Foliar Application of Melatonin Improves the Salt Tolerance, Ion and Redox Homeostasis and Seed Oil Fatty Acid Profile in Camelina sativa.

Salinity affects the yield and quality of oilseed crops. The effects of a single foliar application of solutions with different concentrations (0, 30, 60 or 90 µM) of melatonin (MEL) to camelina (Camelina sativa) plants grown in soil in a greenhouse and irrigated at four salinity levels (0.5, 4, 8 and 16 dS m-1) were assessed. Increasing salinity decreased leaf chlorophyll and photosynthetic rates, decreased K concentrations and increased Na concentrations in roots and shoots, and increased oxidative marker levels and the activity of protective antioxidant enzymes in leaves. Under severe salinity stress, the MEL90 treatment resulted in increases in chlorophyll, gas exchange attributes, leaf antioxidant enzyme activities, and decreases in leaf oxidative markers and Na. Salinity decreased seed yield, with no seeds being produced at salinities above 8 dS m-1. The MEL90 treatment resulted in increases in seed yield and poly- and mono-unsaturated fatty acid contents and decreases in saturated fatty acid contents. The MEL90 treatment was more effective in alleviating salinity effects than those including lower MEL concentrations. The highest concentrations of K and K/Na ratios were observed with the MEL90 treatment under non-stressed conditions. Data suggest that MEL foliar applications could increase salinity stress tolerance in camelina.

Iron deficient Medicago scutellata grown in nutrient solution at high pH accumulates and secretes large amounts of flavins.

Flavin synthesis and secretion is an integral part of the toolbox of root-borne Fe facilitators used by Strategy I species upon Fe deficiency. The Fe-deficiency responses of the wild legume Medicago scutellata grown in nutrient solution have been studied at two different pH values (5.5 and 7.5). Parameters studied include leaf chlorophyll, nutrient solution pH, concentrations and contents of micronutrients, flavin accumulation in roots, flavin export to the medium, and root ferric chelate reductase and acidification activities. Results show that M. scutellata behaves upon Fe deficiency as a Strategy I species, with a marked capacity for synthesizing flavins (riboflavin and three hydroxylated riboflavin derivatives), which becomes more intense at high pH. Results also show that this species is capable of exporting a large amount of flavins to the external medium, both at pH 5.5 and 7.5. This is the first report of a species having a major flavin secretion at pH 7.5, in contrast with the very low flavin secretion found in other flavin-producing species such as Beta vulgaris and M. truncatula. These results provide further support to the hypothesis that flavin secretion is relevant for Fe acquisition at high pH, and open the possibility to improve the Fe-efficiency responses in legumes of agronomic interest.

Chloroplasts preferentially take up ferric-citrate over iron-nicotianamine complexes in Brassica napus.

MAIN CONCLUSION: Fe uptake machinery of chloroplasts prefers to utilise Fe(III)-citrate over Fe-nicotianamine complexes. Iron uptake in chloroplasts is a process of prime importance. Although a few members of their iron transport machinery were identified, the substrate preference of the system is still unknown. Intact chloroplasts of oilseed rape (Brassica napus) were purified and subjected to iron uptake studies using natural and artificial iron complexes. Fe-nicotianamine (NA) complexes were characterised by 5 K, 5 T Mössbauer spectrometry. Expression of components of the chloroplast Fe uptake machinery was also studied. Fe(III)-NA contained a minor paramagnetic Fe(II) component (ca. 9%), a paramagnetic Fe(III) component exhibiting dimeric or oligomeric structure (ca. 20%), and a Fe(III) complex, likely being a monomeric structure, which undergoes slow electronic relaxation at 5 K (ca. 61%). Fe(II)-NA contained more than one similar chemical Fe(II) environment with no sign of Fe(III) components. Chloroplasts preferred Fe(III)-citrate compared to Fe(III)-NA and Fe(II)-NA, but also to Fe(III)-EDTA and Fe(III)-o,o'EDDHA, and the Km value was lower for Fe(III)-citrate than for the Fe-NA complexes. Only the uptake of Fe(III)-citrate was light-dependent. Regarding the components of the chloroplast Fe uptake system, only genes of the reduction-based Fe uptake system showed high expression. Chloroplasts more effectively utilize Fe(III)-citrate, but hardly Fe-NA complexes in Fe uptake.

