Tobacco as an efficient metal accumulator.

Tobacco (Nicotiana tabacum L.) is an important industrial crop plant. However, it efficiently accumulates metals, primarily cadmium (Cd) and also zinc (Zn), in its leaves. Therefore, it could be a source of cadmium intake by smokers. On the other hand, as a high leaf metal accumulator, it is widely used for phytoremediation of metal-contaminated soil. Both issues provide an important rationale for investigating the processes regulating metal homeostasis in tobacco. This work summarizes the results of research to date on the understanding of the molecular mechanisms determining the effective uptake of Zn and Cd, their translocation into shoots and accumulation in leaves. It also discusses the current state of research to improve the phytoremediation properties of tobacco through genetic modification and to limit leaf Cd content for the tobacco industry.

Regulation and Function of Metal Uptake Transporter NtNRAMP3 in Tobacco.

Natural resistance-associated macrophage protein (NRAMP) genes encode proteins with low substrate specificity, important for maintaining metal cross homeostasis in the cell. The role of these proteins in tobacco, an important crop plant with wide application in the tobacco industry as well as in phytoremediation of metal-contaminated soils, remains unknown. Here, we identified NtNRAMP3, the closest homologue to NRAMP3 proteins from other plant species, and functionally characterized it. A NtNRAMP3-GFP fusion protein was localized to the plasma membrane in tobacco epidermal cells. Expression of NtNRAMP3 in yeast was able to rescue the growth of Fe and Mn uptake defective Δfet3fet4 and Δsmf1 mutant yeast strains, respectively. Furthermore, NtNRAMP3 expression in wild-type Saccharomyces cerevisiae DY1457 yeast strain increased sensitivity to elevated concentrations of iron (Fe), manganese (Mn), copper (Cu), cobalt (Co), nickel (Ni), and cadmium (Cd). Taken together, these results point to a possible role in the uptake of metals. NtNRAMP3 was expressed in the leaves and to a lesser extent in the roots of tobacco plants. Its expression occurred mainly under control conditions and decreased very sharply in deficiency and excess of the tested metals. GUS-based analysis of the site-specific activity of the NtNRAMP3 promoter showed that it was primarily expressed in the xylem of leaf blades. Overall, our data indicate that the main function of NtNRAMP3 is to maintain cross homeostasis of Fe, Mn, Co, Cu, and Ni (also Cd) in leaves under control conditions by controlling xylem unloading.

Suppression of NtZIP4A/B Changes Zn and Cd Root-to-Shoot Translocation in a Zn/Cd Status-Dependent Manner.

In tobacco, the efficiency of Zn translocation to shoots depends on Zn/Cd status. Previous studies pointed to the specific contribution of root parts in the regulation of this process, as well as the role of NtZIP4A/B (from the ZIP family; Zrt Irt-like Proteins). Here, to verify this hypothesis, NtZIP4A/B RNAi lines were generated. Then, in plants exposed to combinations of Zn and Cd concentrations in the medium, the consequences of NtZIP4A/B suppression for the translocation of both metals were determined. Furthermore, the apical, middle, and basal root parts were examined for accumulation of both metals, for Zn localization (using Zinpyr-1), and for modifications of the expression pattern of ZIP genes. Our results confirmed the role of NtZIP4A/B in the control of Zn/Cd-status-dependent transfer of both metals to shoots. Furthermore, they indicated that the middle and basal root parts contributed to the regulation of this process by acting as a reservoir for excess Zn and Cd. Expression studies identified several candidate ZIP genes that interact with NtZIP4A/B in the root in regulating Zn and Cd translocation to the shoot, primarily NtZIP1-like in the basal root part and NtZIP2 in the middle one.

Identification and characterization of a tobacco metal tolerance protein, NtMTP2.

