Graziano Zocchi

A drought resistance-promoting microbiome is selected by root system under desert farming

Background Traditional agro-systems in arid areas are a bulwark for preserving soil stability and fertility, in the sight of “reverse desertification”. Nevertheless, the impact of desert farming practices on the diversity and abundance of the plant associated microbiome is poorly characterized, including its functional role in supporting plant development under drought stress. Methodology/Principal Findings We assessed the structure of the microbiome associated to the drought-sensitive pepper plant (Capsicum annuum L.) cultivated in a traditional Egyptian farm, focusing on microbe contribution to a crucial ecosystem service, i.e. plant growth under water deficit. The root system was dissected by sampling root/soil with a different degree of association to the plant: the endosphere, the rhizosphere and the root surrounding soil that were compared to the uncultivated soil. Bacterial community structure and diversity, determined by using Denaturing Gradient Gel Electrophoresis, differed according to the microhabitat, indicating a selective pressure determined by the plant activity. Similarly, culturable bacteria genera showed different distribution in the three root system fractions. Bacillus spp. (68% of the isolates) were mainly recovered from the endosphere, while rhizosphere and the root surrounding soil fractions were dominated by Klebsiella spp. (61% and 44% respectively). Most of the isolates (95%) presented in vitro multiple plant growth promoting (PGP) activities and stress resistance capabilities, but their distribution was different among the root system fractions analyzed, with enhanced abilities for Bacillus and the rhizobacteria strains. We show that the C. annuum rhizosphere under desert farming enriched populations of PGP bacteria capable of enhancing plant photosynthetic activity and biomass synthesis (up to 40%) under drought stress. Conclusions/Significance Crop cultivation provides critical ecosystem services in arid lands with the plant root system acting as a “resource island” able to attract and select microbial communities endowed with multiple PGP traits that sustain plant development under water limiting conditions.

The effect of commercial humic acid on tomato plant growth and mineral nutrition

Abstract The effects of humic acids extracted from two commercially‐available products (CP‐A prepared from peat and CP‐B prepared from leonardite) on the growth and mineral nutrition of tomato plants (Lycopersicon esculentum L.) in hydroponics culture were tested at concentrations of 20 and 50 mg L‐1. Both the humic acids tested stimulated plants growth. The CP‐A stimulated only root growth, especially at 20 mg L‐1 [23% and 22% increase over the control, on fresh weight basis (f.w.b.), and dry weight basis (d.w.b.), respectively]. In contrast, CP‐B showed a positive effect on both shoots and roots, especially at 50 mg L‐1 (shoots: 8% and 9% increase over the control; roots: 18% and 16% increase over the control, on f.w.b. and d.w.b., respectively). Total ion uptake by the plants was affected by the two products. In particular, CP‐A showed an increase in the uptake of nitrogen (N), phosphorus (P), iron (Fe), and copper (Cu), whereas, CP‐B showed positive effects for N, P, and Fe uptake. The change in the F...

Improved plant resistance to drought is promoted by the root‐associated microbiome as a water stress‐dependent trait

Although drought is an increasing problem in agriculture, the contribution of the root-associated bacterial microbiome to plant adaptation to water stress is poorly studied. We investigated if the culturable bacterial microbiome associated with five grapevine rootstocks and the grapevine cultivar Barbera may enhance plant growth under drought stress. Eight isolates, over 510 strains, were tested in vivo for their capacity to support grapevine growth under water stress. The selected strains exhibited a vast array of plant growth promoting (PGP) traits, and confocal microscopy observation of gfp-labelled Acinetobacter and Pseudomonas isolates showed their ability to adhere and colonize both the Arabidopsis and grapevine rhizoplane. Tests on pepper plants fertilized with the selected strains, under both optimal irrigation and drought conditions, showed that PGP activity was a stress-dependent and not a per se feature of the strains. The isolates were capable of increasing shoot and leaf biomass, shoot length, and photosynthetic activity of drought-challenged grapevines, with an enhanced effect in drought-sensitive rootstock. Three isolates were further assayed for PGP capacity under outdoor conditions, exhibiting the ability to increase grapevine root biomass. Overall, the results indicate that PGP bacteria contribute to improve plant adaptation to drought through a water stress-induced promotion ability.

Changes in the proteomic and metabolic profiles of Beta vulgaris root tips in response to iron deficiency and resupply

BackgroundPlants 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.ResultsRoot 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.ConclusionsThe 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.

Metabolic Implications in the Biochemical Responses to Iron Deficiency in Cucumber (Cucumis sativus L.) Roots.

