Glechoma hederocea agglutinin (Gleheda) is a novel glycosylated lectin isolated from the leaves of G. hederacea. Like other glycosylated proteins, the detection of Gleheda by immunological methods is often hampered by the cross-reactivity of the polyclonal antibodies with unrelated glycoproteins. Hence a protocol to purify monospecific polyclonal antibodies from a crude antiserum raised against Gleheda was developed. After selective ammonium sulfate precipitation and successive affinity chromatography on columns of Sepharose 4B with immobilized Gleheda and Robinia pseudoacacia agglutinin (RPA), respectively, ion-exchange chromatography on a column of Q Fast Flow was used for further purification. The specificity of the antibody fractions from each step was tested by double immunodiffusion assay and analyzed by Western blot. Results revealed that affinity chromatography of the immunoglobulin fraction on the immobilized Gleheda antigen yielded an antibody preparation that still cross-reacted with many proteins in leaf extracts. Depletion of nonspecific cross-reacting antibodies directed against the glycan part of the glycoprotein by affinity chromatography on immobilized RPA removed most but not all nonspecifically reacting antibodies. Only upon further purification by ion exchange chromatography an IgG fraction of monospecific antibodies that reacted exclusively with Gleheda could be obtained and accordingly was suitable for immunodetection studies. This antibody purification procedure promises simplicity and efficiency. In addition, this method does not require expensive facilities.

How root system responds to various environmental factors has not yet been fully elucidated. In root, the expression of OsPK1 is mainly in the maturation zone and the root-hair zone of root tip. It is unknown whether the uptake of exogenous sugars by rice seedlings is affected by downregulation of OsPK1. In this study, we used wild-type (WT) and ospk1 rice mutant plants to investigate the uptake of exogenous sugars and the responses of rice seedlings by adding sucrose to 1/2 MS medium or not. The contents of sucrose, glucose, fructose and galactose in leaf blades, sheathes and roots of rice seedlings were measured by GC-MS analysis. The result revealed that direct contact between root and exogenous sugars greatly elevates sugar levels of rice seedlings. And the root length of these seedlings is much longer than that of the seedlings grown in medium omitting exogenous sugars, suggesting that uptake of exogenous sugars by root promotes root elongation. Downregulation of OsPK1 has effects on sugar metabolism and the uptake of exogenous sugars. Semi-quantitative RT-PCR result showed that the expressions of OsPIP2;4, OsPIP2;5 and OsTIP2;1 (three aquaporin genes) in root were greatly upregulated by the direct contact between root and exogenous sugars.
Absorption ; Aquaporins ; genetics ; metabolism ; Carbohydrate Metabolism ; Gene Expression Regulation, Plant ; Genes, Plant ; Genetic Variation ; Oryza ; genetics ; metabolism ; Plant Proteins ; genetics ; metabolism ; Plant Roots ; metabolism ; Protein-Serine-Threonine Kinases ; genetics ; metabolism ; Seedlings ; genetics ; metabolism

The preliminary role of respiratory burst oxidase homolog (Rboh) in plant immune response is defined, but the exact function of OsRboh gene in rice immune response and its expression pattern is yet unclear. In order to clarify the role of OsRboh in rice immune response, we screened seven OsRboh genes from the latest rice genome annotation database. The result of tissue specific expression analysis demonstrated that OsRbohD was expressed only in spike and calli, and OsRbohE and OsRbohF were only expressed in calli. The rest of OsRboh genes were constitutively expressed in rice. In addition, the expression level of OsRboh gene family was analyzed in the rice leaves respectively treated with salicylic acid (SA), methyl jasmonic acid (MeJA) and Xanthomonas oryzae PV. oryzae (Xoo) PXO99 strain by Real-time PCR, and H2O2 content was also quantified by spectrophotometry after the three treatments. The result shows that the expression of OsRbohA, B, C and D was increased under the treatments of SA, the expression of OsRbohA, B, C and G was increased under the treatments of MeJA, and the expression of OsRbohA and OsRbohB was induced by Xoo PXO99 strain. However, the levels of expression and responsive times of these genes were different. Moreover, all three treatments led to H2O2 accumulation. These OsRboh genes have functional roles in rice native immune response.
Acetates ; pharmacology ; Amino Acid Sequence ; Cyclopentanes ; pharmacology ; Hydrogen Peroxide ; metabolism ; Molecular Sequence Data ; Multigene Family ; NADPH Oxidases ; genetics ; immunology ; metabolism ; Oryza ; genetics ; immunology ; metabolism ; Oxylipins ; pharmacology ; Plant Immunity ; genetics ; Salicylic Acid ; pharmacology ; Xanthomonas ; pathogenicity