Molecular and physiological strategies to increase aluminum resistance in plants

Aluminum (Al) toxicity is a primary limitation to plant growth on acid soils. Root meristems are the first site for toxic Al accumulation, and therefore inhibition of root elongation is the most evident physiological manifestation of Al toxicity. Plants may resist Al toxicity by avoidance (Al exclusion) and/or tolerance mechanisms (detoxification of Al inside the cells). The Al exclusion involves the exudation of organic acid anions from the root apices, whereas tolerance mechanisms comprise internal Al detoxification by organic acid anions and enhanced scavenging of free oxygen radicals. One of the most important advances in understanding the molecular events associated with the Al exclusion mechanism was the identification of the ALMT1 gene (Al-activated malate transporter) in Triticum aestivum root cells, which codes for a plasma membrane anion channel that allows efflux of organic acid anions, such as malate, citrate or oxalate. On the other hand, the scavenging of free radicals is dependent on the expression of genes involved in antioxidant defenses, such as peroxidases (e.g. in Arabidopsis thaliana and Nicotiana tabacum), catalases (e.g. in Capsicum annuum), and the gene WMnSOD1 from T. aestivum. However, other recent findings show that reactive oxygen species (ROS) induced stress may be due to acidic (low pH) conditions rather than to Al stress. In this review, we summarize recent findings regarding molecular and physiological mechanisms of Al toxicity and resistance in higher plants. Advances have been made in understanding some of the underlying strategies that plants use to cope with Al toxicity. Furthermore, we discuss the physiological and molecular responses to Al toxicity, including genes involved in Al resistance that have been identified and characterized in several plant species. The better understanding of these strategies and mechanisms is essential for improving plant performance in acidic, Al-toxic soils.     

植物耐铝的生物化学与分子机理

某些耐铝植物在铝胁迫下分泌有机酸被认为是一个重要的抗性机制.从根系分泌出来的有机酸能与根际的Al3 结合,形成无毒性的螯合物,从而减轻了铝对根系的毒害.但是,铝诱导有机酸分泌的中间环节及调节机制至今仍不清楚.一些证据表明,铝能激活根尖细胞质膜内的阴离子通道,因而可以调节有机酸的分泌.近年来,人们开始注意一些信号分子如蛋白激酶、水杨酸等介导铝诱导有机酸的分泌,已经获得一些成果.同时,铝胁迫基因的分离和鉴定也为人们从分子水平上研究和认识铝胁迫下植物的抗性机制奠定了基础.     

Role of organic acids in detoxification of aluminum in higher plants

Phytotoxicity of aluminum ion (Al3+) is a serious problem limiting crop production on acid soils. Organic acids with Al-chelating ability play an important role in the detoxification of Al both externally and internally. Al is detoxified externally by the secretion of organic acids such as citric, oxalic, and/or malic acids from the roots. The secretion of organic acids is highly specific to Al and the site of secretion is localized to the root apex. The kind of organic acids secreted as well as secretion pattern differ among plant species. There are two patterns of Al-induced secretion of organic acids: In pattern I, there is no discernible delay between the addition of Al and the onset of the release of organic acids. Activation of the anion channel seems to be involved in this pattern; In pattern II, there is a marked lag phase between the addition of Al and the onset of organic acid release. The action of genes related to the metabolism and secretion of organic acids seems to be involved in this pattern. Internal detoxification of Al in Al-accumulating plants is achieved by the formation of Al-organic acid complex. For instance, the complex of Al-citrate (1:1) in hydrangea and Al-oxalate (1:3) in buckwheat has been identified.     

酸性土壤上植物应对铝胁迫的过程与机制

铝胁迫是酸性土壤上影响作物产量最重要的因素之一.目前,全球土壤酸化程度进一步加剧了铝胁迫.植物可通过将铝离子与有机酸螯合储藏于液泡和从根系中排出铝毒.排出铝毒主要通过苹果酸转运蛋白ALMT和柠檬酸转运蛋白MATE的跨膜运输来实现.编码ABC转运蛋白和锌指转录因子的基因与植物抗铝胁迫有关.这些抗铝毒基因的鉴别使得通过转基因和分子标记辅助育种等生物技术来提高农作物的抗铝毒能力成为可能.最后提出了植物抗铝胁迫研究中需要解决的关键问题及今后的研究方向.     

