Changes in expression of proteins involved in alleviation of Fe-deficiency by sulfur nutrition in Brassica napus L.

In order to characterize the significance of sulfur (S) nutrition in protein expression under iron (Fe)-deficient conditions, gel-based proteomic analysis was performed with the leaves of Brassica napus exposed to S and Fe combined treatments: sufficient in S and Fe (+S/+Fe, control), sufficient S but Fe deprived (+S/?Fe), deprived S but sufficient Fe (?S/+Fe), and deprived S and Fe (?S/?Fe). The resulting data showed that 15 proteins were down-regulated due to production of oxidative damage as indicated by H₂O₂ and O ₂ ^?1 localizations and due to leaf chlorosis in leaves in S-deprived leaves either in presence (?S/+Fe) or absence of Fe (?S/?Fe), whereas these down-regulated proteins were well expressed in the presence of S (+S/?Fe) compared to control (+S/+Fe). In addition, two proteins were up-regulated under S-deprived condition in presence (?S/+Fe) and absence of (?S/?Fe) Fe. The functional classification of these identified proteins was estimated that 40 % of the proteins belong to chloroplast precursor, and rest of the proteins belongs to hypothetical proteins, RNA binding, secondary metabolism and unknown proteins. On the other hand, five protein spots from S deprived (?S/+Fe) and ten spots from Fe deprived (?S/?Fe) conditions were absent, whereas they were well expressed in presence of S (+S/?Fe) compared to control plants (+S/+Fe). These results suggest that sulfur nutrition plays an important role in alleviating protein damage in Fe-deficient plants and adaptation to Fe-deficiency in oilseed rape.     

Limited sulfur resource forces Arabidopsis thaliana to shift towards non-sulfur tolerance under cadmium stress

Soils in some geographical regions suffer with combined stress of sulfur (S) deficiency and cadmium (Cd) contamination. Although the independent impacts of Cd and S-deficiency on plants are well studied but there are rare reports on synergistic effects of S-deficiency and Cd stress. Thus, this study focuses to investigate the response of Arabidopsis thaliana in terms of defense and growth as influenced by Cd under limited S regime. A. thaliana (Col-0) was grown on S-sufficient MS media for 2 weeks and then subjected to S-deficiency for 15 days. Control (+S/−Cd) and S-starved (−S/−Cd) plants were exposed to Cd (50 μM CdCl₂) for 3–5 days. Results show that S-deficiency (−S/−Cd) induces oxidative stress which was much lesser than Cd (+S/+Cd) but highest in combined stress of S-deficiency along with Cd (−S/+Cd). Interestingly, plant was found to elevate glutathione (GSH) biosynthetic pathway and also improved growth and antioxidative status when sulfur was present during Cd stress (+S/+Cd). Important studies in terms of photosynthetic parameters also support limited loss in +S plants as S-assimilation pathway was up-regulated. Proline accumulation was not influenced much by S-deficiency but stimulated with Cd stress strongly suggesting defense shift towards non-sulfur tolerance mechanism. Levels of glutathione and H₂O₂ removing catalase were also modulated to cope with oxidative stress in a better manner during S-sufficient conditions. Chloroplast ultrastructure showed loss of grana under S-deficiency, however, −S/+Cd resulted in severe disintegration of thylakoids too. Biomass accumulation was also most adversely affected with −S/+Cd followed by Cd stress alone (+S/+Cd) and S-deficiency (−S/−Cd). In conclusion, Arabidopsis maintains equilibrium between defense and growth and thus survive under limited S resource. Also S-assimilation is modulated by Cd stress and Cd-induced stress is prevented by S-nutrition.     

Three-year field response of drip-irrigated grapevine (Vitis vinifera L., cv. Tempranillo) to soil salinity

The aim of this work was to evaluate the effects of Fe deficiency on the activity of several metabolic enzymes (PEPC, PK, PFK, G6PDH and G3PDH), along with the function of the antioxidant enzymes (SK, SDH and PAL) in two lines of Medicago ciliaris, TN11.11 and TN8.7. Plants were grown in a greenhouse under controlled conditions. After germination and pre-treatment, plants were transferred for hydroponic culture. Three treatments were used: 30 μM Fe (+Fe), 0 μM Fe (?Fe) and 30 μM Fe + 10 mM NaHCO₃ (+Bic.). Our results showed that all the enzymatic activities increased in extracts of Fe-deficient roots when compared to the control. The above increases in the activity were particularly evident for the bicarbonate-treated roots of TN11.11. PEPC activity was increased by 277% in TN11.11 plants with the addition of bicarbonate to the nutrient solution. Our results indicate also that, in the two lines of Medic, the activity of SK, SDH and PAL in leaves and roots were increased under Fe deficiency (either direct or induced by bicarbonate), to a greater extent in TN11.11 plants. Furthermore, a considerable increase in lipid peroxidation of roots and leaves of Fe-deficient plants was observed in TN8.7 when compared to TN11.11 plants. Our data suggest that the TN11.11 line is more effective in overcoming Fe deficiency than TN8.7. The tolerance of TN11.11 to Fe deficiency is related to its ability to modulate the carbohydrate metabolism and to increase secondary metabolism pathways.     

