Identification of growth regulators using cross-species network analysis in plants.

With the need to increase plant productivity, one of the challenges plant scientists are facing is to identify genes that play a role in beneficial plant traits. Moreover, even when such genes are found, it is generally not trivial to transfer this knowledge about gene function across species to identify functional orthologs. Here, we focused on the leaf to study plant growth. First, we built leaf growth transcriptional networks in Arabidopsis (Arabidopsis thaliana), maize (Zea mays), and aspen (Populus tremula). Next, known growth regulators, here defined as genes that when mutated or ectopically expressed alter plant growth, together with cross-species conserved networks, were used as guides to predict novel Arabidopsis growth regulators. Using an in-depth literature screening, 34 out of 100 top predicted growth regulators were confirmed to affect leaf phenotype when mutated or overexpressed and thus represent novel potential growth regulators. Globally, these growth regulators were involved in cell cycle, plant defense responses, gibberellin, auxin, and brassinosteroid signaling. Phenotypic characterization of loss-of-function lines confirmed two predicted growth regulators to be involved in leaf growth (NPF6.4 and LATE MERISTEM IDENTITY2). In conclusion, the presented network approach offers an integrative cross-species strategy to identify genes involved in plant growth and development.

MicroRNAs in durum wheat seedlings under chronic and short-term nitrogen stress.

Nitrogen is an essential macronutrient for plant growth and reproduction. In durum wheat, an appropriate nitrogen soil availability is essential for an optimal seed development. miRNAs contribute to the environmental change adaptation of plants through the regulation of important genes involved in stress processes. In this work, nitrogen stress response was evaluated in durum wheat seedlings of Ciccio and Svevo cultivars. Eight small RNA libraries from leaves and roots of chronically stressed plants were sequenced to detect conserved and novel miRNAs. A total of 294 miRNAs were identified, 7 of which were described here for the first time. The expression level of selected miRNAs and target genes was analyzed by qPCR in seedlings subjected to chronic (Ciccio and Svevo, leaves and roots) or short-term (Svevo roots) stress conditions. Some miRNAs showed an immediate stress response, and their level of expression was either maintained or returned to a basal level during a long-term stress. Other miRNAs showed a gradual up- or downregulation during the short-term stress. The newly identified miRNA ttu-novel-106 showed an immediate strongly downregulation after nitrogen stress, which was negatively correlated with the expression of MYB-A, its putative target gene. PHO2 gene was significantly upregulated after 24-48-h stress, corresponding to a downregulation of miR399b. Ttu-miR399b putative binding sites in the 5' UTR region of the Svevo PHO2 gene were identified in the A and B genomes. Both MYB-A and PHO2 genes were validated for their cleavage site using 5' RACE assay.

A Survey of the Transcriptomic Resources in Durum Wheat: Stress Responses, Data Integration and Exploitation.

Durum wheat (Triticum turgidum subsp. durum (Desf.) Husn.) is an allotetraploid cereal crop of worldwide importance, given its use for making pasta, couscous, and bulgur. Under climate change scenarios, abiotic (e.g., high and low temperatures, salinity, drought) and biotic (mainly exemplified by fungal pathogens) stresses represent a significant limit for durum cultivation because they can severely affect yield and grain quality. The advent of next-generation sequencing technologies has brought a huge development in transcriptomic resources with many relevant datasets now available for durum wheat, at various anatomical levels, also focusing on phenological phases and environmental conditions. In this review, we cover all the transcriptomic resources generated on durum wheat to date and focus on the corresponding scientific insights gained into abiotic and biotic stress responses. We describe relevant databases, tools and approaches, including connections with other "omics" that could assist data integration for candidate gene discovery for bio-agronomical traits. The biological knowledge summarized here will ultimately help in accelerating durum wheat breeding.

High-density linkage mapping and genetic dissection of resistance to broomrape (Orobanche crenata Forsk.) in pea (Pisum sativum L.).