Metal tolerance proteins (MTPs) from the CDF (Cation Diffusion Facilitator) family are efflux transporters that play a crucial role in metal homeostasis by maintaining optimal metal concentrations in the cytoplasm. Here, a novel tobacco NtMTP2 transporter was cloned and characterized. It encodes a 512 aa protein containing all specific CDF family domains. A GFP-NtMTP2 fusion protein localizes to the tonoplast in tobacco cells. NtMTP2 expression in yeast conferred tolerance to Co and Ni, indicating that the protein mediates transport of both metals, but not Zn, Mn, Cu, Fe, or Cd. Nonetheless, the expression level was not affected by Co or Ni, except for an increase in leaves at high Co concentrations. Its expression in plant parts remained stable during development, but increased in the leaves of older plants. Analysis of tobacco expressing a promoter-GUS construct indicates that the main sites of promoter activity are the conductive tissue throughout the plant and the palisade parenchyma in leaves. Our results suggest that NtMTP2 is a tonoplast transporter mediating sequestration of Co and Ni into vacuoles and an important housekeeping protein that controls the basal availability of micronutrients and plays a role in the sequestration of metal excess, specifically in leaves.

Zn/Cd status-dependent accumulation of Zn and Cd in root parts in tobacco is accompanied by specific expression of ZIP genes.

BACKGROUND: Root-to-shoot translocation of zinc (Zn) and cadmium (Cd) depends on the concentrations of both metals in the medium. A previous study on tobacco (Nicotiana tabacum) pointed to the contribution of NtZIP1, NtZIP2, NtZIP4 and NtIRT1-like in the regulation of this phenomenon. To learn more, Zn and Cd accumulation, root/shoot distribution and the expression of ZIP genes were investigated in the apical, middle and basal root parts. RESULTS: We show that Zn/Cd status-dependent root-shoot distribution of both metals was related to distinct metal accumulation in root parts. At low Zn and Cd in the medium, the apical part contained the highest metal level; at higher concentrations, the middle and basal parts were the major sink for excess metal. The above were accompanied by root part-specific expression pattern modifications of ZIPs (NtZIP1-like, NtZIP2, NtZIP4A/B, NtZIP5A/B, NtZIP5-like, NtZIP8, NtZIP11, NtIRT1, and NtIRT1-like) that fell into four categories with respect to the root part. Furthermore, for lower Zn/Cd concentrations changes were noted for NtZIP5A/B and NtZIP5-like only, but at higher Zn and Cd levels for NtZIP1-like, NtZIP5-like, NtZIP8, NtZIP11, NtIRT1, and NtIRT1-like. NtZIP1, here renamed to NtZIP5B, was cloned and characterized. We found that it was a zinc deficiency-inducible transporter involved in zinc and cadmium uptake from the soil solution primarily by the middle root part. CONCLUSIONS: We conclude that regulation of the longitudinal distribution of Zn and Cd is highly specific, and that the apical, middle and basal root parts play distinct roles in Zn/Cd status-dependent control of metal translocation efficiency to shoots, including the stimulation of Zn translocation to shoots in the presence of Cd. These results provide new insight into the root part-specific unique role of NtZIP5B and other ZIP genes in the longitudinal distribution of zinc and cadmium and their contribution to the regulation of root-to-shoot translocation.

Functional Analysis of NtZIP4B and Zn Status-Dependent Expression Pattern of Tobacco ZIP Genes.

Tobacco is frequently considered as a plant useful for phytoremediation of metal-contaminated soil, despite the mechanisms for regulation of uptake and accumulation being largely unknown. Here we cloned and characterized a new tobacco Zn and Cd transporter NtZIP4B from the ZIP family (ZRT-IRT-Like proteins). It complemented the Zn-uptake defective yeast mutant zrt1zrt2, and rendered the wild type DY1457 yeast more sensitive to Cd. Bioinformatic analysis and transient expression of the NtZIP4B-GFP fusion protein in tobacco leaves indicated its localization to the plasma membrane. Real-time q-PCR based analysis showed that it is expressed in all vegetative organs with the highest level in leaves. The Zn status determined transcript abundance; NtZIP4B was upregulated by Zn-deficiency and downregulated by Zn excess. At the tissue level, in roots NtZIP4B is expressed in the vasculature of the middle part of the roots and in surrounding tissues including the root epidermis; in leaves primarily in the vasculature. Bioinformatic analysis identified two copies of ZIP4 in tobacco, NtZIP4A and NtZIP4B with 97.57% homology at the amino acid level, with the same expression pattern for both, indicating a high degree of functional redundancy. Moreover, the present study provides new insights into the coordinated function of NtZIP1, NtZIP2, NtZIP4, NtZIP5, NtZIP8, NtIRT1, and NtIRT1-like in response to low-to-high Zn status. Leaves were the major site of NtZIP4, NtZIP5, and NtZIP8 expression, and roots for NtZIP1, NtZIP2, NtIRT1, and NtIRT1-like. Contrasting expression level in the apical and basal root parts indicates distinct roles in root-specific processes likely contributing to the regulation of Zn root-to-shoot translocation. In summary, new insight into the role of ZIP genes in Zn homeostasis pointing to their overlapping and complementary functions, offers opportunities for strategies to modify Zn and Cd root/shoot partition in tobacco.

