Non-invasive Micro-test Technique Research Articles

The role of plasma membrane H(+) -ATPase in jasmonate-induced ion fluxes and stomatal closure in Arabidopsis thaliana.

Methyl jasmonate (MeJA) elicits stomatal closure in many plant species. Stomatal closure is accompanied by large ion fluxes across the plasma membrane (PM). Here, we recorded the transmembrane ion fluxes of H(+) , Ca(2+) and K(+) in guard cells of wild-type (Col-0) Arabidopsis, the CORONATINE INSENSITIVE1 (COI1) mutant coi1-1 and the PM H(+) -ATPase mutants aha1-6 and aha1-7, using a non-invasive micro-test technique. We showed that MeJA induced transmembrane H(+) efflux, Ca(2+) influx and K(+) efflux across the PM of Col-0 guard cells. However, this ion transport was abolished in coi1-1 guard cells, suggesting that MeJA-induced transmembrane ion flux requires COI1. Furthermore, the H(+) efflux and Ca(2+) influx in Col-0 guard cells was impaired by vanadate pre-treatment or PM H(+) -ATPase mutation, suggesting that the rapid H(+) efflux mediated by PM H(+) -ATPases could function upstream of the Ca(2+) flux. After the rapid H(+) efflux, the Col-0 guard cells had a longer oscillation period than before MeJA treatment, indicating that the activity of the PM H(+) -ATPase was reduced. Finally, the elevation of cytosolic Ca(2+) concentration and the depolarized PM drive the efflux of K(+) from the cell, resulting in loss of turgor and closure of the stomata.

O2 and Ca(2+) fluxes as indicators of apoptosis induced by rose bengal-mediated photodynamic therapy in human oral squamous carcinoma cells.

Photodynamic therapy (PDT) triggers various cellular responses and induces cell death via necrosis and/or apoptosis. This study evaluated the feasibility of using O2 and Ca(2+) fluxes as indicators of apoptosis induced by rose bengal (RB)-mediated PDT in human oral squamous carcinoma cells (Cal27 cells). Intracellular reactive oxygen species (ROS) generation was assessed by the dichloro-dihydro-fluorescein diacetate (DCFH-DA) method. Real-time O2 and Ca(2+) flux measurements were performed using the noninvasive micro-test technique (NMT). Apoptosis of the PDT-treated cells was confirmed by 4'6-diamidino-2-phenylindole-dilactate staining. The activation of apoptosis-related molecules was examined using Western blot. We assayed the effects of the fluctuation of O2 and Ca(2+) flux in response to PDT and the apoptotic mechanism, by which ROS, O2, and Ca(2+) synergistically may trigger apoptosis in PDT-treated cells. Real-time O2 and Ca(2+) flux measurements revealed that these indicators were involved in the timely regulation of apoptosis in the PDT-treated cells and were activated 2 h after PDT treatment. RB-mediated PDT significantly elicited the generation of ROS by approximately threefold, which was critical for PDT-induced apoptosis. Cytochrome c and cleaved caspase-3, caspase-9 and poly ADP ribose polymerase (PARP) were overexpressed, and the data provided evidence that 2 h was considered to be the key observation time in RB-mediated PDT-induced apoptosis in Cal27 cells. Our collective results indicated that the effects of O2 and Ca(2+) fluxes may act as a real-time biomonitoring system of apoptosis in the RB-PDT-treated cells. Also, RB-mediated PDT can be a potential and effective therapeutic modality in oral squamous cell carcinoma.

Physiological fluxes and antioxidative enzymes activities of immobilized Phanerochaete chrysosporium loaded with TiO2 nanoparticles after exposure to toxic pollutants in solution

