The Distal Part of the Transition Zone Is the Most Aluminum-Sensitive Apical Root Zone of Maize

For a better understanding of Al inhibition of root elongation, knowledge of the morphological and functional organization of the root apex is a prerequisite. We developed a polyvinyl chloride-block technique to supply Al (90 μm monomeric Al) in a medium containing agarose to individual 1-mm root zones of intact seedlings of maize (Zea mays L. cv Lixis). Root elongation was measured during a period of 5 h. After Al treatment, callose (5 h) and Al (1 h) contents of individual 1-mm apical root segments were determined. For comparison, callose and Al levels were also measured in root segments after uniform Al supply in agarose blocks to the 10-mm root apex. Only applying Al to the three apical 1-mm root zones inhibited root elongation after 1 h. The order of sensitivity was 1 to 2 > 0 to 1 > 2 to 3 mm. In the 1- to 2-mm root zone high levels of Al-induced callose formation and accumulation of Al was found, independently of whether Al was applied to individual apical root zones or uniformly to the whole-root apex. We conclude from these results that the distal part of the transition zone of the root apex, where the cells are undergoing a preparatory phase for rapid elongation (F. Baluška, D. Volkmann, P.W. Barlow [1996] Plant Physiol 112: 3–4), is the primary target of Al in this Al-sensitive maize cultivar.     

Does aluminium affect root growth of maize through interaction with the cell wall – plasma membrane – cytoskeleton continuum?

The mechanism of aluminium-induced inhibition of root elongation is still not well understood. It is a matter of debate whether the primary lesions of Al toxicity are apoplastic or symplastic. The present paper summarises experimental evidence which offers new avenues in the understanding of Al toxicity and resistance in maize. Application of Al for 1 h to individual 1 mm sections of the root apex only inhibited root elongation if applied to the first 3 apical mm. The most Al-sensitive apical root zone appeared to be the 1–2 mm segment. Aluminium-induced prominent alterations in both the microtubular (disintegration) and the actin cytoskeleton (altered polymerisation patterns) were found especially in the apical 1–2 mm zone using monoclonal antibodies. Since accumulation of Al in the root apoplast is dependent on the properties of the pectic matrix, we investigated whether Al uptake and toxicity could be modulated by changing the pectin content of the cell walls through pre-treatment of intact maize plants with 150 mM NaCl for 5 days. NaCl-adapted plants with higher pectin content accumulated more Al in their root apices and they were more Al-sensitive as indicated by more severe inhibition of root elongation and enhanced callose induction by Al. This special role of the pectic matrix of the cell walls in the modulation of Al toxicity is also indicated by a close positive correlation between pectin, Al, and Al-induced callose contents of 1 mm root segments along the 5 mm root apex. On the basis of the presented data we suggest that the rapid disorganisation of the cytoskeleton leading to root growth inhibition may be mediated by interaction of Al with the apoplastic side of the cell wall – plasma membrane – cytoskeleton continuum. This revised version was published online in June 2006 with corrections to the Cover Date.     

Induction of callose formation is a sensitive marker for genotypic aluminium sensitivity in maize

The screening of 37 Zea mays L. cultivars in nutrient solution using root elongation (24 h) as a parameter showed large genotypic differences in Al resistance among the genetic material evaluated.Callose concentrations in root tips were closely and positively related to Al-induced inhibition of root elongation. Therefore, Al-induced callose formation in root tips appears to be an excellent indicator of Al injury and can be used as a selection criteria for Al sensitivity. In contrast, aluminium concentrations in root tips were not related to Al-induced inhibition of root elongation, nor to Al-induced callose formation. Callose formation was also induced by short-term A1 treatment in root tip protoplasts, and the response of protoplasts clearly reflected the cultivar-specific response to Al of intact roots. This indicates that in maize, Al sensitivity is expressed on the protoplast level.     