NO-Dependent programmed cell death is involved in the formation of Zn-related lesions in tobacco leaves.

A recent study indicated that the development of lesions on the leaf blades of tobacco exposed to zinc (Zn) excess can be considered a manifestation of a Zn-tolerance strategy at the organ level. Here, we investigated whether cell death leading to the formation of localized lesions is destructive in character (necrosis type) or results from programmed self-induced cell death (PCD). Selected parameters, including PCD markers, were determined in the leaves from tobacco plants grown in the presence of 200 µM Zn and compared with control conditions. TUNEL assay results showing internucleosomal DNA fragmentation in the nuclei of the cells from Zn-exposed leaves, together with an enhanced expression of three PCD marker genes (NtBI-1, Ntrboh, and NtSIPK), indicated the involvement of PCD in the formation of Zn-related lesions. It is known that NO is a key factor in the execution of PCD. Interestingly, upon exposure to high Zn, in situ localization of NO (visualized using DAF-2DA fluorescence) was restricted to groups of mesophyll cells, and was correlated with the pattern of Zn localization (determined using the fluorophore Zinpyr-1), similarly limited primarily to groups of "Zn accumulating cells". Furthermore, inhibition of the formation of lesions in the presence of l-NAME (an NO synthase inhibitor) was accompanied by the delayed appearance of Zn and by NO localization limited to these groups of cells. Altogether, we provide the first demonstration that Zn-related lesions in leaves develop from groups of mesophyll cells in which accumulation of high concentrations of Zn contributes to enhancement of the NO level and to initiation of PCD processes.

The ratio of Zn to Cd supply as a determinant of metal-homeostasis gene expression in tobacco and its modulation by overexpressing the metal exporter AtHMA4.

This study links changes in the tobacco endogenous metal-homeostasis network caused by transgene expression with engineering of novel features. It also provides insight into the concentration-dependent mutual interactions between Zn and Cd, leading to differences in the metal partitioning between wild-type and transgenic plants. In tobacco, expression of the export protein AtHMA4 modified Zn/Cd root/shoot distribution, but the pattern depended on their concentrations in the medium. To address this phenomenon, the expression of genes identified by suppression subtractive hybridization and the Zn/Cd accumulation pattern were examined upon exposure to six variants of low/high Zn and Cd concentrations. Five tobacco metal-homeostasis genes were identified: NtZIP2, NtZIP4, NtIRT1-like, NtNAS, and NtVTL. In the wild type, their expression depended on combinations of low/high Zn and Cd concentrations; co-ordinated responses of NtZIP1, NtZIP2, and NtVTL were shown in medium containing 4 µM Cd, and at 0.5 µM versus 10 µM Zn. In transgenics, qualitative changes detected for NtZIP1, NtZIP4, NtIRT1-like, and NtVTL are considered crucial for modification of Zn/Cd supply-dependent Zn/Cd root/shoot distribution. Notwithstanding, NtVTL was the most responsive gene in wild-type and transgenic plants under all concentrations of Zn and Cd tested; thus it is a candidate gene for the regulation of metal cross-homeostasis processes involved in engineering new metal-related traits.

Engineering high Zn in tomato shoots through expression of AtHMA4 involves tissue-specific modification of endogenous genes.

