Keitarou Tawaraya

Plasma membrane of younger and outer cells is the primary specific site for aluminium toxicity in roots

Experiments were carried out to identify the primary site for aluminium (Al) toxicity in roots. Al accumulated in large amounts in the younger and outer cells in roots of pea and was retarded when the ionic strength of the Al solution was high. Cell destruction was extensive in the regions with high Al accumulation. The accumulation of Al in, and potassium (K) leakage from, the root tip were in the order pea>maize>rice, the same order as their sensitivity to Al.The protoplasts from the root tip portion of pea incubated with Al showed a wrinkled and uneven surface. The protoplasts progressively shrank and eventually collapsed. Viability decreased in this process. In the control protoplasts of maize, β-glucan formation was uniform on the spherical surfaces, whereas it was spotty in the Al-treated protoplasts; the cell wall material of the latter contained partly 1, 3-β-glucan which is known to be synthesised by 1, 3-β-glucan synthase embedded in the plasma membrane. These results suggest that the specific site for Al toxicity is the plasma membrane of younger and outer cells in roots and that Al tolerance depends largely on the integrity of the plasma membrane.

Relative abundance of Δ5-sterols in plasma membrane lipids of root-tip cells correlates with aluminum tolerance of rice

We investigated variations in aluminum (Al) tolerance among rice plants, using ancestor cultivars from the family line of the Al-tolerant and widely cultivated Japonica cultivar, Sasanishiki. The cultivar Rikuu-20 was Al sensitive, whereas a closely related cultivar that is a descendant of Rikuu-20, Rikuu-132, was Al tolerant. These two cultivars were compared to determine mechanisms underlying variations in Al tolerance. The sensitive cultivar Rikuu-20 showed increased permeability of the plasma membrane (PM) and greater Al uptake within 1 h of Al treatment. This could not be explained by organic acid release. Lipid composition of the PM differed between these cultivars, and may account for the difference in Al tolerance. The tolerant cultivar Rikuu-132 had a lower ratio of phospholipids to Delta(5)-sterols than the sensitive cultivar Rikuu-20, suggesting that the PM of Rikuu-132 is less negatively charged and less permeabilized than that of Rikuu-20. We used inhibitors of Delta(5)-sterol synthesis to alter the ratio of phospholipids to Delta(5)-sterols in both cultivars. These inhibitors reduced Al tolerance in Rikuu-132 and its Al-tolerant ancestor cultivars Kamenoo and Kyoku. In addition, Rikuu-132 showed a similar level of Al sensitivity when the ratio of phospholipids to Delta(5)-sterols was increased to match that of Rikuu-20 after treatment with uniconazole-P, an inhibitor of obtusifoliol-14alpha-demethylase. These results indicate that PM lipid composition is a factor underlying variations in Al tolerance among rice cultivars.

Methylene Blue Stainability of Root‐Tip Protoplasts as an Indicator of Aluminum Tolerance in a Wide Range of Plant Species, Cultivars and Lines

We previously developed a new simple technique of methylene blue (MB) staining for the discrimination of aluminum (Al)-tolerant protoplasts from 4 plant species (rice, oats, maize and pea). The objectives of the present study were to confirm the applicability of this technique to a wider range of plant species, cultivars and lines, and to identify a common strategy for the early stage of Al tolerance. A total of 10 plant species, two Brachiaria spp., two Oryza spp., buckwheat, maize, pea, triticale, wheat and barley, corresponding to 18 different plant samples (species, cultivars, and lines), were used. Al tolerance (relative net root elongation of the longest root), which was screened at 20 μM AICI3 in 0.2 mM CaCl2 (pH 4.9) for 24 h, ranged widely from 10 to 88. Among cultivars and lines within the same species, Al accumulation in the root tip portion was higher in Al-sensitive plants, corresponding to more severe permeabilization of the plasma membranes (PM). Protoplasts isolated mainly from the epidermis, and outer and central parts of the cortex were stained to different degrees by MB, and the blue color was observed both on the surface and inside the protoplasts. Color pictures obtained after staining for 3 min with 0.1% MB were analyzed by Image Hyper II. The ratio of the heavily stained area at threshold 95 to the entire area stained with MB at threshold 125 was defined as MB stainability. MB stainability was negatively correlated with Al tolerance (y= 48.6e−0.02x , R 2= 0.676**) suggesting the common importance of permeation characteristics of PM, in addition to PM negativity for Al tolerance in a wide range of plant species, cultivars and lines. Analysis of the PM lipid composition was proposed as an important topic for future studies on the negativity and permeation of PM.

Plasma Membrane Lipids Are the Powerful Components for Early Stage Aluminum Tolerance in Triticale

The order of aluminum (Al) tolerance in triticale lines (ST2>ST22) after re-elongation in an Al-free 0.2 mM calcium (Ca) solution for 9 h (Ca period) following 1 h pretreatment with 20 μM Al (Al period) agreed with that after 24 h of Al treatment. Permeability of the plasma membrane (PM) of root-tip cells after the Ca period was significantly increased in Al-sensitive ST22. Al was accumulated more heavily in the root-tip portion of ST22 than in that of ST2, although similar amounts of malic and citric acid anions were released from both triticale lines. We established a new system examining lipid permeability using synthesized nylon-2,8 ultrathin and porous capsules trapped previously with 0.1% (w/v) methylene blue solution and coated thereafter with PM lipid isolated from root tips by a newly developed technique. Permeability of the PM lipid measured with time in 0.2 mM Ca with or without 50 μM Al photometrically (A 680) was significantly greater in Al-sensitive ST22 after 5 min of Al treatment. This is the first report to directly show the primary and early role of PM lipid in Al tolerance in triticale.

