Plasticity, exudation and microbiome-association of the root system of Pellitory-of-the-wall plants grown in environments impaired in iron availability.

The investigation of the adaptive strategies of wild plant species to extreme environments is a challenging issue, which favors the identification of new traits for plant resilience. We investigated different traits which characterize the root-soil interaction of Parietaria judaica, a wild plant species commonly known as "Pellitory-of-the-wall". P. judaica adopts the acidification-reduction strategy (Strategy I) for iron (Fe) acquisition from soil, and it can complete its life cycle in highly calcareous environments without any symptoms of chlorosis. In a field-to-lab approach, the microbiome associated with P. judaica roots was analyzed in spontaneous plants harvested from an urban environment consisting in an extremely calcareous habitat. Also, the phenolics and carboxylates content and root plasticity and exudation were analyzed in P. judaica plants grown under three different controlled conditions mimicking the effect of calcareous environments on Fe availability: results show that P. judaica differentially modulates root plasticity under different Fe availability-impaired conditions, and that it induces, to a high extent, the exudation of caffeoylquinic acid derivatives under calcareous conditions, positively impacting Fe solubility.

Modulation of photorespiration and nitrogen recycling in Fe-deficient cucumber leaves.

Low Fe availability affects plant production mainly by impairing the photosynthetic pathway, since Fe plays an essential role in chlorophyll synthesis as well as in the photosynthetic electron transport chain. Under these conditions, plant cells require the activation of protective mechanisms to prevent photo-inhibition. Among these mechanisms, photorespiration (PR) has been relatively little investigated in Fe-deficient plants. The aim of this work was to investigate the effect of Fe deficiency on photorespiration by performing in vivo analysis in leaves as well as biochemical characterization of some PR-related enzyme activities in a peroxisome-purified fraction from cucumber leaves. Modelling of light response curves at both 21 and 2% pO2 revealed a slowing down of PR under Fe deficiency. The activity of some PR-involving enzymes as well as the contents of glycine and serine were affected under Fe deficiency. Furthermore, nitrate reductase, the glutamine synthetase-glutamate synthase (GS-GOGAT) cycle and hydroxypyruvate dehydrogenase isoform activities were differentially altered under Fe deficiency. The dataset indicates that, in Fe-deficient cucumber leaves, the modulation of PR involves the induction of some PR-related pathways, such as the photorespiratory N recycling and cytosolic photorespiratory bypass processes.

Root bacterial endophytes confer drought resistance and enhance expression and activity of a vacuolar H+ -pumping pyrophosphatase in pepper plants.

It has been previously shown that the transgenic overexpression of the plant root vacuolar proton pumps H+ -ATPase (V-ATPase) and H+ -PPase (V-PPase) confer tolerance to drought. Since plant-root endophytic bacteria can also promote drought tolerance, we hypothesize that such promotion can be associated to the enhancement of the host vacuolar proton pumps expression and activity. To test this hypothesis, we selected two endophytic bacteria endowed with an array of in vitro plant growth promoting traits. Their genome sequences confirmed the presence of traits previously shown to confer drought resistance to plants, such as the synthesis of nitric oxide and of organic volatile organic compounds. We used the two strains on pepper (Capsicuum annuum L.) because of its high sensitivity to drought. Under drought conditions, both strains stimulated a larger root system and enhanced the leaves' photosynthetic activity. By testing the expression and activity of the vacuolar proton pumps, H+ -ATPase (V-ATPase) and H+ -PPase (V-PPase), we found that bacterial colonization enhanced V-PPase only. We conclude that the enhanced expression and activity of V-PPase can be favoured by the colonization of drought-tolerance-inducing bacterial endophytes.

Transcriptional Characterization of a Widely-Used Grapevine Rootstock Genotype under Different Iron-Limited Conditions.

