Antonio Michele Stanca

A combined strategy of “in silico” transcriptome analysis and web search engine optimization allows an agile identification of reference genes suitable for normalization in gene expression studies

Traditionally housekeeping genes have been employed as endogenous reference (internal control) genes for normalization in gene expression studies. Since the utilization of single housekeepers cannot assure an unbiased result, new normalization methods involving multiple housekeeping genes and normalizing using their mean expression have been recently proposed. Moreover, since a gold standard gene suitable for every experimental condition does not exist, it is also necessary to validate the expression stability of every putative control gene on the specific requirements of the planned experiment. As a consequence, finding a good set of reference genes is for sure a non-trivial problem requiring quite a lot of lab-based experimental testing. In this work we identified novel candidate barley reference genes suitable for normalization in gene expression studies. An advanced web search approach aimed to collect, from publicly available web resources, the most interesting information regarding the expression profiling of candidate housekeepers on a specific experimental basis has been set up and applied, as an example, on stress conditions. A complementary lab-based analysis has been carried out to verify the expression profile of the selected genes in different tissues and during heat shock response. This combined dry/wet approach can be applied to any species and physiological condition of interest and can be considered very helpful to identify putative reference genes to be shortlisted every time a new experimental design has to be set up.

TOCOLS IN HULL-LESS AND HULLED BARLEY GENOTYPES GROWN IN CONTRASTING ENVIRONMENTS

Abstract The content of tocols is a parameter of increasing interest in evaluating the quality of plant-based food. Cereal grains are amongst the most widely occurring plant food components and their potential for enriching the content of vitamin E and/or hypocholesterolemic active compounds in food is therefore of interest. We investigated the presence of tocol homologues in hulled and hull-less barley, as influenced by genotype and location. Both factors significantly influenced the amount of tocols in the barley kernel, with genotype having the greater effect for most homologues. Significant genotype×location interaction was observed for six out of eight homologues, but not for total tocotrienols and total tocols; however, the coefficient of determination for genotype was high for most homologues, so that selection for this traits should be possible. The hull-less trait negatively affected the content of total tocols, influencing both tocopherols (positively) and tocotrienols (negatively). Statistical analysis suggests this is due to a different sub-set of homologues, which is preferentially accumulated in hull-less vs. hulled barleys. As hulled barley had a greater accumulation of more bioactive homologues, the selection of hull-less barleys for this trait should be considered for enhancing food quality.

Studies for assessing the influence of hardening on cold tolerance of barley genotypes

SummaryFrost tolerance of 30 barley (Hordeum vulgare L.) cultivars have been field evaluated in North Italy during the 1990/1991 winter season that was characterized by exceptionally low temperatures without snow cover. The results showed a significant correlation between cold injury and grain yield loss (r=0.61**). Five cultivars chosen for their varying degree of frost tolerance were further evaluated using laboratory tests. Measurements of survival rate and membrane damage were used to assess the influence of hardening on frost resistance. The reliability of the tests is shown by the high correlation to the field data. For both the laboratory temperature regimes and field conditions, the tested cultivars showed the same order of classification. The effect of a rise in temperature at the end of the hardening treatment on frost tolerance is also reported. The laboratory tests here proposed can be integrated in a breeding programme for improving frost tolerance in barley.

