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.     

Factors responsible for genotypic manganese tolerance in cowpea (Vigna unguiculata)

In experiments with 29 cowpea genotypes considerable variation in Mn tolerance could be found. Ranking according to Mn tolerance was almost the same in sand and water culture. Mn tolerance is not related to greater vigour or exclusion of Mn from uptake and translocation, but depends mainly on the internal tolerance to excess Mn especially in the leaf tissue.Growth depression by Mn excess is characterized by local accumulatiòn of Mn, deposition of Mn oxides, and typical macro-symptoms on the older leaves (brown spotsclorosisshedding of the leaves). Autoradiographic studies with⁵⁴Mn and extraction of the leaves with methanol and H₂O indicate a causal relationship between Mn tolerance and the more homogenous distribution of Mn in the tissue. In tolerant genotypes local accumulation and deposition of Mn is inhibited or retarded.Mn applied to the petioles of fully expanded leaves induces the same toxicity symptoms on the leaf blades as Mn absorbed by the roots. There is a good agreement between the rankings of the different genotypes for Mn tolerance according to the depression of shoot dry matter production by Mn excess in long term pot experiments and the appearance of toxicity symptoms after application of Mn to the petioles.The regulation of Mn tolerance at the leaf tissue level allows a quick and non-destructive screening of large numbers of genotypes for Mn tolerance.     

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.     

Manganese accumulation in the leaf mesophyll of four tree species: a PIXE/EDAX localization study

Little is known about the spatial distribution of excess manganese (Mn) in the leaves of tolerant plants. Recently, the first such study of a Mn hyperaccumulator showed that the highest localized Mn concentrations occur in the photosynthetic tissue. This is in contrast to reports based on localization of foliar accumulation of other heavy metals. Here, four tree species, Gossia bidwillii, Virotia neurophylla, Macadamia integrifolia and Macadamia tetraphylla, which hyperaccumulate or strongly accumulate Mn, were studied. Cross-sectional foliar Mn localization was carried out in situ using proton-induced X-ray emission/energy dispersive X-ray analysis (PIXE/EDAX). All four species contained photosynthetic tissues with multiple palisade layers. These were shown to be the primary sequestration sites for Mn. Mn was not detected in the epidermal tissues. The findings of this study demonstrate a concurrence of three traits in four tree species, that is, accumulation of excess Mn in the leaves, its primary sequestration in the photosynthetic tissues, and multiple-layer palisade mesophyll.     

Maize csmd1 exhibits pre-meiotic somatic and post-meiotic microspore and somatic defects but sustains anther growth

Maize male reproductive development is complex and lengthy, and anther formation and pollen maturation are precisely and spatiotemporally regulated. Here, we document that callose, somatic, and microspore defect 1 (csmd1), a new male-sterile mutant, has both pre-meiotic somatic and post-meiotic gametophyte and somatic defects. Chromosome behavior and cell developmental events were monitored by nuclear staining viewed by bright field microscopy; cell dimensions were charted by Volocity analysis of confocal microscopy images. Aniline blue staining and quantitative assays were performed to record callose deposition, and expression of three callose synthase genes was measured by qRT-PCR. Despite numerous defects and unlike other maize male-sterile mutants that show growth arrest coincident with locular defects, csmd1 anther elongation is nearly normal. Pre-meiotically and during prophase I, there is excess callose surrounding the meiocytes. Post-meiotically csmd1 epidermal cells have impaired elongation but excess longitudinal divisions, and uninucleate microspores cease growth; the microspore nucleoli degrade followed by cytoplasmic vacuolization and haploid cell collapse. The single vascular bundle within csmd1 anthers senesces precociously, coordinate with microspore death. Although csmd1 anther locules contain only epidermal and endothecial cells at maturity, locules are oval rather than collapsed, indicating that these two cell types suffice to maintain an open channel within each locule. Our data indicate that csmd1 encodes a crucial factor important for normal anther development in both somatic and haploid cells, that excess callose deposition does not cause meiotic arrest, and that developing pollen is not required for continued maize anther growth.     

