Aluminum-tolerant Protoplasts from Roots Can be Collected with Positively Charged Silica Microbeads: a Method Based on Differences in Surface Negativity

A new technique was developed for the collection of Al-tolerantplant cells. The procedure takes advantage of the differencein surface charge between Al-tolerant and Al-sensitive protoplasts.Equal volumes of freshly prepared, positively charged silicamicrobeads (0.05% w/v) and purified protoplasts derived fromroot tips (2x10⁵ ml^-1) of rice, maize or pea were mixed andthen centrifuged on a discontinuous Ficoll gradient. Intactprotoplasts from the Al-tolerant plant were recovered mostlyin the bottom fraction, those from the Al-sensitive plant wererecoverd at the uppermost interface, and those from the plantwith intermediate tolerance to Al were collected at the middleinterface. Moreover, the pattern of fractionation was unrelatedto the diameter of the protoplasts. The technique should beuseful in future investigations of the physiological and biochemicalproperties of Al-tolerant cells or their plasma membranes. (Received April 19, 1995; Accepted September 1, 1995)     

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 Δ⁵-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 Δ⁵-sterol synthesis to alter the ratio of phospholipids to Δ⁵-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 Δ⁵-sterols was increased to match that of Rikuu-20 after treatment with uniconazole-P, an inhibitor of obtusifoliol-14α-demethylase. These results indicate that PM lipid composition is a factor underlying variations in Al tolerance among rice cultivars.     

Plasma Membrane Permeability of Root-Tip Cells Following Temporary Exposure to Al Ions Is a Rapid Measure of Al Tolerance among Plant Species

No correlations were recognized between Al tolerance among fourplant species, rice (Oryza sativa L.), maize (Zea mays L.),pea (Pisum sativum L.), and barley (Hordeum vulgare L.), inrank order of Al tolerance, and cation exchange capacities ofroot-tip (0-1 cm) cells or of their cell walls. The plasma membraneof root-tip of Al sensitive plant species (pea and barley) wasconsiderably permeabilized with elongation of root in Al-freesolution following 0.5 h pretreatment with Al. K⁺ release fromand Al permeation into the protoplasts isolated from the root-tipof Al-sensitive plant species were more significant than thosefor Al-tolerant plant species (rice and maize) on 10 or 30 mintreatment with Al. The permeability of the plasma membrane forprotoplasts isolated from Al sensitive plant species was considerablyincreased by treatment with hy-potonic Al-free control solutionfollowing 10 min pretreatment with Al. To our knowlege, theseare the most rapid responses to Al ions reported to date, i.e.,within 0.5 h in whole plant and within 10 min in protoplast.These results suggest that a temporary contact with Al ionsirreversibly alters the plasma membrane of root-tip cells ofAl-sensitive plant species: the cells become more leaky andrigid due to binding of Al ions to the plasma membrane. (Received January 5, 1998; Accepted February 26, 1998)