Enhancement of Zn tolerance and accumulation in plants mediated by the expression of Saccharomyces cerevisiae vacuolar transporter ZRC1.

MAIN CONCLUSION: Transgenic Arabidopsis thaliana and Populus alba plants overexpressing the zinc transporter ScZRC1 in shoots exhibit Zn tolerance. Increased Zn concentrations were observed in shoots of P. alba, a species suitable for phytoremediation. Genetic engineering of plants for phytoremediation is worth to consider if genes leading to heavy metal accumulation and tolerance are expressed in high biomass producing plants. The Saccharomyces cerevisiae ZRC1 gene encodes a zinc transporter which is primarily involved in the uptake of Zn into the vacuole. The ZRC1 gene was expressed in the model species A. thaliana and P. alba (cv. Villafranca). Both species were transformed with constructs carrying ScZRC1 under the control of either the CaMV35S promoter for constitutive expression or the active promoter region of the tobacco Rubisco small subunit (pRbcS) to limit the expression to the above-ground tissues. In hydroponic cultures, A. thaliana and poplar ScZRC1-expressing plants accumulated more Zn in vegetative tissues and were more tolerant than untransformed plants. No differences were found between plants carrying the CaMV35::ScZRC1 or pRbcS::ScZRC1 constructs. The higher Zn accumulation in transgenic plants was accompanied by an increased superoxide dismutase (SOD) activity, indicating the activation of defense mechanisms to prevent cellular damage. In the presence of cadmium in addition to Zn, plants did not show symptoms of metal toxicity, neither in hydroponic cultures nor in soil. Zn accumulation increased in shoots, while no differences were observed for Cd accumulation, in comparison to control plants. These data suggest that ectopic expression of ScZRC1 can increase the potential of poplar for the remediation of Zn-polluted soils, although further tests are required to assay its application in remediating multimetal polluted soils.

TCMP-2 affects tomato flowering and interacts with BBX16, a homolog of the arabidopsis B-box MiP1b.

Flowering and fruiting are processes subject to complex control by environmental and endogenous signals. Endogenous signals comprise, besides classical phytohormones, also signaling peptides and miniproteins. Tomato cystine-knot miniproteins (TCMPs), which belong to a Solanaceous-specific group of Cys-rich protein family, have been recently involved in fruit development. TCMP-1 and TCMP-2 display a highly modulated expression pattern during flower and fruit development. A previous study reported that a change in the ratio of the two TCMPs affects the timing of fruit production. In this work, to investigate TCMP-2 mode of action, we searched for its interacting partners. One of the interactors identified by a yeast two hybrid screen, was the B-box domain-containing protein 16 (SlBBX16), whose closest homolog is the Arabidopsis microProtein 1b implicated in flowering time control. We demonstrated the possibility for the two proteins to interact in vivo in tobacco epidermal cells. Arabidopsis plants ectopically overexpressing the TCMP-2 exhibited an increased level of FLOWERING LOCUS T (FT) mRNA and anticipated flowering. Similarly, in previously generated transgenic tomato plants with increased TCMP-2 expression in flower buds, we observed an augmented expression of SINGLE-FLOWER TRUSS gene, the tomato ortholog of FT, whereas the expression of the antiflorigen SELF-PRUNING was unchanged. Consistently, these transgenic plants showed alterations in the flowering pattern, with an accelerated termination of the sympodial units. Overall, our study reveals a novel function for TCMP-2 as regulatory factor that might integrate, thanks to its capacity to interact with SlBBX16, into the signaling pathways that control flowering, and converge toward florigen regulation.

Plants expressing murine pro-apoptotic protein Bid do not have enhanced PCD.

OBJECTIVES: The purpose of this study was to explore whether plant programmed cell death (PCD) cascade can sense the presence of the animal-only BH3 protein Bid, a BCL-2 family protein known to play a regulatory role in the signaling cascade of animal apoptosis. RESULTS: We have expressed the mouse pro-apoptotic protein Bid in Arabidopsis thaliana and in Nicotiana tabacum. We did not obtain any transformed plant constitutively expressing the truncated protein (tBid-i.e. the caspase-activated form) whereas ectopic expression of the full-length protein (flBid) does not interfere with growth and development of the transformed plants. To verify whether the presence of this animal pro-apoptotic protein modified stress responses and PCD execution, both N. tabacum and A. thaliana plants constitutively expressing flBid have been studied under different stress conditions triggering cell death activation. The results show that the presence of flBid in transgenic plants did not significantly change the responses to abiotic stress (H2O2 or NO) and biotic stress treatments. Moreover, the finding that no Bid active form was present in treated tobacco plants suggests an absence of a proper activation of Bid.

Evolution of the metal hyperaccumulation and hypertolerance traits.