Differential Responses of Two Wheat Varieties Differing in Salt Tolerance to the Combined Stress of Mn and Salinity

The effect of Mn and NaCl on growth, mineral nutrients and antioxidative enzymes in two tetroploid wheat genotypes differing in salt tolerance was investigated in this study. 100 mM NaCl and Mn stress significantly inhibited plant growth, photosynthesis and Ca uptake, while stimulated ROS accumulation, MDA and proline content in wheat plants, Mn stress also increased SOD, APX, GR and DHAR activities. Durum wheat (AS780) was less affected by 100 mM NaCl and Mn stress than emmer wheat (AS847) due to more proline production, higher antioxidative enzymes activities and less-affected mineral nutrients. Application of 10 mM NaCl to Mn-stressed durum wheat alleviated Mn-induced damage by reducing Mn accumulation and translocation, while promoting proline accumulation and SOD, APX and GR activities. Irrespective of NaCl level, the combined stress of Mn and NaCl caused more severe oxidative stress, result in further reduction of photosynthetic rate and plant growth in emmer wheat as compared to Mn stress alone. The additively negative effects of NaCl and Mn stress on growth of emmer wheat results from reduced SOD and APX activities as well as Ca, Cu and Fe accumulation in both shoots and roots. These results suggest that salt-tolerant durum wheat is superior to emmer in adapting to Mn stress and the combined stress of salinity and Mn.

     

Manganese uptake and transportation as well as antioxidant response to excess manganese in plants]

Manganese (Mn) is an essential micronutrient throughout all stages of plant development. Mn plays an important role in many metabolic processes in plants. It is of particular importance to photosynthetic organisms in the chloroplast of which a cluster of Mn atoms at the catalytic centre function in the light-induced water oxidation by photosystem II, and also function as a cofactor for a variety of enzymes, such as Mn-SOD. But excessive Mn is toxic to plants which is one of the most toxic metals in acid soils. The knowledge of Mn(2+) uptake and transport mechanisms, especially the genes responsible for transition metal transport, could facilitate the understanding of both Mn tolerance and toxicity in plants. Recently, several plant genes were identified to encode transporters with Mn(2+) transport activity, such as zinc-regulated transporter/iron-regulated transporter (ZRT/IRT1)-related protein (ZIP) transporters, natural resistance-associated macrophage protein (Nramp) transporters, cation/H(+) antiporters, the cation diffusion facilitator (CDF) transporter family, and P-type ATPase. In addition, excessive Mn frequently induces oxidative stress, then several defense enzymes and antioxidants are stimulated to scavenge the superoxide and hydrogen peroxide formed under stress. Mn-induced oxidative stress and anti-oxidative reaction are very important mechanisms of Mn toxicity and Mn tolerance respectively in plants. This article reviewed the transporters identified as or proposed to be functioning in Mn(2+) transport, Mn toxicity-induced oxidative stress, and the response of antioxidants and antioxidant enzymes in plants to excessive Mn to facilitate further study. Meanwhile, basing on our research results, new problems and views are brought forward.     

Isolation of aluminum-tolerant cell lines of tobacco in a simple calcium medium and their responses to aluminum