Pea (Pisum sativum L.) is a widely cultivated legume of major importance for global food security and agricultural sustainability. Crenate broomrape (Orobanche crenata Forsk.) (Oc) is a parasitic weed severely affecting legumes, including pea, in the Mediterranean Basin and the Middle East. Previously, the identification of the pea line "ROR12", displaying resistance to Oc, was reported. Two-year field trials on a segregant population of 148 F7 recombinant inbred lines (RILs), originating from a cross between "ROR12" and the susceptible cultivar "Sprinter", revealed high heritability (0.84) of the "ROR12" resistance source. Genotyping-by-sequencing (GBS) on the same RIL population allowed the construction of a high-density pea linkage map, which was compared with the pea reference genome and used for quantitative trait locus (QTL) mapping. Three QTLs associated with the response to Oc infection, named PsOcr-1, PsOcr-2, and PsOcr-3, were identified, with PsOcr-1 explaining 69.3% of the genotypic variance. Evaluation of the effects of different genotypic combinations indicated additivity between PsOcr-1 and PsOcr-2, and between PsOcr-1 and PsOcr-3, and epistasis between PsOcr-2 and PsOcr-3. Finally, three Kompetitive Allele Specific PCR (KASP) marker assays were designed on the single-nucleotide polymorphisms (SNPs) associated with the QTL significance peaks. Besides contributing to the development of pea genomic resources, this work lays the foundation for the obtainment of pea cultivars resistant to Oc and the identification of genes involved in resistance to parasitic Orobanchaceae.

R2R3-MYBs in Durum Wheat: Genome-Wide Identification, Poaceae-Specific Clusters, Expression, and Regulatory Dynamics Under Abiotic Stresses.

MYB transcription factors (TFs) represent one of the biggest TF families in plants, being involved in various specific plant processes, such as responses to biotic and abiotic stresses. The implication of MYB TFs in the tolerance mechanisms to abiotic stress is particularly interesting for crop breeding, since environmental conditions can negatively affect growth and productivity. Wheat is a worldwide-cultivated cereal, and is a major source of plant-based proteins in human food. In particular, durum wheat plays an important role in global food security improvement, since its adaptation to hot and dry conditions constitutes the base for the success of wheat breeding programs in future. In the present study, a genome-wide identification of R2R3-MYB TFs in durum wheat was performed. MYB profile search and phylogenetic analyses based on homology with Arabidopsis and rice MYB TFs led to the identification of 233 R2R3-TdMYB (Triticum durum MYB). Three Poaceae-specific MYB clusters were detected, one of which had never been described before. The expression of eight selected genes under different abiotic stress conditions, revealed that most of them responded especially to salt and drought stress. Finally, gene regulatory network analyses led to the identification of 41 gene targets for three TdR2R3-MYBs that represent novel candidates for functional analyses. This study provides a detailed description of durum wheat R2R3-MYB genes and contributes to a deeper understanding of the molecular response of durum wheat to unfavorable climate conditions.

Durum wheat miRNAs in response to nitrogen starvation at the grain filling stage.

Durum wheat highly depends on nitrogen for seed development and yield, and the obtainment of varieties with a better nitrogen use efficiency is crucial to reduce production costs and environmental pollution. In this study, sequencing of two small RNA libraries obtained from tissues of Ciccio and Svevo cultivars grown under nitrogen starvation conditions produced 84 novel, and 161 conserved miRNAs. Of these, 7 novel and 13 known miRNAs were newly identified in this work. Quantitative PCR analysis of selected miRNAs highlighted that the expression levels of some of them depends on the tissue and on the cultivar, Svevo being the most responsive to nitrogen starvation. A number of target genes were predicted to be involved in nitrogen metabolism. An inverse correlation for the qPCR expression data of miRNA/target pairs miR399b/PHO2, miR393c/AFB2, ttu-novel-61/CCAAT-TF was observed in specific tissues or cultivar. Especially, ttu-novel-61 was down-regulated and its target CCAAT-TF up-regulated in almost all tissues both in Svevo and in Ciccio. Moreover, CCAAT-TF was confirmed to be cleaved by ttu-novel-61 at the expected site. The discovery of miRNAs involved in the response to nitrogen stress represents an important step towards functional analyses, with the final aim to design strategies for improving nitrogen use efficiency in durum wheat.

