Delia Tarantino

Antisense reduction of thylakoidal ascorbate peroxidase in Arabidopsis enhances Paraquat-induced photooxidative stress and Nitric Oxide-induced cell death

The production and characterization of Arabidopsis plants containing a transgene in which the Arabidopsis tAPX is inserted in antisense orientation, is described. tAPX activity in these transgenic tAPX plants is around 50% of control level. The tAPX antisense plants are phenotypically indistinguishable from control plants under normal growth conditions; they show, however, enhanced sensitivity to the O2−-generating herbicide, Paraquat. Interestingly, the tAPX antisense plants show enhanced symptoms of damage when cell death is triggered through treatment with the nitric oxide-donor, SNP. These results are in accordance with the ones recently obtained with transgenic plants overexpressing tAPX; altogether, they suggest that tAPX, besides the known ROS scavenging role, is also involved in the fine changes of H2O2 concentration during signaling events.

Preparation and functional characterization of thylakoids from Arabidopsis thaliana

A protocol for the isolation of functional thylakoids from Arabidopsis thaliana leaves was developed. The critical factor in obtaining active, coupled and stable preparation is the inclusion of EDTA and EGTA in the grinding buffer. Preparations were characterized with respect to the whole or partial electron transport chain, ATP/NADPH, ATP/O2 and PS II/chlorophyll ratios. Sensitivity to a light-chill photoinhibitory treatment was also determined by evaluating the decrease in both maximal photochemical efficiency (Fv/Fm) and in electron transport rate.

Differential involvement of the IDRS cis-element in the developmental and environmental regulation of the AtFer1 ferritin gene from Arabidopsis

Four different ferritin genes have been identified in Arabidopsis thaliana, namely AtFer1, 2, 3 and 4. AtFer1, which strongly accumulates in leaves treated with excess iron, contains in its promoter an Iron-Dependent Regulatory Sequence (IDRS). The IDRS sequence is responsible for repression of AtFer1 transcription under conditions of low iron supply. Arabidopsis plants transformed with a 1,400-bp AtFer1 promoter, with either a wild-type or a mutated IDRS fused to the β-glucuronidase (GUS) reporter gene, enabled us to analyze the activity of the AtFer1 promoter in different tissues as well as during age-dependent or dark-induced senescence. Our results show that IDRS mediates AtFer1 expression during dark-induced senescence while it does not affect AtFer1 expression during age-dependent senescence or in young seedlings. Photoinhibition promoted either by high light or chilling temperature, or wounding, does not activate the AtFer1 promoter. In contrast, AtFer2, AtFer3, AtFer4 transcript abundances are increased in response to photoinhibition and AtFer3 transcript abundance is increased upon wounding. Taken together, our results indicate that other cis-elements, different from the IDRS, regulate the territory-specific or developmental expression of AtFer1 gene. Expression of this gene appears insensitive to some of the environmental stresses tested, which instead up-regulate other members of the Arabidopsis ferritin gene family.

Structure-Based Inhibition of Norovirus RNA-Dependent RNA Polymerases.

Caliciviridae are RNA viruses with a single-stranded, positively oriented polyadenylated genome, responsible for a broad spectrum of diseases such as acute gastroenteritis in humans. Recently, analyses on the structures and functionalities of the RNA-dependent RNA polymerase (RdRp) from several Caliciviruses have been reported. The RdRp is predicted to play a key role in genome replication, as well as in synthesis and amplification of additional subgenomic RNA. Starting from the crystal structures of human Norovirus (hNV) RdRp, we performed an in silico docking search to identify synthetic compounds with predicted high affinity for the enzyme active site. The best-ranked candidates were tested in vitro on murine Norovirus (MNV) and hNV RdRps to assay their inhibition of RNA polymerization. The results of such combined computational and experimental screening approach led to the identification of two high-potency inhibitors: Suramin and NF023, both symmetric divalent molecules hosting two naphthalene-trisulfonic acid heads. We report here the crystal structure of MNV RdRp alone and in the presence of the two identified inhibitors. Both inhibitory molecules occupy the same RdRp site, between the fingers and thumb domains, with one inhibitor head close to residue 42 and to the protein active site. To further validate the structural results, we mutated Trp42 to Ala in MNV RdRp and the corresponding residue (i.e., Tyr41 to Ala) in hNV RdRp. Both NF023 and Suramin displayed reduced inhibitory potency versus the mutated hNV RdRp, thus hinting at a conserved inhibitor binding mode in the two polymerases.

