Recent years have seen a flurry of research activity concerning the hyperaccumulation of heavy metals by plants. Much of the interest in hyperaccumulation has been fueled by the commercial potential of phytoremediation, the use of plants to clean up contaminated soils (Baker et al., 1994; Salt et al., 1995; Chaney et al., 1997, 2000). These applications have in turn spurred many studies of the genetics and physiology of metal uptake (e.g. Krämer et al., 1996; Lasat et al., 1996; Salt & Krämer, 1999; Baker et al., 2000; see also Krämer, 2000; Lombi et al., 2000). Although phytoremediation provides an intriguing and potentially profitable backdrop, the ecology and evolution of hyperaccumulation in natural populations are interesting subjects in their own right. Two papers in this issue (Ghaderian et al., pp. 219–224; Davis & Boyd, pp. 211–217) are exciting contributions to the growing consensus that hyperaccumulation may act as a defense against herbivores and pathogens, and suggest that hyperaccumulation may become a model system for research in this area.

How plants gather their resources, harvest solar energy and convert raw materials into an increased biomass is one of the enduring fascinations of biological science. Just about every step has been charted by biochemists and few processes have escaped the determination of a rate constant by physiologists. Even the molecular biologist is busy identifying the genes that govern the processes of synthesis and assembly. Nevertheless, at higher levels of organization, of building populations and, especially, communities, the prediction of growth continues to be problematic. Thus, the development by Bright & Walsby (pp. 301–316 in this issue) of a method for calculating the daily growth of a photoautotroph in its natural environment is bound to command our attention. Their approach is ingenious and the veracity of their calculations compelling. This is exciting science.

Summary 185

I. INTRODUCTION 186

II. EFFECT OF ZINC ON PRODUCTION OF REACTIVE OXYGEN SPECIES 186

1. Superoxide-generating NADPH oxidase 186

2. Zinc deficiency potentiates iron-mediated free radical production 189

(a) Iron accumulation in zinc-deficient plants 189

(b) Iron-induced production of free radicals 189

3. Zinc deficiency-enhanced photooxidation 191

(a) Decrease in photosynthesis 191

(b) Light-induced leaf chlorosis 192

(c) Decrease in indole-3-acetic acid 192

III. MEMBRANE DAMAGE BY REACTIVE OXYGEN SPECIES 193

1. Impairments in membrane structure 193

2. Phospholipids and –SH groups 195

3. Alterations in ion absorption 195

(a) Membrane-bound ATPases 195

(b) Nutrient uptake 197

(c) Changes in activity of ion channels 197

IV. DETOXIFICATION OF REACTIVE OXYGEN SPECIES 198

1. Superoxide dismutases 198

2. H2O2-scavenging enzymes 198

V. INVOLVEMENT OF ZINC IN PLANT STRESS TOLERANCE 199

VI. CONCLUSIONS 199

Acknowledgements 200

References 200

Zinc deficiency is one of the most widespread micronutrient deficiencies in plants and causes severe reductions in crop production. There are a number of physiological impairments in Zn-deficient cells causing inhibition of the growth, differentiation and development of plants. Increasing evidence indicates that oxidative damage to critical cell compounds resulting from attack by reactive O2 species (ROS) is the basis of disturbances in plant growth caused by Zn deficiency. Zinc interferes with membrane-bound NADPH oxidase producing ROS. In Zn-deficient plants the iron concentration increases, which potentiates the production of free radicals. The Zn nutritional status of plants influences photooxidative damage to chloroplasts, catalysed by ROS. Zinc-deficient leaves are highly light-sensitive, rapidly becoming chlorotic and necrotic when exposed to high light intensity. Zinc plays critical roles in the defence system of cells against ROS, and thus represents an excellent protective agent against the oxidation of several vital cell components such as membrane lipids and proteins, chlorophyll, SH-containing enzymes and DNA. The cysteine, histidine and glutamate or aspartate residues represent the most critical Zn- binding sites in enzymes, DNA-binding proteins (Zn-finger proteins) and membrane proteins. In addition, animal studies have shown that Zn is involved in inhibition of apoptosis (programmed cell death) which is preceded by DNA and membrane damage through reactions with ROS.

Plants use chemical defences to reduce damage from herbivores and the effectiveness of these defences can be altered by biotic and abiotic factors, such as herbivory and soil resource availability. Streptanthus polygaloides, a nickel (Ni) hyperaccumulator, possesses both Ni-based defences and organic defences (glucosinolates), but the extent to which these defences interact and respond to environmental conditions is unknown. S. polygaloides plants were grown on high-Ni and low-Ni soil and concentrations of Ni and glucosinolates were compared with those of the congeneric non-hyperaccumulator, S. insignus spp. insignus, grown under the same conditions. Ni contents were highest (4000 μg g−1 dry tissue) in S. polygaloides plants grown on high-Ni soil. Glucosinolate content was significantly higher in S. insignus than in S. polygaloides suggesting that plants defended by Ni produce a lower concentration of organic defences. In a separate experiment, high-Ni S. polygaloides plants were exposed to simulated herbivory or live folivores to determine the inducibility of Ni-based and organic defences. Contents of Ni were not affected by either herbivory treatment, whereas glucosinolate concentrations were >30% higher in damaged plants. We concluded that the Ni-based defence of S. polygaloides is not induced by herbivory.

