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Microsporogenesis is an indispensable period for investigating microspore development and cytoplasmic male sterility (CMS) occurrence. Radish CMS line plays a critical role in elite F hybrid seed production and heterosis utilization. However, the molecular mechanisms of microspore development and CMS occurrence have not been thoroughly uncovered in radish. In this study, a comparative analysis of radish floral buds from a CMS line (NAU-WA) and its maintainer (NAU-WB) was conducted using next generation sequencing (NGS) technology. Digital gene expression (DGE) profiling revealed that 3504 genes were significantly differentially expressed between NAU-WA and NAU-WB library, among which 1910 were upregulated and 1594 were downregulated. Gene ontology (GO) analysis showed that these differentially expressed genes (DEGs) were mainly enriched in extracellular region, catalytic activity, and response to stimulus. KEGG enrichment analysis revealed that the DEGs were predominantly associated with flavonoid biosynthesis, glycolysis, and biosynthesis of secondary metabolites. Real-time quantitative PCR analysis showed that the expression profiles of 13 randomly selected DEGs were in high agreement with results from Illumina sequencing. Several candidate genes encoding ATP synthase, auxin response factor (ARF), transcription factors (TFs), chalcone synthase (CHS), and male sterility (MS) were responsible for microsporogenesis. Furthermore, a schematic diagram for functional interaction of DEGs from NAU-WA vs. NAU-WB library in radish plants was proposed. These results could provide new information on the dissection of the molecular mechanisms underlying microspore development and CMS occurrence in radish. [ABSTRACT FROM AUTHOR]

Cholinergic neurons in the medial habenula (MHb) modulate anxiety during nicotine withdrawal although the molecular neuroadaptation(s) within the MHb that induce affective behaviors during nicotine cessation is largely unknown. MHb cholinergic neurons are unique in that they robustly express neuronal nicotinic acetylcholine receptors (nAChRs), although their behavioral role as autoreceptors in these neurons has not been described. To test the hypothesis that nAChR signaling in MHb cholinergic neurons could modulate anxiety, we expressed novel “gain of function” nAChR subunits selectively in MHb cholinergic neurons of adult mice. Mice expressing these mutant nAChRs exhibited increased anxiety-like behavior that was alleviated by blockade with a nAChR antagonist. To test the hypothesis that anxiety induced by nicotine withdrawal may be mediated by increased MHb nicotinic receptor signaling, we infused nAChR subtype selective antagonists into the MHb of nicotine naïve and withdrawn mice. While antagonists had little effect on nicotine naïve mice, blocking α4β2 or α6β2, but not α3β4 nAChRs in the MHb alleviated anxiety in mice undergoing nicotine withdrawal. Consistent with behavioral results, there was increased functional expression of nAChRs containing the α6 subunit in MHb neurons that also expressed the α4 subunit. Together, these data indicate that MHb cholinergic neurons regulate nicotine withdrawal-induced anxiety via increased signaling through nicotinic receptors containing the α6 subunit and point toward nAChRs in MHb cholinergic neurons as molecular targets for smoking cessation therapeutics. [ABSTRACT FROM AUTHOR]

Key message: Transcriptome-based gene expression analysis identifies many critical salt-responsive genes in radish and facilitates further dissecting the molecular mechanism underlying salt stress response. Abstract: Salt stress severely impacts plant growth and development. Radish, a moderately salt-sensitive vegetable crop, has been studied for decades towards the physiological and biochemical performances under salt stress. However, no systematic study on isolation and identification of genes involved in salt stress response has been performed in radish, and the molecular mechanism governing this process is still indistinct. Here, the RNA-Seq technique was applied to analyze the transcriptomic changes on radish roots treated with salt (200 mM NaCl) for 48 h in comparison with those cultured in normal condition. Totally 8709 differentially expressed genes (DEGs) including 3931 up- and 4778 down-regulated genes were identified. Functional annotation analysis indicated that many genes could be involved in several aspects of salt stress response including stress sensing and signal transduction, osmoregulation, ion homeostasis and ROS scavenging. The association analysis of salt-responsive genes and miRNAs exhibited that 36 miRNA-mRNA pairs had negative correlationship in expression trends. Reverse-transcription quantitative PCR (RT-qPCR) analysis revealed that the expression profiles of DEGs were in line with results from the RNA-Seq analysis. Furthermore, the putative model of DEGs and miRNA-mediated gene regulation was proposed to elucidate how radish sensed and responded to salt stress. This study represents the first comprehensive transcriptome-based gene expression profiling under salt stress in radish. The outcomes of this study could facilitate further dissecting the molecular mechanism underlying salt stress response and provide a valuable platform for further genetic improvement of salt tolerance in radish breeding programs. [ABSTRACT FROM AUTHOR]

