The molecular identity of the characean OH- transporter: a candidate related to the SLC4 family of animal pH regulators.

Characeae are closely related to the ancient algal ancestors of all land plants. The long characean cells display a pH banding pattern to facilitate inorganic carbon import in the acid zones for photosynthetic efficiency. The excess OH-, generated in the cytoplasm after CO2 is taken into the chloroplasts, is disposed of in the alkaline band. To identify the transporter responsible, we searched the Chara australis transcriptome for homologues of mouse Slc4a11, which functions as OH-/H+ transporter. We found a single Slc4-like sequence CL5060.2 (named CaSLOT). When CaSLOT was expressed in Xenopus oocytes, an increase in membrane conductance and hyperpolarization of resting potential difference (PD) was observed with external pH increase to 9.5. These features recall the behavior of Slc4a11 in oocytes and are consistent with the action of a pH-dependent OH-/H+ conductance. The large scatter in the data might reflect intrinsic variability of CaSLOT transporter activation, inefficient expression in the oocyte due to evolutionary distance between ancient algae and frogs, or absence of putative activating factor present in Chara cytoplasm. CaSLOT homologues were found in chlorophyte and charophyte algae, but surprisingly not in related charophytes Zygnematophyceae or Coleochaetophyceae.

Salinity-induced Changes in Gene Expression in the Streptophyte Alga Chara: The Critical Role of a Rare Na+ -ATPase.

The primarily freshwater genus Chara is comprised of many species that exhibit a wide range of salinity tolerance. The range of salt tolerance provides a good platform for investigating the role of transport mechanisms in response to salt stress, and the close evolutionary relationship between Charophytes and land plants can provide broader insights. We investigated the response to salt stress of previously identified transport mechanisms in two species of Chara, Chara longifolia (salt-tolerant), and Chara australis (salt-sensitive): a cation transporter (HKT), a Na+ /H+ antiport (NHX), H+ -ATPase (AHA), and a Na+ -ATPase (ENA). The presence of these candidate genes has been confirmed in both species of Chara, with the exception of the Na+ -ATPase, which is present only in salt-tolerant Chara longifolia. Time-course Illumina transcriptomes were created using RNA from multiple time points (0, 6, 12, 24 and 48 h) after freshwater cultures for each species were exposed to salt stress. These transcriptomes verified our hypotheses of these mechanisms conferring salt tolerance in the two species examined and also aided in identification of specific transcripts representing our genes of interest in both species. The expression of these transcripts was validated through use of qPCR, in a similar experimental set-up used for the RNAseq data described above. The RNAseq and qPCR data showed significant changes of expression mechanisms in C. longifolia (respectively), a down-regulation of HKT and a substantial up-regulation of ENA. Significant responses to salt stress in salt-sensitive C. australis show up-regulation of NHX and AHA.

The role of ion-transporting proteins in the evolution of salt tolerance in charophyte algae.

Species within the genus Chara have variable salinity tolerance. Their close evolutionary relationship with embryophytes makes their study crucial to understanding the evolution of salt tolerance and key evolutionary processes shared among the phyla. We examined salt-tolerant Chara longifolia and salt-sensitive Chara australis for mechanisms of salt tolerance and their potential role in adaptation to salt. We hypothesize that there are shared mechanisms similar to those in embryophytes, which assist in conferring salt tolerance in Chara, including a cation transporter (HKT), a Na+ /H+ antiport (NHX), a H+ -ATPase (AHA), and a Na+ -ATPase (ENA). Illumina transcriptomes were created using cultures grown in freshwater and exposed to salt stress. The presence of these candidate genes, identified by comparing with genes known from embryophytes, has been confirmed in both species of Chara, with the exception of ENA, present only in salt-tolerant C. longifolia. These transcriptomes provide evidence for the contribution of these mechanisms to differences in salt tolerance in the two species and for the independent evolution of the Na+ -ATPase. We also examined genes that may have played a role in important evolutionary processes, suggested by previous work on the Chara braunii genome. Among the genes examined, cellulose synthase protein (GT43) and response regulator (RRB) were confirmed in both species. Genes absent from all three Chara species were members of the GRAS family, microtubule-binding protein (TANGLED1), and auxin synthesizers (YUCCA, TAA). Results from this study shed light on the evolutionary relationship between Chara and embryophytes through confirmation of shared salt tolerance mechanisms, as well as unique mechanisms that do not occur in angiosperms.