Seedling mortality of Alyssum serpyllifolium ssp. lusitanicum and A. murale, both nickel hyperaccumulators, was reduced by increasing concentrations of metal within plant tissues when inoculated with the fungi Pythium mamillatum or P. ultimum, both of which cause damping-off disease of seedlings. Pythium mamillatum, isolated from nickel-rich serpentine soil, was more tolerant of nickel than P. ultimum, isolated from low-metal control soil, and was more pathogenic than P. ultimum towards seedlings containing high concentrations of metal. These results support the hypothesis that metal hyperaccumulation by plants is closely linked to increased protection against disease.

Zinc tolerance was investigated in five populations of Arabidopsis halleri (syn.: Cardaminopsis halleri) raised from seeds collected from contaminated and uncontaminated sites. Tolerance was measured by determining the concentration which inhibited root growth (EC100). A. halleri populations from contaminated and uncontaminated sites were found to be Zn-tolerant compared with the Zn-nontolerant species Arabidopsis thaliana and A. lyrata subsp. petraea. At very high Zn concentrations, populations of A. halleri from uncontaminated sites were slightly less Zn-tolerant than those from contaminated sites. These observations support the hypothesis that in A. halleri, Zn tolerance is largely a constitutive property. One population from an uncontaminated site and one population from a contaminated site were studied for Zn uptake. Zinc content was measured in shoots and roots using a colorimetric test under laboratory conditions. The results showed that whatever their origin, individuals from both populations are Zn accumulators compared with the nonaccumulator species A. thaliana. Moreover, the population from the uncontaminated area accumulated Zn in its shoots and roots more quickly than the population from the contaminated site. These results suggest that, in A. halleri, Zn accumulation to very high concentration is a constitutive property.

Xylem maturation in elongating leaf blades of tall fescue (Festuca arundinacea) was studied using staining and microcasting. Three distinctive regions were identified in the blade: (1) a basal region, in which elongation was occurring and protoxylem (PX) vessels were functioning throughout; (2) a maturation region, in which elongation had stopped and narrow (NMX) and large (LMX) metaxylem vessels were beginning to function; (3) a distal, mature region in which most of the longitudinal water movements occurred in the LMX. The axial hydraulic conductivity (Kh) was measured in leaf sections from all these regions and compared with the theoretical axial hydraulic conductivity (Kt) computed from the diameter of individual inner vessels. Kt was proportional to Kh throughout the leaf, but Kt was about three times Kh. The changes in Kh and Kt along the leaf reflected the different stages of xylem maturation. In the basal 60 mm region, Kh was about 0.30±0.07 mmol s−1 mm MPa−1. Beyond that region, Kh rapidly increased with metaxylem element maturation to a maximum value of 5.0±0.3 mmol s−1 mm MPa−1, 105 mm from the leaf base. It then decreased to 3.5±0.2 mmol s−1 mm MPa−1 near the leaf tip. The basal expanding region was observed to restrict longitudinal water movement. There was a close relationship between the water deposition rate in the elongation zone and the sum of the perimeters of PX vessels. The implications of this longitudinal vasculature on the partitioning of water between growth and transpiration is discussed.

Increased leaf phosphorus (P) concentration improved the water-use efficiency (WUE) and drought tolerance of regularly defoliated white clover plants by decreasing the rate of daily transpiration per unit leaf area in dry soil. Night transpiration was around 17% of the total daily transpiration. The improved control of transpiration in the high-P plants was associated with an increased individual leaf area and WUE that apparently resulted from net photosynthetic assimilation rate being reduced less than the reductions in the transpiration (27% vs 58%). On the other hand, greater transpiration from low-P plants was associated with poor stomatal control of transpirational loss of water, less ABA in the leaves when exposed to dry soil, and thicker and smaller leaf size compared with high-P leaves. The leaf P concentration was positively related with leaf ABA, and negatively with transpiration rates, under dry conditions (P < 0.001). However, leaf ABA was not closely related to the transpiration rate, suggesting that leaf P concentration has a greater influence than ABA on the transpiration rates.