An Exogenous Source of Nitric Oxide Modulates Iron Nutritional Status in Peanut Seedlings (<Emphasis Type="Italic">Arachis hypogaea</Emphasis> L.)

Iron (Fe) deficiency chlorosis is a common and severe nutritional deficiency in plants, and nitric oxide (NO) is an important signaling molecule in regulating Fe homeostasis in plants. We studied the effect of sodium nitroprusside (SNP, an NO donor) on Fe uptake, translocation, storage, and activation in a greenhouse. The concentrations of active Fe, total Fe, and the ratio of active Fe to total Fe, the activities of key enzymes, and chlorophyll concentration were determined, and resistance to oxidative stress and mineral element distribution in peanut plants grown in Fe sufficiency and Fe deficiency (an absence of Fe and low level of Fe concentration) conditions were also investigated. The results showed that NO significantly increased the concentration of active Fe and the ratio of active Fe to total Fe in Fe-deficient plants, and increased active Fe concentration in leaves and stems of Fe-sufficient plants. NO application also increased Fe translocation from roots to the shoots and the accumulation of Fe in cell organelles and the soluble fraction in leaves, especially in the low-level Fe concentration condition, thus increased available Fe and chlorophyll concentration in leaves of Fe-deficient plants. The activities of key enzymes were regulated by NO, which effectively mitigated oxidative damages by enhancing the activities of antioxidant enzymes (SOD, POD, CAT), increasing H⁺-ATPase and Ca²⁺-ATPase activities to balance the ion (Fe, Ca, Mg and Zn) uptake and distribution in Fe-deficient plants. However, NO application had no obvious effect on these variables in Fe-sufficient plants. These results indicated that NO application can improve Fe uptake, translocation, and activation of related enzymes in Fe-deficient plants, thus mitigating the adverse effect of Fe deficiency.     

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 Fe²⁺ 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 reprogramming in nodules,roots, and leaves of symbiotic soybean in response to iron deficiency

To elucidate the mechanism of adaptation of leguminous plants to iron (Fe)‐deficient environment, comprehensive analyses of soybean (Glycine max) plants (sampled at anthesis) were conducted under Fe‐sufficient control and Fe‐deficient treatment using metabolomic and physiological approach. Our results show that soybeans grown under Fe‐deficient conditions showed lower nitrogen (N) fixation efficiency; however, ureides increased in different tissues, indicating potential N‐feedback inhibition. N assimilation was inhibited as observed in the repressed amino acids biosynthesis and reduced proteins in roots and nodules. In Fe‐deficient leaves, many amino acids increased, accompanied by the reduction of malate, fumarate, succinate, and α‐ketoglutarate, which implies the N reprogramming was stimulated by the anaplerotic pathway. Accordingly, many organic acids increased in roots and nodules; however, enzymes involved in the related metabolic pathway (e.g., Krebs cycle) showed opposite activity between roots and nodules, indicative of different mechanisms. Sugars increased or maintained at constant level in different tissues under Fe deficiency, which probably relates to oxidative stress, cell wall damage, and feedback regulation. Increased ascorbate, nicotinate, raffinose, galactinol, and proline in different tissues possibly helped resist the oxidative stress induced by Fe deficiency. Overall, Fe deficiency induced the coordinated metabolic reprogramming in different tissues of symbiotic soybean plants.     

Role of sulfate in detoxification of arsenate-induced toxicity in Zea mays L. (SRHM 445): nutrient status and antioxidants

The study highlights the role of sulfur (S) in detoxification of arsenate-induced toxicity and the shift in essential element homeostasis in Zea mays L (SRHM 445). Overall growth of arsenate-treated plants under sulfur starvation (?S) was lower than that in the presence of excess sulfur (+S). Translocation of arsenate from roots to shoots, increased under As(?S) and decreased with As(+S). The level of micronutrients (Cu, Zn, Fe) increased in As(?S) plants. Whereas, the level of K and PO₄ was higher in As(?S) plants than in As(+S) plants. Higher malondialdehyde, protein carbonyl, and H₂O₂ levels in As(?S) plants are indicative of higher oxidative stress. Higher superoxide dismutase (SOD) and ascorbate peroxidase (APX) activities, in As(?S) plants coincided with higher H₂O₂ levels showing the activity of these enzymes are independent of S availability. Absence of reduced glutathione/oxidized glutathione pool in (?S) plants manifested into failure of ascorbate–glutathione detoxification pathway. Hence, S has dual role of protecting the plant against arsenate-induced toxicity (1) by restricting arsenic (As) translocation to the upper parts and (2) by increasing the activity SOD and APX.     

