Plant cell nucleolus as a hot spot for iron

Many central metabolic processes require iron as a cofactor and take place in specific subcellular compartments such as the mitochondrion or the chloroplast. Proper iron allocation in the different organelles is thus critical to maintain cell function and integrity. To study the dynamics of iron distribution in plant cells, we have sought to identify the different intracellular iron pools by combining three complementary imaging approaches, histochemistry, micro particle-induced x-ray emission, and synchrotron radiation micro X-ray fluorescence. Pea (Pisum sativum) embryo was used as a model in this study because of its large cell size and high iron content. Histochemical staining with ferrocyanide and diaminobenzidine (Perls/diaminobenzidine) strongly labeled a unique structure in each cell, which co-labeled with the DNA fluorescent stain DAPI, thus corresponding to the nucleus. The unexpected presence of iron in the nucleus was confirmed by elemental imaging using micro particle-induced x-ray emission. X-ray fluorescence on cryo-sectioned embryos further established that, quantitatively, the iron concentration found in the nucleus was higher than in the expected iron-rich organelles such as plastids or vacuoles. Moreover, within the nucleus, iron was particularly accumulated in a subcompartment that was identified as the nucleolus as it was shown to transiently disassemble during cell division. Taken together, our data uncover an as yet unidentified although abundant iron pool in the cell, which is located in the nuclei of healthy, actively dividing plant tissues. This result paves the way for the discovery of a novel cellular function for iron related to nucleus/nucleolus-associated processes.     

Straightforward histochemical staining of Fe by the adaptation of an old-school technique: Identification of the endodermal vacuole as the site of Fe storage in Arabidopsis embryos

