Patrizia De Nisi

Metabolic responses in cucumber (Cucumis sativus L.) roots under Fe-deficiency: a 31P-nuclear magnetic resonance in-vivo study

Abstract. The metabolic responses occurring in cucumber (Cucumis sativus L.) roots (a strategy-I plant) grown under iron-deficiency conditions were studied in-vivo using 31P-nuclear magnetic resonance spectroscopy. Iron starvation induced activation of metabolism leading to the consumption of stored carbohydrates to produce the NAD(P)H, ATP and phosphoenolpyruvate necessary to sustain the increased activity of the NAD(P)H:Fe3+-reductase, the H+-ATPase (EC 3.6.1.35) and phosphoenolpyruvate carboxylase (EC 4.1.1.31). Activation of catabolic pathways was supported by the enhancement of glycolytic enzymes and concentrations of the metabolites glucose-6-phosphate and fructose-6-phosphate, and by enhancement of the respiration rate. Moreover, Fe-deficiency induced a slight increase in the cytoplasmic (pHc) and vacuolar (pHv) pHs as well as a dramatic decrease in the vacuolar phosphate (Pi) concentration. A comparison was done using fusicoccin (FC), a fungal toxin which stimulates proton extrusion. Changes in pHc and pHv were measured after addition of FC. Under these conditions, a dramatic alkalinization of the pHv of −Fe roots was observed, as well as a concomitant Pi movement from the vacuole to the cytoplasm. These results showed that Fe starvation was indeed accompanied by the activation of metabolic processes useful for sustaining the typical responses occurring at the plasma-membrane level (i.e. increases in the NAD(P)H:Fe3+-reductase and H+-ATPase activities) as well as those involved in the homeostasis of pHc. The decrease in vacuolar Pi levels induced by Fe-deficiency and FC and movement of Pi from the vacuole to the cytoplasm suggest a possible involvement of this compound in the cellular pH-stat system.

The role of calcium in the cold shock responses.

Abstract Brief cold shock induces several physiological responses in corn root tissue. Ion fluxes across the membrane are greatly affected and cold shock application leads to a rapid influx of calcium towards the cytosol. Given the second messenger role claimed for this ion in the transduction of environmental and hormonal signals in the living cells, we have studied the effect of Ca 2+ -channel blockers during the response to cold shock. Ca 2+ influx was inhibited or reduced demonstrating that its influx was not simply due to an increased membrane permeability but rather to the opening of Ca 2+ channels which, under the conditions of a highly favourable electrochemical gradient, permitted its passive entrance into the cytosol. Other parameters are also influenced by the application of these compounds, showing a cause-effect linkage to the increased cytosolic Ca 2+ concentration. In fact, potassium efflux is greatly diminished and there is also a decrease in the hydrolysis of membrane phospholipids. A tentative model to explain how calcium might be implicated in the physiological responses to cold shock is discussed.

Responses of two ecotypes of Medicago ciliaris to direct and bicarbonate-induced iron deficiency conditions

Our study investigates the effect of iron deficiency on morpho-physiological and biochemical parameters of two Medicago ciliaris ecotypes (Mateur TN11.11 and Soliman TN8.7). Iron deficiency was imposed by making plants grow, either in an iron free or by the addition of CaCO3/NaHCO3 to the Hoagland nutrient solution. Our results showed that both true and bicarbonate Fe-deficiency induced the characteristic iron-chlorosis symptoms, although the intensity of the symptoms was ecotype-dependent. This variability in tolerance to iron deficiency was also displayed by other morphological parameters such as root biomass and chlorophyll concentration. Besides, iron chlorosis induced an increase in biochemical parameters: the iron reducing capacity (measured in vivo on root segments and in vitro on plasma membrane enriched vesicles) and rhizosphere acidification by enhancement of H+-ATPase activity were more pronounced in Mateur ecotype. These findings suggest that Soliman ecotype was more sensitive than Mateur one to iron chlorosis.

Fe Deficiency Responses in Parietaria diffusa: A Calcicole Plant

Abstract Pellitory of the wall (Parietaria diffusa L.), a dicotyledonous wild plant belonging to the family of Urticaceae, is widespread on calcareous soils, and also on walls and debris, were lime concentration, sometimes, is extremely high; it may then be considered a calcicole plant. Since high pH values and the presence of CaCO3 and HCO3 − cause low Fe solubility, its availability in such substrates could be the ecological factor limiting the distribution of spontaneous plants in calcareous soils, and a calcareous soil‐born plant should be characterized by a higher Fe‐efficiency in comparison with calcifuge ones. Parietaria diffusa was grown in nutrient solutions in the presence and in the absence of Fe, and in the presence of CaCO3 and bicarbonate at two concentrations (5 and 15 mM), in order to simulate a natural substrate with different lime contents. Some biochemical parameters were determined and the morphological and hystological modifications of the root system were evaluated in order to verify whether Parietaria is a Fe‐efficient plant and adopts the adaptive mechanisms of Strategy I Fe‐efficient plants.

Effect of iron deficiency on RNA and protein synthesis in cucumber roots

Abstract Strategy I is a multifaceted mechanism developed by plants to overcome iron deficiency. Beyond the main responses based on the Fe(III) reduction and rhizosphere acidification, there are other morphological, physiological, and biochemical responses that enable plants belonging to this class to respond in a more complex way to iron starvation. Most of these responses are catalyzed by enzymes, so the synthesis of mRNA and protein must occur rapidly to support these changes. Increase in the Fe(III) reductase and H+‐ATPase activities at the plasma membrane level, increase in some respiration enzymes and of phosphoenolpyruvate carboxylase (PEPC) are well acknowledged. In this paper we provide more direct evidence that both RNA and protein synthesis are increased under Fe deficiency and that the protein synthesis machinery is better developed in this condition. This hypothesis seems to be sustained also by the greater availability of free aminoacids and in particular of aspartate and glutamate in Fe deficient plant roots.

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

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

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.

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

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