Time change of aluminium toxicity in the acid bulk soil and the rhizosphere in Norway spruce (Picea abies (L.) Karst.) and beech (Fagus sylvatica L.) stands

Context

In acidic forest soils, aluminium can alter tree health due to its potential toxicity. Aluminium phytotoxicity is mainly influenced by its chemical form and its availability.

Methods

As physical-chemical indicators of Al toxicity in soil, Al speciation in soil solutions and in the exchange complex was measured in the rhizosphere and the bulk soil of two tree species (Norway spruce (Picea abies (L.) Karst.) and European Beech (Fagus sylvatica L.) in an acidic soil and in 4 months (November, February, May and August) representing the four seasons in a year.

Results

In the bulk soil, Al toxicity was generally higher under Norway spruce than under beech. Furthermore, temporal changes in Al behaviour were identified under Norway spruce but not under beech. The monomeric Al in the soil solutions and the exchangeable Al in the solid soil increased significantly in February under Norway spruce and were positively correlated with nitrate concentration, suggesting that nitrate influence Al speciation and mobility under Norway spruce. In the rhizosphere, Al toxicity was restricted through Al complexation by organic compounds and by nutrient contents independently from the season. The ecological importance of the rhizosphere in Al detoxification is discussed.

Conclusions

This study suggests that plant specific differences as well as seasonal changes in plant physiology, microbial activity and microclimatology influence aluminum toxicity in acid forest soils.     

The amelioration of aluminium toxicity by silicon in higher plants: Solution chemistry or an in planta mechanism?

Aluminium (Al) toxicity is a very important factor limiting the growth of plants on acidic soils. Recently, a number of workers have shown that, under certain conditions, silicon (Si) can ameliorate the toxic effects of A1 in hydroponic culture. The mechanism of the amelioration is unclear, but three suggestions have been put forward: Si‐induced increase in solution pH during the preparation of hydroponic solutions; reduced availability of Al due to the formation of hydroxyaluminosilicate (HAS) species in those solutions during plant growth; or in planta detoxification. It is now known that it is possible to make up Al and Si solutions in an order in which pH is lowered prior to Al addition; in these cases amelioration has still been observed. Amelioration has also been noted in experiments where HAS formation is minimal. These observations would suggest that, at least under some circumstances, there is an in planta component to the amelioration phenomenon. Several microanalytical investigations have noted codeposition of Al and Si in root cell walls. We propose a model in which root cell walls are the main internal sites of aluminosilicate (AS) and/or HAS formation and of Al detoxification. Factors promoting AS/HAS formation in this compartment include: high apoplastic pH; the presence of organic substances (e.g. malate); and the presence of suitable local concentrations of reactive forms of Al and Si, on or within the surfaces of the wall matrix. All these are likely to be important in the amelioration of Al toxicity.     

Silicon Alleviation of Cadmium Toxicity in Mangrove (Avicennia marina) in Relation to Cadmium Compartmentation

Mangrove plants seem to be highly tolerant of high levels of heavy-metal pollution. Recently, some researchers have focused on the mechanisms involved in their metal uptake and tolerance. However, the important mechanisms involved are still only partly understood. This investigation studied whether silicon (Si) affected cadmium (Cd) subcellular distribution in the leaves and root tips of Avicennia marina (Forsk.) Vierh seedlings, resulting in the amelioration of the toxicity of Cd. The results showed that Si partly overcame the reduction in growth due to Cd. This amelioration was correlated with a reduction in Cd uptake and alteration of Cd subcellular distribution. The mechanisms of Si amelioration of Cd stress were tissue dependent. In the leaves and root tips, Si reduced Cd concentration in subcellular fractions, Cd mobility, and the concentration of biologically active Cd in the cell wall active space. Si did not change the distribution of Cd between compartments in the leaves, but it increased the proportion of Cd in the cell walls and reduced the proportion of Cd in the symplast of the root tips.     