Plant ferritin accumulates in response to photoinhibition but its ectopic overexpression does not protect against photoinhibition

Ferritin is an iron storage protein, involved in iron homeostasis in plant and animal cells. Ferritin could play an important role in protecting cells against the most noxious ROS known, the hydroxyl radicals, whose production is catalysed by Fe(II) and Fe(III). An increased ferritin mRNA abundance was observed in Arabidopsis thaliana leaves photoinhibited with high light at room temperature and also in leaves of A. thaliana plants fumigated with ozone. Very low protein accumulation followed mRNA accumulation in photoinhibited samples. We then investigated the role played by ferritin during photoinhibition or ozone treatment in Nicotiana tabacum and we showed that tobacco lines overexpressing a soybean ferritin gene were not more protected from oxidative stress than the control line. Indeed, at moderate light and chilling temperature, the line overexpressing ferritin in the chloroplast is more affected by a photoinhibitory treatment than its control line and its recovery from stress is impaired.

Structural bases of norovirus RNA dependent RNA polymerase inhibition by novel suramin-related compounds.

Noroviruses (NV) are +ssRNA viruses responsible for severe gastroenteritis; no effective vaccines/antivirals are currently available. We previously identified Suramin (9) as a potent inhibitor of NV-RNA dependent RNA polymerase (NV-RdRp). Despite significant in vitro activities versus several pharmacological targets, Suramin clinical use is hampered by pharmacokinetics/toxicity problems. To improve Suramin access to NV-RdRp in vivo, a Suramin-derivative, 8, devoid of two sulphonate groups, was synthesized, achieving significant anti-human-NV-RdRp activity (IC50 = 28 nM); the compound inhibits also murine NV (mNV) RdRp. The synthesis process led to the isolation/characterization of lower molecular weight intermediates (3–7) hosting only one sulphonate head. The crystal structures of both hNV/mNV-RdRps in complex with 6, were analyzed, providing new knowledge on the interactions that a small fragment can establish with NV-RdRps, and establishing a platform for structure-guided optimization of potency, selectivity and drugability.

Knocking out of the mitochondrial AtFer4 ferritin does not alter response of Arabidopsis plants to abiotic stresses

Ferritins are iron-storage proteins which, in Arabidopsis, have a clear role in protection against oxidative stress. Plant ferritins are localized mainly in chloroplasts, but they can also be targeted to mitochondria; the ATFER4 ferritin isoform, according to bioinformatic subcellular predictors, has the highest scores for such localization in Arabidopsis. We isolated atfer4-2 mutant KO in the AtFer4 gene and we characterized it together with a second, independent mutant atfer4-1. We show that ATFER4 is indeed localized in mitochondria of Fe-treated Arabidopsis plants; when grown under Fe excess, atfer4 plants manifest, however, the same toxicity symptoms and O(2) consumption rates as wt plants. No enhanced sensitivity to oxidative conditions was observed in atfer4 seedlings exposed to salinity, osmotic stress, cold stress or oxidative stress elicited by paraquat. The growth response of roots and aerial parts in atfer4 plants under different light conditions was the same as wt. Also, the process of natural senescence, in which AtFer1 takes active part, was not perturbed in atfer4 plants. We conclude that the ATFER4 ferritin role in counteracting the environmental or developmental oxidative conditions in Arabidopsis plants is ancillary to that of the other isoforms, regardless of its mitochondrial localization.

