Rhizobial nitrogen fixation efficiency shapes endosphere bacterial communities and Medicago truncatula host growth.

BACKGROUND: Despite the knowledge that the soil-plant-microbiome nexus is shaped by interactions amongst its members, very little is known about how individual symbioses regulate this shaping. Even less is known about how the agriculturally important symbiosis of nitrogen-fixing rhizobia with legumes is impacted according to soil type, yet this knowledge is crucial if we are to harness or improve it. We asked how the plant, soil and microbiome are modulated by symbiosis between the model legume Medicago truncatula and different strains of Sinorhizobium meliloti or Sinorhizobium medicae whose nitrogen-fixing efficiency varies, in three distinct soil types that differ in nutrient fertility, to examine the role of the soil environment upon the plant-microbe interaction during nodulation. RESULTS: The outcome of symbiosis results in installment of a potentially beneficial microbiome that leads to increased nutrient uptake that is not simply proportional to soil nutrient abundance. A number of soil edaphic factors including Zn and Mo, and not just the classical N/P/K nutrients, group with microbial community changes, and alterations in the microbiome can be seen across different soil fertility types. Root endosphere emerged as the plant microhabitat more affected by this rhizobial efficiency-driven community reshaping, manifested by the accumulation of members of the phylum Actinobacteria. The plant in turn plays an active role in regulating its root community, including sanctioning low nitrogen efficiency rhizobial strains, leading to nodule senescence in particular plant-soil-rhizobia strain combinations. CONCLUSIONS: The microbiome-soil-rhizobial dynamic strongly influences plant nutrient uptake and growth, with the endosphere and rhizosphere shaped differentially according to plant-rhizobial interactions with strains that vary in nitrogen-fixing efficiency levels. These results open up the possibility to select inoculation partners best suited for plant, soil type and microbial community. Video Abstract.

Regulation of Resource Partitioning Coordinates Nitrogen and Rhizobia Responses and Autoregulation of Nodulation in Medicago truncatula.

Understanding how plants respond to nitrogen in their environment is crucial for determining how they use it and how the nitrogen use affects other processes related to plant growth and development. Under nitrogen limitation the activity and affinity of uptake systems is increased in roots, and lateral root formation is regulated in order to adapt to low nitrogen levels and scavenge from the soil. Plants in the legume family can form associations with rhizobial nitrogen-fixing bacteria, and this association is tightly regulated by nitrogen levels. The effect of nitrogen on nodulation has been extensively investigated, but the effects of nodulation on plant nitrogen responses remain largely unclear. In this study, we integrated molecular and phenotypic data in the legume Medicago truncatula and determined that genes controlling nitrogen influx are differently expressed depending on whether plants are mock or rhizobia inoculated. We found that a functional autoregulation of nodulation pathway is required for roots to perceive, take up, and mobilize nitrogen as well as for normal root development. Our results together revealed that autoregulation of nodulation, root development, and the location of nitrogen are processes balanced by the whole plant system as part of a resource-partitioning mechanism.

Does a voltage-sensitive outer envelope transport mechanism contributes to the chloroplast iron uptake?

MAIN CONCLUSION: Based on the effects of inorganic salts on chloroplast Fe uptake, the presence of a voltage-dependent step is proposed to play a role in Fe uptake through the outer envelope. Although iron (Fe) plays a crucial role in chloroplast physiology, only few pieces of information are available on the mechanisms of chloroplast Fe acquisition. Here, the effect of inorganic salts on the Fe uptake of intact chloroplasts was tested, assessing Fe and transition metal uptake using bathophenantroline-based spectrophotometric detection and plasma emission-coupled mass spectrometry, respectively. The microenvironment of Fe was studied by Mössbauer spectroscopy. Transition metal cations (Cd2+, Zn2+, and Mn2+) enhanced, whereas oxoanions (NO3-, SO42-, and BO33-) reduced the chloroplast Fe uptake. The effect was insensitive to diuron (DCMU), an inhibitor of chloroplast inner envelope-associated Fe uptake. The inorganic salts affected neither Fe forms in the uptake assay buffer nor those incorporated into the chloroplasts. The significantly lower Zn and Mn uptake compared to that of Fe indicates that different mechanisms/transporters are involved in their acquisition. The enhancing effect of transition metals on chloroplast Fe uptake is likely related to outer envelope-associated processes, since divalent metal cations are known to inhibit Fe2+ transport across the inner envelope. Thus, a voltage-dependent step is proposed to play a role in Fe uptake through the chloroplast outer envelope on the basis of the contrasting effects of transition metal cations and oxoaninons.