Callose in roots of Norway spruce (Picea abies (L.) Karst.) is a sensitive parameter for aluminium supply at a forest site (Höglwald)

Under controlled environmental conditions in nutrient solution experiments induction of non-constitutive callose in roots has been shown to be a symptom of aluminium (Al) toxicity. In the present study roots of Norway spruce were sampled from a forest site where soil conditions had been modified by acidic irrigation and liming (Höglwald Experiment in Bavaria, Germany). A significant positive relationship was found between the callose content in short roots and the Al concentration in the soil solution, particularly if free Al, rather than total concentrations of soluble Al, were used for prediction. At the same sites root growth of Norway spruce was not affected by free Al concentrations in the range of 2.5 to 199 µM Al. The results show that also under field conditions a positive relationship between Al supply and callose content can be established. In Norway spruce callose content in roots is a much more sensitive parameter for Al supply than root growth.     

Si amelioration of Al toxicity in barley (Hordeum vulgare L.) growing in two Andosols

The influence of silicon on aluminium toxicity in barley (Hordeum vulgare L. cv. Shunrai) was studied in two Andosols. Silicon sources were a solution of sodium metasilicate with pH adjusted to 5.0, silica gel, and an industrial waste, porous hydrated calcium silicate. The waste is produced in large amounts in the manufacturing processes of autoclaved light concrete, and has been used as a silicon source for rice plants. The addition of the waste increased the concentration of Si in the soil solution, soil pH and amelioration of aluminium toxicity was observed. The addition of silica gel and sodium metasilicate solution to both soils increased significantly (p<0.05) the Si concentration of the soil solutions, but no amelioration of aluminium toxicity was observed. An amelioration of aluminium toxicity by the waste porous hydrated calcium silicate was probably due to the increase in soil pH rather than to the increase of silicon concentration in the soil solution.     

Aluminium/silicon interactions in barley (Hordeum vulgare L.) seedlings

The response of seedlings of the monocot Hordeum vulgare L. cv. Bronze to 0,25 and 50 M aluminium in factorial combination with 0, 1.4, 2.0 and 2.8 mM Si was tested in hydroponic culture at pH 4.5. Nutrient solution (500 M calcium nitrate) and Al/Si treatments were designed to avoid the precipitation of Al from solution. Silicon treatments gave significant amelioration of the toxic effects of Al on root and shoot growth and restored calcium levels in roots and shoots at harvest to levels approaching those of control plants. Aluminium uptake by roots was also significantly diminished in the presence of Si. Silicon alone gave a slight stimulation of growth, insufficient to explain its ameliorative effect on Al toxicity. The mechanism of the Si effect on Al toxicity in monocotyledons awaits further investigation.Abbreviations ICP inductively coupled plasma     

COMPARATIVE EFFECTS OF pH AND ALUMINUM ON SILICA-LIMITED GROWTH AND NUTRIENT UPTAKE IN ASTERIONELLA RALFSII VAR. AMERICANA (BACILLARIOPHYCEAE)1

The acidophilic diatom Asterionella ralfsii cf. var. americana Körn. was grown in continuous culture to examine the influences of both pH and Al on Si-limited growth and uptake kinetics. In contrast to nutrient-replete cultures of A. ralfsii, lowering pH from approximately 6 to 5 reduced algal cell density, chlorophyll a concentration, and intensity of in vivo fluorescence (IVF) at steady state. The lower pH treatments were also characterized by lower Si cell quotas and higher residual dissolved Si concentrations in chemostats with similar nutrient supply rates. Physiological responses to Al stress differed from those to pH reduction when cultures were Si-limited. Nominal Al additions of 20 μmol·L^?1 reduced chlorophyll a concentration and IVF values at higher pH, but all other biomass and chemical parameters remained constant at steady state. The combined efects of Al and reduced pH were more severe than either stress alone, inducing culture washout at pH 4.8. Short-term Si uptake experiments performed at pH 6 showed that Al influenced Michaelis-Menten parameter estimates. Half-saturation (K_s and maximum uptake rate (V_m) constants increased approximately 8- and 2-fold in the presence of Al, respectively, but this difference was only significant for V_m. Similar to previously observed effects of Al on cell morphology in A. ralfsii, Si uptake kinetics were more sensitive to Al additions than to Silimited growth per se.     

