ABA modulates the degradation of phosphoenolpyruvate carboxylase kinase in sorghum leaves

Salt stresses strongly enhance the phosphoenolpyruvate carboxylase kinase (PEPC-k) activity of sorghum leaves. This work shows that (1) abscisic acid (ABA) increased the rise in kinase activity in illuminated leaf disks of the non-stressed plant, (2) ABA decreased the disappearance of PEPC-k activity in the dark, (3) two PEPC-k genes expressed in sorghum leaves, PPCK1 and PPCK2, were not up-regulated by the phytohormone and, (4) ABA effects were mimicked by MG132, a powerful inhibitor of the ubiquitin-proteasome pathway. Collectively these data support a role for the ubiquitin-proteasome pathway in the rapid turnover of PEPC-k. The negative control by ABA on this pathway might account for the increase of kinase activity observed in salt-treated plants.     

Effects of moderate and severe iron deficiency chlorosis on fruit yield, appearance and composition in pear (Pyrus communis L.) and peach (Prunus persica (L.) Batsch)

The effects of different levels of Fe-deficiency chlorosis on the fruit yield, appearance and composition of pear and peach trees grown in field orchards have been studied. The major effect of Fe deficiency in both species was a large yield reduction, even when chlorosis was moderate, associated to decreases in fruit tree load. Fruit size increased with moderate chlorosis in both species and decreased with severe chlorosis in peach. In peach, moderate or severe chlorosis affected uniformly all branches, leading to firmer fruits with higher acidity, total phenolics and carboxylates. This indicates a delayed maturity that can be attributed to a low C-availability for fruits. In Fe-deficient pear trees, the majority of fruits (98%) were on non-chlorotic or moderately chlorotic branches, and fruits were less green and firm with an increased sugars/acids ratio. This indicates an advanced fruit maturity that can be attributed to an increased C-availability for fruits. All chlorosis levels increased within-tree variation in fruit appearance.     

Metabolic changes of iron uptake in N2-fixing common bean nodules during iron deficiency

Iron is an important nutrient in N₂-fixing legume nodules. The demand for this micronutrient increases during the symbiosis establishment, where the metal is utilized for the synthesis of various iron-containing proteins in both the plant and the bacteroid. Unfortunately, in spite of its importance, iron is poorly available to plant uptake since its solubility is very low when in its oxidized form Fe(III). In the present study, the effect of iron deficiency on the activity of some proteins involved in Strategy I response, such as Fe-chelate reductase (FC-R), H⁺-ATPase, and phosphoenolpyruvate carboxylase (PEPC) and the protein level of iron regulated transporter (IRT1) and H⁺-ATPase proteins has been investigated in both roots and nodules of a tolerant (Flamingo) and a susceptible (Coco blanc) cultivar of common bean plants. The main results of this study show that the symbiotic tolerance of Flamingo can be ascribed to a greater increase in the FC-R and H⁺-ATPase activities in both roots and nodules, leading to a more efficient Fe supply to nodulating tissues. The strong increase in PEPC activity and organic acid content, in the Flamingo root nodules, suggests that under iron deficiency nodules can modify their metabolism in order to sustain those activities necessary to acquire Fe directly from the soil solution.     

Bicarbonate blocks iron translocation from cotyledons inducing iron stress responses in Citrus roots

The effect of bicarbonate ion (HCO₃⁻) on the mobilization of iron (Fe) reserves from cotyledons to roots during early growth of citrus seedlings and its influence on the components of the iron acquisition system were studied. Monoembryonic seeds of Citrus limon (L.) were germinated “in vitro” on two iron-deprived media, supplemented or not with 10 mM HCO₃⁻ (−Fe+Bic and −Fe, respectively). After 21 d of culture, Fe concentration in seedling organs was measured, as well as gene expression and enzymatic activities. Finally, the effect of Fe resupply on the above responses was tested in the presence and absence of HCO₃⁻ (+Fe+Bic or +Fe, respectively). −Fe+Bic seedlings exhibited lower Fe concentration in shoots and roots than −Fe ones but higher in cotyledons, associated to a significative inhibition of NRAMP3 expression. HCO₃⁻ upregulated Strategy I related genes (FRO1, FRO2, HA1 and IRT1) and FC-R and H⁺-ATPase activities in roots of Fe-starved seedlings. PEPC1 expression and PEPCase activity were also increased. When −Fe+Bic pre-treated seedlings were transferred to Fe-containing media for 15 d, Fe content in shoots and roots increased, although to a lower extent in the +Fe+Bic medium. Consequently, the above-described root responses became markedly repressed, however, this effect was less pronounced in +Fe+Bic seedlings. In conclusion, it appears that HCO₃⁻ prevents Fe translocation from cotyledons to shoot and root, therefore reducing their Fe levels. This triggers Fe-stress responses in the root, enhancing the expression of genes related with Fe uptake and the corresponding enzymatic activities.     