BACKGROUND: To increase the Zn level in shoots, AtHMA4 was ectopically expressed in tomato under the constitutive CaMV 35S promoter. However, the Zn concentration in the shoots of transgenic plants failed to increase at all tested Zn levels in the medium. Modification of Zn root/shoot distribution in tomato expressing 35S::AtHMA4 depended on the concentration of Zn in the medium, thus indicating involvement of unknown endogenous metal-homeostasis mechanisms. To determine these mechanisms, those metal-homeostasis genes that were expressed differently in transgenic and wild-type plants were identified by microarray and RT-qPCR analysis using laser-assisted microdissected RNA isolated from two root sectors: (epidermis + cortex and stele), and leaf sectors (upper epidermis + palisade parenchyma and lower epidermis + spongy parenchyma). RESULTS: Zn-supply-dependent modification of Zn root/shoot distribution in AtHMA4-tomato (increase at 5 µM Zn, no change at 0.5 µM Zn) involved tissue-specific, distinct from that in the wild type, expression of tomato endogenous genes. First, it is suggested that an ethylene-dependent pathway underlies the detected changes in Zn root/shoot partitioning, as it was induced in transgenic plants in a distinct way depending on Zn exposure. Upon exposure to 5 or 0.5 µM Zn, in the epidermis + cortex of the transgenics' roots the expression of the Strategy I Fe-uptake system (ethylene-dependent LeIRT1 and LeFER) was respectively lower or higher than in the wild type and was accompanied by respectively lower or higher expression of the identified ethylene genes (LeNR, LeACO4, LeACO5) and of LeChln. Second, the contribution of LeNRAMP2 expression in the stele is shown to be distinct for wild-type and transgenic plants at both Zn exposures. Ethylene was also suggested as an important factor in a pathway induced in the leaves of transgenic plants by high Zn in the apoplast, which results in the initiation of loading of the excess Zn into the mesophyll of "Zn accumulating cells". CONCLUSIONS: In transgenic tomato plants, the export activity of ectopically expressed AtHMA4 changes the cellular Zn status, which induces coordinated tissue-specific responses of endogenous ethylene-related genes and metal transporters. These changes constitute an important mechanism involved in the generation of the metal-related phenotype of transgenic tomato expressing AtHMA4.

Determination the Usefulness of AhHMA4p1::AhHMA4 Expression in Biofortification Strategies.

AhHMA4 from Arabidopsis thaliana encodes Zn/Cd export protein that controls Zn/Cd translocation to shoots. The focus of this manuscript is the evaluation of AhHMA4 expression in tomato for mineral biofortification (more Zn and less Cd in shoots and fruits). Hydroponic and soil-based experiments were performed. Transgenic and wild-type plants were grown on two dilution levels of Knop's medium (1/10, 1/2) with or without Cd, to determine if mineral composition affects the pattern of root/shoot partitioning of both metals due to AhHMA4 expression. Facilitation of Zn translocation to shoots of 19-day-old transgenic tomato was noted only when plants were grown in the more diluted medium. Moreover, the expression pattern of Zn-Cd-Fe cross-homeostasis genes (LeIRT1, LeChln, LeNRAMP1) was changed in transgenics in a medium composition-dependent fashion. In plants grown in soil (with/without Cd) up to maturity, expression of AhHMA4 resulted in more efficient translocation of Zn to shoots and restriction of Cd. These results indicate the usefulness of AhHMA4 expression to improve the growth of tomato on low-Zn soil, also contaminated with Cd.

Approach to engineer tomato by expression of AtHMA4 to enhance Zn in the aerial parts.

The aim of this work was to assess the potential for using AtHMA4 to engineer enhanced efficiency of Zn translocation to shoots, and to increase the Zn concentration in aerial tissues of tomato. AtHMA4, a P1B-ATPase, encodes a Zn export protein known to be involved in the control of Zn root-to-shoot translocation. In this work, 35S::AtHMA4 was expressed in tomato (Lycopersicon esculentum var. Beta). Wild-type and transgenic plants were tested for Zn and Cd tolerance; Zn, Fe and Cd accumulation patterns, and for the expression of endogenous Zn/Fe-homeostasis genes. At 10µM Zn exposure, a higher Zn concentration was observed in leaves of AtHMA4-expressing lines compared to wild-type, which is promising in terms of Zn biofortification. AtHMA4 also transports Cd and at 0.25µM Cd the transgenic plants showed similar levels of this element in leaves to wild-type but lower levels in roots, therefore indicating a reduction of Cd uptake due to AtHMA4 expression. Expression of this transgene AtHMA4 also resulted in distinct changes in Fe accumulation in Zn-exposed plants, and Fe/Zn-accumulation in Cd-exposed plants, even though Fe is not a substrate for AtHMA4. Analysis of the transcript abundance of key Zn/Fe-homeostasis genes showed that the pattern was distinct for transgenic and wild-type plants. The reduction of Fe accumulation observed in AtHMA4-transformants was accompanied by up-regulation of Fe-deficiency marker genes (LeFER, LeFRO1, LeIRT1), whereas down-regulation was detected in plants with the status of Fe-sufficiency. Furthermore, results strongly suggest the importance of the up-regulation of LeCHLN in the roots of AtHMA4-expressing plants for efficient translocation of Zn to the shoots. Thus, the modifications of Zn/Fe/Cd translocation to aerial plant parts due to AtHMA4 expression are closely related to the alteration of the endogenous Zn-Fe-Cd cross-homeostasis network of tomato.