Iron (Fe) is an essential metal ion, required for basic cellular processes such as respiration, photosynthesis and cell division. Therefore, Fe has to be stored and distributed to several organelles to fulfill its roles. The molecular basis of Fe distribution is poorly understood. In this context, elemental imaging approaches are becoming essential for a better understanding of metal homeostasis in plants. Recently, several genes have been involved in Fe storage (VIT1) and remobilization (NRAMP3 and NRAMP4) in the seed of Arabidopsis, mostly with the help of sophisticated imaging techniques. We have adapted an histochemical procedure to detect Fe in plant tissues, based on Perls staining coupled to diaminobenzidine (DAB) intensification. The Perls/DAB technique, quick and inexpensive, was shown to be specific for Fe and highly sensitive. We have applied this procedure to Arabidopsis embryos and shown that Fe is stored in the vacuoles of a specific cell layer surrounding the pro-vascular system, the endodermis. Our results have revealed a new role for the endodermis in Fe storage in the embryo and established the Perls/DAB technique as a powerful tool to detect Fe in plant tissues and cells.Key words: iron, vacuole, Arabidopsis, endodermis, embryoIron (Fe) is a very important essential metal for all living organisms. The function of Fe in biological processes relies on its ability to exist in two redox states (ferrous and ferric iron). Consequently, Fe is crucial for metabolic reactions of respiration and photosynthesis. Although at the whole plant level the molecular mechanisms of Fe acquisition and storage are now well documented, the control of Fe distribution at organ, cell and sub-cellular levels is extremely poorly understood and represents an important stake. Recent discoveries on Fe storage and remobilization in the embryo have unequivocally shown that genetic approaches are not sufficient to unravel the function of genes, unless coupled to high-resolution metal imaging. The use of energy dispersive X-ray spectroscopy (EDX) and inelastically scattered electrons (ESI) on electron micrographs clearly showed that in Arabidopsis seeds Fe is accumulated in vacuoles and remobilized during germination.¹ Likewise, the analysis of Arabidopsis seeds by XRF and tomography has provided three-dimensional mapping of Fe that has showed that VIT1 (for Vacuolar Iron Transporter) is required for proper allocation of Fe in the vascular tissue of the embryo, a distribution that is altered in a vit1-1 mutant.² Noteworthy is the fact that these alterations of Fe distribution do not induce a change in total Fe content and thus would not have been detected by measurements of total Fe concentration. There is therefore an increasing interest for imaging techniques that are becoming a must-have to understand the function of metal transporters in plants. Metal imaging technologies are mostly based on X ray absorption/emission/fluorescence, often requiring expensive and rare equipments (synchrotron for instance). Taking advantage on the high reactivity of metal ions for organic ligands, several reagents have been used as chromophores for histochemical staining of metals. Among those, potassium ferrocyanide, also known as Perls reagent, was used since the late XIX^th century to produce the Prussian blue, after reaction with ferric iron. The Perls reagent has been widely used to stain Fe in tissues, but only occasionally in plants, due to its low sensitivity and poor penetration in hydrophobic tissues. Nevertheless, it is possible to increase the sensitivity of the staining by secondary reactions with diaminobenzidine (DAB) and hydrogen peroxide (H₂O₂). Indeed, since the Fe-Perls complex is redox active, the addition of DAB and H₂O₂ triggers the oxidative polymerization of DAB, producing brown pigments.³ This reaction is the basis of the intensification of the Perls staining (Perls/DAB from now on). To adapt this procedure to plants, we have first established that the staining was specific for Fe and did not practically cross-react with other metal ions. In doing so, we also showed that Perls/DAB could stain both Fe^II and Fe^III. We have chosen Arabidopsis seeds as model, in order to compare our Fe staining procedure with the imaging already available by XRF.² Compared to Perls stain alone, the Perls/DAB protocol appeared to be much more sensitive. Iron appeared to be concentrated around the provascular system of mature Arabidopsis embryos.Staining of the vit1-1 mutant showed a modified pattern compatible with the available μXRF-tomography data, thus clearly establishing that the staining procedure is specific for Fe in vivo and represents a new, quick and simple tool to detect Fe in plant samples.⁴ Thin sections of Perls/DAB embryos uncovered that, in mature embryos, Fe was concentrated in a single cell layer, apparently corresponding to the endodermis. This observation was further confirmed by the analysis of longitudinal sections of the radicle-hypocotyl region. In this particular zone, a second periclinal division occurs, giving rise to a cortex cell layer and the endodermis,⁵ the latter one alone being intensively stained with Perls/DAB. Other developmental mutants were used, such as for example the vein patterning mutant SCARFACE (scf),⁶ which presents Fe staining in cotyledons as small segments corresponding to discontinuous veins characteristic of the scf mutant (Fig. 1). Thus, the Perls/DAB protocol represents a very useful tool not only to study Fe homeostasis but also in the field of developmental research, as a marker of endodermis and provascular system of the embryo. Finally, the Perls/DAB procedure can be greatly improved by staining directly the histological thin sections instead of whole embryos, thereby (i) increasing tremendously the resolution and (ii) solving the problem of low penetration of the dyes in hydrophobic plant samples. This modification enabled us to show that in endodermal cells Fe is actually located in vacuoles. Remobilization of the vacuolar pool of iron by AtNRAMP3 and AtNRAMP4 is crucial during germination.¹ Since we found that Fe is blocked in the endodermis of the nramp3nramp4 mutant,⁴ we can now propose that in mature embryos Fe is mainly stored in the vacuoles of the endodermis.Open in a separate windowFigure 1Iron distribution in cotyledons. Wild-type and scf1 dry seed embryos were dissected and stained with Perls/DA B according to Roschzttardtz et al.⁴In conclusion, we have adapted an histochemical staining procedure, easy to set up and inexpensive, that fills the gap between the Perls reagent and X ray-based elemental imaging. The resolution, at the sub-cellular scale, makes it a valuable tool to investigate the distribution of Fe in plant tissues without employing electron microscopy or synchrotron X ray fluorescence. Furthermore, the possibility of direct staining on histological sections makes this technique applicable to virtually any plant material, after fixation and embedding in resin.     

The PAP/SAL1 retrograde signaling pathway is involved in iron homeostasis

Plant Molecular Biology - There is a link between PAP/SAL retrograde pathway, ethylene signaling and Fe metabolism in Arabidopsis. Nuclear gene expression is regulated by a diversity of retrograde...     

