Caspase-like proteases regulate aluminum-induced programmed cell death in peanut

Recent evidence has proved that caspase protease activities are detected in both mammals and plants during programmed cell death (PCD). The characteristics and functions of caspase-like proteases play important roles in understanding the mechanisms of PCD in plants. In this work, we report firstly the involvement of caspase-like protease activities and effects in aluminum (Al) stress in two contrasting peanut genotypes. Caspase-like activities in the root tip cells of ‘Zhonghua 2’ (Al-sensitive) and ‘99-1507’ (Al-tolerant) were detected using synthetic caspase substrates during Al-triggered PCD. Caspase-1-, -2-, -3-, -4-, -5-, -6-, -8- and -9-like proteases were found in peanut root tip cells. VDQQDase (caspase-2-like) and WEHD (caspase-5-like) were the first detected in the plants, and almost all of the caspase-like proteases were activated during Al-induced PCD, especially caspase-3-like and caspase-1-like, which was higher in ‘Zhonghua 2’ than in ‘99-1507’. The highest activity levels of caspase-3- and caspase-1-like proteases occurred 8 and 4 h after 100 µM Al treatment, respectively. Compared with 100 µM AlCl₃ treatment alone, specific caspase-3 protease inhibitor Ac-DEVD-CHO inhibited the increase of caspase-3-like protease activity, Al content, Hsr203j expression, cell death and DNA fragmentation, and the decrease in root growth induced by 100 µM AlCl₃ treatment, but it was more obvious in ‘Zhonghua 2’ than in ‘99-1507’. In conclusion, there were different caspase-like proteases in root tips of peanut, and caspase-3-like protease was a crucial executioner in Al-induced PCD. Its effects in the ‘Zhonghua 2’ genotype were higher than in ‘99-1507’. An improved model of the mechanism of Al-induced PCD and Al tolerance differences in different genotypes is proposed.     

Mechanistic study of mitochondria-dependent programmed cell death induced by aluminium phytotoxicity using fluorescence techniques

Recent studies have suggested that aluminium (Al) induces programmed cell death (PCD) in plants. To investigate possible mechanisms, fluorescence techniques were used to monitor the behaviour of mitochondria in vivo, as well as the activation of caspase-3-like activity during protoplast PCD induced by Al. A quick burst of mitochondrial reactive oxygen species (ROS) was detected in Al-treated protoplasts. The mitochondrial swelling and mitochondrial transmembrane potential (MTP) loss occurred prior to cell death. Pre-incubation with ascorbic acid (AsA, antioxidant molecule) retarded mitochondrial swelling and MTP loss. The real-time detection of caspase-3-like activation was achieved by measuring the degree of fluorescence resonance energy transfer (FRET). At 30 min after exposure to Al, caspase-3-like protease activation, indicated by the decrease in the FRET ratio, occurred, taking about 1 h to reach completion in single living protoplasts. The mitochondrial permeability transition pore (MPTP) inhibitor, cyclosporine (CsA) gave significant protection against MTP loss and subsequent caspase-3-like activation. Our data also showed that Al-induced mitochondrial ROS possibly originated from complex I and III damage in the respiratory chain through the interaction between Al and iron-sulphur (Fe-S) protein. Alternative oxidase (AOX), the unique respiratory terminal oxidase in plants, was demonstrated to play protective roles in Al-induced protoplast death. Our results showed that mitochondrial swelling and MTP loss, as well as the generation of mitochondrial ROS play important roles in Al-induced caspase-3-like activation and PCD, which provided new insight into the signalling cascades that modulate Al phytotoxicity mechanism.     