Iron chlorosis is a serious deficiency that affects orchards and vineyards reducing quality and yield production. Chlorotic plants show abnormal photosynthesis and yellowing shoots. In grapevine iron uptake and homeostasis are most likely controlled by a mechanism known as "Strategy I," characteristic of non-graminaceous plants and based on a system of soil acidification, iron reduction and transporter-mediated uptake. Nowadays, grafting of varieties of economic interest on tolerant rootstocks is widely used practice against many biotic and abiotic stresses. Nevertheless, many interspecific rootstocks, and in particular those obtained by crossing exclusively non-vinifera genotypes, can show limited nutrient uptake and transport, in particular for what concerns iron. In the present study, 101.14, a commonly used rootstock characterized by susceptibility to iron chlorosis was subjected to both Fe-absence and Fe-limiting conditions. Grapevine plantlets were grown in control, Fe-deprived, and bicarbonate-supplemented hydroponic solutions. Whole transcriptome analyses, via mRNA-Seq, were performed on root apices of stressed and unstressed plants. Analysis of differentially expressed genes (DEGs) confirmed that Strategy I is the mechanism responsible for iron uptake in grapevine, since many orthologs genes to the Arabidopsis "ferrome" were differentially regulated in stressed plant. Molecular differences in the plant responses to Fe absence and presence of bicarbonate were also identified indicating the two treatments are able to induce response-mechanisms only partially overlapping. Finally, we measured the expression of a subset of genes differentially expressed in 101.14 (such as IRT1, FERRITIN1, bHLH38/39) or known to be fundamental in the "strategy I" mechanism (AHA2 and FRO2) also in a tolerant rootstock (M1) finding important differences which could be responsible for the different degrees of tolerance observed.

Knocking down mitochondrial iron transporter (MIT) reprograms primary and secondary metabolism in rice plants.

Iron (Fe) is an essential micronutrient for plant growth and development, and its reduced bioavailability strongly impairs mitochondrial functionality. In this work, the metabolic adjustment in the rice (Oryza sativa) mitochondrial Fe transporter knockdown mutant (mit-2) was analysed. Biochemical characterization of purified mitochondria from rice roots showed alteration in the respiratory chain of mit-2 compared with wild-type (WT) plants. In particular, proteins belonging to the type II alternative NAD(P)H dehydrogenases accumulated strongly in mit-2 plants, indicating that alternative pathways were activated to keep the respiratory chain working. Additionally, large-scale changes in the transcriptome and metabolome were observed in mit-2 rice plants. In particular, a strong alteration (up-/down-regulation) in the expression of genes encoding enzymes of both primary and secondary metabolism was found in mutant plants. This was reflected by changes in the metabolic profiles in both roots and shoots of mit-2 plants. Significant alterations in the levels of amino acids belonging to the aspartic acid-related pathways (aspartic acid, lysine, and threonine in roots, and aspartic acid and ornithine in shoots) were found that are strictly connected to the Krebs cycle. Furthermore, some metabolites (e.g. pyruvic acid, fumaric acid, ornithine, and oligosaccharides of the raffinose family) accumulated only in the shoot of mit-2 plants, indicating possible hypoxic responses. These findings suggest that the induction of local Fe deficiency in the mitochondrial compartment of mit-2 plants differentially affects the transcript as well as the metabolic profiles in root and shoot tissues.

Three-Dimensional Reconstruction, by TEM Tomography, of the Ultrastructural Modifications Occurring in Cucumis sativus L. Mitochondria under Fe Deficiency.

BACKGROUND: Mitochondria, as recently suggested, might be involved in iron sensing and signalling pathways in plant cells. For a better understanding of the role of these organelles in mediating the Fe deficiency responses in plant cells, it is crucial to provide a full overview of their modifications occurring under Fe-limited conditions. The aim of this work is to characterize the ultrastructural as well as the biochemical changes occurring in leaf mitochondria of cucumber (Cucumis sativus L.) plants grown under Fe deficiency. METHODOLOGY/RESULTS: Mitochondrial ultrastructure was investigated by transmission electron microscopy (TEM) and electron tomography techniques, which allowed a three-dimensional (3D) reconstruction of cellular structures. These analyses reveal that mitochondria isolated from cucumber leaves appear in the cristae junction model conformation and that Fe deficiency strongly alters both the number and the volume of cristae. The ultrastructural changes observed in mitochondria isolated from Fe-deficient leaves reflect a metabolic status characterized by a respiratory chain operating at a lower rate (orthodox-like conformation) with respect to mitochondria from control leaves. CONCLUSIONS: To our knowledge, this is the first report showing a 3D reconstruction of plant mitochondria. Furthermore, these results suggest that a detailed characterization of the link between changes in the ultrastructure and functionality of mitochondria during different nutritional conditions, can provide a successful approach to understand the role of these organelles in the plant response to Fe deficiency.

Improved plant resistance to drought is promoted by the root-associated microbiome as a water stress-dependent trait.

