Insertion of YFP at P5CS1 and AFL1 shows the potential, and potential complications, of gene tagging for functional analyses of stress-related proteins.

Crispr/CAS9-enabled homologous recombination to insert a tag in frame with an endogenous gene can circumvent difficulties such as context-dependent promoter activity that complicate analysis of gene expression and protein accumulation patterns. However, there have been few reports examining whether such gene targeting/gene tagging (GT) can alter expression of the target gene. The enzyme encoded by Δ1-pyrroline-5-carboxylate synthetase 1 (P5CS1) is key for stress-induced proline synthesis and drought resistance, yet its expression pattern and protein localisation have been difficult to assay. We used GT to insert YFP in frame with the 5' or 3' ends of the endogenous P5CS1 and At14a-Like 1 (AFL1) coding regions. Insertion at the 3' end of either gene generated homozygous lines with expression of the gene-YFP fusion indistinguishable from the wild type allele. However, for P5CS1 this occurred only after selfing and advancement to the T5 generation allowed initial homozygous lethality of the insertion to be overcome. Once this was done, the GT-generated P5CS1-YFP plants revealed new information about P5CS1 localisation and tissue-specific expression. In contrast, insertion of YFP at the 5' end of either gene blocked expression. The results demonstrate that GT can be useful for functional analyses of genes that are problematic to properly express by other means but also show that, in some cases, GT can disrupt expression of the target gene.

Transcriptome analysis of iron over-accumulating Arabidopsis genotypes uncover putative novel regulators of systemic and retrograde signaling.

On account of its competence to accept and donate electrons, iron (Fe) is an essential element across all forms of life, including plants. Maintaining Fe homeostasis requires precise orchestration of its uptake, trafficking, and translocation in order to meet the demand for Fe sinks such as plastids. Plants harboring defects in the systemic Fe transporter OPT3 (OLIGOPEPTIDE TRANSPORTER 3) display constitutive Fe deficiency responses and accumulate toxic levels of Fe in their leaves. Similarly, ectopic expression of IRONMAN (IMA) genes, encoding a family of phloem-localized signaling peptides, triggers the uptake and accumulation of Fe by inhibiting the putative Fe sensor BRUTUS. This study aims at elucidating the mechanisms operating between OPT3-mediated systemic Fe transport, activation of IMA genes in the phloem, and activation of Fe uptake in the root epidermis. Transcriptional profiling of opt3-2 mutant and IMA1/IMA3 overexpressing (IMA Ox) lines uncovered a small subset of genes that were consistently differentially expressed across all three genotypes and Fe-deficient control plants, constituting potential novel regulators of cellular Fe homeostasis. In particular, expression of the the F-box protein At1g73120 was robustly induced in all genotypes, suggesting a putative function in the posttranslational regulation of cellular Fe homeostasis. As further constituents of this module, two plastid-encoded loci that putatively produce transfer ribonucleic acid (tRNA)-derived small ribonucleic acids are possibly involved in retrograde control of root Fe uptake.

L-DOPA induces iron accumulation in roots of Ipomoea aquatica and Arabidopsis thaliana in a pH-dependent manner.

BACKGROUND: Iron deficiency is the leading cause of anemia worldwide, particularly in countries with predominant plant-based diets. Plants constitute the main source of dietary iron. Increasing their iron concentration could reduce the occurrence of anemia. The water spinach Ipomoea aquatica is consumed as a vegetable throughout Asia and tolerates high iron concentrations making it an attractive candidate for iron biofortification. L-DOPA is an allelopathic molecule secreted by some legumes. L-DOPA can trigger the expression of Fe deficiency-inducible genes, and could potentially be used as a biostimulant to increase Fe concentration. RESULTS: L-DOPA significantly affected root growth of water spinach, and triggered a massive accumulation of Fe in roots. Both effects were exacerbated when L-DOPA was dissolved in KOH, which is surprising given that L-DOPA is less stable at high pH. To check whether a higher pH could indeed increase the bioactivity of L-DOPA, we used Arabidopsis thaliana, which grows at lower pH than water spinach, and subjected the plants to L-DOPA treatments at pH 5.5 and pH 6.0, which are both within the optimal range for Arabidopsis nutrition. At pH 6.0, the root growth of Arabidopsis was more strongly inhibited than at pH 5.5. We found that at higher pH, L-DOPA oxidizes to form a melanin precipitate. CONCLUSIONS: We concluded that the oxidation of L-DOPA that we observed upon solubilization in KOH, or in nutrient solutions at slightly higher pH produces melanin-related molecules that are more potent than L-DOPA itself to trigger the primary root growth inhibition, Fe uptake and root Fe accumulation in water spinach and Arabidopsis.