FePt–C, FePt–SiO 2 and FePt–C/FePt–SiO 2 films with different segregants concentration on glass substrate with polycrystalline MgO underlayer have been successfully fabricated by magnetron sputtering system. It was found that the FePt–C films had larger coercivity( H c ), chemical order parameter(S) and smaller diameter of the local interaction zone( D n ) but wider magnetic coercive force distribution(Δ H c / H c ) than that of the FePt–SiO 2 films. By using FePt–C/FePt–SiO 2 bilayer structure, the low D n and Δ H c / H c were obtained. FePt–C30 vol %(4 nm)/FePt–SiO 2 45 vol %(4 nm) film with H c higher than 29 kOe and columnar FePt grains of 10 nm in diameter and 13 nm in height were obtained, which would be very suitable for heat assisted magnetic recording media. [ABSTRACT FROM AUTHOR]

microRNAs (miRNAs) are a class of single-stranded endogenous non-coding RNAs that play critical roles in plant growth, development, and environmental stress responses. Temperature is one of the major physical parameters disturbing cellular homeostasis and causing leaf etiolation in plants. Previous studies have reported that several conserved and novel miRNAs were responsive to heat stress in plants. However, the characterization of miRNAs responsive to heat stress in radish remains poorly understood. To better understand miRNAs and their target genes under heat stress, two small RNA libraries were constructed from heat-treated (Heat24) and heat-untreated (CK) radish roots. Using Solexa system, totally, 26 known and 19 novel miRNAs were identified as differentially expressed under heat stress. Expression patterns of a set of heat-responsive miRNAs were validated by quantitative real-time PCR (qRT-PCR). Furthermore, 422 sliced targets for 25 known miRNAs were identified by degradome sequencing technology, and most of the identified targets are involved in multiple biological processes including transcriptional regulation and response to biotic and abiotic stresses. Moreover, some miRNAs and their corresponding targets, which are related to the accumulation of heat stress transcription factors and heat shock proteins, played important roles in thermo-tolerance in radish. These findings could enhance the understanding of molecular mechanisms underlying miRNAs and their targets in regulating plant responses to heat stress. [ABSTRACT FROM AUTHOR]

Increasing evidence has revealed that microRNA (miRNA)-mediated gene regulation plays a significant role in response to heavy metal stresses. However, there is little information available about the expression patterns or roles of miRNAs under lead toxicity stress in plants. The radish is an important root vegetable crop with a fleshy taproot as the edible part. It was of vital importance to investigate the response mechanisms and explore the regulatory network at the molecular level under the heavy metal stresses in radish. In the present study, using high-throughput sequencing and degradome analysis, a genome-wide identification of radish miRNA and their targets under the exposure of Pb stress was conducted. A total of 74 known and 173 potential novel miRNAs were successfully identified from two radish root libraries of one untreated control (CK) and one Pb-stressed (Pb500). Of these, 25 known and nine novel miRNAs were significantly differentially expressed and identified as Pb-responsive miRNAs. Degradome analysis revealed that 1,979 miRNA-mRNA target transcripts could potentially be cleaved. Gene Ontology (GO) analysis revealed that these target transcripts were predominately involved in the regulation of transcription, defense responses, and binding related terms. The identified target genes for Pb-responsive miRNAs were mainly involved in stress-related signal sensing and transduction, specific metal uptake and homeostasis mechanisms. Additionally, the expression patterns of 20 Pb-responsive miRNAs and six target genes were validated by quantitative real-time PCR (qRT-PCR). These results provide fundamental insights into the miRNA-mediated regulatory networks and molecular mechanisms underlying plant responsiveness to Pb stresses. [ABSTRACT FROM AUTHOR]

