Response of antioxidative enzymes and apoplastic bypass transport in Thlaspi caerulescens and Raphanus sativus to cadmium stress

A hydroponics experiment using hyperaccumulator Thlaspi caerulescens (alpine pennycress) and non-specific accumulator Raphanus sativus (common radish) was conducted to investigate the short-term effect of increasing Cd concentrations (0, 25, 50, 75, 100 microM) on metal uptake, chlorophyll content, antioxidative enzymes, and apoplastic bypass flow. As expected, T. caerulescens generally showed better resistance to metal stress, which was reflected by higher Cd accumulation within plant tissues with no signs of chlorosis, or wilt. Glutathione reductase (GR) and superoxide dismutase (SOD) activities in fresh leaves were monitored as the plant metal-detoxifying response. In general, both plant species exhibited an increase trend of GR activity before declining at 100 microM likely due to excessive levels of phytotoxic Cd. SOD activity exhibited almost a similar variation pattern to GR and decreased also at 100 microM Cd. For both plant species, fluorescent PTS uptake (8-hydroxy-1,3,6-pyrenetrisulphonic acid) increased significantly with metal level in exposure solutions indicating that Cd has a comparable effect to drought or salinity in terms of the gain of relative importance in apoplastic bypass transport under such stress conditions.     

Silicon alleviates cadmium toxicity in peanut plants in relation to cadmium distribution and stimulation of antioxidative enzymes

Silicon (Si) is generally considered a beneficial element for the growth of higher plants, especially for those grown under stressed environments. Recently, the mitigating role of Si in cadmium (Cd) stress has received some attention. However, its mechanisms involved remain poorly understood. We studied the effects of Si on tissue and subcellular distribution of Cd, as well as the activities of major antioxidant enzymes (SOD, POD and CAT) with two contrasting peanut (Arachis hypogaea L.) cultivars (Luhua 11 and Luzi 101) differing in their Cd tolerance. The results showed that Cd exposure alone depressed plant growth and caused oxidative stress for both cultivars, and this toxicity was more obvious in Cd-sensitive cultivar (Luhua 11) than in Cd-tolerant cultivar (Luzi 101). Si supply significantly alleviated the toxicity of Cd in peanut seedlings; this was correlated with a reduction of shoot Cd accumulation, an alteration of Cd subcellular distribution in leaves, and a stimulation of antioxidative enzymes. The mechanisms of Si amelioration of Cd stress were cultivar and tissue dependent. For Luhua 11, Si-mediated inhibition of Cd transport from roots to shoots, reduction of Cd content in cell organelle fractions of leaves, and enhancement of the SOD, POD and CAT activities in roots, might responsible for the role of Si in alleviating Cd toxicity. For Luzi 101, Si alleviation of Cd toxicity is mainly attributed to the decrease in Cd concentration in shoot and stimulation of antioxidants systems.     

Rhizobacterial inoculants can improve nickel phytoextraction by the hyperaccumulator Alyssum pintodasilvae

Aim

Rhizobacteria can influence plant growth and metal accumulation. The aim of this study was to evaluate the effect of rhizobacterial inoculants on the Ni phytoextraction efficiency of the Ni-hyperaccumulator Alyssum pintodasilvae.

Method

In a preliminary screening 15 metal-tolerant bacterial strains were tested for their plant growth promoting (PGP) capacity or effect on Ni bioaccumulation. Strains were selected for their Ni tolerance, plant growth promoting traits and Ni solubilizing capacity. In a re-inoculation experiment five of the previously screened bacterial isolates were used to inoculate A. pintodasilvae in two contrasting Ni-rich soils (a serpentine (SP) soil and a sewage sludge-affected agricultural (LF) soil).

Results

Plant growth was greater in serpentine soil (where it grows naturally) than in the LF soil, probably due to Cd phytotoxicity. Rhizobacterial inoculants influenced plant growth and Ni uptake and accumulation, but the effect of the strains was dependent upon soil type. The increase in plant biomass and/or Ni accumulation significantly promoted shoot Ni removal.

Conclusion

One strain (Arthrobacter nicotinovorans SA40) was able to promote plant growth and phytoextraction of Ni in both soil types and could be a useful candidate for future field-based trials.     