Aluminum (Al)-tolerant cell lines (ALT301 and ALT401) of tobacco were isolated in a simple calcium (Ca) solution from ethyl methane sulfonate (EMS)-treated suspension cultured tobacco cells ( Nicotiana tabacum L. cv. Samsun, a cell line SL) at the logarithmic phase of growth. A highly tolerant cell line ALT301 exhibited the accumulation of Al and the deposition of callose to the same extent as the parental SL cells during the exposure to Al. However, the Al-treated ALT301 cells grew much better than the Al-treated SL cells after transfer to Al-free growth medium. Compared to SL cells, ALT301 cells were more tolerant to toxicity of copper and iron, but not to that of lanthanum. These results suggest that ALT301 cells have an internal tolerance mechanism, which makes cells grow normally in spite of Al accumulation and Al-induced lesion represented by the deposition of callose. This tolerance mechanism seems also to be effective against copper and iron toxicity. A slightly tolerant cell line ALT401 also accumulated Al to the same degree as SL cells, but deposited significantly less callose than did SL cells (43% of SL). The growth of ALT401 cells after Al treatment was only slightly better than that of SL cells. Thus, it seems that ALT401 cells have a mechanism to protect cells only from the Al-induced deposition of callose, which is not enough to overcome the Al-induced inhibition of growth. The different phenotypes exhibited by these Al-tolerant cell lines suggest that the deposition of callose is not directly related to the inhibition of growth in Al-treated cells.     

Differential tolerance to Mn toxicity in perennial ryegrass genotypes: involvement of antioxidative enzymes and root exudation of carboxylates

Mechanisms underlying differential tolerance to Manganese (Mn) toxicity in perennial ryegrass (Lolium perenne L.) cultivars are poorly understood. We evaluated activity of antioxidative enzymes and root exudation of carboxylates in four ryegrass cultivars subjected to increasing Mn supply under nutrient solution conditions. A growth reduction caused by Mn toxicity was smaller in Jumbo and Kingston than Nui and Aries cultivars. Shoot Mn accumulation varied in the order Nui > Aries > Kingston > Jumbo. Ascorbate peroxidase and guaiacol peroxidase activities increased with Mn excess. Mn-tolerant Jumbo and Kingston had high activity of these enzymes and relatively low lipid peroxidation. Kingston was most tolerant to high tissue Mn concentrations and had the highest superoxide dismutase activity. Increased activity of antioxidative enzymes in Mn-tolerant cultivars could protect their tissues against oxidative stress triggered by Mn excess. Mn toxicity induced root exudation of carboxylates; oxalate and citrate may decrease Mn availability in the rhizosphere, thus enhancing Mn tolerance in ryegrass.     

Development of ultrastructural damage to chloroplasts in a plastoquinone-deficient mutant of maize

The ultrastructure of a recently discovered mutant of maize (mutant hcf103-114) that is completely lacking plastoquinone (Cook, W.B., Miles, D., 1992. Nuclear mutations affecting plastoquinone accumulation in maize. Photosyn. Res. 31, 99–111) was investigated. This mutant fails to green and dies at an early age. Tissues along a developmental gradient (from base to tip of a maize leaf) were fixed and prepared for examination via electron microscopy. Initial development was normal in both mesophyll cell (MC) and bundle sheath cell (BSC) chloroplasts. Starch, which was abundant in BSC chloroplasts of wild type maize, did not accumulate in the mutant. As tissue aging progressed, both plastid types exhibited symptoms typical of photooxidative injury. Injury, seen as chloroplast swelling, lipid accumulation and envelope disruption, appeared sooner in BSC chloroplasts than in MC chloroplasts. Chloroplasts in guard cells possessed starch granules and only showed ultrastructural injury after the starch granules disappeared. Stomata developed normally in the hcf103-114 mutant. The results are discussed in terms of the known roles of plastoquinone in chloroplast metabolism.     

Effect of light intensity on manganese toxicity symptoms and callose formation in cowpea (Vigna unguiculata (L.) Walp.)

