Functional characterization of the basic helix-loop-helix transcription factor LeFER essential for upregulation of iron responses in tomato roots

Iron (Fe) deficiency in humans is the most prevalent nutritional disorder in the world. Since plants serve as the primary source of dietary Fe, improving the Fe content of crops represents an important step towards a better public health. Tomato responds to low Fe availability by an enhanced proton extrusion from the root and upregulation of the genes encoding FeIII-chelate reductase (LeFRO1) and FeII transporter (LeIRT1). As a result, more Fe is rendered soluble and thus accessible for the plant. Previously, we identified the tomato gene LeFER as one of the major regulators of Fe uptake in the root under Fe-deficiency conditions. LeFER encodes a bHLH transcription factor that is responsible for the induction of Fe-mobilization genes. The aim of the presented work was to study upstream regulatory events of LeFER action, and the effects of LeFER function on the network of metabolic pathways in the cell under Fe-deficiency conditions. First, we examined the control of LeFER gene and LeFER protein expression in response to Fe-nutritional status in wild type, mutant plants with defects in Fe-uptake regulation, and 35S transgenic plants overexpressing LeFER. Both LeFER gene and LeFER protein were found consistently downregulated in roots after generous (100 µM, physiologically optimal) compared to low (0.1 µM) and sufficient (10 µM) Fe supply, and occasionally downregulated at sufficient compared to low Fe supply. Second, downregulation of LeFER by high Fe was found additionally controlled at posttranscriptional level. LeFER showed nuclear localisation and transcriptional activation in yeast. Third, LeFER protein regulation in the Fe-accumulation mutant chloronerva indicated that LeFER protein expression was not directly controlled by signals derived from Fe transport. Thus, we concluded that LeFER is able to affect transcription in the nucleus and its action is controlled by Fe supply at multiple regulatory levels. Fourth, we investigated the changes in the tomato root proteome when different plant genotypes were grown under different Fe-supply conditions (as indicated above). Using proteomics tools, differentially expressed proteins have been identified — dependent and independent on LeFER protein expression. Our data show major changes in the proteome as a result of exposure to low Fe in the medium, affecting an array of metabolic pathways ultimately involved in, among others, energy balance, stress response, and phytohormone signaling.Eisenmangel ist die häufigste Ernährungskrankheit von Menschen. Da Pflanzen die primäre Quelle von Eisen (Fe) in unserer Ernährung sind, sind Kulturpflanzen mit verbessertem Eisengehalt ein wichtiger Schritt in Richtung einer besseren Gesundheit der Bevölkerung. Tomatenpflanzen reagieren auf niedrige Eisenverfügbarkeit mit Erhöhung der FeII Konzentration in der Wurzelumgebung aufgrund der erhöhten Protonenausscheidung und der Induktion der FeIII-Chelatreduktase (LeFRO1) und des FeII Transporters (LeIRT1). Infolgedessen wird mehr Fe lösbar gemacht und steht der Pflanze zur Verfügung. Vorangegangene Arbeiten der Arbeitsgruppe haben LeFER als hauptsächliches Regulatorgen identifziert, welches die Eisenaufnahme in Wurzeln bei Eisenmangel kontrolliert. LeFER kodiert für einen Transkriptionsfaktor der basischen Helix-Loop-Helix Familie, welcher für die Induktion von Eisenmangelantworten verantwortlich ist. Ziel der vorliegenden Arbeit war es, oberhalb liegende Regulationsmechanismen von LeFER näher zu untersuchen, und die Auswirkungen der LeFER Funktion auf das Netzwerk von metabolischen Wegen in der Zelle unter Eisenmangel zu untersuchen. Zuerst untersuchten wir die Kontrolle der Expression des LeFER Gens und LeFER Proteins als Antwort auf den Eisenhaushalt in Wildtyp, Mutanten mit Defekten der Eisenaufnahmeregulation, und in 35S transgenen Pflanzen, welche LeFER überexprimieren. LeFER mRNA und LeFER Protein waren herunterreguliert in Wurzeln, die großzügig mit Fe versorgt waren (100 µM, physiologisch optimal) verglichen mit Wurzeln, die normal oder unterversorgt waren mit Fe (10 µM, 0.1 µM). Gelegentlich war eine niedrige Expression auch bei normaler Eisenversorgung zu sehen. Zweitens, wir haben gefunden, dass LeFER zusätzlich auf posttranskriptioneller Ebene herunterreguliert war durch viel Fe. LeFER zeigte Zellkernlokalisation und transkriptionelle Aktivierung in Hefe. Drittens, die LeFER Proteinregulation in der Eisenakkumulationsmutante chloronerva zeigte, dass LeFER Proteinexpression nicht direkt durch Signale des Eisentransports reguliert war. Wir schlussfolgerten, dass LeFER die Transkription im Zellkern beeinflusst, und seine Aktivität durch die Eisenversorgung auf verschiedenen Ebenen reguliert wird. Viertens, wir untersuchten Veränderungen im Tomate Wurzelproteom, wenn Pflanzen mit unterschiedlichen Genotypen verschiedenen Eisenbedingungen ausgesetzt waren. Mit Proteomics Werkzeugen haben wir Proteine identifiziert, welche abhängig oder unabhängig von LeFER differentiell durch Fe exprimiert waren. Unsere Daten zeigten, dass große Veränderungen im Wurzelproteom als Antwort auf Eisenmangel auftreten, welche eine Reihe von metabolischen Wegen beeinflussten, die mit Energieversorgung, Stressantworten und Phytohormonsignalwegen in Verbindung stehen

