Non-tolerant Plants Research Articles

Assessment of arbuscular mycorrhizal fungi diversity in the rhizosphere of Viola calaminaria and effect of these fungi on heavy metal uptake by clover

The ability of arbuscular mycorrhizal (AM) fungi from a metal-tolerant plant (Viola calaminaria, violet) to colonise and reduce metal uptake by a non-tolerant plant (Trifolium subterraneum, subterranean clover) in comparison to a metal-tolerant AM fungus isolated from a non-tolerant plant was studied. AM spores from the violet rhizosphere and from violet roots were characterised by polymerase chain reaction (PCR) amplification of the SSU rDNA, and sequencing. Subterranean clover was grown in pots containing a soil supplemented with Cd and Zn salts and inoculated either with a mixture of spores extracted from the violet rhizosphere or with spores of a Cd-tolerant Glomus mosseae P2 (BEG 69), or non-inoculated. The diversity of fungi, including AM fungi, colonising clover roots was assessed and analysed using terminal-restriction fragment length polymorphism. At least four different Glomus species were found in the violet rhizosphere. After 8 weeks in a growth chamber, colonisation of clover roots with spores from the violet rhizosphere increased Cd and Zn concentrations in clover roots without significantly affecting the concentrations of metals in the shoot and plant growth. G. mosseae P2 reduced plant growth and slightly increased the Cd concentration. Only one AM fungus (Glomus b) from the violet rhizosphere colonised clover roots, but other fungi were present. AM fungi from heavy metal-contaminated soils and associated with metal-tolerant plants may be effective in accumulating heavy metals in roots in a non-toxic form.

Hypostatic modifiers cause variation in degree of copper tolerance in Mimulus guttatus

Previous research into copper tolerance in the monkey flower Mimulus guttatus has found that: (i) it is primarily determined by a single dominant gene; and (ii) there is variation between tolerant plants that has been ascribed to ‘modifiers’. Modifiers can be either nonspecific, which act additively on both tolerant (T) and nontolerant (NT) genotypes, or specific, which act only on the tolerant genotype, and are thus hypostatic to the tolerance locus. We show here that there are hypostatic modifiers of tolerance in this species. Two selection lines that differ in degree of tolerance (and thus in the presence of putative modifiers) were crossed to a single NT plant. The F1s were selfed to produce F2s that segregated 3:1 T:NT. NT F2 individuals were crossed to a single homozygous T plant of low tolerance. The families differed in tolerance, showing that the NT F2 individuals differed in genes that only have an effect on tolerance phenotype in the presence of the tolerance gene. F3 individuals from F2s of contrasting phenotype were crossed to a second tester tolerant plant, and these F3s also varied, confirming the presence of the specific modifiers. There was no evidence of the segregation that would suggest a single modifier gene, but there is evidence from the F2s of at least one additive, nonspecific modifier in addition to the specific modifiers.

Hypostatic modifiers cause variation in degree of copper tolerance in Mimulus guttatus

Previous research into copper tolerance in the monkey flower Mimulus guttatus has found that: (i) it is primarily determined by a single dominant gene; and (ii) there is variation between tolerant plants that has been ascribed to ‘modifiers’. Modifiers can be either nonspecific, which act additively on both tolerant (T) and nontolerant (NT) genotypes, or specific, which act only on the tolerant genotype, and are thus hypostatic to the tolerance locus. We show here that there are hypostatic modifiers of tolerance in this species. Two selection lines that differ in degree of tolerance (and thus in the presence of putative modifiers) were crossed to a single NT plant. The F1s were selfed to produce F2s that segregated 3:1 T:NT. NT F2 individuals were crossed to a single homozygous T plant of low tolerance. The families differed in tolerance, showing that the NT F2 individuals differed in genes that only have an effect on tolerance phenotype in the presence of the tolerance gene. F3 individuals from F2s of contrasting phenotype were crossed to a second tester tolerant plant, and these F3s also varied, confirming the presence of the specific modifiers. There was no evidence of the segregation that would suggest a single modifier gene, but there is evidence from the F2s of at least one additive, nonspecific modifier in addition to the specific modifiers.

