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Metal hyperaccumulation is an uncommon but highly distinctive adaptation found in certain plants that can grow on metalliferous soils. Here we review what is known about evolution of metal hyperaccumulation in plants and describe a population-genetic analysis of the Alyssum serpyllifolium (Brassicaceae) species complex that includes populations of nickel-hyperaccumulating as well as non-accumulating plants growing on serpentine (S) and non-serpentine (NS) soils, respectively. To test whether the S and NS populations belong to the same or separate closely related species, we analysed genetic variation within and between four S and four NS populations from across the Iberian peninsula. Based on microsatellites, genetic variation was similar in S and NS populations (average Ho=0.48). The populations were significantly differentiated from each other (overall FST=0.23), and the degree of differentiation between S and NS populations was similar to that within these two groups. However, high S versus NS differentiation was observed in DNA polymorphism of two genes putatively involved in adaptation to serpentine environments, IREG1 and NRAMP4, whereas no such differentiation was found in a gene (ASIL1) not expected to play a specific role in ecological adaptation in A. serpyllifolium. These results indicate that S and NS populations belong to the same species and that nickel hyperaccumulation in A. serpyllifolium appears to represent a case of adaptation to growth on serpentine soils. Further functional and evolutionary genetic work in this system has the potential to significantly advance our understanding of the evolution of metal hyperaccumulation in plants.

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Metal-hyperaccumulating plants, which are hypothesized to use metals for defence against pests and pathogens, provide a unique context in which to study plant-pathogen coevolution. Previously, we demonstrated that the high concentrations of zinc found in leaves of the hyperaccumulator Noccaea caerulescens provide protection against bacterial pathogens, with a potential trade-off between metal-based and pathogen-induced defences. We speculated that an evolutionary arms race between zinc-based defences in N. caerulescens and zinc tolerance in pathogens might have driven the development of the hyperaccumulation phenotype. Here, we investigate the possibility of local adaptation by bacteria to the zinc-rich environment of N. caerulescens leaves and show that leaves sampled from the contaminated surroundings of a former mine site harboured endophytes with greater zinc tolerance than those within plants of an artificially created hyperaccumulating population. Experimental manipulation of zinc concentrations in plants of this artificial population influenced the zinc tolerance of recovered endophytes. In laboratory experiments, only endophytic bacteria isolated from plants of the natural population were able to grow to high population densities in any N. caerulescens plants. These findings suggest that long-term coexistence with zinc-hyperaccumulating plants leads to local adaptation by endophytic bacteria to the environment within their leaves.

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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.

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In this study we examine the effects of polycyclic aromatic hydrocarbons (PAHs) on the ability of the hyperaccumulator plant Alyssum lesbiacum to phytoextract nickel from co-contaminated soil. Planted and unplanted mesocosms containing the contaminated soils were repeatedly amended with sorbitan trioleate, salicylic acid and histidine in various combinations to enhance the degradation of two PAHs (phenanthrene and chrysene) and increase nickel phytoextraction. Plant growth was negatively affected by PAHs; however, there was no significant effect on the phytoextraction of Ni per unit biomass of shoot. Exogenous histidine did not increase nickel phytoextraction, but the histidine-extractable fraction of soil nickel showed a high correlation with phytoextractable nickel. These results indicate that Alyssum lesbiacum might be effective in phytoextracting nickel from marginally PAH-contaminated soils. In addition, we provide evidence for the broader applicability of histidine for quantifying and predicting Ni phytoavailability in soils.

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The organic anion malate is accumulated in the central vacuole of most plant cells. Malate has several important roles in plant vacuoles, such as the maintenance of charge balance and pH regulation, as an osmolyte involved in the generation of cell turgor, and as a storage form of CO2. Transport of malate across the vacuolar membrane is important for the regulation of cytoplasmic pH and the control of cellular metabolism, particularly in plants showing crassulacean acid metabolism (CAM), in which large fluxes of malate occur during the day/night cycle. By applying the patch-clamp technique, in the whole-vacuole configuration, to isolated vacuoles from leaf mesophyll cells of the CAM plant Kalanchoë daigremontiana, we studied the regulation of the vacuolar malate channel by pH and Ca2+, as well as its sensitivity to anion-channel blockers. Malate currents were found to be insensitive to Ca2+ on the cytoplasmic side of the membrane over a range from approximately 10(-8) M to 10(-4) M. In contrast, decreasing cytoplasmic pH below 7.5 had a significant modulatory effect on channel activity, reducing malate currents by 40%, whereas increasing cytoplasmic pH above 7.5 resulted in no change in current. Several known Cl?-channel blockers inhibited the vacuolar malate currents: niflumic acid and indanoyloxyacetic acid (IAA-94) proved to be the most effective inhibitors, exerting half-maximal effects at concentrations of approximately 20 mM, suggesting that the plant vacuolar malate channel may share certain similarities with other classes of known anion channels.