Callose formation as parameter for assessing genotypical plant tolerance of aluminium and manganese

Callose ((1,3)--glucan) formation in plant tissues is induced by excess Al and Mn. In the present study callose was spectrophotometrically quantified in order to evaluate whether it could be used as a parameter to identify genotypical differences in Al and Mn tolerance. Mn leaf-tissue tolerance of cowpea and linseed genotypes was assessed using the technique of isolated leaf tissue floating on Mn solution. Genotypical differences in the density of brown speckles on the leaf tissue (Mn toxicity symptoms) correlated closely with the concentrations of callose for both plant species. In cell suspension cultures Mn excess also induced callose formation. However, differences in tolerance of cowpea genotypes using callose formation as a parameter could only be found in cultured cowpea cells if controls cultured at optimum Mn supply showed low background callose. As soon as after 1 h, Al supply (50 M) induced callose formation predominantly in the 5-mm root tip of soybean seedlings. Callose concentration in the 0–30 mm root tips was inversely related to the root elongation rate when roots were subjected to an increasing Al supply above 10 M. Three soybean genotypes differed in inhibition of root-elongation rate and induction of callose formation when treated with 50 M Al for 8 h. Relative callose concentrations and relative root-elongation rates for these genotypes were significantly negatively correlated.     

Callose in roots of Norway spruce (Picea abies (L.) Karst.) is a sensitive parameter for aluminium supply at a forest site (Höglwald)

Under controlled environmental conditions in nutrient solution experiments induction of non-constitutive callose in roots has been shown to be a symptom of aluminium (Al) toxicity. In the present study roots of Norway spruce were sampled from a forest site where soil conditions had been modified by acidic irrigation and liming (Höglwald Experiment in Bavaria, Germany). A significant positive relationship was found between the callose content in short roots and the Al concentration in the soil solution, particularly if free Al, rather than total concentrations of soluble Al, were used for prediction. At the same sites root growth of Norway spruce was not affected by free Al concentrations in the range of 2.5 to 199 µM Al. The results show that also under field conditions a positive relationship between Al supply and callose content can be established. In Norway spruce callose content in roots is a much more sensitive parameter for Al supply than root growth.     

Aluminum-induced deposition of (1,3)-β-glucans (callose) inTriticum aestivum L.

Aluminum (Al)-induced damage to leaves and roots of two Al-resistant (cv. Atlas 66, experimental line PT741) and two Al-sensitive (cv. Scout 66, cv. Katepwa) lines ofTriticum aestivum L. was estimated using the deposition of (1, 3)--glucans (callose) as a marker for injury. Two-day-old seedlings were grown for forty hours in nutrient solutions with or without added Al, and callose deposition was quantified by spectrofluorometry (0–1000 µM Al) and localized by fluorescence microscopy (0 and 400 µM Al). Results suggested that Al caused little damage to leaves. No callose was observed in leaves with up to 400 µM Al treatment. In contrast, root callose concentration increased with Al treatment, especially in the Al-sensitive lines. At 400 µM Al, root callose concentration of Al-sensitive Scout 66 was nearly four-fold that of Al-resistant Atlas 66. After Al treatment, large callose deposits were observed in the root cap, epidermis and outer cortex of root tips of Scout 66, but not Atlas 66. The identity of callose was confirmed by a reduced fluorescence in Al-treated roots: firstly, after adding an inhibitor of callose synthesis (2-deoxy-D-glucose) to the nutrient solution, and secondly, after incubating root sections with the callosedegrading enzyme -D-glucoside glucohydrolase [EC 3.2.1.21]. Root callose deposition may be a good marker for Al-induced injury due to its early detection by spectrofluorometry and its close association with stress perception.Abbreviations DDG 2-deoxy-D-glucose - PAS periodic acid - Schiffs reagent - PE pachyman equivalents     

Zonal responses of sensitive vs. tolerant wheat roots during Al exposure and recovery