Changes of photosynthetic activities of maize (<Emphasis Type="Italic">Zea mays</Emphasis> L.) seedlings in response to cadmium stress

Maize (Zea mays L.) seedlings were grown in nutrient solution culture containing 0, 5, and 20 μM cadmium (Cd) and the effects on various aspects of photosynthesis were investigated after 24, 48, 96 and 168 h of Cd treatments. Photosynthetic rate (P _N) decreased after 48 h of 20 μM Cd and 96 h of 5μM Cd addition, respectively. Chl a and total Chl content in leaves declined under 48 h of Cd exposure. Chl b content decreased on extending the period of Cd exposure to 96 h. The maximum quantum efficiency and potential photosynthetic capacity of PSII, indicated by F_v/F_m and F_v/F_o, respectively, were depressed after 96 h onset of Cd exposure. After 48 h of 5μM Cd and 24 h of 20 μM Cd treatments, the activities of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco, EC 4.1.39) and phosphoenolpyruvate carboxylase (PEPC, EC 4.1.1.31) in the leaves started to decrease, respectively. We found that the limitation of photosynthetic capacity in Cd stressed maize leaves was associated with Cd toxicity on the light and the dark stages. However, Cd stress initially reduced the activities of Rubisco and PEPC and subsequently affected the PSII electron transfer, suggesting that the Calvin cycle reactions in maize plants are the primary target of the Cd toxic effect rather than PSII.     

Nickel-induced changes in carbon metabolism in wheat shoots

In this study, we analyzed the toxic effect of Ni during the development of wheat shoots. Typical developmental alterations in carbon metabolism-related parameters reflecting changes associated with the transition of the seedlings from heterotrophic to autotrophic metabolism were observed in the control shoots between the 1st and the 4th days. Adverse effects of 50 and 100 μM Ni became evident starting from the 4th day of growth on the metal-containing media. We found that Ni-induced stimulation of phosphoenolpyruvate carboxylase (PEPC) activity coincided with decrease in the ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) level and with declines in net photosynthetic rate (P_N) and stomatal conductance (g_s). Application of Ni resulted in increased activities of several dehydrogenases: glucose-6-phosphate dehydrogenase (G6PDH), 6-phosphogluconate dehydrogenase (6PGDH), isocitrate dehydrogenase (NADP-ICDH) and malate dehydrogenase (NADH-MDH). In contrast, the activities of malic enzymes (NADP-ME and NAD-ME) decreased due to Ni stress. Treatment with Ni led to accumulation of glucose and declined concentration of sucrose as well as considerable increases in concentrations of malic and citric acids. Our results indicate that Ni stress redirects the carbon metabolism of developing wheat shoots to provide carbon skeletons for synthesis of amino acids and organic acids as well as to supply reducing power to sustain normal metabolic processes and to support defense mechanisms against oxidative stress.     

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 profiling using combined GC-MS and LC-MS provides a systems understanding of aristolochic acid-induced nephrotoxicity in rat

We present here a combined GC-MS and LC-MS metabolic profiling approach to unraveling the pathological outcomes of aristolochic acid (AA)-induced nephrotoxicity. Urine samples were analyzed by GC-MS and LC-MS in combination with pattern recognition techniques, e.g. principal component analysis (PCA), orthogonal projection to latent structure-discriminant analysis. The work indicates that AA-induced acute renal toxicity as evidenced by histopathological examinations could be characterized by systemic disturbance of metabolic network involving free fatty acids generation, energy and amino acids metabolism, and alteration in the structure of gut microbiota. Therefore, this method is potentially applicable to the toxicological study, providing a comprehensive understanding of systems response to xenobiotic intervention.     

Metabolic profiling reveals disorder of amino acid metabolism in four brain regions from a rat model of chronic unpredictable mild stress

Chronic stress is closely linked to clinical depression, which could be assessed by a chronic unpredictable mild stress (CUMS) animal model. We present here a GC/MS-based metabolic profiling approach to investigate neurochemical changes in the cerebral cortex, hippocampus, thalamus, and remaining brain tissues. Multi-criteria assessment for multivariate statistics could identify differential metabolites between the CUMS-model rats versus the healthy controls. This study demonstrates that the significantly perturbed metabolites mainly involving amino acids play an indispensable role in regulating neural activity in the brain. Therefore, results obtained from such metabolic profiling strategy potentially provide a unique perspective on molecular mechanisms of chronic stress.     