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A comparison of the performance of two epiphytes with crassulacean acid metabolism (CAM) was made during the rainy season and dry season at the Ciénega el Ostional, Chichiriviche in northern Venezuela. The epiphytic bromeliad, Tillandsia flexuosa has water-retaining tanks and leaf trichomes, and propagates mainly vegetatively to produce large populations in the shrubby island vegetation. The epiphytic orchid, Schomburgkia humboldtiana formed smaller populations, and had large succulent leaves with uniform chlorenchyma and no distinct water-storage parenchyma, unlike T. flexuosa. Both epiphytes were myrmecophilous. Leaf succulence (kg m-2 ) declined by ∼ 10% in the dry season for both plants. Both epiphytes showed reduced CO2 uptake during Phase I (dark period) and (dawn-dusk) titratable acidity (ΔH+ ) in the dry season. Water-use efficiency (WUE) was higher for S. humboldtiana (16.0 × 10-3 mol CO2 per mol H2 O compared with 5.0 × 10 -3 for T. flexuosa) although WUE remained constant during rainy and dry season for each species. Sixty to seventy per cent of the dawn dusk titratable acidity was derived internally from respiratory CO2 (recycling) for both species, and in absolute terms, recycling decreased in the dry season, in contrast to the expected progression under drought stress. Recycling is an important facet of carbon balance for both species in both rainy and dry seasons. Leaf Na+ concentration was higher than values quoted for terrestrial salt stressed CAM plants. Fructose and glucose declined in leaf bases of T. flexuosa during the dark period, but not in the more distal regions of the leaf. S. humboldtiana showed a decrease in sucrose at night, and mannitol was also an important constituent of the leaves. Xylem sap tension maxima increased from 0.38 ± 0.09 MPa (rainy season) to only 0.55 ± 0.06 MPa (dry season) for T. flexuosa, these values being much lower than those found for terrestrial shrubs and herbs at the same site, the two epiphytes use CAM in conjunction with differing morphological adaptations to maintain growth throughout the year at the Ciénega el Ostional, but it would seem that T. flexuosa has better physiological characteristics for maintenance of carbon acquisition during the dry season.

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Seasonal effects on leaf gas exchange and water relations were compared for Avicennia germinans, a true mangrove, and Conocarpus erectus, a mangrove associate, at coastal sites in northern Venezuela. On the Ciénega el Ostional at Chichiriviche, A. germinans was most abundant around lagoons on the seaward side of the vegetation-free alluvial sand plain. C. erectus was the dominant shrub in inland communities, but the two species co-occurred on vegetation islands at the landward edge of the alluvial plain. On the vegetation islands of the Ciénega el Ostional, gas exchange in A. germinans (a species with foliar salt glands) was less severely curtailed in the dry season compared with the rainy season than was gas exchange in C. erectus (a species lacking salt glands). Average rates of photosynthesis at near-saturating light intensities and total diurnal CO2 uptake were reduced in the dry season to 69 and 61%, respectively, of their values in the rainy for A. germinans, but to 48 and 30%, respectively, of their rainy-season values for C. erectus. Similarly, stomatal conductance and transpirational water loss were more reduced in the dry season for C. erectus than for A. germinans, with the result that C. erectus showed a 3.4-fold increase in water-use efficiency in the dry season compared with the rainy season. The importance of the soil environment in determining plant gas-exchange Patterns was evidenced by large seasonal shifts in dawn xylem tension for the two species (which increased from 1.34 MPa in the rainy season to 5.50 MPa in the dry season for A. germinans, and from 0.40 to 5.78 MPa for C. erectus). These values reflected changes in the soil environment caused by inundation of the upper soil layers by fresh water in the rainy season and a progressive increase in salt concentrations (to almost twice those in sea water) by evaporation from the soil in the dry season. Large changes in xylem tension were observed for both species during individual day-night cycles, reaching a maximum of 2.36 MPa for A. germinans. For C. erectus, the magnitude of these day-night changes was greatly reduced in the dry season, consistent with its very low transpiration rates at this time of year. Leaf-cell osmotic pressures also tended to be higher in A. germinans than C. erectus (attaining a maximum of 8.3 MPa for A. germinans in the dry season), and were related to the more seaward distribution of the true mangrove on the alluvial plain. Whereas leaves of A. germinans did not show any changes in succulence, leaf succulence in C. erectus increased with leafage and was slightly higher in the dry season than the rainy season. The more succulent leaves also had higher cell-sap osmotic pressures and NaCl concentrations. The most succulent leaves of C. erectus were observed for exposed shrubs growing on the shoreline. During the dry season, these shoreline plants showed high rates of gas exchange and low values for dawn xylem tension (0.89 MPa), indicating that they had access to relatively non-saline water from the shallow water table. On individual plants, exposed shoots had more succulent leaves and higher osmotic pressure and NaCl concentrations than sheltered leaves, demonstrating the importance of foliar absorption of salt borne in sea spray for the ionic relations of C. erectus. Thus, although the distribution of C. erectus is centred on brackish-water zones, this species can apparently extend from habitats with permanent access to a shallow water table through to areas where it is seasonally exposed to low soil water potentials and high salt concentrations in the substratum.

