Differential regulation of serine acetyltransferase is involved in nickel hyperaccumulation in Thlaspi goesingense

When growing in its native habitat, Thlaspi goesingense can hyperaccumulate 1.2% of its shoot dry weight as nickel. We reported previously that both constitutively elevated activity of serine acetyltransferase (SAT) and concentration of glutathione (GSH) are involved in the ability of T. goesingense to tolerate nickel. A feature of SAT is its feedback inhibition by L-cysteine. To understand the role of this regulation of SAT by Cys on GSH-mediated nickel tolerance in T. goesingense, we characterized the enzymatic properties of SATs from T. goesingense. We demonstrate that all three isoforms of SAT in T. goesingense are insensitive to inhibition by Cys. Further, two amino acids (proline and alanine) in the C-terminal region of the cytosolic SAT (SAT-c) from T. goesingense are responsible for converting the enzyme from a Cys-sensitive to a Cys-insensitive form. Furthermore, the Cys-insensitive isoform of SAT-c confers elevated resistance to nickel when expressed in Escherichia coli and Arabidopsis thaliana, supporting a role for altered regulation of SAT by Cys in nickel tolerance in T. goesingense.     

Constitutively elevated salicylic acid signals glutathione-mediated nickel tolerance in Thlaspi nickel hyperaccumulators

Progress is being made in understanding the biochemical and molecular basis of nickel (Ni)/zinc (Zn) hyperaccumulation in Thlaspi; however, the molecular signaling pathways that control these mechanisms are not understood. We observed that elevated concentrations of salicylic acid (SA), a molecule known to be involved in signaling induced pathogen defense responses in plants, is a strong predictor of Ni hyperaccumulation in the six diverse Thlaspi species investigated, including the hyperaccumulators Thlaspi goesingense, Thlaspi rosulare, Thlaspi oxyceras, and Thlaspi caerulescens and the nonaccumulators Thlaspi arvense and Thlaspi perfoliatum. Furthermore, the SA metabolites phenylalanine, cinnamic acid, salicyloyl-glucose, and catechol are also elevated in the hyperaccumulator T. goesingense when compared to the nonaccumulators Arabidopsis (Arabidopsis thaliana) and T. arvense. Elevation of free SA levels in Arabidopsis, both genetically and by exogenous feeding, enhances the specific activity of serine acetyltransferase, leading to elevated glutathione and increased Ni resistance. Such SA-mediated Ni resistance in Arabidopsis phenocopies the glutathione-based Ni tolerance previously observed in Thlaspi, suggesting a biochemical linkage between SA and Ni tolerance in this genus. Intriguingly, the hyperaccumulator T. goesingense also shows enhanced sensitivity to the pathogen powdery mildew (Erysiphe cruciferarum) and fails to induce SA biosynthesis after infection. Nickel hyperaccumulation reverses this pathogen hypersensitivity, suggesting that the interaction between pathogen resistance and Ni tolerance and hyperaccumulation may have played a critical role in the evolution of metal hyperaccumulation in the Thlaspi genus.     

The Role of Metal Transport and Tolerance in Nickel Hyperaccumulation by Thlaspi goesingense Halacsy

Metal hyperaccumulators are plants that are capable of extracting metals from the soil and accumulating them to extraordinary concentrations in aboveground tissues (greater than 0.1% dry biomass Ni or Co or greater than 1% dry biomass Zn or Mn). Approximately 400 hyperaccumulator species have been identified, according to the analysis of field-collected specimens. Metal hyperaccumulators are interesting model organisms to study for the development of a phytoremediation technology, the use of plants to remove pollutant metals from soils. However, little is known about the molecular, biochemical, and physiological processes that result in the hyperaccumulator phenotype. We investigated the role of Ni tolerance and transport in Ni hyperaccumulation by Thlaspi goesingense, using plant biomass production, evapotranspiration, and protoplast viability assays, and by following short- and long-term uptake of Ni into roots and shoots. As long as both species (T. goesingense and Thlaspi arvense) were unaffected by Ni toxicity, the rates of Ni translocation from roots to shoots were the same in both the hyper- and nonaccumulator species. Our data suggest that Ni tolerance is sufficient to explain the Ni hyperaccumulator phenotype observed in hydroponically cultured T. goesingense when compared with the Ni-sensitive nonhyperaccumulator T. arvense.     