Physical Mapping of Bread Wheat Chromosome 5A: An Integrated Approach

The huge size, redundancy, and highly repetitive nature of the bread wheat [Triticum aestivum (L.)] genome, makes it among the most difficult species to be sequenced. To overcome these limitations, a strategy based on the separation of individual chromosomes or chromosome arms and the subsequent production of physical maps was established within the frame of the International Wheat Genome Sequence Consortium (IWGSC). A total of 95,812 bacterial artificial chromosome (BAC) clones of short‐arm chromosome 5A (5AS) and long‐arm chromosome 5A (5AL) arm‐specific BAC libraries were fingerprinted and assembled into contigs by complementary analytical approaches based on the FingerPrinted Contig (FPC) and Linear Topological Contig (LTC) tools. Combined anchoring approaches based on polymerase chain reaction (PCR) marker screening, microarray, and sequence homology searches applied to several genomic tools (i.e., genetic maps, deletion bin map, neighbor maps, BAC end sequences (BESs), genome zipper, and chromosome survey sequences) allowed the development of a high‐quality physical map with an anchored physical coverage of 75% for 5AS and 53% for 5AL with high portions (64 and 48%, respectively) of contigs ordered along the chromosome. In the genome of grasses, Brachypodium [Brachypodium distachyon (L.) Beauv.], rice (Oryza sativa L.), and sorghum [Sorghum bicolor (L.) Moench] homologs of genes on wheat chromosome 5A were separated into syntenic blocks on different chromosomes as a result of translocations and inversions during evolution. The physical map presented represents an essential resource for fine genetic mapping and map‐based cloning of agronomically relevant traits and a reference for the 5A sequencing projects.

The CC-NB-LRR-type Rdg2a resistance gene confers immunity to the seed-borne barley leaf stripe pathogen in the absence of hypersensitive cell death.

Background Leaf stripe disease on barley (Hordeum vulgare) is caused by the seed-transmitted hemi-biotrophic fungus Pyrenophora graminea. Race-specific resistance to leaf stripe is controlled by two known Rdg (Resistance to Drechslera graminea) genes: the H. spontaneum-derived Rdg1a and Rdg2a, identified in H. vulgare. The aim of the present work was to isolate the Rdg2a leaf stripe resistance gene, to characterize the Rdg2a locus organization and evolution and to elucidate the histological bases of Rdg2a-based leaf stripe resistance. Principal Findings We describe here the positional cloning and functional characterization of the leaf stripe resistance gene Rdg2a. At the Rdg2a locus, three sequence-related coiled-coil, nucleotide-binding site, and leucine-rich repeat (CC-NB-LRR) encoding genes were identified. Sequence comparisons suggested that paralogs of this resistance locus evolved through recent gene duplication, and were subjected to frequent sequence exchange. Transformation of the leaf stripe susceptible cv. Golden Promise with two Rdg2a-candidates under the control of their native 5′ regulatory sequences identified a member of the CC-NB-LRR gene family that conferred resistance against the Dg2 leaf stripe isolate, against which the Rdg2a-gene is effective. Histological analysis demonstrated that Rdg2a-mediated leaf stripe resistance involves autofluorescing cells and prevents pathogen colonization in the embryos without any detectable hypersensitive cell death response, supporting a cell wall reinforcement-based resistance mechanism. Conclusions This work reports about the cloning of a resistance gene effective against a seed borne disease. We observed that Rdg2a was subjected to diversifying selection which is consistent with a model in which the R gene co-evolves with a pathogen effector(s) gene. We propose that inducible responses giving rise to physical and chemical barriers to infection in the cell walls and intercellular spaces of the barley embryo tissues represent mechanisms by which the CC-NB-LRR-encoding Rdg2a gene mediates resistance to leaf stripe in the absence of hypersensitive cell death.

From single genes to co-expression networks: extracting knowledge from barley functional genomics.

The paper reports an ‘in silico’ approach to gene expression analysis based on a barley gene co-expression network resulting from the study of several publicly available cDNA libraries. The work is an application of Systems Biology to plant science: at the end of the computational step we identified groups of potentially related genes. The communities of co-expressed genes constructed from the network are remarkably characterized from the functional point of view, as shown by the statistical analysis of the Gene Ontology annotations of their members. Experimental, lab-based testing has been carried out to check the relationship between network and biological properties and to identify and suggest effective strategies of information extraction from the network-derived data.

