Towards Practical Cadmium Phytoextraction with Noccaea Caerulescens

A series of field trials were conducted to investigate the potential of Noccaea caerulescens F.K. Mey [syn. Thlaspi caerulescens J &C Presl. (see Koch and Al-Shehbaz 2004)] populations (genotypes) derived from southern France to phytoextract localized Cd/Zn contamination in Thailand. Soil treatments included pH variation and fertilization level and application of fungicide. N. caerulescens populations were transplanted to the field plots three months after germination and harvested in May, prior to the onset of seasonal rains. During this period growth was rapid with shoot biomass ranging from 0.93–2.2 g plant^–1 (280–650 kg ha^–1) DW. Shoot Cd and Zn concentrations for the four populations evaluated ranged from 460–600 and 2600–2900 mg kg^–1 DW respectively. Cadmium and Zn Translocation Factors (shoot/root) for the populations tested ranged from 0.91–1.0 and 1.7–2.1 and Bioaccumulation Factors ranged from 12–15 and 1.2–1.3. We conclude that optimizing the use of fungicidal sprays, acidic soil pH, planting density and increasing the effective cropping period will increase rates of Cd and Zn removal enough to facilitate practical Cd phytoextraction from rice paddy soils in Thailand.     

Generation of nonvernal-obligate, faster-cycling Noccaea caerulescens lines through fast neutron mutagenesis

Noccaea caerulescens (formerly Thlaspi caerulescens) is a widely studied metal hyperaccumulator. However, molecular genetic studies are challenging in this species because of its vernal-obligate biennial life cycle of 7-9months. Here, we describe the development of genetically stable, faster cycling lines of N. caerulescens which are nonvernal-obligate. A total of 5500 M(0) seeds from Saint Laurent Le Minier (France) were subjected to fast neutron mutagenesis. Following vernalization of young plants, 79% of plants survived to maturity. In all, 80,000 M(2) lines were screened for flowering in the absence of vernalization. Floral initials were observed in 35 lines, with nine flowering in <12wk. Two lines (A2 and A7) were selfed to the M(4) generation. Floral initials were observed 66 and 87d after sowing (DAS) in A2 and A7, respectively. Silicle development occurred for all A2 and for most A7 at 92 and 123 DAS, respectively. Floral or silicle development was not observed in wild-type (WT) plants. Leaf zinc (Zn) concentration was similar in WT, A2 and A7 lines. These lines should facilitate future genetic studies of this remarkable species. Seed is publicly available through the European Arabidopsis Stock Centre (NASC).     

A new species of Noccaea (Brassicaceae) from South Anatolia, Turkey

Noccaea camlikensis sp. nov. (Brassicaceae) is described and illustrated from a localized serpentine range in southern Turkey. Its close relationship to N. cariensis , from which it differs in leaf and carpological characters, is discussed. The protologue of this fairly unknown species can now be amended to cover better the morphological variability of this taxon. A comparative study of seed-coat anatomy advocates its move from Thlaspi to Noccaea ; the relevant combination N. cariensis (Carlström) Parolly, Nordt & Aytaç is made. The ecology of both vicarious species is discussed and their distribution mapped.  © 2006 The Linnean Society of London, Botanical Journal of the Linnean Society , 2006, 150 , 409–416.     

Metal concentration and metal mass of metallicolous, non metallicolous and serpentine Noccaea caerulescens populations, cultivated in different growth media

Aims

Evaluate the genetic and environmental variability of metal concentration and metal mass of Noccaea caerulescens, from metalliferous (MET), non metalliferous (NMET) and serpentine (SERP) soils.

Methods

18 populations were cultivated in 18 different growth conditions, such as a soil mine tailing, soils amended with zinc (Zn), cadmium (Cd) and nickel (Ni) salts (in mixtures or in monometallic salts) and a hydroponic solution with two Zn concentrations.

Results

MET populations had Zn concentrations lower than NMET and SERP in the different soils but higher Cd mass (the product of aerial biomass and foliar metal concentration). SERP had the highest Ni concentration and Ni mass values. The addition of Cd or Ni to a Zn-contaminated soil significantly decreases Zn concentration. In hydroponics, MET and NMET had equivalent Zn concentrations but these were three times higher than those obtained in soil experiments. Zn mass of NMET was significantly lower than MET with the latter having Zn mass values largely above those obtained in mine soil.