Immobilized Phanerochaete chrysosporium loaded with TiO2 nanoparticles (PTNs) are novel high-value bioremediation materials for adsorbing cadmium and for degrading 2,4-dichlorophenol (2,4-DCP). The real-time changes in H+ and O2 fluxes were measured using the noninvasive microtest technique (NMT). The H+ influx increased after the addition of 2,4-DCP, and shifted to efflux following the addition of Cd2+. The O2 flux decreased after the addition of both 2,4-DCP and Cd2+. A larger Cd2+ flux was immediately observed after exposure to 0.5mM Cd2+ (−351.25pmolcm−2s−1) than to 0.1mM Cd2+ (−107.47pmolcm−2s−1). The removal of Cd2+ by the PTNs increased more after treatment with the 0.5mM exposure solution (27.6mgg−1) than with the 0.1mM exposure solution (3.49mgg−1). The enzyme activities were analyzed to review the antioxidative defense system of PTNs in a solution containing various concentrations of Cd2+. The activities of the coenzyme nicotinamide adenine dinucleotide (NADH) oxidase as well as the enzyme catalase (CAT) plateaued at 6.5Ug−1 FW and 9.7Ug−1 FW, respectively, after exposure to 0.25mM Cd2+. The activity of superoxide dismutase (SOD) increased gradually in solutions containing 0.1–0.6mM Cd2+, and eventually reached a maximum (68.86Ug−1 FW). These results illustrate how the antioxidative defense system and the physiological fluxes of PTNs respond to the stress caused by toxic pollutants.

Heterotrimeric G-proteins involved in the MeJA regulated ion flux and stomatal closure in Arabidopsis thaliana.

Heterotrimeric G-proteins play an important role in plant signalling pathways. The plant hormone methyl jasmonate (MeJA) can induce stomatal closure in many plant species. The signal cascade in MeJA-induced stomatal closure has been studied previously. However, the function of G proteins in this process has not yet been evaluated. In this study, the stomatal movement induced by MeJA in the wild-type Arabidopsis thaliana (L. Heynh.) (WS), Gα subunit loss-of-function mutant gpa1-1 and gpa1-2 guard cells were measured. Further, the transmembrane ion flux (H+, Ca2+ and K+) and reactive oxygen species (ROS) experiments were performed in guard cells from WS, GDP-β-S pre-treated WS, gpa1-1 and gpa1-2 using non-invasive micro-test technique (NMT) and confocal technique. It was observed that the MeJA-induced stomatal closure was abolished in guard cells of gpa1 mutants. GDP-β-S pre-treatment and gpa1 mutants impaired the MeJA-activated H+ efflux, Ca2+ influx and K+ efflux. The accumulation of ROS in gpa1-1 and gpa1-2 guard cells was also lower than that in WS guard cells under MeJA treatment. These results suggested that Gα subunits are involved in regulating the signal events in JA signal pathway and stomatal closure.

Indole derivatives inhibited the formation of bacterial biofilm and modulated Ca2+ efflux in diatom

Marine biofouling is a serious environmental problem worldwide. As an effort to find environmental friendly antifoulants, indole derivatives were determined for their activities to inhibit the growth of bacteria and diatom. The minimum inhibitory concentrations (MICs) of indole derivatives against bacteria were very low, especially for 6-chloroindole. It was proved that 6-chloroindole obviously inhibited the growth of bacteria, interfered with the formation of bacterial biofilm, destroyed bacterial cell morphology and also inhibited the growth of diatom Cylindrotheca sp. as well. By using noninvasive micro-test technique (NMT), 6-chloroindole triggered algal cellular Ca2+ efflux. The highest value was 72.03pmolcm−2s−1, 10.6 times of the control group. The present studies indicated that indole derivatives might have the potential to be new antifouling agents because of their excellent antibacterial and anti-algal activities. At the same time, Ca2+ efflux might be one of the mechanisms that indole derivatives inhibited the growth of diatom.

Net NH4 + and NO3 − fluxes, and expression of NH4 + and NO3 − transporter genes in roots of Populus simonii after acclimation to moderate salinity