Pattern of aluminum-induced secretion of organic acids differs between rye and wheat

Al-Induced secretion of organic acids from the roots has been considered as a mechanism of Al tolerance, but the processes leading to the secretion of organic acids are still unknown. In this study, the secretion pattern and alteration in the metabolism of organic acids under Al stress were examined in rye (Secale cereale L. cv King) and wheat (Triticum aestivum L. cv Atlas 66). Al induced rapid secretion of malate in the wheat, but a lag (6 and 10 h for malic and citric acids, respectively) between the exposure to Al and the secretion of organic acids was observed in the rye. The activities of isocitrate dehydrogenase, phosphoenolpyruvate carboxylase, and malate dehydrogenase were not affected by Al in either plant. The activity of citrate synthase was increased by the exposure to Al in the rye, but not in the wheat. The secretion of malate was not suppressed at low temperature in the wheat, but that of citrate was stopped in the rye. The Al-induced secretion of citrate from roots of the rye was inhibited by the inhibitors of a citrate carrier, which transports citrate from the mitochondria to the cytoplasm. All of these results suggest that alteration in the metabolism of organic acids is involved in the Al-induced secretion of organic acids in rye, but only activation of an anion channel seems to be responsible for the rapid secretion of malate in the wheat.     

有机酸在植物解铝毒中的作用及生理机制

酸性土壤上铝毒是限制作物产量的一个重要障碍因子,具有螯合能力的有机酸在植物铝的外部排斥机制和内部耐受机制均具有重要作用,在铝的外部排斥解毒过程中,植物通过根系分泌有机酸进入根际,如柠檬酸,草酸,苹果酸等与铝形成稳定的复合体,阻止铝进入共质体,从而达到植物体外解除铝毒害效应的目的,且分泌的有机酸对铝的胁迫诱导表现出高度的专一性,分泌的关键点位于根尖,不同的物种间分泌的有机酸种类,分泌的模式及生理机理存在差异,在铝积累型植物的内部解毒过程中,有机酸与铝形成稳定的化合物,降低植物体内铝离子的生理活性,从而降低细胞内铝离子的毒害效应,如绣球花中铝与柠檬酸形成1：1的复合体,荞麦内铝与草酸形成1：3的复合体,本文就有机酸在植物忍耐和积累铝中的作用及生理机制作一简要综述。     

Protecting Cell Walls from Binding Aluminum by Organic Acids Contributes to Aluminum Resistance

Aluminum-induced secretion of organic acids from the root apex has been demonstrated to be one major AI resistance mechanism in plants. However, whether the organic acid concentration is high enough to detoxify AI in the growth medium is frequently questioned. The genotypes of Al-resistant wheat, Cassia tora L. and buckwheat secrete malate, citrate and oxalate, respectively. In the present study we found that at a 35% inhibition of root elongation, the AI activities in the solution were 10, 20, and 50 μM with the corresponding malate, citrate, and oxalate exudation at the rates of 15, 20 and 21 nmol/cm2 per 12 h, respectively, for the above three plant species. When exogenous organic acids were added to ameliorate Al toxicity, twofold and eightfold higher oxalate and malate concentrations were required to produce the equal effect by citrate. After the root apical cell walls were isolated and preincubated in 1 mM malate, oxalate or citrate solution overnight, the total amount of AI adsorbed to the cell walls all decreased significantly to a similar level, implying that these organic acids own an equal ability to protect the cell walls from binding AI. These findings suggest that protection of cell walls from binding Al by organic acids may contribute significantly to AI resistance.     

有机酸在植物解铝毒中的作用及生理机制

酸性土壤上铝毒是限制作物产量的一个重要障碍因子。具有螯合能力的有机酸在植物铝的外部排斥机制和内部耐受机制均具有重要作用。在铝的外部排斥解毒过程中,植物通过根系分泌有机酸进入根际,如柠檬酸、草酸、苹果酸等与铝形成稳定的复合体,阻止铝进入共质体,从而达到植物体外解除铝毒害效应的目的,且分泌的有机酸对铝的胁迫诱导表现出高度的专一性,分泌的关键点位于根尖。不同的物种间分泌的有机酸种类、分泌的模式及生理机理存在差异。在铝积累型植物的内部解毒过程中,有机酸与铝形成稳定的化合物,降低植物体内铝离子的生理活性,从而降低细胞内铝离子的毒害效应,如绣球花中铝与柠檬酸形成1:1的复合体,荞麦内铝与草酸形成1:3的复合体。本文就有机酸在植物忍耐和积累铝中的作用及生理机制作一简要综述。     

Secretion of malate and citrate from roots is related to high Al-resistance in Lespedeza bicolor

Lespedeza bicolor (Lespedeza bicolor Turcz. cv. Jiangxi) is a leguminous shrub that is well adapted to acid infertile soils. However, the mechanisms of aluminum resistance in this species have not been established. This study aimed to assess the possible resistance mechanisms of this plant to Al. An Al-sensitive species of Lespedeza, sericea lespedeza [Lespedeza cuneata (Dum.-Cours.) G. Don cv. Zhejiang], was used as a reference. The roots of L. bicolor secreted both malate and citrate after exposure to Al, but roots of L. cuneata did not. The secretion of organic acids from L. bicolor was specific to Al; neither 15-day P starvation nor 50 μM lanthanum induced the secretion of these organic acid anions. Secretion of organic acid anions in L. bicolor was detected after 3–6 h exposure to Al, and the amount increased significantly after 6 h exposure, suggesting that this plant shows a pattern II-type organic acid secretion. This is supported by the finding that the secretion was significantly inhibited by a protein-synthesis inhibitor, cycloheximide. Two kinds of anion-channel inhibitors had different effects on Al-induced secretion of organic acids: 9-anthracene carboxylic acid completely inhibited secretion, phenylglyoxal had no effect. Root elongation in L. bicolor was more severely inhibited by Al in the presence of 9-anthracene carboxylic acid. All these results indicated that the secretion of malate and citrate is a specialized response to Al stress in L. bicolor roots, which might be one of the Al-resistance mechanisms in this species.     

The ScAACT1 gene at the Q alt5 locus as a candidate for increased aluminum tolerance in rye (Secale cereale L.)

Soluble aluminum (Al³⁺) is a major constraint to plant growth in highly acidic soils, which comprise up to 50% of the world??s arable land. The primary mechanism of Al resistance described in plants is the chelation of Al³⁺ cations by release of organic acids into the rhizosphere. Candidate aluminum tolerance genes encoding organic acid transporter of the ALMT (aluminum-activated malate transporter) and MATE (multi-drug and toxic compound extrusion) families have been characterized in several plant species. In this study, we have isolated in five different cultivars the rye ScAACT1 gene, homolog to barley aluminum activated citrate transporter HvAACT1. This gene mapped to the 7RS chromosome arm, 25?cM away from the ScALMT1 aluminum tolerance gene. The gene consisted of 13 exons and 12 introns and encodes a predicted membrane protein that contains the MatE domain and at least seven putative transmembrane regions. Expression of the ScAACT1 gene is Al-induced, but there were differences in the levels of expression among the cultivars analyzed. A new quantitative trait locus for Al tolerance in rye that co-localizes with the ScAACT1 gene was detected in the 7RS chromosome arm. These results suggest that the ScAACT1 gene is a candidate gene for increased Al tolerance in rye. The phylogenetic relationships between different MATE proteins are discussed.     

Aluminum resistance mechanisms in oat (Avena sativa L.)

Background and aims

Enhanced aluminum (Al) resistance has been observed in dicots over-expressing enzymes involved in organic acid synthesis; however, this approach for improving Al resistance has not been investigated in monocots. Among the cereals, oat (Avena sativa L.) is considered to be Al resistant, but the basis of resistance is not known.

Methods

A hydroponic assay and hematoxylin staining for Al accumulation in roots were used to evaluate Al resistance in 15 oat cultivars. Malate and citrate release from roots was measured over a 24?h period. A malate dehydrogenase gene, neMDH, from alfalfa (Medicago sativa L.) was used to transform oat.