Conclusions

Results showed a large heterogeneity of responses among populations depending on the substrate used, and it was not possible to correctly assign a single population to its accurate origin with only one experiment. Finally, data on metal concentration obtained in culture soils are closer to those in field soils than those from hydroponics so that they could give a more accurate information on the accumulating capacity of Noccaea caerulescens and its use in phytoextraction of metals in field conditions.     

Lead Accumulation Ability of Selected Plants of Noccaea spp.

Several species of the Noccaea genus are known for their hyperaccumulation ability especially in the case of Cd, Ni, and Zn. However, ambiguous observations were previously published concerning their accumulation properties for Pb. The Pb accumulation properties of Noccaea rotundifolia, Noccaea montana, and Noccaea jankae hungarica plants were tested in field and pot experiments in soils differing in the mobile pool of Pb, as well as in soilless hydroponic culture. The Pb content in the dry biomass of plant shoots reached up to 54 mg/kg in field conditions and 84 mg/kg in pots regardless of the bioavailable pool of Pb in the pots. The hydroponic experiment showed a stepwise increase in Pb content in plant biomass with increasing Pb concentration in the solution, but the predominant proportion of plant Pb was retained in the roots. Although the hyperaccumulation ability of some of the Noccaea species is widely discussed in the literature, our results are in agreement with those suggesting no Pb hyperaccumulation potential in these plants.     

Growth and Cadmium Phytoextraction by Swiss Chard,Maize, Rice,Noccaea caerulescens,and Alyssum murale in pH Adjusted Biosolids Amended Soils

Past applications of biosolids to soils at some locations added higher Cd levels than presently permitted. Cadmium phytoextraction would alleviate current land use constraints. Unamended farm soil, and biosolids amended farm and mine soils were obtained from a Fulton Co., IL biosolids management facility. Soils contained 0.16, 22.8, 45.3 mg Cd kg^–1 and 43.1, 482, 812 mg Zn kg^–1 respectively with initial pH 6.0, 6.1, 6.4. In greenhouse studies, Swiss chard (Beta vulgaris var. cicla), a Cd-accumulator maize (inbred B37 Zea mays) and a southern France Cd-hyperaccumulator genotype of Noccaea caerulescens were tested for Cd accumulation and phytoextraction. Soil pH was adjusted from ～5.5–7.0. Additionally 100 rice (Oryza sativa) genotypes and the Ni-hyperaccumulator Alyssum murale were screened for potential phytoextraction use.

Chard suffered phytotoxicity at low pH and accumulated up to 90 mg Cd kg^–1 on the biosolids amended mine soil. The maize inbred accumulated up to 45 mg Cd kg^–1 with only mild phytotoxicity symptoms during early growth at pH > 6.0. N. caerulescens did not exhibit phytotoxicity symptoms at any pH, and accumulated up to 235 mg Cd kg^–1 in 3 months. Reharvested N. caerulescens accumulated up to 900 mg Cd kg^–1 after 10 months. Neither Alyssum nor 90% of rice genotypes survived acceptably.

Both N. caerulescens and B37 maize show promise for Cd phytoextraction in IL and require field evaluation; both plants could be utilized for nearly continuous Cd removal. Other maize inbreds may offer higher Cd phytoextraction at lower pH, and mono-cross hybrids higher shoot biomass yields. Further, maize grown only for biomass Cd maximum removal could be double-cropped.     

Nitrate nutrition enhances zinc hyperaccumulation in Noccaea caerulescens (Prayon)

Nitrate fertilization has been shown to increase Zn hyperaccumulation by Noccaea caerulescens (Prayon) (formerly Thlaspi caerulescens). However, it is unknown whether this increased hyperaccumulation is a direct result of NO₃ ^? nutrition or due to changes in rhizosphere pH as a result of NO₃ ^? uptake. This paper investigated the mechanism of NO₃ ^?-enhanced Zn hyperaccumulation in N. caerulescens by assessing the response of Zn uptake to N form and solution pH. Plants were grown in nutrient solution with 300 μM Zn and supplied with either (NH₄)₂SO₄, NH₄NO₃ or Ca(NO₃)₂. The solutions were buffered at either pH 4.5 or 6.5. The Zn concentration and content were much higher in shoots of NO₃ ^?-fed plants than in NH₄ ⁺-fed plants at pH 4.5 and 6.5. The Zn concentration in the shoots was mainly enhanced by NO₃ ^?, whereas the Zn concentration in the roots was mainly enhanced by pH 6.5. Nitrate increased Zn uptake in the roots at pH 6.5 and increased apoplastic Zn at pH 4.5. Zinc and Ca co-increased and was found co-localized in leaf cells of NO₃ ^?-fed plants. We conclude that NO₃ ^? directly enhanced Zn uptake and translocation from roots to shoots in N. caerulescens.     