Ced-9 inhibits Al-induced programmed cell death and promotes Al tolerance in tobacco

Our previous data showed that apoptotic suppressors inhibit aluminum (Al)-induced programmed cell death (PCD) and promote Al tolerance in yeast cells, however, very little is known about the underlying mechanisms, especially in plants. Here, we show that the Caenorhabditis elegans apoptotic suppressor Ced-9, a Bcl-2 homologue, inhibited both the Al-induced PCD and Al-induced activity of caspase-like vacuolar processing enzyme (VPE), a crucial executioner of PCD, in tobacco. Furthermore, we show that Ced-9 significantly alleviated Al inhibition of root elongation, decreased Al accumulation in the root tip and greatly inhibited Al-induced gene expression in early response to Al, leading to enhancing the tolerance of tobacco plants to Al toxicity. Our data suggest that Ced-9 promotes Al tolerance in plants via inhibition of Al-induced PCD, indicating that conserved negative regulators of PCD are involved in integrated regulation of cell survival and Al-induced PCD by an unidentified mechanism.     

Ultrastructural investigations and EDX-analyses of Al-treated oat (Avena sativa) roots

Seedlings of Avena sativa were precultivated hydroponically at pH 4.1 and subsequently treated with solutions containing 0 or 400 µM Al. Electron microscopic and X-ray microanalytic (EDXA) investigations were carried out on root tips after 1 to 10 days of treatment. Root growth and mitotic activity decreased rapidly upon application of Al. The meristematic tissues of Al-treated roots showed enhanced vacuolation. The cells, however, remained intact after a longer period of Al-treatment and no alterations in ultrastructure (for example of the nucleus) were visible. The EDX analyses of bulk frozen hydrated tissues showed that Al was predominantly localised in the walls of the outermost cortex cells. One day after onset of Al-stress no intracellular Al was detectable. Even after 10 days with continuous Al-stress, only small amounts of the absorbed Al were localised within the cells. These results suggest that the plasma membrane is a very effective barrier for Al in oats. It is improbable that impairments of cytoplasmatic functions are primary effects of Al-intoxication.     

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.     

Sorting out the role of nitric oxide in cadmium-induced Arabidopsis thaliana programmed cell death

As a vital cell-signaling molecule, nitric oxide (NO) has been reported to regulate toxic metal responses in plants. Our recent report has suggested that caspase-3-like protease activation was detected in Arabidopsis (Arabidopsis thaliana) after Cd²⁺ treatment. NO contributed caspase-3-like protease activation in Cd²⁺ induced Arabidopsis thaliana programmed cell death (PCD), which was mediated by MPK6. It was first shown that NO promotes Cd²⁺-induced Arabidopsis PCD by promoting MPK6-mediated caspase-3-like activation. Our study contributed to the understanding of NO signaling pathway in Cd²⁺-induced Arabidopsis thaliana PCD. Although several studies have revealed that NO regulates plant PCD, compared with the study of signaling pathways involved in animal cell apoptosis, the mechanism of NO function still remains elusive and the molecular mechanisms of MAPK are far from clear in Cd²⁺-induced PCD. By using the fluorescence techniques and the Arabidopsis seedlings as the reference model, the subsequent researches have been performed to obtain comprehensive understanding of Cd²⁺-induced plant PCD.     

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.     

Aluminium Toxicity in Roots: An Investigation of Spatial Sensitivity and the Role of the Root Cap

The primary symptom of aluminium (Al) toxicity in higher plantsis inhibition of root growth. In this study, we investigatedthe spatial sensitivity of maize (Zea mays L.) roots to Al.A divided-chamber technique indicated that only exposure ofthe terminal 10 to 15 mm of the root to Al resulted in inhibitionof growth. Application of Al to all but this apical region ofthe root had little or no effect on growth for 24 h and causedminimal damage to the root tissue. Small agar blocks infusedwith Al were then applied to discrete areas of the apex of maizeroots to determine which section (root cap, meristem or elongationzone) was more important to Al-induced inhibition of growth.The terminal 20 to 30 mm of root (root cap and meristem) mustbe exposed to Al for inhibition. Application of Al to the 30mm of root proximal to this terminal zone (elongation zone)resulted in damage to the root tissue but no significant inhibitionof growth. Therefore, the visible injuries incurred by rootsduring Al-stress are not associated directly with the inhibitionof root growth. Furthermore, removal of the root cap had noeffect on the Al-induced inhibition of root growth in solutionexperiments and argues against the root cap providing protectionfrom Al stress or serving an essential role in the mechanismof toxicity. We suggest that the meristem is the primary siteof Al-toxicity. Key words: Aluminium, toxicity, root growth, root cap     

