Knockout of frataxin gene causes embryo lethality in Arabidopsis.

Frataxin is present in mitochondria of all eukaryotes as well as in the cytoplasm of bacteria. In humans, reduced expression of frataxin is associated with Friedreich's ataxia, a recessive inherited neurodegenerative and cardiac disorder leading to reduced life expectancy. Experimental evidences suggest that frataxin acts as an iron-chaperone protein, donating iron to the proteins involved in [Fe-S] cluster assembly and heme synthesis. It also possibly contributes to the process of iron detoxification and storage. 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.

Knock-out of ferritin AtFer1 causes earlier onset of age-dependent leaf senescence in Arabidopsis.

Ferritins are iron-storage proteins involved in the regulation of free iron levels in the cells. Arabidopsis thaliana AtFer1 ferritin, one of the best characterized plant ferritin isoforms to date, strongly accumulates upon treatment with excess iron, via a nitric oxide-mediated pathway. However other environmental factors, such as exposure to oxidative stress or to pathogen attack, as well as developmental factors regulate AtFer1 transcript levels. In particular, recent findings have highlighted an accumulation of the ferritin transcript during senescence. To investigate the physiological relevance of AtFer1 ferritin during senescence we isolated an Arabidopsis mutant knock-out in the AtFer1 gene, which we named atfer1-2. We analyzed it together with a second, independent AtFer1 KO mutant, the atfer1-1 mutant. Interestingly, both atfer1-1 and atfer1-2 mutants show symptoms of accelerated natural senescence; the precocious leaf yellowing is accompanied by accelerated decrease of maximal photochemical efficiency and chlorophyll degradation. However, no accelerated senescence upon dark treatment was observed in the atfer1 mutants with respect to their wt. These results suggest that AtFer1 ferritin isoform is functionally involved in events leading to the onset of age-dependent senescence in Arabidopsis and that its iron-detoxification function during senescence is required when reactive oxygen species accumulate.

Comparative effects of various nitric oxide donors on ferritin regulation, programmed cell death, and cell redox state in plant cells.

Past studies investigating the regulatory functions of nitric oxide (NO) in plant cells have utilized various NO-donors that release NO in different redox forms, which has lead to problems in the interpretation of data. In the present study, the effects of different NO-donors releasing NO with either NO+ (SNP) or NO' (SNAP, GSNO, NOC-18) character have been compared in plant cells. In particular, ferritin regulation, programmed cell death, cellular redox state, and ROS-scavenging enzymes in Arabidopsis thaliana and Nicotiana tabacum cells were examined. The results show that SNP behaves differently than the other NO-donors tested; indeed, SNP induces accumulation of ferritin transcripts in Arabidopsis, whereas SNAP inhibits its accumulation. Moreover, among the assortment of donors tested, only SNP caused programmed cell death and suppression of ROS-scavenging systems.

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.

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.

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.

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.

Suppression of both ELIP1 and ELIP2 in Arabidopsis does not affect tolerance to photoinhibition and photooxidative stress.

ELIPs (early light-induced proteins) are thylakoid proteins transiently induced during greening of etiolated seedlings and during exposure to high light stress conditions. This expression pattern suggests that these proteins may be involved in the protection of the photosynthetic apparatus against photooxidative damage. To test this hypothesis, we have generated Arabidopsis (Arabidopsis thaliana) mutant plants null for both elip genes (Elip1 and Elip2) and have analyzed their sensitivity to light during greening of seedlings and to high light and cold in mature plants. In particular, we have evaluated the extent of damage to photosystem II, the level of lipid peroxidation, the presence of uncoupled chlorophyll molecules, and the nonphotochemical quenching of excitation energy. The absence of ELIPs during greening at moderate light intensities slightly reduced the rate of chlorophyll accumulation but did not modify the extent of photoinhibition. In mature plants, the absence of ELIP1 and ELIP2 did not modify the sensitivity to photoinhibition and photooxidation or the ability to recover from light stress. This raises questions about the photoprotective function of these proteins. Moreover, no compensatory accumulation of other ELIP-like proteins (SEPs, OHPs) was found in the elip1/elip2 double mutant during high light stress. elip1/elip2 mutant plants show only a slight reduction in the chlorophyll content in mature leaves and greening seedlings and a lower zeaxanthin accumulation in high light conditions, suggesting that ELIPs could somehow affect the stability or synthesis of these pigments. On the basis of these results, we make a number of suggestions concerning the biological function of ELIPs.

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.

Mutational and expression analysis of ELIP1 and ELIP2 in Arabidopsis thaliana.

Plants exposed to photoinhibitory conditions respond by accumulation of the early light-induced proteins (ELIPs) with a potential photoprotective function. In Arabidopsis thaliana two genes (Elip1 and Elip2) encode for two ELIP proteins: evidence exists that the two genes are differentially regulated but their precise function is unclear. Mutants null for one or the other Elip gene can help in elucidating ELIPs role and here we describe the expression profile of ELIP1 and ELIP2, and the phenotype of such null mutants. Both ELIPs accumulate during greening of etiolated seedlings and in mature plants the transcripts fluctuate diurnally without protein accumulation. Steady-state transcript level of both genes increases in response to high light with transcription of Elip1 much more sensitive than that of Elip2 to increasing irradiation at 22 degrees C. At 4 degrees C instead Elip2 is strongly transcribed even at growing light. Furthermore, only ELIP1 accumulates under high light at 22 degrees C while both proteins accumulate at 4 degrees C. These results indicate the existence of a differential regulation of ELIPs expression in response to light or chilling stress with mechanisms active either at transcriptional and post-transcriptional level. Phenotypically, the mutants behave as the wild type as far as sensitivity to light- or light and cold-induced short-term photoinhibition, while both ELIPs are necessary to ensure a high rate of chlorophyll accumulation during deetiolation in continuous high light.