Protein profile of Beta vulgaris leaf apoplastic fluid and changes induced by Fe deficiency and Fe resupply.

The fluid collected by direct leaf centrifugation has been used to study the proteome of the sugar beet apoplastic fluid as well as the changes induced by Fe deficiency and Fe resupply to Fe-deficient plants in the protein profile. Plants were grown in Fe-sufficient and Fe-deficient conditions, and Fe resupply was carried out with 45 µM Fe(III)-EDTA for 24 h. Protein extracts of leaf apoplastic fluid were analyzed by two-dimensional isoelectric focusing-SDS-PAGE electrophoresis. Gel image analysis revealed 203 consistent spots, and proteins in 81% of them (164) were identified by nLC-MS/MS using a custom made reference repository of beet protein sequences. When redundant UniProt entries were deleted, a non-redundant leaf apoplastic proteome consisting of 109 proteins was obtained. TargetP and SecretomeP algorithms predicted that 63% of them were secretory proteins. Functional classification of the non-redundant proteins indicated that stress and defense, protein metabolism, cell wall and C metabolism accounted for approximately 75% of the identified proteome. The effects of Fe-deficiency on the leaf apoplast proteome were limited, with only five spots (2.5%) changing in relative abundance, thus suggesting that protein homeostasis in the leaf apoplast fluid is well-maintained upon Fe shortage. The identification of three chitinase isoforms among proteins increasing in relative abundance with Fe-deficiency suggests that one of the few effects of Fe deficiency in the leaf apoplast proteome includes cell wall modifications. Iron resupply to Fe deficient plants changed the relative abundance of 16 spots when compared to either Fe-sufficient or Fe-deficient samples. Proteins identified in these spots can be broadly classified as those responding to Fe-resupply, which included defense and cell wall related proteins, and non-responsive, which are mainly protein metabolism related proteins and whose changes in relative abundance followed the same trend as with Fe-deficiency.

Canopy light heterogeneity drives leaf anatomical, eco-physiological, and photosynthetic changes in olive trees grown in a high-density plantation.

In the field, leaves may face very different light intensities within the tree canopy. Leaves usually respond with light-induced morphological and photosynthetic changes, in a phenomenon known as phenotypic plasticity. Canopy light distribution, leaf anatomy, gas exchange, chlorophyll fluorescence, and pigment composition were investigated in an olive (Olea europaea, cvs. Arbequina and Arbosana) orchard planted with a high-density system (1,250 trees ha(-1)). Sampling was made from three canopy zones: a lower canopy (<1 m), a central one (1-2 m), and an upper one (>2 m). Light interception decreased significantly in the lower canopy when compared to the central and top ones. Leaf angle increased and photosynthetic rates and non-photochemical quenching (NPQ) decreased significantly and progressively from the upper canopy to the central and the lower canopies. The largest leaf areas were found in the lower canopy, especially in the cultivar Arbequina. The palisade and spongy parenchyma were reduced in thickness in the lower canopy when compared to the upper one, in the former due to a decrease in the number of cell layers from three to two (clearly distinguishable in the light and fluorescence microscopy images). In both cultivars, the concentration of violaxanthin-cycle pigments and ß-carotene was higher in the upper than in the lower canopy. Furthermore, the de-epoxidized forms zeaxanthin and antheraxanthin increased significantly in those leaves from the upper canopy, in parallel to the NPQ increases. In conclusion, olive leaves react with morphological and photosynthetic changes to within-crown light gradients. These results strengthen the idea of olive trees as "modular organisms" that adjust the modules morphology and physiology in response to light intensity.

The effects of foliar fertilization with iron sulfate in chlorotic leaves are limited to the treated area. A study with peach trees (Prunus persica L. Batsch) grown in the field and sugar beet (Beta vulgaris L.) grown in hydroponics.

