Giuseppina Rea

Copper amine oxidase expression in defense responses to wounding and Ascochyta rabiei invasion.

Wounding chickpea (Cicer arietinum) internodes or cotyledons resulted in an increase in the steady-state level of copper amine oxidase (CuAO) expression both locally and systemically. Dissection of the molecular mechanisms controlling CuAO expression indicated that jasmonic acid worked as a potent inducer of the basal and wound-inducible CuAO expression, whereas salicylic acid and abscisic acid caused a strong reduction of the wound-induced CuAO expression, without having any effect on the basal levels. Epicotyl treatment with the CuAO mechanism-based inhibitor 2-bromoethylamine decreased hydrogen peroxide (H2O2) levels in all the internodes, as evidenced in vivo by 3,3′-diaminobenzidine oxidation. Moreover, inhibitor pretreatment of wounded epicotyls resulted in a lower accumulation of H2O2 both at the wound site and in distal organs. In vivo CuAO inhibition by 2-bromoethylamine after inoculation of resistant chickpea cv Sultano with Ascochyta rabiei resulted in the development of extended necrotic lesions, with extensive cell damage occurring in sclerenchyma and cortical parenchyma tissues. These results, besides stressing the fine-tuning by key signaling molecules in wound-induced CuAO regulation, demonstrate that local and systemic CuAO induction is essential for H2O2 production in response to wounding and indicate the relevance of these enzymes in protection against pathogens.

Developmentally and wound-regulated expression of the gene encoding a cell wall copper amine oxidase in chickpea seedlings.

A chickpea cDNA encoding a cell wall copper amine oxidase (CuAO) was cloned and characterised. The 2010 bp open reading frame encodes a protein of 76.5 kDa which shares significant primary structure homology with other known CuAOs. Southern blot analysis indicates that in chickpea CuAO is encoded by a single gene or a small gene family. This cDNA was essential for studying the role of CuAO during seedling development and wound healing in chickpea seedlings. CuAO transcript level and activity were modulated during seedling development in parallel with cell maturation. Moreover, mechanical wounding induced a rapid increase of CuAO mRNA accumulation and enzyme activity which remained high during the wound‐healing process. Aminoguanidine, a specific CuAO inhibitor, decreased the deposition of lignin‐suberin barrier along the lesion. CuAO may be a limiting factor in H2O2 production in the cell wall of chickpea seedlings and its expression seems to integrate with the remodelling of plant cell wall occurring during ontogenesis and wound healing.

Optical biosensors for environmental monitoring based on computational and biotechnological tools for engineering the photosynthetic D1 protein of Chlamydomonas reinhardtii

Homology-based protein modelling and computational screening followed by virtual mutagenesis analyses were used to identify functional amino acids in the D1 protein of the photosynthetic electron transfer chain interacting with herbicides. A library of functional mutations in the unicellular green alga Chlamydomonas reinhardtii for preparing biomediators was built and their interactions with herbicides were calculated. D1 proteins giving the lowest and highest binding energy with herbicides were considered as suitable for preparing the environmental biosensors for detecting specific herbicide classes. Arising from the results of theoretical calculations, three mutants were prepared by site-directed mutagenesis and characterized by fluorescence analysis. Their adsorption and selective recognition ability were studied by an equilibrium-adsorption method. The S268C and S264K biomediators showed high sensitivity and resistance, respectively, to both triazine and urea classes of herbicides. When immobilized on a silicon septum, the biomediators were found to be highly stable, remaining so for at least 1-month at room temperature. The fluorescence properties were exploited and a reusable and portable multiarray optical biosensor for environmental monitoring was developed with limits of detection between 0.8 x 10(-11) and 3.0 x 10(-9), depending on the target analyte. In addition, biomediator regeneration without obvious deterioration in performance was demonstrated.

