Identification and Dynamic Regulation of microRNAs Involved in Salt Stress Responses in Functional Soybean Nodules by High-Throughput Sequencing

Both symbiosis between legumes and rhizobia and nitrogen fixation in functional nodules are dramatically affected by salt stress. Better understanding of the molecular mechanisms that regulate the salt tolerance of functional nodules is essential for genetic improvement of nitrogen fixation efficiency. microRNAs (miRNAs) have been implicated in stress responses in many plants and in symbiotic nitrogen fixation (SNF) in soybean. However, the dynamic regulation of miRNAs in functioning nodules during salt stress response remains unknown. We performed deep sequencing of miRNAs to understand the miRNA expression profile in normal or salt stressed-soybean mature nodules. We identified 110 known miRNAs belonging to 61 miRNA families and 128 novel miRNAs belonging to 64 miRNA families. Among them, 104 miRNAs were dramatically differentially expressed (>2-fold or detected only in one library) during salt stress. qRT-PCR analysis of eight miRNAs confirmed that these miRNAs were dynamically regulated in response to salt stress in functional soybean nodules. These data significantly increase the number of miRNAs known to be expressed in soybean nodules, and revealed for the first time a dynamic regulation of miRNAs during salt stress in functional nodules. The findings suggest great potential for miRNAs in functional soybean nodules during salt stress.     

Proteins of Unknown Function in the Protein Data Bank (PDB): An Inventory of True Uncharacterized Proteins and Computational Tools for Their Analysis

Proteins of uncharacterized functions form a large part of many of the currently available biological databases and this situation exists even in the Protein Data Bank (PDB). Our analysis of recent PDB data revealed that only 42.53% of PDB entries (1084 coordinate files) that were categorized under “unknown function” are true examples of proteins of unknown function at this point in time. The remainder 1465 entries also annotated as such appear to be able to have their annotations re-assessed, based on the availability of direct functional characterization experiments for the protein itself, or for homologous sequences or structures thus enabling computational function inference.     

A Heavy Metal-Associated Protein (AcHMA1) from the Halophyte,Atriplex canescens (Pursh) Nutt., Confers Tolerance to Iron and Other Abiotic Stresses When Expressed in Saccharomyces cerevisiae

Many heavy metals are essential for metabolic processes, but are toxic at elevated levels. Metal tolerance proteins provide resistance to this toxicity. In this study, we identified and characterized a heavy metal-associated protein, AcHMA1, from the halophyte, Atriplex canescens. Sequence analysis has revealed that AcHMA1 contains two heavy metal binding domains. Treatments with metals (Fe, Cu, Ni, Cd or Pb), PEG6000 and NaHCO₃ highly induced AcHMA1 expression in A. canescens, whereas NaCl and low temperature decreased its expression. The role of AcHMA1 in metal stress tolerance was examined using a yeast expression system. Expression of the AcHMA1 gene significantly increased the ability of yeast cells to adapt to and recover from exposure to excess iron. AcHMA1 expression also provided salt, alkaline, osmotic and oxidant stress tolerance in yeast cells. Finally, subcellular localization of an AcHMA1/GFP fusion protein expressed in tobacco cells showed that AcHMA1 was localized in the plasma membrane. Thus, our results suggest that AcHMA1 encodes a membrane-localized metal tolerance protein that mediates the detoxification of iron in eukaryotes. Furthermore, AcHMA1 also participates in the response to abiotic stress.     

Identification and Functional Analysis of MicroRNAs and Their Targets in Platanus acerifolia under Lead (Pb) Stress

MicroRNAs (miRNAs) play important regulatory roles in development and stress responses in plants. Lead (Pb) is a non-essential element that is highly toxic to living organisms. Platanus acerifolia is grown as a street tree in cities throughout temperate regions for its importance in improving the urban ecological environment. MiRNAs that respond to abiotic stresses have been identified in plants; however, until now, the influence of Pb stress on P. acerifolia miRNAs has not been reported. To identify miRNAs and predict their target genes under Pb stress, two small RNA and two degradome libraries were constructed from Pb-treated and Pb-free leaves of P. acerifolia seedlings. After sequencing, 55 known miRNAs and 129 novel miRNAs were obtained, and 104 target genes for the miRNAs were identified by degradome sequencing. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway analyses were performed to predict the functions of the targets. The expressions of eight differentially expressed miRNAs were validated by quantitative real-time polymerase chain reaction (qRT-PCR). This is the first report about P. acerifolia miRNAs and their target genes under Pb stress. This study has provided data for further research into molecular mechanisms involved in resistance of P. acerifolia to Pb stress.     