Effect of drought stress on growth parameters, osmolyte contents, antioxidant enzymes and glycyrrhizin synthesis in licorice (Glycyrrhiza glabra L.) grown in the field.

Glycyrrhiza glabra L. (licorice) is a medicinal species rich in the specialised plant metabolite glycyrrhizin. It has been previously proposed that drought, which is increasing in importance due to the climatic change and scarcity of water resources, can promote the synthesis of glycyrrhizin. The effects of slight, moderate and intense drought (70, 35 and 23% of the regular irrigation, respectively) on growth parameters, osmolyte content, oxidative stress markers, antioxidant enzymes, glycyrrhizin biosynthesis genes and root glycyrrhizin concentration and contents, have been assessed in five Iranian licorice genotypes grown in the field. Drought decreased progressively biomass and leaf relative water contents, and increased progressively osmolyte (proline, glycine-betaine and soluble sugars) concentrations in leaves and roots. Drought caused oxidative stress in leaves, as indicated by lipid peroxidation and hydrogen peroxide concentrations, and increased the activities of antioxidant enzymes in leaf extracts (catalase, peroxidase, superoxide dismutase and pholyphenoloxidase). Drought promoted the synthesis of glycyrrhizin, as indicated by the increases in the expression of the glycyrrhizin biosynthesis pathway genes SQS1, SQS2, bAS, CYP88D6, CYP72A154 and UGT73, and increased the root concentrations of glycyrrhizin with drought in some genotypes. However, the large decreases in root biomass caused by drought led to general decreases in the amount of glycyrrhizin per plant with moderate and intense drought, whereas the slight drought treatment led to significant decreases in glycyrrhizin content in only one genotype. Under intense drought two of the genotypes were still capable to maintain half of the control glycyrrhizin yield, whereas in the other three genotypes glycyrrhizin yield was 22-33% of the control values. Results indicate that under intense drought, with only 23% of the normal water dose being applied, an appropriate choice of genotype can still lead to acceptable glycyrrhizin yields.

Iron and Zinc in the Embryo and Endosperm of Rice (Oryza sativa L.) Seeds in Contrasting 2'-Deoxymugineic Acid/Nicotianamine Scenarios.

Iron and Zn deficiencies are worldwide nutritional disorders that can be alleviated by increasing the metal concentration of rice (Oryza sativa L.) grains via bio-fortification approaches. The overproduction of the metal chelator nicotianamine (NA) is among the most effective ones, but it is still unclear whether this is due to the enrichment in NA itself and/or the concomitant enrichment in the NA derivative 2'-deoxymugineic acid (DMA). The endosperm is the most commonly consumed portion of the rice grain and mediates the transfer of nutrients from vegetative tissues to the metal rich embryo. The impact of contrasting levels of DMA and NA on the metal distribution in the embryo and endosperm of rice seeds has been assessed using wild-type rice and six different transgenic lines overexpressing nicotianamine synthase (OsNAS1) and/or barley nicotianamine amino transferase (HvNAATb). These transgenic lines outlined three different DMA/NA scenarios: (i) in a first scenario, an enhanced NA level (via overexpression of OsNAS1) would not be fully depleted because of a limited capacity to use NA for DMA synthesis (lack of -or low- expression of HvNAATb), and results in consistent enrichments in NA, DMA, Fe and Zn in the endosperm and NA, DMA and Fe in the embryo; (ii) in a second scenario, an enhanced NA level (via overexpression of OsNAS1) would be depleted by an enhanced capacity to use NA for DMA synthesis (via expression of HvNAATb), and results in enrichments only for DMA and Fe, both in the endosperm and embryo, and (iii) in a third scenario, the lack of sufficient NA replenishment would limit DMA synthesis, in spite of the enhanced capacity to use NA for this purpose (via expression of HvNAATb), and results in decreases in NA, variable changes in DMA and moderate decreases in Fe in the embryo and endosperm. Also, quantitative LA-ICP-MS metal map images of the embryo structures show that the first and second scenarios altered local distributions of Fe, and to a lesser extent of Zn. The roles of DMA/NA levels in the transport of Fe and Zn within the embryo are thoroughly discussed.

