Anna Manara

Plant Responses to Heavy Metal Toxicity

Plants, like all other organisms, have evolved different mechanisms to maintain physiological concentrations of essential metal ions and to minimize exposure to non-essential heavy metals. Some mechanisms are ubiquitous because they are also required for general metal homeostasis, and they minimize the damage caused by high concentrations of heavy metals in plants by detoxification, thereby conferring tolerance to heavy metal stress. Other mechanisms target individual metal ions (indeed some plants have more than one mechanism to prevent the accumulation of specific metals) and these processes may involve the exclusion of particular metals from the intracellular environment or the sequestration of toxic ions within compartments to isolate them from sensitive cellular components. As a first line of defense, many plants exposed to toxic concentrations of metal ions attempt to prevent or reduce uptake into root cells by restricting metal ions to the apoplast, binding them to the cell wall or to cellular exudates, or by inhibiting long distance transport. If this fails, metals already in the cell are addressed using a range of storage and detoxification strategies, including metal transport, chelation, trafficking, and sequestration into the vacuole. When these options are exhausted, plants activate oxidative stress defense mechanisms and the synthesis of stress-related proteins and signaling molecules, such as heat shock proteins, hormones, and reactive oxygen species.

Insight into the apoptosis-inducing action of α-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.

Plants and Heavy Metals

Plants are sessile organisms that must cope with the surrounding soil composition in order to survive and reproduce. Soils often contain excessive levels of essential and non-essential elements, which may be toxic at high concentrations depending on the plant species and the soil characteristics. Many metals share common toxicity mechanisms, and plants deal with these metals using similar scavenging pathways. The impact of metal toxicity is made more complex by competition, since high levels of one metal may imbalance the uptake and transport of others, therefore contributing to the toxicity symptoms. Here, the toxicity symptoms and mechanisms of the most common essential and nonessential heavy metals will be considered.

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.

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.

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.

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.

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.

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.