SEC14-GOLD protein PATELLIN2 binds IRON-REGULATED TRANSPORTER1 linking root iron uptake to vitamin E

Organisms require micronutrients, and Arabidopsis (Arabidopsis thaliana) IRON-REGULATED TRANSPORTER1 (IRT1) is essential for iron (Fe2+) acquisition into root cells. Uptake of reactive Fe2+ exposes cells to the risk of membrane lipid peroxidation. Surprisingly little is known about how this is avoided. IRT1 activity is controlled by an intracellular variable region (IRT1vr) that acts as a regulatory protein interaction platform. Here, we describe that IRT1vr interacted with peripheral plasma membrane SEC14-Golgi dynamics (SEC14-GOLD) protein PATELLIN2 (PATL2). SEC14 proteins bind lipophilic substrates and transport or present them at the membrane. To date, no direct roles have been attributed to SEC14 proteins in Fe import. PATL2 affected root Fe acquisition responses, interacted with ROS response proteins in roots, and alleviated root lipid peroxidation. PATL2 had high affinity in vitro for the major lipophilic antioxidant vitamin E compound α-tocopherol. Molecular dynamics simulations provided insight into energetic constraints and the orientation and stability of the PATL2-ligand interaction in atomic detail. Hence, this work highlights a compelling mechanism connecting vitamin E with root metal ion transport at the plasma membrane with the participation of an IRT1-interacting and α-tocopherol-binding SEC14 protein

SEC14-GOLD protein PATELLIN2 binds IRON-REGULATED TRANSPORTER1 linking root iron uptake to vitamin E