Evidence for nickel/proton antiport activity at the tonoplast of the hyperaccumulator plant Alyssum lesbiacum

The mechanism of nickel uptake into vacuoles isolated from leaf tissue of Alyssum lesbiacum was investigated to help understand the ability of this species to hyperaccumulate Ni. An imaging system was designed to monitor Ni uptake by single vacuoles using the metal-sensitive fluorescent dye, Newport Green. Nickel uptake into isolated vacuoles from leaf tissue of A. lesbiacum was enhanced by the presence of Mg/ATP, presumably via energisation of the vacuolar H(+)-ATPase (V-ATPase). This ATP-stimulated Ni uptake was abolished by bafilomycin (a diagnostic inhibitor of the V-ATPase) and by dissipation of the transmembrane pH difference with an uncoupler. These observations are consistent with Ni(2+)/nH(+) antiport activity at the tonoplast driven by a proton electrochemical gradient established by the V-ATPase, which would provide a mechanism for secondary active transport of Ni(2+) into the vacuole. This study provides insights into the molecular basis of Ni tolerance in Alyssum, and may aid in the identification of genes involved in Ni hyperaccumulation.     

Response to cadmium in higher plants

The paper summarizes present knowledge in the field of higher plant responses to cadmium, an important environmental pollutant. The principal mechanisms reviewed here include phytochelatin-based sequestration and compartmentalization processes, as well as additional defense mechanisms, based on cell wall immobilization, plasma membrane exclusion, stress proteins, stress ethylene, peroxidases, metallothioneins, etc. An analysis of data taken from the international literature has been carried out, in order to highlight possible ‘qualitative’ and ‘quantitative’ differences in the response of wild-type (non-tolerant) plants to chronic and acute cadmium stress. The dose-response relationships indicate that plant response to low and high cadmium level exposures is a very complex phenomenon indeed: cadmium evokes a number of parallel and/or consecutive events at molecular, physiological and morphological levels. We propose that, above all in response to acute cadmium stress, various mechanisms might operate both in an additive and in a potentiating way. Thus, a holistic and integrated approach seems to be necessary in the study of the response of higher plants to cadmium. This multi-component model, which we would call ‘fan-shaped’ response, may accord with the Selyean ‘general adaptation syndrome’ hypothesis. While cadmium detoxification is a complex phenomenon, probably under polygenic control, cadmium ‘real’ tolerance—found in mine plants or in plant systems artificially grown under long-term selection pressure, exposed to high levels of cadmium—seems to be a simpler phenomenon, possibly involving only monogenic/oligogenic control. We conclude that, following a ‘pyramidal’ model, (adaptive) tolerance is supported by (constitutive) detoxification mechanisms, which in turn rely on (constitutive) homeostatic processes. The shift between homeostasis and ‘fan-shaped’ response can be rapid and involve quick changes in (poly)gene expression. Differently, the slow shift from ‘fan-shaped’ response to ‘real’ cadmium tolerance is caused and affected by long-term selection pressure, which may increase the frequency (and promote the expression) of one or a few tolerance gene(s).     

Response to cytosolic nickel of Slow Vacuolar channels in the hyperaccumulator plant <Emphasis Type="Italic">Alyssum bertolonii</Emphasis>

We applied the patch-clamp technique to investigate the transport properties of the Slow Vacuolar (SV) channel identified in leaf vacuoles of Alyssum bertolonii Desv., a nickel hyperaccumulator plant growing in serpentine soil of the northern Apennines (Italy). SV currents recorded in vacuoles from adult plants collected in their natural habitat showed high sensitivity towards cytosolic nickel. Dose-response analyses indicated half-maximal current inhibition at submicromolar concentrations, i.e. up to three orders of magnitude lower than previously reported values from other plant species. The voltage-dependent increase of residual currents at saturating nickel concentrations could be interpreted as relief of channel block by nickel permeation at high positive membrane potentials. Including young plants of A. bertolonii into the study, we found that SV channels from these plants did not display elevated nickel sensitivity. This difference may be related to age-dependent changes in nickel hyperaccumulation of A. bertolonii leaf cells.     