Arabidopsis Micro-grafting to Study the Systemic Signaling of Nutrient Status.

Grafting enables the study of systemic signals that plants use to maintain their homeostasis at the level of the whole organism. Several protocols of Arabidopsis grafting have been published over the years. These methods are limited because they either affect the overall behavior of the plant, or their throughput is low. The method presented here is based on grafting 3- to 4-days-old seedlings directly on an agar plate, without the use of hormone or collar, and can produce consistently over a hundred grafted plants per day and operator.

Gallium maltolate shows synergism with cisplatin and activates nucleolar stress and ferroptosis in human breast carcinoma cells.

PURPOSE: Breast cancer is the most common cancer in women. Triple-negative breast cancer (TNBC) is an aggressive disease with poor outcomes. TNBC lacks effective targeted treatments, and the development of drug resistance limits the effectiveness of chemotherapy. It is crucial to identify new drugs that can enhance the efficacy of traditional chemotherapy to reduce drug resistance and side effects. METHODS: TNBC cell lines, MDA-MB-231 and Hs 578T, and a normal cell line, MCF-10 A, were included in this study. The cells were treated with gallium maltolate (GaM), and their transcriptome was analyzed. Ferroptosis and nucleolar stress markers were detected by qPCR, western blotting, fluorescence microscopy, and flow cytometry. The impairment of ribosome synthesis was evaluated by northern blotting and sucrose gradients. RESULTS: GaM triggered cell death via apoptosis and ferroptosis. In addition, GaM impaired translation and activated nucleolar stress. Cisplatin (DDP) is a chemotherapeutic agent for advanced breast cancer. While single treatment with GaM or DDP at low concentrations did not impact cell growth, co-administration enhanced cell death in TNBC but not in normal breast cells. The enhancement of ferroptosis and nucleolar stress could be observed in TNBC cell lines after co-treatment. CONCLUSIONS: These results suggest that GaM synergizes with cisplatin via activation of nucleolar stress and ferroptosis in human breast carcinoma cells. GaM is marginally toxic to normal cells but impairs the growth of TNBC cell lines. Thus, GaM has the potential to be used as a therapeutic agent against TNBC.

Medicago truncatula Yellow Stripe-Like7 encodes a peptide transporter participating in symbiotic nitrogen fixation.

Yellow Stripe-Like (YSL) proteins are a family of plant transporters that are typically involved in transition metal homeostasis. Three of the four YSL clades (I, II and IV) transport metals complexed with the non-proteinogenic amino acid nicotianamine or its derivatives. No such capability has been shown for any member of clade III, but the link between these YSLs and metal homeostasis could be masked by functional redundancy. We studied the role of the clade III YSL protein MtSYL7 in Medicago truncatula nodules. MtYSL7, which encodes a plasma membrane-bound protein, is mainly expressed in the pericycle and cortex cells of the root nodules. Yeast complementation assays revealed that MtSYL7 can transport short peptides. M. truncatula transposon insertion mutants with decreased expression of MtYSL7 had lower nitrogen fixation rates and showed reduced plant growth whether grown in symbiosis with rhizobia or not. YSL7 mutants accumulated more copper and iron in the nodules, which is likely to result from the increased expression of iron uptake and delivery genes in roots. Taken together, these data suggest that MtYSL7 plays an important role in the transition metal homeostasis of nodules and symbiotic nitrogen fixation.

Iron acquisition strategies in land plants: not so different after all.

