Study of the role of the DOF transcription factor DAG1 in the control of seedling development in Arabidopsis thaliana

Seedling development relies on environmental conditions; indeed, once seeds have germinated, they undergo photomorphogenesis or skotomorphogenesis, depending on the presence or absence of light. Photomorphogenesis is a multi-traits process characterised by inhibition of hypocotyl elongation, open and expanded cotyledons, and chloroplast development, whereas skotomorphogenesis is characterised by long hypocotyls and small unfolded cotyledons. Hypocotyl elongation is influenced by both environmental and hormonal cues and it has been extensively studied as a model for cell expansion. Nevertheless, the molecular network underlying this process is not yet fully elucidated. The Arabidopsis Dof protein DAG1 (Dof Affecting Germination1) is a repressor of seed germination, and a key player of the seed-to seedling transition, a crucial developmental phase positively controlled by light, as well as by the phytohormones ABA (abscissic acid) and GA (gibberellins). Indeed, DAG1 controls the ratio of ABA and GA, which play opposite roles, as ABA represses germination whereas GAs promote it. We have previously shown that inactivation of DAG1 affects inhibition of hypocotyl elongation. Indeed, light-grown dag1 mutant seedlings show significant shorter hypocotyls compared to the wild-type, suggesting that DAG1 is a negative component of this light-mediated process. To gain some insight into the molecular network in which DAG1 is involved, we have analysed the transcriptome profile of both dag1 and wild-type hypocotyls and seedlings. We have identified more than 250 genes that are differentially expressed in dag1 hypocotyls, and the analysis of this data suggests that DAG1 is mainly involved in promoting hypocotyl elongation. In addition, a number of the DE genes identified are correlated to the response to ABA stimulus. ABA plays a role in inhibition of hypocotyl elongation, although the molecular mechanism remains unclear. Therefore, we investigated the effect of ABA on hypocotyl development, and our results showed that ABA negatively controls cell expansion in hypocotyls, by acting on GA metabolism, and repressing auxin biosynthesis. Consistently, addition of exogenous ABA can revert the hypocotyl phenotype of dag1 mutant seedlings. In conclusion, our results prove that DAG1 is likely to be an element of a molecular network which controls cell expansion by modulating hormonal response, namely auxin, ABA and GAs

Host cell wall damage during pathogen infection: mechanisms of perception and role in plant-pathogen interactions

The plant cell wall (CW) is a complex structure that acts as a mechanical barrier, restricting the access to most microbes. Phytopathogenic microorganisms can deploy an arsenal of CWdegrading enzymes (CWDEs) that are required for virulence. In turn, plants have evolved proteins able to inhibit the activity of specific microbial CWDEs, reducing CW damage and favoring the accumulation of CW-derived fragments that act as damage-associated molecular patterns (DAMPs) and trigger an immune response in the host. CW-derived DAMPs might be a component of the complex system of surveillance of CW integrity (CWI), that plants have evolved to detect changes in CW properties. Microbial CWDEs can activate the plant CWI maintenance system and induce compensatory responses to reinforce CWs during infection. Recent evidence indicates that the CWI surveillance system interacts in a complex way with the innate immune system to fine-tune downstream responses and strike a balance between defense and growth

Assessing Background Values of Regulated Parameters in Groundwater Bodies of Sardinia (Italy)

Abstract The groundwater bodies in the European Union should be classified on the basis of their chemical status according to the European regulations. To this purpose, the background values for electrical conductivity, chloride, sulfate, fluoride and lead in groundwater bodies hosted in carbonatic rocks in Sardinia (Italy) were estimated. Background values were dependent on geological (lithology and mineralization) and geographical (distance from the coast) features of aquifers. Results indicate that statistical methods should be integrated with hydrogeochemical investigations for a correct assessment of the background values

DOF AFFECTING GERMINATION 2 is a positive regulator of light-mediated seed germination and is repressed by DOF AFFECTING GERMINATION 1