Results

Oat seedlings were highly resistant to Al, as a concentration of 325?μM AlK(SO₄)₂ was needed to cause a 50% decrease in root growth. Most oat cultivars tested are naturally resistant to high concentrations of Al and effectively excluded Al from roots. Al-dependent release of malate and Al-independent release of citrate was observed. Al resistance was enhanced in a transgenic oat line with the highest accumulation of neMDH protein. However, overall root growth of this line was reduced and expression of neMDH in transgenic oat did not enhance malate secretion.

Conclusions

Release of malate from oat roots was associated with Al resistance, which suggests that malate plays a role in Al resistance of oat. Over-expression of alfalfa neMDH enhanced Al resistance in some lines but was not effective alone for crop improvement.     

Possible involvement of protein phosphorylation in aluminum-responsive malate efflux from wheat root apex

In many plants, efflux of organic anions from roots has been proposed as one of the major Al resistance mechanisms. However it remains unknown how plants regulate efflux of organic anions in response to Al. In this study, the regulatory mechanisms of Al-responsive malate efflux in wheat (Triticum aestivum) were characterized focusing on the role of protein phosphorylation. Al-resistant wheat (cv Atlas) initiated malate efflux at 5 min after addition of Al, and this response was sensitive to temperature. K-252a, a broad range inhibitor of protein kinases, effectively blocked the Al-induced malate efflux accompanied with an increased accumulation of Al and intensified Al-induced root growth inhibition. A transient activation of a 48-kD protein kinase and an irreversible repression of a 42-kD protein kinase were observed preceding the initiation of malate efflux, and these changes were canceled by K-252a. Malate efflux was accompanied with a rapid decrease in the contents of organic anions in the root apex, such as citrate, succinate, and malate but with no change in the contents of inorganic anions such as chloride, nitrate, and phosphate. These results suggest that protein phosphorylation is involved in the Al-responsive malate efflux in the wheat root apex and that the organic anion-specific channel might be a terminal target that responds to Al signaling mediated by phosphorylation.     

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.     

The roles of organic anion permeases in aluminium resistance and mineral nutrition

Soluble aluminium (Al(3+)) is the major constraint to plant growth on acid soils. Plants have evolved mechanisms to tolerate Al(3+) and one type of mechanism relies on the efflux of organic anions that protect roots by chelating the Al(3+). Al(3+) resistance genes of several species have now been isolated and found to encode membrane proteins that facilitate organic anion efflux from roots. These proteins belong to the Al(3+)-activated malate transporter (ALMT) and multi-drug and toxin extrusion (MATE) families. We review the roles of these proteins in Al(3+) resistance as well as their roles in other aspects of mineral nutrition.     

植物地上部对铝毒的生理响应及其耐性

全世界50％以上潜在的可耕地属于酸性土壤,铝毒害是酸性土壤上植物生长最有害因素之一。近年来,为了阐明植物铝毒害及其耐性,前人已进行了大量的研究,并有一些综述性文章发表。然而,大多数文章主要综述铝对植物根系的影响及其耐性,因为根生长受抑是最早的铝毒害症状之一和溶液培养时最容易辨认的铝毒害症状。为此,本文综述了铝对植物地上部光合作用、光保护系统、水分利用效率、含水量、碳水化合物含量、矿质营养、有机酸和氮代谢的影响,并对富铝植物的解铝毒机制（铝与小分子有机酸螯合和把铝隔离在对铝不敏感的表皮细胞和液泡内）进行了综述。本文还对植物耐铝遗传学和分子生物学及今后需要研究的问题进行了讨论。     

Continuous secretion of organic acids is related to aluminium resistance during relatively long‐term exposure to aluminium stress

