Transcriptomic Studies in Non-Model Plants: Case of Pisum sativum L. and Medicago lupulina L.

Transcriptomics is a dynamically developing branch of biology highly important for geneticists and molecular ecologists alike. A large number of studies concerning differential gene expression, mapping of genes and quantitative trait loci (QTL), analysis of genotyping variations and so on has been conducted recently on several non‐model plants using next‐generation sequencing techniques. One example of non‐model legumes is garden pea (Pisum sativum L.), a valuable pulse crop capable of forming nitrogen‐fixing nodules and arbuscular mycorrhiza. Adaptation of standardised RNA‐seq approaches and data analysis developed for model plants to P. sativum should facilitate both studying of pea molecular genetics and breeding of new cultivars possessing agriculturally important traits. Another non‐model legume is black medick Medicago lupulina L. (a close relative of model legume plant barrel medick, Medicago truncatula Gaertn.), for which unique genetic lines almost obligatory dependent on arbuscular mycorrhiza symbiosis formation have been obtained. Such lines show promise as the perfect model for studying the genetic bases of arbuscular mycorrhiza development. In this chapter, we give a brief description of the current developments in the field of garden pea and black medick transcriptomics. Our aim is to provide a quick start guide to the non‐expert researchers for next‐generation sequencing (NGS)‐based transcriptome analysis

The DELLA Proteins Influence the Expression of Cytokinin Biosynthesis and Response Genes During Nodulation

The key event that initiates nodule organogenesis is the perception of bacterial signal molecules, the Nod factors, triggering a complex of responses in epidermal and cortical cells of the root. The Nod factor signaling pathway interacts with plant hormones, including cytokinins and gibberellins. Activation of cytokinin signaling through the homeodomain-containing transcription factors KNOX is essential for nodule formation. The main regulators of gibberellin signaling, the DELLA proteins are also involved in regulation of nodule formation. However, the interaction between the cytokinin and gibberellin signaling pathways is not fully understood. Here, we show in Pisum sativum L. that the DELLA proteins can activate the expression of KNOX and BELL transcription factors involved in regulation of cytokinin metabolic and response genes. Consistently, pea la cry-s (della1 della2) mutant showed reduced ability to upregulate expression of some cytokinin metabolic genes during nodulation. Our results suggest that DELLA proteins may regulate cytokinin metabolism upon nodulation

High-Throughput Fingerprinting of Rhizobial Free Fatty Acids by Chemical Thin-Film Deposition and Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry

Fatty acids (FAs) represent an important class of metabolites, impacting on membrane building blocks and signaling compounds in cellular regulatory networks. In nature, prokaryotes are characterized with the most impressing FA structural diversity and the highest relative content of free fatty acids (FFAs). In this context, nitrogen-fixing bacteria (order Rhizobiales), the symbionts of legumes, are particularly interesting. Indeed, the FA profiles influence the structure of rhizobial nodulation factors, required for successful infection of plant root. Although FA patterns can be assessed by gas chromatography—(GC-) and liquid chromatography—mass spectrometry (LC-MS), sample preparation for these methods is time-consuming and quantification suffers from compromised sensitivity, low stability of derivatives and artifacts. In contrast, matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF-MS) represents an excellent platform for high-efficient metabolite fingerprinting, also applicable to FFAs. Therefore, here we propose a simple and straightforward protocol for high-throughput relative quantification of FFAs in rhizobia by combination of Langmuir technology and MALDI-TOF-MS featuring a high sensitivity, accuracy and precision of quantification. We describe a step-by-step procedure comprising rhizobia culturing, pre-cleaning, extraction, sample preparation, mass spectrometric analysis, data processing and post-processing. As a case study, a comparison of the FFA metabolomes of two rhizobia species—Rhizobium leguminosarum and Sinorhizobium meliloti, demonstrates the analytical potential of the protocol

Concept, opportunities and challenges of urban tourism in the Arab world: Case studies of Dubai, Cairo and Amman

This paper aims to provide a better understanding of the current situation of urban tourism with referring to the experiences of the Arab World. By using bibliographic documentation and descriptive-analytic method, this paper addresses the main opportunities, impediments, and challenges of urban tourism in three Arab cities (Dubai, Cairo and Amman). This paper indicates that the three cities have many opportunities stemming from their location and their rich tourism resources. Moreover, urban tourism in these cities should confront some internal (country based) and external (global and regional) challenges such as seasonality, pollution, congestion, competition, funding and instability. Some recommendations and policy implications were suggested. The paper concludes, by arguing, that common internal and external challenges need to be addressed in a systematic manner within the broader cultural and tourism policy context in which urban tourism is now implicated. There is a lack of literature on urban tourism within the Arab countries, which is attributed to that tourism in the Arab world is heritage dominated type. Therefore, this research also attempts to bridge the gap in the existing literature about urban tourism in the Arab cities

Mapping-by-sequencing using NGS-based 3′-MACE-Seq reveals a new mutant allele of the essential nodulation gene Sym33 (IPD3) in pea (Pisum sativum L.)

Large collections of pea symbiotic mutants were accumulated in the 1990s, but the causal genes for a large portion of the mutations are still not identified due to the complexity of the task. We applied a Mapping-by-Sequencing approach including Bulk Segregant Analysis and Massive Analysis of cDNA Ends (MACE-Seq) sequencing technology for genetic mapping the Sym11 gene of pea which controls the formation of symbioses with both nodule bacteria and arbuscular-mycorrhizal fungi. For mapping we developed an F2-population from the cross between pea line N24 carrying the mutant allele of sym11 and the wild type NGB1238 (=JI0073) line. Sequencing libraries were prepared from bulks of 20 plants with mutant and 12 with wild-type phenotype. MACE-Seq differential gene expression analysis between mutant-phenotype and wild-type-phenotype bulks revealed 2,235 genes, of which 514 (23%) were up-regulated and 1,721 (77%) were down-regulated in plant roots inoculated with rhizobia as a consequence of sym11 mutation. MACE-Seq also detected single nucleotide variants between bulks in 217 pea genes. Using a novel mathematical model we calculated the recombination frequency (RF) between the Sym11 gene and these 217 polymorphic genes. Six genes with the lowest RF were converted into CAPS or dCAPS markers and genetically mapped on the complete mapping population of 108 F2-plants which confirmed their tight linkage to Sym11 and to each other. The Medicago truncatula Gaertn. (Mt) homologs of these genes are located in a distinct region of Mt chromosome 5, which corresponds to linkage group I of pea. Among 94 candidate genes from this region only one was down-regulated—the pea Sym33 homolog of the Mt IPD3 gene which is essential for nodulation. Sequencing of the Sym33 allele of the N24 (sym11) mutant revealed a single nucleotide deletion (c.C319del) in its third exon resulting in a codon shift in the open reading frame and premature translation termination. Thus, we identified a novel mutant allele sym33-4 most probably responsible for the mutant phenotype of the N24 (sym11) line, thereby demonstrating that mapping by MACE-Seq can be successfully used for genetic mapping of mutations and identification of candidate genes in pea