De-etiolation causes a phytochrome-mediated increase of polyamine oxidase expression in outer tissues of the maize mesocotyl: a role in the photomodulation of growth and cell wall differentiation

Abstract. The photomodulation of polyamine oxidase (PAO) expression during de-etiolation of the maize (Zea mays L.) mesocotyl was used as the experimental model to investigate a possible correlation with the photomodulation of growth and with wall differentiation and stiffening. The accumulation of PAO transcript and enzyme activity were enhanced by light treatment in cortical and epidermal (outer) tissues of the mesocotyl. Histochemical analysis revealed that this phenomenon is mostly due to the increased level of PAO activity in epidermal and sub-epidermal tissues. The photomodulation of PAO activity upon de-etiolation in outer tissues is mediated by phytochrome. A close correlation was found between the time course of red-light-elicited increase of PAO activity and that of growth inhibition in the outer tissues of the apical, growing zone of the mesocotyl. Light exposure of etiolated, sub-apical mesocotyl segments resulted in a higher production of hydrogen peroxide (H2O2) in the incubation medium compared with segments incubated in the dark. The latter phenomenon was inhibited by the specific PAO inhibitor guazatine. A short pre-treatment of mesocotyl and coleoptile segments with 1 mM spermidine inhibited IAA-induced elongation growth, this phenomenon being reversed by catalase. Pre-treatment with catalase alone resulted in a higher extent of IAA-induced elongation. Moreover, pre-incubation with 1,3-diaminopropane, a product of spermidine oxidation catalysed by PAO, had no effect on IAA-induced elongation growth of either coleoptile or mesocotyl segments, while H2O2 pre-treatment was effective. These results indicate that PAO activity is important in producing H2O2 in vivo for peroxidase-catalysed wall-stiffening reactions and may be involved in the modulation of growth and cell wall differentiation in the maize mesocotyl.

Nanotechnology in Agriculture: Which Innovation Potential Does It Have?

Recent scientific data indicate that nanotechnology has the potential to positively impact the agrifood sector, minimizing adverse problems of agricultural practices on environment and human health, improving food security and productivity (as required by the predicted rise in global population), while promoting social and economic equity. In this context, we select and report on recent trends in nanomaterial-based systems and nanodevices that could provide benefits on the food supply chain specifically on sustainable intensification, and management of soil and waste. Among others, nanomaterials for controlled-release of nutrients, pesticides and fertilizers in crops are described as well as nanosensors for agricultural practices, food quality and safety.

Technological applications of chlorophyll a fluorescence for the assessment of environmental pollutants.

Chlorophyll a fluorescence has been extensively studied over the last few years. As demonstrated, this phenomenon is closely related to the state of photosystem II, which plays a leading role in the photosynthetic process, and therefore it has become a powerful tool to investigate this complex and any damage occurring in it as a result of physical or chemical stresses. This means that by using photosynthetic organisms as biological probes, one can consider chlorophyll a fluorescence as one of the techniques of choice to reveal the presence of some hazardous toxicants widely spread in the environment. Herbicides, pesticides, and heavy metals, whose concentration in water and food products is generally subject to extremely severe restrictions, are a concrete example of compounds detectable by chlorophyll a fluorescence. These dangerous substances react with the photosystem II, modifying the fluorescence emitted and giving responses which vary in a concentration-dependent manner. The possibility of performing easy, fast, and direct measurements of the fluorescence, even under light conditions, has opened new frontiers for the analysis in situ of pollutants. The aim of this review is to give an overview of the different techniques based on chlorophyll a fluorescence spectrometry, focusing in particular on those which represented the starting point for applications addressed to the assessment of toxic compounds in environmental samples.

Structure‐based design of novel Chlamydomonas reinhardtii D1‐D2 photosynthetic proteins for herbicide monitoring

