In vitro simulation studies of silica deposition induced by lignin from rice

In vitro simulation studies of silica deposition induced by lignin from rice 1 2 first-author †E-mail:fjy@hsu.edu.cn †E-mail:fjy@hsu.edu.cn fjy@hsu.edu.cn 1 Institute of Biodiversity Research, Huangshan University, Huangshan 245021, China Institute of Biodiversity Research, Huangshan University, Huangshan 245021, China Tea Biochemistry and Biotechnology Laboratory of Ministry of Agriculture, Anhui Agricultural University, Hefei 230036, China Tea Biochemistry and Biotechnology Laboratory of Ministry of Agriculture, Anhui Agricultural University, Hefei 230036, China To reveal the possible mechanism of silica deposition in higher plants, lignin was isolated from rice straw following a modified method to conduct a simulation experiment in vitro. UV and infrared absorption spectra showed that the substance had the unique characteristics of pure lignin. The presence of silicon in the precipitation was revealed by TEM (transmission electron microscopy) with EDXA (energy dispersive X-ray analysis) device. It was found that in the borax solution where lignin precipitation occurred silica-lignin co-precipitation was produced but not in the DMSO solution where lignin was broken into its composition compounds and did not precipitate. This means that it is macromolecular lignin itself but not its compounds that could induce silica deposition in higher plants. Lignin Silica precipitation Higher plants INTRODUCTION 1 Many scientists have confirmed that monocotyledon like grasses deposit opaline silica to regulate biochemical reactions, support the plant and enhance its tolerance to salt, heavy metals and pathogenic fungi (Epstein, 1994 1994 ; Liang, 1999 1999 ; Parry and Smithson, 1964 1964 ; Kaufman et al., 1970 1970 ; Xing and Zhang, 1998 1998 ; Inanaga et al., 2002 2002 ; Datnoff et al., 2005 2005 ; Datnoff and Rutherford, 2004 2004 ; Rodrigues et al., 2003 2003 ). Evidence has shown that silica accumulates mainly in a species’ epidermis of the upper part, in the central sclerenchyma tissue of the stele, in the vessel walls, the endodermal walls and the epidermis and subepidermal cells of the proximal end of the thick cord roots (Parry and Kelso, 1975 1975 ). The common characteristic of the silica deposition in these tissues is that it begins at the time when the cell stops growth to synthesize and accumulate lignin in the secondary wall. Montgomery and Parry ( 1979 1979 ) agreed that during this procedure, lignin is permeable to water and some solutes, but it is highly unlikely that it is permeable to a relatively large molecule such as monosilicic acid, resulting in the deposition of silica. Inanaga et al.( 1995 1995 ) also observed that silica was present in the rice cell wall along with phenol-polysaccharide compound or lignin-polysaccharide compound. They presumed that silica might be associated with phenol or lignin such as the element calcium (Inanaga and Okasaka, 1995 1995 ). We also showed in a simulation experiment that nanostructural silica deposition can be induced by the polymerization of phenols (Fang et al., 2003 2003 ). To obtain direct evidence of the relationship between silica depositions and lignification, a simulation experiment including silicon and lignin from rice, one of the heavy silica accumulation plants, was carried out. The simplest and most credible method to extract lignin from grass was established by Sun et al.( 2000 2000 ). In the isolation, ball-milling process was cancelled due to the very long time and the demand for special equipment, and the characteristics of the extracted substance were examined with UV and infrared absorption. Transmission electron microscopy (TEM) with energy dispersive X-ray analysis (EDXA) device was used to detect the presence of silica in the deposition powder. EXPERIMENTAL DETAILS 1 Materials 2 Mature rice straw ( Oryza sativar L., cultivar upland 297, provided by the School of Agronomy and Biotechnology, China Agricultural University) was ground with a laboratory mill to pass a 60-mesh sieve. The grease contained in the straw powder was removed by toluene-methanol (2:1, v/v) solution and evaporated in a Soxhlet apparatus for 5 h. Lignin isolation 2 Alkali lignin was prepared from the extractive grease-free powder according to modified Sun et al.