The Endosomal Protein CHARGED MULTIVESICULAR BODY PROTEIN1 Regulates the Autophagic Turnover of Plastids in Arabidopsis

Endosomal Sorting Complex Required for Transport (ESCRT)-III proteins mediate membrane remodeling and the release of endosomal intraluminal vesicles into multivesicular bodies. Here, we show that the ESCRT-III subunit paralogs CHARGED MULTIVESICULAR BODY PROTEIN1 (CHMP1A) and CHMP1B are required for autophagic degradation of plastid proteins in Arabidopsis thaliana. Similar to autophagy mutants, chmp1a chmp1b (chmp1) plants hyperaccumulated plastid components, including proteins involved in plastid division. The autophagy machinery directed the release of bodies containing plastid material into the cytoplasm, whereas CHMP1A and B were required for delivery of these bodies to the vacuole. Autophagy was upregulated in chmp1 as indicated by an increase in vacuolar green fluorescent protein (GFP) cleavage from the autophagic reporter GFP-ATG8. However, autophagic degradation of the stromal cargo RECA-GFP was drastically reduced in the chmp1 plants upon starvation, suggesting that CHMP1 mediates the efficient delivery of autophagic plastid cargo to the vacuole. Consistent with the compromised degradation of plastid proteins, chmp1 plastids show severe morphological defects and aberrant division. We propose that CHMP1 plays a direct role in the autophagic turnover of plastid constituents.     

The FRD3 citrate effluxer promotes iron nutrition between symplastically disconnected tissues throughout Arabidopsis development

We present data supporting a general role for FERRIC REDICTASE DEFECTIVE3 (FRD3), an efflux transporter of the efficient iron chelator citrate, in maintaining iron homeostasis throughout plant development. In addition to its well-known expression in root, we show that FRD3 is strongly expressed in Arabidopsis thaliana seed and flower. Consistently, frd3 loss-of-function mutants are defective in early germination and are almost completely sterile, both defects being rescued by iron and/or citrate supply. The frd3 fertility defect is caused by pollen abortion and is associated with the male gametophytic expression of FRD3. Iron imaging shows the presence of important deposits of iron on the surface of aborted pollen grains. This points to a role for FRD3 and citrate in proper iron nutrition of embryo and pollen. Based on the findings that iron acquisition in embryo, leaf, and pollen depends on FRD3, we propose that FRD3 mediated-citrate release in the apoplastic space represents an important process by which efficient iron nutrition is achieved between adjacent tissues lacking symplastic connections. These results reveal a physiological role for citrate in the apoplastic transport of iron throughout development, and provide a general model for multicellular organisms in the cell-to-cell transport of iron involving extracellular circulation.     

Lysine 213 and histidine 233 participate in Mn(II) binding and catalysis in Saccharomyces cerevisiae phosphoenolpyruvate carboxykinase

Saccharomyces cerevisiae phosphoenolpyruvate (PEP) carboxykinase catalyses the reversible metal-dependent formation of oxaloacetate and ATP from PEP, ADP, and CO2 and plays a key role in gluconeogenesis. This enzyme also has oxaloacetate decarboxylase and pyruvate kinase-like activities. Mutations of PEP carboxykinase have been constructed where the residues Lys213 and His233, two residues of the putative Mn2+ binding site of the enzyme, were altered. Replacement of these residues by Arg and by Gln, respectively, generated enzymes with 1.9 and 2.8 kcal/mol lower Mn2+ binding affinity. Lower PEP binding affinity was inferred for the mutated enzymes from the protection effect of PEP against urea denaturation. Kinetic studies of the altered enzymes show at least a 5000-fold reduction in V(max) for the primary reaction relative to that for the wild-type enzyme. V(max) values for the oxaloacetate decarboxylase and pyruvate kinase-like activities of PEP carboxykinase were affected to a much lesser extent in the mutated enzymes. The mutated enzymes show a decreased steady-state affinity for Mn2+ and PEP. The results are consistent with Lys213 and His233 being at the Mn2+ binding site of S. cerevisiae PEP carboxykinase and the Mn2+ affecting the PEP interaction. The different effects of mutations in V(max) for the main reaction and the secondary activities suggest different rate-limiting steps for these reactions.     

Thermal effects vary predictably across levels of organization: empirical results and theoretical basis

Thermal performance curves have provided a common framework to study the impact of temperature in biological systems. However, few generalities have emerged to date. Here, we combine an experimental approach with theoretical analyses to demonstrate that performance curves are expected to vary predictably with the levels of biological organization. We measured rates of enzymatic reactions, organismal performance and population viability in Drosophila acclimated to different thermal conditions and show that performance curves become narrower with thermal optima shifting towards lower temperatures at higher levels or organization. We then explain these results on theoretical grounds, showing that this pattern reflects the cumulative impact of asymmetric thermal effects that piles up with complexity. These results and the proposed framework are important to understand how organisms, populations and ecological communities might respond to changing thermal conditions.