Effect of sequential deletion of extra N-terminal residues on the structure and stability of yeast iso-1-cytochrome-c

A sequence alignment of yeast cytochrome-c (y-cyt-c) with mammalian cyts-c shows that the yeast protein has a five residue long N-terminal extension. A question arises: Does this N-terminal extension play any roles in the stability, structure, and folding of the yeast protein? To answer this question, in silico and in vitro studies were carried out on the wild type (WT) protein and its five deletants (Δ(?5/?5), Δ(?5/?4), Δ(?5/?3), Δ(?5/?2), and Δ(?5/?1) where Δ denotes the deletion and the numbers refer to the residues deleted, e.g. Δ(?5/?1) denotes the deletion of residues numbered from ?5 to ?1 (TEFKA), while Δ(?5/?2) denotes the deletion of resides numbered from ?5 to ?2 (TEFK) and so on). The main conclusion of the in silico study is that the order of stability of deletants and WT protein is Δ(?5/?4) > WT > Δ(?5/?3) > Δ(?5/?5) > Δ(?5/?1) ~ Δ(?5/?2). In vitro studies involved (i) measurements of thermodynamic stability of all proteins by differential scanning calorimetry and from sigmoidal curves of two different structural properties ([θ]₂₂₂, a probe for detecting change in secondary structure, and Δε₄₀₅, a probe for detecting alteration in the heme environment), and (ii) characterization of all proteins by various spectral properties. The main conclusions of the in vitro studies are as follows: (i) The order of thermodynamic stability of all proteins is in excellent agreement with that predicted by in silico studies, and (ii) A sequential deletion of the N-terminal extension has no effects on protein structure and folding.     

Formate Dehydrogenase, an Enzyme of Anaerobic Metabolism, Is Induced by Iron Deficiency in Barley Roots

To identify the proteins induced by Fe deficiency, we have compared the proteins of Fe-sufficient and Fe-deficient barley (Hordeum vulgare L.) roots by two-dimensional polyacrylamide gel electrophoresis. Peptide sequence analysis of induced proteins revealed that formate dehydrogenase (FDH), adenine phosphoribosyltransferase, and the Ids3 gene product (for Fe deficiency-specific) increased in Fe-deficient roots. FDH enzyme activity was detected in Fe-deficient roots but not in Fe-sufficient roots. A cDNA encoding FDH (Fdh) was cloned and sequenced. Fdh expression was induced by Fe deficiency. Fdh was also expressed under anaerobic stress and its expression was more rapid than that induced by Fe deficiency. Thus, the expression of Fdh observed in Fe-deficient barley roots appeared to be a secondary effect caused by oxygen deficiency in Fe-deficient plants.     

Response of barley plants to Fe deficiency and Cd contamination as affected by S starvation

Both Fe deficiency and Cd exposure induce rapid changes in the S nutritional requirement of plants. The aim of this work was to characterize the strategies adopted by plants to cope with both Fe deficiency (release of phytosiderophores) and Cd contamination [production of glutathione (GSH) and phytochelatins] when grown under conditions of limited S supply. Experiments were performed in hydroponics, using barley plants grown under S sufficiency (1.2 mM sulphate) and S deficiency (0 mM sulphate), with or without Fe(III)-EDTA at 0.08 mM for 11 d and subsequently exposed to 0.05 mM Cd for 24 h or 72 h. In S-sufficient plants, Fe deficiency enhanced both root and shoot Cd concentrations and increased GSH and phytochelatin levels. In S-deficient plants, Fe starvation caused a slight increase in Cd concentration, but this change was accompanied neither by an increase in GSH nor by an accumulation of phytochelatins. Release of phytosiderophores, only detectable in Fe-deficient plants, was strongly decreased by S deficiency and further reduced after Cd treatment. In roots Cd exposure increased the expression of the high affinity sulphate transporter gene (HvST1) regardless of the S supply, and the expression of the Fe deficiency-responsive genes, HvYS1 and HvIDS2, irrespective of Fe supply. In conclusion, adequate S availability is necessary to cope with Fe deficiency and Cd toxicity in barley plants. Moreover, it appears that in Fe-deficient plants grown in the presence of Cd with limited S supply, sulphur may be preferentially employed in the pathway for biosynthesis of phytosiderophores, rather than for phytochelatin production.     