The Nicotiana tabacum ABC transporter NtPDR3 secretes O-methylated coumarins in response to iron deficiency.

Although iron is present in large amounts in the soil, its poor solubility means that plants have to use various strategies to facilitate its uptake. In this study, we show that expression of NtPDR3/NtABCG3, a Nicotiana tabacum plasma-membrane ABC transporter in the pleiotropic drug resistance (PDR) subfamily, is strongly induced in the root epidermis under iron deficiency conditions. Prevention of NtPDR3 expression resulted in N. tabacum plants that were less tolerant to iron-deficient conditions, displaying stronger chlorosis and slower growth than those of the wild-type when not supplied with iron. Metabolic profiling of roots and root exudates revealed that, upon iron deficiency, secretion of catechol-bearing O-methylated coumarins such as fraxetin, hydroxyfraxetin, and methoxyfraxetin to the rhizosphere was compromised in NtPDR3-silenced plants. However, exudation of flavins such as riboflavin was not markedly affected by NtPDR3-silencing. Expression of NtPDR3 in N. tabacum Bright Yellow-2 (BY-2) cells resulted in altered intra- and extracellular coumarin pools, supporting coumarin transport by this transporter. The results demonstrate that N. tabacum secretes both coumarins and flavins in response to iron deficiency and that NtPDR3 plays an essential role in the plant response to iron deficiency by mediating secretion of O-methylated coumarins to the rhizosphere.

Phytosiderophores determine thresholds for iron and zinc accumulation in biofortified rice endosperm while inhibiting the accumulation of cadmium.

Nicotianamine (NA) and 2'-deoxymugenic acid (DMA) are metal-chelating ligands that promote the accumulation of metals in rice endosperm, but it is unclear how these phytosiderophores regulate the levels of different metals and limit their accumulation. In this study, transgenic rice plants producing high levels of NA and DMA accumulated up to 4-fold more iron (Fe) and 2-fold more zinc (Zn) in the endosperm compared with wild-type plants. The distribution of Fe and Zn in vegetative tissues suggested that both metals are sequestered as a buffering mechanism to avoid overloading the seeds. The buffering mechanism involves the modulation of genes encoding metal transporters in the roots and aboveground vegetative tissues. As well as accumulating more Fe and Zn, the endosperm of the transgenic plants accumulated less cadmium (Cd), suggesting that higher levels of Fe and Zn competitively inhibit Cd accumulation. Our data show that although there is a strict upper limit for Fe (~22.5 µg g-1 dry weight) and Zn (~84 µg g-1 dry weight) accumulation in the endosperm, the careful selection of strategies to increase endosperm loading with essential minerals can also limit the accumulation of toxic metals such as Cd, thus further increasing the nutritional value of rice.

Accumulation and Secretion of Coumarinolignans and other Coumarins in Arabidopsis thaliana Roots in Response to Iron Deficiency at High pH.