IDENTICAL MAJOR GENE LOCI FOR HEAVY METAL TOLERANCES THAT HAVE INDEPENDENTLY EVOLVED IN DIFFERENT LOCAL POPULATIONS AND SUBSPECIES OF SILENE VULGARIS.

Heavy metal tolerant Silene vulgaris plants, originating from different metalliferous sites in Germany and one in Ireland, were crossed to each other and to nontolerant plants from a nonmetalliferous site in The Netherlands. Analysis of the crosses suggested that there were two distinct major gene loci for zinc tolerance among a total of five tolerant populations. The tolerance loci for zinc, copper, and cadmium in the Irish plants were shown to be identical with those in the German populations. It is argued that the occurrence of common major genes for tolerance among different geographically isolated populations must have resulted from independent parallel evolution in local nontolerant ancestral populations. Each of the tolerances studied seems to be controlled by only a few specific major genes.

Metal Resistance in Trees: The Role of Mycorrhizae

On metal contaminated soils, the colonization and survival of plants is known to occur primarily through the selection of genotypes with heritable resistance traits that occur naturally within some species and populations (Bradshaw and McNeilly 1982, Bradshaw 1984, Baker 1987, Shaw 1990, Ernst et al. 1992). Recent studies have pointed to the additional importance of phenotypic plasticity, itself under normal genetic control, which potentially allows the survival of individual plants in contaminated environments until favourable conditions are encountered either spatially or temporally (Bradshaw 1991, Dickinson et al. 1991); this may involve an alteration of gene expression within an individual plant (Dickinson et al. 1992, Turner and Dickinson 1993a, b). It has also been suggested, however, that the ability to withstand pollution stress may be due to mycorrhizal colonization of roots ameliorating the toxicity of heavy metals (Dickinson et al. 1991, Wilkins 1991). The evolutionary significance of this possibility has not really been considered, but there is considerably more opportunity for genetic change in the shorter lifecycle of a fungus than in even a short-lived higher plant. We argue that elucidation of genetic changes within mycorrhizal communities is crucial to a proper understanding of the survival of long-lived and non-tolerant plants in metal-contaminated soils. Mycorrhizae are found associated with most higher plants and the fossil record suggests that even the earliest land plants were heavily infected (Begon et al. 1990). Perennial species that are normally or occasionally arbuscular mycorrhizal occur in almost twice as many habitat types as those that are infrequently mycorrhizal, which suggests that mycorrhizae increase the niche width of plants (Peat and Fitter 1993). The widespread arbuscular mycorrhizae make up three quarters of the mycorrhizal associations recorded in the British flora; long-lived woody species are almost invariably mycorrhizal. Over 150 taxa of mycorrhizae have been found to be associated with Salix (willows) alone in Britain (Watling 1992). As obligate symbionts, mycorrhizal fungi receive carbon and perhaps other resources from the plant host but it is often difficult to demonstrate that infected plants receive any benefit from the association in the field (Dosskey et al. 1990, Stenstrom and Ek 1990, Colpaert et al. 1992, Colpaert and Van Assche 1993). However, benefits that have been identified include improved uptake of less mobile nutrients such as phosphate, improved water relations, protection from fungal pathogens and protection from soil toxins (Harley and Smith 1983, Fitter 1991, Abbott et al. 1992, Fitter and Merryweather 1992, Gadd 1993). It is thought that the detrimental effect of air pollution on mycorrhizae is a likely causal factor of forest decline in Europe and North America (Jakucs 1988, Fellner 1989), further indicating the likely importance of the symbiotic association. Communities of mycorrhizal fungi change with time after planting trees (Last et al. 1992), and the absence of sheathing (ecto-)mycorrhizae may account for poor establishment of planted trees on some soils, including metal-contaminated soils (Burton and Morgan 1984, Ernst et al. 1990, Kahle 1993). Other studies have shown that, despite an apparent protective role of mycorrhizae, tree roots in mineralized forest soils naturally enriched with metals have a reduced active mycorrhizal root tip count (Bell et al. 1988). Metal-tolerant fungi may be more abundant in soils with extremely high concentrations of metals, but metal-tolerant fungi can also be isolated from clean soil, which suggests that wide genetic variation for metal tolerance may exist in many normal mycorrhizal populations. Mycorrhizal isolates from nickel-, copperand zinc-contaminated soils that have been grown on axenic media containing elevated metals have been found not to grow any better than the same species isolated from clean soils; it appears to be the behaviour of the fungus in the presence of roots that is particularly critical (Brown and Wilkins 1985a,b, Jones and Hutchinson 1986a, b). Seedlings of birch, pine and spruce are less susceptible to toxic concentrations of zinc, copper, nickel and aluminium in the growth medium in the presence of sheath-

