Ruiyu Lin

Characterization of metaproteomics in crop rhizospheric soil.

Soil rhizospheric metaproteomics is a powerful scientific tool to uncover the interactions between plants and microorganisms in the soil ecosystem. The present study established an extraction method suitable for different soils that could increase the extracted protein content. Close to 1000 separate spots with high reproducibility could be identified in the stained 2-DE gels. Among the spots, 189 spots representing 122 proteins on a 2-DE gel of rice soil samples were successfully identified by MALDI-TOF/TOF-MS. These proteins mainly originated from rice and microorganisms. They were involved in protein, energy, nucleotide, and secondary metabolisms, as well as signal transduction and resistance. Three characteristics of the crop rhizospheric metaproteomics seemed apparent: (1) approximately one-third of the protein spots could not be identified by MALDI-TOF/TOF/MS, (2) the conservative proteins from plants formed a feature distribution of crop rhizospheric metaproteome, and (3) there were very complex interactions between plants and microorganisms existing in a crop rhizospheric soil. Further functional analysis on the identified proteins unveiled various metabolic pathways and signal transductions involved in the soil biotic community. This study provides a paradigm for metaproteomic research on soil biology.

Analysis of gene expressions associated with increased allelopathy in rice (Oryza sativa L.) induced by exogenous salicylic acid.

The defense characteristics of allelopathic rice accession PI312777 and its counterpart Lemont induced by exogenous salicylic acid (SA) to suppress troublesome weed barnyardgrass (BYG) were investigated using the methods of suppression subtractive hybridization (SSH) and real-time fluorescence quantitative PCR (qRT-PCR). The results showed that exogenous SA could induce the allelopathic effect of rice on BYG and this inducible defense was SA dose-respondent and treatment time-dependent. PI312777 exhibited higher inhibitory effect than Lemont on BYG after treated with different concentrations of SA. The activities of cell protective enzymes including SOD, POD and CAT in the BYG plants co-cultured with PI312777 treated by SA were highly depressed compared with the control (co-cultured with rice without SA-treatment). Similar but lower depression on these enzymes except for CAT was also observed in the BYG plants when co-cultured with Lemont treated by SA. It is therefore suggested that allelopathic rice should be more sensitive than non-allelopathic rice to exogenous SA. Seventeen genes induced by SA were obtained by SSH analysis from PI312777. These genes encode receptor-kinase proteins, ubiquitin carrier proteins, proteins related to phenylpropanoid metabolism, antioxidant related proteins and some growth-mediating proteins. The differential expressions of these genes were validated in part by qRT-PCR in the two rice accessions. Our work elucidated that allelopathic rice possesses an active chemical defense and auto-detoxifying enzyme system such as the up-regulated enzymes involved in de novo biosynthesis of phenolic allelochemicals and the glutathione-S-transferase (GST) associated with xenobiotic detoxification.

Metaproteomic analysis of ratoon sugarcane rhizospheric soil

BackgroundThe current study was undertaken to elucidate the mechanism of yield decline in ratoon sugarcane using soil metaproteomics combined with community level physiological profiles (CLPP) analysis.ResultsThe available stalk number, stalk diameter, single stalk weight and theoretical yield of ratoon cane (RS) were found to be significantly lower than those of plant cane (NS). The activities of several carbon, nitrogen and phosphorus processing enzymes, including invertase, peroxidase, urease and phosphomonoesterase were found to be significantly lower in RS soil than in NS soil. BIOLOG analysis indicated a significant decline in average well-color development (AWCD), Shannon’s diversity and evenness indices in RS soil as compared to NS soil. To profile the rhizospheric metaproteome, 109 soil protein spots with high resolution and repeatability were successfully identified. These proteins were found to be involved in carbohydrate/energy, amino acid, protein, nucleotide, auxin and secondary metabolisms, membrane transport, signal transduction and resistance, etc. Comparative metaproteomics analysis revealed that 38 proteins were differentially expressed in the RS soil as compared to the control soil or NS soil. Among these, most of the plant proteins related to carbohydrate and amino acid metabolism and stress response were up-regulated in RS soil. Furthermore, several microbial proteins related to membrane transport and signal transduction were up-regulated in RS soil. These proteins were speculated to function in root colonization by microbes.ConclusionsOur experiments revealed that sugarcane ratooning practice induced significant changes in the soil enzyme activities, the catabolic diversity of microbial community, and the expression level of soil proteins. They influenced the biochemical processes in the rhizosphere ecosystem and mediated the interactions between plants and soil microbes.