Aluminium (Al) irreversibly inhibits root growth in sensitive, but not in some tolerant genotypes. To better understand tolerance mechanisms, seedlings from tolerant ('Barbela 7/72' line) and sensitive ('Anahuac') Triticum aestivum L. genotypes were exposed to AlCl(3) 185 μM for: (a) 24 h followed by 48 h without Al (recovery); (b) 72 h of continuous exposure. Three root zones were analyzed (meristematic (MZ), elongation (EZ) and hairy (HZ)) for callose deposition, reserves (starch and lipids) accumulation, endodermis differentiation and tissue architecture. Putative Al-induced genotoxic or cytostatic/mytogenic effects were assessed by flow cytometry in root apices. Tolerant plants accumulated less Al, presented less root damage and a less generalized callose distribution than sensitive ones. Starch and lipid reserves remained constant in tolerant roots but drastically decreased in sensitive ones. Al induced different profiles of endodermis differentiation: differentiation was promoted in EZ and HZ, respectively, in sensitive and tolerant genotypes. No ploidy changes or clastogenicity were observed. However, differences in cell cycle blockage profiles were detected, being less severe in tolerant roots. After Al removal, only the 'Barbela 7/72' line reversed Al-induced effects to values closer to the control, mostly with respect to callose deposition and cell cycle progression. We demonstrate for the first time that: (a) cell cycle progression is differently regulated by Al-tolerant and Al-sensitive genotypes; (b) Al induces callose deposition >3 cm above root apex (in HZ); (c) callose deposition is a transient Al-induced effect in tolerant plants; and (d) in HZ, endodermis differentiation is also stimulated only in tolerant plants, probably functioning in tolerant genotypes as a protective mechanism in addition to callose.     

Pectin methylesterase modulates aluminium sensitivity in Zea mays and Solanum tuberosum

Cell suspension cultures of Zeamays L. were adapted to grow under conditions of NaCl stress, which increased the cell‐wall pectin content of these cells by 31% compared with unadapted cells (controls). Both cultures were treated for 5 or 10 min with pectin methylesterase (PME) and afterwards incubated in the presence of Al for 2 h. The different capabilities of the cells to synthesise callose due to pre‐treatment were taken into account by calculating relative Al‐induced callose induction (digitonin=100%). Only in salt‐adapted cells with a degree of methylation of cell‐wall pectin (DM) decreasing from 34% (control) to 13%, did PME treatment enhance total and BaCl₂‐non‐exchangeable Al contents and Al sensitivity as indicated by increased callose formation. In a further step, a wider variation in DM was achieved by subculturing the NaCl‐adapted cells for up to 3 weeks without NaCl supply and adapting them to the cellulose‐synthesis inhibitor 2,6‐dichlorbenzonitrile (DCB). This reduced DM to 26%, while short‐term treatment with pectolyase resulted in the lowest DM (12%). After the 2 h Al treatment, there was a close negative relationship between DM and relative callose formation of Al contents, with the exception of pectolyase‐treated cells. In addition, intact plants of Solanumtuberosum L. genotypes were characterised for their Al sensitivity in hydroponics using root elongation, Al‐induced callose formation and Al contents of root tips as parameters. Based on all three parameters, the transgenic potato mutant overexpressing PME proved to be more Al‐sensitive than the wild type, the Al‐resistant and even the Al‐sensitive potato cultivar. Especially in the root tips (1 cm), Al treatment (2 h, 50 μM) increased the activity of PME more in the Al‐sensitive than in the Al‐resistant genotypes. The presented data emphasise the importance of the DM of the pectin matrix and the activity of PME for the expression of Al toxicity and Al resistance.     

Genotypical differences in aluminum resistance of maize are expressed in the distal part of the transition zone. Is reduced basipetal auxin flow involved in inhibition of root elongation by aluminum?

Short-term Al treatment (90 microM Al at pH 4.5 for 1 h) of the distal transition zone (DTZ; 1-2 mm from the root tip), which does not contribute significantly to root elongation, inhibited root elongation in the main elongation zone (EZ; 2.5-5 mm from the root tip) to the same extent as treatment of the entire maize (Zea mays) root apex. Application of Al to the EZ had no effect on root elongation. Higher genotypical resistance to Al applied to the entire root apex, and specifically to the DTZ, was expressed by less inhibition of root elongation, Al accumulation, and Al-induced callose formation, primarily in the DTZ. A characteristic pH profile along the surface of the root apex with a maximum of pH 5.3 in the DTZ was demonstrated. Al application induced a substantial flattening of the pH profile moreso in the Al-sensitive than in the Al-resistant cultivar. Application of indole-3-acetic acid to the EZ but not to the meristematic zone significantly alleviated the inhibition of root elongation induced by the application of Al to the DTZ. Basipetal transport of exogenously applied [(3)H]indole-3-acetic acid to the meristematic zone was significantly inhibited by Al application to the DTZ in the Al-sensitive maize cv Lixis. Our results provide evidence that the primary mechanisms of genotypical differences in Al resistance are located within the DTZ, and suggest a signaling pathway in the root apex mediating the Al signal between the DTZ and the EZ through basipetal auxin transport.     

