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1.  Iron deficiency affects nitrogen metabolism in cucumber (Cucumis sativus L.) plants 
BMC Plant Biology  2012;12:189.
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.
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 activity decreased both at the root and leaf level, whilst for glutamine synthetase (EC and glutamate synthase (EC 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.
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.
PMCID: PMC3539955  PMID: 23057967
C/N metabolism; Cucumis sativus L.; Fe deficiency; GS/GOGAT cycle; Isocitrate dehydrogenase; Nitrate reductase
2.  Microarray analysis of iron deficiency chlorosis in near-isogenic soybean lines 
BMC Genomics  2007;8:476.
Iron is one of fourteen mineral elements required for proper plant growth and development of soybean (Glycine max L. Merr.). Soybeans grown on calcareous soils, which are prevalent in the upper Midwest of the United States, often exhibit symptoms indicative of iron deficiency chlorosis (IDC). Yield loss has a positive linear correlation with increasing severity of chlorotic symptoms. As soybean is an important agronomic crop, it is essential to understand the genetics and physiology of traits affecting plant yield. Soybean cultivars vary greatly in their ability to respond successfully to iron deficiency stress. Microarray analyses permit the identification of genes and physiological processes involved in soybean's response to iron stress.
RNA isolated from the roots of two near isogenic lines, which differ in iron efficiency, PI 548533 (Clark; iron efficient) and PI 547430 (IsoClark; iron inefficient), were compared on a spotted microarray slide containing 9,728 cDNAs from root specific EST libraries. A comparison of RNA transcripts isolated from plants grown under iron limiting hydroponic conditions for two weeks revealed 43 genes as differentially expressed. A single linkage clustering analysis of these 43 genes showed 57% of them possessed high sequence similarity to known stress induced genes. A control experiment comparing plants grown under adequate iron hydroponic conditions showed no differences in gene expression between the two near isogenic lines. Expression levels of a subset of the differentially expressed genes were also compared by real time reverse transcriptase PCR (RT-PCR). The RT-PCR experiments confirmed differential expression between the iron efficient and iron inefficient plants for 9 of 10 randomly chosen genes examined. To gain further insight into the iron physiological status of the plants, the root iron reductase activity was measured in both iron efficient and inefficient genotypes for plants grown under iron sufficient and iron limited conditions. Iron inefficient plants failed to respond to decreased iron availability with increased activity of Fe reductase.
These experiments have identified genes involved in the soybean iron deficiency chlorosis response under iron deficient conditions. Single linkage cluster analysis suggests iron limited soybeans mount a general stress response as well as a specialized iron deficiency stress response. Root membrane bound reductase capacity is often correlated with iron efficiency. Under iron-limited conditions, the iron efficient plant had high root bound membrane reductase capacity while the iron inefficient plants reductase levels remained low, further limiting iron uptake through the root. Many of the genes up-regulated in the iron inefficient NIL are involved in known stress induced pathways. The most striking response of the iron inefficient genotype to iron deficiency stress was the induction of a profusion of signaling and regulatory genes, presumably in an attempt to establish and maintain cellular homeostasis. Genes were up-regulated that point toward an increased transport of molecules through membranes. Genes associated with reactive oxidative species and an ROS-defensive enzyme were also induced. The up-regulation of genes involved in DNA repair and RNA stability reflect the inhospitable cellular environment resulting from iron deficiency stress. Other genes were induced that are involved in protein and lipid catabolism; perhaps as an effort to maintain carbon flow and scavenge energy. The under-expression of a key glycolitic gene may result in the iron-inefficient genotype being energetically challenged to maintain a stable cellular environment. These experiments have identified candidate genes and processes for further experimentation to increase our understanding of soybeans' response to iron deficiency stress.
PMCID: PMC2253546  PMID: 18154662
3.  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.
PMCID: PMC3722493  PMID: 23898336
iron; metabolism; proteomics; root; thylakoid
4.  Changes in the proteomic and metabolic profiles of Beta vulgaris root tips in response to iron deficiency and resupply 
BMC Plant Biology  2010;10:120.
Plants grown under iron deficiency show different morphological, biochemical and physiological changes. These changes include, among others, the elicitation of different strategies to improve the acquisition of Fe from the rhizosphere, the adjustment of Fe homeostasis processes and a reorganization of carbohydrate metabolism. The application of modern techniques that allow the simultaneous and untargeted analysis of multiple proteins and metabolites can provide insight into multiple processes taking place in plants under Fe deficiency. The objective of this study was to characterize the changes induced in the root tip proteome and metabolome of sugar beet plants in response to Fe deficiency and resupply.
Root tip extract proteome maps were obtained by 2-D isoelectric focusing polyacrylamide gel electrophoresis, and approximately 140 spots were detected. Iron deficiency resulted in changes in the relative amounts of 61 polypeptides, and 22 of them were identified by mass spectrometry (MS). Metabolites in root tip extracts were analyzed by gas chromatography-MS, and more than 300 metabolites were resolved. Out of 77 identified metabolites, 26 changed significantly with Fe deficiency. Iron deficiency induced increases in the relative amounts of proteins and metabolites associated to glycolysis, tri-carboxylic acid cycle and anaerobic respiration, confirming previous studies. Furthermore, a protein not present in Fe-sufficient roots, dimethyl-8-ribityllumazine (DMRL) synthase, was present in high amounts in root tips from Fe-deficient sugar beet plants and gene transcript levels were higher in Fe-deficient root tips. Also, a marked increase in the relative amounts of the raffinose family of oligosaccharides (RFOs) was observed in Fe-deficient plants, and a further increase in these compounds occurred upon short term Fe resupply.
The increases in DMRL synthase and in RFO sugars were the major changes induced by Fe deficiency and resupply in root tips of sugar beet plants. Flavin synthesis could be involved in Fe uptake, whereas RFO sugars could be involved in the alleviation of oxidative stress, C trafficking or cell signalling. Our data also confirm the increase in proteins and metabolites related to carbohydrate metabolism and TCA cycle pathways.
PMCID: PMC3017792  PMID: 20565974
5.  Proteomic analysis of the effects of exogenous calcium on hypoxic-responsive proteins in cucumber roots 
Proteome Science  2012;10:42.
Hypoxia acts as a plant stress factor, particularly in cucumbers plants under hydroponic culture. Calcium is involved in stress signal transmission and in the growth of plants. To determine the effect of exogenous calcium on hypoxic-responsive proteins in cucumber (Cucumis sativus L. cv. Jinchun No.2) roots, proteomic analysis was performed using two-dimensional electrophoresis (2-DE) and mass spectrometry.
Cucumber roots were used to analyze the influence of hypoxia on plants. The expressions of 38 protein spots corresponding to enzymes were shown to change in response to hypoxia. Of these, 30 spots were identified by matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF/TOF MS analysis). The proteins were categorized according to functional groups, including glycolysis, the tricarboxylic acid (TCA) cycle, fermentative metabolism, nitrogen metabolism, energy metabolism, protein synthesis and defense against stress. Exogenous calcium appeared to alleviate hypoxic stress via these metabolic and physiological systems. Western blotting was used to analyze the accumulation of alcohol dehydrogenase (ADH) and pyruvate decarboxylase (PDC); calcium further increased the expression of ADH and PDC under hypoxia. In addition, semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) was used to assess the transcript levels of differentially expressed proteins.
