Silicon amelioration of aluminium toxicity and cell death in suspension cultures of Norway spruce (Picea abies (L.) Karst.)

A role for silicon (Si) in the amelioration of aluminium (Al) toxicity in gymnosperms is suggested by their codeposition in planta, including within needles. This study was designed to investigate Al/Si interactions at the cellular level using suspension cultures of Norway spruce. Toxic effects of Al were dependent on duration of Al exposure, concentration of Al, and pH. Toxicity was reduced when Si was present, and the effect was enhanced at pH 5.0 compared to pH 4.2. Study of the ultrastructure of Al-treated cells indicated that changes in cell wall thickening, degree of vacuolation, and the degeneration of mitochondria, Golgi bodies, ER and nucleus preceded cell death, and significant amelioration was noted when Si was also present. When the fluorescent dye Morin was employed to localise free Al, cells treated with Al and Si in combination showed less fluorescence than the cells treated with Al alone. Intensity of fluorescence depended on the concentration of Al, duration of treatment and pH. Notably, presence of Si reduced the concentration of free Al in the cell wall in parallel with amelioration of Al toxicity. We therefore propose that formation of aluminosilicate complexes in the wall and apoplasm provide a significant barrier to Al penetration and cell damage in Norway spruce.     

Effects of aluminium on calcium and magnesium uptake and translocation by root segments of whole seedlings of Norway spruce (Picea abies Karst.)

In the Ardennes, spruce decline is correlated with Mg deficiency caused by acid rain leaching of soil nutrients, associated with solubilization of Al-containing soil minerals. Laboratory experiments were carried out to measure the uptake and translocation of 45Ca and 28 Mg by intact roots of spruce seedlings in solutions containing various amounts of added AlCl3. Translocation rates in the various organs of the seedlings were higher for magnesium than for calcium. A 1 mt M Al nutrient solution had a much stronger inhibitory effect on uptake and translocation of Mg than it had on Ca. These rate differences result largely from differences in the chemical characteristics of these two elements.     

Effects of aluminium on growth and nutrient uptake of small Picea abies and Pinus sylvestris plants

Summary The effects of aluminium concentrations between 0.2 and 30 mM at pH 3.8 ±0.2 on small plants of Norway spruce [(Picea abies (L.) Karst], Scots pine (Pinus sylvestris L.), and Scots pine infected with the ectomycorrhizal fungus Suillus bovinus (L. ex Fr.) O. Kuntze were investigated. The plants were grown at maximum relative growth rate (R_G % day^–1) with free access but very low external concentrations of nutrients. Steady-state conditions with respect to relative growth rate (R_G) and internal nutrient concentrations were achieved before addition of aluminium, which was added as AlCl₃ and/or Al(NO₃)₃. There were reductions in rg at aluminium concentrations of 0.3 mM in spruce, 6 mM in pine and 10 mM in ectomycorrhizal pine, i. e. at aluminium concentrations considerably higher than those normally occurring in the top layer of the mineral soil where most fine roots are found. Nutrient uptake rate per unit root growth rate was calculated for different nutrient elements. The uptake rate of calcium and magnesium was reduced at aluminium concentrations of 0.2 mM (spruce), 1 mM (pine) and 3 mM (ectomycorrhizal pine), without influencing R_g. The results question the validity of the hypothesis of aluminium toxicity to forest tree species at low external concentrations.     

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.     

Differential energization of the tonoplast in suspension cells and seedlings from Picea abies