Trade-off between genetic variation and ecological adaptation of metallicolous and non-metallicolous Noccaea and Thlaspi species

Forty-seven accessions of Noccaea and Thlaspi grown in the presence of Ni were phenotyped for leaf mineral concentrations and morphology. Sequences of 9 target genes involved in metal homeostasis were compared in a Ni-adapted population of Noccaea caerulescens grown on the serpentine Monte Prinzera (MP), and in accessions from metalliferous and non-metalliferous soils. The MP population, which resembled most the Zn/Cd accumulators from Austria, showed little genetic variation. Higher levels of sequence variation found in target genes compared to non-target genes, suggest that selective pressure by exposure to high concentrations of metals may have led to adaptation to metalliferous environments, in trade-off with genetic variation. The ZNT2 zinc deficiency responsive zinc transporter gene showed significant variation in a selection test. The hydrophobicity profile of ZNT2 variants indicates, in this case, sequence variation is likely to affect the function of the encoded protein.     

Natural variation of nickel,zinc and cadmium (hyper)accumulation in facultative serpentinophytes Noccaea kovatsii and N. praecox

Aims

Data on the variability of hyperaccumulation potential of the facultative serpentinophytes Noccaea kovatsii and N. praecox on different geological substrates are scarce. The aim of this study was to assess the accumulation potential of these two species from ultramafic and non-ultramafic substrates, with special emphasis on the hyperaccumulation of Ni, Zn and Cd.

Methods

Samples of plants and corresponding soils were collected from 16 sites covering a wide range of geochemistry. Elemental concentrations were determined in the roots, shoots and the rhizosphere soils. The pH, particle size distribution and content of organic matter were also determined in the soil samples.

Results

All ultramafic accessions of both species hyperaccumulated Ni with high intraspecific variability. Only one accession of N. kovatsii from a schist soil hyperaccumulated Zn, with also a high Cd accumulation. Accumulation and translocation of Ni in both species were much higher in the ultramafic accessions, whereas Zn accumulation and translocation was found in both ultramafic and non-ultramafic accessions.

Conclusions

Ni accumulation and translocation was restricted to ultramafic populations of both species, whereas it is a species-wide trait for Zn. This study provides new and comprehensive information on the natural variation of hyperaccumulation of Ni, Zn and Cd in N. kovatsii and N. praecox.

     

Variation of trace metal accumulation,major nutrient uptake and growth parameters and their correlations in 22 populations of Noccaea caerulescens

Background and aims

Noccaea caerulescens is a model plant for the understanding of trace metal accumulation and a source of cultivars for phytoextraction. The aim of this study was to investigate natural variation for trace metal accumulation, major nutrient uptake and growth parameters in 22 populations. The correlations among these traits were particularly examined to better understand the eco-physiology and the phytoextraction potential of the species.

Methods

Populations from three edaphic groups, i.e. calamine (CAL), serpentine (SERP) and non metalliferous (NMET) sites were grown in hydroponics for seven weeks at moderate trace metal exposure. Growth indicators, element contents and correlations between these variables were compared.

Results

All the phenotypic characteristics showed a wide variability among groups and populations. The SERP populations showed a smaller plant size, higher cation contents and strong correlations between all element concentrations. NMET populations did not differ in plant size from the CAL ones, but had higher Zn and Ni contents. The CAL populations showed higher Cd and Mn accumulations and lower Ca contents. The trade-off between biomass production and Cd, Ni and Zn accumulation was high in SERP populations and low in the CAL and NMET ones.

Conclusions

N. caerulescens is a genetically diverse species, showing specific features depending on the group and the population. These features may reflect the wide adaptive capacities of the species, and also reveal promising potential for phytoextraction of Cd, Ni and Zn.     