Although drought is an increasing problem in agriculture, the contribution of the root-associated bacterial microbiome to plant adaptation to water stress is poorly studied. We investigated if the culturable bacterial microbiome associated with five grapevine rootstocks and the grapevine cultivar Barbera may enhance plant growth under drought stress. Eight isolates, over 510 strains, were tested in vivo for their capacity to support grapevine growth under water stress. The selected strains exhibited a vast array of plant growth promoting (PGP) traits, and confocal microscopy observation of gfp-labelled Acinetobacter and Pseudomonas isolates showed their ability to adhere and colonize both the Arabidopsis and grapevine rhizoplane. Tests on pepper plants fertilized with the selected strains, under both optimal irrigation and drought conditions, showed that PGP activity was a stress-dependent and not a per se feature of the strains. The isolates were capable of increasing shoot and leaf biomass, shoot length, and photosynthetic activity of drought-challenged grapevines, with an enhanced effect in drought-sensitive rootstock. Three isolates were further assayed for PGP capacity under outdoor conditions, exhibiting the ability to increase grapevine root biomass. Overall, the results indicate that PGP bacteria contribute to improve plant adaptation to drought through a water stress-induced promotion ability.

Are drought-resistance promoting bacteria cross-compatible with different plant models?

The association between plant and plant growth promoting bacteria (PGPB) contributes to the successful thriving of plants in extreme environments featured by water shortage. We have recently shown that, with respect to the non-cultivated desert soil, the rhizosphere of pepper plants cultivated under desert farming hosts PGPB communities that are endowed with a large portfolio of PGP traits. Pepper plants exposed to bacterial isolates from plants cultivated under desert farming exhibited a higher tolerance to water shortage, compared with untreated control. This promotion was mediated by a larger root system (up to 40%), stimulated by the bacteria, that enhanced plant ability to uptake water from dry soil. We provide initial evidence that the nature of the interaction can have a limited level of specificity and that PGPB isolates may determine resistance to water stress in plants others than the one of the original isolation. It is apparent that, in relation to plant resistance to water stress, a feature of primary evolutionary importance for all plants, a cross-compatibility between PGPB and different plant models exists at least on a short-term.

Fe deficiency differentially affects the vacuolar proton pumps in cucumber and soybean roots.

Iron uptake in dicots depends on their ability to induce a set of responses in root cells including rhizosphere acidification through H(+) extrusion and apoplastic Fe(III) reduction by Fe(III)-chelate reductase. These responses must be sustained by metabolic rearrangements aimed at providing the required NAD(P)H, ATP and H(+). Previous results in Fe-deficient cucumber roots showed that high H(+) extrusion is accompanied by increased phosphoenolpyruvate carboxylase (PEPC) activity, involved in the cytosol pH-stat; moreover (31)P-NMR analysis revealed increased vacuolar pH and decreased vacuolar [inorganic phosphate (Pi)]. The opposite was found in soybean: low rhizosphere acidification, decreased PEPC activity, vacuole acidification, and increased vacuolar [Pi]. These findings, highlighting a different impact of the Fe deficiency responses on cytosolic pH in the two species, lead to hypothesize different roles for H(+) and Pi movements across the tonoplast in pH homeostasis. The role of vacuole in cytosolic pH-stat involves the vacuolar H(+)-ATPase (V-ATPase) and vacuolar H(+)-pyrophosphatase (V-PPase) activities, which generating the ΔpH and ΔΨ, mediate the transport of solutes, among which Pi, across the tonoplast. Fluxes of Pi itself in its two ionic forms, H2PO4 (-) predominating in the vacuole and HPO4 (2-) in the cytosol, may be involved in pH homeostasis owing to its pH-dependent protonation/deprotonation reactions. Tonoplast enriched fractions were obtained from cucumber and soybean roots grown with or without Fe. Both V-ATPase and V-PPase activities were analyzed and the enrichment and localization of the corresponding proteins in root tissues were determined by Western blot and immunolocalization. V-ATPase did not change its activity and expression level in response to Fe starvation in both species. V-PPase showed a different behavior: in cucumber roots its activity and abundance were decreased, while in Fe-deficient soybean roots they were increased. The distinct role of the two H(+) pumps in Pi fluxes between cytoplasm and vacuole in Fe-deficient cucumber and soybean root cells is discussed.

Low iron availability and phenolic metabolism in a wild plant species (Parietaria judaica L.).

