further indicates that the uptake system for silicic acid is different from that for water in rice roots. Exposure to 50 µm for 1 h immediately resulted in a 73% inhibition of Si uptake in the wild-type rice (Fig. 5a). However, the inhibition of water uptake was not observed at 1 h. At 2 h, water uptake was inhibited by by 35% (Fig. 5b), in contrast to a 83% inhibition of Si uptake (Fig. 5a). In the mutant, Si uptake was hardly inhibited by although the water uptake was inhibited as in the wild-type (Fig. 5). The effect of 2-mercaptoethanol on the recovery of - inhibited Si and water uptake was examined. 2-Mercaptoethanol has a high affinity for Hg ions and can remove Hg ions that are bound to cysteine residues in transporter proteins. The -inhibited Si uptake was gradually recovered with increasing concentration of 2-mercaptoethanol in the uptake solution (Fig. 6a). However, the -inhibited Si uptake was not completely recovered in the presence of 10 mm 2-mercaptoethanol. By contrast, the -inhibited water uptake was fully recovered by the presence of 10 mm 2- mercaptoethanol. These results confirm that Cys residue(s) is contained in the Si transporter and that the uptake system for Si differs from that for water. As can also affect the metabolic status of the plant and the Si uptake is an energydependent active process (Ma & Takahashi, 2002), partial recovery of the -inhibited Si uptake by 2-mercaptoethanol could be attributed to the inhibitory effect of on metabolism. Boron is also taken up by the roots in the form of an undissociated molecule, boric acid. Therefore, there is a possibility that B is taken up by the same transporter as Si. We examined the Si uptake in the presence of various B concentrations. In the presence of B up to 10 times of Si, the uptake of Si was unaffected (Fig. 7). It is unlikely that the same transporter works for B and Si. It has recently been reported that B has been transported across the plasma membrane through aquaporins from a series of inhibitor experiment in squash (Dordas & Brown, 2001) and a transporter gene encoding for xylem loading of B has been cloned in Arabidopsis thaliana (Takano et al., 2002). For example, the inhibition Fig. 6 Effect of 2-mercaptoethanol on the recovery of - inhibited Si (a) and water (b) uptake by rice roots. Seedlings were exposed to a nutrient solution containing 0.5 mm Si as silicic acid in the presence or absence of 50 mm, various concentrations of 2-mercaptoethanol. The uptake period was 6 h. Data shown are means ± SD. Fig. 7 Effect of B as boric acid on the uptake of Si by rice roots. Seedlings were allowed to take up Si for 6 h from a solution containing 0.5 mm Si as silicic acid in the presence of various B concentrations. Data shown are means ± SD. of B uptake by is similar to that of water uptake. However, our inhibitor experiments clearly showed that Si uptake is much more inhibited by than water uptake is (Figs 5 and 6). New Phytologist (2003) 158:

6 436 Research In conclusion, Si uptake by rice roots is a transportermediated process and this transporter contains Cys residues but not Lys residues and has a low affinity for silicic acid. Acknowledgements This study was supported in part by a grant for Rice Genome Research projects from The Ministry of Agriculture, Forestry and Fisheries of Japan and by NSFC (no to J. F. Ma). References Dordas C, Brown PH Evidence for channel mediated transport of boric acid in aquash (Cucurbita pepo). Plant and Soil 235: Epstein E The anomaly of silicon in plant biology. Proceedings of National Academic Sciences, USA 91: Epstein E, Rains DW, Elzam OE Resolution of dual mechanisms of potassium absorption by barley roots. Proceedings of National Academic Science, USA 49: Kochian LE Molecular physiology of mineral nutrient acquisition, transport, and utilization. In: Buchanan BB, Gruissem W, Jones RL, eds. Biochemistry and molecular biology of plants. Rockville, MD, USA: American Society of Plant Physiologists, Krupka RM Assymmetrical binding of phloretin to the glucose transport system of human erythrocytes. Journal of Membrane Biology 83: Ma JF Beneficial Elements: Si and Na. In: Rattan L, ed. Encyclopedia of soil science. New York, USA: Marcel Dekker, Inc., Ma JF, Goto S, Tamai K, Ichii M. 2001a. Role of root hairs and lateral roots in silicon uptake by rice. Plant Physiology 127: Ma JF, Higashitani A, Sato H, Takeda K Genotypical variation in Si concentration of barley grain. Plant and Soil (In press.) Ma JF, Miyake Y, Takahashi E. 2001b. Silicon as a beneficial element for crop plants. In: Datnoff LE, Snyder GH, Korndorfer GH, eds. Silicon in agriculture. Amsterdam, The Netherlands: Elsevier Science, Ma JF, Takahashi E Soil, fertilizer and plant silicon research in Japan. Amsterdam, The Netherlands: Elsevier Science. Ma JF, Tamai K, Ichii M, Wu G A rice mutant defective in Si uptake. Plant Physiology 130: Macey RI Transport of water and urea in red blood cells. American Journal of Physiology 246: C195 C203. Maurel C Aquaporins and water permeability of plant membranes. Annual Review of Plant Physiology and Molecular Biology 48: Okuda A, Takahashi E. 1962a. Studies on the physiological role of silicon in crop plant. Part 8 Some examination on the specific behavior of low land rice in silicon uptake. Japanese Journal of Soil Science and Manure 33: Okuda A, Takahashi E. 1962b. Studies on the physiological role of silicon in crop plant. Part9 Effect of various metabolic inhibitors on the silicon uptake by rice plant. Japanese Journal of Soil Science and Manure 33: Raven JA Silicon transport at the cell and tissue level. In: Datnoff LE, Snyder GH, Korndorfer GH, eds. Silicon in agriculture. Amsterdam, The Netherlands: Elsevier Science, Savant NK, Snyder GH, Datnoff LE Silicon management and sustainable rice production. Advance in Agronomy 58: Siddiqi MY, Glass ADM, Ruth TJ, Fernando M Studies of the regulation of nitrate influx by barley seedlings using 13 NO 3 1. Plant Physiology 90: Sitsapesan R Similarities in the effects of DIDS, DBDS and sumarin on cardiac ryanodine receptor function. Journal of Membrane Biology 168: Takahashi E Effect of co-existing inorganic ions on silicon uptake by rice seedling. Comparative studies on silica nutrition in plants (Part20). Japanese Journal of Soil Science and Plant Nutrition 53: Takahashi E, Hino K Silica uptake by plant with special reference to the forms of dissolved silica. Japanese Journal of Soil Science and Manure 49: Takahashi E, Ma JF, Miyake Y The possibility of silicon as an essential element for higher plants. Comments Agricultural and Food Chemistry 2: Takano J, Noguchi K, Yasumori M, Kobayashi M, Gajdos Z, Miwa K, Hayashi H, Yoneyama T, Fujiwara T Arabidopsis boron transporter for xylem loading. Nature 420: New Phytologist (2003) 158: