An intracellular mechanism of aluminum tolerance associated with high antioxidant status in cultured tobacco cells

An aluminum (Al) tolerance mechanism, together with oxidative stress tolerance, was investigated in an Al tolerant cell line (ALT301) and the parental Al sensitive cell line (SL) of tobacco. During Al exposure in a simple calcium solution for 24 h, Al triggered the evolution of a reactive oxygen species (ROS) in SL much higher than ALT301 [Plant Physiol. 128 (2002) 63]. Under the conditions, Al enhanced comparable rates of citrate secretion from both cell lines to the same extent. Al enhanced the gene expression of manganese superoxide dismutase (MnSOD) in both cell lines, but at a significantly higher rate in SL than in ALT301, and also enhanced the enzyme activity of MnSOD in both cell lines to nearly the same level. These results suggest that the extracellular chelation of Al with organic acids and MnSOD is not involved in the mechanism of Al tolerance of ALT301. ALT301 contained ascorbate (ASA) and glutathione (GSH) levels that were higher than SL under normal growth conditions. During 24 h of post-Al treatment culture in growth medium, but not during 24-h Al exposure in a simple Ca(2+) solution, lipid peroxidation was enhanced in SL much higher than in ALT301, and the average SL amounts of ASA and GSH were exhausted compared to ALT301. Pre-loading of ASA prior to Al treatment improved the growth of SL during the post-Al treatment culture. ALT301 also exhibited cross-tolerance to H(2)O(2), Fe(2+) and Cu(2+). Under these oxidant exposures, ALT301 contained lower levels of intracellular H(2)O(2) or lipid peroxides, and maintained higher amounts of ASA and GSH than SL. Taken together, we conclude that the accumulation of Al in cells enhances the peroxidation of lipids exclusively under growing conditions, and that the higher content of ASA and GSH in ALT301 than in SL seems to be in part responsible for the tolerance mechanism of ALT301 to Al by protecting cells from either lipid peroxidation or H(2)O(2) commonly enhanced by Al or other oxidants.     

Impact of the auxin signaling inhibitor p-chlorophenoxyisobutyric acid on short-term Cd-induced hydrogen peroxide production and growth response in barley root tip

Short-term treatment (30min) of barley roots with a low 10μM Cd concentration induced significant H(2)O(2) production in the elongation and differentiation zone of the root tip 3h after treatment. This elevated H(2)O(2) production was accompanied by root growth inhibition and probably invoked root swelling in the elongation zone of the root tip. By contrast, a high 60μM Cd concentration induced robust H(2)O(2) production in the elongation zone of the root tip already 1h after short-term treatment. This robust H(2)O(2) generation caused extensive cell death 6h after short-term treatment. Similarly to low Cd concentration, exogenously applied H(2)O(2) caused marked root growth inhibition, which at lower H(2)O(2) concentration was accompanied by root swelling. The auxin signaling inhibitor p-chlorophenoxyisobutyric acid effectively inhibited 10μM Cd-induced root growth inhibition, H(2)O(2) production and root swelling, but was ineffective in the alleviation of 60μM Cd-induced root growth inhibition and H(2)O(2) production. Our results demonstrated that Cd-induced mild oxidative stress caused root growth inhibition, likely trough the rapid reorientation of cell growth in which a crucial role was played by IAA signaling in the root tip. Strong oxidative stress induced by high Cd concentration caused extensive cell death in the elongation zone of the root tip, resulting in the cessation of root growth or even in root death.     

Dependence of the Extent and Direction of Average Stomatal Response in Zea mays L. and Phaseolus vulgaris L. on the Frequency of Fluctuations in Environmental Stimuli

Three-day-old seedlings of an Al-sensitive (Neepawa) and an Al-resistant (PT741) cultivar of Triticum aestivum were subjected to Al concentrations ranging from 0 to 100 [mu]M for 72 h. At 25 [mu]M Al, growth of roots was inhibited by 57% in the Al-sensitive cultivar, whereas root growth in the Al-resistant cultivar was unaffected. A concentration of 100 [mu]M Al was required to inhibit root growth of the Al-resistant cultivar by 50% and resulted in almost total inhibition of root growth in the sensitive cultivar. Cytoplasmic and microsomal membrane fractions were isolated from root tips (first 5 mm) and the adjacent 2-cm region of roots of both cultivars. When root cytoplasmic proteins were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, no changes in polypeptide patterns were observed in response to Al stress. Analysis of microsomal membrane proteins revealed a band with an apparent molecular mass of 51 kD, which showed significant accumulation in the resistant cultivar following Al exposure. Two-dimensional gel analysis revealed that this band comprises two polypeptides, each of which is induced by exposure to Al. The response of the 51-kD band to a variety of experimental conditions was characterized to determine whether its pattern of accumulation was consistent with a possible role in Al resistance. Accumulation was significantly greater in root tips when compared to the rest of the root. When seedlings were subjected to Al concentrations ranging from 0 to 150 [mu]M, the proteins were evident at 25 [mu]M and were fully accumulated at 100 [mu]M. Time-course studies from 0 to 96 h indicated that full accumulation of the 51-kD band occurred within 24 h of initiation of Al stress. With subsequent removal of stress, the polypeptides gradually disappeared and were no longer visible after 72 h. When protein synthesis was inhibited by cycloheximide, the 51-kD band disappeared even when seedlings were maintained in Al-containing media. Other metals, including Cu, Zn, and Mn, failed to induce this band, and Cd and Ni resulted in its partial accumulation. These results indicate that synthesis of the 51-kD microsomal membrane proteins is specifically induced and maintained during Al stress in the Al-resistant cultivar, PT741.     

Preliminary results from a microscopic examination on the effects of aluminium on the root tips of wheat

Root tips from aluminium (Al) tolerant (Waalt) and Al sensitive (Warigal) wheat (Triticum aestivum (L). Thell.) cultivars exposed to low concentrations of Al (10 M) for 10, 24 and 72 hours were examined under the light and electron microscope. After fixing and embedding, longitudinal and transverse thin and ultrathin sections were cut. There was no evidence of Al damage to the root tips of the Al tolerant cultivar under both the light and electron microscope. For the Al sensitive cultivar, Al had no observable effect on the root tips 10 hours after Al addition when examined under the light microscope. When examined under an electron microscope, electron dense globular deposits were observed between the cell wall and cell membrane of the epidermal cells. There was not obvious damage to the cell cytoplasm. Two or 3 days after Al addition, light microscopy showed that the cells in the root tips had become swollen and extensively vacuolated. The tissues appeared disorganised and degenerate, particularly in the epidermis and outer cortical cells. The electron microscope also revealed a thickening of the cell wall. The cell wall was broken down, particularly in the epidermis in the region 4–6 mm from the root tip. The tissue in the meristematic area was largely intact.     

Isolation and characterization of wheat aluminum-regulated genes: possible involvement of aluminum as a pathogenesis response elicitor

Using differential screening of a root tip cDNA library prepared from an Al-tolerant wheat cultivar (Triticum aestivum L. cv. Atlas-66) exposed to Al, we have isolated and characterized several wheat aluminum-regulated (War) cDNAs. Sequence comparison revealed that genes up-regulated by Al correspond to peroxidase (war4.2), cysteine proteinase (war5.2), phenylalanine-ammonia lyase (war7.2), and oxalate oxidase (war13.2). Two wheat cultivars that differ in their level of tolerance (cv. Atlas-66: tolerant, and cv. Fredrick: sensitive) were used to evaluate the relationship between the accumulation of War mRNAs and Al toxicity, as measured by root growth inhibition (RGI). The mRNA accumulation was modulated to similar levels in both cultivars compared at equivalent RGIs. This indicates that War mRNA accumulation is associated with the toxicity of Al rather than with the cultivar's tolerance. It appears that most of the genes found to be up-regulated by Al share homologies with genes induced by pathogens. This suggests that Al may act as an elicitor of a pathogenesis-related transduction pathway. The potential functions of the up-regulated war genes in cell wall strengthening and Al trapping are discussed. Received: 24 October 1997 / Accepted: 21 January 1998     

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.     

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.     

Interaction between Aluminum Toxicity and Calcium Uptake at the Root Apex in Near-Isogenic Lines of Wheat (Triticum aestivum L.) Differing in Aluminum Tolerance

Aluminum (Al) is toxic to plants at pH < 5.0 and can begin to inhibit root growth within 3 h in solution experiments. The mechanism by which this occurs is unclear. Disruption of calcium (Ca) uptake by Al has long been considered a possible cause of toxicity, and recent work with wheat (Triticum aestivum L. Thell) has demonstrated that Ca uptake at the root apex in an Al-sensitive cultivar (Scout 66) was inhibited more than in a tolerant cultivar (Atlas 66) (J.W. Huang, J.E. Shaff, D.L. Grunes, L.V. Kochian [1992] Plant Physiol 98: 230-237). We investigated this interaction further in wheat by measuring root growth and Ca uptake in three separate pairs of near-isogenic lines within which plants exhibit differential sensitivity to Al. The vibrating calcium-selective microelectrode technique was used to estimate net Ca uptake at the root apex of 6-d-old seedlings. Following the addition of 20 or 50 [mu]M AlCl3, exchange of Ca for Al in the root apoplasm caused a net Ca efflux from the root for up to 10 min. After 40 min of exposure to 50 [mu]M Al, cell wall exchange had ceased, and Ca uptake in the Al-sensitive plants of the near-isogenic lines was inhibited, whereas in the tolerant plants it was either unaffected or stimulated. This provides a general correlation between the inhibition of growth by Al and the reduction in Ca influx and adds some support to the hypothesis that a Ca/Al interaction may be involved in the primary mechanism of Al toxicity in roots. In some treatments, however, Al was able to inhibit root growth significantly without affecting net Ca influx. This suggests that the correlation between inhibition of Ca uptake and the reduction in root growth may not be a mechanistic association. The inhibition of Ca uptake by Al is discussed, and we speculate about possible mechanisms of tolerance.     

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.     

Differences in the metabolic responses of root tips of wheat and rye to aluminium stress

The effects of aluminium (Al) ions on the metabolism of root apical meristems were examined in 4-day-old seedlings of two cereals which differed in their tolerance to Al: wheat cv. Grana (Al-sensitive) and rye cv. Dakowskie Nowe (Al tolerant). During a 24 h incubation period in nutrient solutions containing 0.15 mM and 1.0 mM of Al for wheat and rye, respectively, the activity of first two enzymes in the pentose phosphate pathway (G-6-PDH and 6-PGDH) decreased in the sensitive cultivar. In the tolerant cultivar activities of these enzymes increased initially, then decreased slightly, and were at control levels after 24 h. In the Al-sensitive wheat cultivar a 50% reduction in the activity of 6-phosphogluconate dehydrogenase was observed in the presence of Al. Changes in enzyme activity were accompanied by changes in levels of G-6-P- the initial substrate in the pentose phosphate pathway. When wheat was exposed for 16 h to a nutrient solution containing aluminium, a 90% reduction in G-6-P concentration was observed. In the Al-tolerant rye cultivar, an increase and subsequently a slight decrease in G-6-P concentration was detected, and after 16 h of Al-stress the concentration of this substrate was still higher than in control plants. This dramatic Al-induced decrease in G-6-P concentration in the Al-sensitive wheat cultivar was associated with a decrease in both the concentration of glucose in the root tips as well as the activity of hexokinase, an enzyme which is responsible for phosphorylation of glucose to G-6-P. However, in the Al-tolerant rye cultivar, the activity of this enzyme remained at the level of control plants during Al-treatment, and the decrease in the concentration of glucose occurred at a much slower rate than in wheat. These results suggest that aluminium ions change cellular metabolism of both wheat and rye root tips. In the Al-sensitive wheat cultivar, irreversible disturbances induced by low doses of Al in the nutrient solution appear very quickly, whereas in the Al-tolerant rye cultivar, cellular metabolism, even under severe stress conditions, is maintained for a long time at a level which allows for root elongation to continue.Abbreviations G-6-PDH glucose-6-phosphate dehydrogenase - 6-PGDH 6-phosphogluconate dehydrogenase - G-6-P glucose-6-phosphate - TEA triethanolamine     

Aluminum-induced cell death in root-tip cells of barley

Aluminum-induced cell death was investigated in root-tip cells of barley (Hordeum vulgare). The growth of roots in 0.1-50 mM Al treatments was inhibited after 8 h treatments, and could not be recovered after 24 h recovery culture without Al. Viable detection with fluorescein diacetate-propidium iodide (FDA-PI) staining shows that most of the root-tip cells have lost viability. These results suggest that the irreversible inhibition of root growth after 8 h Al treatments or 24 h recovery culture is mainly caused by cell death. DNA ladders occurred in root tips only after 8 h Al treatments (0.1-1.0 mM), but no apoptotic bodies in root tips were observed. Thus, the cell death caused by Al stress is likely to be Al-induced programmed cell death (PCD). The reactive oxygen species (ROS) in root-tip cells measured by ultraweak luminescence indicated that the oxidation status in root-tip cells basically ceased after exposure to 10-50 mM Al for 24 h, but was very violent in the root-tip cells treated with 0.1-1.0 mM for 24 h. Exposure to 0.1-1.0 mM Al for 3-12 h led to ROS burst. Therefore, our results suggest that 0.1-1.0 mM Al treatments for 8 h induce cell death (Al-induced PCD) possibly via a ROS-activated signal transduction pathway, whereas 10-50 mM Al treatments may cause necrosis in the root-tip cells. These results have an important role for further studies on the mechanism of Al toxicity in plants.     

Effects of aluminium on root growth and apical root cells in rice (<Emphasis Type="Italic">Oryza sativa</Emphasis> L.) cultivars. Reliability of screening tests to detect Al resistance at the seedling stage

The effects of Al₂(SO₄)₃·18H₂O on growth of root and apical root cells were studied in seedlings of rice cultivars differing in Al resistance including I Kong Pao and Aiwu (Al-sensitive) and IRAT 112 and IR6023-10-1-1 (Al-resistant). Inhibition of root growth was a typical effect of Al, and the extent of the inhibition depended on both cultivar and Al concentration. Al impaired the activity of the root meristem as indicated by reductions in its size, mitotic activity and the diameter of the meristematic cell nucleoli. Cell size in the elongation zone of the root was also reduced by Al. The reliability of the haematoxylin staining method to classify rice cultivars according to their Al-sensitivity failed to discriminate the Al-resistant IR6023-10-1-1 cultivar from the two sensitive cultivars. The results are discussed in relation to the Al resistance mechanisms operating in rice.     

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.     

Aluminum Tolerance in Wheat (Triticum aestivum L.) (I. Uptake and Distribution of Aluminum in Root Apices)

We investigated the uptake and distribution of Al in root apices of near-isogenic wheat (Triticum aestivum L.) lines differing in Al tolerance at a single locus (Alt1: aluminum tolerance). Seedlings were grown in nutrient solution that contained 100 [mu]M Al, and the roots were subsequently stained with hematoxylin, a compound that binds Al in vitro to form a colored complex. Root apices of Al-sensitive genotypes stained after short exposures to Al (10 min and 1 h), whereas apices of Al-tolerant seedlings showed less intense staining after equivalent exposures. Differential staining preceded differences observed in either root elongation or total Al concentrations of root apices (terminal 2-3 mm of root). After 4 h of exposure to 100 [mu]M Al in nutrient solution, Al-sensitive genotypes accumulated more total Al in root apices than Al-tolerant genotypes, and the differences became more marked with time. Analysis of freeze-dried root apices by x-ray microanalysis showed that Al entered root apices of Al-sensitive plants and accumulated in the epidermal layer and in the cortical layer immediately below the epidermis. Long-term exposure of sensitive apices to Al (24 h) resulted in a distribution of Al coinciding with the absence of K. Quantitation of Al in the cortical layer showed that sensitive apices accumulated 5- to 10-fold more Al than tolerant apices exposed to Al solutions for equivalent times. These data are consistent with the hypothesis that Alt1 encodes a mechanism that excludes Al from root apices.     

Tolerance to ion toxicities enhances wheat (Triticum aestivum L.) grain yield in waterlogged acidic soils

Aims

The high concentrations of Mn, Fe and Al in acid soils during waterlogging impair root and shoot growth more severely in intolerant than tolerant wheat genotypes. This study aims to establish whether this difference in vegetative growth and survival during waterlogging (1) is verifiable across a range of tolerant/intolerant genotypes and acid soils, and (2) results in improved recovery after cessation of waterlogging and enhanced grain yield.

Methods

Wheat genotypes contrasting in their tolerance to ion toxicities were grown in four acid soils until 63DAS and maturity, with a 42-day waterlogging treatment imposed at 21 DAS.

Results

The shoot Al, Mn and Fe concentrations increased by up to 5-, 3- and 9-fold respectively due to waterlogging in various soils. Compared to the intolerant lines, Al-, Mn- and Fe-tolerant genotypes maintained a relatively lower increase in shoot concentrations of Al (79 vs. 117%), Mn (90 vs. 101%) and Fe (171 vs. 252%) and demonstrated better waterlogging tolerance at the vegetative stage expressed in relative root (38% vs. 25%) and shoot (62% vs. 52%) growth. After cessation of waterlogging and the continued growth to maturity, tolerant genotypes maintained a relatively lower plant concentration of Al, Mn and Fe, but produced a higher above-ground biomass (74% vs. 56%) and most importantly demonstrated improved waterlogging tolerance (a relative grain yield of 78% vs. 54%) compared to intolerant genotypes. Maturity following waterlogging stress was delayed less in tolerant than intolerant genotypes (114 vs. 124%, respectively), which would reduce the potential yield loss where post-anthesis coincides with drought.

Conclusions

The results confirm the validity of a novel approach of enhancing waterlogging tolerance of wheat genotypes grown in acid soil via increased tolerance to ion toxicities.     

Aluminum inhibits the H(+)-ATPase activity by permanently altering the plasma membrane surface potentials in squash roots

Although aluminum (AL) toxicity has been widely studied in monocotyledonous crop plants, the mechanism of Al impact on economically important dicotyledonous plants is poorly understood. Here, we report the spatial pattern of Al-induced root growth inhibition, which is closely associated with inhibition of H(+)-ATPase activity coupled with decreased surface negativity of plasma membrane (PM) vesicles isolated from apical 5-mm root segments of squash (Cucurbita pepo L. cv Tetsukabuto) plants. High-sensitivity growth measurements indicated that the central elongation zone, located 2 to 4 mm from the tip, was preferentially inhibited where high Al accumulation was found. The highest positive shifts (depolarization) in zeta potential of the isolated PM vesicles from 0- to 5-mm regions of Al-treated roots were corresponded to pronounced inhibition of H(+)-ATPase activity. The depolarization of PM vesicles isolated from Al-treated roots in response to added Al in vitro was less than that of control roots, suggesting, particularly in the first 5-mm root apex, a tight Al binding to PM target sites or irreversible alteration of PM properties upon Al treatment to intact plants. In line with these data, immunolocalization of H(+)-ATPase revealed decreases in tissue-specific H(+)-ATPase in the epidermal and cortex cells (2--3 mm from tip) following Al treatments. Our report provides the first circumstantial evidence for a zone-specific depolarization of PM surface potential coupled with inhibition of H(+)-ATPase activity. These effects may indicate a direct Al interaction with H(+)-ATPase from the cytoplasmic side of the PM.     

Progressive apoptotic cell death triggered by transient oxidative insult in H9c2 rat ventricular cells: a novel pattern of apoptosis and the mechanisms

Many pathophysiological processes are associated with oxidative stress and progressive cell death. Oxidative stress is an apoptotic inducer that is known to cause rapid cell death. Here we show that a brief oxidative insult (5-min exposure to 400 microM H(2)O(2)), although it did not kill H9c2 rat ventricular cells during the exposure, triggered an intracellular death cascade leading to delayed time-dependent cell death starting from 1 h after the insult had been withdrawn, and this post-H(2)O(2) cell death cumulated gradually, reaching a maximum level 8 h after H(2)O(2) withdrawal. By comparison, sustained exposure to H(2)O(2) caused complete cell death within a narrow time frame (2 h). The time-dependent post-H(2)O(2) cell death was typical of apoptosis, both morphologically (cell shrinkage and nuclear condensation) and biochemically (DNA fragmentation, extracellular exposure of phosphatidylserines, and caspase-3 activation). A dichlorofluorescein fluorescent signal showed a time-dependent endogenous increase of reactive oxygen species (ROS) production, which was almost abolished by inhibition of the mitochondrial electron transport chain. Application of antioxidants (vitamin E or DTT) before H(2)O(2) addition or after H(2)O(2) withdrawal prevented the H(2)O(2)-triggered progressive ROS production and apoptosis. Sequential appearance of events associated with activation of the mitochondrial death pathway was found, including progressive dissipation of mitochondrial membrane potential, cytochrome c release, and late activation of caspase-3. In conclusion, transient oxidative stress triggers an intrinsic program leading to self-sustained apoptosis in H9c2 cells via cumulative production of mitochondrial ROS and subsequent activation of the mitochondrial death pathway. This pattern of apoptosis may contribute to the progressive and long-lasting cell loss in some degenerative diseases.     

Tornado1 and tornado2 are required for the specification of radial and circumferential pattern in the Arabidopsis root