Cell membrane integrity,callose accumulation,and root growth in aluminum-stressed sorghum seedlings

Aluminum stress usually reduces plant root growth due to the accumulation of Al in specific zones of the root apex. The objectives of this study were to determine the localization of Al in the root apex of Sorghum bicolor (L.) Moech. and its effects on membrane integrity, callose accumulation, and root growth in selected cultivars. Seedlings were grown in a nutrient solution containing 0, 27, or 39 μM Al³⁺ for 24, 48, and 120 h. The Al stress significantly reduced root growth, especially after 48 and 120 h of exposure. A higher Al accumulation, determined by fluorescence microscopy after staining with a Morin dye, occurred in the root extension zone of the sensitive cultivar than in the tolerant cultivar. The membrane damage and callose accumulation were also higher in the sensitive than resistant cultivar. It was concluded that the Al stress significantly reduced root growth through the accumulation of Al in the root extension zone, callose accumulation, and impairment of plasma membrane integrity.     

Protective role of mucilage against Al toxicity to root apex of pea (Pisum sativum)

Mucilage can strongly bind Al in the rhizosphere. Although there are still debates about the role of mucilage in protection of the root apex from Al toxicity, we considered that it might be associated with the characteristics of Al adsorption in mucilage. When the mucilage was kept intact, the accumulation of Al and induction of callose in root tips of pea (Pisum sativum) remained lower; thus root elongation was less inhibited than when mucilage was removed under Al exposure in mist culture. Size exclusion chromatography showed both a high and a low molecular weight polysaccharide fraction from root mucilage. Aluminum was predominately detected in high molecular weight polysaccharides, which strongly bound cations. The results indicate that the persistence of mucilage does protect the root apex from Al toxicity by immobilizing Al in high molecular weight polysaccharides.     

Response of the plant root to aluminum stress: Analysis of the inhibition of the root elongation and changes in membrane function

Pea root elongation was strongly inhibited in the presence of a low concentration of Al (5 μM). In Al-treated root, the epidermis was markedly injured and characterized by an irregular layer of cells of the root surface. Approximately 30% of total absorbed Al accumulated in the root tip and Al therein was found to cause the inhibition of whole root elongation. Increasing concentrations of Ca²⁺ effectively ameliorated the inhibition of root elongation by Al and 1 mM of CaCl₂ completely repressed the inhibition of root elongation by 50 μM Al. The ameliorating effect of Ca²⁺ was due to the reduction of Al uptake. H⁺-ATPase and H⁺-PPase activity as well as ATP and PPidependent H⁺ transport activity of vacuolar membrane vesicles prepared from barley roots increased to a similar extent by the treatment with 50 μM AlCl₃. The rate of increase of the amount of H⁺-ATPase and H⁺-PPase was proportional to that of protein content measured by immunoblot analysis with antibodies against the catalytic subunit of the vacuolar H⁺-ATPase and H⁺-PPase of mung bean. The increase of both activities was discussed in relation to the physiological tolerance mechanism of barley root against Al stress.     

Spatial coordination of aluminium uptake, production of reactive oxygen species, callose production and wall rigidification in maize roots

Aluminium (Al) toxicity associated with acid soils represents one of the biggest limitations to crop production worldwide. Although Al specifically inhibits the elongation of root cells, the exact mechanism by which this growth reduction occurs remains controversial. The aim of this study was to investigate the spatial and temporal dynamics of Al migration into roots of maize (Zea mays L.) and the production of the stress response compound callose. Using the Al-specific fluorescent probe morin, we demonstrate the gradual penetration of AI into roots. Al readily accumulates in the root's epidermal and outer cortical cell layers but does not readily penetrate into the inner cortex. After prolonged exposure times (12-24 h), Al had entered all areas of the root apex. The spatial and temporal accumulation of Al within the root is similarly matched by the production of the cell wall polymer callose, which is also highly localized to the epidermis and outer cortical region. Exposure to Al induced the rapid production of reactive oxygen species and induced a significant rigidification of the cell wall. Our results suggest that Al-induced root inhibition in maize occurs by rigidification of the epidermal layers.     