Overexpression of <Emphasis Type="Italic">Citrus junos</Emphasis> mitochondrial citrate synthase gene in <Emphasis Type="Italic">Nicotiana benthamiana</Emphasis> confers aluminum tolerance

Aluminum (Al) toxicity is one of the major factors that limit plant growth in acid soils. Al-induced release of organic acids into rhizosphere from the root apex has been identified as a major Al-tolerance mechanism in many plant species. In this study, Al tolerance of Yuzu (Citrus Junos Sieb. ex Tanaka) was tested on the basis of root elongation and the results demonstrated that Yuzu was Al tolerant compared with other plant species. Exposure to Al triggered the exudation of citrate from the Yuzu root. Thus, the mechanism of Al tolerance in Yuzu involved an Al-inducible increase in citrate release. Aluminum also elicited an increase of citrate content and increased the expression level of mitochondrial citrate synthase (CjCS) gene and enzyme activity in Yuzu. The CjCS gene was cloned from Yuzu and overexpressed in Nicotiana benthamiana using Agrobacterium tumefaciens-mediated methods. Increased expression level of the CjCS gene and enhanced enzyme activity were observed in transgenic plants compared with the wild-type plants. Root growth experiments showed that transgenic plants have enhanced levels of Al tolerance. The transgenic Nicotiana plants showed increased levels of citrate in roots compared to wild-type plants. The exudation of citrate from roots of the transgenic plants significantly increased when exposed to Al. The results with transgenic plants suggest that overexpression of mitochondrial CS can be a useful tool to achieve Al tolerance.     

Root phosphate exudation and pH shift in the rhizosphere are not responsible for aluminum resistance in rice

A series of hydroponic experiments and an agar culture experiment were carried out to investigate aluminum (Al) accumulation and translocation in two rice (Oryza sativa L.) cultivars (Kasalath and Koshihikari) that differ in Al resistance. Al-resistance mechanisms, including Pi exudation under Al stress and pH shifts in the rhizosphere, were also studied. Al content in rice shoots was 41 mg kg⁻¹ on average and did not differ between the two cultivars, which demonstrated that the rice cultivars were not Al accumulators. The majority of Al (95–97%) accumulated in roots. Al content in roots in the resistant cultivar (Koshihikari) was lower than that in the sensitive cultivar (Kasalath), which indicated that Al-exclusion mechanisms were mainly acting in rice. However, the rate of Pi exudation from the whole root or root tips was very low in both cultivars and was not significantly influenced by Al exposure, and thus seemed not to be the main Al-resistance mechanism. On the other hand, experiments with pH-buffered solution and color changes following culture in agar medium containing bromocresol purple revealed that the Al-induced pH increase could not explain the high Al resistance of rice. In addition, the Al content in shoots of Koshihikari was lower after the formation of iron plaque on the root surface, whereas that of Kasalath was not lower. These results suggested that rice roots cell wall components or root surfaces such as iron plaque, rather than pH changes and/or root exudates including organic acids and phosphate, play important roles in Al resistance in rice.     

The dual role of a yeast metacaspase: What doesn't kill you makes you stronger

Recent reports suggest that the yeast Saccharomyces cerevisiae caspase‐related metacaspase, Mca1, is required for cell‐autonomous cytoprotective functions that slow cellular aging. Because the Mca1 protease has previously been suggested to be responsible for programmed cell death (PCD) upon stress and aging, these reports raise the question of how the opposing roles of Mca1 as a protector and executioner are regulated. One reconciling perspective could be that executioner activation may be restricted to situations where the death of part of the population would be beneficial, for example during colony growth or adaptation into specialized survival forms. Another possibility is that metacaspases primarily harbor beneficial functions and that the increased survival observed upon metacaspase removal is due to compensatory responses. Herein, we summarize data on the role of Mca1 in cell death and survival and approach the question of how a metacaspase involved in protein quality control may act as killer protein.     