In cowpea typical Mn toxicity symptoms are brown speckles on mature leaves representing depositions mainly in the cell walls and formation of non-constitutive callose. The histochemical charecterization of the brown speckles indicates the presence of oxidized Mn. However, the reducing agent hydroxylamine hydrochloride only slightly while thioglycolic acid almost completely decolorized the speckles. Brown boron-deficient roots treated with hydroxylamine hydrochloride and thioglycolic acid showed the same pattern of decoloration suggesting that the brown color of the Mn toxicity symptoms derives mainly from oxidized phenolics. To evaluate the effect of light on the formation of brown speckles by high Mn concentrations and non-constitutive callose in leaves, three approaches were used: (i) comparison of shaded and unshaded plants at different Mn supplies via the roots, (ii) local application of Mn to leaves in the light and in the dark, (iii) local application of Mn to leaves in the dark with subsequent light and dark treatments. Shading of whole plants (i) aggravated formation of both brown speckles and callose at similar Mn concentrations in the leaves. When the Mn application and the light treatments were locally confined (ii, iii), light had no effect on formation of either brown speckles or callose. The present results are in contradiction to the available reports in the literature showing aggravation of Mn toxicity by high light intensities.     

锰对锰超积累植物美洲商陆抗氧化系统的影响

采用水培法,研究了美洲商陆在不同锰浓度下的生长、锰积累、H₂O₂含量、脂质过氧化以及抗氧化系统的响应.结果表明：植株锰含量随锰浓度增大而显著增加,依次为叶＞茎＞根.低浓度锰（5 mmol·L^-1）显著促进植株生长,叶片中H₂O₂含量明显降低,丙二醛（MDA）含量与对照相当;高浓度锰（≥10 mmol·L^-1）抑制植株生长,叶片中H₂O₂和MDA水平显著增加,表明叶片中发生了明显的氧化损伤.抗坏血酸过氧化物酶、脱氢抗坏血酸还原酶、谷胱甘肽还原酶的活性和还原型抗坏血酸水平随锰处理浓度上升;超氧化物歧化酶活性在低浓度锰时显著降低,高浓度锰时则显著上升;过氧化氢酶和过氧化物酶活性、还原型谷胱甘肽含量在锰浓度为5～10 mmol·L^-1时显著上升,而在20 mmol·L^-1时明显回落.说明氧化损伤及锰积累可能是高浓度锰下美洲商陆生长受抑的生理原因.锰处理下,抗氧化系统效率提高可以部分解释美洲商陆耐锰和超积累锰的特性,不同抗氧化剂活性或含量随介质锰浓度变化的模式不同,反映了其在美洲商陆耐受不同浓度范围锰时的作用不同.     

Silicon ameliorates manganese toxicity in cucumber by decreasing hydroxyl radical accumulation in the leaf apoplast

This work was focused on the role of silicon (Si) in amelioration of manganese (Mn) toxicity caused by elevated production of hydroxyl radicals (·OH) in the leaf apoplast of cucumber (Cucumis sativus L.). The plants were grown in nutrient solutions with adequate (0.5 μM) or excessive (100 μM) Mn concentrations with or without Si being supplied. The symptoms of Mn toxicity were absent in the leaves of Si-treated plants subjected to excess Mn, although the leaf Mn concentration remained extremely high. The apoplastic concentration of free Mn(2+) and H(2)O(2) of high Mn-treated plants was significantly decreased by Si treatment. Si supply suppressed the Mn-induced increased abundance of peroxidase (POD) isoforms in the leaf apoplastic fluid, and led to a rapid suppression of guaiacol-POD activity under excess Mn. The spin-trapping reagent 5-(diethoxyphosphoryl)-5-methyl-1-pyrroline-N-oxide was used to detect ·OH by electron paramagnetic resonance spectroscopy. Although supplying Si markedly decreased the accumulation of ·OH in the leaf apoplast with excess Mn, adding monosilicic acid to the Mn(2+)/H(2)O(2) reaction mixture did not directly affect the Fenton reaction in vitro. The results indicate that Si contributes indirectly to a decrease in ·OH in the leaf apoplast by decreasing the free apoplastic Mn(2+), thus regulating the Fenton reaction. A direct inhibitory effect of Si on guaiacol-POD activity (demonstrated in vitro) may also contribute to decreasing the POD-mediated generation of ·OH.     

