Localisation of nickel and mineral nutrients Ca,K, Fe,Mg by Scanning Electron Microscopy microanalysis in tissues of the nickel-hyperaccumulator Alyssum bertolonii Desv. and the non-accumulator Alyssum montanum L.

Plants of the nickel-hyperaccumulator Alyssum bertolonii Desv. and of the non-accumulator A. montanum L. growing on a serpentine site in Tuscany, Italy, and plants of A. montanum from a nearby non-serpentine site were analysed for metal concentration and localisation. The leaves of A. bertolonii contained 160 times more nickel than those of A. montanum from the same site, thus demonstrating its hyperaccumulation capacity towards this metal. On the other hand, both species showed an inversion of the Ca/Mg ratio in their organs relative to the soil. Nickel localisation in plant tissues was examined by Scanning Electron Microanalysis (SEM/EDX). In A. bertolonii, a specific pattern of nickel distribution was detected, with the highest concentrations present in parenchyma and sclerenchyma cells for the roots; in the shoots, the highest amounts of nickel were found in the stem epidermis, the leaf epidermal surface, and the leaf trichome base. This particular nickel tissue distribution pattern was not found in the non-accumulator A. montanum growing on serpentine soil. Other mineral nutrients, namely Mg, Ca, K, Fe, instead, had a similar distribution in the two species. The A. montanum plants from the non-serpentine site had very low nickel levels in their tissues, and these were of the same magnitude as those found in A. bertolonii plants grown in a greenhouse on commercial horticultural soil with low nickel concentration. In A. bertolonii plants, the tissue-specific allocation patterns appeared to depend on the degree of nickel hyperaccumulation, which is, in turn, directly linked to the soil characteristics.     

Comparison of two serpentine species with different nickel tolerance strategies

The effects of nickel were studied in two serpentine species with different metal tolerance strategies:Silene italica L., which limits nickel uptake and translocation, andAlyssum bertolonii Desv., a serpentine endemic, which accumulates nickel mostly in the leaves. InS. italica, nickel 7.5 μM inhibited root growth and depressed mitotic activity in root tips. Peroxidase activity and phenol concentration both in roots and shoots were increased; under the same conditions nickel did not produce any relevant effect onA. bertolonii. InS. italica an adequate calcium concentration (25 mM) was able to reverse the effects of nickel on root growth and metabolism. InA. bertolonii the same calcium concentration reduced root growth, confirming this species adaptation also to low calcium concentrations, typical of serpentines.     

Calcineurin,a Type 2B Protein Phosphatase,Modulates the Ca2+-Permeable Slow Vacuolar Ion Channel of Stomatal Guard Cells

The slowly activating vacuolar (SV) channel of plant vacuoles is gated open by cytosolic free Ca2+ and by cytosol-positive potentials. Using vacuoles isolated from broad bean guard cell protoplasts, SV-mediated currents could be measured in the whole-vacuole configuration of a patch clamp as the time-dependent increase in current at cytosol-positive voltages. Time-dependent deactivation of the SV currents when changing from activating to nonactivating voltages (tail currents) was used to calculate the selectivity of the channel to Ca2+ and Cl- with respect to K+. Changing the equilibrium potential for each permeant ion (Ca2+, Cl-, and K+) at least once for individual vacuoles allowed the relative permeabilities (P) of each of these ions to be calculated in a single experiment. The resulting Pca:Pcl:Pk ratio was close to 3:0.1:1. In accord with its characterization as a weakly selective Ca2+ channel, the SV-mediated current density decreased with increasing Ca2+ activity in the vacuole lumen. SV currents were potently modulated by the Ca2+-dependent, calmodulin-stimulated protein phosphatase 2B (calcineurin). At low concentrations ([less than or equal to]0.4 units per mL), calcineurin stimulated SV currents by ~60%, whereas at higher concentrations the phosphatase was inhibitory, reaching ~90% inhibition at 3 units per mL. Bovine calmodulin had no direct effect on SV-mediated currents, although calcineurin stimulated by exogenous calmodulin inhibited SV currents at all concentrations tested with half-maximal inhibition for calcineurin at 0.16 units per mL. The inhibitory effect of calcineurin could be blocked by the pyrethroid deltamethrin, indicating inhibition of SV channels by calcineurin via dephosphorylation. A model is discussed in which vacuolar Ca2+ release through SV channels is subject to both positive feedforward and negative feedback control through cytosolic Ca2+ and dephosphorylation, respectively.     

Ion transport and metal sensitivity of vacuolar channels from the roots of the aquatic plant Eichhornia crassipes

Using the patch‐clamp technique, we investigated the transport properties of vacuolar ion channels from the roots of water hyacinth, Eichhornia crassipes (Mart. Solms, Pontederiacae). Eichhornia crassipes vacuoles displayed large voltage‐dependent rectifying slow‐vacuolar (SV) currents, which activated in a few seconds at positive potentials and deactivated at negative voltages in a few hundreds of millseconds. Similarly to SV channel previously identified in the tonoplast of terrestrial plants, SV currents in E. crassipes were activated by micromolar concentrations of Ca²⁺ and current slightly increased (25%) on addition (10 mm ) of the reducing agent dithiothreitol (DTT). Eichhornia crassipes SV channels were equally permeable to K⁺ and Na⁺. The permeability sequence derived from current values is: K⁺ ≈ Na⁺ > Rb⁺ > NH₄⁺ ≈ Cs⁺ >> TEA⁺. Excised membrane patches displayed single channel transitions typical of SV‐type single channel openings with a conductance of (83·0 ± 5·6) pS; a smaller channel with a conductance of (31·0 ± 2·7) pS was also identified. Metals such as Ni²⁺ and Zn²⁺ decreased the vacuolar current in a reversible manner. However, although Zn²⁺ inhibition is comparable to that induced by the same metal in vacuoles from the main root of sugar beet (Beta vulgaris L.), the inhibition of the SV currents by Ni²⁺ is not as substantial in E. crassipes as in sugar beet. To our knowledge, this is the first electrophysiological characterization of ionic transport in E. crassipes, a pervasive troublesome aquatic weed, which has exceptional absorption properties of several water contaminants such as heavy metals, pesticides and phenols.     

Simple method to isolate vacuoles and protoplasts for patch-clamp experiments

It was not possible to obtain protoplasts or vacuoles from the thallus of the liverwortConocephalum conicum by applying cell-wall-degrading enzymes. Therefore, a surgical method was developed to isolate protoplasts and vacuoles. A thallus was plasmolyzed and cut. The few protoplasts along the cutting edge that were not destroyed emerged from the edge under deplasmolysis and became thus accessible for a patch pipette. Whereas under slightly hypoosmolar conditions the emerging protoplast remained largely intact, more hypoosmolar conditions gave rise to isolated vacuoles. This method to isolate protoplasts and vacuoles could also be applied to other plant tissues like leaves ofArabidopsis thaliana. Patch-clamp measurements were performed with isolated vacuoles and excised tonoplast patches. A slowly activating vacuolar channel inC. conicum displayed the characteristic features of higher-plant slowly activating vacuolar channels.Abbreviations AP action potential - SV channel slowly activating vacuolar channel     

Genetic Diversity of Bacterial Communities of Serpentine Soil and of Rhizosphere of the Nickel-Hyperaccumulator Plant <Emphasis Type="Italic">Alyssum bertolonii</Emphasis>

Serpentine soils are characterized by high levels of heavy metals (Ni, Co, Cr), and low levels of important plant nutrients (P, Ca, N). Because of these inhospitable edaphic conditions, serpentine soils are typically home to a very specialized flora including endemic species as the nickel hyperaccumulator Alyssum bertolonii. Although much is known about the serpentine flora, few researches have investigated the bacterial communities of serpentine areas. In the present study bacterial communities were sampled at various distances from A. bertolonii roots in three different serpentine areas and their genetic diversity was assessed by terminal restriction fragment length polymorphism (T-RFLP) analysis. The obtained results indicated the occurrence of a high genetic diversity and heterogeneity of the bacterial communities present in the different serpentine areas. Moreover, TRFs (terminal restriction fragments) common to all the investigated A. bertolonii rhizosphere samples were found. A new cloning strategy was applied to 27 TRFs that were sequenced and taxonomically interpreted as mainly belonging to Gram-positive and -Proteobacteria representatives. In particular, cloned TRFs which discriminated between rhizosphere and soil samples were mainly interpreted as belonging to Proteobacteria representatives.This revised version was published online in November 2004 with corrections to Volume 48.     