HMA4 expression in tobacco reduces Cd accumulation due to the induction of the apoplastic barrier.

Ectopic expression in tobacco (Nicotiana tabacum v. Xanthi) of the export protein AtHMA4 (responsible in Arabidopsis for the control of Zn/Cd root to shoot translocation) resulted in decreased Cd uptake/accumulation in roots and shoots. This study contributes to understanding the mechanisms underlying this Cd-dependent phenotype to help predict the consequences of transgene expression for potential phytoremediation/biofortification-based strategies. Microarray analysis was performed to identify metal homeostasis genes that were differentially expressed in roots of Cd-exposed AtHMA4-expressing tobacco relative to the wild type. It was established that down-regulation of genes known to mediate Cd uptake was not responsible for reduced Cd uptake/accumulation in AtHMA4 transformants. The transcript levels of NtIRT1 and NtZIP1 were higher in transgenic plants, indicating an induction of the Fe and Zn deficiency status due to AtHMA4 expression. Interestingly, upon exposure to Cd, genes involved in cell wall lignification (NtHCT, NtOMET, and NtPrx11a) were up-regulated in transformants. Microscopic analysis of roots demonstrated that expression of AtHMA4 caused an induction of cell wall lignification in the external cell layers that was accompanied by enhanced H2O2 accumulation. Further study showed that the concentration of other elements (B, Co, Cu, Ni, Mo, and Zn) was reduced in AtHMA4 transformants in the presence of Cd. In conclusion, due to ectopic expression of 35S::AtHMA4, the physical apoplastic barrier within the external cell layer developed, which is likely to be responsible for the reduction of Cd uptake/accumulation.

Expression of HvHMA2 in tobacco modifies Zn-Fe-Cd homeostasis.

HvHMA2 is a plasma membrane P1B-ATPase from barley that functions in Zn/Cd root-to-shoot transport. To assess the usefulness of HvHMA2 for modifying the metal content in aerial plant parts, it was expressed in tobacco under the CaMV35S promoter. Transformation with HvHMA2 did not produce one unique pattern of Zn and Cd accumulation; instead it depended on external metal supply. Thus Zn and Cd root-to-shoot translocation was facilitated, but not at all applied Zn/Cd concentrations. Metal uptake was restricted in HvHMA2-transformed plants and the level in the shoot was not enhanced. It was shown that HvHMA2 localizes to the plasma membrane of tobacco cells, and overloads the apoplast with Zn, which could explain the overall decrease in metal uptake observed. Despite the lower levels in the shoot, HvHMA2 transformants showed increased Zn sensitivity. Moreover, introduction of HvHMA2 into tobacco interfered with Fe metabolism and Fe accumulation was modified in HvHMA2-transformants in a Zn- and Cd-concentration dependent manner. The results indicate that ectopic expression of the export protein HvHMA2 in tobacco interferes with tobacco metal Zn-Cd-Fe cross-homeostasis, inducing internal mechanisms regulating metal uptake and tolerance.

Development of Zn-related necrosis in tobacco is enhanced by expressing AtHMA4 and depends on the apoplastic Zn levels.

AtHMA4 was previously shown to contribute to the control of Zn root-to-shoot translocation and tolerance to high Zn. However, heterologous expression of 35S::AtHMA4 in tobacco (Nicotiana tabacum cv. Xanthi) results in enhanced Zn sensitivity. This study provides a better understanding of the development of this Zn-sensitive phenotype and demonstrates that substantial modifications of Zn homeostasis occur due to AtHMA4 expression. We show that ectopically expressing AtHMA4 in tobacco results in overloading the root and leaf apoplast with Zn. The tissue and cellular distribution of Zn, monitored using Zinpyr-1, was altered in the AtHMA4-expressing plants compared with wild type. Increased loading of the leaf apoplast with Zn in AtHMA4 transformants induced necrosis; this appeared at lower levels of Zn supply in the transgenics compared with wild type. This study suggests that Zn concentration may be sensed in the apoplast of leaves, and if concentrations are above a certain threshold then particular groups of cells accumulate Zn and necrosis is initiated. Therefore, this could be considered as a mechanism for protecting the other parts of the photosynthetically active leaf from Zn toxicity.