Net NH flux was higher in fine roots of P. simonii after NaCl treatment compared to the NO flux which was consistent with the expression levels of AMTs and NRTs. The characteristics of nitrogen uptake under salt stress would provide insights into the mechanisms of salt tolerance in plants. In the present study, net NH4 + and NO3 − fluxes were measured using the non-invasive micro-test technique, and transcript abundance of ammonium/nitrate transporter genes (AMTs/NRTs) were determined by real time PCR in the fine roots of Populus simonii after treatment for 21 days with 75 mM NaCl. The NH4 + and NO3 − fluxes showed heterogeneous spatial patterns along the root tip in nutrient solution. The maximum influx of NH4 + occurred near the root tip, while that of NO3 − was 15 mm from the root tip. The net NO3 − flux was inhibited by ca. 25 % with the provision of NH4 +. On average, the newly grown fine roots took up more NH4 + after the 75 mM NaCl treatment than did the control. Consistent with the NH4 + and NO3 − influxes, most AMTs were up-regulated in the salt-treated roots relative to the control, while most NRTs were down-regulated. Our study provides insights into nitrogen uptake and the AMT/NRT expression levels in the fine roots of P. simonii after acclimation to moderate salinity. This may be valuable for understanding nitrogen uptake mechanisms after adaptation to salinity.

Behavior detection and activity recovery of damaged anammox bacteria culture after accidental overheating

Abstract Anaerobic ammonium-oxidizing (anammox) is a novel wastewater treatment process, which can realize efficient nitrogen removal with low operational cost. The anammox process is usually executed at 35 °C by heating devices. The potential effects of overheating on anammox culture are still remained as an open question. In this study, we share experiences about behavior detection and activity recovery of damaged anammox culture after accidental overheating. When anammox culture suffered 48 °C for 1 h, flocculating and deflocculating occurred, resulting in the suspended culture and the settled. Fluorescence in situ Hybridization, Flow Cytometry and Non-invasive Micro-test Technique showed that the settled culture contained more anammox bacteria than the suspended, leading to its higher death probability as time course, higher bulk liquid H + flux rate and faster activity restoration for nitrogen removal after overheating. The autofluorescence of Candidatus Brocadia fulgida varied during this process and could be considered as a characteristic fingerprint for these anammox bacteria. Experimental results also indicated the shear force produced by 150 rpm stir would contribute to the further damage of the heated bacteria. Reducing shear stress by low stirring rates and static cultivation might potentially be used as effective strategies in the repair of damaged anammox culture.

Over-expression of the MxIRT1 gene increases iron and zinc content in rice seeds.

Iron and zinc are essential in plant and human nutrition. Iron deficiency has been one of the causes of human mortality, especially in developing countries with high rice consumption. MxIRT1 is a ferrous transporter that has been screened from an iron-efficient genotype of the apple tree, Malus xiaojinensis Cheng et Jiang. In order to produce Fe-biofortified rice with MxIRT1 to solve the Fe-deficiency problem, plant expression vectors of pCAMBIA1302-MxIRT1:GFP and pCAMBIA1302-anti MxIRT1:GFP were constructed that led to successful production of transgenic rice. The transgenic plant phenotypes showed that the expression of endogenous OsIRT1 was suppressed by anti-MxIRT1 in antisense lines that acted as an opposing control, while sense lines had a higher tolerance under Zn- and Fe-deficient conditions. The iron and zinc concentration in T3 seeds increased by three times in sense lines when compared to the wild type. To understand the MxIRT1 cadmium uptake, the MxIRT1 cadmium absorption trait was compared with AtIRT1 and OsIRT1 in transgenic rice protoplasts, and it was found that MxIRT1 had the lowest Cd uptake capacity. MxIRT1 transgenic tobacco-cultured bright yellow-2 (BY-2) cells and rice lines were subjected to different Fe conditions and the results from the non-invasive micro-test technique showed that iron was actively transported compared to cadmium as long as iron was readily available in the environment. This suggests that MxIRT1 is a good candidate gene for plant Fe and Zn biofortification.

The fluxes of H2O2 and O2 can be used to evaluate seed germination and vigor of Caragana korshinskii

Seed deterioration is detrimental to plant germplasm conservation, and predicting seed germination and vigor with reliability and sensitivity means is urgently needed for practical problems. We investigated the link between hydrogen peroxide (H2O2) flux, oxygen influx and seed vigor of Caragana korshinskii by the non-invasive micro-test technique (NMT). Some related physiological and biochemical changes in seeds were also determined to further explain the changes in the molecular fluxes. The results showed that there was a good linear relationship between germination and H2O2 flux, and that O2 influx was more suitable for assessing seed vigor. H2O2 flux changed relatively little initially, mainly affected by antioxidants (APX, CAT and GSH) and H2O2 content; afterward, the efflux increased more and more rapidly due to high membrane permeability. With the damage of mitochondrial respiration and membrane integrity, O2 influx was gradually reduced. We propose that monitoring H2O2 and O2 fluxes by NMT may be a reliable and sensitive method to evaluate seed germination and vigor.