Osmotic adjustment (OA) and increased cell-wall extensibility required for expansive leaf growth are well defined components of adaptation to water stress in dry soil, which might interact with soil phosphorus (P) concentration and defoliation frequency for intensively grazed white clover in legume-based pastures. Experiments were conducted with frequently and infrequently defoliated mini-swards of white clover growing in dry soil with low and high P concentrations. The higher yielding high-P plants were able to dry the soil to greater soil water suctions; their leaves had lower water potential values, yet they showed fewer water stress symptoms and underwent a more complete recovery from the water stress symptoms on rewatering, than the low-P plants. High- P plants had greater OA, proline concentration and leaf expansion rate. On the other hand, low-P plants showed an increased osmotic concentration when there was no change in the total solute content per unit of leaf d. wt, indicating more loss of water from the leaf tissue. The key measures that appeared to be directly associated with plant recovery over a short period following water stress were increased proline concentration and leaf expansion rate, probably resulting from increased cell-wall extensibility rather than increased production of cells for the high-P plants.

The effects of ambient and elevated ozone levels on growth and photosynthesis of beech (Fagus sylvatica) were studied by exposing seedlings in open-top chambers for one growing season to three treatments: charcoal-filtered (CF), non-filtered (NF) and non-filtered air with addition of ozone (30 ppb ozone) on clear days for 8–10 h d−1 (NF +). Ambient levels were relatively low and accumulated to an AOT40 (accumulated exposure over a threshold of 40 ppb) of 4055 ppb h (for the period 23 Apr–30 Sept). The NF + chambers received an AOT40 of 8880 ppb h. Throughout the growing season we measured growth and photosynthetic properties. The treatments did not cause strong effects: measurements of gas exchange (light-saturated assimilation rate, CO2 and light-response curves) and chlorophyll fluorescence showed slight and mostly non-significant reductions of several parameters. No significant differences were found for growth, though in the NF + treatment (AOT40 8880 ppb h) the relative growth rate for diameter increment was at times reduced by 12% compared with the control treatment.

In the northeastern part of Belgium, the Centaurea jacea complex shows extensive morphological variation and is represented by a diploid (2n = 22) and a tetraploid (2n = 44) cytotype. Polysomic inheritance of allozyme markers in the tetraploids, suggesting autopolyploidy, is here demonstrated for the first time. In order to test whether the two cytotypes occupy distinct habitats and possess different gene pools, patterns of allozymic and morphological variation were investigated in relation to ploidy level and site characteristics in 26 populations from the Belgian Ardennes. The two cytotypes showed a parapatric distribution, the diploids occurring at higher elevations (mostly above 500 m) than the tetraploids (mostly below 500 m). Three mixed populations were found near the contact zone of the two cytotypes. Within the mixed populations no triploid plant and no evidence for gene flow between cytotypes were found, despite widely overlapping flowering periods. The two cytotypes can be distinguished on the basis of morphological traits and enzymatic gene pools. The congruence of morphological and allozymic variation with chromosome numbers suggests a secondary contact between the two cytotypes with limited gene flow between them. The origin and persistence of the parapatric distribution are discussed.

Patterns of photobiont diversity were examined in some Nostoc-containing lichens using the nucleotide sequence of the cyanobacterial tRNALeu (UAA) intron. Lichen specimens collected in northwestern USA were analysed and the sequence data were compared with tRNALeu(UAA) intron sequences previously obtained from lichens in northern Europe. Generally, it is the species identity of a lichen rather than the geographical origin of the specimen that determines the identity of the cyanobiont. Identical intron sequences were found in Peltigera membranacea specimens collected in Oregon (USA) and in Sweden, and very similar sequences were also found in Nephroma resupinatum thalli collected in Oregon and Finland. Furthermore, in mixed assemblages where two Peltigera species grew in physical contact with each other, the different lichen species housed different photobiont strains. There is however not a one-to-one relation between mycobiont and photobiont as some intron sequences were found in more than one lichen species, and different intron sequences were found in different samples of some lichen taxa. Peltigera venosa exhibited a higher level of photobiont diversity than any other lichen species studied, and several intron sequences could for the first time be obtained from a single thallus. It is not clear whether this is evidence of lower cyanobiont specificity, or reflects an ability to exhibit different degrees of lichenization with different Nostoc strains. In one specimen of P. venosa, which contained bipartite cyanosymbiodemes and tripartite, cephalodiate thalli, both thallus types contained the same intron sequence.

