Identification And Functional Analysis Of A Silica-Binding Protein In Rice Plant

The paper was the first time to report the method of isolating and extracting the silica-binding protein, its molecular character, the function and its distribution. We elucidated the mechanism of silica deposition in rice from molecular level. In addition, we investigated the relationship between the silica-binding protein and the mechanism that silica alleviated the toxicity of cadmium in rice. The main results are as following:We succeeded in isolating the native silica bodies from rice leaves by low temperature homogenizing and sieving method. From native silica bodies we extracted a 117 kDa protein which is tightly binding with silica, namely SBP117 (Silica-Binding Protein 117 kDa). By analyzing the N-terminal amino acid sequence and searching in NCBI database, we found SBP117 is a member of proline-rich protein family and its function is unknown. The registered number is AAO72557.1. The composition of amino acids of this protein is similar with those proteins found in diatom and cucumber, but the sequence of amino acids is different. It means that SBPl 17 is a special protein for graminaceous plants to control the silica deposition.We found SBP117 could catalyze the silica polymerization in vitro. The amount of silica precipitated was proportional to the amount of SBP117 present, and also to the amount of silica present. At pH 5.0, which is a range of physiological range of plants, protein is rather active.Specific polyclonal antibodies against SBP117 are successfully raised by synthesized peptides which were conjugated with Keyhole Limpet Hemocyanin and used as antigens to immunize rabbits. Immunoblot results indicate the antibody can also cross react with the proteins of other silica-accumulated graminaceous plants, while it does not react with the proteins of dicotyledonous plants (such as tomato leaves). These findings indicate that the homologous proteins of SBP117 are widely existed in the graminaceous plantss. This indicated silica accumulated in higher plants posses the character of systematic development. Immunocytochemistry localization shows SBP117 is mainly located at the bulliform cell in the leaf. Tissue printing study showed that SBPl 17 is mainly located at the epidermis of roots, shoots and leaves as well as in the vascular bundle of the rice roots and leaves. The distribution of SBPl 17 is coincided with the sites of Si accumulated in rice plants. Therefore it is concluded that SBPl 17 is involved in the control of silica deposition in rice plants.In addition, we also investigated the relationship between the silica-binding protein and the mechanism that silica increased the resistance of rice seedlings to toxic metals (Cd). Si added significantly alleviated the toxicity symptom of Cd in rice seedlings grown hydroponically. Si dramatically reduced the transport of Cd from roots to shoots. The result of the apoplastic fluorescence tracer PTS (tri-sodium-8-hydroxy-1, 3, 6-pyrenesulphonate) showed the significant reduction of apoplastic transport in +Si plants. Energy-dispersive X-ray analysis and immunocytochemistry localization showed the silica-binding protein, Si and Cd were mainly deposited in the vicinity of the endodermis and the fiber cell, which suggests SBP117 maybe induceSi precipitated in above two sites and restrain the apoplastic transport of Cd, and alleviated the toxicity of Cd.