Exogenous calcium enhanced the expression of enzymes involved in glycolysis, the TCA cycle, fermentative metabolism, nitrogen metabolism, and reactive oxygen species (ROS) defense in plants under hypoxia. Calcium appears to induce hypoxic tolerance of cucumber seedlings. These phenomena have prompted us to further investigate the mechanisms by which cucumbers respond to exogenous calcium under hypoxia.
PMCID: PMC3576256  PMID: 22788869
Cucumber; Calcium; Hypoxic stress; Proteomics
6.  A small-scale proteomic approach reveals a survival strategy, including a reduction in alkaloid biosynthesis, in Hyoscyamus albus roots subjected to iron deficiency 
Hyoscyamus albus is a well-known source of the tropane alkaloids, hyoscyamine and scopolamine, which are biosynthesized in the roots. To assess the major biochemical adaptations that occur in the roots of this plant in response to iron deficiency, we used a small-scale proteomic approach in which 100 mg of root tips were treated with and without Fe, respectively, for 5 days. Two-dimensional mini gels showed that 48 spots were differentially accumulated between the two conditions of Fe availability and a further 36 proteins were identified from these spots using MALDI-QIT-TOF mass spectrometry. The proteins that showed elevated levels in the roots lacking Fe were found to be associated variously with carbohydrate metabolism, cell differentiation, secondary metabolism, and oxidative defense. Most of the proteins involved in carbohydrate metabolism were increased in abundance, but mitochondrial NAD-dependent malate dehydrogenase was decreased, possibly resulting in malate secretion. Otherwise, all the proteins showing diminished levels in the roots were identified as either Fe-containing or ATP-requiring. For example, a significant decrease was observed in the levels of hyoscyamine 6β-hydroxylase (H6H), which requires Fe and is involved in the conversion of hyoscyamine to scopolamine. To investigate the effects of Fe deficiency on alkaloid biosynthesis, gene expression studies were undertaken both for H6H and for another Fe-dependent protein, Cyp80F1, which is involved in the final stage of hyoscyamine biosynthesis. In addition, tropane alkaloid contents were determined. Reduced gene expression was observed in the case of both of these proteins and was accompanied by a decrease in the content of both hyoscyamine and scopolamine. Finally, we have discussed energetic and Fe-conservation strategies that might be adopted by the roots of H. albus to maintain iron homeostasis under Fe-limiting conditions.
PMCID: PMC3755260  PMID: 24009619
Hyoscyamus albus; roots; Fe deficiency; small-scale proteomics; hyoscyamine 6β-hydroxylase; tropane alkaloid biosynthesis; malic acid secretion
7.  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 31P-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 HPO42- 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.
PMCID: PMC3753452  PMID: 23986768
V-ATPase; V-PPase; Fe deficiency; cucumber; soybean
8.  Proteome-wide systems analysis of a cellulosic biofuel-producing microbe 
We apply mass spectrometry-based ReDi proteomics to quantify the Clostridium phytofermentans proteome during fermentation of cellulosic substrates. ReDi proteomics gives accurate, low-cost quantification of an extra and intracellular microbial proteome. When combined with physiological measurements, these methods form a general systems biology strategy to evaluate the efficiency of cellulosic bioconversion and to identify enzyme targets to engineer for improving this process.C. phytofermentans expressed more than 100 carbohydrate-active enzymes, of which distinct subsets were upregulated on cellulose and hemicellulose. Numerous extracellular enzymes cleave insoluble plant polysaccharides into oligosaccharides, which are transported into the cell to be further degraded by intracellular carbohydratases. Sugars are catabolized by EMP glycolysis incorporating alternative glycolytic enzymes to maximize the ATP yield of anaerobic metabolism.During cellulosic fermentation, cells adhered to the substrate and altered metabolic processes such as upregulation of tryptophan and nicotinamide synthesis proteins and repression of proteins for fatty acid metabolism and cell motility. These diverse metabolic changes highlight how a systems approach can identify novel ways to optimize cellulosic fermentation.
Cellulose is the world's most abundant renewable, biological energy source (Leschine, 1995). Microbial fermentation of cellulosic biomass could sustainably provide enough ethanol for 65% of US ground transportation fuel at current levels (Somerville, 2006). However, cellulose in plant biomass is packaged into a crystalline matrix, making biomass deconstruction a key roadblock to using it as a feedstock (Houghton et al, 2006). A promising strategy to overcome biomass recalcitrance is consolidated bioprocessing (Lynd et al, 2002), which uses microbes such as Clostridium phytofermentans to both secrete enzymes to depolymerize biomass and then ferment the resulting hexose and pentose sugars to a biofuel such as ethanol. The C. phytofermentans genome encodes 161 carbohydrate-active enzymes (CAZy) including 108 glycoside hydrolases spread across 39 families (Cantarel et al, 2009), highlighting the elaborate set of enzymes needed to breakdown different cellulosic polysaccharides. Faced with the complexity of metabolizing biomass, systems biology strategies are needed to comprehensively identify which cellulolytic and metabolic enzymes are used to ferment different cellulosic substrates.
This study presents a systems-level analysis of how C. phytofermentans ferments different cellulosic substrates that incorporates quantitative mass spectrometry-based proteomics of over 2500 proteins. Protein concentrations within each carbon source treatment were calculated by machine learning-supported spectral counting (Absolute Protein EXpression, APEX) (Lu et al, 2007). Protein levels on hemicellulose and cellulose relative to glucose were determined using reductive methylation (Hsu et al, 2003; Boersema et al, 2009), here called ReDi labeling, to chemically incorporate hydrogen or deuterium isotopes at lysines and N-terminal amines of tryptic peptides. We show that ReDi proteomics gives accurate, low-cost quantification of a microbial proteome and can be used to discern extracellular proteins. Further, we combine these quantitative proteomics with detailed measurements of growth, biomass consumption, fermentation product analyses, and electron microscopy. Together, these methods form a general strategy to evaluate the efficiency of cellulosic bioconversion and to identify enzyme targets to engineer for improving this process (Figure 1).
We found that fermentation of cellulosic substrates by C. phytofermentans involves secretion of numerous CAZy as well as proteins for binding of extracellular solutes, proteolysis, and motility. The most highly expressed protein in the proteome is a secreted protein that appears to compose a surface layer to support the cell and anchor cell surface proteins, including some enzymes for plant degradation. Once the secreted CAZy cleave insoluble plant polysaccharides into oligosaccharides, they are taken into the cell to be further degraded by intracellular CAZy, enabling more efficient sugar transport, conserving energy by phosphorolytic cleavage, and ensuring the sugar monomers were not available to competing microbes. Sugars are catabolized by EMP glycolysis incorporating reversible, PPi-dependent glycolytic enzymes, and pyruvate ferredoxin oxidoreductase. The genome encodes seven alcohol dehydrogenases, among which two iron-dependent enzymes are highly expressed and likely facilitate the high ethanol yields. Growth on cellulose also resulted in indirect changes such as increased tryptophan and nicotinamide synthesis and repression of fatty acid synthesis. We distilled the data into a model showing the highly expressed enzymes enabling efficient cellulosic fermentation by C. phytofermentans (Figure 7). Collectively, these data help understand how bacteria recycle plant biomass works towards enabling the use of plant biomass as a low-cost chemical feedstock.