Evolution of fluoride tolerance in Plantago lanceolata

Fluoride tolerance evolution was investigated by means of simple solution culture experiments. Seeds of Plantago lanceolata were collected at different distances from a fluoride manufacturing works and germinated in Petri dishes. A previous study had demonstrated an exponential type decrease in soil fluoride concentrations with distance from the factory. Plants grown from seeds collected near the factory were slower growing than susceptible plants in the absence of fluoride, but faster growing in the presence of fluoride. This is the first study providing evidence of fluoride tolerance in plants. As in studies demonstrating tolerance to other pollutants, tolerance evolution seems to involve a ‘cost’ in terms of a lower productivity of tolerant compared with non-tolerant plants.

Effects of copper concentration on mineral nutrient uptake and copper accumulation in protein of copper-tolerant and nontolerant Lotus purshianus L.

One copper-tolerant and one copper-sensitive inbred line of Lotus purshianus L. derived from a copper mine waste site in Northern California and one inbred line of the same species derived from a pasture next to the mine waste were examined for the effects of excessive copper concentrations on mineral nutrient uptake and accumulation of copper in protein fractions. Plants were grown from seeds for a period of 24 days in a modified Hoagland nutrient solution culture supplemented with 3, 6, and 10 μ M copper as copper sulfate. The basal nutrient solution without copper amendment was used as the control treatment. The uptake of Cu found in the roots was 100 times or more than that in the leaves. The root tissue copper concentrations reached a plateau under 6 μ M copper treatment. The leaf tissue copper concentrations increased with the increase of copper concentration in the solution culture. No difference in pattern of copper uptake was detected between the copper-tolerant and nontolerant plants. The effects of excessive copper concentrations caused reduction of Ca uptake in the leaf tissue and P uptake in both the root and leaf tissues, and no difference was found between the copper-tolerant and nontolerant plants. Increased tissue copper concentration caused greater reduction of Fe, Mn, and Zn uptake in the nontolerant plants than in the tolerant plants; this difference may be important for the growth of the tolerant plants under conditions of excessive copper concentrations. Protein extracted from the roots and leaves of both the copper tolerant and nontolerant plants was subjected to Sephadex G-75 column separation. Two major peaks of protein fractions were detected. Under low (normal level) copper concentration treatment, the copper-tolerant and nontolerant plants had similar Cu/protein ratios. However, under higher copper concentration challenged conditions the copper-tolerant plant had a considerably greater Cu/protein ratio (peak II protein) than the nontolerant plants. The amino acid composition of the copper-rich protein fraction (peak II) extracted from both the tolerant and nontolerant plants demonstrated a high asparate (about 25%) content. The contents of glutamate, cystine, and glycine were about 11, 2.5, and 10%, respectively, and the rest of the amino acids were in a range of 2 to 6%. This pattern of amino acid composition is different from the amino acid composition of the phytochelatin metallothionein-like proteins found in copper-tolerant plants which are very high in cysteine. Instead, the copper rich protein found in the L. purshianus resembles the copper-binding protein found in spinach which has high acidic amino acids and asparate content. More studies are needed to characterize this copper-binding protein and discover its possible role in copper tolerance of L. purshianus.

The genetics of arsenate tolerance in Yorkshire fog, Holcus lanatus L.