Elevated oxalate oxidase activity is correlated with Al-induced plasma membrane injury and root growth inhibition in young barley roots

In order to characterise the possible mechanisms involved in Al toxicity some functional characteristics were analysed in young barley (Hordeum vulgare L.) seedlings cultivated between moistened filter paper. Transfer of germinated barley seeds into hydroponic culture system caused significant stress, which was manifested by root-growth inhibition and elevated Evans blue uptake of root tips. Hydroponics caused stress unabled the analysis of Al-induced stress in the young barley roots during the first day of cultivation. Several (3–4) days are required for adaptation of barley seedlings to hydroponics in spite of strong aeration of the medium. Using filter paper compared to cultivation in solution application of much higher Al concentrations were required to inhibit root growth. Al-induced root growth inhibition, Al uptake, damage of plasma-membrane (PM) permeability of root cells, as well as elevated oxalate oxidase - OxO (EC 1.2.3.4) activity were significantly correlated. While 1 mM Al concentration had no effect on barley roots growing on filter paper, 5 to 100 mM Al concentration inhibited root growth, enhanced cell death and induced oxalate oxidase activity with increasing intensity. The time course analysis of OxO gene expression and OxO activity showed that 10 mM Al increased OxO activity as soon as 3 h after exposure of roots to Al reaching its maximum at about 18 h after Al application. These results indicate that expression of OxO is activated very early after exposure of barley to Al, suggesting its role in oxidative stress and subsequent cell death caused by Al toxicity in plants.     

Proteasome function is required for activation of programmed cell death in heat shocked tobacco Bright-Yellow 2 cells

To find out whether and how proteasome is involved in plant programmed cell death (PCD) we measured proteasome function in tobacco cells undergoing PCD as a result of heat shock (HS-PCD). Reactive oxygen species (ROS) production, cytochrome c levels and caspase-3-like protease activation were also measured in the absence or presence of MG132, a proteasome inhibitor. We show that proteasome activation occurs in early phase of HS-PCD upstream of the caspase-like proteases activation; moreover inhibition of proteasome function by MG132 results in prevention of PCD perhaps due to the prevention of ROS production, cytochrome c release and caspase-3-like protease activation.     

Aluminum distribution and reactive oxygen species accumulation in root tips of two Melaleuca trees differing in aluminum resistance

To elucidate the mechanism of the high aluminum (Al) resistance of a Myrtaceae tree, Melaleuca cajuputi Powell, we investigated the responses of root tips to Al and compared them with those of an Al-sensitive species, M. bracteata F. Muell. Roots of seedlings of both species were treated with a calcium solution (pH 4.0) containing 0 or 1 mM AlCl₃. After 3 h of Al treatment, inhibition of root elongation and deposition of callose and lignin in root tips, typical signs of Al injury, were induced in M. bracteata but not in M. cajuputi, yet Al accumulation in root tips was similar in both species. These results indicate that internal Al tolerance mechanisms, not Al exclusion mechanisms, are responsible for the Al resistance of M. cajuputi. After 3 h of Al treatment, amount of Al tightly bound to root tips, Al remaining after washing with a desorbing solution, was less in M. cajuputi than in M. bracteata. In M. bracteata, 6 h of Al treatment triggered the accumulation of hydrogen peroxide (H₂O₂) in root tips despite the upregulation of antioxidant mechanisms, activity of peroxidase and concentration of reduced glutathione. In M. cajuputi, 6 h of Al treatment did not affect the concentration of H₂O₂, but decreased activity of peroxidase, and increased concentration of reduced glutathione in root tips. These results suggest that the less Al tightly bound to root tips is involved in the suppressing the H₂O₂ accumulation and the internal Al tolerance in M. cajuputi, and that the H₂O₂ accumulation or changes in cellular environment that bring about H₂O₂ accumulation despite the upregulation of antioxidant mechanisms results in Al-induced inhibition of root elongation in M. bracteata.     