Due to its ability to accept and donate electrons, iron (Fe) is an indispensable component of electron transport chains and a cofactor in many vital enzymes. Except for waterlogged conditions, under which the lack of oxygen prevents oxidation and precipitation of iron as Fe3+ hydroxides, the availability of iron in soils is generally far below the plant's demand for optimal growth. Plants have evolved two phylogenetically separated and elaborately regulated strategies to mobilize iron from the soil, featuring mechanisms which are thought to be mutually exclusive. However, recent studies uncovered several shared components of the two strategies, questioning the validity of the concept of mutual exclusivity. Here, we use publicly available data obtained from the model species rice (Oryza sativa) to unveil similarities and incongruities between co-expression networks derived from transcriptomic profiling of iron-deficient rice and Arabidopsis plants. This approach revealed striking similarities in the topographies of the resulting co-expression networks with relatively minor deviations in the molecular attributes of the comprised genes, which nonetheless lead to different physiological functions. The analysis also discovered several novel players that are possibly involved in the regulation plant adaptation to iron deficiency.

IRON MAN is a ubiquitous family of peptides that control iron transport in plants.

Iron (Fe) is an essential mineral nutrient that severely affects the growth, yield and nutritional quality of plants if not supplied in sufficient quantities. Here, we report that a short C-terminal amino-acid sequence consensus motif (IRON MAN; IMA) conserved across numerous, highly diverse peptides in angiosperms is essential for Fe uptake in plants. Overexpression of the IMA sequence in Arabidopsis induced Fe uptake genes in roots, causing accumulation of Fe and manganese in all plant parts including seeds. Silencing of all eight IMA genes harboured in the Arabidopsis genome abolished Fe uptake and caused severe chlorosis; increasing the Fe supply or expressing IMA1 restored the wild-type phenotype. IMA1 is predominantly expressed in the phloem, preferentially in leaves, and reciprocal grafting showed that IMA1 peptides in shoots positively regulate Fe uptake in roots. IMA homologues are highly responsive to the Fe status and functional when heterologously expressed across species. IMA constitutes a novel family of peptides that are critical for the acquisition and cellular homeostasis of Fe across land plants.

The multiple facets of root iron reduction.

The biological significance of iron (Fe) is based on its propensity to oscillate between the ferric and ferrous forms, a transition that also affects its phyto-availability in soils. With the exception of grasses, Fe3+ is unavailable to plants. Most angiosperms employ a reduction-based Fe uptake mechanism, which relies on enzymatic reduction of ferric iron as an obligatory, rate-limiting step prior to uptake. This system functions optimally in acidic soils. Calcicole plants are, however, exposed to environments that are alkaline and/or have suboptimal availability of phosphorous, conditions under which the enzymatic reduction mechanism ceases to work effectively. We propose that auxiliary, non-enzymatic Fe reduction can be of critical importance for conferring fitness to plants thriving in alkaline soils with low bioavailability of Fe and/or phosphorus.

Inventory of metal complexes circulating in plant fluids: a reliable method based on HPLC coupled with dual elemental and high-resolution molecular mass spectrometric detection.

Description of metal species in plant fluids such as xylem, phloem or related saps remains a complex challenge usually addressed either by liquid chromatography-mass spectrometry, X-ray analysis or computational prediction. To date, none of these techniques has achieved a complete and true picture of metal-containing species in plant fluids, especially for the least concentrated complexes. Here, we present a generic analytical methodology for a large-scale (> 10 metals, > 50 metal complexes) detection, identification and semiquantitative determination of metal complexes in the xylem and embryo sac liquid of the green pea, Pisum sativum. The procedure is based on direct injection using hydrophilic interaction chromatography with dual detection by elemental (inductively coupled plasma mass spectrometry) and molecular (high-resolution electrospray mass spectrometry) mass spectrometric detection. Numerous and novel complexes of iron(II), iron(III), copper(II), zinc, manganese, cobalt(II), cobalt(III), magnesium, calcium, nickel and molybdenum(IV) with several ligands including nicotianamine, citrate, malate, histidine, glutamine, aspartic acid, asparagine, phenylalanine and others are observed in pea fluids and discussed. This methodology provides a large inventory of various types of metal complexes, which is a significant asset for future biochemical and genetic studies into metal transport/homeostasis.

