Chiara Pucciariello

Glutathione synthesis is regulated by nitric oxide in Medicago truncatula roots

Glutathione (GSH) is one of the main antioxidants in plants. Legumes have the specificity to produce a GSH homolog, homoglutathione (hGSH). We have investigated the regulation of GSH and hGSH synthesis by nitric oxide (NO) in Medicago truncatula roots. Analysis of the expression level of gamma-glutamylcysteine synthetase (γ-ECS), glutathione synthetase (GSHS) and homoglutathione synthetase (hGSHS) after treatment with sodium nitroprusside (SNP) and nitrosoglutathione (GSNO), two NO-donors, showed that γ-ecs and gshs genes are up regulated by NO treatment whereas hgshs expression is not. Differential accumulation of GSH was correlated to gene expression in SNP-treated roots. Our results provide the first evidence that GSH synthesis pathway is regulated by NO in plants and that there is a differential regulation between gshs and hgshs in M. truncatula.

Arabidopsis thaliana MYB75/PAP1 transcription factor induces anthocyanin production in transgenic tomato plants

Tomato (Solanum lycopersicum L.) cv. Micro-Tom plants were transformed with the Arabidopsis thaliana (L.)Heyhn. MYB75/PAP1 (PRODUCTION OF ANTHOCYANIN PIGMENT 1) gene. This gene encodes for a well known transcription factor, which is involved in anthocyanin production and is modulated by light and sucrose. Transgenic tomato plants expressing AtMYB75 were characterised by a significantly higher anthocyanin production in leaves, stems, roots and flowers under normal growth conditions. Further, they also exhibited anthocyanins in fruits. Anthocyanin accumulation was not widespread but took place in specific groups of cells located in epidermal or cortical regions or in proximity of vascular bundles. In all the organs of the transgenic plants, where AtMYB75 overexpression was determined, a clear increase in the accumulation of DFR (DIHYDROFLAVONOL 4-REDUCTASE) transcript was also detected. The expression of the tomato MYB-gene ANT1 (ANTHOCYANIN1), which had previously been identified as a transcriptional endogenous regulator of anthocyanin biosynthesis, was not altered. The higher basal content of anthocyanins in the leaves of the transgenic plants could be further increased in the presence of high light conditions and contributed to mitigate photobleaching damages under high irradiance.

Expression of Medicago truncatula Genes Responsive to Nitric Oxide in Pathogenic and Symbiotic Conditions

Nitric oxide (NO) is involved in diverse physiological processes in plants, including growth, development, response to pathogens, and interactions with beneficial microorganisms. In this work, a dedicated microarray representing the widest database available of NO-related transcripts in plants has been produced with 999 genes identified by a cDNA amplified fragment length polymorphism analysis as modulated in Medicago truncatula roots treated with two NO donors. The microarray then was used to monitor the expression of NO-responsive genes in M. truncatula during the incompatible interaction with the foliar pathogen Colletotrichum trifolii race 1 and during the symbiotic interaction with Sinorhizobium meliloti 1,021. A wide modulation of NO-related genes has been detected during the hypersensitive reaction or during nodule formation and is discussed with special emphasis on the physiological relevance of these genes in the context of the two biotic interactions. This work clearly shows that NO-responsive genes behave differently depending on the plant organ and on the type of interaction, strengthening the need to consider regulatory networks, including different signaling molecules.