Effects of aluminium treatment on Norway spruce roots: Aluminium binding forms,element distribution,and release of organic substances

In order to investigate if Al resistance in Norway spruce (Picea abies[L.] Karst.) can be attributed to similar exclusion mechanisms as they occur in several crop plants, three-year-old Norway spruce plants were treated for one week in hydroculture with either 500 μM AlCl₃ or CaCl₂ solutions at pH 4. Sequential root extraction with 1 M NH₄Cl and 0.01 M HCl and EDX microanalysis revealed that Al and Ca in cell walls and on the surface participated in exchange processes. About half of the Al extracted by the sequential extraction was not exchangeable by 1 M NH₄Cl. Phenolics and phosphate present in the root extracts are possible ligands for Al adsorbed to or precipitated at the root in a non-exchangeable form. In both treatments, C release during the first period of 2 d was much higher than during the remaining time of the experiment. Al treated plants released less total C, carbohydrates and phenolics than did Ca treated plants. Acetate was the only organic acid anion that could be detected in some samples of both treatments. Free amino acids were present at micromolar concentrations but as hydrolysis did not increase their yield, there was no evidence of peptide release. One to two thirds of the released C were large enough not to pass a 1 kDa ultrafilter. The results suggest that exudation of soluble organic complexors is not a major Al tolerance mechanism in Norway spruce, although complexation of Al by phenolic substances released by the root could be detected by fluorescence spectroscopy. Aluminium tolerance could rather be attributed to immobilization in the root apoplast, where strong binding sites are available or precipitation may occur. This revised version was published online in June 2006 with corrections to the Cover Date.     

Cell wall pectin content modulates aluminium sensitivity of Zea mays (L.) cells grown in suspension culture

The pectin content of the cell walls of maize suspension cells was modified to investigate its role in the expression of aluminium (Al) toxicity. Long‐term adaptation to NaCl or to the cellulose synthesis inhibitor 2,6‐dichlorbenzonitirle (DCB) increased the pectin content by 31 and 86%, respectively. Subculturing salt‐adapted cells for up to 3 weeks without NaCl supply or treatment of cells with pectolyase for up to 15 min reduced pectin contents by up to 46%. Such pre‐cultured cells were incubated for 2 h in presence of Al. There was a close positive correlation between pectin and both total and BaCl₂ non‐exchangeable Al contents. Aluminium‐induced callose formation as an indicator of Al injury was closely positively correlated to the loss of cell viability. In NaCl‐adapted and pectolyase‐treated cells, Al‐induced callose formation was reduced when compared with normal cells. However, there was a close positive relationship between pectin contents and relative callose induction (digitonin‐induced callose formation, reflecting the different capabilities of cells to synthesize callose set to 100%) indicating that cells with higher pectin contents are more Al‐sensitive. The results presented support our view that the binding of Al to the cell wall pectin‐matrix represents an important step in the expression of Al toxicity.     

Effect of aluminium on membrane properties of soybean (Glycine max) cells in suspension culture

Short-term responses of soybean (Glycine max) cells to aluminium (Al) were studied in suspension culture. Formation of callose was the most sensitive indicator of Al effects. As low as 5 µM Al induced callose formation and an increase in callose concentration could be measured as early as 15 min after beginning the Al treatment. Also membrane permeability was rapidly affected by Al. Potassium net-efflux was reduced by increasing Al concentrations up to 300 µM Al. Increasing the pH of the external solution from 4.3 to 5.3 enhanced callose formation, indicating more severe Al damage at pH 5.3, which is in agreement with a model on H⁺ amelioration of Al toxicity. Al did not initiate or enhance ferrous sulfate (FeSO₄)-promoted lipid peroxidation. The results indicate that the plasma membrane is a primary target of Al and that cell suspension culture is a powerful tool to study effects of Al on plant roots.     

The dibenzodioxocin lignin substructure is abundant in the inner part of the secondary wall in Norway spruce and silver birch xylem

A specific condensed lignin substructure, dibenzodioxocin, was immunolocalized in differentiating cell walls of Norway spruce (Picea abies (L.) H. Karsten) and silver birch (Betula pendula Roth) xylem. A fluorescent probe, Alexa 488 was used as a marker on the dibenzodioxocin-specific secondary antibody. For the detection of this lignin substructure, 25-m cross-sections of xylem were viewed with a confocal laser-scanning microscope with fluorescein isothiocyanate fluorescence filters. In mature cells, fluorescence was detected in the S3 layer of the secondary wall in both tree species, but it was more intense in Norway spruce than in silver birch. In silver birch most of the signal was detected in vessel walls and less in fiber cell walls. In very young tracheids of Norway spruce and vessels and fibers of silver birch, where secondary cell wall layers were not yet formed, the presence of the dibenzodioxocin structure could not be shown.Abbreviation CLSM confocal laser-scanning fluorescence microscopy     

