Silvia Donnini

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

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

Differential responses in pear and quince genotypes induced by Fe deficiency and bicarbonate

Most of the studies carried out on Fe deficiency condition in arboreous plants have been performed, with the exception of those carried out on plants grown in the field, in hydroponic culture utilizing a total iron depletion growth condition. This can cause great stress to plants. By introducing Fe deficiency induced by the presence of bicarbonate, we found significant differences between Pyrus communis L. cv. Conference and Cydonia oblonga Mill. BA29 and MA clones, characterized by different levels of tolerance to chlorosis. Pigment content and the main protein-pigment complexes were investigated by HPLC and protein gel blot analysis, respectively. While similar changes in the structural organization of photosystems (PSs) were observed in both species under Fe deficiency, a different reorganization of the photosynthetic apparatus was found in the presence of bicarbonate between tolerant and susceptible genotypes, in agreement with the photosynthetic electron transport rate measured in isolated thylakoids. In order to characterize the intrinsic factors determining the efficiency of iron uptake in a tolerant genotype, the main mechanisms induced by Fe deficiency in Strategy I species, such as Fe3+-chelate reductase (EC 1.16.1.7) and H+-ATPase (EC 3.6.3.6) activities, were also investigated. We demonstrate that physiological and biochemical root responses in quince and pear are differentially affected by iron starvation and bicarbonate supply, and we show a high correlation between tolerance and Strategy I activation.

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

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

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.

Leaf Responses to Reduced Iron Availability in Two Tomato Genotypes: T3238FER (Iron Efficient) and T3238fer (Iron Inefficient)

Abstract The present work is aimed at evaluating some effects induced by different levels of iron availability in the growth medium for two different tomato (Lycopersicon esculentum Mill.) genotypes, the T3238fer (Tfer), unable to activate mechanisms for iron mobilization and uptake known as “strategy I,” and its correspondent wild‐type T3238FER (TFER). By using different iron concentration in the growth solution, the most suitable iron level to induce phenotypic differences between the two genotypes without being lethal for the mutant was found to be 40 µM Fe‐Na‐EDTA. The analyses were carried out also on plants grown with 80 µM Fe‐Na‐EDTA, an iron concentration at which the two genotypes showed no phenotypic differences. A significant decrease in total leaf iron and chlorophyll content was detected in both genotypes following reduction of iron concentration in the nutrient solution, and was particularly evident in Tfer40, which showed symptoms of chlorosis. The photo‐electron transport rate of the whole chain was significantly affected by growth conditions as well as by genotype, the lowest activity being detected in Tfer40 plants. Chlorophyll a fluorescence analysis revealed an increase in non‐photochemical quenching (q NP) of Tfer plants grown at both iron concentrations, indicating the activation of photoprotective mechanisms, which, however, were not sufficient to prevent photoinhibition when plants were grown at 40 µM iron, as indicated by significant reduction in PSII photochemistry (F v/F m) and photochemical quenching (q P). The actual quantum yield of PSII (ΦPSII) and the intrinsic PSII efficiency (ΦEXC) showed the same behavior of q P and F v/F m ratio. A significant effect of mutation and iron supply on all the pigments was detected, and was particularly evident in the mutant grown at 40 µM iron. A different behavior was shown by the three pigments involved in the xantophyll cycle, violaxanthin being less affected than chlorophylls and the other carotenoids, and zeaxanthin even increasing, due to the xanthophyll cycle activation. In conclusion, the interaction between iron deprivation and fer mutation induced functional alterations to the photosynthetic apparatus. Anyway, as far as concerns the photo‐electron transport activity, the influence of fer mutation seemed to occur independently from iron supply.

Root antioxidant responses of two Pisum sativum cultivars to direct and induced Fe deficiency

The contribution of antioxidant defence systems in different tolerance to direct and bicarbonate-induced Fe deficiency was evaluated in two pea cultivars (Kelvedon, tolerant and Lincoln, susceptible). Fe deficiency enhanced lipid peroxidation and H2 O2 concentration in roots of both cultivars, particularly in the sensitive one grown under bicarbonate supply. The results obtained on antioxidant activities (SOD, CAT, POD) suggest that H2 O2 accumulation could be due to an overproduction of this ROS and, at the same time, to a poor capacity to detoxify it. Moreover, under bicarbonate supply the activity of POD isoforms was reduced only in the sensitive cultivar, while in the tolerant one a new isoform was detected, suggesting that POD activity might be an important contributor to pea tolerance to Fe deficiency. The presence of bicarbonate also resulted in stimulation of GR, MDHAR and DHAR activities, part of the ASC-GSH pathway, which was higher in the tolerant cultivar than in the sensitive one. Overall, while in the absence of Fe only slight differences were reported between the two cultivars, the adaptation of Kelvedon to the presence of bicarbonate seems to be related to its greater ability to enhance the antioxidant response at the root level.

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.

Adaptation to iron-deficiency requires remodelling of plant metabolism: an insight in chloroplast biochemistry and functionality

Iron-deficiency is a widespread plant disorder particularly frequent in neutral or alkaline soils, where iron availability often falls below the threshold required to meet plant needs. Plants have developed effi cient mechanisms to acquire iron: strategy I, adopted by all dicots and non-graminaceous monocots, and strategy II, adopted by graminaceous plants. The activation of co-ordinated mechanisms able to perceive and respond to changes in cell iron levels is of main importance in maintaining iron homeostasis, and, consequently, to prevent biochemical and physiological alterations.

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

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

Identification of morphological, biochemical and physiological parameters for characterizing nutritional stress status in arboreous species differently tolerant to chlorosis

Lime-induced chlorosis is one of the major abiotic stresses affecting fruit tree crops in the Mediterranean area. However, fruit tree species have been the object of only few studies and the results obtained are insufficient to supply parameters for breeding. Here we report the results of a study carried out to identify morphological and biochemical modifications induced by low iron availability and a high level of bicarbonate in the medium in pear (cv Conference; tolerant genotype) and quince rootstocks (MA and BA29; susceptible genotypes) cultured by in vitro and hydroponic culture. Morphological parameters of in vitro plantlets were differently influenced by the two stress conditions depending on plant genotype and parameter analyzed, and suggested that the pear cv carried out an adaptive strategy to warrant sufficient iron supply, whereas the two quince root-stocks failed to adapt to conditions typical of calcareous soil. The strong and generalized reduction in chlorophyll and carotenoid content observed only in quince plantlets suggests a down-regulation of the whole chloroplast machinery in iron-deficient quince. Measurement of Fe(III)-chelate reductase activity (FCR) of rooted cuttings from in vitro culture grown in hydroponic solution suggests the probable involvement of enhanced FCR activity in the major tolerance of cv Conference to iron chlorosis. Cv Conference was also less sensitive to bicarbonate supply than quince rootstocks in terms of reduction of leaf pigment content and activation of the photoprotective xanthophyll cycle. In conclusion, this study shows that the mechanisms of differential Fe efficiency are associated to differences in leaf pigment content and photoprotective process and that in vitro culture could be a valid technique to test rootstock susceptibility to iron chlorosis.