To succeed in life, living organisms have to adapt to the environmental issues to which they are subjected. Some plants, defined as hyperaccumulators, have adapted to metalliferous environments, acquiring the ability to tolerate and accommodate high amounts of toxic metal into their shoot, without showing symptoms of toxicity. The determinants for these traits and their mode of action have long been the subject of research, whose attention lately moved to the evolution of the hypertolerance and hyperaccumulation traits. Genetic evidence indicates that the evolution of both traits includes significant evolutionary events that result in species-wide tolerant and accumulating backgrounds. Different edaphic environments are responsible for subsequent refinement, by local adaptive processes, leading to specific strategies and various degrees of hypertolerance and hyperaccumulation, which characterize metallicolous from non-metallicolous ecotypes belonging to the same genetic unit. In this review, we overview the most updated concepts regarding the evolution of hyperaccumulation and hypertolerance, highlighting also the ecological context concerning the plant populations displaying this fascinating phenomenon.

The Tomato Metallocarboxypeptidase Inhibitor I, which Interacts with a Heavy Metal-Associated Isoprenylated Protein, Is Implicated in Plant Response to Cadmium.

Metallocarboxypeptidases are metal-dependent enzymes, whose biological activity is regulated by inhibitors directed on the metal-containing active site. Some metallocarboxypeptidase inhibitors are induced under stress conditions and have a role in defense against pests. This paper is aimed at investigating the response of the tomato metallocarboxypeptidase inhibitor (TCMP)-1 to Cd and other abiotic stresses. To this aim, the tomato TCMP-1 was ectopically expressed in the model species Arabidopsis thaliana, and a yeast two-hybrid analysis was performed to identify interacting proteins. We demonstrate that TCMP-1 is responsive to Cd, NaCl, and abscisic acid (ABA) and interacts with the tomato heavy metal-associated isoprenylated plant protein (HIPP)26. A. thaliana plants overexpressing TCMP-1 accumulate lower amount of Cd in shoots, display an increased expression of AtHIPP26 in comparison with wild-type plants, and are characterized by a modulation in the expression of antioxidant enzymes. Overall, these results suggest a possible role for the TCMP-1/HIPP26 complex in Cd response and compartmentalization.

Heavy Metal Pollutions: State of the Art and Innovation in Phytoremediation.

Mineral nutrition of plants greatly depends on both environmental conditions, particularly of soils, and the genetic background of the plant itself. Being sessile, plants adopted a range of strategies for sensing and responding to nutrient availability to optimize development and growth, as well as to protect their metabolisms from heavy metal toxicity. Such mechanisms, together with the soil environment, meaning the soil microorganisms and their interaction with plant roots, have been extensively studied with the goal of exploiting them to reclaim polluted lands; this approach, defined phytoremediation, will be the subject of this review. The main aspects and innovations in this field are considered, in particular with respect to the selection of efficient plant genotypes, the application of improved cultural strategies, and the symbiotic interaction with soil microorganisms, to manage heavy metal polluted soils.

The potential of genetic engineering of plants for the remediation of soils contaminated with heavy metals.

The genetic engineering of plants to facilitate the reclamation of soils and waters contaminated with inorganic pollutants is a relatively new and evolving field, benefiting from the heterologous expression of genes that increase the capacity of plants to mobilize, stabilize and/or accumulate metals. The efficiency of phytoremediation relies on the mechanisms underlying metal accumulation and tolerance, such as metal uptake, translocation and detoxification. The transfer of genes involved in any of these processes into fast-growing, high-biomass crops may improve their reclamation potential. The successful phytoextraction of metals/metalloids and their accumulation in aerial organs have been achieved by expressing metal ligands or transporters, enzymes involved in sulfur metabolism, enzymes that alter the chemical form or redox state of metals/metalloids and even the components of primary metabolism. This review article considers the potential of genetic engineering as a strategy to improve the phytoremediation capacity of plants in the context of heavy metals and metalloids, using recent case studies to demonstrate the practical application of this approach in the field.

Functional components of the bacterial CzcCBA efflux system reduce cadmium uptake and accumulation in transgenic tobacco plants.

Cadmium (Cd) is a toxic trace element released into the environment by industrial and agricultural practices, threatening the health of plants and contaminating the food/feed chain. Biotechnology can be used to develop plant varieties with a higher capacity for Cd accumulation (for use in phytoremediation programs) or a lower capacity for Cd accumulation (to reduce Cd levels in food and feed). Here we generated transgenic tobacco plants expressing components of the Pseudomonas putida CzcCBA efflux system. Plants were transformed with combinations of the CzcC, CzcB and CzcA genes, and the impact on Cd mobilization was analysed. Plants expressing PpCzcC showed no differences in Cd accumulation, whereas those expressing PpCzcB or PpCzcA accumulated less Cd in the shoots, but more Cd in the roots. Plants expressing both PpCzcB and PpCzcA accumulated less Cd in the shoots and roots compared to controls, whereas plants expressing all three genes showed a significant reduction in Cd levels only in shoots. These results show that components of the CzcCBA system can be expressed in plants and may be useful for developing plants with a reduced capacity to accumulate Cd in the shoots, potentially reducing the toxicity of food/feed crops cultivated in Cd-contaminated soils.