Reactive Oxygen Species-Driven Transcription in Arabidopsis under Oxygen Deprivation

Reactive oxygen species (ROS) play an important role as triggers of gene expression during biotic and abiotic stresses, among which is low oxygen (O2). Previous studies have shown that ROS regulation under low O2 is driven by a RHO-like GTPase that allows tight control of hydrogen peroxide (H2O2) production. H2O2 is thought to regulate the expression of heat shock proteins, in a mechanism that is common to both O2 deprivation and to heat stress. In this work, we used publicly available Arabidopsis (Arabidopsis thaliana) microarray datasets related to ROS and O2 deprivation to define transcriptome convergence pattern. Our results show that although Arabidopsis response to anoxic and hypoxic treatments share a common core of genes related to the anaerobic metabolism, they differ in terms of ROS-related gene response. We propose that H2O2 production under O2 deprivation is a trait present in a very early phase of anoxia, and that ROS are needed for the regulation of a set of genes belonging to the heat shock protein and ROS-mediated groups. This mechanism, likely not regulated via the N-end rule pathway for O2 sensing, is probably mediated by a NADPH oxidase and it is involved in plant tolerance to the stress.

SUB1A‐dependent and ‐independent mechanisms are involved in the flooding tolerance of wild rice species

Crop tolerance to flooding is an important agronomic trait. Although rice (Oryza sativa) is considered a flood-tolerant crop, only limited cultivars display tolerance to prolonged submergence, which is largely attributed to the presence of the SUB1A gene. Wild Oryza species have the potential to unveil adaptive mechanisms and shed light on the basis of submergence tolerance traits. In this study, we screened 109 Oryza genotypes belonging to different rice genome groups for flooding tolerance. Oryza nivara and Oryza rufipogon accessions, belonging to the A-genome group, together with Oryza sativa, showed a wide range of submergence responses, and the tolerance-related SUB1A-1 and the intolerance-related SUB1A-2 alleles were found in tolerant and sensitive accessions, respectively. Flooding-tolerant accessions of Oryza rhizomatis and Oryza eichingeri, belonging to the C-genome group, were also identified. Interestingly, SUB1A was absent in these species, which possess a SUB1 orthologue with high similarity to O. sativa SUB1C. The expression patterns of submergence-induced genes in these rice genotypes indicated limited induction of anaerobic genes, with classical anaerobic proteins poorly induced in O. rhizomatis under submergence. The results indicated that SUB1A-1 is not essential to confer submergence tolerance in the wild rice genotypes belonging to the C-genome group, which show instead a SUB1A-independent response to submergence.

ROS signaling as common element in low oxygen and heat stresses

The activation of the oxidative metabolism in plants under low oxygen conditions has prompted controversial views. The presence of a ROS component in the transcriptome in response to low oxygen has been observed and an overlap with heat stress has been proved. It has been also demonstrated that ROS are produced during both anoxia and heat, but the site of their production remain contentious. Membrane NADPH oxidase and mitochondrial electron transport flow have been indicated as possible ROS generation systems. Both anoxia and heat have been shown to induce the transcription of Heat Shock Factors (HSFs) and Heat Shock Proteins (HSPs), among which HSFA2 and some of its targets. HSFA2 over-expressing plant has been shown to be more tolerant to anoxia, while the knockout hsfa2 lose the capability of wild type plants to cross-acclimate to anoxia through mild heat pre-treatment. The production of ROS seems to be an integral part of the anoxia and heat response, where HSFs likely play a central role in activating the HSP pathway. This mechanism is suggested to result in enhanced plant tolerance to both anoxia and heat.

Ozone distribution in central Italy and its effect on crop productivity

The focus of the present study was to assess the ozone levels in a typical area of the Mediterranean basin, viz. Tuscany (central Italy). Eighty-thousand hourly mean ozone concentrations were recorded by 10 automatic analysers in the districts of Florence, Pisa, Lucca and Prato, from May to September 1995 to 1997. The highest daily mean concentrations were reached in Florence, with a maximum hourly average of 197 ppb. In Lucca and Pisa, the peaks were close to 100 ppb. Data from Prato were much lower. Long-term critical levels for vegetation, as set by United Nations Economic Commission for Europe (UNECE), were constantly exceeded in Florence and Pisa, occasionally in Lucca, never in Prato. The results were used to fit exposure/yield response relationships proposed by UNECE and US National Crop Loss Assessment Network for some important crops. The estimated yield losses varied in Florence from 8% for corn and alfalfa to 27% for soybean, in Pisa from 5% for corn to 24% for soybean, in Lucca from 3% for corn to 17% for soybean. A preliminary economic estimate for corn, wheat, barley, soybean, tomato and alfalfa, calculated annual damage to be 4.6 M Euro in Florence, 0.5 M Euro in Lucca and 3 M Euro in Pisa. The picture must be regarded as only partial, as exposure/yield response relationships for important Italian crops (such as grapevine and vegetables) are not available.