The genetics of arsenate tolerance in Holcus lanatus is investigated. Three tolerant plants from an abandoned arsenic mine, and three non-tolerant plants and one less tolerant plant (C9) from an uncontaminated site were crossed. Five polycrosses between plants from F1 crosses between mine and non-tolerants were set up. Four polycrosses between tolerant F2 progeny, and four polycrosses between non-tolerant F2 progeny, were established. A polycross involving the progeny of a single tolerant plant allowed to cross at random with a normal population was also established. The results are broadly compatible with a single-gene model for tolerance, with tolerance being dominant. The majority of F2 crosses segregated in to 3:1 ratios, and backcrosses gave 1:1 ratios. The crosses between C9 and non-tolerants gave 1:1 ratios, which suggests that the less tolerant C9 was heterozygous for tolerance. All crosses between non-tolerants gave all non-tolerant offspring. In one cross a major gene for albinism also segregated, and linkage of the tolerance gene to this gene (r.f. = 35%) was demonstrated. A number of families produced progeny ratios incompatible with the simple major gene model. Possible causes of these anomalous crosses are discussed and it is suggested that the tolerance gene may show variable penetrance depending on the genetic background.

Concerns a Weed Scientist Might Have About Herbicide-Tolerant Crops

There are three primary activities that characterize the discipline of Weed Science. These activities are weed technology, weed biology, and the ethics of weed control. Each of these activities needs to be considered as herbicide-tolerant crops (HTCs) are introduced. HTCs are the most recent refinement in the existing technology to control weeds. The potential benefits from the improved weed control must be weighed against possible increased costs of production and potential for genes that control herbicide tolerance to escape into non-tolerant plant populations. These questions about herbicide resistance are primarily technological and biological. They demonstrate the paucity of information in Weed Science on weed genetics, gene flow, fitness, and other aspects of weed-crop population dynamics. Other questions about HTCs are ethical. They require that we ask who benefits from the technology and what are the economic, ecological, and social consequences of it.

Are Phytochelatins Involved in Differential Metal Tolerance or Do They Merely Reflect Metal-Imposed Strain?

Plants from nontolerant and copper-tolerant populations of Silene vulgaris both produce phytochelatins upon exposure to copper. The threshold copper concentration for induction of phytochelatin and the copper concentration at which maximum phytochelatin contents occurs increase proportionally with the level of tolerance to copper. When exposed to their own highest no-effect concentration or 50%-effect concentration of copper for root growth, tolerant and nontolerant plants exhibit equal phytochelatin contents in the root apex, which is the primary copper target. This also holds for distinctly tolerant nonsegregating F(3) families, derived from a single cross of a nontolerant plant to a tolerant one. Therefore, the phytochelatin content of the root apex can be used as a quantitative tolerance-independent measure of the degree of toxicity experienced by the plant. Differential copper tolerance in S. vulgaris does not appear to rely on differential phytochelatin production.

Copper tolerance and copper accumulation of herbaceous plants colonizing inactive California copper mines

Herbaceous plant species colonizing four copper mine waste sites in northern California were investigated for copper tolerance and copper accumulation. Copper tolerance was found in plant species colonizing soils with high concentrations of soil copper. Seven of the eight plant species tested were found at more than one copper mine. The mines are geographically isolated, which makes dispersal of seeds from one mine to another unlikely. Tolerance has probably evolved independently at each site. The nontolerant field control population of Vulpia microstachya displays significantly higher tolerance to copper at all copper concentration levels tested than the nontolerant Vulpia myrous population, and the degree of copper tolerance attained by V. microstachya at the two copper mines was much greater than that found in V. myrous. It suggests that even in these two closely related species, the innate tolerance in their nontolerant populations may reflect their potential for evolution of copper tolerance and their ability to initially colonize copper mine waste sites. The shoot tissue of the copper mine plants of Arenaria douglasii, Bromous mollis, and V. microstachya accumulated less copper than those plants of the same species from the field control sites when the two were grown in identical conditions in nutrient solution containing copper. The root tissue of these mine plants contain more copper than the roots of the nonmine plants. This result suggests that exclusion of copper from the shoots, in part by immobilization in the roots, may be a feature of copper tolerance. No difference in the tissue copper concentration was detected between tolerant and nontolerant plants of Lotus purshianus, Lupinus bicolor, and Trifolium pratense even though the root tissue had more copper than the leaves. It suggests that copper tolerance in these legume species is not due to a mechanism of differential capacity for copper accumulation in the roots. Different mechanisms of copper tolerance may have evolved among the plant species colonizing the northern California copper mine waste sites.

Arsenic, cobalt and nickel tolerances in two populations of Silene vulgaris (Moench) Garcke from Ontario, Canada.