The D1‐D2 heterodimer in the reaction center core of phototrophs binds the redox plastoquinone cofactors, QA and QB, the terminal acceptors of the photosynthetic electron transfer chain in the photosystem II (PSII). This complex is the target of the herbicide atrazine, an environmental pollutant competitive inhibitor of QB binding, and consequently it represents an excellent biomediator to develop biosensors for pollutant monitoring in ecosystems. In this context, we have undertaken a study of the Chlamydomonas reinhardtii D1‐D2 proteins aimed at designing site directed mutants with increased affinity for atrazine. The three‐dimensional structure of the D1 and D2 proteins from C. reinhardtii has been homology modeled using the crystal structure of the highly homologous Thermosynechococcus elongatus proteins as templates. Mutants of D1 and D2 were then generated in silico and the atrazine binding affinity of the mutant proteins has been calculated to predict mutations able to increase PSII affinity for atrazine. The computational approach has been validated through comparison with available experimental data and production and characterization of one of the predicted mutants. The latter analyses indicated an increase of one order of magnitude of the mutant sensitivity and affinity for atrazine as compared to the control strain. Finally, D1‐D2 heterodimer mutants were designed and selected which, according to our model, increase atrazine binding affinity by up to 20 kcal/mol, representing useful starting points for the development of high affinity biosensors for atrazine.

Structure/function/dynamics of photosystem II plastoquinone binding sites.

Photosystem II (PSII) continuously attracts the attention of researchers aiming to unravel the riddle of its functioning and efficiency fundamental for all life on Earth. Besides, an increasing number of biotechnological applications have been envisaged exploiting and mimicking the unique properties of this macromolecular pigment-protein complex. The PSII organization and working principles have inspired the design of electrochemical water splitting schemes and charge separating triads in energy storage systems as well as biochips and sensors for environmental, agricultural and industrial screening of toxic compounds. An intriguing opportunity is the development of sensor devices, exploiting native or manipulated PSII complexes or ad hoc synthesized polypeptides mimicking the PSII reaction centre proteins as bio-sensing elements. This review offers a concise overview of the recent improvements in the understanding of structure and function of PSII donor side, with focus on the interactions of the plastoquinone cofactors with the surrounding environment and operational features. Furthermore, studies focused on photosynthetic proteins structure/function/dynamics and computational analyses aimed at rational design of high-quality bio-recognition elements in biosensor devices are discussed.

Healthy and adverse effects of plant-derived functional metabolites: the need of revealing their content and bioactivity in a complex food matrix.

In recent years, both food quality and its effect on human health have become a fundamental issue all over the world. As a consequence of this new and increased awareness, American, European, and Asian policymakers have strongly encouraged the research programs on food quality and safety thematic. Attempts to improve human health and to satisfy peoples desire for healthcare without intake of pharmaceuticals, has led the food industry to focus attention on functional or nutraceutical food. For a long time, compounds with nutraceutical activity have been produced chemically, but the new demands for a sustainable life have gradually led the food industry to move towards natural compounds, mainly those derived from plants. Many phytochemicals are known to promote good health, but, sometimes, undesirable effects are also reported. Furthermore, several products present on the market show few benefits and sometimes even the reverse - unhealthy effects; the evidence of efficacy is often unconvincing and epidemiological studies are necessary to prove the truth of their claims. Therefore, there is a need for reliable analytical control systems to measure the bioactivity, content, and quality of these additives in the complex food matrix. This review describes the most widespread nutraceutics and an analytical control of the same using recently developed biosensors which are promising candidates for routine control of functional foods.

Photosynthesis at the forefront of a sustainable life.

The development of a sustainable bio-based economy has drawn much attention in recent years, and research to find smart solutions to the many inherent challenges has intensified. In nature, perhaps the best example of an authentic sustainable system is oxygenic photosynthesis. The biochemistry of this intricate process is empowered by solar radiation influx and performed by hierarchically organized complexes composed by photoreceptors, inorganic catalysts, and enzymes which define specific niches for optimizing light-to-energy conversion. The success of this process relies on its capability to exploit the almost inexhaustible reservoirs of sunlight, water, and carbon dioxide to transform photonic energy into chemical energy such as stored in adenosine triphosphate. Oxygenic photosynthesis is responsible for most of the oxygen, fossil fuels, and biomass on our planet. So, even after a few billion years of evolution, this process unceasingly supports life on earth, and probably soon also in outer-space, and inspires the development of enabling technologies for a sustainable global economy and ecosystem. The following review covers some of the major milestones reached in photosynthesis research, each reflecting lasting routes of innovation in agriculture, environmental protection, and clean energy production.