Greater contribution of low‐nutrient tolerance to sorghum and maize growth under combined stress conditions with high aluminum and low nutrients in solution culture simulating the nutrient status of tropical acid soils

Abstract Aluminum is usually regarded as the determining factor for plant growth in acid soils and nutrient deficiencies are often additional growth-limiting factors in tropical acid soils. Taking into account the potential interactions between Al toxicity and nutrient deficiencies, the present study investigated sorghum (Sorghum bicolor Moench [L.]) and maize (Zea mays L.) cultivar differences for: (1) Al tolerance (relative growth in a one-fifth strength nutrient solution [low-nutrient medium, ionic strength: 4.5 mmol L−1] with Al and without Al), (2) low-nutrient tolerance (relative growth in a low-nutrient medium compared with growth in a full-strength nutrient solution), (3) combined tolerance (relative growth in a low-nutrient medium containing Al compared with a full-strength medium lacking Al). The goal of the present study was to identify the predominant growth-limiting factor using a solution culture medium that simulates the nutrient status of tropical acid soils. Differential Al tolerance among 15 cultivars of sorghum and 10 cultivars of maize in short-term assays (2.5 or 20 µmol L−1 AlCl3 in 0.2 mmol L−1 CaCl2 at pH 5.0 or 4.9, respectively, for 24 h) was positively correlated with Al tolerance in long-term cultures (11.1 or 42.6 µmol L−1 soluble Al in the low-nutrient medium at pH 4.5 or 4.3, respectively, for 29 days). However, the level of Al tolerance in the short-term assays was not correlated with the combined tolerance, suggesting that a short-term screening technique may not be practically useful for estimating cultivar adaptation to a combination of stress factors in tropical acid soils. In sorghum, a less Al-tolerant plant species, higher Al tolerance was associated with less Al absorption by the roots and greater K translocation into the shoots. In maize, a more Al-tolerant plant species, there was no correlation between the accumulation or transport of elements and Al tolerance. Standardized partial regression coefficients suggested that low-nutrient tolerance contributed more to combined tolerance than Al tolerance under most conditions (except for Al-sensitive sorghum at 42.6 µmol L−1 AlCl3). A greater combined tolerance was associated with a higher K shoot concentration in sorghum and a higher Ca shoot level in maize. Plant nutritional characteristics linked to low-nutrient tolerance should be evaluated as an important strategy for plant production in tropical acid soils, both for Al-tolerant plant species and for Al-sensitive plant species under low-Al conditions.

Effects of temporary contact with Al ions on the plasma-membrane permeability of root-tip cells in seven plant species

Seven plant species were subjected to long-term (2 months) and short-term treatments (1 h) with Al to evaluate differences in Al resistance. In long-term experiments, Al resistance was associated with Al accumulation in root-tips and K concentration in shoots and roots. Short-term treatment with Al for 1 h was enough to inhibit subsequent root elongation of Al-sensitive species in Al-free solution. Also, Al increased the plasma-membrane permeability of root-tip cells in Alsensitive species. These results suggest that Al resistance depends on the intactness of the plasma membrane under Al stress.

Mechanisms of higher tolerance of Al stress in phosphorus deficient maize seedlings: the significance of phenolics in Al resistance.

Maize seedlings grown preliminarily without P were more Al-resistant than plants pre-cultured in complete or in nutrient solutions deficient in N, Mg, or K. Aluminium resistance of roots was negatively correlated with root Al concentrations. Phosphorus-deficient roots contained approximately twice the amounts of soluble phenolic compounds as control roots. Exudation of phenolic compounds was generally proportional to their concentration in the roots. Chlorogenic acid (CA), lumogallion (LU), morin (MO), pyrogallol (PG) or hematoxylin (HX) added in the same concentration of 15 µM together with the same concentration of Al to the medium (pH 4.7) ameliorated Al toxicity to a similar extent as malic acid. Excluding CA and HX from the regression, the increase in root elongation by the addition of phenolic compounds to the Alcontaining medium was negatively correlated with the decrease in Al absorption by roots. This suggests that the ameliorative effect of phenolic compounds on Al rhizotoxicity is due to the decrease of Al absorption by roots as a result of the formation of stable Al complexes in the growth medium as has been reported for organic acids.

A New and Simple Technique for the Isolation of Plasma Membrane Lipids from Root-Tips

It has been suggested that plasma membrane (PM) lipids play a major role in aluminum (Al) tolerance; however, no direct investigations have been carried out using PM lipids from root-tips. Here we report a new technique for PM isolation as an alternative to the laborious two-polymer phase partitioning method that is commonly applied, as follows: 1) separation of protoplasts from 1-cm root-tip portions by enzymatic digestion, 2) attachment of the purified protoplasts to glass plates coated with polylysine, 3) preparation of PM ghosts by successive burst of the attached protoplasts using three separate buffer solutions (25 mM PIPES, 5 mM EDTA, and 2 mM MgCl2, at pH 7.0) with slow stirring for 60 s. The PMs were confirmed to be devoid of organelle membranes by fluorescence microscopy, thin layer chromatography (TLC) and western blot analysis. The PM lipids obtained were found to be useful for studies on their differential permeability and lipid composition between lines of triticale or cultivars of maize under Al stress.