Iron in seeds - loading pathways and subcellular localization.

Iron (Fe) is one of the most abundant elements on earth, but its limited bioavailability poses a major constraint for agriculture and constitutes a serious problem in human health. Due to an improved understanding of the mechanisms that control Fe homeostasis in plants, major advances toward engineering biofortified crops have been made during the past decade. Examples of successful biofortification strategies are, however, still scarce and the process of Fe loading into seeds is far from being well understood in most crop species. In particular in grains where the embryo represents the main storage compartment such as legumes, increasing the seed Fe content remains a challenging task. This review aims at placing the recently identified actors in Fe transport into the unsolved puzzle of grain filling, taking the differences of Fe distribution between various species into consideration. We summarize the current knowledge on Fe transport between symplasmic and apoplasmic compartments, and provide models for Fe trafficking and localization in different seed types that may help to develop high seed Fe germplasms.

Ascorbate efflux as a new strategy for iron reduction and transport in plants.

Iron (Fe) is essential for virtually all living organisms. The identification of the chemical forms of iron (the speciation) circulating in and between cells is crucial to further understand the mechanisms of iron delivery to its final targets. Here we analyzed how iron is transported to the seeds by the chemical identification of iron complexes that are delivered to embryos, followed by the biochemical characterization of the transport of these complexes by the embryo, using the pea (Pisum sativum) as a model species. We have found that iron circulates as ferric complexes with citrate and malate (Fe(III)3Cit2Mal2, Fe(III)3Cit3Mal1, Fe(III)Cit2). Because dicotyledonous plants only transport ferrous iron, we checked whether embryos were capable of reducing iron of these complexes. Indeed, embryos did express a constitutively high ferric reduction activity. Surprisingly, iron(III) reduction is not catalyzed by the expected membrane-bound ferric reductase. Instead, embryos efflux high amounts of ascorbate that chemically reduce iron(III) from citrate-malate complexes. In vitro transport experiments on isolated embryos using radiolabeled (55)Fe demonstrated that this ascorbate-mediated reduction is an obligatory step for the uptake of iron(II). Moreover, the ascorbate efflux activity was also measured in Arabidopsis embryos, suggesting that this new iron transport system may be generic to dicotyledonous plants. Finally, in embryos of the ascorbate-deficient mutants vtc2-4, vtc5-1, and vtc5-2, the reducing activity and the iron concentration were reduced significantly. Taken together, our results identified a new iron transport mechanism in plants that could play a major role to control iron loading in seeds.

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.

Genetic and physiological analysis of the relationship between partial resistance to clubroot and tolerance to trehalose in Arabidopsis thaliana.

In Arabidopsis thaliana the induction of plant trehalase during clubroot disease was proposed to act as a defense mechanism in the susceptible accession Col-0, which could thereby cope with the accumulation of pathogen-synthesized trehalose. In the present study, we assessed trehalose activity and tolerance to trehalose in the clubroot partially resistant accession Bur-0. We compared both accessions for several trehalose-related physiological traits during clubroot infection. A quantitative trait loci (QTLs) analysis of tolerance to exogenous trehalose was also conducted on a Bur-0xCol-0 RIL progeny. Trehalase activity was not induced by clubroot in Bur-0 and the inhibition of trehalase by validamycin treatments resulted in the enhancement of clubroot symptoms only in Col-0. In pathogen-free cultures, Bur-0 showed less trehalose-induced toxicity symptoms than Col-0. A QTL analysis identified one locus involved in tolerance to trehalose overlapping the confidence interval of a QTL for resistance to Plasmodiophora brassicae. This colocalization was confirmed using heterogeneous inbred family (HIF) lines. Although not based on trehalose catabolism capacity, partial resistance to clubroot is to some extent related to the tolerance to trehalose accumulation in Bur-0. These findings support an original model where contrasting primary metabolism-related regulations could contribute to the partial resistance to a plant pathogen.