In Lake Zürich, populations of the cyanobacterium Planktothrix rubescens develop in the metalimnion during the summer and become gradually entrained in the deepening surface mixed layer during the autumn. It had previously been demonstrated that the daily integrals of photosynthetic production accounted for the growth observed in the metalimnion and greatly exceeded the smaller increase during the autumn. We have now determined the relationship between growth rate (μ) and irradiance (I) in cultures of P. rubescens strain Pla 9316 maintained at 20 °C on a 12[ratio ]12 h light[ratio ]dark cycle: the highest net growth rate averaged over the 24 h (μh) was 0.123 d−1, the dark rate (μD) was −0.020 d−1, the gross rate (ϕm = μh−μD) was 0.144 d−1, the affinity coefficient (α) was 0.0273 (d μmol m−2 s−1)−1 and the compensation point (IC) was 1.76 μmol m−2 s−1. Using the corresponding coefficients calculated for the light period (μLh = 0.267 d−1, ϕLm = 0.287 d−1 and αL = 0.0547 (d μmol m−2 s−1)−1), instantaneous growth rates could be calculated from the irradiance. Comparison with growth rates at 10 °C indicated a Q10 of 1.48. These coefficients were used in a modification of the Smith equation to calculate potential growth rates of Planktothrix from the irradiance and temperature at each time and depth in Lake Zürich. Data on irradiance, vertical light attenuation and temperature were used to calculate the daily integrals of biomass increase over a period of 136 d. These growth integrals gave a closer correspondence to the observed population increase than the photosynthetic integrals calculated previously from measurements made with lakewater samples dominated by Planktothrix. Photosynthetic measurements made with the Planktothrix culture indicated a maximum rate of carbon increase (0.467 d−1 that exceeds the maximum growth rate, which suggests that other factors limit growth over long periods.

Soilborne pathogens, especially Fusarium oxysporum, are responsible for damping-off and root necrosis in Eucalyptus nurseries. New technologies are increasingly considering strategies for plant disease control other than chemical fungicides. Among these, natural fungal antagonists, which are colonizers of the root cortex, are potential biocontrol agents. An in vitro system was used: (1) to test the pathogenic effects of F. oxysporum strain Foeu1 which was recovered from a forest nursery soil; (2) to explore the potential of the nonpathogenic F. oxysporum strain Fo47, which is known for its efficiency in biological control, to suppress damping-off of Eucalyptus seedlings; (3) to compare the patterns of root colonization and host response to invasion by the two Fusarium strains inoculated separately in a time-course study. Root inoculation of E. viminalis with F. oxysporum strain Foeu1 caused damping-off in young seedlings in vitro, whilst disease symptoms were not visible in plants inoculated with F. oxysporum strain Fo47 or when both strains (Foeu1 + Fo47) were inoculated simultaneously. Each strain showed similarities in patterns of root tissue colonization, and in the processes of root penetration and initial colonization. Differential effects on root tissue were observed with fungal development within the cortex: ingress of strain Foeu1 was accompanied by severe host-cell alterations whilst no tissue damage occurred with development of strain Fo47.

In previously reported laboratory experiments, infection of Rumex obtusifolius by the rust fungus Uromyces rumicis was decreased on leaves which had prior herbivory by the beetle Gastrophysa viridula. In this paper we investigate whether this interaction is robust for natural infection by a variety of fungi in field experiments carried out in spring and autumn with plants given different levels of nitrogen fertilization. Grazing by G. viridula led to a decrease in lesion density of Ramularia rubella and Venturia rumicis in the spring and V. rumicis and U. rumicis in the autumn experiment. For V. rumicis and U. rumicis significant reductions in lesion density occurred on the undamaged leaves of damaged plants, compared with similar leaves on undamaged plants, suggesting systemic induced resistance. This induced resistance was usually independent of the amount of nitrogen fertilization, although the inhibitory effect of grazing on R. rubella in the spring and V. rumicis in the autumn experiment was enhanced by increasing nitrogen fertilization and was inhibited by increasing nitrogen fertilization for V. rumicis in the spring. In both experiments, the lesion density of V. rumicis was greater on leaves on which R. rubella was also present, and the presence of U. rumicis in the autumn experiment was linked to a similar but greater effect on V. rumicis lesion density. We found no evidence of induced resistance by fungi against fungi in these experiments. We highlight the complex interactions between inhibitory and facilitatory processes acting on leaf fungal infection. These results are compared with the proposed molecular mechanisms of induced resistance(s) and we consider the benefits of closer integration between molecular and ecological investigations of induced resistances that occur in the field.

Using genetic crosses between single chs mutants of Ustilago maydis inoculated into maize (Zea mays) seedlings, two classes of double mutants affected in genes coding for chitin synthetases were isolated: chs3/chs4, and chs4/chs5. Analysis of the mutants showed almost no change in their phenotype compared with wild-type strains. Growth rate, effect of stress conditions, dimorphic transition and mating were not affected. The only salient differences were increased sensitivity to osmotics at acid pH, and decrease in chitin synthetase activity, especially when measured with CO2+, and in chitin content. Most significant was a decrease in virulence, although this appeared to be due a factor unrelated to CHS genes. These data can be taken as further evidence that multigenic control of chitin synthetase in fungi operates as a safety mechanism to guarantee fungal viability in changing and hostile environmental conditions.