Danuta Maria Antosiewicz

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 μM 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 γ-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 γ-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.

Ectopic expression of Arabidopsis ABC transporter MRP7 modifies cadmium root-to-shoot transport and accumulation

Arabidopsis MRPs/ABCCs have been shown to remove various organic and inorganic substrates from the cytosol to other subcellular compartments. Here we first demonstrate that heterologous expression of AtMRP7 in tobacco (Nicotiana tabacum var. Xanthi) modifies cadmium accumulation, distribution and tolerance. Arabidopsis MRP7 was localized both in the tonoplast and in the plasma membrane when expressed in tobacco. Its overexpression increased tobacco Cd-tolerance and resulted in enhanced cadmium concentration in leaf vacuoles, indicating more efficient detoxification by means of vacuolar storage. Heterologous AtMRP7 expression also led to more efficient retention of Cd in roots, suggesting a contribution to the control of cadmium root-to-shoot translocation. The results underscore the use of AtMRP7 in plant genetic engineering to modify the heavy-metal accumulation pattern for a broad range of applications.

Arsenic response of AtPCS1- and CePCS-expressing plants : Effects of external As(V) concentration on As-accumulation pattern and NPT metabolism

Phytochelatins (PCs) are small, cysteine-rich peptides, known to play a major role in detoxification of both cadmium and arsenic. The aim of this study was to determine whether overexpression of either of two PC synthase (PCS) genes, AtPCS1 and CePCS in Nicotiana tabacum (previously shown to cause decrease and increase, respectively, of cadmium tolerance of tobacco - Wojas et al., 2008) also contributes to such contrasting phenotypes with respect to arsenic (As) tolerance and accumulation, and how observed responses relate to non-protein thiol (NPT) metabolism. The expression of both genes resulted in an increase of As-tolerance, with CePCS plants most tolerant. We showed for the first time that the response of PCS overexpressing plants to As qualitatively depends on the external As(V) concentration. At the less toxic 50muM As(V), AtPCS1 and CePCS transformants accumulated more As in roots and leaves than WT. An increase in PC production and the level of PC2 species was detected in leaves of AtPCS1 and CePCS plants, which might explain their enhanced As-accumulation and tolerance. In contrast, at the highly toxic 200muM As(V), several disturbances in thiol metabolism of PCS overexpressing plants were found, surprisingly, including decrease of PC levels both in roots and leaves of transgenic plants relative to WT. The increase in As-tolerance and accumulation due to AtPCS1 and CePCS overexpression, observed at the As(V) concentrations similar to those found in As-contaminated soils, makes these genes promising candidates for plant engineering for phytoremediation.

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 P1B-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 μM 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.

Expression of the P1B‐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.

The relationships between constitutional and inducible Pb-tolerance and tolerance to mineral deficits in Biscutella laevigata and Silene inflata

Abstract Four populations, two each of Silene inflata and Biscutella laevigata, characterized by different tolerances to lead (inducible and constitutional) were studied in terms of possible relationship among Pb-tolerance and tolerance to Ca-, Mg- and P-deficits, general nutrient stress and the respective Ca-, Mg- and P-status of the plants. Plants were grown in a growth chamber in solution cultures containing 5, 10 or 20 mg Pb dm−3. Both Silene populations were more Pb-tolerant than Biscutella. Root lead concentrations in all plants were several times higher than in shoots. Shoot lead concentrations were significantly higher in both Silene populations than in Biscutella. The high lead tolerance of the Silene populations was accompanied by their high Ca-status and high tolerance to calcium deficit. These features of calcium metabolism are characteristic of this species and were not found in the Biscutella populations, which indicates that it did not evolve with the development of inducible tolerance to lead. The possible role of calcium in lead tolerance is discussed.

Mineral status of dicotyledonous crop plants in relation to their constitutional tolerance to lead

Abstract Four crop species—mustard, flax, tomato and sunflower—used in experiments, were grown in the growth chamber in solution culture. The plants were used in the following studies: (i) constitutional tolerance to Pb (index of tolerance was estimated using Wilkins root test with 0, 5, 10 and 20 mg dm −3 Pb concentrations in Knops medium deficient in PO 4 −3 ions); (ii) tolerance to deficit of P, Ca and Mg and to general nutrient stress (50 × diluted Knops medium) as determined by dry matter yield; and (iii) effect of Pb and used nutrient regimes on mineral status of the plants, by estimation of internal ion concentration. The species most tolerant to Pb was tomato. Tomato was also the least tolerant to P deficit (although the test for Pb tolerance was conducted in medium without PO 4 −3 ions). The least Pb-tolerant species, mustard and sunflower, were moderately tolerant or non-tolerant to the mineral deficiencies used in the study. High constitutional tolerance to Pb in tomato was associated with the highest tissue of level of Ca during adminstration of Pb, and with the highest tolerance to Ca deficit. The magnitude of the Pb concentration in the media and its absolute amounts in the roots and shoots were not proportional to the degree of Pb tolerance. Two of the least Pb-tolerant species had the highest (mustard) and lowest (sunflower) root Pb concentrations. The absolute amount of Pb in the roots was highest in mustard. However, the amount of Pb which was transported to the shoot in relation to the total amount of Pb taken up by the plant was the highest for the least tolerant species, sunflower (43.4%).

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.

Metal response of transgenic tomato plantsexpressing P1B-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.