Bradyrhizobium elkanii Induces Outer Cortical Root Swelling in Soybean

When soybean roots were inoculated with Bradyrhizobium elkaniiUSDA76, USDA94 or USDA31, a localized swelling of the root outercortical cells was observed within 10 days after inoculation.This phenotype was designated outer cortical swelling (OCS).OCS was not observed in roots inoculated with B.japonicum USDA110or USDA122. Therefore, the OCS response appears to be specificfor B. elkanii. To determine the involvement of the nodulationgenes in OCS formation, we constructed a nod^- mutant, strainUSDA94     

Annealing control primer system for identification of differentially expressed genes on agarose gels

We developed GeneFishing technology, an improved method for the identification of differentially expressed genes (DEGs) using our novel annealing control primer (ACP) system. Because of high annealing specificity during PCR using the ACP system, the application of the ACP to DEG discovery generates reproducible, authentic, and long (100 bp to 2 kb) PCR products that are detectable on agarose gels. To demonstrate this method for gene expression profiling, Gene-Fishing technology was used to detect genes that are differentially expressed during development using total RNAs isolated from mouse conceptus tissues at 4.5-18.5 days of gestation. Ten DEGs (DEG1-10) were isolated and confirmed by Northern blot hybridization. The sequence analysis of these DEGs showed that DEG6 and DEG10 are unknown genes.     

Induction of thioredoxin is required for nodule development to reduce reactive oxygen species levels in soybean roots

Nodules are formed on legume roots as a result of signaling between symbiotic partners and in response to the activities of numerous genes. We cloned fragments of differentially expressed genes in spot-inoculated soybean (Glycine max) roots. Many of the induced clones were similar to known genes related to oxidative stress, such as thioredoxin and beta-carotene hydroxylase. The deduced amino acid sequences of full-length soybean cDNAs for thioredoxin and beta-carotene hydroxylase were similar to those in other species. In situ RNA hybridization revealed that the thioredoxin gene is expressed on the pericycle of 2-d-old nodules and in the infected cells of mature nodules, suggesting that thioredoxin is involved in nodule development. The thioredoxin promoter was found to contain a sequence resembling an antioxidant responsive element. When a thioredoxin mutant of yeast was transformed with the soybean thioredoxin gene it became hydrogen peroxide tolerant. These observations prompted us to measure reactive oxygen species levels. These were decreased by 3- to 5-fold in 7-d-old and 27-d-old nodules, coincident with increases in the expression of thioredoxin and beta-carotene hydroxylase genes. Hydrogen peroxide-producing regions identified with cerium chloride were found in uninoculated roots and 2-d-old nodules, but not in 7-d-old and 27-d-old nodules. RNA interference-mediated repression of the thioredoxin gene severely impaired nodule development. These data indicate that antioxidants such as thioredoxin are essential to lower reactive oxygen species levels during nodule development.     

Efficient release of ferulic acid from sweet potato (<Emphasis Type="Italic">Ipomoea batatas</Emphasis>) stems by chemical hydrolysis

An efficient method for release of ferulic acid from sweet potato stems was developed. Ferulic acid along with phenolic compounds were released from stems by acid and alkaline treatments. The base hydrolysis with 0.1 N NaOH yielded the highest quantity of total extracts (471.1 mg/g). The stems released more phenolic compounds when 0.0125∼0.025 N NaOH was employed. Where as ferulic acid release was maximal with 0.05 N H₂SO₄ (0.32 mg/g). Ferulic acid was separated from phenolics by column chromatography. Among the elution solvents, ethyl acetate fractions (80%) contained ferulic acid. Ethyl acetate eluants were further fractionated with n-hexane/ethyl acetate/formic acid (100/50/0.5, v/v/v). All fractions showed ferulic acid and phenolic compounds. Fraction V among them was ascribed to ferulic acid with an yield of 5.41 mg/g of dry sweet potato tissue.     