Fermentation of plant biomass by microbes like Clostridium phytofermentans recycles carbon globally and can make biofuels from inedible feedstocks. We analyzed C. phytofermentans fermenting cellulosic substrates by integrating quantitative mass spectrometry of more than 2500 proteins with measurements of growth, enzyme activities, fermentation products, and electron microscopy. Absolute protein concentrations were estimated using Absolute Protein EXpression (APEX); relative changes between treatments were quantified with chemical stable isotope labeling by reductive dimethylation (ReDi). We identified the different combinations of carbohydratases used to degrade cellulose and hemicellulose, many of which were secreted based on quantification of supernatant proteins, as well as the repertoires of glycolytic enzymes and alcohol dehydrogenases (ADHs) enabling ethanol production at near maximal yields. Growth on cellulose also resulted in diverse changes such as increased expression of tryptophan synthesis proteins and repression of proteins for fatty acid metabolism and cell motility. This study gives a systems-level understanding of how this microbe ferments biomass and provides a rational, empirical basis to identify engineering targets for industrial cellulosic fermentation.
PMCID: PMC3049413  PMID: 21245846
bioenergy; clostridium; proteomics
9.  Brassinosteroids are involved in response of cucumber (Cucumis sativus) to iron deficiency 
Annals of Botany  2012;110(3):681-688.
Background and Aims
Brassinosteroids (BR) are a class of plant polyhydroxysteroids with diverse functions in plant growth and development. However, there is little information on the role of BRs played in the response to nutrient deficiency.
To evaluate the role of BR in the response of plants to iron (Fe) deficiency, the effect of 24-epibrassinolide (EBR) on ferric reductase (FRO) activity, acidification of the rhizosphere and Fe content in cucumber (Cucumis sativus) seedlings under Fe-deficient (1 µm FeEDTA) and Fe-sufficient (50 µm FeEDTA) conditions were investigated.
Key Results
There was a significant increase in FRO activity upon exposure of cucumber seedlings to an Fe-deficient medium, and the Fe deficiency-induced increase in FRO activity was substantially suppressed by EBR. In contrast, application of EBR to Fe-sufficient seedlings stimulated FRO activity. Ethylene production evoked by Fe deficiency was suppressed by EBR, while EBR induced ethylene production from Fe-sufficient seedlings. Fe contents in shoots were reduced by treatment with EBR, while Fe contents in roots were markedly increased under both Fe-deficient and Fe-sufficient conditions. The reductions in Fe contents of shoots were independent of chlorophyll (CHL) contents under Fe-sufficient conditions, but they were positively correlated with CHL contents under Fe-deficient conditions. At the transcriptional level, transcripts encoding FRO (CsFRO1) and Fe transporter (CsIRT1) were increased upon exposure to the Fe-deficient medium, and the increases in transcripts were reversed by EBR.
The results demonstrate that BRs are likely to play a negative role in regulating Fe-deficiency-induced FRO, expressions of CsFRO1 and CsIRT1, as well as Fe translocation from roots to shoots.
PMCID: PMC3400454  PMID: 22684685
Brassinosteroids; iron deficiency; cucumber; Cucumis sativus; ferric reductase activity; Fe translocation
10.  Understanding the physiology of Lactobacillus plantarum at zero growth 
The physiology of Lactobacillus plantarum at extremely low growth rates, through cultivation in retentostats, is much closer to carbon-limited growth than to stationary phase, as evidenced from transcriptomics data, metabolic fluxes, and biomass composition and viability.Using a genome-scale metabolic model and constraint-based computational analyses, amino-acid fluxes—in particular, the rather paradoxical excretion of Asp, Arg, Met, and Ala—could be rationalized as a means to allow extensive metabolism of other amino acids, that is, that of branched-chain and aromatic amino acids.Catabolic products from aromatic amino acids are known to have putative plant-hormone action. The metabolism of amino acids, as well as transcription data, strongly suggested a plant environment-like response in slow-growing L. plantarum, which was confirmed by significant effects of fermented medium on plant root formation.
Natural ecosystems are usually characterized by extremely low and fluctuating nutrient availability. Hence, microorganisms in these environments live a ‘feast-and-famine' existence, with famine the most habitual state. As a result, extremely slow or no growth is the most common state of bacteria, and maintenance processes dominate their life.
In the present study, Lactobacillus plantarum was used as a model microorganism to investigate the physiology of slow growth. Besides fermented foods, this microorganism can be observed in a variety of environmental niches, including plants and lakes, in which nutrient supply is limited. To mimic these conditions, L. plantarum was grown in a glucose-limited chemostat with complete biomass retention (retentostat). During cultivation, biomass progressively accumulated, resulting in steadily decreasing specific substrate availability. Less energy was thus available for growth, and the specific growth rate decreased accordingly, with a final calculated doubling time greater than one year. Detailed measurements of metabolic fluxes were used as constraints in a genome-scale metabolic model to precisely calculate the amount of energy used for net biomass synthesis and for maintenance purposes: at the lowest growth rate investigated (μ=0.0002 h−1), maintenance accounted for 94% of all energy expenses.
Genome-scale metabolic analysis was used in combination with transcriptomics to study the adaptation of L. plantarum to extremely slow growth under limited carbon and energy supply. Importantly, slow growth as investigated here was fundamentally different from the widely studied carbon starvation-induced stationary phase: non-growing cells in retentostat conditions were glucose limited rather than starved, and the transition from a growing to a non-growing state under retentostat conditions was progressive, in contrast with the abrupt transition in batch cultures. These differences were reflected in various aspects of the cell physiology.
The metabolic behavior was remarkably stable during adaptation to slow growth. Although carbon catabolite repression was clearly relieved, as indicated by the upregulation of genes for the utilization of alternative carbohydrates, the metabolism remained largely based on the conversion of glucose to lactate.
Stress resistance mechanisms were also not massively induced. In particular, analysis of the biomass composition—which remained similar to fast-growing cells even under virtually non-growing conditions—and of the gene expression profile, failed to reveal clear stringent or general stress responses, which are generally triggered in glucose-starved cells. The observation that genes involved in growth-associated processes were not downregulated suggested that active synthesis of biomass components (RNA, proteins, and membranes) was required to account for the observed stable biomass and that turnover of macromolecules was high in slow-growing cells. Biomass viability or morphology was also not affected, compared with faster growth conditions. The only typical stress response was the induction of an SOS response—in particular, the upregulation of the two error-prone DNA polymerases—suggesting an increased potential for genetic diversity under adverse conditions. Although diversity was not apparent under the conditions studied here, such mechanisms of increased rates of mutagenesis are likely to have an important role in the adaptation of L. plantarum to slow growth.
