Synchrotron-Based Techniques Shed Light on Mechanisms of Plant Sensitivity and Tolerance to High Manganese in the Root Environment

Plant species differ in response to high available manganese (Mn), but the mechanisms of sensitivity and tolerance are poorly understood. In solution culture, greater than or equal to 30 µm Mn decreased the growth of soybean (Glycine max), but white lupin (Lupinus albus), narrow-leafed lupin (Lupin angustifolius), and sunflower (Helianthus annuus) grew well at 100 µm Mn. Differences in species’ tolerance to high Mn could not be explained simply by differences in root, stem, or leaf Mn status, being 8.6, 17.1, 6.8, and 9.5 mmol kg^–1 leaf fresh mass at 100 µm Mn. Furthermore, x-ray absorption near edge structure analyses identified the predominance of Mn(II), bound mostly to malate or citrate, in roots and stems of all four species. Rather, differences in tolerance were due to variations in Mn distribution and speciation within leaves. In Mn-sensitive soybean, in situ analysis of fresh leaves using x-ray fluorescence microscopy combined with x-ray absorption near edge structure showed high Mn in the veins, and manganite [Mn(III)] accumulated in necrotic lesions apparently through low Mn sequestration in vacuoles or other vesicles. In the two lupin species, most Mn accumulated in vacuoles as either soluble Mn(II) malate or citrate. In sunflower, Mn was sequestered as manganite at the base of nonglandular trichomes. Hence, tolerance to high Mn was ascribed to effective sinks for Mn in leaves, as Mn(II) within vacuoles or through oxidation of Mn(II) to Mn(III) in trichomes. These two mechanisms prevented Mn accumulation in the cytoplasm and apoplast, thereby ensuring tolerance to high Mn in the root environment.Manganese (Mn) is an essential element for plant growth, but its availability differs greatly in space and time, depending largely on the nature and amount of Mn minerals present and on the soil’s pH and redox potential. With an elaborate chemistry, Mn forms complexes with many organic and inorganic ligands. In soils, Mn has three common oxidation states, Mn(II), Mn(III), and Mn(IV), which form hydrated oxides of mixed valency; Mn is present also as numerous carbonates, silicates, sulfates, and phosphates (Lindsay, 1979). Cationic Mn²⁺ is the most common form readily absorbed by plant roots (Clarkson, 1988). The toxicity of Mn occurs in acid or waterlogged soils high in Mn minerals.Many plants have mechanisms to accommodate the large differences in Mn²⁺ in soils. At low available Mn, uptake is increased in some Poaceae by excretion of phytosiderophores of the mugineic acid family (Takahashi et al., 2003), with root phytase exudation also potentially important for acquisition of Mn when Mn availability is limited (George et al., 2014). Mechanisms in other plants include the ability of roots to decrease rhizosphere pH or excrete organic ligands (Neumann and Romheld, 2012; Lambers et al., 2015). However, the relative importance of the many complexes on Mn uptake remains unclear. Toxicity results from high Mn in leaf cell walls (Wissemeier et al., 1992; Wissemeier and Horst, 1992) and through adverse effects on symplastic proteins (Führs et al., 2008). Many plants have mechanisms that limit the adverse effects of high Mn²⁺ in soils, with numerous ligands involved in its translocation and that of other essential cations (Haydon and Cobbett, 2007). Edwards and Asher (1982) classified a range of crop and pasture species based on their ability to deal with high Mn as those that (1) limit Mn from entering the roots, (2) retain Mn in the roots, or (3) tolerate high Mn in the shoots. At the extreme are plants that hyperaccumulate more than 10,000 mg Mn kg^–1 on a dry mass (DM) basis in foliar tissues without metabolic damage (Fernando et al., 2013; van der Ent et al., 2013). Based on 15% DM of leaves, this equates to 12.1 mmol kg^–1 on a fresh mass (FM) basis. Celosia argentia, a species adapted to growth on Mn-contaminated mine tailings, accumulated more than 20,000 mg kg^–1 Mn in leaves (Liu et al., 2014). Tolerance of high Mn in shoots of some Mn hyperaccumulators has been found to occur through binding to ligands (such as malate or citrate) or sequestration in the vacuole (Fernando et al., 2010).Characteristic symptoms of Mn toxicity include chlorotic and distorted leaves with small necrotic lesions. These lesions have been shown in cowpea (Vigna unguiculata) to contain oxidized Mn and callose (Wissemeier et al., 1992), which forms as a reaction to high intracellular Ca (Kartusch, 2003). The necrotic lesions result mainly from oxidized phenolics (Wissemeier and Horst, 1992) and increased peroxidase activity in the apoplast (Horst et al., 1999). With a critical solution concentration for toxicity (10% growth reduction) of no more than 9 µm Mn, Edwards and Asher (1982) found that cotton (Gossypium hirsutum), bean (Phaseolus vulgaris), cowpea, and soybean (Glycine max) were the most sensitive species of 13 crop and pasture plants grown for 18 to 31 d at constant Mn in solution culture. By contrast, the critical concentration for sunflower (Helianthus annuus) was 7 times higher at 65 µm Mn. Sunflower was the first species found to tolerate high Mn through its sequestration in the trichomes on stems, petioles, and leaves (Blamey et al., 1986). The suspected accumulation of Mn was confirmed using wavelength dispersive x-ray spectroscopy with darkening inferred as due to insoluble higher oxides of Mn. Similarly, high Mn results in darkened trichomes of cucumber (Cucumis sativus) leaves due to oxidized Mn, as shown by the colorimetric benzidine test (Horiguchi, 1987). Watermelon (Citrullus lanatus; Elamin and Wilcox, 1986b), but not muskmelon (Citrullus melo; Elamin and Wilcox, 1986a), grown at high Mn also develops small dark spots around the leaf trichomes. Other species that sequester Mn in the trichomes include common nettle (Urtica dioica; Hughes and Williams, 1988) and Alyssum murale, a Ni hyperaccumulator (Broadhurst et al., 2009; McNear and Küpper, 2014). Thus, some plants in four families, Asteraceae, Cucurbitaceae, Urticaceae, and Brassicaceae, tolerate high Mn in shoots through Mn sequestration in or around the trichomes. The mechanisms may differ, however, because the high Mn present during development of common nettle stinging hairs decreases as plants mature (Hughes and Williams, 1988).Recently developed techniques, including those based on synchrotron radiation, allow investigations of the distribution and speciation of Mn in planta, with most research to date focused on Mn hyperaccumulators (Fernando et al., 2013). For example, Fernando et al. (2010) used x-ray absorption near-edge spectroscopy (XANES) to confirm the widely accepted view that Mn(II) predominates in seven Mn hyperaccumulators. Synchrotron-based x-ray fluorescence microspectroscopy (µ-XRF) was used by McNear and Küpper (2014) to show that the basal region of trichomes of A. murale plants grown at no more than 10 µm Mn contained Mn(II) complexed with phosphate. At 50 µm Mn in solution, however, the increased amount of Mn that had accumulated around the trichomes was present as Mn(III). Few studies, however, have used synchrotron-based techniques to investigate the mechanisms of Mn toxicity and tolerance in agronomic species despite their importance for food production in regions where soils are acidic or intermittently waterlogged. One study on cowpea, with a critical toxicity concentration of only 2 µm Mn (Edwards and Asher, 1982), has shown an accumulation of Mn-citrate in the root cap and associated mucigel within 5 min of exposure to 150 µm Mn (Kopittke et al., 2013).This study aimed to determine the distribution and speciation of Mn in fresh roots, stems, and leaves of four crop species, soybean, white lupin (Lupinus albus), narrow-leafed lupin (Lupinus angustifolius), and sunflower, which differ in tolerance to high Mn. It was hypothesized that Mn distribution and speciation would differ between Mn-sensitive soybean and the three other species. Furthermore, we considered it likely that the Mn tolerance mechanism of sunflower would differ from those of the two lupin species, which do not have darkened trichomes when grown at high Mn.     