High-resolution mass spectrometry-based metabolomics reveal the disruption of jasmonic pathway in Arabidopsis thaliana upon copper oxide nanoparticle exposure.

Mass-spectrometry based metabolomics has recently emerged as a valuable technique in understanding the ecotoxicity and mode of action of a wide range of xenobiotics in the environment, including engineered nanomaterials (ENMs). However, the applications of metabolomics in elucidating the biochemical pathways affected by xenobiotics have been mostly performed using targeted analysis. In this study, the effects of copper oxide nanoparticles (CuO NPs) on Arabidopsis thaliana, a model plant, was investigated using untargeted metabolite profiling based on two platforms of high-resolution mass spectrometry (MS): (1) liquid chromatography quadrupole time-of-flight mass spectrometry (LC-QToF-MS) and (2) LC Q Exactive™ Hybrid Quadrupole-Orbitrap™-MS (LC-Orbitrap-MS). This approach was performed to identify specific features (mass-to-charge ratios, m/z's) that are significantly changed in a reproducible manner regardless of the MS platform used in metabolomics. In addition, the total copper concentrations taken up in plant tissues were quantified using inductively coupled plasma mass spectrometry (ICP-MS), which provided evidence of translocation of CuO NPs from roots to leaves and flowering shoots. Results from untargeted metabolomics showed that there were 65 plant metabolites that were altered commonly in both LC/MS platforms resulting from CuO NPs exposure of Arabidopsis thaliana. These metabolites belong to the jasmonic acid and glucosinolates pathways, suggesting the stress response induced by CuO NPs in Arabidopsis. This study demonstrated the effectiveness of high-resolution LC/MS in providing insight on the mechanism of nanotoxicity of CuO NPs in plants.

Uptake and transformations of engineered nanomaterials: Critical responses observed in terrestrial plants and the model plant Arabidopsis thaliana.

With the applications of engineered nanomaterials (ENMs) continually expanding and production quickly growing, residues of ENMs will end up in the environment at levels that may be harmful to non-target organisms. Many of the tunable properties that have made them desirable, such as type, size, charge, or coating, also contribute to the current difficulties in understanding the fate of ENMs in the environment. This review article focuses on studies that investigate plant-ENM interactions, including techniques used to study these interactions and documented plant responses due to the phytotoxic effects of ENMs. The many variables which can be altered for an experiment, such as type, size, and concentration of ENMs, make it difficult to formulate generalizations about the uptake mechanism involved, or to make an inference on the subcellular localization and distribution of the internalized ENMs in plant tissue. In order to avoid these challenges, studies can utilize a model organism such as Arabidopsis thaliana, and a combination of analytical techniques that can reveal complementary information in order to assess how the different experimental conditions influence the uptake and phytotoxicity of ENMs. This review presents recent studies regarding plant-ENM interactions employing Arabidopsis to demonstrate how the use of this model plant can advance our understanding of plant-ENM interactions and guide additional studies using other plant species. Overarching results suggest that more sensitive tests and consistency in experimental designs are needed to fully assess and understand the phytotoxic effects of ENMs in the environment.

Salinity-induced noise in membrane potential of Characeae Chara australis: effect of exogenous melatonin.

Salt sensitive Characeae Chara australis responds to 50 mM NaCl by a prompt appearance of noise in the trans-membrane potential difference (PD). The noise diminishes with time in saline and PD depolarization, leading to altered current-voltage characteristics that could be modeled with H(+)/OH(-) channels. Beilby and Al Khazaaly (JMB 230:21-34, 2009) suggested that the noise might arise from cooperative transient opening of H(+)/OH(-) channels. Presoaking cells in 10 µM melatonin over 24 h abolished the noise in some cells, postponed its appearance in others or changed its characteristics. As melatonin is a very effective antioxidant, we postulated opening of H(+)/OH(-) channels by reactive oxygen species (ROS). Measurement of ROS using dihydrodichlorofluorescein diacetate confirmed substantial reduction in ROS production in melatonin-treated cells in saline and sorbitol media. However, ROS concentration decreased as a function of time in saline medium. Possible schemes for activation of H(+)/OH(-) channels under salinity stress are considered.