Differential representation of albumins and globulins during grain development in durum wheat and its possible functional consequences.

Durum wheat (Triticum turgidum ssp. durum (Desf.) Husn.) is an economically important crop used for the production of semolina, which is the basis of pasta and other food products. Its grains provide proteins and starch for human consumption. Grain development is a key process in wheat physiology; it is highly affected by a number of enzymes that control the metabolic processes governing accumulation of starch and storage proteins and ultimately grain weight. Most of these enzymes are present in the albumin/globulin grain fraction, which represents about a quarter of total seed proteins. With the aim to describe the dynamic profile of the albumin/globulin fraction during durum wheat grain development, we performed a proteomic analysis of this subproteome using a two-dimensional differential gel electrophoresis (2D-DIGE)-based approach and compared six developmental stages. A total of 285 differentially (237 over- and 48 under-) represented spots was identified by nanoLC-ESI-LIT-MS/MS, which were associated with 217 non-redundant Triticum sequence entries. Quantitative protein dynamics demonstrated that carbon metabolism, energy, protein destination/storage, disease/defense and cell growth/division functional categories were highly affected during grain development, concomitantly with progressive grain size increase and starch/protein reserve accumulation. Bioinformatic interaction prediction revealed a complex network of differentially represented proteins mainly centered at enzymes involved in carbon and protein metabolism. A description of 18 proteins associated with wheat flour human allergies was also obtained; these components showed augmented levels at the last developmental stages. By providing a comprehensive understanding of the molecular basis of durum wheat grain development, yield and quality formation, this study provides the foundation and reveals potential biomarkers for further investigations of durum wheat breeding and semolina quality. BIOLOGICAL SIGNIFICANCE: A 2D-DIGE-based comparative analysis of the albumin/globulin fraction from durum wheat caryopses at six developmental stages was performed to describe the dynamic subproteomic changes associated with grain development. Quantitative variations of 217 differentially proteins demonstrated that highly affected are the functional categories of carbon metabolism, energy, protein destination/storage, disease/defense and cell growth/division, which displayed a general over-representation, consistently with concomitant occurrence of grain size increase and starch/protein reserve accumulation. Bioinformatics revealed a complex protein network centered mainly at enzymes involved in carbon and protein metabolism. Differentially represented proteins and corresponding functional categories highly resembled those previously identified as variable in developing bread wheat grain. This suggests that the main differences in kernel hardness between durum and bread wheat probably do not depend on proteomic changes in corresponding albumins/globulins, but on other specific factors affecting the interaction between the starch granules and the endosperm protein matrix in the kernel.

The complete chloroplast genome of Cynara humilis.

The complete chloroplast genome of the wild thistle Cynara humilis L. (Asteraceae) is presented in this study. The genome is 152,585 bp in length and has a quadripartite structure composed by a large single-copy (LSC) of 83,622 bp, a small single-copy (SSC) of 18,651 bp and two inverted repeats (IRb/a) of 25,156 bp each. The GC content corresponds to 37.7%. The amount of unique genes is 114, in which 17 are duplicated in the IRs, for a total of 131 genes. A maximum parsimony phylogenetic analysis revealed that C. humilis chloroplast genome is closely related to that of the globe artichoke within the Carduoideae subfamily.

How a Small Double-Stranded Trick Can Mislead Sanger Sequencing.

Notwithstanding the arrival of "third-generation sequencing," Sanger sequencing, developed in 1980, is still the most accurate and used method for sequencing, although on a smaller scale. It is a powerful resource for studying sequences and discovering polymorphisms and genes, as well as regulatory elements. There has already been described a wide range of possible problems with this very sensitive and accurate technology. Here, we show that a specific event, related to genomes rich in repetitive sequences, can mislead operators working with Sanger sequencing.