Physiological mechanisms for high salt tolerance in wild soybean (Glycine soja) from Yellow River Delta, China: photosynthesis, osmotic regulation, ion flux and antioxidant capacity.

Glycine soja (BB52) is a wild soybean cultivar grown in coastal saline land in Yellow River Delta, China. In order to reveal the physiological mechanisms adapting to salinity, we examined photosynthesis, ion flux, antioxidant system and water status in Glycine soja under NaCl treatments, taking a cultivated soybean, ZH13, as control. Upon NaCl exposure, higher relative water content and water potential were maintained in the leaf of BB52 than ZH13, which might depend on the more accumulation of osmotic substances such as glycinebetaine and proline. Compared with ZH13, activities of antioxidant enzymes including superoxide dismutase, catalase, ascorbate peroxidase and contents of ascorbate, glutathione and phenolics were enhanced to a higher level in BB52 leaf under NaCl stress, which could mitigate the salt-induced oxidative damage in BB52. Consistently, lipid peroxidation indicated by malondialdehyde content was lower in BB52 leaf. Photosynthetic rate (Pn) was decreased by NaCl stress in BB52 and ZH13, and the decrease was greater in ZH13. The decreased Pn in BB52 was mainly due to stomatal limitation. The inhibited activation of rubisco enzyme in ZH13 due to the decrease of rubisco activase content became an important limiting factor of Pn, when NaCl concentration increased to 200 mM. Rubisco activase in BB52 was not affected by NaCl stress. Less negative impact in BB52 derived from lower contents of Na+ and Cl- in the tissues, and non-invasive micro-test technique revealed that BB52 roots had higher ability to extrude Na+ and Cl-. Wild soybean is a valuable genetic resource, and our study may provide a reference for molecular biologist to improve the salt tolerance of cultivated soybean in face of farmland salinity.

Responses of Phanerochaete chrysosporium to Toxic Pollutants: Physiological Flux, Oxidative Stress, and Detoxification

The white-rot fungus Phanerochaete chrysosporium has been widely used for the treatment of waste streams containing heavy metals and toxic organic pollutants. The development of fungal-based treatment technologies requires detailed knowledge of the relationship between bulk water quality and the physiological responses of fungi. A noninvasive microtest technique was used to quantify real-time changes in proton, oxygen, and cadmium ion fluxes following the exposure of P. chrysosporium to environmental toxic (2,4-dichlorophenol and cadmium). Significant changes in H(+) and O(2) flux occurred after exposure to 10 mg/L 2,4-dichlorophenol and 0.1 mM cadmium. Cd(2+) flux decreased with time. Reactive oxygen species formation and antioxidant levels increased after cadmium treatment. Superoxide dismutase activity correlated well with malondialdehyde levels (r(2) = 0.964) at low cadmium concentrations. However, this correlation diminished and malondialdehyde levels significantly increased at the highest cadmium concentration tested. Real-time microscale signatures of H(+), O(2), and Cd(2+) fluxes coupled with oxidative stress analysis can improve our understanding of the physiological responses of P. chrysosporium to toxic pollutants and provide useful information for the development of fungal-based technologies to improve the treatment of wastes cocontaminated with heavy metals and organic pollutants.