Plant phenolics encompass a wide range of aromatic compounds and functions mainly related to abiotic and biotic environmental responses. In calcareous soils, the presence of bicarbonate and a high pH cause a decrease in iron (Fe) bioavailability leading to crop yield losses both qualitatively and quantitatively. High increases in phenolics were reported in roots and root exudates as a consequence of decreased Fe bioavailability suggesting their role in chelation and reduction of inorganic Fe(III) contributing to the mobilization of Fe oxides in soil and plant apoplast. Shikimate pathway represents the main pathway to provide aromatic precursors for the synthesis of phenylpropanoids and constitutes a link between primary and secondary metabolism. Thus the increased level of phenolics suggests a metabolic shift of carbon skeletons from primary to secondary metabolism. Parietaria judaica, a spontaneous plant well adapted to calcareous environments, demonstrates a high metabolic flexibility in response to Fe starvation. Plants grown under low Fe availability conditions showed a strong accumulation of phenolics in roots as well as an improved secretion of root exudates. P. judaica exhibits enhanced enzymatic activities of the shikimate pathway. Furthermore, the non-oxidative pentose phosphate pathway, through the transketolase activity supplies erythrose-4-phosphate, is strongly activated. These data may indicate a metabolic rearrangement modifying the allocation of carbon skeletons between primary and secondary metabolism and the activation of a nonoxidative way to overcome a mitochondrial impairment. We suggest that high content of phenolics in P. judaica play a crucial role in its adaptive strategy to cope with low Fe availability.

Signals from chloroplasts and mitochondria for iron homeostasis regulation.

Iron (Fe) is an essential element for human nutrition. Given that plants represent a major dietary source of Fe worldwide, it is crucial to understand plant Fe homeostasis fully. A major breakthrough in the understanding of Fe sensing and signaling was the identification of several transcription factor cascades regulating Fe homeostasis. However, the mechanisms of activation of these cascades still remain to be elucidated. In this opinion, we focus on the possible roles of mitochondria and chloroplasts as cellular Fe sensing and signaling sites, offering a new perspective on the integrated regulation of Fe homeostasis and its interplay with cellular metabolism.

A drought resistance-promoting microbiome is selected by root system under desert farming.

BACKGROUND: Traditional agro-systems in arid areas are a bulwark for preserving soil stability and fertility, in the sight of "reverse desertification". Nevertheless, the impact of desert farming practices on the diversity and abundance of the plant associated microbiome is poorly characterized, including its functional role in supporting plant development under drought stress. METHODOLOGY/PRINCIPAL FINDINGS: We assessed the structure of the microbiome associated to the drought-sensitive pepper plant (Capsicum annuum L.) cultivated in a traditional Egyptian farm, focusing on microbe contribution to a crucial ecosystem service, i.e. plant growth under water deficit. The root system was dissected by sampling root/soil with a different degree of association to the plant: the endosphere, the rhizosphere and the root surrounding soil that were compared to the uncultivated soil. Bacterial community structure and diversity, determined by using Denaturing Gradient Gel Electrophoresis, differed according to the microhabitat, indicating a selective pressure determined by the plant activity. Similarly, culturable bacteria genera showed different distribution in the three root system fractions. Bacillus spp. (68% of the isolates) were mainly recovered from the endosphere, while rhizosphere and the root surrounding soil fractions were dominated by Klebsiella spp. (61% and 44% respectively). Most of the isolates (95%) presented in vitro multiple plant growth promoting (PGP) activities and stress resistance capabilities, but their distribution was different among the root system fractions analyzed, with enhanced abilities for Bacillus and the rhizobacteria strains. We show that the C. annuum rhizosphere under desert farming enriched populations of PGP bacteria capable of enhancing plant photosynthetic activity and biomass synthesis (up to 40%) under drought stress. CONCLUSIONS/SIGNIFICANCE: Crop cultivation provides critical ecosystem services in arid lands with the plant root system acting as a "resource island" able to attract and select microbial communities endowed with multiple PGP traits that sustain plant development under water limiting conditions.

Iron deficiency affects nitrogen metabolism in cucumber (Cucumis sativus L.) plants.