Cadmium-stress mitigation through gene expression of rice and silicon addition

Cadmium (Cd) pollution is one of the major concerns in the development of sustainable agriculture, particularly for rice production. Silicon (Si) was recently recognized for its ability to mitigate a variety of abiotic stresses including that caused by Cd on rice. However, mechanism of the complex process is still not fully understood. Under Cd-stress, the low Si-influx 1-RNAi transgenic Lemont rice (Lsi1-RNAi line) exhibited an increased Cd-uptake over its counterparts, the wild type and the Lsi1-overexpression transgenic rice (Lsi1-OE line). In contrast, the Lsi1 expression-enhanced Lsi1-OE line showed the greatest Si-uptake among the three lines, with the highest activities on anti-oxidants (such as, superoxide dismutase) and the lowest content of malondialdehyde. Lsi1 also displayed a negative regulation on Low Cd gene and the natural resistance-associated macrophage proteins, Nramp5, indicating its capacity to alleviate Cd-stress on rice. The results obtained by this study suggested that the mitigation of Cd-toxicity on rice by Si might involve functions, such as the inhibition on Cd-uptake and transport and the enhancement on anti-oxidative enzyme activities, as well as the Lsi1-related expression on regulation of Si-uptake in rice. A new avenue might become available for overcoming the rampant pollution that threatens the rice production in China.

Electrochemical sensor of 4-aminobutyric acid based on molecularly imprinted electropolymer

A simple and effective procedure based on a molecularly imprinted polymer (MIP) was developed for preparing a selective 4-aminobutyric acid (4-ABA) sensor. The sensitive layer was prepared by electropolymerization of o-phenylenediamine (o-PD) on a gold electrode in the presence of 4-ABA, which acts as a template. Cyclic voltammetry (CV), differential pulse voltammetry (DPV), linear sweep voltammetry (LSV) and electrochemical impedance spectroscopy (EIS) measurements were used to monitor the process of electropolymerization. The molecularly imprinted sensor was tested by CV as well as DPV to verify the changes of redox currents of hexacyanoferrate. The concentration of 4-ABA in the range of 0.2–20.0 μmol L−1 can be determined with a detection limit of 0.08 μmol L−1 (defined as S/N = 3) under the optimum conditions. The MIP sensor shows high selectivity, sensitivity and reproducibility. The results from sample analysis indicate that the MIP-4-ABA sensor can be used for quantitative analysis.

Rice allelopathy and its properties of molecular ecology

Crop allelopathy is a promising and environmentally friendly method in weed control; however, the inducible genetic trait for allelopathy in the suppression of weeds needs to be overcome for practical use. Further study needs to be directed to this end to elucidate the molecular genetics and its physiologic mechanism. In this paper, the authors review recent advances in the investigation of rice allelopathy and its molecular regulatory mechanism, especially in responses to stressful conditions including biotic and abiotic factors in China. Previous studies show that rice allelopathy could be enhanced when the rice accession was exposed to stressful conditions, and further analysis by the transcriptomics and proteomics approaches conducted in our laboratory indicated that the increase in allelopathic potential of rice, when exposed to the stresses, was attributed to increased expression level of genes involved in phenolic synthetic metabolism. The increasing phenolic compounds have been confirmed as the main allelochemicals and they jointly act to suppress the target, especially in responses to stressful condition, but it seems to be the primary effect in phenolic allelopathy. We still wonder how the exudates from rice root, which were released into rhizosphere soil, are transformed by soil microorganism to produce the higher secondary effect of phenolic allelopathy in the suppression of weeds. Therefore, the authors suggest that rhizosphere biologic properties of allelopathy in rice and its mechanism are being the key research areas in the world now, and systems biology and its approaches, such as metagenomics and metaproteomics, would be helpful to reveal the process and its molecular ecological mechanism regarding rhizospheric biology of rice allelopathy.