丛枝菌根真菌对枳根净离子流及锌污染下枳苗矿质营养的影响

盆栽实验研究了不同施Zn水平(0、300 mg/kg和600 mg/kg)下,接种丛枝菌根真菌<em>Glomus intraradices</em>对枳苗生长、Zn、Cu、P、K、Ca、Mg分布的影响,并采用非损伤微测技术测定分析了菌根化与非菌根化枳根净Ca²⁺、H⁺、NO₃^-离子流动态。结果表明:(1)在不同施Zn水平下,接种菌根真菌显著提高了枳苗地上部及根部鲜重;随着施Zn水平的提高,菌根侵染率呈降低趋势,枳苗地上部与根部Zn含量呈增加趋势,且接种株根部Zn含量显著高于未接种株。(2)接种株未施Zn处理的地上部Cu、P、K、Mg和根部Cu含量、施600 mg/kg Zn处理的根部Cu及施300 mg/kg Zn处理的根部P含量均显著高于对照,而菌根真菌侵染对枳苗Ca含量并无显著性影响。(3)接种株未施Zn处理的根部距根尖端0 μm和600 μm处净Ca²⁺流出速率、600 μm处净H⁺流入速率、2400 μm处净NO₃^-流入速率均显著高于未接种株。;We investigated the effects of arbuscular mycorrhizal fungus inoculation on net ion flux in the roots of trifoliate orange (<em>Poncirus trifoliata</em> (L.) Raf.) and mineral nutrition in seedlings under zinc contamination. A pot experiment was conducted to study plant growth as well as the distribution of zinc, copper, phosphorus, potassium, calcium, and magnesium in trifoliate orange seedlings inoculated by the arbuscular mycorrhizal fungus, <em>Glomus intraradices</em>, under greenhouse conditions. Plants were grown in yellow soil:quartz sand (9∶1, v/v) medium and were irrigated with solutions containing three different zinc concentrations (0, 300 and 600 mg/kg), which corresponded to control, medium and high zinc contamination treatments, respectively. Meanwhile, a non-invasive micro-test technique was used to determine the net fluxes of Ca²⁺, H⁺ and NO₃^- along mycorrhizal and non-mycorrhizal roots of trifoliate orange seedlings grown in medium with no zinc added. Arbuscular mycorrhizal colonization significantly increased the fresh weight of shoots and roots exposed to different zinc levels. With increasing zinc concentrations, the mycorrhizal colonization percentage decreased, while the zinc levels in the shoots and roots progressively increased. Zinc concentrations in the roots were significantly higher in arbuscular mycorrhizal seedlings than in non-arbuscular mycorrhizal seedlings. The shoot/root ratios of the zinc translocation coefficient gradually decreased in arbuscular mycorrhizal seedlings with increasing zinc concentrations. This indicated that at medium or high levels of zinc contamination, a lot of zinc was immobilized in roots through the establishment of mycorrhizae. Therefore, phytotoxicity might be alleviated. Copper, phosphorus, potassium, and magnesium concentrations in shoots of plants grown in medium with no added zinc were significantly higher in arbuscular mycorrhizal seedlings than those in non-arbuscular mycorrhizal seedlings. Similarly, copper concentrations in the roots of plants in medium with 600 mg/kg of added zinc and phosphorus concentrations in roots of plants in medium with 300 mg/kg added zinc were higher in arbuscular mycorrhizal seedlings. Arbuscular mycorrhizal colonization had no significant effects on calcium concentrations in seedlings. Copper and phosphorus concentrations gradually decreased in both arbuscular mycorrhizal and non-arbuscular mycorrhizal seedlings with increasing zinc levels, which demonstrated that zinc levels in seedlings are negatively correlated with copper or phosphorus. With no added zinc, phosphorus, potassium, magnesium and copper uptake was promoted by arbuscular mycorrhizal fungus infection. Under zinc contamination, phosphorus, and copper uptake was still accelerated by mycorrhizal colonization. Thus, the effects of mycorrhizal colonization were not only related to the degree of zinc pollution, but were also correlated with the species of fungi and host plants. Additionally, net Ca²⁺ efflux at 0 μm and 600 μm, net H⁺ influx at 600 μm, and net NO₃^- influx at 2400 μm from the root tip of arbuscular mycorrhizal seedlings in medium with no added zinc were significantly higher than those of non-arbuscular mycorrhizal seedlings. These results suggest that mycorrhizal symbiosis activates Ca²⁺-ATPase, Ca²⁺/H⁺ antiporters and NO₃^-/H⁺ symporters in root plasma membranes. Nutrient uptake and stimulation of growth are mediated by arbuscular mycorrhizal fungi. Furthermore, the variations detected in arbuscular mycorrhizal roots for Ca²⁺, H⁺ and NO₃^- fluxes point to a significant involvement of the fungus.