Aluminium accumulation in root cell walls coincides with inhibition of root growth but not with inhibition of magnesium uptake in Norway spruce

High levels of aluminium in the soil solution of forest soils cause stress to forest trees. Within the soil profile, pH and aluminium concentration in the soil solution vary considerably with soil depth. pH strongly influences the speciation of A1 in solution, and is a factor when considering toxicity of A1 to roots. Norway spruce ( Picea abies [L.] Karst.) seedlings were grown for 7 weeks in nutrient solutions at pH 3.2, 4.0 or 5.0 containing 0, 100 or 400 µ M A1. At the end of this period, seedling growth, the cation exchange capacity of the roots and the amount of exchangeable Ca and Mg in roots were determined. A1 concentrations in whole roots, root segments, and in needles were measured. Using X‐ray microanalysis, the concentrations of Al, Ca, Mg and P were determined in cortical cell walls. We wanted to test the hypotheses that (1) the amount of Al bound to cation exchange sites can be used as a marker for Al toxicity and (2) the Mg concentration of needles is controlled by the amount of Mg bound to cation exchange sites. Low pH reduced the inhibition of Al on root growth and shoot length. Both low pH and Al lowered the concentration of Ca and Mg in needles. Al concentrations in the roots decreased as the pH decreased. In the roots, Al displaced Mg and Ca from binding sites at the root cortical cell walls. A comparison of the effects of Al at the different pH values on root growth and Mg concentration in the needles, suggests that, at pH 5.0, an Al fraction in the apoplast inhibits root growth, but does not affect Mg uptake. This fraction of Al is not available for transport to the shoots. In contrast, Mg uptake is strongly affected by Al at pH 3.2, although only very low levels of Al were detected in the roots. Thus, Al accumulation in the apoplast is a positive marker for Al effects on root growth, but not Mg uptake. The Mg concentration of needles is not controlled by the amount of Mg bound to cation exchange sites.     

The role of root exudates in aluminium resistance and silicon-induced amelioration of aluminium toxicity in three varieties of maize (Zea mays L.).

Aluminium (Al) toxicity is widely considered to be the most important growth-limiting factor for plants in strongly acid soils (pH<5.0). The inhibition of root elongation in three varieties of maize (Zea mays L. vars Clavito, HS701b and Sikuani) was followed over the first 48 h of Al treatment, and during the initial 10 h elongation was determined on an hourly basis. The silicon (Si)-induced amelioration of Al toxicity was investigated by pre-treating seedlings for 72 h in nutrient solutions with 1000 microM Si before transfer into solutions with 0, 20 or 50 microM Al (without Si). Plants were either grown in complete low ionic strength nutrient solutions (CNS) or in low salt solutions of 0.4 mM CaCl2 (LSS). In addition, the role of root exudation of organic compounds as a mechanism of Si-induced alleviation of Al toxicity was investigated. Aluminium-induced inhibition of root elongation in the maize var. HS701b was observed within 1 h of Al exposure. After a lag time of at least 8 h, Si-induced alleviation of Al toxicity was observed in this variety when grown in LSS. In the Al-resistant var. Sikuani, Al-resistance was only observed after exposure to 50 microM Al, and not after exposure to 20 microM Al, suggesting that there exists a threshold Al concentration before the mechanisms of Al resistance are activated. Aluminium stimulated root exudation of oxalic acid in all three varieties, but exudate concentrations did not increase with either Al resistance or with Si pretreatment. Aluminium and Si triggered release of catechol and of the flavonoid-type phenolics: catechin, and quercetin. In the Al-resistant variety, Sikuani, Al-exposed plants pretreated with Si exuded up to 15 times more phenolics than those plants not pretreated with Si. The flavonoid-type phenolics, to date unconsidered, appear to play a role in the mechanism(s) of Si-induced amelioration of Al toxicity.     