Organisms require micronutrients, and Arabidopsis (Arabidopsis thaliana) IRON-REGULATED TRANSPORTER1 (IRT1) is essential for iron (Fe2+) acquisition into root cells. Uptake of reactive Fe2+ exposes cells to the risk of membrane lipid peroxidation. Surprisingly little is known about how this is avoided. IRT1 activity is controlled by an intracellular variable region (IRT1vr) that acts as a regulatory protein interaction platform. Here, we describe that IRT1vr interacted with peripheral plasma membrane SEC14-Golgi dynamics (SEC14-GOLD) protein PATELLIN2 (PATL2). SEC14 proteins bind lipophilic substrates and transport or present them at the membrane. To date, no direct roles have been attributed to SEC14 proteins in Fe import. PATL2 affected root Fe acquisition responses, interacted with ROS response proteins in roots, and alleviated root lipid peroxidation. PATL2 had high affinity in vitro for the major lipophilic antioxidant vitamin E compound α-tocopherol. Molecular dynamics simulations provided insight into energetic constraints and the orientation and stability of the PATL2-ligand interaction in atomic detail. Hence, this work highlights a compelling mechanism connecting vitamin E with root metal ion transport at the plasma membrane with the participation of an IRT1-interacting and α-tocopherol-binding SEC14 protein

Effects of metal-contaminated soils on the accumulation of heavy metals in gotu kola (Centella asiatica) and the potential health risks: a study in Peninsular Malaysia

Centella asiatica is a commonly used medicinal plant in Malaysia. As heavy metal accumulation in medicinal plants which are highly consumed by human is a serious issue, thus the assessment of heavy metals in C. asiatica is important for the safety of consumers. In this study, the heavy metal accumulation in C. asiatica and the potential health risks were investigated. Samples of C. asiatica and surface soils were collected from nine different sites around Peninsular Malaysia. The concentration of six heavy metals namely Cd, Cu, Ni, Fe, Pb and Zn were determined by air-acetylene flame atomic absorption spectrophotometer (AAS). The degree of anthropogenic influence was assessed by calculating the enrichment factor (EF) and index of geoaccumulation (Igeo). The heavy metal uptake into the plant was estimated through the calculation of translocation factor (TF), bioconcentration factor (BCF) and correlation study. Estimated daily intakes (EDI) and target hazard quotients (THQ) were used to determine the potential health risk of consuming C. asiatica. The results showed that the overall surface soil was polluted by Cd, Cu and Pb, while the uptake of Zn and Ni by the plants was high. The value of EDI and THQ showed that the potential of Pb toxicity in C. asiatica was high as well. As heavy metal accumulation was confirmed in C. asiatica, daily consumption of the plant derived from polluted sites in Malaysia was not recommended

Stress response of lettuce (Lactuca sativa) to environmental contamination with selected pharmaceuticals: A proteomic study

Pharmaceutical compounds have been found in rivers and treated wastewaters. They often contaminate irrigation waters and consequently accumulate in edible vegetables, causing changes in plants metabolism The main objective of this work is to understand how lettuce plants cope with the contamination from three selected pharmaceuticals using a label free proteomic analysis. A lettuce hydroponic culture, grown for 36 days, was exposed to metformin, acetaminophen and carbamazepine (at 1 mg/L), during 8 days, after which roots and leaves were sampled and analysed using a liquid chromatography-mass spectrometry proteomics-based approach. In roots, a total of 612 proteins showed differentially accumulation while in leaves 237 proteins were identified with significant differences over controls. Carbamazepine was the contaminant that most affected protein abundance in roots, while in leaves the highest number of differentially accumulated proteins was observed for acetaminophen. In roots under carbamazepine, stress related protein species such as catalase, superoxide dismutase and peroxidases presented higher abundance. Ascorbate peroxidase increased in roots under metformin. Cell respiration protein species were affected by the presence of the three pharmaceuticals suggesting possible dysregulation of the Krebs cycle. Acetaminophen caused the main differences in respiration pathways, with more emphasis in leaves. Lettuce plants revealed different tolerance levels when contaminants were compared, being more tolerant to metformin presence and less tolerant to carbamazepineinfo:eu-repo/semantics/acceptedVersio

Funktionelle Charakterisierung des für die Up-Regulation der Eisenantworten in Tomatenwurzeln essentiellen basischen Helix-Loop-Helix-Transkriptionsfaktors LeFER