Effects and mechanisms of store-operated calcium channel blockade on hepatic ischemia-reperfusion injury in rats

To further investigate the important role of store-operated calcium channels (SOCs) in rat hepatocytes and to explore the effects of SOC blockers on hepatic ischemia-reperfusion injury (HIRI). Using freshly isolated hepatocytes from a rat model of HIRI (and controls), we measured cytosolic free Ca(2+) concentration (by calcium imaging), net Ca(2+) fluxes (by a non-invasive micro-test technique), the SOC current (I(SOC); by whole-cell patch-clamp recording), and taurocholate secretion [by high-performance liquid chromatography and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assays]. Ca(2+) oscillations and net Ca(2+) fluxes mediated by Ca(2+) entry via SOCs were observed in rat hepatocytes. I(SOC) was significantly higher in HIRI groups than in controls (57.0 ± 7.5 pA vs 31.6 ± 2.7 pA, P < 0.05) and was inhibited by La(3+). Taurocholate secretion by hepatocytes into culture supernatant was distinctly lower in HIRI hepatocytes than in controls, an effect reversed by SOC blockers. SOCs are pivotal in HIRI. SOC blockers protected against HIRI and assisted the recovery of secretory function in hepatocytes. Thus, they are likely to become a novel class of effective drugs for prevention or therapy of HIRI patients in the future.

A DExD/H box RNA helicase is important for K+ deprivation responses and tolerance in Arabidopsis thaliana

The molecular mechanism for sensing and transducing the stress signals initiated by K(+) deprivation in plants remains unknown. Here, we found that the expression of AtHELPS, an Arabidopsis DExD/H box RNA helicase gene, was induced by low-K(+), zeatin and cold treatments, and downregulated by high-K(+) stress. To further investigate the expression pattern of AtHELPS, pAtHELPS::GUS transgenic plants were generated. Histochemical staining indicated that AtHELPS is mainly expressed in the young seedlings and vascular tissues of leaves and roots. Using both helps mutants and overexpression lines, we observed that, in the low-K(+) condition, AtHELPS affected Arabidopsis seed germination and plant weight. Interestingly, the mRNA levels of AKT1, CBL1/9 and CIPK23 in the helps mutants were much higher than in the overexpression lines under low-K(+) stress. Moreover, under low-K(+) stress, the helps mutants displayed increased K(+) influx, whereas the overexpression line of AtHELPS had a lower flux rate in the roots by the noninvasive micro-test technique. Taken together, these results provide information for the functional analysis of plant DEVH box RNA helicases, and suggest that AtHELPS, as an important negative regulator, plays a role in K(+) deprivation stress.

Measuring Ca2+influxes of TRPC1-dependent Ca2+channels in HL-7702 cells with Non-invasive Micro-test Technique

To explore the possibility of using the Non-invasive Micro-test Technique (NMT) to investigate the role of Transient Receptor Potential Canonical 1 (TRPC1) in regulating Ca(2+) influxes in HL-7702 cells, a normal human liver cell line. Net Ca(2+) fluxes were measured with NMT, a technology that can obtain dynamic information of specific/selective ionic/molecular activities on material surfaces, non-invasively. The expression levels of TRPC1 were increased by liposomal transfection, whose effectiveness was evaluated by Western-blotting and single cell reverse transcription-polymerase chain reaction. Ca(2+) influxes could be elicited by adding 1 mmol/L CaCl(2) to the test solution of HL-7702 cells. They were enhanced by addition of 20 micromol/L noradrenaline and inhibited by 100 micromol/L LaCl(3) (a non-selective Ca(2+) channel blocker); 5 micromol/L nifedipine did not induce any change. Overexpression of TRPC1 caused increased Ca(2+) influx. Five micromoles per liter nifedipine did not inhibit this elevation, whereas 100 micromol/L LaCl(3) did. In HL-7702 cells, there is a type of TRPC1-dependent Ca(2+) channel, which could be detected via NMT and inhibited by La(3+).