High-nickel insects and nickel hyperaccumulator plants: A review

Insects can vary greatly in whole‐body elemental concentrations. Recent investigations of insects associated with Ni hyperaccumulator plants have identified insects with relatively elevated whole‐body Ni levels. Evaluation of the limited data available indicates that a whole‐body Ni concentration of 500 μg Ni/g is exceptional: I propose that an insect species with a mean value of 500 μg Ni/g or greater, in either larval/nymphal or adult stages, be considered a “high‐Ni insect”. Using the 500 μg Ni/g criterion, 15 species of high‐Ni insects have been identified to date from studies in Mpumalanga (South Africa), New Caledonia and California (USA). The highest mean Ni concentration reported is 3 500 μg Ni/g for nymphs of a South African Stenoscepa species (Orthoptera: Pyrgomorphidae). The majority of high‐Ni insects (66%) are heteropteran herbivores. Studies of high‐Ni insect host preference indicate they are monophagous (or nearly so) on a particular Ni hyperaccumulator plant species. Much of the Ni in bodies of these insects is in their guts (up to 66%–75%), but elevated levels have also been found in Malpighian tubules, suggesting efficient elimination as one strategy for dealing with a high‐Ni diet. Tissue levels of Ni are generally much lower than gut concentrations, but up to 1200 μg Ni/g has been reported from exuviae, suggesting that molting may be another pathway of Ni elimination. One ecological function of the high Ni concentration of these insects may be to defend them against natural enemies, but to date only one experimental test has supported this “elemental defense” hypothesis. Community‐level studies indicate that high‐Ni insects mobilize Ni into food webs but that bioaccumulation of Ni does not occur at either plant‐herbivore or herbivore‐predator steps. Unsurprisingly, Ni bioaccumulation indices are greater for high‐Ni insects compared to other insect species that feed on Ni hyperaccumulator plants. There is some evidence of Ni mobilization into food webs by insect visitors to flowers of Ni hyperaccumulator plants, but no high‐Ni insect floral visitors have been reported.     

Cadmium tolerance and antioxidative defenses in hairy roots of the cadmium hyperaccumulator,Thlaspi caerulescens

Plant species capable of hyperaccumulating heavy metals are of considerable interest for phytoremediation and phytomining. This work aims to identify the role of antioxidative metabolism in heavy metal tolerance in the Cd hyperaccumulator, Thlaspi caerulescens. Hairy roots of T. caerulescens and the non-hyperaccumulator, Nicotiana tabacum (tobacco), were used to test the effects of high Cd environments. In the absence of Cd, endogenous activities of catalase were two to three orders of magnitude higher in T. caerulescens than in N. tabacum. T. caerulescens roots also contained significantly higher endogenous superoxide dismutase activity and glutathione concentrations. Exposure to 20 ppm (178 microM) Cd prevented growth of N. tabacum roots and increased hydrogen peroxide (H(2)O(2)) levels by a factor of five relative to cultures without Cd. In contrast, growth was maintained in T. caerulescens, and H(2)O(2) concentrations were controlled to low, nontoxic levels in association with a strong catalase induction response. Treatment of roots with the glutathione synthesis inhibitor, buthionine sulfoximine (BSO), exacerbated H(2)O(2) accumulation in Cd-treated N. tabacum, but had a relatively minor effect on H(2)O(2) levels and did not reduce Cd tolerance in T. caerulescens. Lipid peroxidation was increased by Cd treatment in both the hyperaccumulator and non-hyperaccumulator roots. This work demonstrates that metal-induced oxidative stress occurs in hyperaccumulator tissues even though growth is unaffected by the presence of heavy metals. It also suggests that superior antioxidative defenses, particularly catalase activity, may play an important role in the hyperaccumulator phenotype of T. caerulescens.     

Comparison of the chemical changes in the rhizosphere of the nickel hyperaccumulator Alyssum murale with the non-accumulator Raphanus sativus

Changes in pH and redox potential were studied in the rhizosphere soil of a nickel hyperaccumulator plant (Alyssum murale) and of a crop plant, radish (Raphanus sativus). Differences in rhizosphere pH and reducing activity were found between the lateral and the main roots of both species, but the pH changes in the rhizosphere were similar in both species. Changes in pH were associated with the relative uptakes of cations and anions; whether the concentrations of heavy metals in the growth medium did not have any effect on the rhizosphere pH. The source of nitrogen (ammonium or nitrate) was the major factor determining the pH of the rhizosphere of both species. The redox potential of the rhizosphere was influenced by both the N-source and the concentrations of heavy metals. When heavy metals were not present in the growth medium, and nitrate was the N-source, the reducing capacity of A. murale roots was enhanced. However, the reducing activity of A. murale was always smaller than that of radish. Therefore, the mechanism of metal solubilization by the hyperaccumulator plant does not involve either the reduction of pH in the rhizosphere or the release of reductants from roots. The acidification and reducing activity of the roots of A. murale was always smaller than that of R. sativus.     