Amino acid contents and transport in oilseed rape (<Emphasis Type="Italic">Brassica napus</Emphasis> L.) under different nitrogen conditions

Oilseed rape (Brassica napus L.) needs very high nitrogen fertilizer inputs. Significant amounts of this nitrogen are lost during early leaf shedding and are a source of environmental and economic concern. The objective of this study was to investigate whether the remobilization of leaf amino acids could be limiting for nitrogen use efficiency. Therefore, amino acid concentrations were analyzed in subcellular compartments of leaf mesophyll cells of plants grown under low (0.5 mM NO₃^–) and high (4 mM NO₃^–) nitrogen supply. With high nitrogen supply, young leaves showed an elevated amino acid content, mainly in vacuoles. In old leaves, however, subcellular concentrations were similar under high and low nitrogen conditions, showing that the excess nitrogen had been exported during leaf development. The phloem sap contained up to 650 mM amino acids, more than four times as much than the cytosol of mesophyll cells, indicating a very efficient phloem-loading process. Three amino acid permeases, BnAAP1, BnAAP2, and BnAAP6, were identified and characterized. BnAAP1 and BnAAP6 mediated uptake of neutral and acidic amino acids into Xenopus laevis oocytes at the actual apoplastic substrate concentrations. All three transporters were expressed in leaves and the expression was still detectable during leaf senescence, with BnAAP1 and BnAAP2 mRNA levels increasing from mature to old leaves. We conclude that phloem loading of amino acids is not limiting for nitrogen remobilization from senescing leaves in oilseed rape.     

Sodium Chloride Compartmentation in Leaf Vacuoles of the Halophyte Suaeda maritima (L.) Dum. and its Relation to Tonoplast Permeability

Single channel properties, whole vacuole currents and protonpumping capacity were investigated in the intact vacuoles andmembrane patches of leaf tonoplast from the halophyte Suaedamaritima. ATP-dependent proton pumping capacity was similarto non-halophytes whether the plants were or were not grownwith added sodium chloride (200 mM). The most abundant ion channelwas inward rectifying and had a single channel conductance of58 pS in symmetrical KCl solutions (100 mM) to 170 pS usingphysiological conditions (50/150 mM KCl/NaCl cytoplasmic side,50/450 mM KCl/NaCl vacuolar side). The channel showed all thecharacteristics of the SV type channel described in many otherspecies. In the open state these channels caused tonoplast conductancesin excess of 0.5 nS m²– but conductances were much lowerusing physiological ion concentrations and membrane potentials.In spite of the poor selectivity and the potentially large tonoplastconductance it is calculated that compartmentation of NaCl inleaf vacuoles can be sustained by about 30% of ATP-dependentproton pumping capacity. The results do not indicate any specialadaptation of the tonoplast ion channels in the halophyte. Key words: Ion-channels, patch-clamp, salt-tolerance, vacuole     

Accumulation of hexoses in leaf vacuoles: Studies with transgenic tobacco plants expressing yeast-derived invertase in the cytosol,vacuole or apoplasm