Phosphorus application and elevated CO2 enhance drought tolerance in field pea grown in a phosphorus-deficient vertisol

Background and Aims Benefits to crop productivity arising from increasing CO₂ fertilization may be offset by detrimental effects of global climate change, such as an increasing frequency of drought. Phosphorus (P) nutrition plays an important role in crop responses to water stress, but how elevated CO₂ (eCO₂) and P nutrition interact, especially in legumes, is unclear. This study aimed to elucidate whether P supply improves plant drought tolerance under eCO₂.Methods A soil-column experiment was conducted in a free air CO₂ enrichment (SoilFACE) system. Field pea (Pisum sativum) was grown in a P-deficient vertisol, supplied with 15 mg P kg⁻¹ (deficient) or 60 mg P kg⁻¹ (adequate for crop growth) and exposed to ambient CO₂ (aCO₂; 380–400 ppm) or eCO₂ (550–580 ppm). Drought treatments commenced at flowering. Measurements were taken of soil and leaf water content, photosynthesis, stomatal conductance, total soluble sugars and inorganic P content (Pi).Key Results Water-use efficiency was greatest under eCO₂ when the plants were supplied with adequate P compared with other treatments irrespective of drought treatment. Elevated CO₂ decreased stomatal conductance and transpiration rate, and increased the concentration of soluble sugars and relative water contents in leaves. Adequate P supply increased concentrations of soluble sugars and Pi in drought-stressed plants. Adequate P supply but not eCO₂ increased root length distribution in deeper soil layers.Conclusions Phosphorus application and eCO₂ interactively enhanced periodic drought tolerance in field pea as a result of decreased stomatal conductance, deeper rooting and high Pi availability for carbon assimilation in leaves.     