Comparative studies of nickel,cobalt, and copper uptake by some nickel hyperaccumulators of the genus Alyssum

The uptake of Ni, Co, and Cu by the nickel hyperaccumulator Alyssum troodii Boiss and the non-accumulator Aurinia saxatilis (L.) Desv. were studied in pot trials using artificial rooting media with varying concentrations of the metals added as soluble salts, singly and in combination. The ability of five other Ni hyperaccumulating species of Alyssum to hyperaccumulate Co was also investigated.Leaves and stems of A. troodii accumulated Ni to almost the same extent (8000–10 000 g g^-1). In roots, the highest Ni concentration was 2000 g g^-1. In leaves of Au. saxatilis, the maximum Ni concentration was only 380 g g^-1 and the level in roots was even lower.In media containing Co, the maximum concentration of this element in A. troodii (2325 g g^-1) was ten times higher than in the non-accumulator species. Slightly less Co was found in stems and roots of both species. Among the other Ni hyperaccumulators, the maximum concentration of Co in leaves ranged from about 1000–8000 g g^-1.Copper concentrations were the same in all organs of both species when they were grown in copper-rich media and were in the range 40–80 g g^-1, showing that neither plant was capable of taking up Cu at levels comparable to those of Ni and Co.When both plants were grown in media containing equal amounts of both Co and Ni, the Co concentrations in plant organs were the same as for specimens grown in media containing Co only. However, the Ni levels were lower in both species. Uptake of Co therefore appeared to suppress Ni uptake.Pot trials showed that the order of tolerance was Ni>Cu>Co for A. troodii and Ni>CoCu for Au. saxatilis, whereas the seedling tests showed the order to be Co>Ni>Cu. At metal concentrations 10 000 g g^-1, the overall tolerance of A. troodii was greater than that of Au. saxatilis which exhibited equally low tolerance to Ni and Cu.We conclude that in A. troodii, A. corsicum Duby, A. heldreichii Hausskn., A. murale Waldstein & Kitaibel, A. pintodasilvae T.R. Dudley, and A. tenium Hálácsy, Ni tolerance and hyperaccumulation conveys the same character towards Co. This behaviour should be investigated in other hyperaccumulators of Ni and/or Co.     

Cadmium-induced inhibition of photosynthesis and long-term acclimation to cadmium stress in the hyperaccumulator Thlaspi caerulescens

Acclimation of hyperaccumulators to heavy metal-induced stress is crucial for phytoremediation and was investigated using the hyperaccumulator Thlaspi caerulescens and the nonaccumulators T. fendleri and T. ochroleucum. Spatially and spectrally resolved kinetics of in vivo absorbance and fluorescence were measured with a novel fluorescence kinetic microscope. At the beginning of growth on cadmium (Cd), all species suffered from toxicity, but T. caerulescens subsequently recovered completely. During stress, a few mesophyll cells in T. caerulescens became more inhibited and accumulated more Cd than the majority; this heterogeneity disappeared during acclimation. Chlorophyll fluorescence parameters related to photochemistry were more strongly affected by Cd stress than nonphotochemical parameters, and only photochemistry showed acclimation. Cd acclimation in T. caerulescens shows that part of its Cd tolerance is inducible and involves transient physiological heterogeneity as an emergency defence mechanism. Differential effects of Cd stress on photochemical vs nonphotochemical parameters indicate that Cd inhibits the photosynthetic light reactions more than the Calvin-Benson cycle. Differential spectral distribution of Cd effects on photochemical vs nonphotochemical quenching shows that Cd inhibits at least two different targets in/around photosystem II (PSII). Spectrally homogeneous maximal PSII efficiency (F(v)/F(m)) suggests that in healthy T. caerulescens all chlorophylls fluorescing at room temperature are PSII-associated.     

植物抗氧化动态平衡研究进展

植物在生长发育的过程中会产生代谢副产物活性氧,其含量在植物生长过程中起双重作用。适量的活性氧可提高植物对逆境胁迫的耐受性,但是过量的活性氧会诱发氧化猝发反应,严重影响植物的生长发育。因此,提高植物的抗氧化能力对于提高植物的抗逆能力来说显得尤为重要,该方面的研究也成为近年来逆境生物学的一大热点。植物体为了应对逆境环境造成的活性氧动态失衡,进化出了含酶和非酶组分的抗氧化系统。本文主要介绍了参与高等植物活性氧代谢的相关物质,对近年来国内外报道的代谢途径进行了综述,为提高植物的抗逆能力提供参考依据。     