Application of rhizosphere interaction of hyperaccumulator Noccaea caerulescens to remediate cadmium-contaminated agricultural soil

There is an urgent requirement for selecting appropriate technologies to solve food safety problems due to soil contamination. In this study, the hyperaccumulator Noccaea caerulescens and a high Cd accumulator pakchoi cultivar (Brassica rapa L. spp. Chinenesis cv.) were grown in a moderately Cd-contaminated soil with three planting systems (monocrop, inter-crop, and crop-rotation) and three growing durations (25, 50, and 75 days) to study the role of rhizosphere interaction of both species on the uptake of Cd. The Cd accumulations in the shoot of pakchoi were significantly reduced in the inter-crop treatment, also the decreased percentage increased with rhizosphere interaction between the two species. In the inter-crop systems of 75 days, the Cd concentration and amount in the shoot of pakchoi represented 54% and 83% reduction, respectively, while the total depletion of Cd decreased by approximate 19%. Although the Cd concentration and amount in the shoot of pakchoi were significantly reduced by 52% and 44%, respectively, in the crop-rotation treatment, the decreased percentage were markedly lower than in the inter-crop treatment. Therefore, the rhizosphere interaction of hyperaccumulator with non-hyperaccumulator may reduce the risk of vegetable contamination during making full use of or remediating the contaminated soil.     

Accumulation of Nickel by Excluder Thlaspi arvense and Hyperaccumulator Noccaea caerulescens upon Short-Term and Long-Term Exposure

Russian Journal of Plant Physiology - The ability to accumulate nickel (Ni) was compared in hyperaccumulator Noccaea сaerulescens F.K.&nbsp;Mey and excluder Thlaspi arvense L. after a...     

Nickel translocation via the phloem in the hyperaccumulator Noccaea caerulescens (Brassicaceae)

Plant and Soil - This study evaluated the effect of phloem translocation on Ni accumulation in the hyperaccumulator Noccaea caerulescens. The first experiment assessed the metal and organic...     

Anthocyanin production in the hyperaccumulator plant Noccaea caerulescens in response to herbivory and zinc stress

Stomatal behavior in response to drought has been the focus of intensive research, but less attention has been paid to stomatal density. In this study, 5-week-old maize seedlings were exposed to different soil water contents. Stomatal density and size as well as leaf gas exchange were investigated after 2-, 4- and 6-week of treatment, which corresponded to the jointing, trumpeting, and filling stages of maize development. Results showed that new stomata were generated continually during leaf growth. Reduced soil water content significantly stimulated stomatal generation, resulting in a significant increase in stomatal density but a decrease in stomatal size and aperture. Independent of soil water conditions, stomatal density and length in the trumpeting and filling stages were greater than in the jointing stage. Irrespective of growth stage, severe water deficit significantly reduced stomatal conductance (G _s), decreasing the leaf transpiration rate (T _r) and net photosynthetic rate (P _n). Stomatal density was significantly negatively correlated with both P _n and T _r but more strongly with T _r, so the leaf instantaneous water use efficiency (WUE _i ) correlated positively with stomatal density. In conclusion, drought led to a significant increase in stomatal density and a reduction in stomatal size and aperture, resulting in decreased P _n and T _r. Because the negative correlation of stomatal density to T _r was stronger than that to P _n, leaf WUE _i tended to increase.     

In vivo speciation of zinc in Noccaea caerulescens in response to nitrogen form and zinc exposure

Nitrate has been shown to enhance Zn hyperaccumulation in the shoots of Noccaea caerulescens (formerly Thlaspi caerulescens) (Prayon); however, the mechanisms beyond the effect of nitrogen form are unknown. This study used synchrotron X-ray absorption near-edge spectroscopy (XANES) on alive and intact plants at room temperature to examine whether enhanced Zn hyperaccumulation in nitrate-fed plants was associated with differences in Zn speciation, and to correlate Zn species with mechanisms of Zn uptake, translocation and hyperaccumulation. The higher Zn concentration in plants supplied with nitrate compared to ammonium, or with high Zn exposure (300 ???), was not due to differences in Zn speciation. The importance of carboxylates for Zn hyperaccumulation in the shoots was supported by a predominance of Zn-malate or Zn-citrate. Zinc-phytate was detected for the first time in this species and may assist Zn-tolerance in the roots. The feasible presence of Zn-histidine in the roots but not in the xylem sap suggests a mechanism for Zn binding and non-toxic transport through the cytoplasm and release of aqueous Zn into the xylem vessels. Zinc was translocated in the xylem as Zn-malate and weakly complexed or aqueous Zn forms. Zinc speciation in roots, shoots and xylem did not differ between nitrate- and ammonium-fed plants.     