Combined effects of cadmium and zinc on growth, tolerance, and metal accumulation in Chara australis and enhanced phytoextraction using EDTA.

Chara australis (R. Br.) is a macrophytic alga that can grow in and accumulate Cd from artificially contaminated sediments. We investigated the effects of Zn independently and in combination with Cd on C. australis growth, metal tolerance, and uptake. Plant growth was reduced at concentrations ≥ 75 mg Zn (kg soil)⁻¹. Zn also increased the concentration of glutathione in the plant, suggesting alleviation of stress. Phytotoxic effects were observed at ≥ 250 mg added Zn (kg soil)⁻¹. At 1.5mg Zn (kg soil)⁻¹, the rhizoid bioconcentration factor (BCF) was >1.0 for both Cd and Zn. This is a criterion for hyperaccumulator status, a commonly used benchmark for utility in remediation of contaminated soils by phytoextraction. There was no significant interaction between Cd and Zn on accumulation, indicating that Chara should be effective at phytoextraction of mixed heavy metal contamination in sediments. The effects of the chelator, ethylenediaminetetraacetic acid (EDTA), were also tested. Moderate levels of EDTA increased Cd and Zn accumulation in rhizoids and Cd BCF of shoots, enhancing Chara's potential in phytoremediation. This study demonstrates for the first time the potential of macroalgae to remove metals from sediments in aquatic systems that are contaminated with a mixture of metals.

Investigating uptake of water-dispersible CdSe/ZnS quantum dot nanoparticles by Arabidopsis thaliana plants.

Interest on the environmental impacts of engineered nanomaterials has rapidly increased over the past years because it is expected that these materials will eventually be released into the environment. The present work investigates the potential root uptake of water-dispersible CdSe/ZnS quantum dots (QDs) by the model plant species, Arabidopsis thaliana. Experiments revealed that Arabidopsis exposed to QDs that are dispersed in Hoagland's solution for 1-7 days did not internalize intact QDs. Analysis of Cd and Se concentrations in roots and leaves by inductively-coupled plasma mass spectrometry indicated that Cd and Se from QD-treated plants were not translocated into the leaves, and remained in the root system of Arabidopsis. Furthermore, fluorescence microscopy showed strong evidence that the QDs were generally on the outside surfaces of the roots, where the amount of QDs adsorbed is dependent on the stability of the QDs in suspension. Despite no evidence of nanoparticle internalization, the ratio of reduced glutathione levels (GSH) relative to the oxidized glutathione (GSSG) in plants decreased when plants were exposed to QD dispersions containing humic acids, suggesting that QDs caused oxidative stress on the plant at this condition.

Cd tolerance and accumulation in the aquatic macrophyte, Chara australis: potential use for charophytes in phytoremediation.

We investigated the potential use of the alga Chara australis (R. Br.) forphytore mediation of Cd-contaminated sediments in aquatic systems. Chara tolerated up to 20 mg added Cd (kg soil)⁻¹ in laboratory culture. Chlorophyll a and b levels were not affected even at Cd concentrations that suppressed growth. Levels of glutathione were suppressed at 2-35 mg added Cd (kg soil)⁻¹ to 200-350 nmol GSH (g DW)⁻¹, while control levels were 660 nmol GSH (g DW)⁻¹). Histochemical studies showed Cd occurred throughout cell walls and cytoplasm in plants grown in 5-20 mg Cd (kg soil)⁻¹. Quantification using ICP-MS showed the maximum concentration in shoots was 72 mg Cd (kg DW)⁻¹ at 35 mg added Cd (kg soil)⁻¹, while the maximum in rhizoids was 116 mg Cd (kg DW)⁻¹ at 25 mg added Cd (kg soil)⁻¹. The bioconcentration factor (BCF, concentration in plant/concentration in soil) exceeded 1.0, the critical value for hyperaccumulators, for shoots exposed to 35 mg Cd (kg soil)⁻¹ and rhizoids exposed to ≥25 mg Cd (kg soil)⁻¹. Translocation factors (TF, shoot concentration/rhizoid concentration) did not exceed 1.0 for any treatment. While Chara cannot be considered a hyperaccumulator, it shows promise for use in phytoremediation efforts.