Short-term aluminium-induced changes in barley root tips

The short-term exposure of barley roots to low Al concentration caused significant root growth inhibition and radial swelling of roots. During Al treatment, the radial expansion of root cells occurred in root tissues representing elongation zone and meristem. Both low pH and Al treatments caused significant disruption of cell membranes in swollen roots. In contrast to Evans blue uptake callose formation was observed only at higher Al concentrations and was detected in both swollen and adjacent root areas. Similarly to Al, exogenous short-term application of indole-3-acetic acid, polar transport inhibitor triiodobenzoic acid, ethylene precursor 1-aminocyclopropane-1-carboxylic acid or H₂O₂ evoked root growth inhibition and radial cell expansion in barley root tip too.     

Aluminum-induced 1-->3-beta-D-glucan inhibits cell-to-cell trafficking of molecules through plasmodesmata. A new mechanism of aluminum toxicity in plants

Symplastic intercellular transport in plants is achieved by plasmodesmata (PD). These cytoplasmic channels are well known to interconnect plant cells to facilitate intercellular movement of water, nutrients, and signaling molecules including hormones. However, it is not known whether Al may affect this cell-to-cell transport process, which is a critical feature for roots as organs of nutrient/water uptake. We have microinjected the dye lucifer yellow carbohydrazide into peripheral root cells of an Al-sensitive wheat (Triticum aestivum cv Scout 66) either before or after Al treatment and followed the cell-to-cell dye-coupling through PD. Here we show that the Al-induced root growth inhibition is closely associated with the Al-induced blockage of cell-to-cell dye coupling. Immunofluorescence combined with immuno-electron microscopic techniques using monoclonal antibodies against 1-->3-beta-D-glucan (callose) revealed circumstantial evidence that Al-induced callose deposition at PD may responsible for this blockage of symplastic transport. Use of 2-deoxy-D-glucose, a callose synthesis inhibitor, allowed us to demonstrate that a reduction in callose particles correlated well with the improved dye-coupling and reduced root growth inhibition. While assessing the tissue specificity of this Al effect, comparable responses were obtained from the dye-coupling pattern in tobacco (Nicotiana tabacum) mesophyll cells. Analyses of the Al-induced expression of PD-associated proteins, such as calreticulin and unconventional myosin VIII, showed enhanced fluorescence and co-localizations with callose deposits. These results suggest that Al-signal mediated localized alterations to calcium homeostasis may drive callose formation and PD closure. Our data demonstrate that extracellular Al-induced callose deposition at PD could effectively block symplastic transport and communication in higher plants.     

Differential Al resistance and citrate secretion in barley (<Emphasis Type="Italic">Hordeum vulgare</Emphasis> L.)

While barley ( Hordeum vulgare L.) is the most sensitive species to Al toxicity among small-grain crops, variation in Al resistance between cultivars does exist. We examined the mechanism responsible for differential Al resistance in 21 barley varieties. Citrate was secreted from the roots in response to Al stress. A positive correlation between citrate secretion and Al resistance [(root elongation with Al)/(root elongation without Al)] and a negative correlation between citrate secretion and Al content of root apices, were obtained, suggesting that citrate secretion from the root apices plays an important role in excluding Al and thereby detoxifying Al. The Al-induced secretion of citrate was characterized using an Al-resistant variety (Sigurdkorn) and an Al-sensitive variety (Kearney). In Sigurdkorn, Al-induced secretion of citrate occurred within 20 min, and the secretion did not increase with increasing external Al concentration. The Al-induced citrate secretion ceased at low temperature (6 degrees C) and was inhibited by anion-channel inhibitors. Internal citrate content of root apices was increased by Al exposure in Sigurdkorn, but was not affected in Kearney. The activity of citrate synthase was unaffected by Al in both Al-resistant and Al-sensitive varieties. The secretion rate of organic acid anions from barley was the lowest among wheat, rye and triticale.     