AtFer4 ferritin is a determinant of iron homeostasis in Arabidopsis thaliana heterotrophic cells

In plants, the iron storage protein ferritin can be targeted to both chloroplasts and mitochondria. To investigate the role of Arabidopsis ATFER4 ferritin in mitochondrial iron trafficking, atfer4-1 and atfer4-2 mutant knock-outs for the AtFer4 gene were grown in heterotrophic suspension cultures. Both mutants showed altered cell size and morphology, reduced viability, higher H₂O₂ content and reduced O₂ consumption rates when compared to wt. Although no reduction in total ferritin or in mitochondrial ferritin was observed in atfer4 mutants, total iron content increased in atfer4 cells and in atfer4 mitochondria. Transcript correlation analysis highlighted a partial inverse relationship between the transcript levels of the mitochondrial ferric reductase oxidase FRO3, putatively involved in mitochondrial iron import/export, and AtFer4. Consistent with this, FRO3 transcript levels were higher in atfer4 cells. We propose that the complex molecular network maintaining Fe cellular homeostasis requires, in Arabidopsis heterotrophic cells, a proper balance of the different ferritin isoforms, and that alteration of this equilibrium, such as that occurring in atfer4 mutants, is responsible for an altered Fe homeostasis resulting in a change of intraorganellar Fe trafficking.

Identification of an Arabidopsis mitoferrinlike carrier protein involved in Fe metabolism.

Iron has a major role in mitochondrial as well as in chloroplast metabolism, however the processes involved in organelle iron transport in plants are only partially understood. To identify mitochondrial iron transporters in Arabidopsis, we searched for proteins homologous to the Danio rerio (zebrafish) Mitoferrin2 MFRN2, a mitochondrial iron importer in non-erythroid cells. Among the identified putative Arabidopsis mitoferrinlike proteins, we focused on that one encoded by At5g42130, which we named AtMfl1 (MitoFerrinLike1). AtMfl1 expression strongly correlates with genes coding for proteins involved in chloroplast metabolism. Such an unexpected result is supported by the identification by different research groups, of the protein encoded by At5g42130 and of its homologs from various plant species in the inner chloroplastic envelope membrane proteome. Notably, neither the protein encoded by At5g42130 nor its homologs from other plant species have been identified in the mitochondrial proteome. AtMfl1 gene expression is dependent on Fe supply: AtMfl1 transcript strongly accumulates under Fe excess, moderately under Fe sufficiency and weakly under Fe deficiency. In order to understand the physiological role of AtMfl1, we isolated and characterized two independent AtMfl1 KO mutants, atmfl1-1 and atmfl1-2: both show reduced vegetative growth. When grown under conditions of Fe excess, atmfl1-1 and atmfl1-2 mutants (seedlings, rosette leaves) contain less total Fe than wt and also reduced expression of the iron storage ferritin AtFer1. Taken together, these results suggest that Arabidopsis mitoferrinlike gene AtMfl1 is involved in Fe transport into chloroplasts, under different conditions of Fe supply and that suppression of its expression alters plant Fe accumulation in various developmental stages.

PPNDS inhibits murine Norovirus RNA-dependent RNA-polymerase mimicking two RNA stacking bases

Norovirus (NV) is a major cause of gastroenteritis worldwide. Antivirals against such important pathogens are on demand. Among the viral proteins that orchestrate viral replication, RNA‐dependent‐RNA‐polymerase (RdRp) is a promising drug development target. From an in silico‐docking search focused on the RdRp active site, we selected the compound PPNDS, which showed low micromolar IC50 vs. murine NV‐RdRp in vitro. We report the crystal structure of the murine NV‐RdRp/PPNDS complex showing that two molecules of the inhibitor bind in antiparallel stacking interaction, properly oriented to block exit of the newly synthesized RNA. Such inhibitor‐binding mode mimics two stacked nucleotide‐bases of the RdRp/ssRNA complex.