Calcium (Ca) currents and the M-type potassium current are modulated by an incompletely characterized diffusible second messenger pathway called the slow pathway. Great enthusiasm has greeted a recent hypothesis that PLC metabolism of phosphatidylinositol 4,5 bis-phosphate ([PIP.sub.2]), constitutively bound to channels, dissociates causing current inhibition; no further downstream processing of [PIP.sub.2] is required (Suh and Hille. 2002. Neuron 35:507-520; Wu et al. 2002. Nature. 419:947-952). In contrast, our data indicate that additional processing of [PIP.sub.2], is required for Ca current modulation. In addition to [M.sub.1] muscarinic receptors ([M.sub.1]Rs), [G.sub.q] and phospholipase C (PLC), we found phospholipase [A.sub.2] ([PLA.sub.2]) and a free fatty acid, most likely arachidonic acid (AA), appear to participate in L- and N-type Ca current inhibition (SGP abstract Liu, Roberts, and Rittenhouse). Demonstrating that [PLA.sub.2] is required tot Ca current inhibition by the slow pathway is critical for resolving the differences, if any, between these two models. Here, we use patch-clamp and molecular methods to determine which [PLA.sub.2] participates in L-current inhibition by the slow pathway in neonatal rat SCG neurons. Antibodies (Abs) selective for different [PLA2]s were dialyzed into neurons treated with [omega]-conotoxin-GVIA to block N-current. Type IV (c[PLA.sub.2]) or type VI (I[PLA.sub.2]) Abs, but not nonimmunized IgG or type IIa [PLA.sub.2] (s[PLA.sub.2]) Abs, minimized current inhibition by the muscarinic agonist oxotremorine-M (Oxo-M). Inhibition of FPL-64176 induced long-lasting L-type tail currents, normally observed with Oxo-M, was also minimized when the c[PLA.sub.2] Ab was dialyzed into cells. Western blot analysis revealed that the c[PLA.sub.2] Ab recognized a single band at the approximate molecular weight of c[PLA.sub.2]. PCR confirmed c[PLA.sub.2]'s presence in SCG consistent with Western blot analysis. These data identify, c[PLA.sub.2] as a necessary participant in [M.sub.1]R inhibition of L-current. We are currently probing the role of c[PLA.sub.2] further using gene knockout technology. (Supported by NIH RO1-NS 34195.)

microRNAs (miRNAs), endogenous non-coding RNAs of approximately 21-24 nucleotides, are important regulators of transcriptional and post-transcriptional gene expression. These regulators play a key role during plant growth and development, including embryogenesis, which is crucial to the life cycle of most plant species. However, although embryogenesis-associated miRNAs have been isolated in a few species, the diversity of these regulatory miRNAs remains largely unexplored, especially in radish. In this study, two small RNA libraries were constructed from radish ovules before and after fertilization. Both libraries were sequenced by next generation sequencing (NGS) technology. This analysis identified 144 conserved and 34 non-conserved miRNAs (representing 43 known miRNA families) and 38 novel miRNAs (representing 28 miRNA families). Comparative analysis revealed that 29 known and 10 novel miRNA families were differentially expressed during embryogenesis. QRT-PCR analysis confirmed miRNA expression patterns and revealed tissue-specific and/or developmental stage-dependent expression for some miRNAs. Moreover, potential target predictions indicated that most of these targets were transcription factors involved in regulating plant growth, development and metabolism. Notably, target transcripts such as squamosa promoter-binding protein, auxin response factor and agamous-like MADS-box protein participated in radish embryogenesis. [ABSTRACT FROM AUTHOR]

Resistance Gene Analogs (RGAs)-based molecular markers can significantly enhance the breeding efficiency for disease resistance in various crops. However, RGAs isolation and RGA-based marker development have not been conducted in radish. In this study, two prevalent approaches, PCR amplification with degenerate primers and data-mining of radish EST databases, were used to isolate radish resistance gene candidate sequences. A total of 26 RsRGAs with high homology to known R-genes or RGAs were obtained by PCR amplification. Meanwhile, 257 R-gene-like unigenes/ESTs were identified using data-mining method. In total, 115 out of 124 designed RGA-specific primer pairs can successfully amplified the target bands. To explore the RGA genetic variability, 32 radish accessions were genotyped and 269 RGA loci were successfully identified. It was found that 44 RGA primers only generated monomorphic band and the remaining 71 RGA primers generated 225 RGA polymorphic bands among the 32 radish accessions. Based on RGA marker analysis, 32 radish accessions were clustered into four main groups at the similarity index of 0.68, which was in a high accordance with disease index from a downy mildew evaluation. This study might be the first report on candidate R-genes identification and RGA-based markers development in radish. These findings will facilitate the disease resistance identification and speed up the genetic improvement of DM resistances in radish breeding programs. [ABSTRACT FROM AUTHOR]