Determination of Phenolic Acids in Root Exudates of Allelopathic Rice by Solid Phase Extraction-Ion Chromatography with Conductivity Detection

A simple, effective, and green ion chromatography method with conductivity detection was developed for the determination of benzoic acid, cinnamic acid, syringic acid, and p-hydroxybenzoic acid in the root exudates of allelopathic rice. The analytes were well separated within 25 min in an anion exchange column (150 mm × 4.0 mm i.d., 5 µm particle size) with mixtures of 6.4 m mol L−1 Na2CO3 and 2.0 m mol L−1 NaHCO3 as the mobile phase at a flow-rate of 0.7 mL min−1. Detection limits of benzoic acid, cinnamic acid, syringic acid, and p-hydroxybenzoic acid were 0.05, 0.20, 0.50, and 0.05 µg mL−1, respectively. Intra- and inter-day precision was ≤4.0% and 3.2%, respectively, and the intra- and inter-day accuracy, indicated by relative error, ranged from −8.0% to 9.0%. The developed method was successfully used to determine phenolic acids in the root exudates of allelopathic rice. The average recoveries of the analytes were between 90.7% and 103.0%.

Highly sensitive determination of lead( ii ) and cadmium( ii ) by a large surface area mesoporous alumina modified carbon paste electrode

Nanosized mesoporous γ-alumina (M-γ-Al2O3) was first prepared and then modified into a carbon paste to fabricate a novel modified carbon paste electrode. The prepared alumina has pores with an amorphous wall and large surface area. The electrochemical behavior of the modified carbon paste electrode was investigated using cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) methods. The modified carbon paste electrode was employed to determine Pb2+ and Cd2+ simultaneously by a differential pulse voltammetry (DPV) method. Amperometric determination was carried out in 0.1 mol L−1 NaAc–HAc buffer solution (pH 6.0) after enriching for 360 s at −1.0 V. The oxidation peak currents of Pb2+ and Cd2+ were proportional to their concentration in the range of 0.001–10 μmol L−1 and 0.01–10 μmol L−1, respectively. The detection limits of Pb2+ and Cd2+ were 0.20 nmol L−1 and 2.0 nmol L−1 (S/N = 3), respectively. The modified carbon paste electrode shows good stability, repeatability and sensitivity. The proposed method was applied to the determination of Pb2+ and Cd2+ in water samples with satisfactory results.

A hydrogen peroxide biosensor based on horseradish peroxidase/poly(L-leucine)/polydopamine modified glassy carbon electrode

A simple and practical sensor of hydrogen peroxide (H2O2) was designed successfully. The mixture of horseradish peroxidase (HRP) and chitosan (Chit) are effectively immobilized on the surface of poly-L-leucine/polydopamine modified glassy carbon electrode (PL-LEU/PDA/GCE). Under the optimum conditions, the biosensor based on HRP exhibits a fast amperometric response (within 3 s) to H2O2. The linear response range of the sensor is 0.5–952.0 μmol L–1, with the detection limit of 0.1 μmol L–1 (S/N = 3) and the sensitivity of 0.23 A L moL–1 cm–2. The apparent Michaelis–Menten constant (kMapp) of the biosensor is evaluated to be 0.12 mmol L–1, which suggests that the HRP-Chit/PL-LEU/PDA/GCE shows a higher affinity for H2O2. The sensor exhibits good sensitivity, selectivity, stability and reproducibility. The proposed method has been successfully applied to the determination of H2O2 in practical samples.