Root secretion of coumarin-phenolic type compounds has been recently shown to be related to Arabidopsis thaliana tolerance to Fe deficiency at high pH. Previous studies revealed the identity of a few simple coumarins occurring in roots and exudates of Fe-deficient A. thaliana plants, and left open the possible existence of other unknown phenolics. We used HPLC-UV/VIS/ESI-MS(TOF), HPLC/ESI-MS(ion trap) and HPLC/ESI-MS(Q-TOF) to characterize (identify and quantify) phenolic-type compounds accumulated in roots or secreted into the nutrient solution of A. thaliana plants in response to Fe deficiency. Plants grown with or without Fe and using nutrient solutions buffered at pH 5.5 or 7.5 enabled to identify an array of phenolics. These include several coumarinolignans not previously reported in A. thaliana (cleomiscosins A, B, C, and D and the 5'-hydroxycleomiscosins A and/or B), as well as some coumarin precursors (ferulic acid and coniferyl and sinapyl aldehydes), and previously reported cathecol (fraxetin) and non-cathecol coumarins (scopoletin, isofraxidin and fraxinol), some of them in hexoside forms not previously characterized. The production and secretion of phenolics were more intense when the plant accessibility to Fe was diminished and the plant Fe status deteriorated, as it occurs when plants are grown in the absence of Fe at pH 7.5. Aglycones and hexosides of the four coumarins were abundant in roots, whereas only the aglycone forms could be quantified in the nutrient solution. A comprehensive quantification of coumarins, first carried out in this study, revealed that the catechol coumarin fraxetin was predominant in exudates (but not in roots) of Fe-deficient A. thaliana plants grown at pH 7.5. Also, fraxetin was able to mobilize efficiently Fe from a Fe(III)-oxide at pH 5.5 and pH 7.5. On the other hand, non-catechol coumarins were much less efficient in mobilizing Fe and were present in much lower concentrations, making unlikely that they could play a role in Fe mobilization. The structural features of the array of coumarin type-compounds produced suggest some can mobilize Fe from the soil and others can be more efficient as allelochemicals.

Does a voltage-sensitive outer envelope transport mechanism contributes to the chloroplast iron uptake?

MAIN CONCLUSION: Based on the effects of inorganic salts on chloroplast Fe uptake, the presence of a voltage-dependent step is proposed to play a role in Fe uptake through the outer envelope. Although iron (Fe) plays a crucial role in chloroplast physiology, only few pieces of information are available on the mechanisms of chloroplast Fe acquisition. Here, the effect of inorganic salts on the Fe uptake of intact chloroplasts was tested, assessing Fe and transition metal uptake using bathophenantroline-based spectrophotometric detection and plasma emission-coupled mass spectrometry, respectively. The microenvironment of Fe was studied by Mössbauer spectroscopy. Transition metal cations (Cd2+, Zn2+, and Mn2+) enhanced, whereas oxoanions (NO3-, SO42-, and BO33-) reduced the chloroplast Fe uptake. The effect was insensitive to diuron (DCMU), an inhibitor of chloroplast inner envelope-associated Fe uptake. The inorganic salts affected neither Fe forms in the uptake assay buffer nor those incorporated into the chloroplasts. The significantly lower Zn and Mn uptake compared to that of Fe indicates that different mechanisms/transporters are involved in their acquisition. The enhancing effect of transition metals on chloroplast Fe uptake is likely related to outer envelope-associated processes, since divalent metal cations are known to inhibit Fe2+ transport across the inner envelope. Thus, a voltage-dependent step is proposed to play a role in Fe uptake through the chloroplast outer envelope on the basis of the contrasting effects of transition metal cations and oxoaninons.

Using Perls Staining to Trace the Iron Uptake Pathway in Leaves of a Prunus Rootstock Treated with Iron Foliar Fertilizers.