Co-expression analysis reveals a group of genes potentially involved in regulation of plant response to iron-deficiency

Iron (Fe) is an essential element for plant growth and development. Iron deficiency results in abnormal metabolisms from respiration to photosynthesis. Exploration of Fe-deficient responsive genes and their networks is critically important to understand molecular mechanisms leading to the plant adaptation to soil Fe-limitation. Co-expression genes are a cluster of genes that have a similar expression pattern to execute relatively biological functions at a stage of development or under a certain environmental condition. They may share a common regulatory mechanism. In this study, we investigated Fe-starved-related co-expression genes from Arabidopsis. From the biological process GO annotation of TAIR (The Arabidopsis Information Resource), 180 iron-deficient responsive genes were detected. Using ATTED-II database, we generated six gene co-expression networks. Among these, two modules of PYE and IRT1 were successfully constructed. There are 30 co-expression genes that are incorporated in the two modules (12 in PYE-module and 18 in IRT1-module). Sixteen of the co-expression genes were well characterized. The remaining genes (14) are poorly or not functionally identified with iron stress. Validation of the 14 genes using real-time PCR showed differential expression under iron-deficiency. Most of the co-expression genes (23/30) could be validated in pye and fit mutant plants with iron-deficiency. We further identified iron-responsive cis-elements upstream of the co-expression genes and found that 22 out of 30 genes contain the iron-responsive motif IDE1. Furthermore, some auxin and ethylene-responsive elements were detected in the promoters of the co-expression genes. These results suggest that some of the genes can be also involved in iron stress response through the phytohormone-responsive pathways.     

Differences in leaf proteome response to cold acclimation between Lolium perenne plants with distinct levels of frost tolerance

Perennial ryegrass (Lolium perenne) is a high quality forage and turf grass mainly due to its excellent nutritive values and rapid establishment rate. However, this species has limited ability to perform in harsh winter climates. Though winter hardiness is a complex trait, it is commonly agreed that frost tolerance (FT) is its main component. Species growing in temperate regions can acquire FT through exposure to low, non-lethal temperatures, a phenomenon known as cold acclimation (CA). The research on molecular basis of FT has been performed on the model plants, but they are not well adapted to extreme winter climates. Thus, the mechanisms of cell response to low temperature in winter crops and agronomically important perennial grasses have yet to be revealed. Here, two L. perenne plants with contrasting levels of FT, high frost tolerant (HFT) and low frost tolerant (LFT) plants, were selected for comparative proteomic research. The work focused on analyses of leaf protein accumulation before and after 2, 8, 26 h, and 3, 5, 7, 14 and 21 days of CA, using a high-throughput two-dimensional electrophoresis, and on the identification of proteins which were accumulated differentially between the selected plants by the application of mass spectrometry (MS). Analyses of 580 protein profiles revealed a total of 42 (7.2%) spots that showed at a minimum of 1.5-fold differences in protein abundance, at a minimum of at one time point of CA between HFT and LFT genotypes. It was shown that significant differences in profiles of protein accumulation between the analyzed plants appeared most often on the 5th (18 proteins) and the 7th (19 proteins) day of CA. The proteins derived from 35 (83.3%) spots were successfully identified by the use of MS and chloroplast proteins were shown to be the major group selected as differentially accumulated during CA. The functions of the identified proteins and their probable influence on the level of FT in L. perenne are discussed.     

A metabonomic investigation of the effects of 60 days exposure of rats to two types of pyrethroid insecticides

Type I and II pyrethroid insecticides display different neurotoxicity. To investigate the long-term (60 days exposure) metabolic effect of the two types of pyrethroid insecticides deltamethrin and permethrin, ¹H nuclear magnetic resonance (NMR) spectroscopy-based metabonomics was used to analyze the biochemical composition of urine and serum samples from rats administrated daily with deltamethrin or permethrin for 60 consecutive days, and principal component analysis used to visualize similarities and differences in the resultant biochemical profiles. Rats treated with either deltamethrin or permethrin displayed increased levels of urinary acetate, dimethylamine, dimethylglycine, trimethylamine and serum free amino acids, and decreased urinary 2-oxoglutarate, all of which are indicative of kidney lesions and nephrotoxicity. The reduced excretion of tricarboxylic acid cycle intermediates, together with increased 3-D-hydroxybutyrate, acetate, and lactate in treated rats could suggest disturbance of the energy metabolism, including an increased rate of anaerobic glycolysis, enhanced fatty acid β-oxidation and ketogenesis. These results show that these two types of insecticides have similarities in the urine and serum spectra, indicating that similar metabolic pathways are perturbed by the insecticides, which induced hepatotoxicity and nephrotoxicity. This approach may lead to the discovery of novel biomarkers of pyrethroids toxicity and thereby provide new insights into the toxicological mechanisms of pesticides pyrethroids.