Secretion of organic acid has been suggested to be one of the mechanisms for Al resistance in short‐term experiments. In the present study, relatively long‐term response of roots to Al stress was investigated in terms of organic acid secretion. Eight plant cultivars belonging to 5 species that exhibited differential sensitivity to Al were used. Ten days of intermittent exposure to Al (one day in 0.5 m M CaCl₂ containing 50 µ M AlCl₃ at pH 4.5, alternating with one day in nutrient solution without Al) inhibited root growth by 65% in an Al‐sensitive cultivar of wheat ( Triticum aestivum L. Scout 66) and by 25‐50% in two cultivars of oilseed rape ( Brassica napus L. 94008 and H166), two cultivars of oat ( Avena sativa L. Tochiyutaka and Heoats), and an Al‐tolerant cultivar of wheat (Atlas 66). However, root growth was hardly affected by the same treatment in buckwheat ( Fagopyrum esculentum Moench Jianxi) and radish ( Raphanus sativus L. Guangxi). Organic acids were monitored during the first 6 h of each day of Al treatment, and both the kind and amount of organic acids secreted were found to differ among different species. Roots of buckwheat secreted oxalic acid, those of wheat exuded malic acid, while those of rapeseed, oats, and radish secreted both citric and malic acids. Three different patterns in response to relatively long‐term treatment of Al were found in terms of total amount of organic acids secreted: (1) the amount secreted was very low during the treatment (wheat cv. Scout 66, oat), (2) the amount gradually decreased with duration of treatment (wheat cv. Atlas 66, oilseed rape), and (3) the amount maintained at a high level during the whole period of Al treatment (buckwheat and radish). Combined with the results of growth inhibition, it is suggested that the continuous secretion of organic acids at a high level is related to high Al resistance.     

Organic acids in the rhizosphere – a critical review

Organic acids, such as malate, citrate and oxalate, have been proposed to be involved in many processes operating in the rhizosphere, including nutrient acquisition and metal detoxification, alleviation of anaerobic stress in roots, mineral weathering and pathogen attraction. A full assessment of their role in these processes, however, cannot be determined unless the exact mechanisms of plant organic acid release and the fate of these compounds in the soil are more fully understood. This review therefore includes information on organic acid levels in plants (concentrations, compartmentalisation, spatial aspects, synthesis), plant efflux (passive versus active transport, theoretical versus experimental considerations), soil reactions (soil solution concentrations, sorption) and microbial considerations (mineralization). In summary, the release of organic acids from roots can operate by multiple mechanisms in response to a number of well-defined environmental stresses (e.g., Al, P and Fe stress, anoxia): These responses, however, are highly stress- and plant-species specific. In addition, this review indicates that the sorption of organic acids to the mineral phase and mineralisation by the soil's microbial biomass are critical to determining the effectiveness of organic acids in most rhizosphere processes.     

The role of organic acids in the short- and long-term aluminum tolerance in maize seedlings (<Emphasis Type="Italic">Zea mays</Emphasis> L.)

Aluminum (Al) affects numerous physiological processes in plants. However, Al tolerance mechanisms mediated by increased synthesis of organic acids (OAs) have been outlined recently. In this study, we examined the role of OAs in the short (1–8 h) and long-term (4 days) Al tolerance in maize seedlings. Exposure to Al stress for 4 days results in a rapid inhibition of root growth. Al induced morphological changes in the maize roots, especially at a higher solution of Al concentration (1,000 μM Al). The increase in Al accumulation in roots, including strongly elevated levels of Al accumulated in root cell walls suggests that Al tolerance in maize is mediated in part by higher accumulation of Al in the roots. The enhanced citrate exudation, which was only observed at 1,000 μM Al may lead to detoxification of Al by formation of OA–Al complexes in the root apoplast. This mechanism has been suggested to play a significant role in Al resistance response in maize. The short-term responses underlying internal detoxification via OA-chelators were also investigated. Succinate, malate, citrate and total root OA contents decreased markedly, 2 h after the Al exposure. At 4 and 8 h time points, OA contents increased or remained unchanged, except for that of malate which decreased. The level of OAs in shoots, on the other hand, showed alterations that were less pronounced in response to Al. Specifically, the citrate and total OA concentrations significantly increased at 4 h, but showed a pronounced decrease at the 8 h time point. Based on our findings, we propose that multiple responses, including Al exclusion by Al accumulation in root cells and citrate efflux, may contribute towards higher Al resistance in maize. The rapid OA changes in responses to short-term Al treatment may not be responsible for Al tolerance. However, increased OA synthesis observed in this study may be involved in diminishing the stress triggered by Al. The molecular aspects underlying Al resistance mechanism via Al-induced expression of the enzymes catalyzing OA synthesis and metabolism remain to be elucidated.