A surprising response of L. plantarum during adaptation to slow growth was the production of several amino acids (Arg, Asp, Met, and Ala). A priori, this metabolic behavior seemed inefficient in a context of energy limitation. However, reduced cost analysis using the genome-scale metabolic model indicated that it had a positive effect on energy generation. In-depth analysis of metabolic flux distributions showed that biosynthesis of these amino acids was connected to the catabolism of branched-chain and aromatic amino acids (BCAAs and AAAs), under conditions of limited ammonium efflux. At a fixed ammonium efflux—fixed at the measured value—flux balance analysis indicated that BCAAs and AAAs were expensive to metabolize, because the regeneration of 2-ketoglutarate through glutamate dehydrogenase was limited by ammonium dissipation. Therefore, alternative pathways had to be active to supply the necessary pool of 2-ketoglutarate. At low growth rates, amino-acid production (Arg, Asp, Ala, and Met) accounted for most of the 2-ketoglutarate regeneration. Although it came at the expense of ATP, this metabolic alternative to glutamate dehydrogenase was less energy costly than other solutions such as purine biosynthesis. This is thus an excellent example in which precise, quantitative modeling results in new insights in physiology that intuition would never have achieved. It also shows that flux balance analysis can be used to accurately predict energetically inefficient metabolism, provided the appropriate fluxes are constrained (here, ammonium efflux).
The observation that BCAAs and AAAs were catabolized at the expense of energy was intriguing. However, several end products of these catabolic pathways can serve as signaling molecules for interactions with other organisms. In particular, precursors of plant hormones were predicted as possible end products in the model simulations. Accordingly, the production of compounds interfering with plant root development was demonstrated in slow-growing L. plantarum. The metabolic analysis thus suggested that slow-growing L. plantarum produced plant hormones—or precursors thereof—as a strategy to divert the plant metabolism towards its own interest. In support of this view, transcriptome analysis indicated the upregulation of genes involved in the catabolism of β-glucosides—typical sugars from plant cell wall—as well as a very high induction of six gene clusters encoding cell-surface protein complexes predicted to have a role in the utilization of plant polysaccharides (csc clusters). In such a plant context, limited ammonium production would also make sense, because of the well-documented toxicity of ammonium for plants: production of amino acids could represent an alternative to ammonium excretion while keeping both parties satisfied.
In conclusion, the physiology of L. plantarum at extremely low growth rates, as studied by genome-scale metabolic modeling and transcriptomics, is fundamentally different from that of starvation-induced stationary phase cells. Excitingly, these conditions seem to trigger responses that favor interactions with the environment, more specifically with plants. The reported observations were made in the absence of any plant-derived material, suggesting that this response might constitute a hardwired behavior.
Situations of extremely low substrate availability, resulting in slow growth, are common in natural environments. To mimic these conditions, Lactobacillus plantarum was grown in a carbon-limited retentostat with complete biomass retention. The physiology of extremely slow-growing L. plantarum—as studied by genome-scale modeling and transcriptomics—was fundamentally different from that of stationary-phase cells. Stress resistance mechanisms were not massively induced during transition to extremely slow growth. The energy-generating metabolism was remarkably stable and remained largely based on the conversion of glucose to lactate. The combination of metabolic and transcriptomic analyses revealed behaviors involved in interactions with the environment, more particularly with plants: production of plant hormones or precursors thereof, and preparedness for the utilization of plant-derived substrates. Accordingly, the production of compounds interfering with plant root development was demonstrated in slow-growing L. plantarum. Thus, conditions of slow growth and limited substrate availability seem to trigger a plant environment-like response, even in the absence of plant-derived material, suggesting that this might constitute an intrinsic behavior in L. plantarum.
PMCID: PMC2964122  PMID: 20865006
Lactobacillus plantarum; metabolic modeling; retentostat; slow growth; transcriptome analysis
11.  Trichoderma-Plant Root Colonization: Escaping Early Plant Defense Responses and Activation of the Antioxidant Machinery for Saline Stress Tolerance 
PLoS Pathogens  2013;9(3):e1003221.
Trichoderma spp. are versatile opportunistic plant symbionts which can colonize the apoplast of plant roots. Microarrays analysis of Arabidopsis thaliana roots inoculated with Trichoderma asperelloides T203, coupled with qPCR analysis of 137 stress responsive genes and transcription factors, revealed wide gene transcript reprogramming, proceeded by a transient repression of the plant immune responses supposedly to allow root colonization. Enhancement in the expression of WRKY18 and WRKY40, which stimulate JA-signaling via suppression of JAZ repressors and negatively regulate the expression of the defense genes FMO1, PAD3 and CYP71A13, was detected in Arabidopsis roots upon Trichoderma colonization. Reduced root colonization was observed in the wrky18/wrky40 double mutant line, while partial phenotypic complementation was achieved by over-expressing WRKY40 in the wrky18 wrky40 background. On the other hand increased colonization rate was found in roots of the FMO1 knockout mutant. Trichoderma spp. stimulate plant growth and resistance to a wide range of adverse environmental conditions. Arabidopsis and cucumber (Cucumis sativus L.) plants treated with Trichoderma prior to salt stress imposition show significantly improved seed germination. In addition, Trichoderma treatment affects the expression of several genes related to osmo-protection and general oxidative stress in roots of both plants. The MDAR gene coding for monodehydroascorbate reductase is significantly up-regulated and, accordingly, the pool of reduced ascorbic acid was found to be increased in Trichoderma treated plants. 1-Aminocyclopropane-1-carboxylate (ACC)-deaminase silenced Trichoderma mutants were less effective in providing tolerance to salt stress, suggesting that Trichoderma, similarly to ACC deaminase producing bacteria, can ameliorate plant growth under conditions of abiotic stress, by lowering ameliorating increases in ethylene levels as well as promoting an elevated antioxidative capacity.
Author Summary
Trichoderma fungi have been developed as biocontrol agents and are applied to protect and improve crop yields. Colonization of plant roots by Trichoderma can protect plants against diseases and environmental stresses such as salinity and drought, and an improve plant growth and development. To better understand the mechanism underlining the plant-Trichoderma interaction we followed changes in global gene expression in colonized Arabidopsis roots. We associate the known gene biological function to the processes of root colonization and abiotic stress tolerance mediated by Trichoderma. Using Arabidopsis mutant lines we show the function of a subset of those genes in root colonization. We show that wrky18 and wrky40 transcription factors activate and suppress the expression of different genes in order to allow successful root colonization. We also combine the gene expression data together with the measurement of ascorbic acid level to demonstrate that salt stress tolerance offered by Trichoderma is dependent on activation of the plant antioxidant defense machinery. Using Trichoderma lines mutated in the ACC deaminase gene, we demonstrate that reduction of ethylene levels is also essential in achieving salt tolerance. This study represents an important step forward in understanding the nature of the non-pathogenic plant Trichoderma interaction, and may contribute to the efforts to improve Trichoderma biocontrol abilities.
