Irene Murgia

An Iron-induced Nitric Oxide Burst Precedes Ubiquitin-dependent Protein Degradation for Arabidopsis AtFer1 Ferritin Gene Expression

Ferritins play an essential role in iron homeostasis by sequestering iron in a bioavailable and non-toxic form. In plants, ferritin mRNAs are highly and quickly accumulated in response to iron overload. Such accumulation leads to a subsequent ferritin protein synthesis and iron storage, thus avoiding oxidative stress to take place. By combining pharmacological and imaging approaches in an Arabidopsis cell culture system, we have identified several elements in the signal transduction pathway leading to the increase of AtFer1 transcript level after iron treatment. Nitric oxide quickly accumulates in the plastids after iron treatment. This compound acts downstream of iron and upstream of a PP2A-type phosphatase to promote an increase of AtFer1 mRNA level. The AtFer1 gene transcription has been previously shown to be repressed under low iron conditions with the involvement of the cis-acting element iron-dependent regulatory sequence identified within the AtFer1 promoter sequence. We show here that the repressor is unlikely a transcription factor directly bound to the iron-dependent regulatory sequence; such a repressor is ubiquitinated upon iron treatment and subsequently degraded through a 26 S proteasome-dependent pathway.

Reactive oxygen intermediates modulate nitric oxide signaling in the plant hypersensitive disease-resistance response

The mechanisms involved in plant defense show several similar characteristics with the innate immune systems of vertebrates and invertebrates. In animals, nitric oxide (NO) cooperates with reactive oxygen intermediates (ROI) to kill tumor cells and is also required for macrophage killing of bacteria. Such cytotoxic events occur because unregulated levels of NO determine its diffusion-limited reaction with O2 – generating peroxynitrite (ONOO – ), a mediator of cellular injury in many biological systems. In soybean suspension cells, unregulated NO production during the onset of a pathogen-induced hypersensitive response (HR) is not sufficient to activate the hypersensitive cell death, which is triggered only by fine tuning the NO/ROI ratio. Furthermore, that hypersensitive cell death is activated following interaction of NO with H2O2, rather than O2 – . Increasing O2 – levels reduces NO-derived toxicity, and the addition of ONOO – to soybean suspensions does not affect cell viability. Consistently with the fact that ONOO – is not an essential mediator of NO/RO-induced cell death, during the HR superoxide dismutase (SOD) accelerates O2 – dismutation to H2O2 and therefore minimizes the loss of NO by reaction with O2 – and triggers hypersensitive cell death through the NO/H2O2 synergism. Consequently, the rates of production and dismutation of O2 – generated during the oxidative burst play a crucial role in modulating NO signaling through the cell death pathway, which proceeds through mechanisms different from those commonly observed in animals.

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.

The functions of nitric oxide-mediated signaling and changes in gene expression during the hypersensitive response.

Nitric oxide (NO) is a highly reactive molecule that rapidly diffuses and permeates cell membranes. In animals, NO is implicated in a number of diverse physiological processes, such as neurotransmission, vascular smooth muscle relaxation, and platelet inhibition. It may have beneficial effects, e.g., as a messenger in immune responses, but it is also potentially toxic when the antioxidant system is overwhelmed and reactive oxygen intermediates (ROI) accumulate. During the last few years, NO has been detected in several plant species, and an increasing number of reports on its function have implicated NO as an important effector in plant growth, development, and defense. The broad chemistry of NO involves an array of interrelated redox forms with different chemical reactivities and numerous potential biological targets in plants. NO signaling functions depend on its reactivity. ROI are key modulators of NO in triggering cell death, but the nature of the mechanisms by which this occurs in plants is different from those commonly observed in animals. This review focuses on the signaling functions of NO, when channeled through the cell death pathway by ROI.

Biofortification for combating ‘hidden hunger’ for iron

Micronutrient deficiencies are responsible for so-called hidden undernutrition. In particular, iron (Fe) deficiency adversely affects growth, immune function and can cause anaemia. However, supplementation of iron can exacerbate infectious diseases and current policies of iron therapy carefully evaluate the risks and benefits of these interventions. Here we review the approaches of biofortification of valuable crops for reducing hidden undernutrition of iron in the light of the latest nutritional and medical advances. The increase of iron and prebiotics in edible parts of plants is expected to improve health, whereas the reduction of phytic acid concentration, in crops valuable for human diet, might be less beneficial for the developed countries, or for the developing countries exposed to endemic infections.