Genome Structure of the Heavy Metal Hyperaccumulator Noccaea caerulescens and Its Stability on Metalliferous and Nonmetalliferous Soils

Noccaea caerulescens (formerly known as Thlaspi caerulescens), an extremophile heavy metal hyperaccumulator model plant in the Brassicaceae family, is a morphologically and phenotypically diverse species exhibiting metal tolerance and leaf accumulation of zinc, cadmium, and nickel. Here, we provide a detailed genome structure of the approximately 267-Mb N. caerulescens genome, which has descended from seven chromosomes of the ancestral proto-Calepineae Karyotype (n = 7) through an unusually high number of pericentric inversions. Genome analysis in two other related species, Noccaea jankae and Raparia bulbosa, showed that all three species, and thus probably the entire Coluteocarpeae tribe, have descended from the proto-Calepineae Karyotype. All three analyzed species share the chromosome structure of six out of seven chromosomes and an unusually high metal accumulation in leaves, which remains moderate in N. jankae and R. bulbosa and is extreme in N. caerulescens. Among these species, N. caerulescens has the most derived karyotype, with species-specific inversions on chromosome NC6, which grouped onto its bottom arm functionally related genes of zinc and iron metal homeostasis comprising the major candidate genes NICOTIANAMINE SYNTHASE2 and ZINC-INDUCED FACILITATOR-LIKE1. Concurrently, copper and organellar metal homeostasis genes, which are functionally unrelated to the extreme traits characteristic of N. caerulescens, were grouped onto the top arm of NC6. Compared with Arabidopsis thaliana, more distal chromosomal positions in N. caerulescens were enriched among more highly expressed metal homeostasis genes but not among other groups of genes. Thus, chromosome rearrangements could have facilitated the evolution of enhanced metal homeostasis gene expression, a known hallmark of metal hyperaccumulation.Noccaea caerulescens (formerly known as Thlaspi caerulescens) is a diploid (2n = 14) biennial or short-living perennial plant from the family Brassicaceae. N. caerulescens is native to Europe, with a patchy distribution from the United Kingdom and France to Slovakia and from Germany and Poland southward to northern Spain and Italy. The widespread occurrence in Scandinavia is thought to represent naturalized populations over the past few hundred years (Koch et al., 1998, and refs. therein). N. caerulescens is one of the 120 Noccaea spp., which, together with two other genera, constitute the tribe Coluteocarpeae (approximately 127 species; Al-Shehbaz, 2012). However, the generic treatment of the tribe is far from settled, and up to 12 genera are recognized in Coluteocarpeae by F.K. Meyer (Meyer, 2001, 2006a, 2006b; Koch and German, 2013).Together with the metal hyperaccumulator species Arabidopsis halleri, N. caerulescens is among the most prominent plant model systems for the study of heavy metal hyperaccumulation and associated hypertolerance (Krämer, 2010; Hanikenne and Nouet, 2011; Pollard et al., 2014). N. caerulescens is a hyperaccumulator of zinc (Zn) on metalliferous as well as nonmetalliferous soils and of cadmium (Cd) on metalliferous soils (Reeves et al., 2001; Krämer, 2010), and its populations on serpentine soils are known to hyperaccumulate nickel (Ni; Reeves and Brooks, 1983; Reeves, 1988; Visioli et al., 2012; Maestri et al., 2013). N. caerulescens is a morphologically highly diverse species comprising at least two, but up to eight (http://www.gbif.org), recognized subspecies with partly overlapping distribution ranges (the simplest treatment includes ssp. caerulescens and sylvestris, the latter including populations formerly described as Thlaspi calaminare or N. caerulescens ssp. calaminare). As the morphological variation often has a clinal character, the taxonomic and biological value of various intraspecific taxa is questionable, and detailed studies will be needed to resolve this issue (for discussion, see Koch and German, 2013). In addition to pervasive morphological variation (Koch and German, 2013) apparently reinforced by geographical constraints (Besnard et al., 2009), there is also pronounced phenotypic variation in metal tolerance and accumulation (Escarré et al., 2000; Reeves et al., 2001; Assunção et al., 2006; Xing et al., 2008; Krämer, 2010; Tuomainen et al., 2010).Although N. caerulescens is the