PMCID: PMC3597500  PMID: 23516362
12.  Cadmium Toxicity Induced Alterations in the Root Proteome of Green Gram in Contrasting Response towards Iron Supplement 
Cadmium signifies a severe threat to crop productivity and green gram is a notably iron sensitive plant which shows considerable variation towards cadmium stress. A gel-based proteomics analysis was performed with the roots of green gram exposed to iron and cadmium combined treatments. The resulting data show that twenty three proteins were down-regulated in iron-deprived roots either in the absence (−Fe/−Cd) or presence (−Fe/+Cd) of cadmium. These down-regulated proteins were however well expressed in roots under iron sufficient conditions, even in the presence of cadmium (+Fe/+Cd). The functional classification of these proteins determined that 21% of the proteins are associated with nutrient metabolism. The other proteins in higher quantities are involved in either transcription or translation regulation, and the rest are involved in biosynthesis metabolism, antioxidant pathways, molecular chaperones and stress response. On the other hand, several protein spots were also absent in roots in response to iron deprivation either in absence (−Fe/−Cd) or presence (−Fe/+Cd) of cadmium but were well expressed in the presence of iron (+Fe/+Cd). Results suggest that green gram plants exposed to cadmium stress are able to change the nutrient metabolic balance in roots, but in the mean time regulate cadmium toxicity through iron supplements.
PMCID: PMC4013632  PMID: 24739807
cadmium toxicity; iron suppliment; proteomics
13.  Proteomic analysis of iron acquisition, metabolic and regulatory responses of Yersinia pestis to iron starvation 
BMC Microbiology  2010;10:30.
The Gram-negative bacterium Yersinia pestis is the causative agent of the bubonic plague. Efficient iron acquisition systems are critical to the ability of Y. pestis to infect, spread and grow in mammalian hosts, because iron is sequestered and is considered part of the innate host immune defence against invading pathogens. We used a proteomic approach to determine expression changes of iron uptake systems and intracellular consequences of iron deficiency in the Y. pestis strain KIM6+ at two physiologically relevant temperatures (26°C and 37°C).
Differential protein display was performed for three Y. pestis subcellular fractions. Five characterized Y. pestis iron/siderophore acquisition systems (Ybt, Yfe, Yfu, Yiu and Hmu) and a putative iron/chelate outer membrane receptor (Y0850) were increased in abundance in iron-starved cells. The iron-sulfur (Fe-S) cluster assembly system Suf, adapted to oxidative stress and iron starvation in E. coli, was also more abundant, suggesting functional activity of Suf in Y. pestis under iron-limiting conditions. Metabolic and reactive oxygen-deactivating enzymes dependent on Fe-S clusters or other iron cofactors were decreased in abundance in iron-depleted cells. This data was consistent with lower activities of aconitase and catalase in iron-starved vs. iron-rich cells. In contrast, pyruvate oxidase B which metabolizes pyruvate via electron transfer to ubiquinone-8 for direct utilization in the respiratory chain was strongly increased in abundance and activity in iron-depleted cells.
Many protein abundance differences were indicative of the important regulatory role of the ferric uptake regulator Fur. Iron deficiency seems to result in a coordinated shift from iron-utilizing to iron-independent biochemical pathways in the cytoplasm of Y. pestis. With growth temperature as an additional variable in proteomic comparisons of the Y. pestis fractions (26°C and 37°C), there was little evidence for temperature-specific adaptation processes to iron starvation.
PMCID: PMC2835676  PMID: 20113483
14.  Iron-Induced Changes in the Proteome of Trichomonas vaginalis Hydrogenosomes 
PLoS ONE  2013;8(5):e65148.
Iron plays a crucial role in metabolism as a key component of catalytic and redox cofactors, such as heme or iron-sulfur clusters in enzymes and electron-transporting or regulatory proteins. Limitation of iron availability by the host is also one of the mechanisms involved in immunity. Pathogens must regulate their protein expression according to the iron concentration in their environment and optimize their metabolic pathways in cases of limitation through the availability of respective cofactors. Trichomonas vaginalis, a sexually transmitted pathogen of humans, requires high iron levels for optimal growth. It is an anaerobe that possesses hydrogenosomes, mitochondrion-related organelles that harbor pathways of energy metabolism and iron-sulfur cluster assembly. We analyzed the proteomes of hydrogenosomes obtained from cells cultivated under iron-rich and iron-deficient conditions employing two-dimensional peptide separation combining IEF and nano-HPLC with quantitative MALDI-MS/MS. We identified 179 proteins, of which 58 were differentially expressed. Iron deficiency led to the upregulation of proteins involved in iron-sulfur cluster assembly and the downregulation of enzymes involved in carbohydrate metabolism. Interestingly, iron affected the expression of only some of multiple protein paralogues, whereas the expression of others was iron independent. This finding indicates a stringent regulation of differentially expressed multiple gene copies in response to changes in the availability of exogenous iron.
PMCID: PMC3669245  PMID: 23741475
15.  Nitrate transport in cucumber leaves is an inducible process involving an increase in plasma membrane H+-ATPase activity and abundance 
BMC Plant Biology  2012;12:66.
The mechanisms by which nitrate is transported into the roots have been characterized both at physiological and molecular levels. It has been demonstrated that nitrate is taken up in an energy-dependent way by a four-component uptake machinery involving high- and low- affinity transport systems. In contrast very little is known about the physiology of nitrate transport towards different plant tissues and in particular at the leaf level.
The mechanism of nitrate uptake in leaves of cucumber (Cucumis sativus L. cv. Chinese long) plants was studied and compared with that of the root. Net nitrate uptake by roots of nitrate-depleted cucumber plants proved to be substrate-inducible and biphasic showing a saturable kinetics with a clear linear non saturable component at an anion concentration higher than 2 mM. Nitrate uptake by leaf discs of cucumber plants showed some similarities with that operating in the roots (e.g. electrogenic H+ dependence via involvement of proton pump, a certain degree of induction). However, it did not exhibit typical biphasic kinetics and was characterized by a higher Km with values out of the range usually recorded in roots of several different plant species. The quantity and activity of plasma membrane (PM) H+-ATPase of the vesicles isolated from leaf tissues of nitrate-treated plants for 12 h (peak of nitrate foliar uptake rate) increased with respect to that observed in the vesicles isolated from N-deprived control plants, thus suggesting an involvement of this enzyme in the leaf nitrate uptake process similar to that described in roots. Molecular analyses suggest the involvement of a specific isoform of PM H+-ATPase (CsHA1) and NRT2 transporter (CsNRT2) in root nitrate uptake. At the leaf level, nitrate treatment modulated the expression of CsHA2, highlighting a main putative role of this isogene in the process.
Obtained results provide for the first time evidence that a saturable and substrate-inducible nitrate uptake mechanism operates in cucumber leaves. Its activity appears to be related to that of PM H+-ATPase activity and in particular to the induction of CsHA2 isoform. However the question about the molecular entity responsible for the transport of nitrate into leaf cells therefore still remains unresolved.
PMCID: PMC3403936  PMID: 22571503
16.  Chill-Induced Decrease in Capacity of RuBP Carboxylation and Associated H2O2 Accumulation in Cucumber Leaves are Alleviated by Grafting onto Figleaf Gourd 
Annals of Botany  2007;100(4):839-848.