Subcellular localization and speciation of nickel in hyperaccumulator and non-accumulator Thlaspi species

The ability of Thlaspi goesingense Hálácsy to hyperaccumulate Ni appears to be governed by its extraordinary degree of Ni tolerance. However, the physiological basis of this tolerance mechanism is unknown. We have investigated the role of vacuolar compartmentalization and chelation in this Ni tolerance. A direct comparison of Ni contents of vacuoles from leaves of T. goesingense and from the non-tolerant non-accumulator Thlaspi arvense L. showed that the hyperaccumulator accumulates approximately 2-fold more Ni in the vacuole than the non-accumulator under Ni exposure conditions that were non-toxic to both species. Using x-ray absorption spectroscopy we have been able to determine the likely identity of the compounds involved in chelating Ni within the leaf tissues of the hyperaccumulator and non-accumulator. This revealed that the majority of leaf Ni in the hyperaccumulator was associated with the cell wall, with the remaining Ni being associated with citrate and His, which we interpret as being localized primarily in the vacuolar and cytoplasm, respectively. This distribution of Ni was remarkably similar to that obtained by cell fractionation, supporting the hypothesis that in the hyperaccumulator, intracellular Ni is predominantly localized in the vacuole as a Ni-organic acid complex.     

Cellular compartmentation of nickel in the hyperaccumulators Alyssum lesbiacum, Alyssum bertolonii and Thlaspi goesingense.

Nickel uptake and cellular compartmentation were investigated in three Ni hyperaccumulators: Alyssum bertolonii (Desv), Alyssum lesbiacum (Candargy) and Thlaspi goesingense (Hálácsy). The three species showed similar hyperaccumulation of Ni, but T. goesingense was less tolerant to Ni than the two Alyssum species. An addition of 500 mg Ni kg(-1) to a nutrient-rich growth medium significantly increased shoot biomass of all three species, suggesting that the Ni hyperaccumulators have a higher requirement for Ni than normal plants. Energy-dispersive X-ray microanalysis (EDXA) was performed on frozen-hydrated tissues of leaves (all species) and stems (Alyssum only). In all species analysed, Ni was distributed preferentially in the epidermal cells, most likely in the vacuoles, of the leaves and stems. In stems, there was a second peak of Ni in the boundary cells between the cortical parenchyma and the vascular cylinder. The non-glandular trichomes on the leaf surfaces of the two Alyssum species were highly enriched with Ca, but contained little Ni except in the base. In the leaves of T. goesingense, the large elongated epidermal cells contained more Ni than the cells of the stomatal complexes. The role of cellular compartmentation in Ni hyperaccumulation is discussed.     

Cadmium accumulation in populations of Thlaspi caerulescens and Thlaspi goesingense

The capacity to accumulate cadmium (Cd) and zinc (Zn) was compared in Thlaspi goesingense and four populations of Thlaspi caerulescens . Two populations of T. caerulescens were grown in hydroponics at five concentrations of Cd. In addition, plants were grown in pots containing compost in which three different concentrations of Cd and two concentrations of Zn were added. A field trial was conducted to compare Zn and Cd uptake by three populations of T. caerulescens on nine selected plots of the Woburn Market Garden Experiment (UK) which had been contaminated to different degrees with heavy metals owing to past applications of sewage sludge. Results show that the four populations of T. caerulescens had the same ability to hyperaccumulate Zn but were significantly different in terms of Cd accumulation. Two populations of T. caerulescens from Southern France accumulated much more Cd than the populations from Prayon (Belgium) and Whitesike (UK). Generally, uptake of Cd was not decreased by increased concentrations of Zn in the substrate. These results indicate that the mechanisms of Cd and Zn hyperaccumulation are not identical in this species. This is the first report of hyperaccumulation of Cd by T. goesingense , but the growth of this species was markedly reduced by the large concentrations of Zn in the substrate. Future work should focus on the differences between Cd and Zn uptake in hyperaccumulator plants at the species and population level.     