BACKGROUND: Nitrogen is a principal limiting nutrient in plant growth and development. Among factors that may limit NO3- assimilation, Fe potentially plays a crucial role being a metal cofactor of enzymes of the reductive assimilatory pathway. Very few information is available about the changes of nitrogen metabolism occurring under Fe deficiency in Strategy I plants. The aim of this work was to study how cucumber (Cucumis sativus L.) plants modify their nitrogen metabolism when grown under iron deficiency. RESULTS: The activity of enzymes involved in the reductive assimilation of nitrate and the reactions that produce the substrates for the ammonium assimilation both at root and at leaf levels in Fe-deficient cucumber plants were investigated. Under Fe deficiency, only nitrate reductase (EC 1.7.1.1) activity decreased both at the root and leaf level, whilst for glutamine synthetase (EC 6.3.1.2) and glutamate synthase (EC 1.4.1.14) an increase was found. Accordingly, the transcript analysis for these enzymes showed the same behaviour except for root nitrate reductase which increased. Furthermore, it was found that amino acid concentration greatly decreased in Fe-deficient roots, whilst it increased in the corresponding leaves. Moreover, amino acids increased in the xylem sap of Fe-deficient plants. CONCLUSIONS: The data obtained in this work provided new insights on the responses of plants to Fe deficiency, suggesting that this nutritional disorder differentially affected N metabolism in root and in leaf. Indeed under Fe deficiency, roots respond more efficiently, sustaining the whole plant by furnishing metabolites (i.e. aa, organic acids) to the leaves.

Application of the split root technique to study iron uptake in cucumber plants.

The regulation exerted by the Fe status in the plant on Fe deficiency responses was investigated in Cucumis sativus L. roots at both biochemical and molecular levels. Besides the two activities strictly correlated with Fe deficiency response, those of the Fe(III)-chelate reductase and the high affinity Fe transporter, we considered also H(+)-ATPase (EC 3.6.3.6) and phosphoenolpyruvate carboxylase (EC 4.1.1.31), that have been shown to be involved in this response. Both enzymatic activities and gene expression were monitored using a split root system. Absence of Fe induced the expression of the four transcripts, accompanied by an increase in the corresponding enzymatic activities. The application of the split root technique gave some information about the regulation of Fe uptake. In fact, 24 h after split root application, transcripts were still high and comparable to those of the -Fe control in the Fe-supplied half side, while in the -Fe side there was a drop in the expression and the relative enzymatic activities. Major changes occurred after 48 and 72 h. The coordinated regulation of these responses is discussed.

Adaptive strategies of Parietaria diffusa (M.&K.) to calcareous habitat with limited iron availability.

The study of native plants growing in hostile environments is useful to understand how these species respond to stress conditions. Parietaria diffusa (M.&K.) is able to survive in highly calcareous soils and extreme environments, such as house walls, without displaying any chlorotic symptoms. Here, we have investigated the existence of Strategy I complementary/alternative mechanism(s) involved in Fe solubilization and uptake and responsible for Parietaria's extraordinary efficiency. After assessing the specific traits involved in a calcicole-behaviour in the field, we have grown plants in conditions of Fe deficiency, either direct (-Fe) or induced by the presence of bicarbonate (+FeBic). Then, the growth performance, physiological and biochemical responses of the plants were investigated. The study shows that in Parietaria+FeBic, the classical responses of Strategy I plants are activated to a lower extent than in -Fe. In addition, there is a greater production of phenolics and organic acids that are both exuded and accumulated in the roots, which in turn show structures similar to 'proteoid-like roots'. We suggest that in the presence of this constraint, Parietaria undergoes some metabolic rearrangements that involve PEP-consuming reactions and an enhancement of the shikimate pathway.

Immunolocalization of H(+)-ATPase and IRT1 enzymes in N(2)-fixing common bean nodules subjected to iron deficiency.

The demand for iron in leguminous plants increases during symbiosis, as the metal is utilised for the synthesis of various Fe-containing proteins in both plant and bacteroids. However, the acquisition of this micronutrient is problematic due to its low bioavailability at physiological pH under aerobic conditions. Induction of root Fe(III)-reductase activity is necessary for Fe uptake and can be coupled to the rhizosphere acidification capacity linked to the H(+)-ATPase activity. Fe uptake is related to the expression of a Fe(2+) transporter (IRT1). In order to verify the possible role of nodules in the acquisition of Fe directly from the soil solution, the localization of H(+)-ATPase and IRT1 was carried out in common bean nodules by immuno-histochemical analysis. The results showed that these proteins were particularly abundant in the central nitrogen-fixing zone of nodules, around the periphery of infected and uninfected cells as well as in the vascular bundle of control nodules. Under Fe deficiency an over-accumulation of H(+)-ATPase and IRT1 proteins was observed especially around the cortex cells of nodules. The results obtained in this study suggest that the increase in these proteins is differentially localized in nodules of Fe-deficient plants when compared to the Fe-sufficient condition and cast new light on the possible involvement of nodules in the direct acquisition of Fe from the nutrient solution.

Metabolic changes of iron uptake in N(2)-fixing common bean nodules during iron deficiency.

