ascorbate peroxidase ͉ chloroplast ͉ oxidative stress ͉ reactive oxygen species

Iron is an essential but poorly bioavailable nutrient because of its low solubility, especially in alkaline soils. Here, we describe the discovery of a previously undescribed redox-active catecholic metabolite, termed sideretin, which derives from the coumarin fraxetin and is the primary molecule exuded by Arabidopsis thaliana roots in response to iron deficiency. We identified two enzymes that complete the biosynthetic pathway of fraxetin and sideretin. Chemical characterization of fraxetin and sideretin, and biological assays with pathway mutants, suggest that these coumarins are critical for iron nutrition in A. thaliana. Further, we show that sideretin production also occurs in eudicot species only distantly related to A. thaliana. Untargeted metabolomics of the root exudates of various eudicots revealed production of structurally diverse redox-active molecules in response to iron deficiency. Our results indicate that secretion of small-molecule reductants by roots may be a widespread and previously underappreciated component of reduction-based iron uptake.

SummaryAscorbate peroxidases (APX), localized in the cytosol, peroxisomes, mitochondria and chloroplasts of plant cells, catalyze the reduction of H 2 O 2 to water by using ascorbic acid (ASA) as speci®c electron donor. The chloroplastic isoenzymes of APX are involved in the water±water cycle, which contributes to the photophosphorylation coupled to the photosynthetic electron transport. In order to better clarify the contribution of thylakoidal APX (tAPX) to the reactive oxygen species (ROS) scavenging activity, as well as to the ®ne modulation of ROS for signaling, we produced Arabidopsis lines overexpressing tAPX. These lines show an increased resistance to treatment with the O 2 À generating herbicide Paraquat (Pq). However, when challenged with photoinhibitory treatments at high light or low temperature, or with iron (Fe) or copper (Cu) overload, the tAPX-overexpressing lines show no increased resistance with respect to controls, indicating that in such experimental conditions, tAPX overexpression does not reinforce plant defenses against the oxidative stresses tested. Interestingly, the nitric oxide (NO)±donor sodium nitroprusside (SNP) represses accumulation of tAPX transcript; SNP also partially inhibits tAPX enzymatic activity. After treatment with SNP, the tAPX-overexpressing lines show reduced symptoms of damage with respect to control plants treated with SNP. These transgenic lines con®rm that H 2 O 2 acts in partnership with NO in causing cell death and highlight the important role of tAPX in the ®ne modulation of H 2 O 2 for signaling.

SummaryNitric oxide (NO) is a signaling molecule that plays a critical role in the activation of innate immune and in¯ammatory responses in animals. During the last few years, NO has also been detected in several plant species and the increasing number of reports on its function in plants have implicated NO as an important effector of growth, development and defense. Analogously to animals, NO has been recently shown to inhibit tobacco aconitase. This suggests that NO may elevate free iron levels in the cells by converting tobacco cytoplasmic aconitase into a mRNA binding protein that negatively regulates accumulation of ferritin.We investigated the possible role of NO as a regulator of ferritin levels in Arabidopsis and found that the NO-donor sodium nitroprusside (SNP) induces accumulation of ferritin both at mRNA and protein level. Iron is not necessary for this NO-mediated ferritin transcript accumulation, since SNP is still able to induce the accumulation of ferritin transcript in Arabidopsis suspension cultures pre-treated with the iron chelants DFO or ferrozine. However, NO is required for iron-induced ferritin accumulation, as the NO scavenger CPTIO prevents ferritin transcript accumulation in Arabidopsis suspension cultures treated with iron. The pathway is ser/thr phosphatase-dependent and necessitates protein synthesis; furthermore, NO mediates ferritin regulation through the IDRS sequence of the Atfer1 promoter responsible for transcriptional repression under low iron supply.NO, by acting downstream of iron in the induction of ferritin transcript accumulation is therefore a key signaling molecule for regulation of iron homeostasis in plants.

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.As the major cofactor of proteins involved in essential processes like photosynthesis, respiration, DNA replication, or nitrogen fixation, iron is an essential element for life. Nonetheless, in the free ionic form, iron is toxic as it can catalyze the formation of reactive oxygen species through the Fenton reaction. These reactive oxygen species damage the cell membranes, DNA, and proteins (1, 2). Thus, iron homeostasis has to be tightly regulated, to avoid starvation that impairs the metabolism, and to avoid excess that may lead to cell death. Iron homeostasis is strongly dependent on ferritins, which are ironstorage proteins, found in bacteria, animals, and plants. Plant and animal ferritin structures are very similar, and are formed by 24 subunits arranged to form a hollow sphere able to sequester iron in a non-toxic and bioavailable form (3).In animals, ferritin synthesis is mainly regulated at the posttranscriptional level (3, 4). Ferritin mRNAs contain iron-responsive elements in their 5Ј-untranslated regions that function as binding sites for two related trans-acting factors, namely iron regulatory proteins IRP1 and IRP2. When bound to the iron-responsive element in the ferritin mRNA, the IRP inhibit translation of the transcript (4). IRP1 is a bifunctional protein that when iron is abundant possesses a 4Fe-4S cluster and acts as cytoplasmic aconitase. When iron levels are low, the 4Fe-4S cluster disassembles and the apoprotein acquires IRP 3 activity, thus repressing ferritin translation. High levels of iron lead to the 4Fe-4S cluster reconstitution and therefore the protein aconitase activity. In contrast to IRP1, IRP2 cannot assemble a iron-sulfur cluster and lacks aconitase activity. IRP2 shares about 60% amino acid sequence identity with IRP1, but differs only in having a 73-amino aci...