( 2000 2000 )’s method, omitting the procedure of ultra grinding by ball milling. The powder was subjected to evaporation under a fume hood to remove the organic solvent. Then, it was stirred in 1.5% NaOH aqueous solution (40 ml solution for 1 g straw powder) at room temperature for 6 d. After filtration, the supernatant was acidified to pH 5.5 with 20% HCl aqueous solution, precipitated with 4 volumes of ethanol to isolate crude hemicelluloses and hemicellulosic-lignin complexes. The supernatant was concentrated by an evaporator (Labconco, Centrivap Cold Trap & Labconco, Centrivap Concentrator) with reduced pressure at 40 °C to obtain the alkali-soluble lignin fraction and then re-precipitated at pH 1.5 with 20% HCl solution. Again after filtration, the isolated lignin fraction was rinsed with pH 2.0 H 2 O and then air-dried. To remove the possible associated silicon, the sample was soaked in 2% HF aqueous solution buffered with 3% NH 4 F and shaken on a gyratory shaker (80 r/min) at 28 °C for 24 h. Centrifugation was employed to wash the sample for 5 times. Lignin identification 2 UV spectra were recorded on a UV-VIS recording spectrophotometer (UV-2201). The lignin sample was dissolved in ethonal or 4% NaOH in ethonal solution. The absorbance at 190 to 500 nm was estimated. Micro IR spectra were recorded on an IR microscope (Nicolet, NIC-PLAN) attached to an IR spectrometer (Nicolet, MAGNA-IR 750), with a BaF 2 slide to support the lignin powder. The measurement range was 800~4000 cm −1 , scanning 5 min and 526 times. Co-precipitation of Si and lignin 2 Method 1: Citrate-phosphate pH 6.4 and 5.4 buffers were prepared to make 20 mmol/L Na 2 SiO 3 solution and pH was maintained with 0.1 mmol/L citric acid solution. Lignin powder (2.5 mg) was added into 400 μl DMSO to get lignin solution. Then Na 2 SiO 3 solution and lignin solution were mixed together according to the proportion in Table 1 1 . pH Proportion 1 Proportion 2 5.4 1:1 4:6 6.4 1:1 4:6 Method 2: Na 2 SiO 3 aqueous solution was prepared in the same way as in Method 1. In 1% borax aqueous solution, pH was adjusted from 9.11 to 10.05 with 1 mol/L NaOH. In 400 μl such borax solution, 26 mg of the lignin powder was slowly added to ensure a little lignin precipitation occurs. Then Na 2 SiO 3 solution and lignin solution were mixed together in the same proportion. For each mixture, two contrasts were set. In CK1, Na 2 SiO 3 solution was mixed with 1% borax solution (without lignin) and in CK2, citrate-phosphate buffer solution (without Na 2 SiO 3 ) was mixed with lignin solution. TEM and EDXA of the precipitation 2 Precipitation from each mixture was suspended in equal amount of water, stirred rapidly, dropped on Cu-grids and air-dried. The image of the precipitation was taken by a TEM (PHILIPS, EM400T) and the silicon composition was investigated by the same TEM with an EDXA detector (ECAMII-S), under 80 kV in 15 μA, with electron beam dot of 100 nm for 200 s. RESULTS 1 Characteristics of lignin 2 The extracted powder dissolving in the ethanol solution had a distinctive absorption in UV area and red shifted when alkali was present, showing that the sample had phenolic groups, which was one of the characteristics of lignin (Sun et al., 2000 2000 ; Harborne, 1991 1991 ; Lewis and Yamamoto, 1990 1990 ). As in previously published data (Sun et al., 2000 2000 ; Lawther et al., 1996 1996 ), the sample showed typical infrared absorption of pure lignin from the grass (Fig. 1 1 ). The details of the characteristic bands were analyzed (Table 2 2 ). Lawther et al.( 1996 1996 ) Sun et al.