The subcellular distribution of hexoses, sucrose and amino acids among the stromal, cytosolic and vacuolar compartments was analysed by a nonaqueous fractionation technique in leaves of tobacco (Nicotiana tabaccum L.) wild-type and transgenic plants expressing a yeast-derived invertase in the cytosolic, vacuolar or apoplasmic compartment. In the wild-type plants the amino acids were found to be located in the stroma and in the cytosol, sucrose mainly in the cytosol and up to 98% of the hexoses in the vacuole. In the leaves of the various transformants, where the contents of hexoses were greater than in wild-type plants, again 97–98% of these hexoses were found in the vacuoles. It is concluded that leaf vacuoles contain transporters for the active uptake of glucose and fructose against a high concentration gradient. A comparison of estimated metabolite concentrations in the subcellular compartments of wild-type and transformant plants indicated that the decreased photosynthetic capacity of the transformants is not due to an osmotic effect on photosynthesis, as was shown earlier to be the case in transformed potato leaves, but is the result of a long-term dedifferentiation of tobacco leaf cells to heterotrophic cells.Abbreviations apo-inv tobacco plant with yeast invertase in the apoplasm - Chl chlorophyll - cy-inv tobacco plant with yeast invertase in the cytosol - vac-inv tobacco plant with yeast invertase in the vacuole - WT wild-type tobacco plant The authors thank A. Großpietsch for her able technical assistance. This work has been supported by the Bundesminister für Forschung und Technologie.     

Ulteriori indagini sul rapporto tra nichel e acidi malico e malonico in Alyssum

Abstract

Further contribution on the relationship between nickel and malic and malonic acids in Alyssum.—Further research has been carried out on extracts of leaves and seeds of A. bertolonii Desv., grown on serpentine and normal garden soil, in order to investigate the nature of the nickel compounds present in this plant. On the purified extracts, obtained with water and formic acid, malic and malonic acids have been determined through gas-chromatography of the trimethyl-sililderivatives; Ni, Ca, Mg and K have been analysed by atomic absorption spectrophotometry.

Both malic and malonic acids are present in the leaves of the plants collected on serpentine and therefore rich in nickel, whilst they are present only in small amounts in the plants grown in garden soil. Seeds contain mainly high levels of malic acid. Remarkable amounts of malic acid, related to a high nickel content, have been observed also in the leaves of another nickel accumulator in the genus Alyssum, i.e. A. serpyllifolium Desf. ssp. lusitanicum Dudley et P. Silva, endemic of Portugal serpentines.     

HEAVY METAL ACCUMULATION AND THE NATURE OF EDAPHIC ENDEMISM IN THE GENUS CALOCHORTUS (LILIACEAE)

Bulb and leaf tissues from five species in the genus Calochortus and the soils from collection sites were analyzed for cation and trace element concentrations. All five species, only three of which are considered ultramafic endemics, possessed high concentrations of nickel and copper in both the bulbs and leaves, but not at levels high enough (> 1% dry wt) to be considered hyperaccumulators. Only moderate to trace amounts of cobalt and chromium were detected in both plant tissues. Calcium-magnesium ratios, typically low in ultramafic soils and plants, were low in bulb tissue, but two to 10 times higher in leaf tissue. The ability to tolerate excessively high levels of nickel and other heavy metals may be a physiological exaptation of the genus Calochortus and not necessarily an evolutionary response by several species to life on an ultramafic substrate. The nature of “serpentine” endemism is discussed in the context of heavy metal accumulation by various plant species.     

Overexpression of a <Emphasis Type="Italic">Panax ginseng</Emphasis> tonoplast aquaporin alters salt tolerance,drought tolerance and cold acclimation ability in transgenic <Emphasis Type="Italic">Arabidopsis</Emphasis> plants

Water movement across cellular membranes is regulated largely by a family of water channel proteins called aquaporins (AQPs). Since several abiotic stresses such as, drought, salinity and freezing, manifest themselves via altering water status of plant cells and are linked by the fact that they all result in cellular dehydration, we overexpressed an AQP (tonoplast intrinsic protein) from Panax ginseng, PgTIP1, in transgenic Arabidopsis thaliana plants to test its role in plant’s response to drought, salinity and cold acclimation (induced freezing tolerance). Under favorable conditions, PgTIP1 overexpression significantly increased plant growth as determined by the biomass production, and leaf and root morphology. PgTIP1 overexpression had beneficial effect on salt-stress tolerance as indicated by superior growth status and seed germination of transgenic plants under salt stress; shoots of salt-stressed transgenic plants also accumulated greater amounts of Na⁺ compared to wild-type plants. Whereas PgTIP1 overexpression diminished the water-deficit tolerance of plants grown in shallow (10 cm deep) pots, the transgenic plants were significantly more tolerant to water stress when grown in 45 cm deep pots. The rationale for this contrasting response, apparently, comes from the differences in the root morphology and leaf water channel activity (speed of dehydration/rehydration) between the transgenic and wild-type plants. Plants overexpressed with PgTIP1 exhibited lower (relative to wild-type control) cold acclimation ability; however, this response was independent of cold-regulated gene expression. Our results demonstrate a significant function of PgTIP1 in growth and development of plant cells, and suggest that the water movement across tonoplast (via AQP) represents a rate-limiting factor for plant vigor under favorable growth conditions and also significantly affect responses of plant to drought, salt and cold stresses.     