The aim of this study was to trace the Fe uptake pathway in leaves of Prunus rootstock (GF 677; Prunus dulcis × Prunus persica) plants treated with foliar Fe compounds using the Perls blue method, which detects labile Fe pools. Young expanded leaves of Fe-deficient plants grown in nutrient solution were treated with Fe-compounds using a brush. Iron compounds used were the ferrous salt FeSO4, the ferric salts Fe2(SO4)3 and FeCl3, and the chelate Fe(III)-EDTA, all of them at concentrations of 9 mM Fe. Leaf Fe concentration increases were measured at 30, 60, 90 min, and 24 h, and 70 µm-thick leaf transversal sections were obtained with a vibrating microtome and stained with Perls blue. In vitro results show that the Perls blue method is a good tool to trace the Fe uptake pathway in leaves when using Fe salts, but is not sensitive enough when using synthetic Fe(III)-chelates such as Fe(III)-EDTA and Fe(III)-IDHA. Foliar Fe fertilization increased leaf Fe concentrations with all Fe compounds used, with inorganic Fe salts causing larger leaf Fe concentration increases than Fe(III)-EDTA. Results show that Perls blue stain appeared within 30 min in the stomatal areas, indicating that Fe applied as inorganic salts was taken up rapidly via stomata. In the case of using FeSO4 a progression of the stain was seen with time toward vascular areas in the leaf blade and the central vein, whereas in the case of Fe(III) salts the stain mainly remained in the stomatal areas. Perls stain was never observed in the mesophyll areas, possibly due to the low concentration of labile Fe pools.

Flavins secreted by roots of iron-deficient Beta vulgaris enable mining of ferric oxide via reductive mechanisms.

Iron (Fe) is abundant in soils but generally poorly soluble. Plants, with the exception of Graminaceae, take up Fe using an Fe(III)-chelate reductase coupled to an Fe(II) transporter. Whether or not nongraminaceous species can convert scarcely soluble Fe(III) forms into soluble Fe forms has deserved little attention so far. We have used Beta vulgaris, one among the many species whose roots secrete flavins upon Fe deficiency, to study whether or not flavins are involved in Fe acquisition. Flavins secreted by Fe-deficient plants were removed from the nutrient solution, and plants were compared with Fe-sufficient plants and Fe-deficient plants without flavin removal. Solubilization of a scarcely soluble Fe(III)-oxide was assessed in the presence or absence of flavins, NADH (nicotinamide adenine dinucleotide, reduced form) or plant roots, and an Fe(II) trapping agent. The removal of flavins from the nutrient solution aggravated the Fe deficiency-induced leaf chlorosis. Flavins were able to dissolve an Fe(III)-oxide in the presence of NADH. The addition of extracellular flavins enabled roots of Fe-deficient plants to reductively dissolve an Fe(III)-oxide. We concluded that root-secretion of flavins improves Fe nutrition in B. vulgaris. Flavins allow B. vulgaris roots to mine Fe from Fe(III)-oxides via reductive mechanisms.

The effects of foliar fertilization with iron sulfate in chlorotic leaves are limited to the treated area. A study with peach trees (Prunus persica L. Batsch) grown in the field and sugar beet (Beta vulgaris L.) grown in hydroponics.

Crop Fe deficiency is a worldwide problem. The aim of this study was to assess the effects of foliar Fe applications in two species grown in different environments: peach (Prunus persica L. Batsch) trees grown in the field and sugar beet (Beta vulgaris L. cv. "Orbis") grown in hydroponics. The distal half of Fe-deficient, chlorotic leaves was treated with Fe sulfate by dipping and using a brush in peach trees and sugar beet plants, respectively. The re-greening of the distal (Fe-treated) and basal (untreated) leaf areas was monitored, and the nutrient and photosynthetic pigment composition of the two areas were also determined. Leaves were also studied using chlorophyll fluorescence imaging, low temperature-scanning electron microscopy microanalysis, scanning transmission ion microscopy-particle induced X-ray emission and Perls Fe staining. The distal, Fe-treated leaf parts of both species showed a significant increase in Fe concentrations (across the whole leaf volume) and marked re-greening, with significant increases in the concentrations of all photosynthetic pigments, as well as decreases in de-epoxidation of xanthophyll cycle carotenoids and increases in photochemical efficiency. In the basal, untreated leaf parts, Fe concentrations increased slightly, but little re-greening occurred. No changes in the concentrations of other nutrients were found. Foliar Fe fertilization was effective in re-greening treated leaf areas both in peach trees and sugar beet plants. Results indicate that the effects of foliar Fe-sulfate fertilization in Fe-deficient, chlorotic leaves were minor outside the leaf surface treated, indicating that Fe mobility within the leaf is a major constraint for full fertilizer effectiveness in crops where Fe-deficiency is established and leaf chlorosis occurs.