( 2000 2000 ) Lit * * Wheat traw ** ** Rice straw Assignment 3400 3405 3405 OH stretching 2925 2923 2937 CH stretching of methyl, methylene or methane group 2850 2850 2852 CH stretching of methyl, methylene or methane group 1651 1655 C=O conjugated ketone stretching 1600 1593 1596 Aromatic skeletal vibrations 1505 1507 1508 Aromatic skeletal vibrations 1461 1463 Aromatic methyl group vibrations 1440 1422 1423 Aromatic skeletal vibrations 1325 1328 1331 Syringyl ring breathing with CO stretching 1280 1265 1290 Guaiacyl ring breathing with CO stretching 1220 1225 1227 Guaiacyl ring breathing with CO stretching 1155 1156 1153 Aromatic CH in-plane deformation, guaiacyl type 1120 1123 1126 Aromatic CH in-plane deformation, syringyl type 1015 1029 1034 Aromatic CH in-plane deformation, guaiacyl type 840 843 838 Aromatic C-H out of plane bending Co-precipitation of Si and lignin 2 For the reaction between Si and lignin, precipitation was not observed in Method 1. Although no precipitation occurred in CK1, there was precipitation in all the other reactions including CK2 in Method 2. This result showed that Si without lignin failed to precipitate in the borax aqueous solution, but lignin could precipitate in the same solution. TEM photographs of the precipitation from lignin (CK2) and the mixture of Si and lignin are shown on Fig. 2 2 . It is distinctive that when Na 2 SiO 3 was present in lignin aqueous solution, the precipitation is much more condensed than that in the lignin solution without silicon, showing that silica might be induced to precipitate by the precipitated lignin. The presence of silicon in the precipitation was directly confirmed by the EDXA (Fig. 3 3 ). BG is background absorbance value and P / B is the ratio of peak to background DISCUSSION 1 Although great efforts have been made since a report on polycationic peptides from diatom cell walls directing silica nanosphere formation (Kröger et al., 1999 1999 ), similar protein has not yet been found in higher plants such as grasses in which silicon is an important element for plant growth. Many scientists believe that silica deposition in higher plants is associated with lignification (Parry and Kelso, 1975 1975 ; Montgomery and Parry, 1979 1979 ; Wang and Naser, 1994 1994 ; Chérif et al., 1994 1994 ). In order to provide direct evidence to support this hypothesis, the first step was to isolate pure lignin from rice straw. UV and infrared spectra of the extracted substance indicated that pure lignin could be isolated following the modified Sun et al.( 2000 2000 )’s method without ball milling for 6 d if the aim was just for qualitative analysis of the compound and not for quantity measurement. The next step was to mix Si with lignin in a proper solution. In both borax and DMSO aqueous solution, Si did not precipitate. However, with the presence of lignin precipitation in borax aqueous solution, Si precipitation occurred. Kinrade et al.( 1999 1999 ) reported that addition of aliphatic polyols to aqueous silicate solutions yielded high concentration of stable polyolate complexes containing five- or six-coordinated silicon. Lignin from the secondary cell wall of the rice has structure similar to that of aliphatic polyols. Therefore, it is estimated that five- and six-coordinated silicon and lignin could play an essential role during the absorption and transport of silicon in rice. 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Review of the studies on silicon nutrition of plants We are grateful to Professor ZHANG Fu-suo for his valuable support and Dr. WANG He for his kind suggestions. 10.1631/jzus.2006.B0267 Projects supported by the National Natural Science Foundation of China (No. 30170550), the Natural Science Foundation of Anhui Provincial Education Department (No. 2005kj398zc) and the Culture Project for “the Tenth Five-year” Learning Leaders of Higher School of Anhui Province, China 2005-12-12 2006-01-06 2006-03-13 2022-01-05 Copyright © 2006, Journal of Zhejiang University Science 2006 Journal of Zhejiang University-SCIENCE B (Biomedicine and Biotechnology) 04 07