By in silicon cloning and genome walking strategies, the gDNA, cDNA and promoter sequences of RsFPF1 gene in radish were isolated, and the expression pattern analysis as well as transgenic functional validation was conducted. Sequence analysis showed that RsFPF1 gene was 330 bp and encoded 109 amino acids, which shared highest homology with FPF1 protein in Arabidopsis thaliana and white mustard. The 5' upstream promoter region of RsFPF1 gene was 1 845 bp, and PLACE and PlantCARE analysis indicated that it contained several typical promoter elements and light response cis-regulatory elements. Moreover, with semi-quantitative RT-PCR the RsFPF1 gene exhibited a high expression level in shoot tip before flowering, whereas it showed relatively maximum expression level in flowers and floral buds after flowering. Furthermore, the positive transgenic tobacco plants from Agrobac terium-mediated leaf-disc transformation exhibited significant early flowering stage as compared with the wild-type plants. These results suggested that RsFPF1 gene can promote early flowering and its expression profile might be regulated by light, which could play critical roles in the regulation of bolting and flowering, and gene expression relative to floral initiation in radish. [ABSTRACT FROM AUTHOR]

Simple sequence repeats (SSRs) are highly polymorphic and co-dominant markers, providing an important genomic resource for genetic research. Recently, large-scale transcriptome sequencing has become a reliable and efficient approach for the identification and development of new genic-SSR markers and has been successfully conducted in a few important plant species. However, SSR development based on transcriptome sequencing remains limited in radish ( Raphanus sativus L.). In the present study, from a total of 73,084 unigenes and 150,455 contigs which were assembled from 71.95 million Illumina sequence reads of a radish taproot library, a collection of 11,928 genic-SSR loci were successfully identified in 11,311 unigene sequences. Trinucleotide repeats were the most abundant repeat units, as in many other plants, with a frequency of 52 %. Furthermore, a total of 5,503 genic-SSR primers were developed, from which 1,052 SSR primers were synthesized, and a subset of 823 (78.23 %) primers could generate stable bands. Moreover, 67 selected informative genic-SSR markers were used to determine the genetic diversity of 32 radish genotypes, in which the polymorphism information content values ranged from 0.49 to 0.89. For effective cultivar identification, a novel strategy called manual cultivar identification diagram was employed. Thirty-two radish accessions were clearly separated by six genic-SSR markers. Additionally, the cross-species/genera transferability of these SSRs was further validated in nine relatives in Brassicaceae. These results suggested that the novel genic-SSR markers, as a basis for future genetic linkage and gene tagging analysis, could be very valuable in facilitating genetic mapping, marker-assisted selection and comparative genome analysis. [ABSTRACT FROM AUTHOR]

Cytoplasmic male sterility (CMS) is a maternally inherited trait, which becomes an important way in heterosis breeding to simplify the seed production procedures and reduce the production cost. Cytological observation of pollen development stages showed a clear difference between the newly developed CMS line and its maintainer; the profiling comparisons between floral buds of CMS line and its maintainer were conducted using differential display reverse transcription PCR (DDRT-PCR) technology. Thirty genes were up-regulated and sixty genes were down-regulated in newly developed CMS line when compared with its maintainer. These genes were involved in cell wall biosynthesis and regulation, transporter and ion channel, flower development and protein metabolism, etc. Expression patterns of six genes encoding RsCAM6, RsGPI, RsPMEI, RsRac, RsCHS, and RsSTP9 were verified by RT-PCR and qRT-PCR in different development stage of floral buds and different organs of CMS line and its maintainer. The expression level of RsRac was higher in stamens and microspores of CMS line than in maintainer. In comparison, the expression levels of the other five genes in CMS line were lower compared with maintainer. This expression profile suggests that these genes played important roles in the development of the pollen and may be closely related to male sterility. The results observed in this study will contribute to understanding the mechanism of pollen abortion during CMS in radish. [ABSTRACT FROM AUTHOR]