Transcript profiling of chitosan-treated Arabidopsis seedlings

In nature, plants can recognize potential pathogens, thus activating intricate networks of defense signals and reactions. Inducible defense is often mediated by the detection of microbe or pathogen associated molecular pattern elicitors, such as flagellin and chitin. Chitosan, the deacetylated form of chitin, plays a role in inducing protection against pathogens in many plant species. We evaluated the ability of chitosan to confer resistance to Botrytis cinerea in Arabidopsis leaves. We subsequently treated Arabidopsis seedlings with chitosan and carried out a transcript profiling analysis using both ATH1 GeneChip microarrays and quantitative RT-PCR. The results showed that defense response genes, including camalexin biosynthesis genes, were up-regulated by chitosan, both in wild-type and in the chitin-insensitive cerk1 mutant, indicating that chitosan is perceived through a CERK1-independent pathway.

Low oxygen response mechanisms in green organisms

Low oxygen stress often occurs during the life of green organisms, mostly due to the environmental conditions affecting oxygen availability. Both plants and algae respond to low oxygen by resetting their metabolism. The shift from mitochondrial respiration to fermentation is the hallmark of anaerobic metabolism in most organisms. This involves a modified carbohydrate metabolism coupled with glycolysis and fermentation. For a coordinated response to low oxygen, plants exploit various molecular mechanisms to sense when oxygen is either absent or in limited amounts. In Arabidopsis thaliana, a direct oxygen sensing system has recently been discovered, where a conserved N-terminal motif on some ethylene responsive factors (ERFs), targets the fate of the protein under normoxia/hypoxia. In Oryza sativa, this same group of ERFs drives physiological and anatomical modifications that vary in relation to the genotype studied. The microalga Chlamydomonas reinhardtii responses to low oxygen seem to have evolved independently of higher plants, posing questions on how the fermentative metabolism is modulated. In this review, we summarize the most recent findings related to these topics, highlighting promising developments for the future.

Universal stress protein HRU1 mediates ROS homeostasis under anoxia

Plant survival is greatly impaired when oxygen levels are limiting, such as during flooding or when anatomical constraints limit oxygen diffusion. Oxygen sensing in Arabidopsis thaliana is mediated by Ethylene Responsive Factor (ERF)-VII transcription factors, which control a core set of hypoxia- and anoxia-responsive genes responsible for metabolic acclimation to low-oxygen conditions. Anoxic conditions also induce genes related to reactive oxygen species (ROS). Whether the oxygen-sensing machinery coordinates ROS production under anoxia has remained unclear. Here we show that a low-oxygen-responsive universal stress protein (USP), Hypoxia Responsive Universal Stress Protein 1 (HRU1), is induced by RAP2.12 (Related to Apetala 2.12), an ERF-VII protein, and modulates ROS production in Arabidopsis. We found that HRU1 is strongly induced by submergence, but that this induction is abolished in plants lacking RAP2.12. Mutation of HRU1 through transfer DNA (T-DNA) insertion alters hydrogen peroxide production, and reduces tolerance to submergence and anoxia. Yeast two-hybrid and bimolecular fluorescence complementation (BiFC) analyses reveal that HRU1 interacts with proteins that induce ROS production, the GTPase ROP2 and the NADPH oxidase RbohD, pointing to the existence of a low-oxygen-specific mechanism for the modulation of ROS levels. We propose that HRU1 coordinates oxygen sensing with ROS signalling under anoxic conditions.