Boron-induced amelioration of aluminium toxicity in a monocot and a dicot species

Previous research has reported inconsistent results from experiments on the influence of boron (B) on plant sensitivity to potentially toxic aluminium (Al) concentrations. Differences in B requirement and cell wall properties among species, especially between Poaceae and dicots, may account for this. This investigation reports amelioration by B of Al-induced inhibition of root elongation in Al-sensitive cucumber (Cucumis sativus), but not in Al-sensitive maize (Zea mays). Vital staining, however, also revealed a positive influence of B supply on Al tolerance in maize. In both species, adequate B supply decreased Al-induced damage of cell integrity. In cucumber, increasing B supply enhanced Al concentrations and haematoxylin staining in root tips. In maize, no differences for root Al among B treatments were observed. These results indicate that the positive effect of B on Al resistance was not due to less Al accumulation in root tips. Enhanced concentrations of reduced glutathione were found in roots of Al-stressed maize plants growing with adequate B. It is concluded that adequate B supply is essential for prevention of Al toxicity in both the dicot and the monocot species. In dicot cucumber, the B-induced amelioration of root elongation, despite higher Al accumulation in root tips, indicates B-induced change in either or both Al speciation and compartmentation in the tips. The protection by an adequate B supply of roots against Al-induced cell death suggests a role for B in the defence against oxidative stress. This is supported by the observation that Al induced enhanced levels of GSH in roots of maize plants growing with adequate B supply but not in those growing with either deficient or excess B concentrations.     

Aluminum-induced secretion of both citrate and malate in rye

Aluminum (Al)-resistant mechanisms responsible for Al-induced secretion of organic acids are poorly understood. In this study, we characterized the Al-induced secretion of both citrate and malate from rye (Secale cereale L. cv. King). Secretion of organic acids increased with increasing concentration (10, 30 and 50 M) and duration of Al treatments. Neither phosphorous (P) deficiency up to 15 days nor addition of 50M lanthanum, 50 M lead, 10 M cadmium, or 200 M manganese caused secretion of organic acids, suggesting that this secretion was a specific response to Al stress. Aluminum activated citrate synthase, the main enzyme for the synthesis of citrate, but its activation occurred only in the root tip. The elongation of roots of an Al-sensitive cultivar of wheat (Tritium aestivum L. cv. Scout 66) was not inhibited by 50 M Al in the presence of externally applied 50 M citrate or 400 M malate. The secretion of citrate and malate from intact rye roots exposed to 50 M Al corresponded to 31.3 ± 1.7 M and 11.5 ± 2.5 M, respectively, in the rhizosphere based on an assumption of a 2 mm thick unstirred layer around root tips. This result indicated that Al-resistance in rye was achieved by the Al-induced synthesis of citrate in root apices followed by Al-induced specific secretion of citrate from root tips.     

Aluminium causes nutrient imbalance and structural changes in the needles of Scots pine without inducing clear root injuries

The effects of aluminium chloride (AICI3) treatments (50 and 150 mg/l) on 3-year-old Scots pine (Pinus sylvestris L.) seedlings were studied in a sand culture during 2 growing periods in an open field experiment. Even by the end of the first growing period, a decline was observed in the concentrations of Ca, Mg and P within the needles, and of Ca and Mg in the roots. After the second growing period, increased N and K concentrations were observed in the needles of Al-treated seedlings. Both the needles and roots of Al-treated seedlings showed, after the second growing period, a decline in growth and increased concentrations of AI as the amount of AICI3 in the nutrient solution increased. Al-induced changes in needle structure were found to be symptomatic of a nutrient imbalance, particularly of Mg and P. Al-stress did not result in any observable changes in root anatomy or in the number of mycorrhizas. Scots pine proved to be rather resistant to Al-stress, indicating that direct Al-injuries are not likely in the field, though Al-stress may be a contributing factor in the formation of nutrient imbalances.