Copper and manganese induce yeast apoptosis via different pathways

Metal ions are essential as well as toxic to the cell. The mechanism of metal-induced toxicity is not well established. Here, for the first time we studied two essential nutritional elements, copper and manganese, for their apoptotic effects in yeast Saccharomyces cerevisiae. Although beneficial at subtoxic levels, we demonstrated that at moderately toxic levels, both metals induce extensive apoptosis in yeast cells. At even higher concentrations, necrosis takes over. Furthermore, we investigated the molecular pathways mediating Cu- and Mn-mediated apoptotic action. Mitochondria-defective yeast exhibit a much reduced apoptotic marker expression and better survival under Cu and Mn stress, indicating mitochondria are involved in both Cu- and Mn-induced apoptosis. Reactive oxygen species (ROS) are generated in high amounts in Cu- but not in Mn-induced cell death, and Cu toxicity can be alleviated by overexpression of superoxide dismutase 2, suggesting ROS mediate Cu but not Mn toxicity. Yeast metacaspase Yca1p is not involved in Cu-induced apoptosis, although it plays an important role in the Mn-induced process. A genetic screen identified Cpr3p, a yeast cyclophilin D homologue, as mediating the Cu-induced apoptotic program. Cpr3p mutant seems to eliminate Cu-induced apoptosis without affecting ROS production, while leaving necrosis intact. These results may provide important insight into a detailed understanding at the molecular and cellular level of metal toxicity and metal accumulation diseases.     

Callose deposition in leaves of cowpea (Vigna unguiculata [L.] Walp.) as a sensitive response to high Mn supply

First macroscopic visible symptoms of Mn toxicity in cowpea (Vegna unguiculata [L.] Walp.) plants grown in solution culture were dark brown spots on the older leaves. Close to these spots, large quantities of substances which fluorescence with aniline blue were deposited, indicating formation of (1,3)-β-glucan (callose). Callose formation in the leaf epidermis was a more sensitive indicator of Mn toxicity than the appearance of macroscopic symptoms, or the Mn concentration in the leaf.     

Screening sorghum for tolerance to excess manganese in solution culture

This study was conducted to define traits to screen sorghum (Sorghum bicolor L. Moench) genotypes for tolerance to excess Mn. Visual Mn toxicity symptoms, net and total root lengths, shoot and root dry matter yields, and shoot and root Mn concentrations were determined for plants grown in nutrient solutions (pH 4.5) at different levels of Mn (0, 3, 6, 9 and 12 mM above the initial 18 M) to assess plant responses to excess Mn. Dry matter yields showed greatest variability among genotypes, and was an effective trait to evaluate sorghum for tolerance to excess Mn. Reductions in dry matter yields did not occur until Mn levels were above 3 mM. Levels of Mn between 3 and 6 mM could effectively be used to screen sorghum for genotypic differences to excess Mn. Manganese levels above 6 mM were too severe to allow good genotypic differentiation. Of genotypes tested, NB9040 and Wheatland showed good tolerance and SC283 and ICA-Nataima were sensitive to excess Mn.     

Manganese neurotoxicity: A bioinorganic chemist’s perspective

Manganese is an essential metal for life, yet chronic exposure to this metal can cause a neurodegenerative disease named manganism, with symptoms that resemble Parkinson’s disease. Mn accumulates in the striatum and damages this brain structure that controls motor function; however, the molecular mechanisms underlying this neurodegenerative disease are poorly understood. In this short review, a summary of the current knowledge on the mechanisms involved in Mn neurotoxicity is given, with a special emphasis on the features that suggest specific protein-manganese interactions. The mechanisms of Mn uptake into the brain are discussed, displaying its similarities to Fe metabolism. Cellular trafficking of Mn is also reviewed, pointing out at its connection to Ca homeostasis, and its relevance for understanding Mn-induced neuronal death. The main purpose of this review is to provide a glimpse of an unexplored bioinorganic facet of a Mn-induced neurodegenerative disease.