 Vacuolar ATPase (EC 3.6.1.3) and PPase (EC 3.6.1.1) were studied in suspension cells and seedlings from spruce [Picea abies (L.) Karst. Proton transport activity and uncoupler (1 μM nigericin) stimulated substrate hydrolysis were measured in tonoplast enriched membrane vesicles. In suspension cells the vacuolar PPase exhibited 1.8-fold activity of the ATPase. In roots and needles from 12-week-old spruce seedlings the vacuolar PPase was inactive, whereas the ATPase was active. Therefore, we investigated whether the preparation of spruce tonoplast vesicles from roots and needles inactivates the vacuolar PPase but not the ATPase. For this purpose, maize (Zea mays L.) tonoplast membranes exhibiting vacuolar PPase as well as ATPase activity were used as a probe and added to the homogenization medium prior to the preparation of spruce vesicles. The preparation of spruce vesicles was more inhibitory to the vacuolar ATPase than to the PPase. The comparison of vacuolar PPases from spruce suspension cells and maize roots revealed similar enzymatic properties. After isopycnic centrifugation on continuous sucrose gradients the vacuolar PPase from spruce suspension cells co-purified with the vacuolar ATPase. Together, these data show: (1) vacuolar PPases from spruce suspension cells and maize roots are similar, (2) the preparation of tonoplast vesicles from spruce roots and needles does not inactivate the vacuolar PPase, (3) tonoplasts of suspension cultured cells and seedlings from spruce are differentially energized by the vacuolar pyrophosphatase that may indicate a difference in pyrophosphate metabolism between embryogenic and differentiated spruce cells, and (4) tonoplast vesicles from spruce seedlings may allow investigations of the effect of pyrophosphate on the vacuolar ATPase in the absence of vacuolar PPase activity. Received: 2 July 1998 / Accepted: 14 September 1998     

The amelioration of aluminium toxicity by silicon in wheat (Triticum aestivum L.): malate exudation as evidence for an in planta mechanism

Two wheat (Triticum aestivum L.) cultivars, one aluminium tolerant (Atlas 66) and one sensitive (Scout 66), were grown in a continuous-flow culture system (≤pH 5.0) containing aluminium (0–100 μM) and silicon (0–2000 μM) in factorial combination. Treatment with silicon resulted in a highly significant amelioration of aluminium toxicity as assessed by root growth in both cultivars. Amelioration was influenced by wheat cultivar and silicon concentration, as 2000 μM silicon significantly ameliorated the toxic effects of 100 μM aluminium in Atlas 66, and only 5 μM silicon alleviated the effect of 1.5 μM aluminium on Scout 66. Nutrient medium pH was critical, as an amelioration by silicon was apparent only at pH > 4.2 for Atlas 66, and at pH > 4.6 for Scout 66. Silicon neither reduced levels of toxic aluminium species in the growth solutions, nor the amount of aluminium taken up by roots. In experiments to assess exudation of malate by Atlas 66 roots treated with 100 μM aluminium, the presence of 2000 μM silicon (pH 4.6) was found to have a negligible effect on exudation. In contrast, citrate, a known aluminium chelator, reduced aluminium-induced exudation of malate at 5–40 μM and completely inhibited it at 100 μM citrate. The results indicate that silicon does not reduce aluminium phytotoxicity as a result of aluminium/silicon interactions in the external media, and that the mechanism of amelioration has an in planta component. Received: 22 April 1997 / Accepted: 16 August 1997     

Chlorophyll formation and the development of photosynthesis in dark grown seedlings of Picea abies

Abstract Seeds of Picea abies were germinated and grown in either darkness or constant light. The chlorophyll content and photosynthetic carbon dioxide uptake of developing seedlings of different ages was determined. Ten-day-old dark grown seedlings showed an instant ability for photosynthetic carbon dioxide uptake and also formed further chlorophyll most rapidly upon subsequent illumination. These activities progressively diminished when the dark growth period was extended. Light grown seedlings reached a maximum chlorophyll level after 15 days growth, and this preceded maximal photosynthetic development.     

Effect of seed size on seedling growth response to elevated CO2 in Picea abies and Picea rubens

Several previous studies have observed that species and individuals with large seeds respond more positively to elevated CO (2) than those with small seeds. We explored the reasons for this pattern by examining the relationship between seed size and CO (2) response in Picea abies and P. rubens using growth analysis. The large seeded species (P. abies) responded more positively to elevated CO (2) than the small seeded species (P. rubens). At the intraspecific level, P. abies individuals from large seeds responded more positively to elevated CO (2) than individuals from small seeds, however, there was no significant intraspecific variation in CO (2) response in P. rubens. The greater CO (2) response of plants from large seeds was not simply the result of a larger starting capital compounded at the same rate as in plants from small seeds. Elevated CO (2) increased relative growth rate to a greater extent in individuals from large seeds. This effect appears to be related to differences in time of establishment, source to sink ratio and nutrient availability with seed size. These results are significant not only in understanding the potential effect of rising atmospheric CO (2) concentrations on plant populations, but also in understanding the factors affecting plant success at current atmospheric CO (2) levels due to the elevation of CO (2) within the litter layer that occurs at many germination sites.     