Iron (Fe) deficiency in humans is the most prevalent nutritional disorder in the world. Since plants serve as the primary source of dietary Fe, improving the Fe content of crops represents an important step towards a better public health. Tomato responds to low Fe availability by an enhanced proton extrusion from the root and upregulation of the genes encoding FeIII-chelate reductase (LeFRO1) and FeII transporter (LeIRT1). As a result, more Fe is rendered soluble and thus accessible for the plant. Previously, we identified the tomato gene LeFER as one of the major regulators of Fe uptake in the root under Fe-deficiency conditions. LeFER encodes a bHLH transcription factor that is responsible for the induction of Fe-mobilization genes. The aim of the presented work was to study upstream regulatory events of LeFER action, and the effects of LeFER function on the network of metabolic pathways in the cell under Fe-deficiency conditions. First, we examined the control of LeFER gene and LeFER protein expression in response to Fe-nutritional status in wild type, mutant plants with defects in Fe-uptake regulation, and 35S transgenic plants overexpressing LeFER. Both LeFER gene and LeFER protein were found consistently downregulated in roots after generous (100 µM, physiologically optimal) compared to low (0.1 µM) and sufficient (10 µM) Fe supply, and occasionally downregulated at sufficient compared to low Fe supply. Second, downregulation of LeFER by high Fe was found additionally controlled at posttranscriptional level. LeFER showed nuclear localisation and transcriptional activation in yeast. Third, LeFER protein regulation in the Fe-accumulation mutant chloronerva indicated that LeFER protein expression was not directly controlled by signals derived from Fe transport. Thus, we concluded that LeFER is able to affect transcription in the nucleus and its action is controlled by Fe supply at multiple regulatory levels. Fourth, we investigated the changes in the tomato root proteome when different plant genotypes were grown under different Fe-supply conditions (as indicated above). Using proteomics tools, differentially expressed proteins have been identified — dependent and independent on LeFER protein expression. Our data show major changes in the proteome as a result of exposure to low Fe in the medium, affecting an array of metabolic pathways ultimately involved in, among others, energy balance, stress response, and phytohormone signaling.Eisenmangel ist die häufigste Ernährungskrankheit von Menschen. Da Pflanzen die primäre Quelle von Eisen (Fe) in unserer Ernährung sind, sind Kulturpflanzen mit verbessertem Eisengehalt ein wichtiger Schritt in Richtung einer besseren Gesundheit der Bevölkerung. Tomatenpflanzen reagieren auf niedrige Eisenverfügbarkeit mit Erhöhung der FeII Konzentration in der Wurzelumgebung aufgrund der erhöhten Protonenausscheidung und der Induktion der FeIII-Chelatreduktase (LeFRO1) und des FeII Transporters (LeIRT1). Infolgedessen wird mehr Fe lösbar gemacht und steht der Pflanze zur Verfügung. Vorangegangene Arbeiten der Arbeitsgruppe haben LeFER als hauptsächliches Regulatorgen identifziert, welches die Eisenaufnahme in Wurzeln bei Eisenmangel kontrolliert. LeFER kodiert für einen Transkriptionsfaktor der basischen Helix-Loop-Helix Familie, welcher für die Induktion von Eisenmangelantworten verantwortlich ist. Ziel der vorliegenden Arbeit war es, oberhalb liegende Regulationsmechanismen von LeFER näher zu untersuchen, und die Auswirkungen der LeFER Funktion auf das Netzwerk von metabolischen Wegen in der Zelle unter Eisenmangel zu untersuchen. Zuerst untersuchten wir die Kontrolle der Expression des LeFER Gens und LeFER Proteins als Antwort auf den Eisenhaushalt in Wildtyp, Mutanten mit Defekten der Eisenaufnahmeregulation, und in 35S transgenen Pflanzen, welche LeFER überexprimieren. LeFER mRNA und LeFER Protein waren herunterreguliert in Wurzeln, die großzügig mit Fe versorgt waren (100 µM, physiologisch optimal) verglichen mit Wurzeln, die normal oder unterversorgt waren mit Fe (10 µM, 0.1 µM). Gelegentlich war eine niedrige Expression auch bei normaler Eisenversorgung zu sehen. Zweitens, wir haben gefunden, dass LeFER zusätzlich auf posttranskriptioneller Ebene herunterreguliert war durch viel Fe. LeFER zeigte Zellkernlokalisation und transkriptionelle Aktivierung in Hefe. Drittens, die LeFER Proteinregulation in der Eisenakkumulationsmutante chloronerva zeigte, dass LeFER Proteinexpression nicht direkt durch Signale des Eisentransports reguliert war. Wir schlussfolgerten, dass LeFER die Transkription im Zellkern beeinflusst, und seine Aktivität durch die Eisenversorgung auf verschiedenen Ebenen reguliert wird. Viertens, wir untersuchten Veränderungen im Tomate Wurzelproteom, wenn Pflanzen mit unterschiedlichen Genotypen verschiedenen Eisenbedingungen ausgesetzt waren. Mit Proteomics Werkzeugen haben wir Proteine identifiziert, welche abhängig oder unabhängig von LeFER differentiell durch Fe exprimiert waren. Unsere Daten zeigten, dass große Veränderungen im Wurzelproteom als Antwort auf Eisenmangel auftreten, welche eine Reihe von metabolischen Wegen beeinflussten, die mit Energieversorgung, Stressantworten und Phytohormonsignalwegen in Verbindung stehen