Matricaria chamomilla is not a hyperaccumulator, but tolerant to cadmium stress

The influence of low (3 μM) and high (60 and 120 μM) cadmium (Cd) concentrations were studied on selected aspects of metabolism in 4-week-old chamomile (Matricaria chamomilla L.) plants. After 10 days’ exposure, dry mass accumulation and nitrogen content were not significantly altered under any of the levels of Cd. However, there was a significant decline in chlorophyll and water content in the leaves. Among coumarin-related compounds, herniarin was not affected by Cd, while its precursors (Z)- and (E)-2-β-d-glucopyranosyloxy-4-methoxycinnamic acids (GMCAs) increased significantly at all the levels of Cd tested. Cd did not have any effect on umbelliferone, a stress metabolite of chamomile. Lipid peroxidation was also not affected by even 120 μM Cd. Cd accumulation was approximately seven- (60 μM Cd treatment) to eleven- (120 μM Cd treatment) fold higher in the roots than that in the leaves. At high concentrations, it stimulated potassium leakage from the roots, while at the lowest concentration it could stimulate potassium uptake. The results supported the hypothesis that metabolism was altered only slightly under high Cd stress, indicating that chamomile is tolerant to this metal. Preferential Cd accumulation in the roots indicated that chamomile could not be classified as a hyperaccumulator and, therefore, it is unsuitable for phytoremediation.     

Response of antioxidative enzymes to plum pox virus in two apricot cultivars

Recent evidence has indicated that activated oxygen species (AOS) may function as molecular signals in the induction of defence genes. In the present work, the response of antioxidative enzymes to the plum pox virus (PPV) was examined in two apricot (Prunus armeniaca L.) cultivars, which behaved differently against PPV infection. In the inoculated resistant cultivar (Goldrich), a decrease in catalase (CAT) as well as an increase in total superoxide dismutase (SOD) and dehydroascorbate reductase (DHAR) activities were observed. Ascorbate peroxidase (APX), glutathione reductase (GR) and monodehydroascorbate reductase (MDHAR) did not change significantly in relation to non-inoculated (control) plants. In the susceptible cultivar (Real Fino), inoculation with PPV brought about a decrease in CAT, SOD and GR, whereas a rise in APX, MDHAR and DHAR activities was found in comparison to non-inoculated (control) plants. Apricot leaves contain only CuZn-SOD isozymes, which responded differently to PPV depending on the cultivar. Goldrich leaves contained 6 SODs and both SOD 1 and SOD 2 increased in the inoculated plants. In leaves from Real Fino, 5 SODs were detected and only SOD 5 was increased in inoculated plants. The different behaviour of SODs (H2O2-generating enzymes) and APX (an H2O2-remover enzyme) in both cultivars suggests an important role for H2O2 in the response to PPV of the resistant cultivar, in which no change in APX activity was observed. This result also points to further studies in order to determine if an alternative H2O2-scavenging mechanism takes place in the resistant apricot cultivar exposed to PPV. On the other hand, the ability of the inoculated resistant cultivar to induce SOD 1 and SOD 2 as well as the important increase of DHAR seems to suggest a relationship between these activities and resistance to PPV. This is the first report about the effect of PPV infection on the antioxidative enzymes of apricot plants. It opens the way for the further studies, which are necessary for a better understanding of the role of antioxidative processes in viral infection by PPV in apricot plants.     

Response of Rhizobium leguminosarum to nickel stress

Rhizobium leguminosarum strain P-5 biovar viciae was sensitive to Ni²⁺ (MIC, 75 M) and showed concentration-dependent Ni²⁺ uptake in a wide concentration range (50–500 M). Ni²⁺ uptake up to a certain threshold limit also increased thiol content (66 nmol mg^–1 protein), proline content (10.85 nmol mg^–1 protein) and urease specific activity (500 nmol min^–1 mg^–1 protein) maximum corresponding to 100 M Ni²⁺ as the external concentration or 151 nmol Ni²⁺ mg^–1 protein as the intracellular buildup. Proline synthesis was stimulated most even at much lower Ni²⁺ concentration (25 M). Higher intracellular Ni²⁺ load neither favoured thiol nor proline biosynthesis nor urease activity. Ni²⁺ requirement of urease was ascertained by using EDTA-grown cells and the addition of bicarbonate (NaHCO₃, 100 mM) to the crude extract. The induction of thiol or proline by Ni²⁺, therefore, reflects the possible strategies adopted by bacterial cells to overcome the environmental stress.