Circumnutation oscillations of the hypocotyl and hypocotyl hook formation: A comparison of Norway spruce (Picea abies (L.)Karst.) and Scots pine (Pinus silvestris L.) seedlings

Circumnutation oscillations of hypocotyls were studied in Norway spruce and Scots pine using time-lapse cinematography. It was found that the circumnutations were specific in species. The analysis of hypocotyl ontogeny (100 h) revealed a different duration of the phases I, II and III of nutation oscillations in the first and second taxon. From the quantitative point of view it can be stated that during the phase I of nutation oscillations the Norway spruce hypocotyls exhibited higher frequency activity (corrections in direction of the hypocotyl by an elongation-controlling system corrected by a feedback mechanism), while in the phase III of nutation oscillations higher growth rate and larger amplitudes were observed in the Scots pine hypocotyl when compared with those of Norway spruce. The hypocotyl hook appeared only in Norway spruce.     

Aluminium/silicon interactions in higher plants

Aluminium and silicon are usually abundant in soil mineral matter,but their availability for plant uptake is limited by low solubilityand, in the case of Al, high soil pH causes precipitation ofthe element in insoluble forms. Al toxicity is a major problemin naturally occurring acid soils and in soils affected by acidicprecipitation. Al has no known role in higher plants, and isgenerally known as a toxic element, whereas Si is generallyregarded as a beneficial element. Recently, it has been suggestedthat Al toxicity can be ameliorated by Si in a variety of animalsystems. In this review the evidence that amelioration of Altoxicity by Si can also occur in plants is assessed. At presentsuch amelioration has been shown in sorghum, barley, teosinte,and soybean, but not in rice, wheat, cotton, and pea. Plantspecies vary considerably in the amounts of Al and Si that theytransport into their tissues, and it seems that very high Siaccumulation and very high Al accumulation are mutually exclusive.The mechanisms considered for amelioration are: solution effects;codeposition of Al and Si within the plant; effects in the cytoplasmand on enzyme activity; and indirect effects. Key words: Aluminium, silicon, biomineralization, codeposition, toxicity, tolerance     

Effects of elevated soil solution Al concentrations on fine roots in a middle-aged Norway spruce (Picea abies (L.) Karst.) stand

Aluminium (Al), mobilized by acidic deposition, has been claimed to be a major threat to forest vitality. Fine root mortality, decreased root growth and reduced nutrient uptake have been observed in controlled laboratory experiments where roots of tree seedlings were exposed to elevated concentrations of Al. Yet, evidence for Al-induced root damage from forest stands is scarcely reported. Nevertheless, Al dissolved in soil water has received a key role in the critical load concept for forests. Here, we present effects of artificially elevated concentrations of Al in the soil solution on fine roots in a middle-aged stand of Norway spruce (Picea abies (L.) Karst.). Although the inorganic Al concentrations about 200 µM and Ca:Al ratio about 0.7 that were established in the soil solution within this experiment have been associated with reduction of root growth and root mortality for spruce seedlings in hydroponic studies, no acute damage on fine roots was observed. Three years of treatment did not cause visual root damage, nor were effects on fine root necromass observed. Fine root necromass made up about 10% of fine root biomass for all treatments. However, significantly lower molar Ca:Al and Mg:Al ratios in living and dead fine roots were found in the plots where Al concentrations were highest and ratios of Ca to Al in the soil solution were lowest. The lack of response on fine root biomass suggests that forest stands tolerate higher Al levels than results from laboratory experiments indicate. We conclude that effect studies in the laboratory have limited value for field conditions. The key role of Al toxicity, expressed as the Ca/Al ratio, in critical load calculations for forests may have to be reconsidered.     

Solution pH modifies the response of Norway spruce seedlings to aluminium

Norway spruce (Picea abies) was exposed to nutrient solutions containing a range of aluminium (Al) concentrations at several pH levels (3.2, 4 and 5). Root growth was reduced by 100 µM and 400 µM Al at pH 4 and 5, but at pH 3.2 only by 400 µM Al. The Al content of the roots increased with increasing pH. The Al content of the roots was higher at the root tips than at the older root parts at all pH values. Using X-ray microanalysis it could be shown that higher levels of Al at increased pH were mainly due to increased Al contents in root cortex cell walls. In seedlings, mycorrhizal with Pisolithus tinctorius or Lactarius rufus, the Al concentration of cortex cell walls was higher when nitrate (NO₃) rather than ammonium (NH₄) was the nitrogen (N) source.     