An atypical soybean leucine-rich repeat receptor-like kinase, GmLRK1, may be involved in the regulation of cell elongation

A leucine-rich repeat receptor-like kinase (LRR-RLK) encoded by one of the genes highly expressed in a specific stage of soybean seed development, referred to as GmLRK1, was identified and characterized. Examination of its kinase domain indicated that GmLRK1 may be a catalytically inactive atypical receptor kinase. An autophosphorylation assay confirmed that GmLRK1 is incapable of autophosphorylation in vitro. However, the phosphorylation of GmRLK1 could be induced after incubation with plant protein extracts, suggesting that some plant proteins may interact with GmLRK1 and phosphorylate the protein in vivo. Analyses of the expression profiles of GmLRK1 and its Arabidopsis ortholog At2g36570 revealed that they may be involved in regulation of more fundamental metabolic and/or developmental pathways, rather than a specialized developmental program such as seed development. Our results further indicate that the GmLRK1 and At2g36570 may play a role in the regulation of certain cellular processes that lead to cell elongation and expansion. S. Kim and S.-J. Kim have contributed equally to this work.     

Overexpression of <Emphasis Type="Italic">GmAKR1</Emphasis>, a stress-induced aldo/keto reductase from soybean,retards nodule development

Development of symbiotic root nodules in legumes involves the induction and repression of numerous genes in conjunction with changes in the level of phytohormones. We have isolated several genes that exhibit differential expression patterns during the development of soybean nodules. One of such genes, which were repressed in mature nodules, was identified as a putative aldo/keto reductase and thus named Glycine max aldo/keto reductase 1 (GmAKR1). GmAKR1 appears to be a close relative of a yeast aldo/keto reductase YakC whose in vivo substrate has not been identified yet. The expression of GmAKR1 in soybean showed a root-specific expression pattern and inducibility by a synthetic auxin analogue 2,4-D, which appeared to be corroborated by presence of the root-specific element and the stress-response element in the promoter region. In addition, constitutive overexpression of GmAKR1 in transgenic soybean hairy roots inhibited nodule development, which suggests that it plays a negative role in the regulation of nodule development. One of the Arabidopsis orthologues of GmAKR1 is the ARF-GAP domain 2 protein, which is a potential negative regulator of vesicle trafficking; therefore GmAKR1 may have a similar function in the roots and nodules of legume plants. These authors contributed equally to this work.     

Dual priming oligonucleotide system for the multiplex detection of respiratory viruses and SNP genotyping of CYP2C19 gene

Successful PCR starts with proper priming between an oligonucleotide primer and the template DNA. However, the inevitable risk of mismatched priming cannot be avoided in the currently used primer system, even though considerable time and effort are devoted to primer design and optimization of reaction conditions. Here, we report a novel dual priming oligonucleotide (DPO) which contains two separate priming regions joined by a polydeoxyinosine linker. The linker assumes a bubble-like structure which itself is not involved in priming, but rather delineates the boundary between the two parts of the primer. This structure results in two primer segments with distinct annealing properties: a longer 5′-segment that initiates stable priming, and a short 3′-segment that determines target-specific extension. This DPO-based system is a fundamental tool for blocking extension of non-specifically primed templates, and thereby generates consistently high PCR specificity even under less than optimal PCR conditions. The strength and utility of the DPO system are demonstrated here using multiplex PCR and SNP genotyping PCR.     

Annealing control primer system for improving specificity of PCR amplification

A novel primer designed to improve the specificity of PCR amplification, called the annealing control primer (ACP), comprises a tripartite structure with a polydeoxyinosine [poly(dI)] linker between the 3' end target core sequence and the 5' end nontarget universal sequence. We show that this ACP linker prevents annealing of the 5' end nontarget sequence to the template and facilitates primer hybridization at the 3' end to the target sequence at specific temperatures, resulting in a dramatic improvement of annealing specificity. The effect of this linker is demonstrated by the incorporation of ACP sequences as primers during the amplification of target nucleotide sequence and as hybridization probes in the genotyping of single nucleotide polymorphisms. This is the first report to show that a poly(dI) linker between two different sequences of ACP forms a bubble-like structure and disrupts or destabilizes DNA duplex formation at certain annealing temperatures.     

Induction of a homeodomain-leucine zipper gene by auxin is inhibited by cytokinin in Arabidopsis roots

Homeobox genes are essential regulators of the development of plants as well as other organisms. We chose eight putative Arabidopsis homeobox genes not previously characterized and examined their expression in response to treatment with auxin/cytokinin. One of them, ATHB53, was further studied because it was auxin-inducible and its induction was inhibited by cytokinin. Its full-length cDNA was cloned and found to encode a protein of the HD-Zip superfamily. Whole-mount in situ hybridization and RT-PCR showed that it was expressed in the root meristem, and auxin treatment increased its expression, especially in a region from 0.3 to 0.6mm from the root tip. These results suggest that ATHB53 plays a regulatory role in auxin/cytokinin signaling during root development.