The role of glutathione in mercury tolerance resembles its function under cadmium stress in Arabidopsis.

Recent research efforts have highlighted the importance of glutathione (GSH) as a key antioxidant metabolite for metal tolerance in plants. Little is known about the mechanisms involved in stress due to mercury (Hg), one of the most hazardous metals to the environment and human health. To understand the implication of GSH metabolism for Hg tolerance, we used two γ-glutamylcysteine synthetase (γECS) Arabidopsis thaliana allele mutants (rax1-1 and cad2-1) and a phytochelatin synthase (PCS) mutant (cad1-3). The leaves of these mutants and of wild type (Col-0) were infiltrated with a solution containing Cd or Hg (0, 3 and 30 µM) and incubated for 24 and 48 h. The formation of phytochelatins (PCs) in the leaf extracts was followed by two different HPLC-based methods and occurred in Col-0, cad2-1 and rax1-1 plants exposed to Cd, whereas in the Hg treatments, PCs accumulated mainly in Col-0 and rax1-1, where Hg-PC complexes were also detected. ASA and GSH/GSSG levels increased under moderate metal stress conditions, accompanied by increased GSH reductase (GR) activity and expression. However, higher metal doses led to a decrease in the analysed parameters, and stronger toxic effects appeared with 30 µM Hg. The GSH concentration was significantly higher in rax1-1 (70% of Col-0) than in cad2-1 (40% of Col-0). The leaves of rax1-1 were less sensitive than cad2-1, in accordance with the greater expression of γECS in rax1-1. Our results underline the existence of a minimal GSH concentration threshold needed to minimise the toxic effects exerted by Hg.

Involvement of the ABCG37 transporter in secretion of scopoletin and derivatives by Arabidopsis roots in response to iron deficiency.

Studies of Iron (Fe) uptake mechanisms by plant roots have focussed on Fe(III)-siderophores or Fe(II) transport systems. Iron deficency also enhances root secretion of flavins and phenolics. However, the nature of these compounds, their transport outside the roots and their role in Fe nutrition are largely unknown. We used HPLC/ESI-MS (TOF) and HPLC/ESI-MS/MS (ion trap) to characterize fluorescent phenolic-type compounds accumulated in roots or exported to the culture medium of Arabidopsis plants in response to Fe deficiency. Wild-type and mutant plants altered either in phenylpropanoid biosynthesis or in the ABCG37 (PDR9) ABC transporter were grown under standard or Fe-deficient nutrition conditions and compared. Fe deficiency upregulates the expression of genes encoding enzymes of the phenylpropanoid pathway and leads to the synthesis and secretion of phenolic compounds belonging to the coumarin family. The ABCG37 gene is also upregulated in response to Fe deficiency and coumarin export is impaired in pdr9 mutant plants. Therefore it can be concluded that: Fe deficiency induces the secretion of coumarin compounds by Arabidopsis roots; the ABCG37 ABC transporter is required for this secretion to take place; and these compounds improved plant Fe nutrition.

Iron deficiency in plants: an insight from proteomic approaches.

Iron (Fe) deficiency chlorosis is a major nutritional disorder for crops growing in calcareous soils, and causes decreases in vegetative growth as well as marked yield and quality losses. With the advances in mass spectrometry techniques, a substantial body of knowledge has arisen on the changes in the protein profiles of different plant parts and compartments as a result of Fe deficiency. Changes in the protein profile of thylakoids from several species have been investigated using gel-based two-dimensional electrophoresis approaches, and the same techniques have been used to investigate changes in the root proteome profiles of tomato (Solanum lycopersicum), sugar beet (Beta vulgaris), cucumber (Cucumis sativus), Medicago truncatula and a Prunus rootstock. High throughput proteomic studies have also been published using Fe-deficient Arabidopsis thaliana roots and thylakoids. This review summarizes the major conclusions derived from these "-omic" approaches with respect to metabolic changes occurring with Fe deficiency, and highlights future research directions in this field. A better understanding of the mechanisms involved in root Fe homeostasis from a holistic point of view may strengthen our ability to enhance Fe-deficiency tolerance responses in plants of agronomic interest.