Phytoextraction potential of the nickel hyperaccumulators Leptoplax emarginata and Bornmuellera tymphaea

Leptoplax emarginata and Bornmuellera tymphaea are nickel hyperaccumulators of the Brassicaceae family endemic to serpentine soils in Greece. The aims of this work were to compare the growth and uptake behavior of these plants with the Ni hyperaccumulator species Thlaspi caerulescens and Alyssum murale, and to evaluate their effect on soil Ni availability. Plants were grown for 3 mo on three soils that differ in Ni availability. Ni availability in soils was measuredby isotopic exchange kinetics and DTPA-TEA extractions. Results showed that L. emarginata produced significantly more biomass than other plants. On the serpentine soil, B. tymphaea showed the highest Ni concentration in shoots. However, Niphytoextraction on the three soils was maximal with L. emarginata. The high initial Ni availability of soil Serp (470.5 mg kg(-1)) was the main explanation for the high Ni concentrations measured in plant shoots grown on this soil, compared to those grown on soils Calc and Silt A. murale was the least efficient in reducing Ni availability on the serpentine soil L. emarginata appeared as the most efficient species for Ni phytoextraction and decrease of the Ni available pool.     

Soil moisture effects on uptake of metals by Thlaspi, Alyssum, and Berkheya

Most commonly used hyperaccumulator plants for phytoextraction of metals evolved on soils where moisture is limited throughout much of the year. As these plant species are commercialized for use, they are frequently moved from the point of evolution to locations where environmental conditions may be significantly different. Greatest among these potential differences is soil moisture. The objective of this study was therefore to determine whether these plants could grow in soils with much higher soil moisture and whether they would continue to hyperaccumulate metals as soils approach saturation. We examined extractable soil metal concentrations, plant growth, and metal accumulation for the Ni hyperaccumulators, Alyssum murale and Berkheya coddii and the Zn hyperaccumulators Thlaspi caerulescens cultivars AB300 and AB336. Non-hyperaccumulating control species for each were also examined. In general, extractable soil concentrations of Ni decreased with increasing soil moisture content. Few significant effects related to Zn extractability were observed for any of the soil moisture treatments. The biomass of all tested species was generally greater at higher soil moisture and inhibited at low soil moisture. Further, plants accumulated large amounts of metals from soil at higher soil moisture. Highest foliar concentrations of Zn or Ni were found at the two highest WHCs of 80 and 100%. These results show that hyperaccumulators grow well under conditions of high soil moisture content and that they continue to hyperaccumulate metals. Thus, growing Thlaspi, Alyssum, and Berkheya for commercial phytoextraction under nonnative conditions is appropriate and suggests that this technology may be applied to a wide and diverse range of soil types, climatic conditions, and irrigation regimes.     

Nickel and cobalt resistance engineered in Escherichia coli by overexpression of serine acetyltransferase from the nickel hyperaccumulator plant Thlaspi goesingense

The overexpression of serine acetyltransferase from the Ni-hyperaccumulating plant Thlaspi goesingense causes enhanced nickel and cobalt resistance in Escherichia coli. Furthermore, overexpression of T. goesingense serine acetyltransferase results in enhanced sensitivity to cadmium and has no significant effect on resistance to zinc. Enhanced nickel resistance is directly related to the constitutive overactivation of sulfur assimilation and glutathione biosynthesis, driven by the overproduction of O-acetyl-L-serine, the product of serine acetyltransferase and a positive regulator of the cysteine regulon. Nickel in the serine acetyltransferase-overexpressing strains is not detoxified by coordination or precipitation with sulfur, suggesting that glutathione is involved in reducing the oxidative damage imposed by nickel.     

The metal tolerance profile of <Emphasis Type="Italic">Thlaspi goesingense</Emphasis> is mimicked in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>heterologously expressing serine acetyl-transferase

Background  

The Ni hyperaccumulator Thlaspi goesingense is tolerant to Ni ≅ Zn, ≅ Co and slightly resistant to > Cd. We previously observed that elevated glutathione, driven by constitutive activation of serine acetyltransferase (SAT), plays a role in the Ni tolerance of T. goesingense.     