The use of 3-amino-1,2,4-triazole to investigate the short-term effects of oxygen toxicity on carbon assimilation by Pisum sativum seedlings

To study the in vivo short-term effect of hydrogen peroxide on plant metabolism, 2 mol m^?3 3-amino-1,2,4-triazole, a catalase inhibitor, was applied through the transpiration stream to Pisum sativum seedlings, and gas exchange characteristics, ascorbate peroxidase, glutathione reductase and catalase activities, and levels of hydrogen peroxide and formate were determined. Carbon dioxide assimilation rates were inhibited after the addition of aminotriazole: photorespiratory conditions exacerbated this inhibition. Carbon dioxide response curves showed that aminotriazole reduced both the RuBP regeneration rate and the efficiency of the carboxylation reaction of Rubisco. Catalase activity was completely inhibited 200 min after the application of this inhibitor, but no concomitant increase in H₂O₂ concentration was found. Under enhanced photorespiratory conditions, H₂O₂ concentrations increased. This suggests that under normal environmental conditions hydrogen peroxide is metabolized via alternative mechanisms. The aminotriazole treatment had no effect on the ascotbate peroxidase and glutathione reductase activities, but caused a substantial increase in the formate pool size. These results suggest that hydrogen peroxide is metabolized by reacting with glyoxylate to produce formate and CO₂. The increased production of formate may reduce the flow of carbon through the normal photorespiratory pathway and may also be used anaplerotically as a precursor of products of 1-C metabolism other than serine. This would prevent the return of photorespiratory carbon to the RPP pathway, leading to a smaller RuBP pool size which would in turn result in a decrease in carboxylation conductance (carboxylation efficiency) and regeneration rate of RuBP.     

Boron-induced amelioration of aluminium toxicity in a monocot and a dicot species

Previous research has reported inconsistent results from experiments on the influence of boron (B) on plant sensitivity to potentially toxic aluminium (Al) concentrations. Differences in B requirement and cell wall properties among species, especially between Poaceae and dicots, may account for this. This investigation reports amelioration by B of Al-induced inhibition of root elongation in Al-sensitive cucumber (Cucumis sativus), but not in Al-sensitive maize (Zea mays). Vital staining, however, also revealed a positive influence of B supply on Al tolerance in maize. In both species, adequate B supply decreased Al-induced damage of cell integrity. In cucumber, increasing B supply enhanced Al concentrations and haematoxylin staining in root tips. In maize, no differences for root Al among B treatments were observed. These results indicate that the positive effect of B on Al resistance was not due to less Al accumulation in root tips. Enhanced concentrations of reduced glutathione were found in roots of Al-stressed maize plants growing with adequate B. It is concluded that adequate B supply is essential for prevention of Al toxicity in both the dicot and the monocot species. In dicot cucumber, the B-induced amelioration of root elongation, despite higher Al accumulation in root tips, indicates B-induced change in either or both Al speciation and compartmentation in the tips. The protection by an adequate B supply of roots against Al-induced cell death suggests a role for B in the defence against oxidative stress. This is supported by the observation that Al induced enhanced levels of GSH in roots of maize plants growing with adequate B supply but not in those growing with either deficient or excess B concentrations.     