Serpentine and non-serpentine Silene dioica plants do not differ in nickel tolerance

Serpentine soils are hostile to plant life. They are dry, contain high concentrations of nickel and have an unfavorable calcium/magnesium ratio. The dioecious plant Silene dioica (L.) Clairv. (Caryophyllaceae) is the most common herb on serpentine soils in the Swedish mountains. It also commonly grows on non-serpentine soils in the subalpine and coastal area. I have compared the germination frequency, plant establishment and growth of serpentine and subalpine non-serpentine populations in serpentine soil under greenhouse conditions. Further more I have studied the specific effect of nickel on root and shoot growth of serpentine and non-serpentine plants from the subalpine and coastal area in solutions with different concentrations of nickel. Plants from serpentine and non-serpentine populations grew well and in a similar fashion in serpentine soil. Moreover, S. dioica plants, irrespective of original habitat, tolerated enhanced concentrations of nickel when grown in solutions. An analysis of metal content in serpentine plants from natural populations shows that S. dioica has a higher nickel concentration in the roots than in the shoots. The growth studies show that S. dioica is constitutively adapted to serpentine, and that all populations have the genetic and ecological tolerance to grow on serpentine.     

Assessing Plant Phytoextraction Potential Through Mathematical Modeling

One of the most serious and long-term consequences of environmental pollution is heavy metal contamination of soils. Elements such as zinc, cadmium, lead, nickel, and chromium are being released into the environment by many industrial processes and have now reached concentrations that are of concern. Phytoremediation is a new, low-cost, and environmentally friendly technique that relies on the natural properties of some plants to clean-up the ground through their ability to take up metals from the soil. Hyperaccumulator plants, capable of accumulating metals far in excess of any normal physiological requirement, represent a most promising tool for metal phytoextraction, but the in field establishment of their conditions for utilization needs a long period because of the plant life-cycle. The use of a mathematical model is proposed to process growth and uptake data from in vitro experiments for a rapid assessment of the time and concentration parameters for the deployment of hyperaccumulator plants for phytoextraction purposes. This preliminary research has been carried out using Alyssum bertolonii Desv., a nickel hyperaccumulator endemic to Italian serpentine soils.     

Nicotianamine Over-accumulation Confers Resistance to Nickel in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

Nicotianamine is a methionine derivative involved in iron homeostasis, able to bind various other metals in vitro. To investigate its role in vivo, we expressed a nicotianamine synthase cDNA (TcNAS1) isolated from the polymetallic hyperaccumulator Thlaspi caerulescens in Arabidopsis thaliana. Transgenic plants expressing TcNAS1 over-accumulated NA, up to 100-fold more than wild type plants. Furthermore, increased NA levels in different transgenic lines were quantitatively correlated with increased nickel tolerance. The tolerance to nickel is expressed at the cellular level in protoplast experiments and is associated with an increased NA content. We have also shown that the most NA-over accumulating line showed a high tolerance to nickel and a significant Ni accumulation in the leaves when grown on nickel-contaminated soil. Our results highlight a new potential role for nicotianamine in heavy metal tolerance at the cellular but also at the whole plant level, easily transposable to a non-tolerant non-hyperaccumulator species. These results open new perspectives for the modulation of nicotianamine content in plants for phytoremediation.     

Sensitivity of the Plant Vacuolar Malate Channel to pH, Ca2+ and Anion-Channel Blockers

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 Kalancho? 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.     