Bacterial communities associated with flowering plants of the Ni hyperaccumulator Thlaspi goesingense

Thlaspi goesingense is able to hyperaccumulate extremely high concentrations of Ni when grown in ultramafic soils. Recently it has been shown that rhizosphere bacteria may increase the heavy metal concentrations in hyperaccumulator plants significantly, whereas the role of endophytes has not been investigated yet. In this study the rhizosphere and shoot-associated (endophytic) bacteria colonizing T. goesingense were characterized in detail by using both cultivation and cultivation-independent techniques. Bacteria were identified by 16S rRNA sequence analysis, and isolates were further characterized regarding characteristics that may be relevant for a beneficial plant-microbe interaction-Ni tolerance, 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase and siderophore production. In the rhizosphere a high percentage of bacteria belonging to the Holophaga/Acidobacterium division and alpha-Proteobacteria were found. In addition, high-G+C gram-positive bacteria, Verrucomicrobia, and microbes of the Cytophaga/Flexibacter/Bacteroides division colonized the rhizosphere. The community structure of shoot-associated bacteria was highly different. The majority of clones affiliated with the Proteobacteria, but also bacteria belonging to the Cytophaga/Flexibacter/Bacteroides division, the Holophaga/Acidobacterium division, and the low-G+C gram-positive bacteria, were frequently found. A high number of highly related Sphingomonas 16S rRNA gene sequences were detected, which were also obtained by the cultivation of endophytes. Rhizosphere isolates belonged mainly to the genera Methylobacterium, Rhodococcus, and Okibacterium, whereas the majority of endophytes showed high levels of similarity to Methylobacterium mesophilicum. Additionally, Sphingomonas spp. were abundant. Isolates were resistant to Ni concentrations between 5 and 12 mM; however, endophytes generally tolerated higher Ni levels than rhizosphere bacteria. Almost all bacteria were able to produce siderophores. Various strains, particularly endophytes, were able to grow on ACC as the sole nitrogen source.     

Seasonal fluctuations of selenium and sulfur accumulation in selenium hyperaccumulators and related nonaccumulators

Some plants hyperaccumulate selenium (Se) up to 1% of dry weight. This study was performed to obtain insight into whole-plant Se fluxes in hyperaccumulators. Selenium hyperaccumulators Astragalus bisulcatus and Stanleya pinnata were monitored over two growing seasons for seasonal fluctuations in concentrations of Se and the chemically similar element sulfur (S). The related nonhyperaccumulators Astragalus sericoleucus, Oxytropis sericea and Thlaspi montanum were included for comparison. In both hyperaccumulators leaf Se decreased from April to October, coinciding with Se hyperaccumulation in flowers and seeds. Root Se levels were lowest in summer. Selenium concentration decreased with leaf age in both hyperaccumulators. Leaf S levels peaked in summer in all plant species, as did Se levels in nonhyperaccumulators. Selenium and S levels tended to be negatively correlated in hyperaccumulators, and positively correlated in nonhyperaccumulators. These results suggest a specific flow of Se in hyperaccumulator plants over the growing season, from root to young leaves in spring, followed by remobilization from aging leaves to reproductive tissues in summer, and back to roots in the autumn.     

Dominant-negative modification reveals the regulatory function of the multimeric cysteine synthase protein complex in transgenic tobacco

Cys synthesis in plants constitutes the entry of reduced sulfur from assimilatory sulfate reduction into metabolism. The catalyzing enzymes serine acetyltransferase (SAT) and O-acetylserine (OAS) thiol lyase (OAS-TL) reversibly form the heterooligomeric Cys synthase complex (CSC). Dominant-negative mutation of the CSC showed the crucial function for the regulation of Cys biosynthesis in vivo. An Arabidopsis thaliana SAT was overexpressed in the cytosol of transgenic tobacco (Nicotiana tabacum) plants in either enzymatically active or inactive forms that were both shown to interact efficiently with endogenous tobacco OAS-TL proteins. Active SAT expression resulted in a 40-fold increase in SAT activity and strong increases in the reaction intermediate OAS as well as Cys, glutathione, Met, and total sulfur contents. However, inactive SAT expression produced much greater enhancing effects, including 30-fold increased Cys levels, attributable, apparently, to the competition of inactive transgenic SAT with endogenous tobacco SAT for binding to OAS-TL. Expression levels of tobacco SAT and OAS-TL remained unaffected. Flux control coefficients suggested that the accumulation of OAS and Cys in both types of transgenic plants was accomplished by different mechanisms. These data provide evidence that the CSC and its subcellular compartmentation play a crucial role in the control of Cys biosynthesis, a unique function for a plant metabolic protein complex.     