The Role of the Atypical Kinases ABC1K7 and ABC1K8 in Abscisic Acid Responses.

The ABC1K family of atypical kinases (activity of bc1 complex kinase) is represented in bacteria, archaea, and eukaryotes. In plants they regulate diverse physiological processes in the chloroplasts and mitochondria, but their precise functions are poorly defined. ABC1K7 and ABC1K8 are probably involved in oxidative stress responses, isoprenyl lipid synthesis and distribution of iron within chloroplasts. Because reactive oxygen species take part in abscisic acid (ABA)-mediated processes, we investigated the functions of ABC1K7 and ABC1K8 during germination, stomatal movement, and leaf senescence. Both genes were upregulated by ABA treatment and some ABA-responsive physiological processes were affected in abc1k7 and abc1k8 mutants. Germination was more severely affected by ABA, osmotic stress and salt stress in the single and double mutants; the stomatal aperture was smaller in the mutants under standard growth conditions and was not further reduced by exogenous ABA application; ABA-induced senescence symptoms were more severe in the leaves of the single and double mutants compared to wild type leaves. Taken together, our results suggest that ABC1K7 and ABC1K8 might be involved in the cross-talk between ABA and ROS signaling.

Loss of the Atypical Kinases ABC1K7 and ABC1K8 Changes the Lipid Composition of the Chloroplast Membrane.

The activity of bc1 complex kinase (ABC1K) family is a large group of atypical protein kinases found in prokaryotes and eukaryotes. In bacteria and mitochondria, ABC1K kinases are necessary for the synthesis of coenzyme Q and are therefore involved in the respiratory pathway. In chloroplasts, they are involved in prenylquinone synthesis and stress responses, but their functional role remains unclear. Plants can respond to biotic and abiotic stress by modifying membrane fluidity in order to create a suitable environment for the activity of integral membrane proteins. Therefore, this work was focused on the analysis of the effect of ABC1K7 and ABC1K8 on the production of polar lipids and their accumulation in Arabidopsis thaliana leaves. A comparison of abc1k7 and abc1k8 single mutants and the abc1k7/abc1k8 double mutant with wild-type plants and transgenic lines overexpressing ABC1K7 and ABC1K8 was performed using untargeted lipidomic analysis based on liquid chromatography coupled to mass spectrometry. Multivariate data analysis identified sets of chloroplast lipids representing the different genotypes. The abc1k7 and abc1k8 single mutants produced lower levels of the highly unsaturated lipid digalactosyldiacylglycerol (DGDG) than wild-type plants and also different forms of monogalactosyldiacylglycerol (MGDG) and kaempferol. The abc1k8 mutant also produced higher levels of oxylipin-conjugated DGDG and sinapates. The double mutant produced even higher levels of oxylipin-conjugated MGDG and DGDG. These results show that ABC1K7 and ABC1K8 influence chloroplast lipid synthesis or accumulation and modulate chloroplast membrane composition in response to stress.

Nutrient metal elements in plants.

Plants need many different metal elements for growth, development and reproduction, which must be mobilized from the soil matrix and absorbed by the roots as metal ions. Once taken up by the roots, metal ions are allocated to different parts of the plant by the vascular tissues. Metals are naturally present in the soil, but human activities, ranging from mining and agriculture to sewage processing and heavy industry, have increased the amount of metal pollution in the environment. Plants are challenged by environmental metal ion concentrations that fluctuate from low to high toxic levels, and have therefore evolved mechanisms to cope with such phenomena. In this review, we focus on recent data that provide insight into the molecular mechanisms of metal absorption and transport by plants, also considering the effect of metal deficiency and toxicity. We also highlight the positive effects of some non-essential metals on plant fitness.

AtSIA1 AND AtOSA1: two Abc1 proteins involved in oxidative stress responses and iron distribution within chloroplasts.

The Abc1 protein kinases are a large family of functionally diverse proteins with multiple roles in the regulation of respiration and oxidative stress tolerance. A functional characterization was carried out for AtSIA1, an Arabidopsis thaliana Abc1-like protein, focusing on its potential redundancy with its homolog AtOSA1. Both proteins are located within chloroplasts, even if a different subplastidial localization seems probable. The comparison of atsia1 and atosa1 mutants, atsia1/atosa1 double mutant and wild-type plants revealed a reduction in plastidial iron-containing proteins of the Cytb6 f complex in the mutants. Iron uptake from soil is not hampered in mutant lines, suggesting that AtSIA1 and AtOSA1 affect iron distribution within the chloroplast. Mutants accumulated more ferritin and superoxide, and showed reduced tolerance to reactive oxygen species (ROS), potentially indicating a basal role in oxidative stress. The mutants produced higher concentrations of plastochromanol and plastoquinones than wild-type plants, but only atsia1 plants developed larger plastoglobules and contained higher concentrations of α- and γ-tocopherol and VTE1. Taken together, these data suggest that AtSIA1 and AtOSA1 probably act in signaling pathways that influence responses to ROS production and oxidative stress.