Metal response of transgenic tomato plants expressing P(1B) -ATPase.

Heterologous expression of HMA4 (P(1B) -ATPase) in plants is a useful strategy to engineer altered metal distribution in tissues for biofortification or phytoremediation purposes. This study contributes to understanding mechanisms underlying complex Zn-dependent phenotypes observed in transgenic plants and to better predict the consequences of transgene expression. Tomato was transformed with AhHMA4(p1) ::AhHMA4 from Arabidopsis halleri encoding the Zn export protein involved in xylem loading of Zn. Homozygous lines were tested for Zn tolerance, Zn and Fe concentrations in organs and in the apoplastic fluid, and for the expression of the transgene and tomato metal homeostasis endogenes. Expression of AhHMA4 facilitates root-to-shoot Zn translocation and induces Zn uptake in a Zn supply-dependent manner. Unexpectedly, it increases Zn excess-triggered Fe deficiency in leaves and transcriptional activation of Fe-uptake systems in roots. Moreover, AhHMA4 expression causes Zn overload of the apoplast, which may contribute to enhanced Zn sensitivity of transgenics and may lead to cell-wall remodeling. This study highlights that alteration of the apoplast/symplast Zn status through introduction of cellular Zn export activity via AhHMA4 may alter tomato metal homeostasis network, thus seems to be crucial in the generation of the phenotype of transgenic tomato.

Expression of the P(1B) -type ATPase AtHMA4 in tobacco modifies Zn and Cd root to shoot partitioning and metal tolerance.

Genetic modification of Zn/Cd accumulation in roots and shoots for biofortification or phytoremediation is a focus of this manuscript. We expressed AtHMA4 (a P(1B) ATPase involved in Zn and Cd transport), AtHMA4-trunc (lacking the C-terminal region) and AtHMA4-C terminus (the C-terminal region alone) in tobacco under the CaMV 35S constitutive promoter and examined accumulation and tolerance to both metals. Expression of AtHMA4 enhanced Zn translocation to the shoots only at 10 µM Zn but not at 0.5, 100 and 200 µM Zn. AtHMA4-trunc did not show this effect and instead reduced Zn translocation to the shoot. AtHMA4-expressing plants showed a decrease in cadmium uptake when exposed to 0.25 and 5 µM Cd; this was also observed with AtHMA4-trunc-expressing lines, although to a lesser extent. Expression of AtHMA4-C-terminus containing potential metal binding sites increased cadmium and zinc concentrations in roots and shoots up to fourfold. We have demonstrated that both AtHMA4 and AtHMA4 C-terminus could be candidate genes/sequences for engineering modifications of zinc and cadmium root/shoot partitioning. However, the phenotype of transformants depended on the external metal concentration, thus it might be difficult to engineer a plant displaying the desired metal-related phenotype when grown under varying conditions of metal supply.

Metal accumulation in tobacco expressing Arabidopsis halleri metal hyperaccumulation gene depends on external supply.

Engineering enhanced transport of zinc to the aerial parts of plants is a major goal in bio-fortification. In Arabidopsis halleri, high constitutive expression of the AhHMA4 gene encoding a metal pump of the P(1B)-ATPase family is necessary for both Zn hyperaccumulation and the full extent of Zn and Cd hypertolerance that are characteristic of this species. In this study, an AhHMA4 cDNA was introduced into N. tabacum var. Xanthi for expression under the control of its endogenous A. halleri promoter known to confer high and cell-type specific expression levels in both A. halleri and the non-hyperaccumulator A. thaliana. The transgene was expressed at similar levels in both roots and shoots upon long-term exposure to low Zn, control, and increased Zn concentrations. A down-regulation of AhHMA4 transcript levels was detected with 10 muM Zn resupply to tobacco plants cultivated in low Zn concentrations. In general, a transcriptional regulation of AhHMA4 in tobacco contrasted with the constitutively high expression previously observed in A. halleri. Differences in root/shoot partitioning of Zn and Cd between transgenic lines and the wild type were strongly dependent on metal concentrations in the hydroponic medium. Under low Zn conditions, an increased Zn accumulation in the upper leaves in the AhHMA4-expressing lines was detected. Moreover, transgenic plants exposed to cadmium accumulated less metal than the wild type. Both modifications of zinc and cadmium accumulation are noteworthy outcomes from the biofortification perspective and healthy food production. Expression of AhHMA4 may be useful in crops grown on soils poor in Zn.