The response of carbon metabolism and antioxidant defenses of alfalfa nodules to drought stress and to the subsequent recovery of plants

Alfalfa (Medicago sativa) plants were exposed to drought to examine the involvement of carbon metabolism and oxidative stress in the decline of nitrogenase (N(2)ase) activity. Exposure of plants to a moderate drought (leaf water potential of -1.3 MPa) had no effect on sucrose (Suc) synthase (SS) activity, but caused inhibition of N(2)ase activity (-43%), accumulation of succinate (+36%) and Suc (+58%), and up-regulation of genes encoding cytosolic CuZn-superoxide dismutase (SOD), plastid FeSOD, cytosolic glutathione reductase, and bacterial MnSOD and catalases B and C. Intensification of stress (-2.1 MPa) decreased N(2)ase (-82%) and SS (-30%) activities and increased malate (+40%), succinate (+68%), and Suc (+435%). There was also up-regulation (mRNA) of cytosolic ascorbate peroxidase and down-regulation (mRNA) of SS, homoglutathione synthetase, and bacterial catalase A. Drought stress did not affect nifH mRNA level or leghemoglobin expression, but decreased MoFe- and Fe-proteins. Rewatering of plants led to a partial recovery of the activity (75%) and proteins (>64%) of N(2)ase, a complete recovery of Suc, and a decrease of malate (-48%) relative to control. The increase in O(2) diffusion resistance, the decrease in N(2)ase-linked respiration and N(2)ase proteins, the accumulation of respiratory substrates and oxidized lipids and proteins, and the up-regulation of antioxidant genes reveal that bacteroids have their respiratory activity impaired and that oxidative stress occurs in nodules under drought conditions prior to any detectable effect on SS or leghemoglobin. We conclude that a limitation in metabolic capacity of bacteroids and oxidative damage of cellular components are contributing factors to the inhibition of N(2)ase activity in alfalfa nodules.     

Influence of cadmium stress and arbuscular mycorrhizal fungi on nodule senescence in Cajanus cajan (L.) Millsp

Cadmium (Cd) causes oxidative damage and affects nodulation and nitrogen fixation process of legumes. Arbuscular mycorrhizal (AM) fungi have been demonstrated to alleviate heavy metal stress of plants. The present study was conducted to assess role of AM in alleviating negative effects of Cd on nodule senescence in Cajanus cajan genotypes differing in their metal tolerance. Fifteen day-old plants were subjected to Cd treatments--25 mg and 50 mg Cd per kg dry soil and were grown with and without Glomus mosseae. Cd treatments led to a decline in mycorrhizal infection (MI), nodule number and dry weights which was accompanied by reductions in leghemoglobin content, nitrogenase activity, organic acid contents. Cd supply caused a marked decrease in nitrogen (N), phosphorus (P), and iron (Fe) contents. Conversely, Cd increased membrane permeability, thiobarbituric acid reactive substances (TBARS), hydrogen peroxide (H2O2), and Cd contents in nodules. AM inoculations were beneficial in reducing the above mentioned harmful effects of Cd and significantly improved nodule functioning. Activities of superoxide dismutase (SOD), catalase (CAT), and peroxidase (POD) increased markedly in nodules of mycorrhizal-stressed plants. The negative effects of Cd were genotype and concentration dependent.     

Unraveling the iron deficiency responsive proteome in <i>Arabidopsis?</i>shoot by iTRAQ-OFFGEL approach

Iron (Fe) is required by plants for basic redox reactions in photosynthesis and respiration, and for many other key enzymatic reactions in biological processes. Fe homeostatic mechanisms have evolved in plants to enable the uptake and sequestration of Fe in cells. To elucidate the network of proteins that regulate Fe homeostasis and transport, we optimized the iTRAQ-OFFGEL method to identify and quantify the number of proteins that respond to Fe deficiency in the model plant Arabidopsis. In this study, Fe deficiency was created using Fe-deficient growth conditions, excess zinc (Zn), and use of the irt1-1 mutant in which the IRT1 Fe transporter is disrupted. Using the iTRAQ-OFFGEL approach, we identified 1139 proteins, including novel Fe deficiency-responsive proteins, in microsomal fractions isolated from 3 different types of Fe-deficient shoots compared with just 233 proteins identified using conventional iTRAQ-CEX. Further analysis showed that greater numbers of low-abundance proteins could be identified using the iTRAQ-OFFGEL method and that proteins could be identified from numerous cellular compartments. The improved iTRAQ-OFFGEL method used in this study provided an efficient means for identifying greater numbers of proteins from microsomal fractions of Arabidopsis shoots. The proteome identified in this study provides new insight into the regulatory cross talk between Fe-deficient and excess Zn conditions.     