An overview of heavy metal challenge in plants: from roots to shoots.

Heavy metals are often present naturally in soils, but many human activities (e.g. mining, agriculture, sewage processing, the metal industry and automobiles) increase their prevalence in the environment resulting in concentrations that are toxic to animals and plants. Excess heavy metals affect plant physiology by inducing stress symptoms, but many plants have adapted to avoid the damaging effects of metal toxicity, using strategies such as metal chelation, transport and compartmentalization. Understanding the molecular basis of heavy metal tolerance in plants will facilitate the development of new strategies to create metal-tolerant crops, biofortified foods and plants suitable for the phytoremediation of contaminated sites.

Pseudomonas putida response to cadmium: changes in membrane and cytosolic proteomes.

Pseudomonas putida is a saprophytic bacterium with remarkable environmental adaptability and the capacity to tolerate high concentrations of heavy metals. The strain P. putida-Cd001 was isolated from soil contaminated with Cd, Zn and Pb. Membrane-associated and cytosolic proteomes were analyzed to identify proteins whose expression was modulated in response to 250 µM CdSO4. We identified 44 protein spots in the membrane and 21 in the cytosolic fraction differentially expressed in Cd-treated samples compared to untreated controls. Outer membrane porins from the OprD and OprI families were less abundant in bacteria exposed to Cd, whereas those from the OprF and OprL, OprH and OprB families were more abundant, reflecting the increased need to acquire energy sources, the need to maintain membrane integrity and the process of adaptation. Components of the efflux system, such as the CzcB subunit of the CBA system, were also induced by Cd. Analysis of the cytosolic proteome revealed that proteins involved in protein synthesis, degradation and folding were induced along with enzymes that combat oxidative stress, showing that the entire bacterial proteome is modulated by heavy metal exposure. This analysis provides new insights into the adaptation mechanisms used by P. putida-Cd001 to survive in Cd-polluted environments.

Bid binding to negatively charged phospholipids may not be required for its pro-apoptotic activity in vivo.

Bid is a ubiquitous pro-apoptotic member of the Bcl-2 family that has been involved in a variety of pathways of cell death. Unique among pro-apoptotic proteins, Bid is activated after cleavage by the apical caspases of the extrinsic pathway; subsequently it moves to mitochondria, where it promotes the release of apoptogenic proteins in concert with other Bcl-2 family proteins like Bak. Diverse factors appear to modulate the pro-apoptotic action of Bid, from its avid binding to mitochondrial lipids (in particular, cardiolipin) to multiple phosphorylations at sites that can modulate its caspase cleavage. This work addresses the question of how the lipid interactions of Bid that are evident in vitro actually impact on its pro-apoptotic action within cells. Using site-directed mutagenesis, we identified mutations that reduced mouse Bid lipid binding in vitro. Mutation of the conserved residue Lys157 specifically decreased the binding to negatively charged lipids related to cardiolipin and additionally affected the rate of caspase cleavage. However, this lipid-binding mutant had no discernable effect on Bid pro-apoptotic function in vivo. The results are interpreted in relation to an underlying interaction of Bid with lysophosphatidylcholine, which is not disrupted in any mutant retaining pro-apoptotic function both in vitro and in vivo.

Insight into the apoptosis-inducing action of alpha-bisabolol towards malignant tumor cells: involvement of lipid rafts and Bid.

In a precedent report we showed that alpha-bisabolol, a sesquiterpene present widely in the plant kingdom, exerts a rapid and efficient apoptosis-inducing action selectively towards human and murine malignant glioblastoma cell lines through mitochondrial damage. The present study extends these data demonstrating the apoptosis-inducing action of alpha-bisabolol towards highly malignant human pancreatic carcinoma cell lines without affecting human fibroblast viability. The present study further shows the preferential incorporation of alpha-bisabolol to transformed cells through lipid rafts on plasma membranes and, thereafter, direct interaction between alpha-bisabolol and Bid protein, one of pro-apoptotic Bcl-2 family proteins, analyzed either by Surface Plasmon Resonance method or by intrinsic fluorescence measurement. Notions that lipid rafts are rich in plasma membranes of transformed cells and that Bid, richly present in lipid rafts, is deeply involved in lipid transport make highly credible the hypothesis that the molecular mechanism of alpha-bisabolol action may include its capacity to interact with Bid protein.