Effects of stump and slash removal on growth and mycorrhization of Picea abies seedlings outplanted on a forest clear-cut

The objectives of this study were to investigate impact of stump and slash removal on growth and mycorrhization of Picea abies seedlings outplanted on a forest clear-cut. Four non-replicated site preparation treatments included: (1) mounding (M), (2) removal of stumps (K), (3) mounding and removal of logging slash (HM) and (4) removal of logging slash and stumps (HK). Results showed that height increment of the seedlings was highest in K and lowest in M after the third growing season, and similar pattern remained after the fourth season. Ectomycorrhizal (ECM) colonisation of seedling roots was highest in M (96.6%) and lowest in K (72.3%), and even in HK (76.0%) and HM (76.3%). Morphotyping and sequencing of internal transcribed spacer of fungal ribosomal DNA revealed a total of 13 ECM species. Among those, Thelephora terrestris and Cenococcum geophilum were the most common, found on 27.4% and 26.3% of roots, respectively. The rest of species colonised 26.6% of roots. Richness of ECM species was highest in M (10 species) and lowest in K (three species). Consequently, stump and slash removal from clear-felled sites had a positive effect on growth of outplanted spruce seedlings, but negative effect on their mycorrhization. This suggests that altered soil conditions due to site disturbance by stump and slash removal might be more favourable for tree growth than more abundant mycorrhization of their root systems in less disturbed soil.     

Effects of aluminium on growth and root reactions of phosphorus stressed Betula pendula seedlings

The impact of two constant non-toxic levels of Al stress (0.2 and 0.4 mM) on growth and ³²P uptake capacity on sub-optimal (P-limited) Betula pendula seedlings grown in sand culture was examined.Seedling growth was under optimum controlled conditions in a growth chamber where nutrient additions were made at a predetermined relative addition rate (R_A) of 10% day^-1. Three treatment groups of seedlings 0, 0.2 and 0.4 mM Al were harvested at 15, 29 and 42 days. The excised roots were exposed to a ³²P-labelled solution for 15 minutes to measure their capacity for P uptake. Growth was determined by weighing the roots, stems and leaves of the seedlings.Growth data showed that relative growth rate (R_G) should equal the R_A of P the most limiting nutrient, which was supplied at P/N 3% instead of an optimal 15%. Therefore, Ingestad's theory can also be used succesfully in sand culture and this may be particularly important for future studies of root and rhizosphere exudates. Low levels of Al (< 0.2 mM) in combination with low P supply significantly lowered the R_G of the birch seedlings by further reducing P supply. However, previous studies of birch seedling growth and nutrient uptake using Ingestad's solution culture technique with optimumal P supply did not show any effect of Al on growth untill the Al was in excess of 3 mM. Aluminium was not directly toxic to the plants and therefore roots could respond to the ³²P bioassay.     

Accumulation of ammonium in Norway spruce (Picea abies) seedlings measured by in vivo 14N-NMR

(14)N-NMR and (31)P-NMR have been used to monitor the in vivo pH in roots, stems, and needles from seedlings of Norway spruce, a typical ammonium-tolerant plant. The vacuolar and cytoplasmic pH measured by (31)P-NMR was found to be c. pH 4.8 and 7.0, respectively, with no significant difference between plants growing with ammonium or nitrate as the N-source. The (1)H-coupled (14) NH 4+ resonance is pH-sensitive: at alkaline pH it is a narrow singlet line and below pH 4 it is an increasing multiplet line with five signals. The pH values in ammonium-containing compartments measured by (14)N-NMR ranged from 3.7 to 3.9, notably lower than the estimated pH values of the P(i) pools. This suggests that, in seedlings of Norway spruce, ammonium is stored in vacuoles with low pH possibly to protect the seedlings against the toxic effects of ammonium ( NH 4+) or ammonia (NH3). It was also found that concentrations of malate were 3-6 times higher in stems than in roots and needles, with nitrate-grown plants containing more malate than plants grown with ammonium.     