The cell layers of the Arabidopsis primary root are arranged in a simple radial pattern. The outermost layer is the lateral root cap and lies outside the epidermis that surrounds the ground tissue. The files of epidermal and lateral root cap cells converge on a ring of initials (lateral root cap/epidermis initial) from which the epidermal and lateral root cap tissues of the seedling are derived, once root growth is initiated after germination. Each initial gives rise to a clone of epidermal cells and a clone of lateral root cap cells. These initial divisions in the epidermal/lateral root cap initial are defective in tornado1 (trn1) and trn2 plants indicating a requirement for TRN1 and TRN2 for initial cell function. Furthermore, lateral root cap cells develop in the epidermal position in trn1 and trn2 roots indicating that TRN1 and TRN2 are required for the maintenance of the radial pattern of cell specification in the root. The death of these ectopic lateral root cap cells in the elongation zone (where lateral root cap cells normally die) results in the development of gaps in the epidermis. These observations indicate that TRN1 and TRN2 are required to maintain the distinction between the lateral root cap and epidermis and suggest that lateral root cap fate is the default state. It also suggests that TRN1 and TRN2 repress lateral root cap fate in cells in the epidermal location. Furthermore, the position-dependent pattern of root hair and non-root hair cell differentiation in the epidermis is defective in trn1 and trn2 mutants. Together these results indicate that TRN1 and TRN2 are required for the maintenance of both the radial pattern of tissue differentiation in the root and for the subsequent circumferential pattern within the epidermis.     

Patterns of Root Respiration Associated with the Induction of Aluminium Tolerance in Phaseolus vulgaris L.

Aluminium (Al) tolerance in an Al-tolerant cultivar of Phaseolusvulgaris L. (‘Dade’) was found to be an inducibletrait. Upon exposure to 10 µM Al, the rate of root elongationwas inhibited in comparison to controls. During the following72 h, the rate of elongation returned to levels comparable tocontrols. In contrast, root elongation of an Al-sensitive cultivar(‘Romano’) did not recover after exposure to Al.In Dade, the resumption of root elongation following exposureto Al was accompanied by increased rates of root respiration,whereas respiration rates slowly declined over the 72 h treatmentperiod in Romano. When partitioned into growth and maintenanceexpenditures, a larger proportion of root respiration of Dadeexposed to Al was allocated to maintenance processes, potentiallyreflecting diversion of energy to metabolic pathways that offsetthe adverse effects of Al toxicity. Romano did not show sucha pattern and respiration associated with both growth and maintenancewas reduced after exposure to Al. Root and shoot growth of bothcultivars were also measured to determine the effects of long-term(21 d) exposure to 10 µM Al. Dade plants exposed to Alexhibited enhanced growth in comparison to controls, whereasRomano plants were characterized by reduced shoot and root growth.Modelling the time-course of root respiration and measuringthe long-term growth responses to Al is a valuable method ofelucidating respiratory costs of stress tolerance. Key words: Aluminium, differential tolerance, maintenance respiration, Phaseolus vulgaris, root respiration     

Disorganized distribution of homogalacturonan epitopes in cell walls as one possible mechanism for aluminium-induced root growth inhibition in maize

Background and Aims

Aluminium (Al) toxicity is one of the most severe limitations to crop production in acid soils. Inhibition of root elongation is the primary symptom of Al toxicity. However, the underlying basis of the process is unclear. Considering the multiple physiological and biochemical functions of pectin in plants, possible involvement of homogalacturonan (HG), one of the pectic polysaccharide domains, was examined in connection with root growth inhibition induced by Al.

Methods

An immunolabelling technique with antibodies specific to HG epitopes (JIM5, unesterified residues flanked by methylesterifed residues; JIM7, methyl-esterified residues flanked by unesterified residues) was used to visualize the distribution of different types of HG in cell walls of root apices of two maize cultivars differing in Al resistance.

Key Results

In the absence of Al, the JIM5 epitope was present around the cell wall with higher fluorescence intensity at cell corners lining the intercellular spaces, and the JIM7 epitope was present throughout the cell wall. However, treatment with 50 µm Al for 3 h produced 10 % root growth inhibition in both cultivars and caused the disappearance of fluorescence in the middle lamella of both epitopes. Prolonged Al treatment (24 h) with 50 % root growth inhibition in ‘B73’, an Al-sensitive cultivar, resulted in faint and irregular distribution of both epitopes. In ‘Nongda3138’, an Al-resistant cultivar, the distribution of HG epitopes was also restricted to the lining of intercellular spaces when a 50 % inhibition to root growth was induced by Al (100 µm Al, 9 h). Altered distribution of both epitopes was also observed when of roots were exposed to 50 µm LaCl₃ for 24 h, resulting in 40 % inhibition of root growth.

Conclusions

Changes in HG distribution and root growth inhibition were highly correlated, indicating that Al-induced perturbed distribution of HG epitopes is possibly involved in Al-induced inhibition of root growth in maize.Key words: Al toxicity, cell wall, homogalacturnonan, immunofluorescence, methylesterification, pectin