Aluminum (Al) tolerance mechanisms have been reported by many researchers for different crop plant species. Organic acid (OA) anion exudation has been clarified as main Al-tolerance mechanism in wheat, maize, buckwheat, soybean and some other crop plant species. On the other hand, it was also suggested that only one mechanism is not enough to explain Al tolerance even in one crop plant species. Rice, one of the primary crop plant species in the world, is famous for Al-tolerant crop plant species, however its tolerance mechanism is almost unknown. In the first stage, Al tolerance mechanism was investigated with special interest in plasma membrane (PM) lipid layer using rice root-tips (1 cm). Thereafter, Al tolerance mechanism for other crop plant species was investigated whether the same tolerance mechanism is working or not.

　In the first stage of experiments, investigations were carried out to determine the Al tolerance mechanism in rice ( Oryza sativa L.) using 23 Bangladeshi ( Indica ) and 6 Japanese ( Japonica ) cultivars. Al tolerance was screened with 20 μM AlCl₃ in 0.2 mM CaCl₂ solution (pH 4.9) for 24h. Of the Bangladeshi cultivars, BRRIdhan41 and Rahmat were found to be most Al-tolerant, and Moyna and BRRIdhan34 to be most Al-sensitive cultivars. Of the Japanese cultivars, Sasanishiki and Domannaka were found to be both extremes of tolerant and sensitive cultivars, respectively. Hematoxylin staining of root-tip portions and root-tip sections from Al-tolerant cv. Sasanishiki and cv. BRRIdhan41, and Al-sensitive cv. Domannaka and cv. BRRIdhan34 indicated that rice possesses an Al exclusion mechanism. Staining with fluorescein diacetate-propidium iodide (FDA-PI) indicated that PM permeability of Al-tolerant rice cultivars remains almost intact, whereas even after 1-h Al treatment, the PM of sensitive rice cultivars becomes permeable. These results suggest that PM permeability is the key factor in early stage of Al tolerance of rice.

　Among many rice cultivars ever screened, Sasanishiki was found to be one of the most tolerant cultivar to high concentration of Al in medium. However, wide variation of Al tolerance was found in almost all pedigree cultivars of Sasanishiki. I selected and investigated further using mainly two cultivars with both extreme tolerances to high Al. The cultivar Rikuu-20 was Al sensitive, whereas a closely related cultivar that is a descendant of Rikuu-20, Rikuu-132, was Al tolerant. The sensitive cultivar Rikuu-20 showed increased permeability of PM within 1 h of Al treatment. Furthermore, greater Al accumulation was observed in the root-tip portion of sensitive Rikuu-20. Differential Al tolerance and Al uptake could not be explained by the difference in the release capability of malate and citrate as sensitive cultivar secreted more malate and citrate than tolerant cultivar even after Al treatment. Lipid composition of the PM differed between these cultivars. Sensitive cultivar contains more phospholipids and less Δ⁵-sterols than tolerant cultivars in control and Al treatment. In general, phospholipids content increased and Δ⁵-sterols decreased after Al treatment compared to that in control irrespective of their Al tolerance. After inclusion of sterol metabolism inhibitors with Al, greatest increase of phospholipids was observed in the Al+uniconazole treatment for sensitive cultivar's root-tip compared to control and Al treatment. Conversely, greater decrease of Δ⁵-sterols was observed in Al+uniconazole treatment for tolerant Rikuu-132. Also, 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. I used inhibitors of Δ⁵-sterol synthesis (uniconazole-P, an inhibitor of obtusifoliol-14α-demethylase [OBT 14DM], and fenpropimorph, an inhibitor of cycloeucalenol obtusifoliol isomerase) to lower the content of Δ⁵-sterols in both cultivar. 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. This inhibitor reduced Al tolerance in Rikuu-132 and its Al-tolerant ancestor cultivars Kamenoo and Kyoku to the same level of Al tolerance for Al-sensitive Rikuu-20 and Aikoku. Al tolerance was negatively correlated with the ratio of phospholipids to Δ⁵-sterols in root-tip portions of both cultivars in the existence of Al and inhibitors. This indicates that greater contribution of this lipid ratio as phospholipids makes more sensitive (negative impact) and Δ⁵-sterols makes intact (positive effect) on PM permeabilization which offers tolerance to Al. Differentially induced permeabilizations could be discussed based on van der Waals conformational differences in phytosterols (stigmasterol) and abnormal sterols (cycloeucalenol and obtusifoliol) synthesized greater after the treatment with inhibitors. This is the first investigation which suggests the significant roles of relative abundance of Δ⁵-sterols within PM and of OBT 14DM in Al tolerance of rice.

　In the second stage of experiments, further investigations were carried out using other crop plant species to clarify whether the same Al tolerance mechanism is working or not. I used the Al-tolerant and Al-sensitive cultivars or lines of sorghum (Super sugar and Kaneko, respectively), wheat (ET8 and ES8, respectively), triticale (ST22 and ST2, respectively), maize (Golddent KD520 and Golddent KD500, respectively) and soybean (Enrei and Ryokuheki, respectively) for these experiments. Greater Al accumulation and PM permeabilization were also observed in the Al-sensitive cultivars of all the crop plant species studied. Greater Δ⁵-sterol and less phospholipids content were also found in tolerant cultivars or lines for all the crop species except for soybean, although this tendency did not show any trend among different crop plant species. Additionally, it was observed that Al treatment increased phospholipids and decreased Δ⁵-sterol for all the crop plant species. This result surely shows the existence of the different mechanism for Al tolerance additionally to the mechanism on OA release for sorghum and wheat. No exceptions have been observed yet in monocot plants on the greater lipid ratio for Al-tolerance.