Root growth analysis: An underutilised approach to understanding aluminium rhizotoxicity

Despite numerous published studies, the mechanistic bases for Al rhizotoxicity and for intraspecific differences in Al tolerance remain elusive. Classical methods of growth analysis offer a means to describe quantitatively the onset of root growth inhibition in response to an Al challenge, and any subsequent recovery in growth rates (i.e. acclimation). Such information could help elucidate tolerance mechanisms (i.e. constitutive vs. inducible). Despite this potential utility, however, published data of high accuracy and precision are few, especially with wheat (Triticum aestivum L.). In this paper, a conceptual framework for analysing root growth responses to Al is presented. Specially designed tanks and a computerised video analysis system were used to monitor root elongation in a simple medium (1 mM CaCl₂, pH 4.3) containing varied Al concentrations. Root lengths of five wheat cultivars were measured every 2 to 5 h for 48 h after Al challenge. Growth rates typically exhibited a brief (1–2 h) lag phase prior to a sharp decline, followed by a recovery phase of 12 h duration. At low levels of Al (i.e. sufficient to cause a 10% reduction in net elongation) post-recovery growth rates generally equalled those of control roots, but were significantly reduced at Al concentrations yielding a 40 to 50% reduction in net elongation. All cultivars exhibited similar inhibition-acclimation response patterns and these responses did not explain differences in sensitivity to Al. Nonetheless, solution Al concentrations required to elicit the same growth responses varied by 20-fold across the five cultivars, consistent with the notion that root exclusion of Al is a significant component of differential Al tolerance in wheat.     

铝胁迫下胡枝子根尖胼胝质形成规律及影响因素

采用水培试验,研究了铝胁迫下两个胡枝子品种根尖产生胼胝质的变化规律及影响因素。结果表明,两个品种的根尖铝吸收量与胼胝质形成量呈正比例关系。品种间差异主要是在根尖0—0.5 cm处。敏感品种胼胝质形成量同铝吸收量的变化趋势相一致,而耐性品种则在铝处理6 h时出现一个高峰值后下降。去除铝胁迫后,耐性品种胼胝质形成量并不显著减少。与单独铝处理相比,阴离子通道抑制剂苯甲酰甲醛加铝处理对两个品种胼胝质形成无影响;尼氟灭酸加铝处理抑制敏感品种胼胝质的形成,对耐性品种无影响;蒽-9-羧酸加铝处理显著抑制两个品种的胼胝质形成。另外,抑制剂2-去氧-D-葡萄糖加铝共同处理与单独铝处理相比,敏感品种的胼胝质形成量显著降低,耐性品种无影响。甘露醇对两个品种胼胝质形成的影响无显著差别。镧处理下胼胝质的形成量是耐性品种显著高于敏感品种,铝、镧同时处理胼胝质的形成量最高。敏感品种胼胝质形成处理间无差别。总之,耐性品种在铝胁迫下胼胝质形成与有机酸分泌可能存在一定的协调关系;铝胁迫下胼胝质形成是敏感指标;在一定条件下,特别是有机酸分泌前胼胝质的形成可能具有一定抗性意义;铝诱导胼胝质的形成受多种外界因素(浓度、时间、有机酸分泌,渗透压等)的影响。     

Transgenic Arabidopsis thaliana plants expressing a β‐1,3‐glucanase from sweet sorghum (Sorghum bicolor L.) show reduced callose deposition and increased tolerance to aluminium toxicity

Seventy‐one cultivars of sweet sorghum (Sorghum bicolor L.) were screened for aluminium (Al) tolerance by measuring relative root growth (RRG). Two contrasting cultivars, ROMA (Al tolerant) and POTCHETSTRM (Al sensitive), were selected to study shorter term responses to Al stress. POTCHETSTRM had higher callose synthase activity, lower β‐1,3‐glucanase activity and more callose deposition in the root apices during Al treatment compared with ROMA. We monitored the expression of 12 genes involved in callose synthesis and degradation and found that one of these, SbGlu1 (Sb03g045630.1), which encodes a β‐1,3‐glucanase enzyme, best explained the contrasting deposition of callose in ROMA and POTCHETSTRM during Al treatment. Full‐length cDNAs of SbGlu1 was prepared from ROMA and POTCHETSTRM and expressed in Arabidopsis thaliana using the constitutive cauliflower mosaic virus (CaMV) 35S promoter. Independent transgenic lines displayed significantly greater Al tolerance than wild‐type plants and vector‐only controls. This phenotype was associated with greater total β‐1,3‐glucanase activity, less Al accumulation and reduced callose deposition in the roots. These results suggest that callose production is not just an early indicator of Al stress in plants but likely to be part of the toxicity pathway that leads to the inhibition of root growth.