Mutually exclusive alterations in secondary metabolism are critical for the uptake of insoluble iron compounds by Arabidopsis and Medicago truncatula.

The generally low bioavailability of iron in aerobic soil systems forced plants to evolve sophisticated genetic strategies to improve the acquisition of iron from sparingly soluble and immobile iron pools. To distinguish between conserved and species-dependent components of such strategies, we analyzed iron deficiency-induced changes in the transcriptome of two model species, Arabidopsis (Arabidopsis thaliana) and Medicago truncatula. Transcriptional profiling by RNA sequencing revealed a massive up-regulation of genes coding for enzymes involved in riboflavin biosynthesis in M. truncatula and phenylpropanoid synthesis in Arabidopsis upon iron deficiency. Coexpression and promoter analysis indicated that the synthesis of flavins and phenylpropanoids is tightly linked to and putatively coregulated with other genes encoding proteins involved in iron uptake. We further provide evidence that the production and secretion of phenolic compounds is critical for the uptake of iron from sources with low bioavailability but dispensable under conditions where iron is readily available. In Arabidopsis, homozygous mutations in the Fe(II)- and 2-oxoglutarate-dependent dioxygenase family gene F6'H1 and defects in the expression of PLEIOTROPIC DRUG RESISTANCE9, encoding a putative efflux transporter for products from the phenylpropanoid pathway, compromised iron uptake from an iron source of low bioavailability. Both mutants were partially rescued when grown alongside wild-type Arabidopsis or M. truncatula seedlings, presumably by secreted phenolics and flavins. We concluded that production and secretion of compounds that facilitate the uptake of iron is an essential but poorly understood aspect of the reduction-based iron acquisition strategy, which is likely to contribute substantially to the efficiency of iron uptake in natural conditions.

Iron-dependent modifications of the flower transcriptome, proteome, metabolome, and hormonal content in an Arabidopsis ferritin mutant.

Iron homeostasis is an important process for flower development and plant fertility. The role of plastids in these processes has been shown to be essential. To document the relationships between plastid iron homeostasis and flower biology further, a global study (transcriptome, proteome, metabolome, and hormone analysis) was performed of Arabidopsis flowers from wild-type and triple atfer1-3-4 ferritin mutant plants grown under iron-sufficient or excess conditions. Some major modifications in specific functional categories were consistently observed at these three omic levels, although no significant overlaps of specific transcripts and proteins were detected. These modifications concerned redox reactions and oxidative stress, as well as amino acid and protein catabolism, this latter point being exemplified by an almost 10-fold increase in urea concentration of atfer1-3-4 flowers from plants grown under iron excess conditions. The mutant background caused alterations in Fe-haem redox proteins located in membranes and in hormone-responsive proteins. Specific effects of excess Fe in the mutant included further changes in these categories, supporting the idea that the mutant is facing a more intense Fe/redox stress than the wild type. The mutation and/or excess Fe had a strong impact at the membrane level, as denoted by the changes in the transporter and lipid metabolism categories. In spite of the large number of genes and proteins responsive to hormones found to be regulated in this study, changes in the hormonal balance were restricted to cytokinins, especially in the mutant plants grown under Fe excess conditions.

Metabolic responses to iron deficiency in roots of Carrizo citrange [Citrus sinensis (L.) Osbeck. x Poncirus trifoliata (L.) Raf].