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The terrestrial CAM plant Bromelia humilis was examined in the salinas of the Ciénega el Ostional, on the north coast of Venezuela, in the rainy and dry seasons. Three colour forms were distinguished; yellow (in full sun), green exposed (also in sun) and green shaded (beneath woodland). Plant size decreased with increasing irradiance. An examination was made of the three phenotypes in terms of CO2 exchange (Jco2 ), dawn-dusk changes in titratable acidity (ΔH+ ) and malate and citrate levels, osmotic pressure, xylem tension, sugar and amino acids levels, nitrogen and ion concentrations and ambient temperature fluorescence. All phenotypes exhibited lowered Jco2 and ΔH+ in the dry as compared to the rainy season. Citrate, as well as malate, showed dawn-dusk fluctuations. Soluble sugars were the major source of carbon skeletons for nocturnal organic acid production. The dawn-dusk changes in osmotic pressure were negligible. Yellow plants performed poorly in contrast to shaded plants in both seasons. The former showed higher dawn-dusk changes of citrate levels and contained much less nitrogen than shaded plants. Nocturnal recycling of respiratory CO2 was more important in yellow plants and, in the dry season, reached 87%. These differences were reflected in the overall productivity, shaded plants showing increases in size whereas yellow plants utilized energy mainly for leaf replacement. Water availability and nitrogen supply appear to be the overriding factors determining higher productivity and CO2 assimilation in partially shaded plants as compared with plants in full sun.

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The perennial halophytes, Batis maritima L., Sesuvium portulacastrum L., and Portulaca rubricaulis H.B.K. which inhabit the extreme environment on parts of the salt plain and at the edges of the vegetation islands of Ciénega el Ostional were examined to determine their strategies for growth and survival in the rainy and dry seasons. All of the three species are leaf-succulents. High leaf-sap osmotic pressures, xylem tensions and Na+ and Cl- levels indicated that S. portulacastrum and B. maritima are salt-accumulating halophytes. Succulence, leaf-sap osmolalities and Na+ and Cl- levels increased in the dry season in B. maritima by a factor of 1.5-2.0 and in S. Portulacastrum by a factor of 1.9-2.7. B. maritima also accumulated sulphate with a two-fold increase of concentrations in the dry season. In S. portulacastrum Na+ accumulation much exceeded Cl- accumulation and oxalate synthesis was found to serve charge balance. In this species the compatible solutes, proline and pinitol, were clearly detectable in both seasons; their levels increased by a factor of about 6 in the dry season. Exchange of water vapour and CO2 was measured with a portable steady-state porometer. Photosynthesis in B. maritima showed little response to the transition from the rainy to the dry season while S. portulacastrum was severely impaired in the dry season, showing pronounced midday depressions of gas exchange and about 40% inhibition of light-saturated rates of CO2 uptake. P. rubricaulis shed its leaves in the dry season. According to carbon isotope ratios (δ13 C), B. maritima (δ13 C =-26.4‰) and S. portulacastrum (δ13 C =-25.8 ‰) are C3 plants while P. rubricaulis (δ13 C =-12.3‰) performed C4 photosynthesis.

