Chemical Mechanisms For Inhibition Of Cadmium Ion Uptake In Rice (Oryza Sativa) Single Cells By Organosilicon And Inorganic Silica On Cell Walls

Rice(Oryza sativa) is a typical Silicon-accumulating plant, taking up 1%- 10% Silicon(Si) of its dry weight. Despite the amorphous inorganic silica presented in rice cell walls or inside specialized silica cells, evidence has suggested that organosilicon may intimately associate with cell walls. The presence of Si in rice plants can alleviate various biotic and abiotic stresses such as cadmium(Cd) toxicity. However, the chemical mechanisms of Cd toxicity alleviation by these two different structures of Si at the single-cell level are poorly understood. Therefore, we cultivated suspended rice cells which provide undifferentiated single cells with the same developmental stage and biological function. As a simplified and effective model system, it will rule out interference arising from the multiscale complexity at the tissue, organ, and whole-plant level. We find that organosilicon covalently crosslink the hemicellulose in cell walls of rice single cells. Moreover, we coated a silica shell on the surfaces of rice single cells by mimicking diatom biosilicification. Using a combination of physical and chemical analysis method, cellular and molecular biological techniques, we investigated the chemical mechanisms of inhibiting Cd adsorption by organosilicon and inorganic silica on cell walls. Meanwhile, we also investigated the differences of the protein expression in rice single cells affected by the organosilicon on cell walls under short- and long-term Cd stress by isobaric tags for relative and absolute quantitation(iTRAQ) method. The main results are summarized as following: 1. Hemicellulose-bound form of Organosilicon exists in the cell walls of ricesuspension cells, results in more net negative charges in the walls andsubsequently can form [Si-hemicellulose matrix]-Cd co-deposition, andsignificantly decreased the Cd uptake into rice cells.Inductively coupled plasma-mass spectrometry(ICP-MS) and X-ray photoelectron spectroscopy(XPS) results revealed that 64% of the total Si was bound to hemicellulose of the cell walls. AFM in KPFM mode demonstrated Si-hemicellulose complex resulted in a heterogeneity net negative charge of the wall surface potential in cell walls, and this net negative charge(charge density) could be neutralized by an increasing of the Cd2+ concentration in the measuring solution and consequently formed [Si-hemicellulose matrix]-Cd co-deposition. In situ non-invasive microtest technology(NMT) measurement of cellular fluxes of the Cd2+ in suspension cells of rice showed that comparing with –Si cells, +Si cells significantly inhibited the net Cd2+ influx, and consequently significantly reduced total Cd concentrations in protoplasts. Polymerase chain reaction(PCR) showed that the expression of the Si transporter gene Low silicon rice 1(Lsi1) was up-regulated when the Si and Cd concentration in the medium was increased, meanwhile, the expression of Cd transporter Natural resistance-associated macrophage protein 5(Nramp5) was down-regulated. 2. 100 proteins differentially regulated by Si under the short- or long-term Cd stressare identified and two different mechanisms for Si-mediated Cd tolerance underthe short- and long-term Cd stress are suggested.iTRAQ revealed 100 proteins differentially regulated by Si under the short- or long-term Cd stress, and 70% of these proteins were down-regulated, suggesting that Si may improve protein use efficiency and maintaining cells in the normal physiological status. Combine with fluorescent staining and ICP-MS, we found the reduced expressions of wall-related glycosidase, cell surface non-specific lipid-transfer proteins(ns LTPs), and several stress-related proteins under the short-term Cd stress. As the stress time goes on, besides the part of Cd inhibited by cell walls, the amount of Cd in the cytoplasm in +Si cells was decreased by compartmentation of Cd into vacuoles, therefore, maintaining the glutathione(GSH) system in a good function status. 3. Besides improving the mechanical property of cells, the biomimetic silicananoshells coated on rice single cells can significantly reduce the surface potentialof rice cells and consequently strongly adsobed the Cd by electrostatic interaction.Using Poly(ethyleneimine)(PEI) as a catalytic template for silicification on cell walls, we catalyzed the hydrolysis of tetramethyl orthosilicate(TMOS) to silica nanoparticles on the rice single cell surface. AFM force measurement revealed that the silica shell can increase the mechanical strength of cells. FE-SEM and AFM-KPFM showed that the silica nanoshell is composed by different sizes silica nanoparticles which has a large surface area, and can take much more negative charges to strongly adsorbe Cd2+. NMT result revealed that the silica nanoshells have Cd2+ adsorption fluxes 6 to 10 times greater than those of the unsilicified cell walls, thus significantly inhibited Cd uptake into cells.