The effects of iron (Fe) deficiency on the low-molecular-weight organic acid (LMWOA) metabolism have been investigated in Carrizo citrange (CC) [Citrus sinensis (L.) Osb. × Poncirus trifoliata (L.) Raf.] roots. Major LMWOAs found in roots, xylem sap and root exudates were citrate and malate and their concentrations increased with Fe deficiency. The activities of several enzymes involved in the LMWOA metabolism were also assessed in roots. In the cytosolic fraction, the activities of malate dehydrogenase (cMDH) and phosphoenolpyruvate carboxylase (PEPC) enzymes were 132 and 100% higher in Fe-deficient conditions, whereas the activity of pyruvate kinase was 31% lower and the activity of malic enzyme (ME) did not change. In the mitochondrial fraction, the activities of fumarase, MDH and citrate synthase enzymes were 158, 117 and 53% higher, respectively, in Fe-deficient extracts when compared with Fe-sufficient controls, whereas no significant differences between treatments were found for aconitase (ACO) activity. The expression of their corresponding genes in roots of Fe-deficient plants was higher than that measured in Fe-sufficient controls, except for ACO and ME. Also, dicarboxylate-tricarboxylate carrier (DTC) expression was significantly increased in Fe-deficient roots. In conclusion, Fe deficiency in CC seedlings causes a reprogramming of the carbon metabolism that involves an increase of anaplerotic fixation of carbon via PEPC and MDH activities in the cytosol and a shift of the Krebs cycle in the mitochondria towards a non-cyclic mode, as previously described in herbaceous species. In this scheme, DTC could play an important role shuttling both malate and reducing equivalents between the cytosol and the mitochondria. As a result of this metabolic switch malate and citrate concentrations in roots, xylem sap and root exudates increase.

Nicotianamine functions in the Phloem-based transport of iron to sink organs, in pollen development and pollen tube growth in Arabidopsis.

The metal chelator nicotianamine promotes the bioavailability of Fe and reduces cellular Fe toxicity. For breeding Fe-efficient crops, we need to explore the fundamental impact of nicotianamine on plant development and physiology. The quadruple nas4x-2 mutant of Arabidopsis thaliana cannot synthesize any nicotianamine, shows strong leaf chlorosis, and is sterile. To date, these phenotypes have not been fully explained. Here, we show that sink organs of this mutant were Fe deficient, while aged leaves were Fe sufficient. Upper organs were also Zn deficient. We demonstrate that transport of Fe to aged leaves relied on citrate, which partially complemented the loss of nicotianamine. In the absence of nicotianamine, Fe accumulated in the phloem. Our results show that rather than enabling the long-distance movement of Fe in the phloem (as is the case for Zn), nicotianamine facilitates the transport of Fe from the phloem to sink organs. We delimit nicotianamine function in plant reproductive biology and demonstrate that nicotianamine acts in pollen development in anthers and pollen tube passage in the carpels. Since Fe and Zn both enhance pollen germination, a lack of either metal may contribute to the reproductive defect. Our study sheds light on the physiological functions of nicotianamine.

Spatially resolved analysis of small molecules by matrix-assisted laser desorption/ionization mass spectrometric imaging (MALDI-MSI).

• Matrix-assisted laser desorption/ionization mass spectrometric imaging (MALDI-MSI) of tissues provides the means to analyse the spatial distributions of small molecules and proteins within tissues. This imaging technique is commonplace in medicinal and pharmaceutical research, but its application in plant science is very recent. Broader introduction requires specific adaptations for plant tissues. Sample preparation is of paramount importance in order to obtain high-quality spectra providing sufficient spatial resolution for compounds. Optimization is required for sectioning, choice of matrix and means of matrix deposition. • Here, we present our current protocols for the detection of small molecules in cryodissected immature barley (Hordeum vulgare) grains and tobacco (Nicotiana tabacum) roots. • Examples of MALDI-MSI measurements are provided, and the level of reproducibility across biological replicates is addressed. Furthermore, our approaches for the validation of distribution patterns and for the identification of molecules are described. • Finally, we discuss how MALDI-MSI can contribute to applied plant research.