The role of subcellular distribution of cadmium and phytochelatins in the generation of distinct phenotypes of AtPCS1- and CePCS3-expressing tobacco.

Exposure to Cd2+ leads to activation of phytochelatin synthase (PCS) and the formation of phytochelatins (PCs) in the cytosol. Binding of Cd by PCs and the subsequent transport of PC-Cd complexes to the vacuole are essential for Cd tolerance. Attempts to improve Cd detoxification by PCS overexpression have resulted in contrasting plant phenotypes, ranging from enhanced Cd tolerance to Cd hypersensitivity. In the present paper, changes in the subcellular phytochelatin, glutathione, gamma-glutamylcysteine and cadmium vacuolar and cytosolic distribution underlying these phenotypes were examined. Cadmium and PCs levels were determined in protoplasts and vacuoles isolated from leaves of Nicotiana tabacum expressing either of two phytochelatin synthase genes, AtPCS1 and CePCS (differing in their level of Cd tolerance; being Cd hypersensitive or more Cd-tolerant as compared to wild-type plants, respectively). We showed that Cd hypersensitivity of AtPCS1-expressing tobacco results from a significant decrease in both the cytosolic and vacuolar pool of PCs, indicating a decreased cadmium detoxification capacity. By contrast, enhanced Cd tolerance of CePCS plants was accompanied by an increased cytosolic and vacuolar SH of PC/Cd ratio, suggesting more efficient Cd detoxification. Surprisingly, the substantially reduced level of PCs did not influence Cd accumulation in vacuoles of AtPCS1-transformed tobacco (relative to the wild-type), which suggests the important role of mechanisms other than PC-Cd transport in Cd translocation to the vacuole. Our data suggest that the key role of the PCs in Cd tolerance is temporary binding of Cd2+ in the cytosol, and contrary to the current view, their contribution to cadmium sequestration seems to be less important.

Overexpression of phytochelatin synthase in tobacco: distinctive effects of AtPCS1 and CePCS genes on plant response to cadmium.

Phytochelatins, heavy-metal-binding polypeptides, are synthesized by phytochelatin synthase (PCS) (EC 2.3.2.15). Previous studies on plants overexpressing PCS genes yielded contrasting phenotypes, ranging from enhanced cadmium tolerance and accumulation to cadmium hypersensitivity. This paper compares the effects of overexpression of AtPCS1 and CePCS in tobacco (Nicotiana tabacum var. Xanthi), and demonstrates how the introduction of single homologous genes affects to a different extent cellular metabolic pathways leading to the opposite of the desired effect. In contrast to WT and CePCS transformants, plants overexpressing AtPCS1 were Cd-hypersensitive although there was no substantial difference in cadmium accumulation between studied lines. Plants exposed to cadmium (5 and 25 muM CdCl2) differed, however, in the concentration of non-protein thiols (NPT). In addition, PCS activity in AtPCS1 transformants was around 5-fold higher than in CePCS and WT plants. AtPCS1 expressing plants displayed a dramatic accumulation of gamma-glutamylcysteine and concomitant strong depletion of glutathione. By contrast, in CePCS transformants, a smaller reduction of the level of glutathione was noticed, and a less pronounced change in gamma-glutamylcysteine concentration. There was only a moderate and temporary increase in phytochelatin levels due to AtPCS1 and CePCS expression. Marked changes in NPT composition due to AtPCS1 expression led to moderately decreased Cd-detoxification capacity reflected by lower SH:Cd ratios, and to higher oxidative stress (assessed by DAB staining), which possibly explains the increase in Cd-sensitivity. The results indicate that contrasting responses to cadmium of plants overexpressing PCS genes might result from species-dependent differences in the activity of phytochelatin synthase produced by the transgenes.