Crop Fe deficiency is a worldwide problem. The aim of this study was to assess the effects of foliar Fe applications in two species grown in different environments: peach (Prunus persica L. Batsch) trees grown in the field and sugar beet (Beta vulgaris L. cv. "Orbis") grown in hydroponics. The distal half of Fe-deficient, chlorotic leaves was treated with Fe sulfate by dipping and using a brush in peach trees and sugar beet plants, respectively. The re-greening of the distal (Fe-treated) and basal (untreated) leaf areas was monitored, and the nutrient and photosynthetic pigment composition of the two areas were also determined. Leaves were also studied using chlorophyll fluorescence imaging, low temperature-scanning electron microscopy microanalysis, scanning transmission ion microscopy-particle induced X-ray emission and Perls Fe staining. The distal, Fe-treated leaf parts of both species showed a significant increase in Fe concentrations (across the whole leaf volume) and marked re-greening, with significant increases in the concentrations of all photosynthetic pigments, as well as decreases in de-epoxidation of xanthophyll cycle carotenoids and increases in photochemical efficiency. In the basal, untreated leaf parts, Fe concentrations increased slightly, but little re-greening occurred. No changes in the concentrations of other nutrients were found. Foliar Fe fertilization was effective in re-greening treated leaf areas both in peach trees and sugar beet plants. Results indicate that the effects of foliar Fe-sulfate fertilization in Fe-deficient, chlorotic leaves were minor outside the leaf surface treated, indicating that Fe mobility within the leaf is a major constraint for full fertilizer effectiveness in crops where Fe-deficiency is established and leaf chlorosis occurs.

Zinc deficiency in field-grown pecan trees: changes in leaf nutrient concentrations and structure.

BACKGROUND: Zinc (Zn) deficiency is a typical nutritional disorder in pecan trees [Carya illinoinensis (Wangenh.) C. Koch] grown under field conditions in calcareous soils in North America, including northern Mexico and south-western United States. The aim of this study was to assess the morphological and nutritional changes in pecan leaves affected by Zn deficiency as well as the Zn distribution within leaves. RESULTS: Zinc deficiency led to decreases in leaf chlorophyll concentrations, leaf area and trunk cross-sectional area. Zinc deficiency increased significantly the leaf concentrations of K and Ca, and decreased the leaf concentrations of Zn, Fe, Mn and Cu. All nutrient values found in Zn-deficient leaves were within the sufficiency ranges, with the only exception of Zn, which was approximately 44, 11 and 9 µg g(-1) dry weight in Zn-sufficient, moderately and markedly Zn-deficient leaves, respectively. Zinc deficiency led to decreases in leaf thickness, mainly due to a reduction in the thickness of the palisade parenchyma, as well as to increases in stomatal density and size. The localisation of Zn was determined using the fluorophore Zinpyr-1 and ratio-imaging technique. Zinc was mainly localised in the palisade mesophyll area in Zn-sufficient leaves, whereas no signal could be obtained in Zn-deficient leaves. CONCLUSION: The effects of Zn deficiency on the leaf characteristics of pecan trees include not only decreases in leaf chlorophyll and Zn concentrations, but also a reduction in the thickness of the palisade parenchyma, an increase in stomatal density and pore size and the practical disappearance of Zn leaf pools. These characteristics must be taken into account to design strategies to correct Zn deficiency in pecan tree in the field.

Mercury uptake by Silene vulgaris grown on contaminated spiked soils.

Mercury is a highly toxic pollutant with expensive clean up, because of its accumulative and persistent character in the biota. The objective of this work was to evaluate the effectiveness of Silene vulgaris, facultative metallophyte which have populations on both non-contaminated and metalliferous soils, to uptake Hg from artificially polluted soils. A pot experiment was carried out in a rain shelter for a full growth period. Two soils (C pH = 8.55 O.M. 0.63% and A pH = 7.07 O.M. 0.16%) were used, previously contaminated with Hg as HgCl(2) (0.6 and 5.5 mg Hg kg(-1) soil). Plants grew healthy and showed good appearance throughout the study without significantly decreasing biomass production. Mercury uptake by plants increased with the mercury concentration found in both soils. Differences were statistically significant between high dosage and untreated soil. The fact that S. vulgaris retains more mercury in root than in shoot and also, the well known effectiveness of these plants in the recovering of contaminated soils makes S. vulgaris a good candidate to phytostabilization technologies.

Natural attenuation of residual heavy metal contamination in soils affected by the Aznalcóllar mine spill, SW Spain.

Non-amended soils affected by pyritic sludge residues were monitored for 7 years to assess the long-term natural attenuation ability of these soils. The decrease in both the total concentration of elements (particularly As) and (NH(4))(2)SO(4)-extractable fractions of Mn, and Zn, below the maximum permissible levels indicate a successful natural ability to attenuate soil pollution. Soil acidification by pyrite oxidation and rainfall-enhanced leaching were the largest contributors to the reduction of metals of high (Mn, Cu, Zn and Cd) and low (Fe, Al, and As) availability. Periodic use of correlation and spatial distribution analysis was useful in monitoring elemental dispersion and soil property/element relationships.

Using Mediterranean shrubs for the phytoremediation of a soil impacted by pyritic wastes in Southern Spain: a field experiment.