Mechanism of luminal Ca<Superscript>2+</Superscript> and Mg<Superscript>2+</Superscript> action on the vacuolar slowly activating channels

The non-selective slow vacuolar (SV) channel can dominate tonoplast conductance, making it necessary to tightly control its activity. Applying the patch-clamp technique to vacuoles from sugar beet (Beta vulgaris L.) taproots we studied the effect of divalent cations on the vacuolar side of the SV channel. Our results show that the SV channel has two independent binding sites for vacuolar divalent cations, (i) a less selective one, inside the channel pore, binding to which impedes channel conductance, and (ii) a Ca²⁺-selective one outside the membrane-spanning part of the channel protein, binding to which stabilizes the channels closed conformations. Vacuolar Ca²⁺ and Mg²⁺ almost indiscriminately blocked ion fluxes through the open channel pore, decreasing measured single-channel current amplitudes. This low-affinity block displays marked voltage dependence, characteristic of a permeable blocker. Vacuolar Ca²⁺—with a much higher affinity than Mg²⁺—slows down SV channel activation and shifts the voltage dependence to more (cytosol) positive potentials. A quantitative analysis results in a model that exactly describes the Ca²⁺-specific effects on the SV channel activation kinetics and voltage gating. According to this model, multiple (approximately three) divalent cations bind with a high affinity at the luminal interface of the membrane to the channel protein, favoring the occupancy of one of the SV channels closed states (C2). Transition to another closed state (C1) diminishes the effective number of bound cations, probably due to mutual repulsion, and channel opening is accompanied by a decrease of binding affinity. Hence, the open state (O) is destabilized with respect to the two closed states, C1 and C2, in the presence of Ca²⁺ at the vacuolar side. The specificity for Ca²⁺ compared to Mg²⁺ is explained in terms of different binding affinities for these cations. In this study we demonstrate that vacuolar Ca²⁺ is a crucial regulator to restrict SV channel activity to a physiologically meaningful range, which is less than 0.1% of maximum SV channel activity.Abbreviation SV Slow vacuolar     

Control of ionic currents in guard cell vacuoles by cytosolic and luminal calcium

Activity of vacuolar ion channels can be regulated by the cytosolic free Ca²⁺ concentration ([Ca²⁺]_cyt). Using the whole-vacuole mode of patch-clamp with Vicia faba guard cell vacuoles, three distinct cation currents were apparent that were differentially regulated by [Ca²⁺]_cyt. At ‘zero’ to 100 nM [Ca²⁺]_cyt, instantaneous currents typical of Fast Vacuolar (FV) channels were activated. A 10 fold KCl gradient directed out of the vacuole increased FV currents (up to fivefold) at negative potentials compared with the currents in symmetrical KCl. At [Ca²⁺]_cyt higher than 100 nM, instantaneous currents became smaller and voltage-independent (non-rectifying) and were typical of Vacuolar K,+-selective (VK) channels. These currents were less sensitive to a KCl gradient than were the FV currents, being stimulated less than twofold at negative potentials. Reversal potentials measured in the presence of a KCl gradient indicated a high K⁺ permeability of both FV and VK currents. At [Ca²⁺]_cyt higher than 600 nM time-dependent currents elicited by positive potentials were typical of Slow Vacuolar (SV) channel activation. When the Ca²⁺ mole fraction in the cytosolic or luminal solution was varied the reversal potential of SV currents (determined by tail current analysis) passed through maximum or minimum values. The resultant calculated apparent permeability ratios varied with ionic conditions but indicated high Ca²⁺ and K⁺ permeabilities. If a Cl^? permeability was assumed then the apparent P_Ca was lower. However, substitution of Cl^? by the larger (impermeant) anion gluconate had no effect on the reversal potential of SV tail currents in the presence of Ca²⁺ and a K⁺ gradient, demonstrating that the assumption of Cl^? permeability of the SV channel is invalid. Single-channel SV currents also decreased with increasing cytosolic Ca²⁺ mole fraction. These data indicate that the SV channel is highly cation selective, shows characteristics typical of a multi-ion pore and derives ion selectivity by Ca²⁺ binding. The SV channel currents could also be Mg²⁺-activated and were demonstrated to be Mg²⁺-permeable in the absence of Ca²⁺. The apparent permeability ratio (P_Mg:P_K) also varied under different ionic conditions. The results indicate not only that FV, VK and SV channels are all present in a single cell type, but also that each is differentially regulated by [Ca²⁺]_cyt. The respective roles of these channels in vacuolar ion release are discussed, and possible conditions are presented in which these channels could be activated by disparate signalling pathways during stomatal closure.     