Abstract BACKGROUND: The transcription factor DOF AFFECTING GERMINATION1 (DAG1) is a repressor of the light-mediated seed germination process. DAG1 acts downstream PHYTOCHROME INTERACTING FACTOR3-LIKE 5 (PIL5), the master repressor, and negatively regulates gibberellin biosynthesis by directly repressing the biosynthetic gene AtGA3ox1. The Dof protein DOF AFFECTING GERMINATION (DAG2) shares a high degree of aminoacidic identity with DAG1. While DAG1 inactivation considerably increases the germination capability of seeds, the dag2 mutant has seeds with a germination potential substantially lower than the wild-type, indicating that these factors may play opposite roles in seed germination. RESULTS: We show here that DAG2 expression is positively regulated by environmental factors triggering germination, whereas its expression is repressed by PIL5 and DAG1; by Chromatin Immuno Precipitation (ChIP) analysis we prove that DAG1 directly regulates DAG2. In addition, we show that Red light significantly reduces germination of dag2 mutant seeds. CONCLUSIONS: In agreement with the seed germination phenotype of the dag2 mutant previously published, the present data prove that DAG2 is a positive regulator of the light-mediated seed germination process, and particularly reveal that this protein plays its main role downstream of PIL5 and DAG1 in the phytochrome B (phyB)-mediated pathway

The DAG1 transcription factor negatively regulates the seed-to-seedling transition in Arabidopsis acting on ABA and GA levels

BACKGROUND: In seeds, the transition from dormancy to germination is regulated by abscisic acid (ABA) and gibberellins (GAs), and involves chromatin remodelling. Particularly, the repressive mark H3K27 trimethylation (H3K27me3) has been shown to target many master regulators of this transition. DAG1 (DOF AFFECTING GERMINATION1), is a negative regulator of seed germination in Arabidopsis, and directly represses the GA biosynthetic gene GA3ox1 (gibberellin 3-β-dioxygenase 1). We set to investigate the role of DAG1 in seed dormancy and maturation with respect to epigenetic and hormonal control. RESULTS: We show that DAG1 expression is controlled at the epigenetic level through the H3K27me3 mark during the seed-to-seedling transition, and that DAG1 directly represses also the ABA catabolic gene CYP707A2; consistently, the ABA level is lower while the GA level is higher in dag1 mutant seeds. Furthermore, both DAG1 expression and protein stability are controlled by GAs. CONCLUSIONS: Our results point to DAG1 as a key player in the control of the developmental switch between seed dormancy and germination

Combining hydrogeochemistry, statistics and explorative mapping to estimate regional threshold values of trace elements in groundwater (Sardinia, Italy)

Assessing geochemical baseline and threshold values of potentially toxic elements at adequate scales is fundamental for distinguishing geogenic contamination from anthropogenic pollution in groundwater. This study was aimed to estimate the regional threshold values of Li, Be, B, Al, V, Cr, Mn, Fe, Co, Ni, Cu, Zn, As, Se, Rb, Sr, Mo, Ag, Cd, Sb, Te, Ba, Hg, Tl, Pb, Bi, and U (elements listed according to atomic numbers) in groundwater, compare results to guidelines established for drinking water and the protection of groundwater from contamination, investigate the geographical distribution of trace elements, and assess the potential influence of water-rock interaction. A pre-selection aimed at excluding groundwater samples affected by known anthropogenic activities was carefully carried out based on hydrogeochemical characteristics of waters and considering the potential sources of contamination. The resulting dataset was comprised of 1227 groundwater sampling sites located in Sardinia (Italy). Undetected values were treated using the Regression on Order Statistics method. For elements containing >75 % of undetected values and/or a limited number of samples in the dataset (Li, Rb, Sr, Mo, Ag, Te, Tl, Sb, Hg and Bi), the threshold values were estimated using either the 95th or 97.7th percentiles. For the other elements the mean + 2SD (Standard Deviation), the median + 2MAD (Median Absolute Deviation), and the TIF (Tukey Inner Fence) estimators were also calculated. Geochemical maps allowed to recognize the threshold value of each element at different scales. Regional threshold values of the regulated elements B, Al, V, Cr, Cu and Cd in groundwater were below the Italian and World Health Organization drinking water guidelines, whereas Mn and As were above them. Regional threshold values estimated with TIF exceeded the drinking water guidelines for Ni, Se, Pb and U. Results of this study showed that high concentrations of trace elements in groundwater were primarily dependent on the corresponding amount in parent materials with which the groundwater came into contact. Physical-chemical parameters and geochemical characteristics may contribute to enhancing concentrations of some trace elements in groundwater, e.g. As via reductive dissolution of Fe(III)-Mn(IV) hydroxides/oxides, Pb via formation of stable aqueous complexes, and other elements via adsorption onto fine particles with size below 0.4 μm (i.e. the pore size of filters used). Maps drawn on the centered log-ratio (clr) transformation of hydrogeochemical data, following the CoDA (Compositional Data Analysis) approach, allowed to pinpoint critical areas to be investigated in more detail. For each geological complex, groundwater samples likely representing nearly pristine conditions were identified. The monitoring of these representative groundwater samples may help to pinpoint eventual changes in environmental conditions