A population of Silene vulgaris (Moench) Garcke collected from mine tailings near the town of Cobalt, Ontario was compared in its metal tolerance with a population collected at an uncontaminated site (Baymouth, Ontario). Seedlings of the Cobalt population were tolerant to elevated levels of arsenic, cobalt and nickel, both compared with the Baymouth population and with other literature reports for this species. However, the tolerance indices in the Cobalt plants for these toxic elements did not correlate well with their concentration in water extractions in the mine soils. Indices of arsenic tolerance, based on the inhibition of root growth in 12 d experiments, showed a similar response to that in a 28 d experiment for both populations. The latter however, appeared to be a more accurate indicator of As tolerance in S. vulgaris, because the longer-term tolerance indices had lower standard deviations and higher probabilities for tolerance than the short-term tolerance indices. Reduced uptake of As occurred in the roots of tolerant individuals compared with non-tolerant individuals at a low As treatment. At a higher arsenic exposure, however, arsenic entered the roots of tolerant individuals. A partial exclusion or reduced translocation of arsenic to the shoots then appeared to operate in the shoots of tolerant individuals, i.e. arsenic levels were higher in shoots of non-tolerant plants. At the highest arsenic exposure, where the individuals from the Cobalt population were no longer tolerant, this exclusion pattern broke down. From a preliminary selection experiment, some arsenic tolerant individuals occurred in the Baymouth population. This suggests that under circumstances of arsenic contamination this population also has the potential to develop a tolerance to high arsenic exposure.

Genetics of arsenic tolerance in Agrostis capillaris L.

The genetic control of arsenic tolerance in Agrostis capillaris is examined by two experiments, one involving natural seed families collected from the wild and one based on the six basic generations from a cross between a tolerant plant and a non-tolerant plant. Arsenic tolerance is shown to be an inherited characteristic, the tolerant trait being dominant. Control by more than one gene locus is indicated by the data, although the number of loci involved appears to be small. The genetic architecture of this character allows for heritable variation in degree of tolerance among tolerant plants.

EVIDENCE THAT ARSENIC TOLERANCE IN HOLCUS LANATUS L. IS CAUSED BY AN ALTERED PHOSPHATE UPTAKE SYSTEM

The uptake of arsenate by arsenic tolerant and non-tolerant plants of Holcus lanatus L., and the interaction of this uptake with phosphate level, was studied. The tolerance of the different plants, as measured by root growth, was also investigated. Arsenate inhibited the root growth of non-tolerant plants much more than that of tolerant ones, but at low levels of arsenate, the high phosphate treatment reduced the toxicity of arsenate in non-tolerants but not in tolerants. Tolerant plants took up less arsenate than non-tolerants. The effect of phosphate on arsenate uptake was very small in the tolerant plants, but in non-tolerants phosphate inhibited arsenate uptake when the phosphate was at a higher concentration than arsenate. These results are compatible with the hypothesis that arsenate is taken up by the phosphate uptake system in non-tolerant plants, but in tolerant plants this system has become altered so that it no longer recognizes arsenate.

Reclamation of wastes contaminated by copper, lead, and zinc

Waste materials containing toxic levels of copper, lead, and zinc, such as mine and smelter wastes, present difficult conditions for the establishment of vegetation. This article reviews the many attempts which have been made to reclaim these wastes. Inert wastes from mining and quarrying operations, such as slate quarry waste and certain colliery shales, seem to be good materials for reclaiming wastes contaminated by copper, lead, and zinc. Organic wastes, such as sewage sludge and domestic refuse, may provide only a temporary visual improvement and stabilization of the toxic materials. Nontolerant plant materials may often be planted directly on modern waste materials, which are less toxic than they were in the past. However, tolerant plant materials are needed for revegetating waste materials produced by early and more primitive extraction methods.