Responses to Mg/Ca balance in an Iranian serpentine endemic plant, Cleome heratensis (Capparaceae) and a related non-serpentine species, C. foliolosa

A soil Ca/Mg quotient greater than unity is generally considered necessary for normal plant growth but some serpentine plants are adapted to much lower Ca/Mg quotients, resulting from a major cation imbalance in their substrata. In order to investigate the growth and tolerance responses of serpentine and non-serpentine species to varied Ca/Mg quotients, controlled nutrient solution experiments were performed using an a newly reported Iranian endemic serpentine plant, Cleome heratensis Bunge et Bien. Ex Boiss. and a related non-serpentine species Cleome foliolosa DC. and a Eurasian Ni-hyperaccumulating species Alyssum murale Waldst. and Kit. Seedlings were grown in modified Hoagland’s solutions with varying Ca and Mg concentrations (0.2–2.5 and 0.5–10 mM, respectively) in a fully factorial randomised block design. The yields of the two serpentine plants increased significantly as Mg concentrations in the nutrient solution were increased from 0.5 to 4 mM but decreased in the 10 mM Mg treatment. For C. foliolosa yields decreased significantly from 0.5 to 10 mM Mg, indicating the sensitivity of this non-serpentine plant, and the relative tolerance of the serpentine plants to extremely high levels of Mg. Shoot and root Mg and Ca concentrations in C. heratensis and A. murale were higher than those in C. foliolosa in the low and moderate Mg treatments, supporting the view that many serpentine plants have a relatively high requirement for Mg. Maximum Mg concentrations were found in the roots of C. heratensis. Yields of C. heratensis and A. murale did not change significantly as Ca levels in nutrient solution increased from 0.2 to 2.5 mM Ca, However the yield of C. foliolosa increased significantly from 0.2 to 1.5 mM Ca, indicating sensitivity in this non-serpentine plant and tolerance of the two serpentine plants to low levels of Ca correlated with tissue Ca concentrations, probably because of a greater ability for Ca uptake at low-Ca availability. Calcium deficiency in the low-Ca treatments could be a reason for reduced yield in the non-serpentine plants.     

Assessment of somaclonal variation in <Emphasis Type="Italic">Dieffenbachia</Emphasis> plants regenerated through indirect shoot organogenesis

The occurrence of somaclonal variation among regenerants derived through indirect shoot organogenesis from leaf explants of three Dieffenbachia cultivars Camouflage, Camille and Star Bright was evaluated. Three types of somaclonal variants (SV1, SV2, and SV3) were identified from regenerated plants of cv. Camouflage, one type from cv. Camille, but none from cv. Star Bright. The three variants had novel and distinct foliar variegation patterns compared to cv. Camouflage parental plants. Additionally, SV1 was taller with a larger canopy and longer leaves than parental plants and SV2. SV2 and SV3 did not produce basal shoots (single stem) but basal shoot numbers between SV1 and parental plants were similar ranging from three to four. The variant type identified from regenerated cv. Camille had lanceolate leaves compared to the oblong leaves of the parent. This variant type also grew taller and had a larger canopy than parental plants. The rates of somaclonal variation were up to 40.4% among regenerated cv. Camouflage plants and 2.6% for regenerated cv. Camille. The duration of callus culture had no effect on somaclonal variation rates of cv. Camouflage as the rates between plants regenerated from 8 months to 16 months of callus culture were similar. The phenotypes of the identified variants were stable as verified by their progenies after cutting propagation. This study demonstrated the potential for new cultivar development by selecting callus-derived somaclonal variants of Dieffenbachia.