Iron-Mediated Control of the Basic Helix-Loop-Helix Protein FER, a Regulator of Iron Uptake in Tomato

Root iron mobilization genes are induced by iron deficiency downstream of an unknown signaling mechanism. The FER gene, encoding a basic helix-loop-helix domain protein and putative transcription factor, is required for induction of iron mobilization genes in roots of tomato (Lycopersicon esculentum). To study upstream regulatory events of FER action, we examined the control of FER gene and FER protein expression in response to iron nutritional status. We analyzed expression of the FER gene and FER protein in wild-type plants, in mutant plants with defects in iron uptake regulation, and in 35S transgenic plants that overexpressed the FER gene. An affinity-purified antiserum directed against FER epitopes was produced that recognized FER protein in plant protein extracts. We found that the FER gene and FER protein were consistently down-regulated in roots after generous (100 μm, physiologically optimal) iron supply compared to low (0.1 μm) and sufficient (10 μm) iron supply. FER gene and FER protein expression were also occasionally down-regulated at sufficient compared to low iron supply. Analysis of FER protein expression in FER overexpression plants, as well as cellular protein localization studies, indicated that FER was down-regulated by high iron at the posttranscriptional level. The FER protein was targeted to plant nuclei and showed transcriptional activation in yeast (Saccharomyces cerevisiae). FER protein regulation in the iron accumulation mutant chloronerva indicated that FER protein expression was not directly controlled by signals derived from iron transport. We conclude that FER is able to affect transcription in the nucleus and its action is controlled by iron supply at multiple regulatory levels

Regulation of ZAT12 protein stability: The role of hydrogen peroxide

Signaling mediated by reactive oxygen species (ROS) has emerged as a key component of plants' responses to environmental stress. The ROS-regulated transcription factor ZAT12 was revealed as a negative regulator of iron (Fe) deficiency responses through its direct interaction with the bHLH protein FIT. In the epidermis of the early root differentiation zone, ZAT12 stability depended on the presence of the ZAT12 EAR motif. It was concluded that ZAT12 may be the target of 2 alternative degradation pathways. Here, we present a model aiming to explain the regulatory mechanisms by which ZAT12 could be targeted for degradation and to predict the types of potential regulators involved. In addition to an E3 ubiquitin ligase, we predict 2 critical regulatory factors, namely a protein interacting with the ZAT12 EAR motif and a ROS-responsive regulatory protein