The impact of elevated carbon dioxide on the phosphorus nutrition of plants: a review

Background Increasing attention is being focused on the influence of rapid increases in atmospheric CO₂ concentration on nutrient cycling in ecosystems. An understanding of how elevated CO₂ affects plant utilization and acquisition of phosphorus (P) will be critical for P management to maintain ecosystem sustainability in P-deficient regions.Scope This review focuses on the impact of elevated CO₂ on plant P demand, utilization in plants and P acquisition from soil. Several knowledge gaps on elevated CO₂-P associations are highlighted.Conclusions Significant increases in P demand by plants are likely to happen under elevated CO₂ due to the stimulation of photosynthesis, and subsequent growth responses. Elevated CO₂ alters P acquisition through changes in root morphology and increases in rooting depth. Moreover, the quantity and composition of root exudates are likely to change under elevated CO₂, due to the changes in carbon fluxes along the glycolytic pathway and the tricarboxylic acid cycle. As a consequence, these root exudates may lead to P mobilization by the chelation of P from sparingly soluble P complexes, by the alteration of the biochemical environment and by changes to microbial activity in the rhizosphere. Future research on chemical, molecular, microbiological and physiological aspects is needed to improve understanding of how elevated CO₂ might affect the use and acquisition of P by plants.     

Growth Medium and Phosphorus Supply Affect Cluster Root Formation and Citrate Exudation by Lupinus albus Grown in a Sand/Solution Split-Root System

We examined cluster root formation and root exudation by white lupin (Lupinus albus L. cv. Kiev Mutant) in response to growth medium and phosphorus supply in a sand/solution split-root system. The split-root system consisted of a nutrient solution compartment and a siliceous sand compartment. Phosphorus was applied at 1 (low-P plants) or 50 (high-P plants) μM as KH₂PO₄ to the solution compartment and at 10, 50 or 250 mg P kg⁻¹ as hydroxyapatite (Ca-P) to the sand compartment. In contrast to the high-P plants, P concentration and P uptake in the low-P plants increased with increasing P supply to the sand compartment. The NaHCO₃-extractable P was lower in the rhizosphere of the low-P plants than the high-P ones. The proton extrusion rate by the solution-grown roots of the low-P plants was higher than that of the high-P plants at the early growth stage. For the low-P plants, the proportion of dry root biomass allocated to cluster roots was higher in the solution compartment than that in the sand compartment. The citrate exudation increased in the sand compartment and decreased in the solution compartment with time, showing a lack of synchronization in citrate exudation by two root halves grown in different media. The cluster root proportion and citrate exudation in both compartments decreased with increasing shoot P concentration. An additional experiment with no P added to either root compartment showed that the proportion of cluster roots was about 9% lower in the sand than solution compartments. The results suggest that cluster root formation and citrate exudation can be significantly affected by the root growth medium in addition to being regulated by shoot P status. More P can be exploited from sparingly available Ca-P by the low-P plants than the high-P ones due to greater citrate exudation under P deficiency.     

Biological amelioration of subsoil acidity through managing nitrate uptake by wheat crops

Subsoil acidity occurs in many agricultural lands in the world, and is considered to be an irreversible constraint due to amelioration difficulties. This field study aimed to develop a biological method to ameliorate subsoil acidity through the root-induced alkalisation resulting from nitrate uptake. Aluminium (Al)-tolerant wheat variety Diamondbird and Al-sensitive variety Janz (Triticum aestivum L.) were grown at two contrasting field sites with mild and severe subsurface acidity, respectively, and were supplied with either Ca(NO₃)₂ at the soil surface, Ca(NO₃)₂ at 10 cm depth or urea at 10 cm depth. Application of nitrate increased rhizosphere pH up to 0.5 units and bulk soil pH to 0.3 units, and to a depth >30 cm in the Kandosol. The placement of nitrate at 10 cm increased subsoil pH more than the surface application. Nitrate application increased nitrate concentration in soil profiles as expected, whereas urea application increased NH ₄ ⁺ concentration which in turn favored acidification processes. Diamondbird generally produced more tillers and shoot biomass at anthesis but the two varieties did not differ in grain yield or rhizosphere alkalisation. Similar grain yields were achieved under supply of nitrate and urea. The results suggest that biological amelioration through managing nitrate uptake is possible as part of an integrated approach to combat subsoil acidity in farming systems.     