　In practical sense, major growth limiting factors in tropical acid soils are not only the toxic level of Al but also low nutrients. After investigation under the combination of high Al and low-nutrient conditions in long-term, it was found that Al tolerance in full nutrient condition is greater than that of low-nutrient conditions indicating simultaneous effect of toxic Al and low-nutrient. Though Al concentration in roots can explain Al tolerance, however, it can not explain combined tolerance (high Al and low nutrient tolerance) or low nutrient tolerance. From the above results, it was suggested that contribution of low-nutrient tolerance is greater than that of Al tolerance for rice. Transport capability of Ca to shoots was suggested as an important component for low-nutrient tolerance in rice.In conclusion, I demonstrated for the first time the significant role of plasma membrane lipid layer (especially, sterol molecule) in Al tolerance and suggested also the significant role of transport capability for Ca to rice shoot in better growth on tropical acid soils.

＜要約＞

　アルミニウム（Al）耐性機構は種々の植物で多数報告されている。有機酸放出はコムギ、トウモロコシ、 ソバ、ダイズなどの植物での主要なＡｌ耐性機構とされている。 しかしながら、Ａｌ耐性機構は単一でないことも解っている。 イネは重要作物であり、Ａｌ耐性作物としても有名であるが、その耐性機構は不明である。 そこで、まず最初にイネを用い、根端細胞膜（ＰＭ）脂質層に注目してＡｌ耐性機構を、 ついで、イネ以外の植物での同様の機構の関与を調査した。

　バングラデシュのイネ２３品種と日本のイネ６品種のＡｌ耐性を調査した。 その結果、Ａｌ耐性はBRRIdhan41、Rhamatが最強で、Moyna、BRRIdhan34が最弱であり、 日本のイネではササニシキが最強で、どまんなかは最弱であった。 根端のヘマトキシリン染色とFDA-PI蛍光染色結果から、Ａｌ耐性品種はＡｌ排除機構が共通して認められ、 また、ＰＭ透過性はＡｌ存在下でも増大しにくいことが明らかとなった。

　Ａｌ耐性の強いササニシキのほぼ全部の系統品種に該当する１８品種に関してＡｌ耐性を調査した結果、広範な耐性差を認めた。 それらのなかで、陸羽２０号は著しく感受性なのに対し、その直系の子孫である陸羽１３２号は著しく耐性であった。 これら両品種間にも上記のＡｌ耐性機構の違いが認められたが、根端からのリンゴ酸やクエン酸放出能には差が認められず、 有機酸放出機構でＡｌ耐性を説明できなかった。

　根端のリン脂質/Δ５-ステロール（ＰＬ／Ｓ）比はＡｌ耐性品種の陸羽１３２号で小さかった。 ウニコナゾールＰは、obtusifoliol-14α-demethylase (OBT 14DM )の 阻害剤であり、 通常は極く微量しか含まれていないobtusifoliolなどの含量を増大させ、 他方最終生成物であるＳの含量を低下させることが既に解っている。 また、フェンプロピモーフは、cycloeucalenol obtusifoliol isomerase (COI)　の阻害剤であり、 同様にcycloeucalenolなどを増やし、他方Ｓを減らす。 これら二つの阻害剤処理で、陸羽１３２号根端部のＳ含量は低下し、他方ＰＬ含量は増加し、その結果PL/S比、 Ａｌ含有率、膜透過性のいずれもが増大し、Ａｌ耐性は低下した。 ステロール合成阻害剤は根端細胞膜脂質層中のステロール含量を低下させることによって、 膜の負荷電性を高め、その結果Ａｌの膜脂質層への結合能を高め、同時にabnormalステロール量の増大により膜の透過性を高めるため、 Ａｌ耐性を低下させると解釈された。 ステロール合成阻害剤とＡｌの同時処理のデータを総合した結果、PL/S比とＡｌ耐性の間に負の相関が認められた。 また、ウニコナゾールＰ処理でＡｌ感受性品種である陸羽２０号、愛国のＡｌ耐性値は変わらないのに対し、 Ａｌ耐性品種である亀の尾、旭、ササニシキのＡｌ耐性値は、感受性品種の値にまで低下した。 以上の結果、イネのＡｌ耐性におけるＰＭ中のΔ５-ステロールと、OBT 14DMの重要な役割が示唆された。 本研究は、Ａｌ耐性における根端細胞膜脂質の重要な意義を示した最初のものである。

　つぎに、このイネのＡｌ耐性機構が他の植物でも関与しているのかを調査した。 まず、Ａｌ耐性の最強（Ｔ）、最弱（Ｓ）をあらかじめ選抜した。 すなわち、ソルゴーではカネコ・ハイブリッド（Ｔ）とスーパーシュガー（Ｓ）、コムギではET8 (T) とES (8) 、 ライコムギではＳＴ（２）とＳＴ（２２）、トウモロコシではＫＤ５２０(Ｔ)とＫＤ８５０(Ｓ)を用いた。 その結果、Ａｌ耐性品種間差と根端のＡｌ集積性、膜透過性、ＰＬ／Ｓ比の品種間差の間に、イネと同様の傾向を認めた。 しかしながら、ダイズ品種間では、この機構の関与が認められなかった。

　最後に、典型的酸性土壌である熱帯酸性土壌での作物生育支配要因を検討した。 実際のこれら酸性土壌に似せたＡｌと養分の濃度に各種組み合わせ、多くのイネ品種を長期間水耕栽培し、生育量と体内養分組成を調査した。 その結果、Ａｌ耐性植物であるイネでは、低養分耐性が生育をより大きく支配し、 カルシウム（Ｃａ）の茎葉部への輸送能力が品種間生育差の大きな要因であることを示唆する結果を得た。