Nitric oxide mediates iron-induced ferritin accumulation in Arabidopsis.

Nitric oxide (NO) is a signaling molecule that plays a critical role in the activation of innate immune and inflammatory 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.

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 beta-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.

Arabidopsis thaliana plants overexpressing thylakoidal ascorbate peroxidase show increased resistance to Paraquat-induced photooxidative stress and to nitric oxide-induced cell death.

Ascorbate 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 specific 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 fine modulation of ROS for signaling, we produced Arabidopsis lines overexpressing tAPX. These lines show an increased resistance to treatment with the O(2)(-) 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 confirm that H(2)O(2) acts in partnership with NO in causing cell death and highlight the important role of tAPX in the fine modulation of H(2)O(2) for signaling.

Role of visible light in the recovery of photosystem II structure and function from ultraviolet-B stress in higher plants.

The effect of visible light on photosystem II reaction centre D1 protein in plants treated with ultraviolet-B light was studied. It was found that a 20 kDa C-terminal fragment of D1 protein generated during irradiation with ultraviolet-B light was stable when plants were incubated in the dark, but was degraded when plants were incubated in visible light. In this condition the recovery of photosynthetic activity was also observed. Even a low level of white light was sufficient to promote both further degradation of the fragment and recovery of activity. During this phase, the D1 protein is the main synthesized thylakoid polypeptide, indicating that other photosystem II proteins are recycled in the recovery process. Although both degradation of the 20 kDa fragment and resynthesis of D1 are light-dependent phenomena, they are not closely related, as degradation of the 20 kDa fragment may occur even in the absence of D1 synthesis. Comparing chemical and physical factors affecting the formation of the fragment in ultraviolet-B light and its degradation in white light, it was concluded that the formation of the fragment in ultraviolet-B light is a photochemical process, whereas the degradation of the fragment in white light is a protease-mediated process.

Evidence for the presence of ferritin in plant mitochondria.

In this work, evidence for the presence of ferritins in plant mitochondria is supplied. Mitochondria were isolated from etiolated pea stems and Arabidopsis thaliana cell cultures. The proteins were separated by SDS/PAGE. A protein, with an apparent molecular mass of approximately 25-26 kDa (corresponding to that of ferritin), was cross-reacted with an antibody raised against pea seed ferritin. The mitochondrial ferritin from pea stems was also purified by immunoprecipitation. The purified protein was analyzed by MALDI-TOF mass spectrometry and the results of both mass finger print and peptide fragmentation by post source decay assign the polypeptide sequence to the pea ferritin (P < 0.05). The mitochondrial localization of ferritin was also confirmed by immunocytochemistry experiments on isolated mitochondria and cross-sections of pea stem cells. The possible role of ferritin in oxidative stress of plant mitochondria is discussed.

A stable LHCII-PSI aggregate and suppression of photosynthetic state transitions in the psae1-1 mutant of Arabidopsis thaliana.

During photosynthetic state transitions, a fraction of the major light-harvesting complex (LHCII) shuttles between photosystems II (PSII) and I (PSI), depending on whether or not it is phosphorylated. Its phosphorylation state in turn depends on the relative activity of the two photosystems, which is a function of redox state and illumination parameters. In the psae1-1 mutant of Arabidopsis thaliana (L.) Heynh., amounts of the PSI subunits E, C, D, H and L are decreased. A fraction of LHCII is stably associated with PSI when plants are exposed to low light conditions, giving rise to a high-molecular-mass protein-pigment complex detectable in native protein gels. The formation of this abnormal LHCII-PSI complex is associated with an almost complete suppression of state transitions, a drastic increase in the levels of phosphorylated LHCII under all light regimes tested, and a permanent reduction in PSII antenna size. All these observations suggest that the altered polypeptide composition of PSI perturbs the docking of phosphorylated LHCII, making psae1-1 a unique mutant for the study of PSI-LHCII interactions and additional effects of the mutation, such as a decrease in grana stacking and increased adenylate kinase activity.

A novel interaction partner for the C-terminus of Arabidopsis thaliana plasma membrane H+-ATPase (AHA1 isoform): site and mechanism of action on H+-ATPase activity differ from those of 14-3-3 proteins.

Using the two-hybrid technique we identified a novel protein whose N-terminal 88 amino acids (aa) interact with the C-terminal regulatory domain of the plasma membrane (PM) H+-ATPase from Arabidopsis thaliana (aa 847-949 of isoform AHA1). The corresponding gene has been named Ppi1 for Proton pump interactor 1. The encoded protein is 612 aa long and rich in charged and polar residues, except for the extreme C-terminus, where it presents a hydrophobic stretch of 24 aa. Several genes in the A. thaliana genome and many ESTs from different plant species share significant similarity (50-70% at the aa level over stretches of 200-600 aa) to Ppi1. The PPI1 N-terminus, expressed in bacteria as a fusion protein with either GST or a His-tag, binds the PM H+-ATPase in overlay experiments. The same fusion proteins and the entire coding region fused to GST stimulate H+-ATPase activity. The effect of the His-tagged peptide is synergistic with that of fusicoccin (FC) and of tryptic removal of a C-terminal 10 kDa fragment. The His-tagged peptide binds also the trypsinised H+-ATPase. Altogether these results indicate that PPI1 N-terminus is able to modulate the PM H+-ATPase activity by binding to a site different from the 14-3-3 binding site and is located upstream of the trypsin cleavage site.