Evidence For The Formation Of Silicon-xyloglucan Complexes And Their Functions Of The Cell Walls Of Rice

As a beneficial element,silicon(Si)could improve the growth of Poaceae plants and enhance the resistance to multiple abiotic and biotic stresses in various higher plants.Rice(Oryza sativa)is a representative Si-accumulating Poaceae plant.It accumulates monomeric silicic acid and disilicic acid and subsequent deposition amorphous Si on the cell walls.In addition to inorganic silica,it has long been suggested that organosilicon might also exist in plant cell walls,where trace amounts of Si was firmly bound to hemicellulose(HC)molecules.This HC-bound form of Si could influence the structural remodeling and the linkages of cell wall components,thereby improving both the mechanical properties and the regeneration of the cell walls.However,it remains unclear which component among HCs is involved in the binding of Si.Therefore,rice suspension cells cultured in the absence(-Si)or presence(+Si)of Si,and their extracted cell walls and hemicellulose components were used as experimental specimens.With the help of new interdisciplinary interdisciplinary techniques,including gas liquid chromatography(GC-MS),high-performance anion-exchange chromatography with pulsed amperometric detection(HPAEC-PAD)and matrix-assisted laser desorption ionization time-of-flight mass spectrometry(MALDI-TOF MS),we have characterized chemical composition of/+Si cell walls.Conbined inductively coupled plasma mass spectrometry(ICP-MS)and X-ray photoelectron spectroscopy(XPS),the Si content and chemical composition in two different components of XyGs and xylans in hecelluloses were analyzed;Additionally,we used in situ atomic force microscopy(AFM)with PeakForce-quantitative nanomechanical mapping(PF-QNM)mode to image the binding difference of XEG or xylanase to the depectinized walls of suspension-cultured cells under physiological solution conditions,revealing the precise crosslinking components of Si in HCs.We further used in situ AFM to image the process of enzymatic degradation and measured the local nanomechanical properties of the cell walls to determined the function of organosilicon at the single-cell level.To further verify the biological effects of the Si,rice suspension cells and plants with xyloglucan 6-xylosyltransferase(OsXXT1)gene mutation were constructed by CRISPR/Case 9 gene editing technology,the existance and biological function of the SiXyG complex were verified.Detailed results were as follows:1.Si is bound to XyGs on the HCs of rice suspension cells.We used GC,HPAEC-PAD and MALDI-TOF MS to quantitatively characterize chemical composition of-/+Si cells walls and its components,as well as the release amounts of XyG or xylan oligosaccharides from the whole cell walls under XEG or xylanase treatment.No significant differences in the chemical composition of-/+Si cells walls was observed,however,the walls of-Si cells released more XyGs than that of+Si cells.Subsequently,ICP-MS was used to analyze the Si content and chemical composition,respectively,in xylans and XyGs.The concentrations of Si in the dry weight(DW)of XyG,xylan were 103.7±10.6 μg g-1,6.1±2.3 μg g-1,respectively,in the+Si walls.The trend of XPS intensities for each component were consistent with the result of ICP-MS,which indicated that Si may covalently crosslink with XyG in the HCs to form Si-XyG complex.We further employed AFM in PF-QNM mode to image the interaction force of XEG binding to the depectinized walls of rice cells at single-molecule scale.The wall surface of+Si cells exhibited significant fewer specific binding sites than that of-Si cells,it is shown that this Si-XyG complex can reduce the recognition of xyloglucanase on the cell wall.This is further demonstrated that Si is covalently bound to XyGs rather than xylans of HCs in the cell walls at the single-molecule scale.Our findings determined the form of organosilicon in plant cell walls,and are critical for further understanding the structure and function of cell walls.2.XyG-bound form of Si enhances the resistance to enzymatic degradation and improves the elastic modulus for the cell walls.To understand how the Si-XyG complex resists enzymatic degradation,we applied AFM to image the in situ degradation kinetic process of-/+Si cell walls.Only one cellulose microfibril was degraded on the surface of+Si cell walls after treating with cellulase for 240 min;whereas four cellulose microfibrils on the-Si cell walls were completely degraded.Using PF-QNM mapping,we measured the in situ elastic modulus of the cell walls in culture solutions.The+Si cells obtained an average wall modulus at 1.4±0.1 1Mpa,which was significantly higher than that of-Si cells at 0.84±0.18 Mpa,it was indicated that the XyG-bound Si can enhances resistance to enzymatic degradation and nanomechanical properties of rice cell walls.3.Constructed OsXXT1 mutant cells and plants to investigate the biological evidence and fuctions of Si-XyG complexesRice suspension cells and plants with xyloglucan 6-xylosyltransferase(OsXXT1)gene mutation were constructed by CRISPR/Case 9 gene editing technology.GC-MS was used to determine the amounts of monosaccharides released by XEG from the wild and OsXXT1 mutation cell walls.The result showed that the content of galactose,glucan,especially xylose in the mutant cell walls was significantly lower than that in the wild type.MALDI-TOF MS failed to detect the xylosyl-containing oligosaccharide fragments in the XyG extracts of mutant cells.The ICP-MS showed that the Si concentrations(DW)of mutant and wild type were 12.9±3.1,33.8±12.5μg g-1,respectively.The trend of XPS intensities were consistent with the result of ICP-MS,suggesting that Si may bind to XyGs especially the xylose of XyG side chain.This organic bound Si contribute to the changes of the cell walls,including altered morphology,increased resistance to enzymatic hydrolysis,and higher mechanical strength.The enhanced cell wall stability at the single-cell level,in turn,will affect seed germination and yields of whole plant.