Geochemistry, stable isotopes and statistic tools to estimate threshold and source of nitrate in groundwater (Sardinia, Italy)

In the European Union, nitrate vulnerable zone (NVZ) should be designed for the mitigation of nitrate (NO3−) contamination caused by agricultural practices. Before establishing new NVZ, the sources of NO3− must be recognized. A geochemical and multiple stable isotopes approach (hydrogen, oxygen, nitrogen, sulfur and boron) and statistical tools were applied to define the geochemical characteristics of groundwater (60 samples), calculate the local NO3− threshold and assess potential sources of NO3− contamination in two study areas (hereafter Northern and Southern), located in a Mediterranean environment (Sardinia, Italy). Results of the integrated approach applied to two case study, permits to highlight the strengths of integrating geochemical and statistical methods to provide nitrate source identification as a reference by decision makers to remediate and mitigate nitrate contamination in groundwater. Hydrogeochemical features in the two study areas were similar: near neutral to slightly alkaline pH, electrical conductivity in the range of 0.3 to 3.9 mS/cm, and chemical composition ranging from Ca-HCO3− at low salinity to Na-Cl− at high salinity. Concentrations of NO3− in groundwater were in the range of 1 to 165 mg/L, whereas the nitrogen reduced species were negligible, except few samples having NH4+ up to 2 mg/L. Threshold values in the studied groundwater samples were between 4.3 and 6.6 mg/L NO3−, which was in agreement with previous estimates in Sardinian groundwater. Values of δ34S and δ18OSO4 of SO42− in groundwater samples indicated different sources of SO42−. Sulfur isotopic features attributed to marine SO42− were consistent with groundwater circulation in marine-derived sediments. Other source of SO42− were recognize due to the oxidation of sulfide minerals, to fertilizers, manure, sewage fields, and SO42− derived from a mix of different sources. Values of δ15N and δ18ONO3 of NO3− in groundwater samples indicated different biogeochemical processes and NO3− sources. Nitrification and volatilization processes might have occurred at very few sites, and denitrification was likely to occur at specific sites. Mixing among various NO3− sources in different proportions might account for the observed NO3− concentrations and the nitrogen isotopic compositions. The SIAR modeling results showed a prevalent NO3− source from sewage/manure. The δ11B signatures in groundwater indicated the manure to be the predominant NO3− source, whereas NO3− from sewage was recognized at few sites. Geographic areas showing either a predominant process or a defined NO3− source where not recognize in the studied groundwater. Results indicate widespread contamination of NO3− in the cultivated plain of both areas. Point sources of contamination, due to agricultural practices and/or inadequate management of livestock and urban wastes, were likely to occur at specific sites

Inhibition of Polycomb Repressive Complex2 activity reduces trimethylation of H3K27 and affects development in Arabidopsis seedlings

Background: Polycomb repressive complex 2 (PRC2) is an epigenetic transcriptional repression system, whose catalytic subunit (ENHANCER OF ZESTE HOMOLOG 2, EZH2 in animals) is responsible for trimethylating histone H3 at lysine 27 (H3K27me3). In mammals, gain-of-function mutations as well as overexpression of EZH2 have been associated with several tumors, therefore making this subunit a suitable target for the development of selective inhibitors. Indeed, highly specific small-molecule inhibitors of EZH2 have been reported. In plants, mutations in some PRC2 components lead to embryonic lethality, but no trial with any inhibitor has ever been reported. Results: We show here that the 1,5-bis (3-bromo-4-methoxyphenyl)penta-1,4-dien-3-one compound (RDS 3434), previously reported as an EZH2 inhibitor in human leukemia cells, is active on the Arabidopsis catalytic subunit of PRC2, since treatment with the drug reduces the total amount of H3K27me3 in a dose-dependent fashion. Consistently, we show that the expression level of two PRC2 targets is significantly increased following treatment with the RDS 3434 compound. Finally, we show that impairment of H3K27 trimethylation in Arabidopsis seeds and seedlings affects both seed germination and root growth. Conclusions: Our results provide a useful tool for the plant community in investigating how PRC2 affects transcriptional control in plant development