Odors of Norway spruce (Picea abies L.) seedlings: differences due to age and chemotype

Small conifer seedlings (mini-seedlings) are less damaged by the large pine weevil Hylobius abietis (L.) (Coleoptera: Curculionidae) compared to conventional seedlings. Chemical difference between the seedling types is one possible explanation for this phenomenon. In the present paper, the emissions of volatile organic compounds (VOC) of 7- to 43-week-old Norway spruce [Picea abies (L.) Karst.] seedlings were analyzed. Collection and identification of the volatiles was made by solid phase micro-extraction and gas chromatography mass spectrometry (SPME–GC–MS). The enantiomers of α-pinene and limonene were separated in a two-dimensional GC (2D-GC). Most of the seedlings represented either a limonene- or a bornyl acetate-chemotype. Only minor changes in the volatile composition of the two types of seedlings were found during the first year. Age-related changes, however, were found in the volatiles released by wounded phloem where green leaf volatiles (GLVs) and borneol were the dominated VOC for young seedling. The attractive compound for the pine weevil, α-pinene, was first detected in the phloem emissions of 18- to 22-week-old seedlings. Different storage conditions of the seedlings during the winter/early spring-phase influenced the volatile composition in the phloem. High amount of GLVs was characteristic for the 43-week-old seedlings stored in naturally changing outdoor temperature, but not present in the seedlings winter-stored at a constant temperature of ?4 °C. The possible role of these observed differences in odor emissions between seedlings of different age and physiological status for the feeding preferences of the large pine weevil is discussed.     

The effects of aluminium and Paxillus involutus Fr. on the growth of Norway spruce [Picea abies (L.) Karst.]

Norway spruce [ Picea aides (L.) Karst.] seed lots were obtained from populations growing on an acid soil in the Black Forest, West Germany (acid), and a calcareous soil in the Schwabische Alb, West Germany (calc). Seedlings were grown in sterile perlite culture containing 0–6 mM aluminium. Hypocotyl extension was inhibited by aluminium in the calcareous seedlings, hut not in the acidic seedlings. In a longer term experiment acidic and calcareous plants were grown for 10 weeks in perlite. Some of the tubes were inoculated with the fungus Paxillus involutus Fr. (designated F +) and some were not (F -). Aluminium sulphate solutions were then added to the tubes to raise the aluminium concentrations to 0–6 mM. Plants were harvested after a further 10 weeks. Fungus was associated with the roots in F+ plants, but mycorrhizas did not form. Growth of acid(F -) was somewhat stimulated by aluminium treatment, but that of calc(F -) was greatly reduced, and the plants were chlorotic. The presence of a rhizospherie fungus (F +) enhanced the growth of the calcareous plants, but had little effect on the acidic plants. Shoot analyses suggested that the greater aluminium sensitivity of the calcareous plants involves an inability to exclude aluminium or to maintain normal levels of calcium and magnesium uptake in its presence. The presence of rhizospheric fungi reduced the effects of aluminium.     

Ethylene effects on cambial activity and cell wall formation in hypocotyls of Picea abies seedlings

Ethylene regulation of cell division in the vascular cambium and cell wall formation was studied in hypocotyls of Norway spruce ( Picea abies [L.] Karst.) seedlings. Cuttings from 6-week-old seedlings were placed in water culture to which compounds affecting the synthesis and action of ethylene were added. After a 3-week treatment period, growth, ethylene production, morphology and cell wall composition of the hypocotyls were determined. Addition of high concentrations of the potent ethylene releasing agent 2-chloroethylphosphonic acid (ethrel), which increased ethylene emission by more than twice compared to control plants, inhibited the expansion of xylem cells while stimulating the incorporation of cell wall material, especially cellulose. Addition of small amounts of ethrel, which slightly stimulated ethylene emission, led to increases in the size of xylem cells, the amount of phloem tissue and the number of intercellular spaces in the cortex, and thus to increased hypocotyl diameter. However, no significant change in cell wall composition was detected. When ethylene production was decreased by adding Co²⁺ to the nutrient solution, differentiation of new xylem was disturbed, but the rate of cell division was not affected. Although the incorporation of cell wall material was inhibited, the proportions of lignin and cellulose in the wall appeared to remain unchanged. Silver ions stimulated the expansion of both xylem and cortex cells, but had no significant effect on cell wall formation. We conclude that ethylene has a role in regulating the incorporation of wall carbohydrates.