Re-vegetation is the main aim of ecological restoration projects, and in Mediterranean environments native plants are desirable to achieve successful restoration. In 1998, the burst of a tailings dam flooded the Guadiamar river valley downstream from Aznalcóllar (Southern Spain) with sludges that contained elevated concentrations of metals and metalloids, polluting soils and waters. A phytoremediation experiment to assess the potential use of native shrub species for the restoration of soils affected by the spillage was performed from 2005 to 2007, with soils divided into two groups: pH < 5 and pH > 5. Four native shrubs (Myrtus communis, Retama sphaerocarpa, Rosmarinus officinalis and Tamarix gallica) were planted and left to grow without intervention. Trace element concentrations in soils and plants, their extractability in soils, transfer factors and plant survival were used to identify the most-interesting species for phytoremediation. Total As was higher in soils with pH < 5. Ammonium sulphate-extractable zinc, copper, cadmium and aluminium concentrations were higher in very-acid soils, but arsenic was extracted more efficiently when soil pH was >5. Unlike As, which was either fixed by Fe oxides or retained as sulphide, the extractable metals showed significant relationships with the corresponding total soil metal concentration and inverse relationships with soil pH. T. gallica, R. officinalis and R. sphaerocarpa survived better in soils with pH > 5, while M. communis had better survival at pH < 5. R. sphaerocarpa showed the highest survival (30%) in all soils. Trace element transfer from soil to harvestable parts was low for all species and elements, and some species may have been able to decrease trace element availability in the soil. Our results suggest that R. sphaerocarpa is an adequate plant species for phytostabilising these soils, although more research is needed to address the self-sustainability of this remediation technique and the associated environmental changes.

Towards a knowledge-based correction of iron chlorosis.

Iron (Fe) deficiency-induced chlorosis is a major nutritional disorder in crops growing in calcareous soils. Iron deficiency in fruit tree crops causes chlorosis, decreases in vegetative growth and marked fruit yield and quality losses. Therefore, Fe fertilizers, either applied to the soil or delivered to the foliage, are used every year to control Fe deficiency in these crops. On the other hand, a substantial body of knowledge is available on the fundamentals of Fe uptake, long and short distance Fe transport and subcellular Fe allocation in plants. Most of this basic knowledge, however, applies only to Fe deficiency, with studies involving Fe fertilization (i.e., with Fe-deficient plants resupplied with Fe) being still scarce. This paper reviews recent developments in Fe-fertilizer research and the state-of-the-art of the knowledge on Fe acquisition, transport and utilization in plants. Also, the effects of Fe-fertilization on the plant responses to Fe deficiency are reviewed. Agronomical Fe-fertilization practices should benefit from the basic knowledge on plant Fe homeostasis already available; this should be considered as a long-term goal that can optimize fertilizer inputs, reduce grower's costs and minimize the environmental impact of fertilization.

Comparative analysis of the contribution of phytochelatins to cadmium and arsenic tolerance in soybean and white lupin.

The biosynthesis of phytochelatins (PCs) plays a crucial role in the detoxification and homeostasis of heavy metals and metalloids in plants. However, in an increasing number of plant species metal(loid) tolerance is not well correlated with the accumulation of PCs: tolerant ecotypes frequently contain lower levels of PCs than non-tolerant ecotypes. In this study we have compared the responses of soybean (Glycine max L. cv. Resnik) and white lupin (Lupinus albus L. cv. Marta) to cadmium and arsenate in order to assess the role of homophytochelatins (hPCs) in the tolerance of soybean to these toxic elements. Soybean plants treated with Cd and As showed a high contribution of homo-glutathione (hGSH) to the pool of thiols in shoots in comparison to white lupin. Higher levels of hPCs in Cd-treated soybeans compared to PCs in lupins did not prevent growth inhibition. In contrast, the role of hPCs in the detoxification mechanism to arsenate in soybean seems to be clearer, showing higher thiol concentrations and lower growth reductions than those present in lupin plants.

Evolution of arsenate toxicity in nodulated white lupine in a long-term culture.

White lupine is an As-resistant legume that is of interest for phytoremediation of As-contaminated soils. To achieve successful phytoremediation, monitoring of the nutritional status of the selected plant species during the entire culture cycle is required to maintain a plant cover with high biomass production. A long-term pot experiment was carried out with nodulated lupine grown on perlite with 10 and 100 microM As concentrations. The reproductive period (from 10 weeks) was the most sensitive phenologic stage of white lupine to long-term As exposure. The 10 microM As treatment increased the uptake and translocation of micronutrients, except for Cu, mainly at flowering with As levels in pods below the statutory limit (1 mg kg (-1) fresh weight). However, the 100 microM As treatment induced significant differences compared to the control. These findings confirm the relatively high resistance of white lupine to arsenate and support the use of this species in phytoremediation and/or revegetation of As-contaminated sites, with special attention on P and Cu nutrition at flowering.