GRIDA3—a shared resources manager for environmental data analysis and applications

GRIDA3 (Shared Resources Manager for Environmental Data Analysis and Applications) is a multidisciplinary project designed to deliver an integrated system to forge solutions to some environmental challenges such as the constant increase of polluted sites, the sustainability of natural resources usage and the forecast of extreme meteorological events. The GRIDA3 portal is mainly based on Web 2.0 technologies and EnginFrame framework. The portal, now at an advanced stage of development, provides end-users with intuitive Web-interfaces and tools that simplify job submission to the underneath computing resources. The framework manages the user authentication and authorization, then controls the action and job execution into the grid computing environment, collects the results and transforms them into an useful format on the client side. The GRIDA3 Portal framework will provide a problem-solving platform allowing, through appropriate access policies, the integration and the sharing of skills, resources and tools located at multiple sites across federated domains

Assessing background values of chloride, sulfate and fluoride in groundwater: A geochemical-statistical approach at a regional scale

The Sardinia island (Italy) is one of the European areas least affected by potentially anthropogenic impacts, such as spreading urbanization, intensive agriculture and regional atmospheric contamination. Such characteristics allow to consider Sardinia a good site for testing an approach that integrates geochemical tools, hierarchical cluster and geographical information system, aimed at estimating background concentrations of chloride, sulfate and fluoride at the regional scale. Analytical data were obtained from several hydrogeochemical surveys and from the groundwater-monitoring program established by the Sardinian Regional Government. Groundwater samples were grouped according to their circulation in the predominant hydrogeologic complex: Quaternary sediments, Quaternary basalts, Tertiary sediments, Tertiary volcanic rocks, Mesozoic carbonatic rocks, Paleozoic carbonatic rocks, granitic rocks and metamorphic rocks. Samples surely affected by anthropogenic inputs, thermal waters, waters collected at wells with unknown construction details and poor quality analyses were excluded. The resulting dataset included 1414 groundwater sampling sites distributed over an area of 24,090 km2 (All data). Another dataset comprised of 641 sampling sites (Selected data) was derived by All data excluding the groundwater with NO3- > 10 mg/L. Hierarchical clustering analysis was performed on both datasets considering Ca2+, Mg2+, Na+, K+, Cl-, HCO3-, SO42-, NO3- and F-. The values of total dissolved solids (TDS) were a major distinguishing factor among clusters, but distinct signatures related to the median nitrate and fluoride concentrations were also recognized. The geographic distribution of clusters reflected the role of geological and geographic characteristics on the geochemistry of groundwater. Background ranges of the regulated parameters chloride, sulfate and fluoride in each cluster, identified either using All data or Selected data, were calculated using the median±2MAD. Although results were found in general agreement, the threshold using the median+2MAD was calculated using the Selected data only, because the Selected data better represents near pristine conditions. Chloride threshold values above the drinking water limit were mainly observed in groundwater located in western Sardinia, where sediments and volcanic rocks prevalently outcrop, and also in some coastal areas. Threshold values of sulfate and fluoride above the limit were related to local conditions. Specifically, high threshold values of sulfate were observed in groundwater interacting with the Tertiary volcanic rocks that host known sulfide mineralization and at sites where evaporitic deposits occur. Threshold values of fluoride above the limit were often observed in the areas where fluoride mineralization occurs. High fluoride values may also result from cation exchange and/or supersaturation with respect to calcite. The results of this study indicate that the integration of hierarchical clustering analysis with the geochemical characteristics of groundwater, also taking into account the geological context, allow the repartition of groundwater samples in distinct hydrogeochemical groups, which in turn allow to calculate the background ranges and reliable threshold values in groundwater. This approach can be applied to assess the background concentrations of chemical parameters at a regional scale when a large dataset is available