Iron is an important nutrient in N(2)-fixing legume nodules. The demand for this micronutrient increases during the symbiosis establishment, where the metal is utilized for the synthesis of various iron-containing proteins in both the plant and the bacteroid. Unfortunately, in spite of its importance, iron is poorly available to plant uptake since its solubility is very low when in its oxidized form Fe(III). In the present study, the effect of iron deficiency on the activity of some proteins involved in Strategy I response, such as Fe-chelate reductase (FC-R), H(+)-ATPase, and phosphoenolpyruvate carboxylase (PEPC) and the protein level of iron regulated transporter (IRT1) and H(+)-ATPase proteins has been investigated in both roots and nodules of a tolerant (Flamingo) and a susceptible (Coco blanc) cultivar of common bean plants. The main results of this study show that the symbiotic tolerance of Flamingo can be ascribed to a greater increase in the FC-R and H(+)-ATPase activities in both roots and nodules, leading to a more efficient Fe supply to nodulating tissues. The strong increase in PEPC activity and organic acid content, in the Flamingo root nodules, suggests that under iron deficiency nodules can modify their metabolism in order to sustain those activities necessary to acquire Fe directly from the soil solution.

Oxidative stress responses and root lignification induced by Fe deficiency conditions in pear and quince genotypes.

We analysed Pyrus communis cv. Conference and Cydonia oblonga BA29, differently tolerant to lime-induced chlorosis, to identify the key mechanisms involved in their different performance under Fe deficiency induced by the absence of Fe (-Fe) or by the presence of bicarbonate (+FeBic). Under our experimental conditions, a decrease in root elongation was observed in BA29 under bicarbonate supply. Superoxide dismutase (SOD) and peroxidase (POD) activities were analysed and the relative isoforms were detected by native electrophoresis. The data obtained for both genotypes under -Fe and for BA29 +FeBic suggest the occurrence of overproduction of reactive oxygen species (ROS) and, at the same time, of a scarce capacity to detoxify them. The detection of ROS (O(2)(-) and H(2)O(2)) through histochemical localization supports these results and suggests that they could account for the modifications of mechanical properties of the cell wall during stress adaptation. On the other hand, in the cv. Conference +FeBic, an increase in non-specific POD activity was detected, confirming its higher level of protection in particular against H(2)O(2) accumulation. Peroxidases involved in lignification were assayed and histochemical analysis was performed. The results suggest that only in BA29 under bicarbonate supply can the presence of ROS in root apoplast be correlated with lignin deposits in external layers and in endodermis as a consequence of the shift of PODs towards a lignification role. We suggest that in BA29 the decrease in root growth could impair mineral nutrition, generating susceptibility to calcareous soils. In the cv. Conference, the allocation of new biomass to the root system could improve soil exploration and consequently Fe uptake.

Proteomic characterization of iron deficiency responses in Cucumis sativus L. roots.

BACKGROUND: Iron deficiency induces in Strategy I plants physiological, biochemical and molecular modifications capable to increase iron uptake from the rhizosphere. This effort needs a reorganization of metabolic pathways to efficiently sustain activities linked to the acquisition of iron; in fact, carbohydrates and the energetic metabolism has been shown to be involved in these responses. The aim of this work was to find both a confirmation of the already expected change in the enzyme concentrations induced in cucumber root tissue in response to iron deficiency as well as to find new insights on the involvement of other pathways. RESULTS: The proteome pattern of soluble cytosolic proteins extracted from roots was obtained by 2-DE. Of about two thousand spots found, only those showing at least a two-fold increase or decrease in the concentration were considered for subsequent identification by mass spectrometry. Fifty-seven proteins showed significant changes, and 44 of them were identified. Twenty-one of them were increased in quantity, whereas 23 were decreased in quantity. Most of the increased proteins belong to glycolysis and nitrogen metabolism in agreement with the biochemical evidence. On the other hand, the proteins being decreased belong to the metabolism of sucrose and complex structural carbohydrates and to structural proteins. CONCLUSIONS: The new available techniques allow to cast new light on the mechanisms involved in the changes occurring in plants under iron deficiency. The data obtained from this proteomic study confirm the metabolic changes occurring in cucumber as a response to Fe deficiency. Two main conclusions may be drawn. The first one is the confirmation of the increase in the glycolytic flux and in the anaerobic metabolism to sustain the energetic effort the Fe-deficient plants must undertake. The second conclusion is, on one hand, the decrease in the amount of enzymes linked to the biosynthesis of complex carbohydrates of the cell wall, and, on the other hand, the increase in enzymes linked to the turnover of proteins.