Molecular dissection of the role of histidine in nickel hyperaccumulation in Thlaspi goesingense (Hálácsy)

To understand the role of free histidine (His) in Ni hyperaccumulation in Thlaspi goesingense, we investigated the regulation of His biosynthesis at both the molecular and biochemical levels. Three T. goesingense cDNAs encoding the following His biosynthetic enzymes, ATP phosphoribosyltransferase (THG1, GenBank accession no. AF003347), imidazoleglycerol phosphate dehydratase (THB1, GenBank accession no. AF023140), and histidinol dehydrogenase (THD1, GenBank accession no. AF023141) were isolated by functional complementation of Escherichia coli His auxotrophs. Northern analysis of THG1, THD1, and THB1 gene expression revealed that each gene is expressed in both roots and shoots, but at the concentrations and dosage times of Ni treatment used in this study, these genes failed to show any regulation by Ni. We were also unable to observe any increases in the concentration of free His in root, shoot, or xylem sap of T. goesingense in response to Ni exposure. X-ray absorption spectroscopy of root and shoot tissue from T. goesingense and the non-accumulator species Thlaspi arvense revealed no major differences in the coordination of Ni by His in these tissues. We therefore conclude that the Ni hyperaccumulation phenotype in T. goesingense is not determined by the overproduction of His in response to Ni.     

MTP1-dependent Zn sequestration into shoot vacuoles suggests dual roles in Zn tolerance and accumulation in Zn-hyperaccumulating plants

The integral membrane protein Thlaspi goesingense metal tolerance protein 1 (TgMTP1) has been suggested to play an important role in Zn hyperaccumulation in T. goesingense . Here, we show that the TgMTP1 protein is accumulated to high levels at the vacuolar membrane in shoot tissue of T. goesingense . TgMTP1 is likely to act in the transport of Zn into the vacuole, enhancing both Zn accumulation and tolerance. By specifically expressing TgMTP1 in Arabidopsis thaliana shoots, we show that TgMTP1, localized at the vacuolar membrane, can drive the enhanced shoot accumulation of Zn by initiating a systemic Zn deficiency response. The systematic response includes increased expression of Zn transporters ( ZIP3 , ZIP4 , ZIP5 and ZIP9 ) in both shoot and root tissue. Furthermore, shoot-specific accumulation of TgMTP1 at the vacuolar membrane also leads to increased resistance to Zn in A. thaliana , probably through enhanced Zn compartmentalization in the vacuole. Such evidence leads to the conclusion that the high levels of TgMTP1 at the vacuolar membrane in shoot tissue of the Zn hyperaccumulator T. goesingense play a role in both Zn tolerance and enhanced Zn uptake and accumulation, via the activation of a systemic Zn deficiency response.     

Mitochondrial serine acetyltransferase functions as a pacemaker of cysteine synthesis in plant cells

Cysteine (Cys) synthesis in plants is carried out by two sequential reactions catalyzed by the rate-limiting enzyme serine acetyltransferase (SAT) and excess amounts of O-acetylserine(thiol)lyase. Why these reactions occur in plastids, mitochondria, and cytosol of plants remained unclear. Expression of artificial microRNA (amiRNA) against Sat3 encoding mitochondrial SAT3 in transgenic Arabidopsis (Arabidopsis thaliana) plants demonstrates that mitochondria are the most important compartment for the synthesis of O-acetylserine (OAS), the precursor of Cys. Reduction of RNA levels, protein contents, SAT enzymatic activity, and phenotype strongly correlate in independent amiSAT3 lines and cause significantly retarded growth. The expression of the other four Sat genes in the Arabidopsis genome are not affected by amiRNA-SAT3 according to quantitative real-time polymerase chain reaction and microarray analyses. Application of radiolabeled serine to leaf pieces revealed severely reduced incorporation rates into Cys and even more so into glutathione. Accordingly, steady-state levels of OAS are 4-fold reduced. Decrease of sulfate reduction-related genes is accompanied by an accumulation of sulfate in amiSAT3 lines. These results unequivocally show that mitochondria provide the bulk of OAS in the plant cell and are the likely site of flux regulation. Together with recent data, the cytosol appears to be a major site of Cys synthesis, while plastids contribute reduced sulfur as sulfide. Thus, Cys synthesis in plants is significantly different from that in nonphotosynthetic eukaryotes at the cellular level.     

