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1.  Zinc Excess Triggered Polyamines Accumulation in Lettuce Root Metabolome, As Compared to Osmotic Stress under High Salinity 
Abiotic stresses such as salinity and metal contaminations are the major environmental stresses that adversely affect crop productivity worldwide. Crop responses and tolerance to abiotic stress are complex processes for which “-omic” approaches such as metabolomics is giving us a newest view of biological systems. The aim of the current research was to assess metabolic changes in lettuce (Lactuca sativa L.), by specifically probing the root metabolome of plants exposed to elevated isomolar concentrations of NaCl and ZnSO4. Most of the metabolites that were differentially accumulated in roots were identified for stress conditions, however the response was more intense in plants exposed to NaCl. Compounds identified in either NaCl or ZnSO4 conditions were: carbohydrates, phenolics, hormones, glucosinolates, and lipids. Our findings suggest that osmotic stress and the consequent redox imbalance play a major role in determining lettuce root metabolic response. In addition, it was identified that polyamines and polyamine conjugates were triggered as a specific response to ZnSO4. These findings help improve understanding of how plants cope with abiotic stresses. This information can be used to assist decision-making in breeding programs for improving crop tolerance to salinity and heavy metal contaminations.
PMCID: PMC4901059  PMID: 27375675
Lactuca sativa L.; metabolomics; heavy metals; abiotic stress; pathways analysis
2.  Comparison of proteome response to saline and zinc stress in lettuce 
Zinc salts occurring in soils can exert an osmotic stress toward plants. However, being zinc a heavy metal, some more specific effects on plant metabolisms can be forecast. In this work, lettuce has been used as a model to investigate salt and zinc stresses at proteome level through a shotgun tandem MS proteomic approach. The effect of zinc stress in lettuce, in comparison with NaCl stress, was evaluated to dissect between osmotic/oxidative stress related effects, from those changes specifically related to zinc. The analysis of proteins exhibiting a fold change of 3 as minimum (on log 2 normalized abundances), revealed the involvement of photosynthesis (via stimulation of chlorophyll synthesis and enhanced role of photosystem I) as well as stimulation of photophosphorylation. Increased glycolytic supply of energy substrates and ammonium assimilation [through formation of glutamine synthetase (GS)] were also induced by zinc in soil. Similarly, protein metabolism (at both transcriptional and ribosomal level), heat shock proteins, and proteolysis were affected. According to their biosynthetic enzymes, hormones appear to be altered by both the treatment and the time point considered: ethylene biosynthesis was enhanced, while production of abscisic acid was up-regulated at the earlier time point to decrease markedly and gibberellins were decreased at the later one. Besides aquaporin PIP2 synthesis, other osmotic/oxidative stress related compounds were enhanced under zinc stress, i.e., proline, hydroxycinnamic acids, ascorbate, sesquiterpene lactones, and terpenoids biosynthesis. Although the proteins involved in the response to zinc stress and to salinity were substantially the same, their abundance changed between the two treatments. Lettuce response to zinc was more prominent at the first sampling point, yet showing a faster adaptation than under NaCl stress. Indeed, lettuce plants showed an adaptation after 30 days of stress, in a more pronounced way in the case of zinc.
PMCID: PMC4399213  PMID: 25932029
osmotic stress; oxidative stress; shotgun MS; photosystem I; phytohormone
3.  Acclimation to different depths by the marine angiosperm Posidonia oceanica: transcriptomic and proteomic profiles 
For seagrasses, seasonal and daily variations in light and temperature represent the mains factors driving their distribution along the bathymetric cline. Changes in these environmental factors, due to climatic and anthropogenic effects, can compromise their survival. In a framework of conservation and restoration, it becomes crucial to improve our knowledge about the physiological plasticity of seagrass species along environmental gradients. Here, we aimed to identify differences in transcriptomic and proteomic profiles, involved in the acclimation along the depth gradient in the seagrass Posidonia oceanica, and to improve the available molecular resources in this species, which is an important requisite for the application of eco-genomic approaches. To do that, from plant growing in shallow (−5 m) and deep (−25 m) portions of a single meadow, (i) we generated two reciprocal Expressed Sequences Tags (EST) libraries using a Suppressive Subtractive Hybridization (SSH) approach, to obtain depth/specific transcriptional profiles, and (ii) we identified proteins differentially expressed, using the highly innovative USIS mass spectrometry methodology, coupled with 1D-SDS electrophoresis and labeling free approach. Mass spectra were searched in the open source Global Proteome Machine (GPM) engine against plant databases and with the X!Tandem algorithm against a local database. Transcriptional analysis showed both quantitative and qualitative differences between depths. EST libraries had only the 3% of transcripts in common. A total of 315 peptides belonging to 64 proteins were identified by mass spectrometry. ATP synthase subunits were among the most abundant proteins in both conditions. Both approaches identified genes and proteins in pathways related to energy metabolism, transport and genetic information processing, that appear to be the most involved in depth acclimation in P. oceanica. Their putative rules in acclimation to depth were discussed.