Background and Aims
Chilling results in a significant decrease in Rubisco content and increased generation of reactive oxygen species (ROS) in cucumber (Cucumis sativus), a chilling-sensitive species. The role of roots in the regulation of the tolerance is unknown. Here, cucumber plants grafted onto figleaf gourd (Cucurbita ficifolia), a chilling-tolerant species were used to study the role of roots in the regulation of shoot functioning and the associated root-to-shoot communication.
Gas exchange and chlorophyll fluorescence were measured using an infrared gas analyser combined with a pulse amplitude fluorimeter during chilling at 14 °C or 7 °C and subsequent recovery. At the same time, Rubisco content and activity and ROS generation were spectrophotometrically assayed. Abscisic acid and cytokinin concentrations in xylem sap were also determined by enzyme-linked immunosorbent assay.
Key Results and Conclusions
Grafted plants showed a significantly higher light-saturated rate of CO2 assimilation (Asat) than own-rooted plants when roots were gradually cooled, but no differences were detected when shoots were cooled. Chill at 7 °C irreversibly reduced Asat, and significantly decreased maximum carboxylation activity, Rubisco content and initial Rubisco activity. However, grafted plants showed weaker inhibition, together with decreased electron flux in the water–water cycle. Higher activity of antioxidant enzymes with less ROS production was found in grafted plants. In addition, ABA concentration increased by 48·4-fold whilst cytokinin concentration decreased by 91·5 % in the xylem sap of own-rooted plants after exposure to a 7 °C chill. In comparison, ABA and cytokinin concentrations increased by 10·5-fold and 36·9 %, respectively, for the grafted plants. Improved plant growth was also observed in grafted plants after the chill. These results suggest that some signals coming from chilling-resistant roots (i.e. ABA and cytokinins) protect leaf photosynthesis in shoots of chilling-sensitive plants.
PMCID: PMC2749637  PMID: 17761689
Chilling; chlorophyll fluorescence; Cucumis sativus; electron transport flux; grafting; photosynthesis; reactive oxygen species; water–water cycle
17.  Body iron delocalization: the serious drawback in iron disorders in both developing and developed countries 
Pathogens and Global Health  2012;106(4):200-216.
Over 2 billion people in both developing as well as developed countries – over 30% of the world’s population – are anaemic. With the classical preconception that oral iron administration or the intake of foods rich in iron increase haemoglobin concentration and reduce the prevalence of anaemia, specific programs have been designed, but iron supplementations have been less effective than expected. Of note, this hazardous simplification on iron status neglects its distribution in the body. The correct balance of iron, defined iron homeostasis, involves a physiological ratio of iron between tissues/secretions and blood, thus avoiding its delocalization as iron accumulation in tissues/secretions and iron deficiency in blood. Changes in iron status can affect the inflammatory response in multiple ways, particularly in the context of infection, an idea that is worth remembering when considering the value of iron supplementation in areas of the world where infections are highly prevalent. The enhanced availability of free iron can increase susceptibility and severity of microbial and parasitic infections.
The discovery of the hepcidin–ferroportin (Fpn) complex, which greatly clarified the enigmatic mechanism that supervises the iron homeostasis, should prompt to a critical review on iron supplementation, ineffective on the expression of the most important proteins of iron metabolism. Therefore, it is imperative to consider new safe and efficient therapeutic interventions to cure iron deficiency (ID) and ID anaemia (IDA) associated or not to the inflammation.
In this respect, lactoferrin (Lf) is emerging as an important regulator of both iron and inflammatory homeostasis. Oral administration of Lf in subjects suffering of ID and IDA is safe and effective in significantly increasing haematological parameters and contemporary decreasing serum IL-6 levels, thus restoring iron localization through the direct or indirect modulation of hepcidin and ferroportin synthesis. Of note, the nuclear localization of Lf suggests that this molecule may be involved in the transcriptional regulation of some genes of host inflammatory response.
We recently also reported that combined administration of oral and intravaginal Lf on ID and IDA pregnant women with preterm delivery threat, significantly increased haematological parameters, reduced IL-6 levels in both serum and cervicovaginal fluid, cervicovaginal prostaglandin PGF2α, and suppressed uterine contractility. Moreover, Lf combined administration blocked further the shortening of cervical length and the increase of foetal fibronectin, thus prolonging the length of pregnancy until the 37th–38th week of gestation.
These new Lf functions effective in curing ID and IDA through the restoring of iron and inflammatory homeostasis and in preventing preterm delivery, could have a great relevance in developing countries, where ID and IDA and inflammation-associated anaemia represent the major risk factors of preterm delivery and maternal and neonatal death.
PMCID: PMC4001586  PMID: 23265420
anaemia; iron deficiency; lactoferrin; pregnancy; malaria; iron homeostasis; inflammatory homeostasis
18.  Analysis of the Proteome of Intracellular Shigella flexneri Reveals Pathways Important for Intracellular Growth 
Infection and Immunity  2013;81(12):4635-4648.
Global proteomic analysis was performed with Shigella flexneri strain 2457T in association with three distinct growth environments: S. flexneri growing in broth (in vitro), S. flexneri growing within epithelial cell cytoplasm (intracellular), and S. flexneri that were cultured with, but did not invade, Henle cells (extracellular). Compared to in vitro and extracellular bacteria, intracellular bacteria had increased levels of proteins required for invasion and cell-to-cell spread, including Ipa, Mxi, and Ics proteins. Changes in metabolic pathways in response to the intracellular environment also were evident. There was an increase in glycogen biosynthesis enzymes, altered expression of sugar transporters, and a reduced amount of the carbon storage regulator CsrA. Mixed acid fermentation enzymes were highly expressed intracellularly, while tricarboxylic acid (TCA) cycle oxidoreductive enzymes and most electron transport chain proteins, except CydAB, were markedly decreased. This suggested that fermentation and the CydAB system primarily sustain energy generation intracellularly. Elevated levels of PntAB, which is responsible for NADPH regeneration, suggested a shortage of reducing factors for ATP synthesis. These metabolic changes likely reflect changes in available carbon sources, oxygen levels, and iron availability. Intracellular bacteria showed strong evidence of iron starvation. Iron acquisition systems (Iut, Sit, FhuA, and Feo) and the iron starvation, stress-associated Fe-S cluster assembly (Suf) protein were markedly increased in abundance. Mutational analysis confirmed that the mixed-acid fermentation pathway was required for wild-type intracellular growth and spread of S. flexneri. Thus, iron stress and changes in carbon metabolism may be key factors in the S. flexneri transition from the extra- to the intracellular milieu.
PMCID: PMC3837999  PMID: 24101689
19.  Genome and low-iron response of an oceanic diatom adapted to chronic iron limitation 
Genome Biology  2012;13(7):R66.
Biogeochemical elemental cycling is driven by primary production of biomass via phototrophic phytoplankton growth, with 40% of marine productivity being assigned to diatoms. Phytoplankton growth is widely limited by the availability of iron, an essential component of the photosynthetic apparatus. The oceanic diatom Thalassiosira oceanica shows a remarkable tolerance to low-iron conditions and was chosen as a model for deciphering the cellular response upon shortage of this essential micronutrient.