Nitric oxide, nitrosyl iron complexes, ferritin and frataxin: A well equipped team to preserve plant iron homeostasis

Iron is a key element in plant nutrition. Iron deficiency as well as iron overload results in serious metabolic disorders that affect photosynthesis, respiration and general plant fitness with direct consequences on crop production. More than 25% of the cultivable land possesses low iron availability due to high pH (calcareous soils). Plant biologists are challenged by this concern and aimed to find new avenues to ameliorate plant responses and keep iron homeostasis under control even at wide range of iron availability in various soils. For this purpose, detailed knowledge of iron uptake, transport, storage and interactions with cellular compounds will help to construct a more complete picture of its role as essential nutrient. In this review, we summarize and describe the recent findings involving four central players involved in keeping cellular iron homeostasis in plants: nitric oxide, ferritin, frataxin and nitrosyl iron complexes. We attempt to highlight the interactions among these actors in different scenarios occurring under iron deficiency or iron overload, and discuss their counteracting and/or coordinating actions leading to the control of iron homeostasis.

Knockout of frataxin gene causes embryo lethality in Arabidopsis

The frataxin homolog from Arabidopsis thaliana (AtFH) is a single nuclear‐encoded gene targeted to mitochondria and sharing 65% similarity with animal frataxin. In the present work, we show that the knocking out of AtFH gene causes arrest of Arabidopsis embryo development at the globular stage. Consistently with that, we also show by in situ hybridization that AtFH is expressed, in wt Arabidopsis plants, in ovule primordia as well as in embryos at various stages of development, suggesting a key role of plant frataxin during embryogenesis.

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.

Arabidopsis CYP82C4 expression is dependent on Fe availability and circadian rhythm, and correlates with genes involved in the early Fe deficiency response

Under conditions of reduced iron availability, most frequent in calcareous soils, plants induce the Fe Deficiency Response to improve root Fe uptake. The transcription factor FIT is essential for such a response in strategy I plants, such as Arabidopsis thaliana. From microarray analysis of Arabidopsis roots, it is known that three different cytochrome P450 genes, CYP82C4, CYP82C3 and CYP71B5 are up-regulated under Fe deficiency through a FIT-dependent pathway. We show that, out of these three P450 genes, only CYP82C4 strongly correlates with genes involved in metal uptake/transport. The CYP82C4 promoter, unlike those of CYP82C3 and CYP71B5, contains several IDE1-like sequences (iron deficiency-responsive element) as well as an RY element. While confirming that the CYP82C4 transcript accumulates in Fe-deficient Arabidopsis seedlings, with circadian fluctuations in a light-dependent way, we also demonstrate that such accumulation is suppressed under Fe excess. Full suppression of CYP82C4 expression, as observed in the atc82c4-1 KO mutant, is associated with longer roots at the seedling stage. We propose that CYP82C4 is involved in the early Fe deficiency response, possibly through an IDE1-like mediated pathway.

Searching iron sensors in plants by exploring the link among 2'-OG-dependent dioxygenases, the iron deficiency response and metabolic adjustments occurring under iron deficiency.

Knowledge accumulated on the regulation of iron (Fe) homeostasis, its intracellular trafficking and transport across various cellular compartments and organs in plants; storage proteins, transporters and transcription factors involved in Fe metabolism have been analyzed in detail in recent years. However, the key sensor(s) of cellular plant “Fe status” triggering the long-distance shoot–root signaling and leading to the root Fe deficiency responses is (are) still unknown. Local Fe sensing is also a major task for roots, for adjusting the internal Fe requirements to external Fe availability: how such sensing is achieved and how it leads to metabolic adjustments in case of nutrient shortage, is mostly unknown. Two proteins belonging to the 2′-OG-dependent dioxygenases family accumulate several folds in Fe-deficient Arabidopsis roots. Such proteins require Fe(II) as enzymatic cofactor; one of their subgroups, the HIF-P4H (hypoxia-inducible factor-prolyl 4-hydroxylase), is an effective oxygen sensor in animal cells. We envisage here the possibility that some members of the 2′-OG dioxygenase family may be involved in the Fe deficiency response and in the metabolic adjustments to Fe deficiency or even in sensing Fe, in plant cells.