Effects of barley β-glucan-enriched flour fractions on the glycaemic index of bread

The aim of this research was to evaluate β-glucan-enriched flours, obtained from barleys with either normal or waxy starch, for their effects on the glycaemic index (GI) and the quality of bread. Rheological results confirmed that when barley flour was included in the dough the overall quality of bread slightly worsened. However, positive consequences on glycaemia were obtained with the normal starch barley: the GI of all-wheat bread (82.8 ± 7.2) was significantly reduced (57.2 ± 7.9) when 40% of wheat flour was substituted with β-glucan-enriched barley flour (6.0% ± 0.1 β-glucan in the final flour blend). In contrast, this positive effect was significantly reduced (GI: 70.1 ± 9.1) when 40% of wheat flour was substituted with the β-glucan-enriched flour of a waxy barley (CDC Alamo; 6.6 ± 0.2 β-glucan in the final flour blend), suggesting that the ability of β-glucans to lower the GI was affected by the barley starch-type.

Identification of a Protein Network Interacting with TdRF1, a Wheat RING Ubiquitin Ligase with a Protective Role against Cellular Dehydration

Plants exploit ubiquitination to modulate the proteome with the final aim to ensure environmental adaptation and developmental plasticity. Ubiquitination targets are specifically driven to degradation through the action of E3 ubiquitin ligases. Genetic analyses have indicated wide functions of ubiquitination in plant life; nevertheless, despite the large number of predicted E3s, only a few of them have been characterized so far, and only a few ubiquitination targets are known. In this work, we characterized durum wheat (Triticum durum) RING Finger1 (TdRF1) as a durum wheat nuclear ubiquitin ligase. Moreover, its barley (Hordeum vulgare) homolog was shown to protect cells from dehydration stress. A protein network interacting with TdRF1 has been defined. The transcription factor WHEAT BEL1-TYPE HOMEODOMAIN1 (WBLH1) was degraded in a TdRF1-dependent manner through the 26S proteasome in vivo, the mitogen-activated protein kinase TdWNK5 [for Triticum durum WITH NO LYSINE (K)5] was able to phosphorylate TdRF1 in vitro, and the RING-finger protein WHEAT VIVIPAROUS-INTERACTING PROTEIN2 (WVIP2) was shown to have a strong E3 ligase activity. The genes coding for the TdRF1 interactors were all responsive to cold and/or dehydration stress, and a negative regulative function in dehydration tolerance was observed for the barley homolog of WVIP2. A role in the control of plant development was previously known, or predictable based on homology, for wheat BEL1-type homeodomain1(WBLH1). Thus, TdRF1 E3 ligase might act regulating the response to abiotic stress and remodeling plant development in response to environmental constraints.

Molecular adaptation of barley to cold and drought conditions

Molecular adaptation to cold and drought involves a series of biochemical and molecular changes leading plants to improve their winter hardiness or drought resistance.

Constitutive differences between steely and mealy barley samples associated with endosperm modification

BACKGROUND Structurally different areas may occur in the endosperm of the barley grain, and they can be visually classified as either mealy or steely. Barleys with a high proportion of grains that are mostly steely often show uneven physical-chemical modification of the endosperm during malting. To study the relationship between steeliness and endosperm modification, two samples of barley cv. Scarlett with contrasting malting quality were analysed. RESULTS The proportions of steely grains were 77% and 46% in the two samples, which were then defined as steely sample and mealy sample, respectively. The steely sample showed slower modification during malting (in terms of beta-glucan degradation, friability increase, and Calcofluor staining), lower hot water extract (HWE) and acrospire growth, and higher extract viscosity. Endosperm permeation to large molecules (tested with the fluorescein isothiocyanate-dextran conjugate, FITC-D) closely followed cell wall modification in the steely sample, but this was not so in the mealy sample. CONCLUSIONS Higher steeliness was associated with higher levels of C hordeins in the grain of barley cv. Scarlett. It is proposed that such hordeins can increase the permeability to large molecules (FITC-D) but slow modification. Like steeliness and the level of C hordeins, permeability to FITC-D appears to be more linked to environmental rather than genetic effects. Although a more general association of C hordeins with steeliness of malting barley still has to be ascertained, the negative role of C hordeins in malting quality has been confirmed.