most commonly studied metal hyperaccumulator model species, with more than 210 studies published on the subject (Pollard et al., 2014), the detailed genome structure of N. caerulescens remains unresolved. Assunção et al. (2006) published the first amplified fragment-length polymorphism-based genetic linkage map and identified quantitative trait loci for Zn accumulation in roots. Another amplified fragment-length polymorphism-based genetic map based on a cross between two accessions with differential Cd accumulation and tolerance was used to identify quantitative trait loci associated with the accumulation of Cd and Zn (Deniau et al., 2006). Both maps comprised the expected seven linkage groups with dense clusters of linked markers located on each linkage group, most likely corresponding to centromeric regions with suppressed recombination rates. Apart from the tentative identification of centromeres, small numbers of orthologous markers shared with Arabidopsis thaliana and A. halleri did not allow the establishment of chromosome collinearity between these genomes for inference of the genome structure of N. caerulescens.More recently, Mandáková and Lysak (2008) reconstructed karyotype evolution in eight Brassicaceae species of tribes in extended lineage II (Franzke et al., 2011) by comparative chromosome painting (CCP) using chromosome-specific bacterial artificial chromosome (BAC) contigs of A. thaliana. They concluded that genomes of all the analyzed species with seven or 14 chromosome pairs (n = 7/14) were derived from the eight chromosomes of the Ancestral Crucifer Karyotype (ACK; n = 8) via an ancestral n = 7 genome named the proto-Calepineae Karyotype (PCK). The genome of N. caerulescens (accession Korenec), analyzed as a representative of the tribe Coluteocarpeae (formerly Noccaeeae) by Mandáková and Lysak (2008), has descended from a PCK-like ancestor but showed a remarkably high number of secondary chromosome rearrangements. By comparison with the ancestral PCK, in N. caerulescens six of the seven ancestral chromosomes were reshuffled by inversions encompassing pericentromeric regions. However, that study did not establish the detailed structure of the N. caerulescens genome, including precise positions of chromosome break points. Consequently, evolutionary steps leading to the origin of the inversion chromosomes were reconstructed only approximately, and gene content could not be estimated.Our study here provides a detailed comparative genome structure of N. caerulescens and relates the genome structure to the evolution of heavy metal-related extreme traits. Based on a more precise definition of ancestral genomic blocks within the ACK (Cheng et al., 2013) and using PCK as the most probable ancestral genome of N. caerulescens, we carried out a detailed analysis of the entire karyotype, including inversions, by means of CCP. Furthermore, considering the unusually high incidence of inversions, we were intrigued to find out whether this variation is unique to the reference accession (Mandáková and Lysak, 2008) or fixed in populations on both nonmetalliferous and metalliferous soils enriched for different heavy metal elements. Toward this aim, we constructed detailed comparative karyotypes for 13 populations of N. caerulescens from metalliferous and nonmetalliferous soils throughout the European species distribution range, including, in particular, populations from southern France known for the extraordinarily high concentrations of Zn and Cd in their leaves (St. Félix de Pallières and Viviez; Reeves et al., 2001). Next, we asked whether the inversions are specific to N. caerulescens or shared by other Coluteocarpeae species by deducing the chromosome structure and evolution of the N. caerulescens genome in comparison with two other species from the tribe Coluteocarpeae, Noccaea jankae and Raparia bulbosa. Finally, we addressed the question of whether chromosome inversions and rearrangements might have affected the physical positions of metal homeostasis candidate genes that were proposed to act in naturally selected metal hyperaccumulation and associated hypertolerance of N. caerulescens. We tested for the clustering of functionally related metal homeostasis genes in closer proximity on the chromosome toward supergene formation as well as for the relation between changes in chromosomal position and gene expression among metal homeostasis genes.     