Effects of the Bradyrhizobium japonicum waaL (rfaL) Gene on Hydrophobicity,Motility, Stress Tolerance,and Symbiotic Relationship with Soybeans

We cloned and sequenced the waaL (rfaL) gene from Bradyrhizobium japonicum, which infects soybean and forms nitrogen-fixing nodules on soybean roots. waaL has been extensively studied in the lipopolysaccharide (LPS) biosynthesis of enteric bacteria, but little is known about its function in (brady)rhizobial LPS architecture. To characterize its role as O-antigen ligase in the LPS biosynthesis pathway, we constructed a waaL knock-out mutant and its complemented strain named JS015 and CS015, respectively. LPS analysis showed that an LPS structure of JS015 is deficient in O-antigen as compared to that of the wild type and complemented strain CS015, suggesting that WaaL ligates the O-antigen to lipid A-core oligosaccharide to form a complete LPS. JS015 also revealed increased cell surface hydrophobicity, but it showed decreased motility in soft agar plates. In addition to the alteration in cell surface properties, disruption of the waaL gene caused increased sensitivity of JS015 to hydrogen peroxide, osmotic pressure, and novobiocin. Specifically, plant tests revealed that JS015 failed to nodulate the host plant soybean, indicating that the rhizobial waaL gene is responsible for the establishment of a symbiotic relationship between soybean and B. japonicum.     

Nitrogen rhizodeposition from soybean (<Emphasis Type="Italic">Glycine max</Emphasis>) and its impact on nutrient budgets in two contrasting environments of the Guinean savannah zone of Nigeria

Nitrogen (N) rhizodeposition by grain legumes such as soybean is potentially a large but neglected source of N in cropping systems of Sub-Saharan Africa. Field studies were conducted to measure soybean N rhizodeposition in two environments of the Guinean savannah of Nigeria using ¹⁵N leaf labelling techniques. The first site was located in Ibadan in the humid derived savannah. The second site was in Zaria in the drier Northern Guinean savannah. Soybean N rhizodeposition in the top 0.30 m of soil varied from 7.5 kg ha⁻¹ on a diseased crop in Ibadan to 33 kg ha⁻¹ in Zaria. More than two-thirds of soybean belowground N was contained in the rhizodeposits at crop physiological maturity, while the rest was found in the recoverable roots. Belowground plant-derived N was found to constitute 16–23% of the total soybean N. Taking rhizodeposited pools into account led to N budgets close to zero when all residues were removed. If residues were left in the field or recycled as manure after being fed to steers, soybean cultivation led to positive N budgets of up to +95 kg N ha⁻¹. The role and potential of grain legumes as N purveyors have been underestimated in the past by neglecting the N contained in their rhizodeposits.     

Identification and determination of the glyceride and tris-(2-hydroxyethyl)isocyanurate structures in the average macromolecule of a polyester with carbon-13-NMR spectroscopy: Analysis of the polyester average structure

The ¹³C-NMR approach to quantitative analysis of a polyester resin synthesized from dimethylterephthalate, ethylene glycol, tris-(2-hydroxyethyl)isocyanurate (THEIC), and glycerol is presented. Various glyceride and isocyanurate structures formed during the esterification have been identified and analyzed, at first in simple model systems and then in the structure of the resin. The contents of particular structural units were determined as well as a hypothetical average macromolecule of the resin defined. The assignments of ¹³C-NMR signals were made and correlated with the ¹H shifts using a 2D-HETCOR experiment. © 1998 John Wiley & Sons, Inc. J. Appl. Polym. Sci. 70: 675–687, 1998