Evidence for copper homeostasis function of metallothionein (MT3) in the hyperaccumulator Thlaspi caerulescens

Metallothioneins chelate metals and consequently may be a control point of metal homeostasis. Homologous to type 3 metallothioneins, TcMT3 cDNA was identified in the Cd/Zn hyperaccumulator, Thlaspi caerulescens. TcMT3 amino acid sequence showed modifications in the Cys positions when compared with its Arabidopsis orthologue. A structural model established that the MT3 carboxyterminal domain is similar to the beta domain of animal metallothioneins and predicts a smaller cavity to chelate metals for A. thaliana than for T. caerulescens. Functional testing in yeast and Northern blot analysis added further evidence for adaptative variations of MT3 for the maintenance of Cu homeostasis in a metal hyperaccumulator.     

The plant CDF family member TgMTP1 from the Ni/Zn hyperaccumulator Thlaspi goesingense acts to enhance efflux of Zn at the plasma membrane when expressed in Saccharomyces cerevisiae

To avoid metal toxicity, organisms have evolved mechanisms including efflux of metal ions from cells and sequestration into internal cellular compartments. Members of the ubiquitous cation diffusion facilitator (CDF) family are known to play an important role in these processes. Overexpression of the plant CDF family member metal tolerance protein 1 (MTP1) from the Ni/Zn hyperaccumulator Thlaspi goesingense (TgMTP1), in the Saccharomyces cerevisiaeDelta zinc resistance conferring (zrc)1Delta cobalt transporter (cot)1 double mutant, suppressed the Zn sensitivity of this strain. T. goesingense was found to contain several allelic variants of TgMTP1, all of which confer similar resistance to Zn in Deltazrc1Deltacot1. Similarly, MTP1 from various hyperaccumulator and non-accumulator species also confer similar resistance to Zn. Deltazrc1Deltacot1 lacks the ability to accumulate Zn in the vacuole and has lower accumulation of Zn after either long- or short-term Zn exposure. Expression of TgMTP1 in Deltazrc1Deltacot1 leads to further lowering of Zn accumulation and an increase in Zn efflux from the cells. Expression of TgMTP1 in a V-type ATPase-deficient S. cerevisiae strain also confers increased Zn resistance. In vivo and in vitro immunological staining of hemagglutinin (HA)-tagged TgMTP1::HA reveals the protein to be localized in both the S. cerevisiae vacuolar and plasma membranes. Taken together, these data are consistent with MTP1 functioning to enhance plasma membrane Zn efflux, acting to confer Zn resistance independent of the vacuole in S. cerevisiae. Transient expression in Arabidopsis thaliana protoplasts also reveals that TgMTP1::green fluorescent protein (GFP) is localized at the plasma membrane, suggesting that TgMTP1 may also enhance Zn efflux in plants.     

The role of free histidine in xylem loading of nickel in Alyssum lesbiacum and Brassica juncea

Exposure of the hyperaccumulator Alyssum lesbiacum to nickel (Ni) is known to result in a dose-dependent increase in xylem sap concentrations of Ni and the chelator free histidine (His). Addition of equimolar concentrations of exogenous L-His to an Ni-amended hydroponic rooting medium enhances Ni flux into the xylem in the nonaccumulator Alyssum montanum, and, as reported here, in Brassica juncea L. cv Vitasso. In B. juncea, reducing the entry of L-His into the root by supplying D-His instead of L-His, or L-His in the presence of a 10-fold excess of L-alanine, did not affect root Ni uptake, but reduced Ni release into the xylem. Compared with B. juncea, root His concentrations were constitutively about 4.4-fold higher in A. lesbiacum, and did not increase within 9 h of exposure to Ni. Cycloheximide did not affect root His or Ni concentrations, but strongly decreased the release of His and Ni from the root into the xylem of A. lesbiacum, whereas xylem sap concentrations of Ca and Mg remained unaffected. Near-quantitative chelation of Ni with nitrilotriacetate in the rooting medium did not enhance Ni flux into the xylem of A. lesbiacum and B. juncea, suggesting the absence of a significant apoplastic pathway for Ni entry into the xylem. The data suggest that in B. juncea roots, Ni(2+) uptake is independent of simultaneous uptake of His. In both species, enhanced release of Ni into the xylem is associated with concurrent release of His from an increased root free His pool.