The combined efforts in genomics, transcriptomics and proteomics reveal an unexpected metabolic flexibility in response to iron availability for T. oceanica CCMP1005. The complex response comprises cellular retrenchment as well as remodeling of bioenergetic pathways, where the abundance of iron-rich photosynthetic proteins is lowered, whereas iron-rich mitochondrial proteins are preserved. As a consequence of iron deprivation, the photosynthetic machinery undergoes a remodeling to adjust the light energy utilization with the overall decrease in photosynthetic electron transfer complexes.
Beneficial adaptations to low-iron environments include strategies to lower the cellular iron requirements and to enhance iron uptake. A novel contribution enhancing iron economy of phototrophic growth is observed with the iron-regulated substitution of three metal-containing fructose-bisphosphate aldolases involved in metabolic conversion of carbohydrates for enzymes that do not contain metals. Further, our data identify candidate components of a high-affinity iron-uptake system, with several of the involved genes and domains originating from duplication events. A high genomic plasticity, as seen from the fraction of genes acquired through horizontal gene transfer, provides the platform for these complex adaptations to a low-iron world.
PMCID: PMC3491386  PMID: 22835381
20.  Identification of candidate genes involved in early iron deficiency chlorosis signaling in soybean (Glycine max) roots and leaves 
BMC Genomics  2014;15(1):702.
Iron is an essential micronutrient for all living things, required in plants for photosynthesis, respiration and metabolism. A lack of bioavailable iron in soil leads to iron deficiency chlorosis (IDC), causing a reduction in photosynthesis and interveinal yellowing of leaves. Soybeans (Glycine max (L.) Merr.) grown in high pH soils often suffer from IDC, resulting in substantial yield losses. Iron efficient soybean cultivars maintain photosynthesis and have higher yields under IDC-promoting conditions than inefficient cultivars.
To capture signaling between roots and leaves and identify genes acting early in the iron efficient cultivar Clark, we conducted a RNA-Seq study at one and six hours after replacing iron sufficient hydroponic media (100 μM iron(III) nitrate nonahydrate) with iron deficient media (50 μM iron(III) nitrate nonahydrate). At one hour of iron stress, few genes were differentially expressed in leaves but many were already changing expression in roots. By six hours, more genes were differentially expressed in the leaves, and a massive shift was observed in the direction of gene expression in both roots and leaves. Further, there was little overlap in differentially expressed genes identified in each tissue and time point.
Genes involved in hormone signaling, regulation of DNA replication and iron uptake utilization are key aspects of the early iron-efficiency response. We observed dynamic gene expression differences between roots and leaves, suggesting the involvement of many transcription factors in eliciting rapid changes in gene expression. In roots, genes involved iron uptake and development of Casparian strips were induced one hour after iron stress. In leaves, genes involved in DNA replication and sugar signaling responded to iron deficiency. The differentially expressed genes (DEGs) and signaling components identified here represent new targets for soybean improvement.
Electronic supplementary material
The online version of this article (doi:10.1186/1471-2164-15-702) contains supplementary material, which is available to authorized users.
PMCID: PMC4161901  PMID: 25149281
Iron deficiency chlorosis; Soybean; Glycine max; RNA-Seq
21.  Genome-Wide Analysis of the bZIP Transcription Factors in Cucumber 
PLoS ONE  2014;9(4):e96014.
bZIP proteins are one of the largest transcriptional regulators playing crucial roles in plant development, physiological processes, and biotic/abiotic stress responses. Despite the availability of recently published draft genome sequence of Cucumis sativus, no comprehensive investigation of these family members has been presented for cucumber. We have identified 64 bZIP transcription factor-encoding genes in the cucumber genome. Based on structural features of their encoded proteins, CsbZIP genes could be classified into 6 groups. Cucumber bZIP genes were expanded mainly by segmental duplication rather than tandem duplication. Although segmental duplication rate of the CsbZIP genes was lower than that of Arabidopsis, rice and sorghum, it was observed as a common expansion mechanism. Some orthologous relationships and chromosomal rearrangements were observed according to comparative mapping analysis with other species. Genome-wide expression analysis of bZIP genes indicated that 64 CsbZIP genes were differentially expressed in at least one of the ten sampled tissues. A total of 4 CsbZIP genes displayed higher expression values in leaf, flowers and root tissues. The in silico micro-RNA (miRNA) and target transcript analyses identified that a total of 21 CsbZIP genes were targeted by 38 plant miRNAs. CsbZIP20 and CsbZIP22 are the most targeted by miR165 and miR166 family members, respectively. We also analyzed the expression of ten CsbZIP genes in the root and leaf tissues of drought-stressed cucumber using quantitative RT-PCR. All of the selected CsbZIP genes were measured as increased in root tissue at 24th h upon PEG treatment. Contrarily, the down-regulation was observed in leaf tissues of all analyzed CsbZIP genes. CsbZIP12 and CsbZIP44 genes showed gradual induction of expression in root tissues during time points. This genome-wide identification and expression profiling provides new opportunities for cloning and functional analyses, which may be used in further studies for improving stress tolerance in plants.
PMCID: PMC3997510  PMID: 24760072
22.  The Genome Sequence of the North-European Cucumber (Cucumis sativus L.) Unravels Evolutionary Adaptation Mechanisms in Plants 
PLoS ONE  2011;6(7):e22728.
Cucumber (Cucumis sativus L.), a widely cultivated crop, has originated from Eastern Himalayas and secondary domestication regions includes highly divergent climate conditions e.g. temperate and subtropical. We wanted to uncover adaptive genome differences between the cucumber cultivars and what sort of evolutionary molecular mechanisms regulate genetic adaptation of plants to different ecosystems and organism biodiversity. Here we present the draft genome sequence of the Cucumis sativus genome of the North-European Borszczagowski cultivar (line B10) and comparative genomics studies with the known genomes of: C. sativus (Chinese cultivar – Chinese Long (line 9930)), Arabidopsis thaliana, Populus trichocarpa and Oryza sativa. Cucumber genomes show extensive chromosomal rearrangements, distinct differences in quantity of the particular genes (e.g. involved in photosynthesis, respiration, sugar metabolism, chlorophyll degradation, regulation of gene expression, photooxidative stress tolerance, higher non-optimal temperatures tolerance and ammonium ion assimilation) as well as in distributions of abscisic acid-, dehydration- and ethylene-responsive cis-regulatory elements (CREs) in promoters of orthologous group of genes, which lead to the specific adaptation features. Abscisic acid treatment of non-acclimated Arabidopsis and C. sativus seedlings induced moderate freezing tolerance in Arabidopsis but not in C. sativus. This experiment together with analysis of abscisic acid-specific CRE distributions give a clue why C. sativus is much more susceptible to moderate freezing stresses than A. thaliana. Comparative analysis of all the five genomes showed that, each species and/or cultivars has a specific profile of CRE content in promoters of orthologous genes. Our results constitute the substantial and original resource for the basic and applied research on environmental adaptations of plants, which could facilitate creation of new crops with improved growth and yield in divergent conditions.