Effects of aluminum in red spruce (Picea rubens) cell cultures: Cell growth and viability, mitochondrial activity, ultrastructure and potential sites of intracellular aluminum accumulation

The effects of Al on red spruce ( Picea rubens Sarg.) cell suspension cultures were examined using biochemical, stereological and microscopic methods. Exposure to Al for 24–48 h resulted in a loss of cell viability, inhibition of growth and a significant decrease in mitochondrial activity. Soluble protein content increased in cells treated with Al. Using energy-dispersive X-ray microanalysis on sections of freeze-substituted cells that had no obvious disruption in cytoplasmic or cell wall structure, Al (always in the presence of P) was detected in dense regions in cell walls, cytoplasm, plastids and vacuoles after 48 h exposure to Al. Stereological quantification of spruce cell structure showed that, after 24 h of Al treatment, intact cells had increased vacuolar and total cell volume, but the nuclear volume did not change. In addition, Al treatment resulted in increased surface area of Golgi membranes and endoplasmic reticulum. The biochemical and ultrastructural alterations in red spruce cells, in combination with the presence of Al in cellular organelles of visually intact cells, suggest that Al movement occurred across the plasma membrane without major cellular disruption. Detailed short-term time course studies are needed to determine if intracellular Al in these cells results from its passage into cells through submicroscopic lesions in the plasma membrane or it is taken up into the symplast through the intact membrane by an active, but slow, process.     

Structural variation of tracheids in Norway spruce (Picea abies [L.] Karst.)

The orientation of cellulose microfibrils in the cell wall and the shape and the dimensions of the cells of earlywood of four Norway spruce (Picea abies [L.] Karst.) stems grown in Finland were studied by X-ray diffraction and optical microscopy. The average microfibril angle (MFA) decreased and the diameter of the cell increased rapidly up to rings 5-10 from the pith and remained at the same level after that. The average MFA close to the pith was over 20 degrees and decreased to about 8 degrees after ring 10 from the pith. The average diameter of the cells was 35 microm in the outer rings. The shape of the cross section of the lumen changed from circular to rectangular from the pith to the bark. The tracheid length increased also as a function of the distance from the pith. The thickness of the cell wall varied between 2.8 and 3.5 microm. Automatic cell lumen and cell wall recognition procedures were developed for the analysis of the images of the cross sections of the cells.     

Effect of pH and nitrogen source on aluminium tolerance of rye (Secale cereale L.) and yellow lupin (Lupinus luteus L.)

Soluble aluminium (Al) is a major factor limiting plant growth in acid mineral soils. Aluminium concentrations in soil solutions are mainly determined by soil pH. However, pH also affects the ratio between activities of protons and cationic Al species and the equilibrium between mono-and polynuclear hydroxy-Al species. The phytotoxicity of these species is not yet clear. The objective of the present study was to clarify the role of minor changes of pH in the rhizosphere on Al phytotoxicity in two Al-tolerant plant species by direct control of the pH in the nutrient solution (4.1, 4.3, 4.5) and in addition by varying the pH in the root apoplast using either nitrate or ammonium as N source. The plants were grown in solution culture at constant external pH. Whereas the Al-sensitive plant species barley and horse bean were damaged at very low Al supplies (1.85 μM and 9.3 μM respectively), 222 μM had to be applied to rye and yellw lupin for a comparable inhibition of root elongation. Yellow lupin was initially severely inhibited in root growth by Al, but then gradually recovered from this ‘Al shock’ within 3 days. In contrast to lupin, rye was hardly affected by Al initially, and it took about 16 h until maximum inhibition of root elongation. In the presence of nitrate, raising the pH from 4.1 to 4.5 aggravated root-growth depression by Al in rye and lupin. Whereas rye roots were severely damaged by ammonium especially at low pH, lupin was rather indifferent to the N source. Aluminium toxicity was less severe in presence of ammonium compared to nitrate N. This effect was less clear with rye at lower pH, because of it's higher proton sensitivity compared to lupin. Less Al injury at lower pH and in presence of ammonium was related to lower Al concentrations in the 1 cm root tips. The results are compatible with data showing high phytotoxicity of mononuclear and polynuclear hydroxy-Al species. However, they could also be interpreted in the light of proton amelioration of Al toxicity owing to competition for Al-sensitive binding sites in the root apoplast.