Bioavailability of metals and As from acidified multicontaminated soils: use of white lupin to validate several extraction methods.

White lupin is an annual crop that has been used for phytostabilization of acidified multicontaminated (heavy metals and As) soils from the Aznalcóllar spill-affected area, Southern Spain. One of the most important factors for successful phytostabilization is monitoring the pollutant bioavailability in the soil. The aim of this work was to determine the best-suited method for assessing the bioavailability of heavy metals together with As in the Aznalcóllar spill-affected area, by means of a systematic comparison between different extraction methods (Ammonium bicarbonate-diethylenetriamine pentaacetic acid (AB-DTPA), CaCl2, NaNO3, BCR, (NH4)2SO4 and rhizo). Both AB-DTPA and the first step of the BCR method were found to be unsuitable for assessing the bioavailability of heavy metals and As to plants growing in acidic soils. However, CaCl2-extractable As, Cu, and Zn and NaNO3-extractable As and Zn were well correlated with their concentrations in plant organs. Rhizo and (NH4)2SO4, with the highest determination coefficients, were the most recommended simple extraction methods to assess the bioavailability of As, Cu, Fe, Mn, and Zn in acidified multicontaminated soils using white lupin as an excluder model plant.

Subcellular compartmentalisation of cadmium in white lupins determined by energy-dispersive X-ray microanalysis.

The microlocalisation of cadmium (Cd) at the tissue-cellular level in Lupinus albus L. cv. Multolupa was determined by energy-dispersive X-ray microanalysis (EDXMA). Experimental plants were grown on Cd-treated (0 and 150 microM) perlite for 35 days. In leaves, Cd was found inside cells (cytoplasm or vacuoles), especially in the vascular bundle cells. Cd-induced damage of the chloroplast structure was also detected. EDXMA of the roots showed the cell wall to be the main area of Cd binding at the cellular level; only a small amount of Cd was found in the vacuoles. At the tissue level, a decreasing Cd gradient was seen from the outer to the inner root cortical parenchyma. Cd and S were found co-localised in the vascular cylinder.

Chelate-assisted phytoextraction of heavy metals in a soil contaminated with a pyritic sludge.

The occurrence of many polluted areas as that affected by the accident of the Aznalcóllar pyrite mine has promoted phytoremediation as a technology able to reduce the risk of heavy metal contamination at low cost. White lupin plant has been considered a good candidate for phytoremediation. We studied the capacity of several complexing agents to improve the ability of white lupin for heavy metal phytoremediation in soils with multi-elemental pollution from acid pyritic sludge. Solution-soil interaction was studied and pot experiments with sludge-affected soil were carried out to this end. The interaction experiments indicated that EDTA and NTA were more efficient than malate and citrate in solubilizing metals (Fe, Mn, Cu, Zn, Cd), with minimum differences between EDTA and NTA. The pot trial showed that NTA was able to mobilize toxic elements from sludge-polluted soil and hence increasing their concentrations in plant (Mn, Cu, Zn, As, Cd). However, the NTA treatment promoted an increase of toxic elements concentrations, especially for As, Cd, Pb, in the lixiviates exceeding the maximum permissible levels, so a careful management of chelate is necessary.

Use of perlite in cadmium plant studies: an approach to polluted soil conditions.

Two different types of hydroponic cultures, "water culture" and "perlite system", were compared using white lupin plants (Lupinus albus L., cv. Marta) under different Cd treatments: 0, 0.2, 0.6, 2, 4, 6, 13, 20, 40 and 60 microM (water culture) and 0.2, 2, 20, 60 and 150 microM (moistened perlite). Fresh weight, shoot and root length, and total Cd concentration in the plants were measured. Moreover, a batch experiment was carried out to study the ability of perlite to adsorb and desorb Cd from nutrient solution. Lupin plants under Cd treatments in "water culture" showed a higher growth inhibition than those grown on perlite. A high positive correlation between Cd concentration in the plant and Cd supply was obtained regardless of the substrate used. Moreover, a high positive correlation between Cd doses with the "perlite system" and their equivalent Cd doses estimated for the "water culture" system was observed. Thus, the "water culture-equivalent" Cd doses were 14 times lower than the Cd doses in the perlite system. On the other hand, desorbed Cd concentrations were calculated giving values 12 times lower than the tested Cd doses.