Chickpea facilitates phosphorus uptake by intercropped wheat from an organic phosphorus source

Pot experiments were conducted to investigate interspecific complementation in utilization of phytate and FePO₄ by plants in the wheat (Triticum aestivum L.)/chickpea (Cicer arietinum L.) intercropping under sterile and non-sterile conditions. The pots were separated into two compartments by either a solid root barrier to eliminate root contact and solute movement, by a nylon mesh (30 M) to prevent root contact but permit solute exchange, or not separated between the compartments. Wheat plants were grown in one compartment and chickpea in the other. Two P sources were tested at 60 mg P kg^–1 soil (sodium phytate or FePO₄). Under non-sterile conditions, the biomass of wheat was significantly greater when the roots were intermingled with chickpea than when the roots were separated from chickpea roots by a solid root barrier or nylon mesh. When phytate–P was applied, P concentrations in wheat (2.9 g kg^–1 in shoots and 1.4 g kg^–1 in roots) without root barrier between the two species were higher than those in the treatments with nylon mesh or with the solid root barrier separation (1.9 g kg^–1 in shoots and 1.0 g kg^–1 in roots). In contrast, P concentrations in wheat supplied with FePO₄ were similar between the root separation treatments. There was no significant difference in P uptake by chickpea between the P sources or between the root separation treatments, except that P uptake was greater in the phytate treatment with the root barrier. Total P uptake from phytate was increased by 25% without root separation compared to the root separation treatments. Under sterile conditions and supply of phytate–P, the biomass of wheat was doubled when the roots were intermingled with chickpea and increased by a third with the nylon mesh separation compared to that with the solid root barrier. Biomass production in wheat at various treatments correlated with P concentration in shoot. Biomass production and P concentration in chickpea were unaffected by root separation. Total P uptake by plants was 68% greater with root intermingling and 37% greater with nylon mesh separation than that with the solid root barrier. The results suggest that chickpea roots facilitate P utilization from the organic P by wheat.     

Cadmium reduces zinc uptake but enhances its translocation in the cadmium-accumulator, <Emphasis Type="Italic">Carpobrotus rossii</Emphasis>, without affecting speciation

Background and aims

Interactions between Cd and Zn occur in soils and plants but are inconsistent. This study examined how Cd/Zn interactions influence the growth of Carpobrotus rossii (Haw.) and the accumulation of Cd and Zn in plants.

Methods

Plants were grown in nutrient solutions containing 5–100 μM Zn and 0, 5 or 15 μM Cd. Plant growth and tissue concentrations were measured, and the speciation of Zn within the plant tissues determined using synchrotron-based X-ray absorption spectroscopy.

Results

There was an additive negative interaction between Cd and Zn on root growth. Only the highest level of Zn (100 μM) decreased Cd concentrations in root and shoot tissues (by 40–64%), whilst 100 μM Zn enhanced Cd translocation at 5 μM Cd but decreased it at 15 μM Cd. In contrast, both 5 and 15 μM Cd decreased Zn concentrations in root and shoot tissues but increased Zn translocation by 30–90%. This interaction was not associated with changes in Zn speciation within the plants, with most Zn associated with oxalate (48–87%).

Conclusions

The presence of Zn and Cd resulted in an additive negative effect on root growth, but an antagonistic pattern in their accumulation in shoots of C. rossii.

     

Nitrate nutrition enhances zinc hyperaccumulation in Noccaea caerulescens (Prayon)

Nitrate fertilization has been shown to increase Zn hyperaccumulation by Noccaea caerulescens (Prayon) (formerly Thlaspi caerulescens). However, it is unknown whether this increased hyperaccumulation is a direct result of NO₃ ^? nutrition or due to changes in rhizosphere pH as a result of NO₃ ^? uptake. This paper investigated the mechanism of NO₃ ^?-enhanced Zn hyperaccumulation in N. caerulescens by assessing the response of Zn uptake to N form and solution pH. Plants were grown in nutrient solution with 300 μM Zn and supplied with either (NH₄)₂SO₄, NH₄NO₃ or Ca(NO₃)₂. The solutions were buffered at either pH 4.5 or 6.5. The Zn concentration and content were much higher in shoots of NO₃ ^?-fed plants than in NH₄ ⁺-fed plants at pH 4.5 and 6.5. The Zn concentration in the shoots was mainly enhanced by NO₃ ^?, whereas the Zn concentration in the roots was mainly enhanced by pH 6.5. Nitrate increased Zn uptake in the roots at pH 6.5 and increased apoplastic Zn at pH 4.5. Zinc and Ca co-increased and was found co-localized in leaf cells of NO₃ ^?-fed plants. We conclude that NO₃ ^? directly enhanced Zn uptake and translocation from roots to shoots in N. caerulescens.     

Elevated CO2 promotes the acquisition of phosphorus in crop species differing in physiological phosphorus-acquiring mechanisms

Plant and Soil - Crop species differ in phosphorus (P) acquisition in P-limiting environments. However, it is not fully understood how elevated atmospheric CO2 concentrations affects these P...