PMCID: PMC3683636  PMID: 23785376
Posidonia oceanica; acclimation; ESTs; proteomic; eco-genomic
4.  Proteomic characterization of the Rph15 barley resistance gene-mediated defence responses to leaf rust 
BMC Genomics  2012;13:642.
Leaf rust, caused by the biotrophic fungal pathogen Puccinia hordei, is one of the most important foliar disease of barley (Hordeum vulgare) and represents a serious threat in many production regions of the world. The leaf rust resistance gene Rph15 is of outstanding interest for resistance breeding because it confers resistance to over 350 Puccinia hordei isolates collected from around the world. Molecular and biochemical mechanisms responsible for the Rph15 effectiveness are currently not investigated. The aim of the present work was to study the Rph15-based defence responses using a proteomic approach.
Protein pattern changes in response to the leaf rust pathogen infection were investigated in two barley near isogenic lines (NILs), Bowman (leaf rust susceptible) and Bowman-Rph15 (leaf rust resistant), differing for the introgression of the leaf rust resistance gene Rph15. Two infection time points, 24 hours and four days post inoculation (dpi), were analysed. No statistically significant differences were identified at the early time point, while at 4 dpi eighteen protein spots were significantly up or down regulated with a fold-change equal or higher than two in response to pathogen infection. Almost all the pathogen-responsive proteins were identified in the Bowman-Rph15 resistant NIL. Protein spots were characterized by LC-MS/MS analysis and found to be involved in photosynthesis and energy metabolism, carbohydrate metabolism, protein degradation and defence. Proteomic data were complemented by transcriptional analysis of the respective genes. The identified proteins can be related to modulation of the photosynthetic apparatus components, re-direction of the metabolism to sustain defence responses and deployment of defence proteins.
The identification of leaf rust infection-modulated defence responses restricted to the resistant NIL support the hypothesis that basal defence responses of Bowman, but not the Rph15 resistance gene-based ones, are suppressed or delayed by pathogen effectors to levels below the detection power of the adopted proteomic approach. Additionally, Rph15-mediated resistance processes identified mainly resides on a modulation of primary metabolism, affecting photosyntesis and carbohydrate pool.
PMCID: PMC3541957  PMID: 23167439
Barley; Leaf rust; Resistance gene; Rph15; Proteomics; Near isogenic lines
5.  On the complexity of miRNA-mediated regulation in plants: novel insights into the genomic organization of plant miRNAs 
Biology Direct  2012;7:15.
MicroRNAs (miRNAs) are endogenous small non-coding RNAs of about 20–24 nt, known to play key roles in post-transcriptional gene regulation, that can be coded either by intergenic or intragenic loci. Intragenic (exonic and intronic) miRNAs can exert a role in the transcriptional regulation and RNA processing of their host gene. Moreover, the possibility that the biogenesis of exonic miRNAs could destabilize the corresponding protein-coding transcript and reduce protein synthesis makes their characterization very intriguing and suggests a possible novel mechanism of post-transcriptional regulation of gene expression.
This work was designed to carry out the computational identification of putative exonic miRNAs in 30 plant species and the analysis of possible mechanisms involved in their regulation.
The results obtained represent a useful starting point for future studies on the complex networks involved in microRNA-mediated gene regulation in plants.
PMCID: PMC3464803  PMID: 22569316
Gene regulation; Exonic miRNA; miRNA self regulation; Plants
6.  A Survey of MicroRNA Length Variants Contributing to miRNome Complexity in Peach (Prunus Persica L.) 
MicroRNAs (miRNAs) are short non-coding RNA molecules produced from hairpin structures and involved in gene expression regulation with major roles in plant development and stress response. Although each annotated miRNA in miRBase ( is a single defined sequence with no further details on possible variable sequence length, isomiRs – namely the population of variants of miRNAs coming from the same precursors – have been identified in several species and could represent a way of broadening the regulatory network of the cell. Next-gen-based sequencing makes it possible to comprehensively and accurately assess the entire miRNA repertoire including isomiRs. The aim of this work was to survey the complexity of the peach miRNome by carrying out Illumina high-throughput sequencing of miRNAs in three replicates of five biological samples arising from a set of different peach organs and/or phenological stages. Three hundred-ninety-two isomiRs (miRNA and miRNA*-related) corresponding to 26 putative miRNA coding loci, have been highlighted by mirDeep-P and analyzed. The presence of the same isomiRs in different biological replicates of a sample and in different tissues demonstrates that the generation of most of the detected isomiRs is not random. The degree of mature sequence heterogeneity is very different for each individual locus. Results obtained in the present work can thus contribute to a deeper view of the miRNome complexity and to better explore the mechanism of action of these tiny regulators.
PMCID: PMC3405489  PMID: 22855688
microRNA; isomiRs; next generation sequencing

Results 1-6 (6)