Cadmium-stress in white lupin: effects on nodule structure and functioning

Cadmium effects on nodule structure and changes in organic and amino acids, proteins, nutrients and some stress indicators were studied in nodules of white lupin (Lupinus albus L., cv. Multolupa). Plants were grown hydroponically on perlite for 49 d with (18 μM) or without Cd in the nutrient solution. Cadmium-treated plants showed decreases in leaf chlorophyll and shoot sucrose concentrations, but sucrose did not change in nodules. Cadmium application produced alterations in nodule cortex and infected zone structure. Furthermore, Cd supply caused a marked decrease in P, K, leghemoglobin, N–amino compounds, malate, succinate and soluble protein in the nodules. Conversely, the levels of lipid peroxidation and total thiols increased strongly. Results obtained suggest that white lupin nodules are Cd sensitive, in spite of Cd sequestering by cell walls and thiols. The main phytotoxic effects of Cd on nodule structure and function were the occlusion with glycoprotein of intracellular spaces of nodule cortex, alterations in symbiosomes, enrichment in Cd of cell walls and oxidative stress. Glycoprotein accumulation and leghemoglobin depletion may be considered useful indicators of Cd stress in white lupin nodules.     

Developmental control of the β-phaseolin gene requires positive, negative, and temporal seed-specific transcriptional regulatory elements and a negative element for stem and root expression

To identify cis-acting regulatory elements responsible for developmental control of the common bean seed storage protein β-phaseolin, a series of 5′-deletion mutants of the 782 bp upstream sequence together with the coding and 3′-regions of the β-phaseolin gene were used to transform tobacco. One major positive regulatory element (?295/?228) and a putative minimal promoter (?64/?14) were indicated by large reductions in phaseolin mRNA and protein concentrations in seeds of plants deficient for these regions. In addition, minor negative (?422/?296) and positive (?782/?423) elements also influenced the level of phaseolin mRNA expression in seeds. One temporal element (?295/?107) governed late expression of phaseolin mRNA in seeds, and possibly a second (?64/?14) regulated early expression. The ?64/?14 promoter containing two TATA boxes conferred spatially regulated gene expression, and was sufficient for a low level of expression in root and stem. Significant levels of phaseolin mRNA and protein were detected in stem cortices and secondary roots of plants lacking the ?295/?107 negative element. No significant expression in leaf tissue was detected. Results demonstrate that developmental control of β-phaseolin requires a minimal promoter, one element for the suppression of expression in root and stem tissue, three elements governing quantitative expression in seeds, and at least one temporal element controlling expression in seeds.     

Influence of iron status on cadmium and zinc uptake by different ecotypes of the hyperaccumulator Thlaspi caerulescens

We have previously identified an ecotype of the hyperaccumulator Thlaspi caerulescens (Ganges), which is far superior to other ecotypes (including Prayon) in Cd uptake. In this study, we investigated the effect of Fe status on the uptake of Cd and Zn in the Ganges and Prayon ecotypes, and the kinetics of Cd and Zn influx using radioisotopes. Furthermore, the T. caerulescens ZIP (Zn-regulated transporter/Fe-regulated transporter-like protein) genes TcZNT1-G and TcIRT1-G were cloned from the Ganges ecotype and their expression under Fe-sufficient and -deficient conditions was analyzed. Both short- and long-term studies revealed that Cd uptake was significantly enhanced by Fe deficiency only in the Ganges ecotype. The concentration-dependent kinetics of Cd influx showed that the V(max) of Cd was 3 times greater in Fe-deficient Ganges plants compared with Fe-sufficient plants. In Prayon, Fe deficiency did not induce a significant increase in V(max) for Cd. Zn uptake was not influenced by the Fe status of the plants in either of the ecotypes. These results are in agreement with the gene expression study. The abundance of ZNT1-G mRNA was similar between the Fe treatments and between the two ecotypes. In contrast, abundance of the TcIRT1-G mRNA was greatly increased only in Ganges root tissue under Fe-deficient conditions. The present results indicate that the stimulatory effect of Fe deficiency on Cd uptake in Ganges may be related to an up-regulation in the expression of genes encoding for Fe(2+) uptake, possibly TcIRT1-G.