Involvement of a metallothionein-like copper complex in the mechanism of copper tolerance in Mimulus guttatus

The possible involvement of a metallothionein-like copper complex in the mechanism of copper tolerance in Mimulus guttatus has been examined. In the roots of copper-tolerant plants, after exposure to copper, a constant proportion of the total copper content is bound to this complex. The complex is present in the roots of both copper-tolerant and non-tolerant plants, and is also induced in the roots of both by increased exposure to copper. Differences in the amount of the complex in the roots of copper-tolerant and non-tolerant plants suggest that it may be involved in the mechanism of copper tolerance. However, only a small proportion (6%) of the copper in the roots of the copper-tolerant clone is bound to the complex after growth in a high concentration (16 μm) of copper; this result suggests that the mechanism of tolerance does not simply involve the sequestration of all excess copper by the complex. The complex may have a transient function in the mechanism of copper tolerance.

Intracellular Localization of Lead in a Lead Tolerant and Sensitive Clone of Anthoxanthum odoratum

Summary Tillers from lead tolerant and non-tolerant plants of Anthoxanthum odoratum were incubated in the presence of 30μM lead for 7 days then the root tips examined for the intracellular distribution of lead. Electron microscopy of sections of both chemically fixed and freeze dried sections of root tips showed an accumulation of electron dense material in the cell walls which was assumed to be lead. An X-ray probe analysis of sections of chemically fixed root tips from tolerant plants also showed lead to be present in the cell walls and excluded from the cytoplasm. Examination of the non-tolerant material by electron microscopy alone again showed electron dense material in the cell walls, and also in the cytoplasm. It was suggested that the tolerance mechanism for lead in A. odoratum was related to the capacity of the tolerant plants to restrict the lead to the cell walls.

Metabolic responses of plants differing in SO 2 sensitivity towards SO 2 fumigation

To study the variations of plant response to sulphur dioxide, fumigation experiments were carried out with plants which have different sensitivities to SO 2. After SO 2 exposure it was obvious that the pea cultivar Späths Violetta (SV) is more sensitive to the pollutant than the pea cultivar Wunder von Kelvedon (WvK, Kelvedon Wonder). In comparison to SV, after 18 days of exposure, the less sensitive WvK showed increases in yield at lower SO 2 concentrations, sulphur accumulation in roots, and higher putrescine concentrations. After a short-term fumigation with 1·5 mg SO 2 m −3 differences between the cultivars could be recognised concerning the contents of sulphite and malondialdehyde and the activities of superoxide dismutase and peroxidase. In a fumigation experiment with deciduous trees, Tilia cordata was more sensitive to SO 2 than Populus nigra. This is revealed by the fresh/dry weight ratio, the sulphur content and the peroxidase activity. Besides that, the variations in susceptibility correlate with the results of a screening of the plants. Both SV and the sensitive Tilia cordata revealed lower buffering capacities for H + ions. Moreover, they showed a lower superoxide dismutase activity than the non-sensitive plants. These findings demonstrate that SO 2 tolerant plants have a more effective mechanism for the compensation of injurious reactions in metabolism than non-tolerant plants.

Cadmiun, lead and zinc interactions and tolerance in two populations of Holcus lanatus L. grown in solution culture

Uptake of Cd, Pb, Zn, Cu and Mn in two populations of Holcus lanatus was studied in a solution culture experiment in which Cd, Pb and Zn were supplied in factorial combination. Results show complex interactions between these three metals and their accumulation in non-tolerant plant shoots. In the non-tolerant population, most interactions are positive and thus tend towards increased metal concentrations. In the tolerant population, apart from a positive cadmiun-zinc interaction on cadmium concentrations, results are generally insignificant. It is apparent that single metal studies of non-tolerant populations should take into account these interactions, while studies of metal-tolerant populations should take into account that, in Holcus lanatus at least, tolerances to Pb, Zn and Cd are closely interlinked. The results confirm that in this population metal tolerance is related to decreased translocation of metals from roots to shoots.

The effects of sodium chloride, potassium chloride and glycerol on the activity of nitrate reductase of a salt-tolerant and two non-tolerant plants

The activity of nitrate reductase from the salt-tolerant alga Dunaliella parva is inhibited by sodium chloride and potassium chloride, but not by glycerol. The activity of the enzyme from Chlorella pyrenoidosa Chick 611-8b and from the XD line of tobacco (Nicotiana tabacum L. cv. Xanthi) cells is inhibited by all three solutes. Salt tolerance in Dunaliella parva, which is due to internal formation of glycerol, is accompanied by the adaptation of the activity of the enzyme to elevated glycerol concentrations.