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This paper describes the ecology of a coastal alluvial plain at Chichiriviche in northern Venezuela. The area supports a great diversity of plant communities, ranging from mangroves on the seaward edge of the plain to non-halophytic, fresh-water communities on the landward side. Small differences on topography result in a mosaic of saline and less-saline environments. Rainfall is strongly seasonal, causing superficial flooding of the alluvial plain in the rainy season and the creation of a hypersaline Substratum during the dry season. As a consequence, much of the plain is devoid or vegetation. Towards the landward side of the plain there are numerous small 'vegetation islands', fringed by halophilic succulent herbs, and made up of deciduous and semi-deciduous shrubs and trees together with non-halophytic CAM plants such as cacti and bromeliads. In subsequent papers the results of ecophysiological studies of these diverse plant species are presented.

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In an ecophysiological field investigation of plant communities of vegetation islands of an alluvial plain in northern Venezuela the members of the Cactaceae, Pereskia guamacho, and a columnar ceroid cactus, Subpilosocereus ottonis, were studied. The alluvial plain was flooded by fresh water in the rainy season in November/December 1985 and was dry and saline in the dry season in March/April 1986. The highly succulent leaves of P. guamacho were shed in the dry season. They performed C3 photosynthesis in the wet season and did not show signs of considerable salt accumulation. P. guamacho avoids stress due to drought and salinity by leaf-shedding. The columnar stem succulent ceroids are salt-excluding plants with crassulacean acid metabolism (CAM). Rapid die back and regeneration of absorptive roots, water parenchyma and CAM, with the possibility of nocturnal stomatal closure and CO2 recycling, are traits of adaptive value under fluctuating conditions between rainy and dry seasons on the alluvial plain.

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A study was made of the bulk-leaf water relations of selected species of epiphytic bromeliads growing in their natural habitat in Trinidad (West Indies). Field measurements were made during the rainy season at three forest sites centred on the wetter part of the island. The epiphytic bromeliads were sampled in situ using modified rock-climbing techniques at 4- to 6-h intervals during complete day-nigh cycles. Eleven species were studied that differed in their photosynthetic pathways and habitat preferences.The C3 species among the epiphytic bromeliads characteristically showed maximum values of xylem tension (measured with the pressure chamber) during the day, whereas the species with crassulacean acid metabolism (CAM) attained maximum values towards the end of the night. In addition, the CAM species showed large nocturnal increases in leaf-cell-sap osmotic pressure and titratable acidity. These nocturnal increases showed mean values of 0.601 MPa and 289 mol H+ m-3, respectively, for four species sampled at an exposed forest clearing (250 m), where CAM species were well represented. At the other two sites, a lowland forest (60 m) and a ridge forest (740 m), CAM bromeliads were found in the forest canopy, but in the lowest strata all the bromeliads were C3 species. This species distribution was associated with a marked vertical stratification of microlimate, the forest canopy being characterized by much bigger day-night changes in temperature and water-vapour-pressure deficit than the undergrowth. The C3-CAM intermediateGuzmania monostachia var.monostachia showed significant nocturnal acidification in the forest clearing but not in the understory of the lowland forest.Taken as a whole, the C3 and CAM bromeliads were very similar in the range of values observed for xylem tension and osmotic pressure, as well as in aspects of their leaf anatomy. However, epidermal trichomes covered a large percentage of the leaf surface area in xeromorphic species (e.g.Tillandsia utriculata), whereas they were poorly developed in shade-tolerant species (e.g.G. lingulata var.lingulata). The absolute values of sylem tension and osmotic pressure were low for all species. Mean minimum xylem tension during the day-night cycles was in the range of 0.18-0.23 MPa and mean maximum in the range 0.41-0.53 MPa; during periods of rain, xylem tension reached a mean minimum of 0.12 MPa. Mean minimum osmotic pressure was in the range 0.449-0.523 MPa. Such between-site and between-species differences as were observed in the water relations of the bromeliads could be related to the microclimatic conditions prevailing in the various epiphytic habitats.