Organic acids are not specifically involved in the nitrate-enhanced Zn hyperaccumulation mechanism in Noccaea caerulescens

Nitrogen form has been shown to affect Zn uptake, translocation and storage in the Zn-hyperaccumulating plant Noccaea caerulescens but the biochemical processes are not fully understood. Organic acids and amino acids have been implicated in Zn transport and storage. This study aimed to examine the effect of N form on concentrations of organic acids and amino acids and how these metabolites correlated with Zn hyperaccumulation. Plants were grown in nutrient solution with NO₃⁻, NH₄NO₃ or NH₄⁺, supplied with 50 or 300 μM Zn, and buffered at either pH 4.5 or 6.5. The metabolomic profile was determined by gas chromatography mass spectroscopy. The concentration of Zn in shoots, xylem and roots was greatest for the NO₃⁻, pH 6.5 and 300 μM Zn treatments. For all N forms, the lower growth-medium pH raised xylem sap pH but had no influence on Zn concentration or exudation rate of the xylem sap. Nitrate enhanced organic acid production while NH₄⁺ increased amino acid production. Organic acids in the xylem were more responsive to changes in growth-medium pH than N form, and did not correlate with Zn concentration in shoots, roots or xylem. Serine might be directly involved in Zn hyperaccumulation. Phosphoric acid was associated with reduced Zn accumulation in the shoots. Malic acid was not detected in the shoots but responded to cation uptake more than to Zn specifically in the roots. Citric acid responded to cation uptake more than to Zn specifically in the shoots but did not correlate with Zn concentration in the roots or the xylem sap, or any other cations in the roots. In conclusion, organic acids in N. caerulescens are not specifically involved in Zn hyperaccumulation but are involved in regulating pH in the xylem and cation–anion balance in plants.     

Biochemical characterization of cell types within leaves of metal-hyperaccumulating Noccaea praecox (Brassicaceae)

Background and aims

Distinct metal distribution patterns within leaves of metal hyperaccumulating plants are repeatedly observed however, the presumable role of key structural biochemical molecules in determining and regulating their allocation remains largely unknown. We aimed to characterise in a spatially resolved manner the distribution of the main biochemical components in leaves of field-collected Cd/Zn-hyperaccumulating Noccaea praecox in order to relate them to metal distribution patterns at tissue level.

Methods

The biomolecular composition of the leaves was spatially analysed using synchrotron radiation Fourier Transform Infrared (FTIR) and the distribution of Zn with synchrotron radiation Low-Energy X-Ray Fluorescence (LEXRF) microspectroscopy was determined on the same tissues of interest (epidermis, sub-epidermis, mesophyll).

Results

In epidermal cells high proportion of free-carboxyl, nitro and phosphate groups standing for pectin, nitroaromatics, phytic and other organic acids were found. Adjacent mesophyll cells had higher proportions of proteins, carbohydrates and cellulosic compounds.

Conclusions

Pectin compounds were indicated as important components of Zn enriched epidermal cell walls. In addition, intense lignification of epidermal cell walls might limit leakage of the trapped metals back to the metabolically active and thus more sensitive mesophyll. Distribution of metal-binding compounds in particular cell types/tissues may therefore predispose metal distribution patterns and tolerance in leaves.     

Characterization of the high affinity Zn transporter from Noccaea caerulescens, NcZNT1, and dissection of its promoter for its role in Zn uptake and hyperaccumulation

? In this paper, we conducted a detailed analysis of the ZIP family transporter, NcZNT1, in the zinc (Zn)/cadmium (Cd) hyperaccumulating plant species, Noccaea caerulescens, formerly known as Thlaspi caerulescens. NcZNT1 was previously suggested to be the primary root Zn/Cd uptake transporter. Both a characterization of NcZNT1 transport function in planta and in heterologous systems, and an analysis of NcZNT1 gene expression and NcZNT1 protein localization were carried out. ? We show that NcZNT1 is not only expressed in the root epidermis, but also is highly expressed in the root and shoot vasculature, suggesting a role in long-distance metal transport. Also, NcZNT1 was found to be a plasma membrane transporter that mediates Zn but not Cd, iron (Fe), manganese (Mn) or copper (Cu) uptake into plant cells. ? Two novel regions of the NcZNT1 promoter were identified which may be involved in both the hyperexpression of NcZNT1 and its ability to be regulated by plant Zn status. ? In conclusion, we demonstrate here that NcZNT1 plays a role in Zn and not Cd uptake from the soil, and based on its strong expression in the root and shoot vasculature, could be involved in long-distance transport of Zn from the root to the shoot via the xylem.