PMCID: PMC3145757  PMID: 21829493
23.  The metal transporter ZIP13 supplies iron into the secretory pathway in Drosophila melanogaster 
eLife  2014;3:e03191.
The intracellular iron transfer process is not well understood, and the identity of the iron transporter responsible for iron delivery to the secretory compartments remains elusive. In this study, we show Drosophila ZIP13 (Slc39a13), a presumed zinc importer, fulfills the iron effluxing role. Interfering with dZIP13 expression causes iron-rescuable iron absorption defect, simultaneous iron increase in the cytosol and decrease in the secretory compartments, failure of ferritin iron loading, and abnormal collagen secretion. dZIP13 expression in E. coli confers upon the host iron-dependent growth and iron resistance. Importantly, time-coursed transport assays using an iron isotope indicated a potent iron exporting activity of dZIP13. The identification of dZIP13 as an iron transporter suggests that the spondylocheiro dysplastic form of Ehlers–Danlos syndrome, in which hZIP13 is defective, is likely due to a failure of iron delivery to the secretory compartments. Our results also broaden our knowledge of the scope of defects from iron dyshomeostasis.
eLife digest
Iron is essential for life. Amongst its many important roles, iron is crucial for producing collagen—the protein that provides both strength and elasticity to bones, tendons, ligaments, and skin. Like many other proteins, collagens are produced inside the endoplasmic reticulum—an organelle inside the cell that is enclosed by a membrane that is similar to the plasma membrane that surrounds the cell itself.
Two enzymes that are critical for producing collagen need to bind with iron in order to work correctly. To do this, iron in the cytoplasm of the cell has to cross the membrane that surrounds the endoplasmic reticulum. Small molecules are commonly transported across membranes by proteins called transporters, which tend to work on specific types of ions or molecules. However, researchers did not know the identity of the membrane transporter responsible for moving iron into the secretory pathway—including the endoplasmic reticulum—to bind with the enzymes that produce collagen.
Xiao, Wan et al. have now investigated the function of the transporter ZIP13 in the fruit fly Drosophila. This transporter was thought to transport zinc across membranes and into the cytoplasm. Instead, Xiao, Wan et al. found that ZIP13 transports iron out of the cytoplasm and into the endoplasmic reticulum.
Ehlers–Danlos syndrome is a condition that causes individuals to suffer from frequent joint dislocations, bone deformities, and fragile skin as a result of their body producing collagen incorrectly. One form of Ehlers–Danlos syndrome is caused by ZIP13 transporters working incorrectly. However, this was difficult to understand when it was thought that ZIP13 only transports zinc. The discovery that ZIP13 mostly transports iron rather than zinc can explain the link between this transporter and Ehlers–Danlos syndrome: if ZIP13 doesn't work, the collagen-building enzymes cannot get the iron they need to work properly.
Disorders caused by iron deficiencies are normally identified by a few tell-tale symptoms, such as anemia, but these are not seen in Ehlers–Danlos syndrome. Xiao, Wan et al. suggest that iron transport problems could therefore be behind a wider range of diseases and disorders than is currently known.
PMCID: PMC4130162  PMID: 25006035
Slc39a13; iron transporter; secretory compartments; Ehlers–Danlos syndrome; Drosophila; D. melanogaster
24.  Early iron-deficiency-induced transcriptional changes in Arabidopsis roots as revealed by microarray analyses 
BMC Genomics  2009;10:147.
Iron (Fe) is an essential nutrient in plants and animals, and Fe deficiency results in decreased vitality and performance. Due to limited bio-availability of Fe, plants have evolved sophisticated adaptive alterations in development, biochemistry and metabolism that are mainly regulated at the transcriptional level. We have investigated the early transcriptional response to Fe deficiency in roots of the model plant Arabidopsis, using a hydroponic system that permitted removal of Fe from the nutrient solution within seconds and transferring large numbers of plants with little or no mechanical damage to the root systems. We feel that this experimental approach offers significant advantages over previous and recent DNA microarray investigations of the Fe-deficiency response by increasing the resolution of the temporal response and by decreasing non-Fe deficiency-induced transcriptional changes, which are common in microarray analyses.
The expression of sixty genes were changed after 6 h of Fe deficiency and 65% of these were found to overlap with a group of seventy-nine genes that were altered after 24 h. A disproportionally high number of transcripts encoding ion transport proteins were found, which function to increase the Fe concentration and decrease the zinc (Zn) concentration in the cytosol. Analysis of global changes in gene expression revealed that changes in Fe availability were associated with the differential expression of genes that encode transporters with presumed function in uptake and distribution of transition metals other than Fe. It appeared that under conditions of Fe deficiency, the capacity for Zn uptake increased, most probably the result of low specificity of the Fe transporter IRT1 that was induced upon Fe deficiency. The transcriptional regulation of several Zn transports under Fe deficiency led presumably to the homeostatic regulation of the cytosolic concentration of Zn and of other transition metal ions such as Mn to avoid toxicity.
The genomic information obtained from this study gives insights into the rapid transcriptional responses to Fe shortage in plants, and is important for understanding how changes in nutrient availability are translated into responses that help to avoid imbalances in ion distribution. We further identified rapidly induced or repressed genes with potential roles in perception and signaling during Fe deficiency which may aid in the elucidation of these processes.
PMCID: PMC2676303  PMID: 19348669
25.  Different effect of cadmium and copper on H+-ATPase activity in plasma membrane vesicles from Cucumis sativus roots 
Journal of Experimental Botany  2012;63(11):4133-4142.
The effect of heavy metals on plasma membrane (PM) H+-ATPase (EC activity in cucumber (Cucumis sativus) roots was studied. The aim of this work was to explain the mechanism of modification of the PM H+-ATPase activity in plants subjected to heavy metals. Plants were treated with 10 μM Cd or Cu for 6 d. After 3 d exposure to the heavy metals, some of the plants were transferred to control conditions for a further 3 d (3/3 plants). The activity of PM H+-ATPase was found to be increased in plants treated with heavy metals. The highest activity measured as proton transport was observed in 3/3 plants. Estimation of transcript levels of C. sativus PM H+-ATPase in roots indicated that the action of Cd, but not Cu, affected the gene expression level. Transcript levels of C. sativus PM H+-ATPase (CsHA2, CsHA3, CsHA4, CsHA8, and CsHA9) genes increased in roots treated with Cd. Moreover, Western blot analysis with antibody against phosphothreonine and 14-3-3 protein indicated that increased activity of PM H+-ATPase under heavy-metal stress resulted from phosphorylation of the enzyme. It was found that Cu markedly increased the activity of catalase and ascorbate peroxidase and reduced the level of H2O2 in cucumber roots. In contrast, Cd did not affect these parameters. These results indicate that Cd and Cu can, in different ways, lead to modification of PM H+-ATPase activity. Additionally, it was observed that treatment of plants with heavy metals led to an increased level of heat-shock proteins in the tissues. This suggests that the plants had started adaptive processes to survive adverse conditions, and increased PM H+-ATPase activity could further enhance the repair processes in heavy-metal-stressed plants.
PMCID: PMC3398447  PMID: 22451724
Cd; Cu; H+-ATPase activity; heavy metals; plasma membrane

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