Strategy I plants respond to Fe deficiency by inducing morphological and biochemical modifications at the root level that are apt to make iron available for uptake. Cucumber (Cucumis sativus L.) grown in the absence of Fe has been shown to increase the capacity to acidify the rhizosphere and Fe3+ reduction activity. We have determined in these roots some metabolic activities that might be correlated with the increased proton extrusion. Proton efflux from roots may be followed by a mechanism regulating the cytosolic pH according to the pH-stat theory. Roots grown in the absence of Fe showed an increase in dark 14CO2 fixation and organic acid synthesis and a 6-fold increase in the extractable phosphoenolpyruvate carboxylase activity with respect to the control roots. Dehydrogenase activities producing cytosolic NAD(P)H were also increased under Fe deficiency. The presence of Fe2+, but not Fe3+, inhibited dark 14CO2 fixation in a range between 24 and 52% but did not show any effect on the in vitro phosphoenolpyruvate carboxylase activity.

Iron availability affects the function of mitochondria in cucumber roots

* In Strategy-I-plants, iron (Fe) deficiency induces processes leading to increased Fe solubilization in the rhizosphere, including reduction by ferric reductases and active proton extrusion. These processes require active respiration to function. In this work we investigated the effect of Fe deficiency on respiratory activities of cucumber (Cucumis sativus) roots. * We compared oxygen consumption rate and the activities of the respiratory chain complexes on purified mitochondria from roots grown in the presence or absence of Fe using biochemical and molecular approaches. * Oxygen consumption rate in apex roots was increased under Fe deficiency that was mostly resistant to KCN and salycilichydroxamic acid (SHAM) inhibitors, indicating other oxygen-consuming reactions could be present. Indeed, enzyme assays revealed that lack of Fe induced a decrease in the activities of respiratory complexes that was proportional to the number of Fe atoms in each complex. A decrease of cyt c, Rieske and NAD9 proteins was also observed. Transmission electron microscopy (TEM) analysis showed that mitochondria undergo structural changes under Fe deficiency. * Our data show that mitochondria and the electron transport chain are an important target of Fe limitation and that mitochondria modify their function to meet higher demands for organic acids while restricting the activity of enzymes with Fe cofactors.

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.

Metabolic Changes in Iron-Stressed Dicotyledonous Plants

The response to iron deficiency stress in dicotyledonous plants is by far more complex than the simple activation of the reduction-based mechanism. In most of the Strategy I plants studied so far there is an associated increase in the activity of a plasma membrane H + -ATPase which actively extrudes the protons necessary both for decreasing the rhizospheric pH and generating the electrochemical proton gradient needed to drive ion uptake. Along with these activities, localised on the root plasma membrane, it has been found that metabolism is involved in sustaining the production of reducing equivalents (NAD(P)H) and ATP. Many processes (glycolysis, respiration, etc.) are increased to efficiently sustain the response to iron depletion. In particular, the activity of phosphoenolpyruvate carboxylase is increased several fold. These increases could be linked to the production of substrates for the ferric chelate reductases and H + -ATPase activities, generation of H + for the cytosolic pH- stat and organic acids. The activation of all these processes makes plants more efficient in the acquisition of iron.

Mechanisms of Fe-efficiency in roots of Vitis spp. in response to iron deficiency stress

Iron chlorosis induced by Fe-deficiency is a widespread nutritional disorder in many woody plants and in particular in grapevine. This phenomenon results from different environmental, nutritional and varietal factors. Strategy I plants respond to Fe-deficiency by inducing physiological and biochemical modifications in order to increase Fe uptake. Among these, acidification of the rhizosphere, membrane redox activities and synthesis of organic acids are greatly enhanced during Fe-deficiency. Grapevine is a strategy I plant but the knowledge on the physiological and biochemical responses to this iron stress deficiency in this plant is still very poor.In this work four different genotypes of grapevine were assayed for these parameters. It was found that there is a good correlation between genotypes which are known to be chlorosis-resistant and increase in both rhizosphere acidification and FeIII reductase activity. In particular, when grown in the absence of iron, Vitis berlandieri and Vitis vinifera cv Cabernet sauvignon and cv Pinot blanc show a higher capacity to acidify the culture medium (pH was decreased by 2 units), a higher concentration of organic acids, a higher resting transmembrane electrical potential and a greater capacity to reduce FeIII-chelates. On the contrary, Vitis riparia, well known for its susceptibility to iron chlorosis, fails to decrease the pH of the medium and shows a lower concentration in organic acids, lower capacity to reduce FeIII and no difference in the resting transmembrane electrical potential. H Marschner Section editor

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.