A conserved oxalyl-coenzyme A decarboxylase in oxalate catabolism

The ability to biosynthesize oxalic acid can provide beneficial functions to plants; however, uncontrolled or prolonged exposure to this strong organic acid results in multiple physiological problems. Such problems include a disruption of membrane integrity, mitochondrial function, metal chelation, and free radical formation. Recent work suggests that a CoA-dependent pathway of oxalate catabolism plays a critical role in regulating tissue oxalate concentrations in plants. Although this CoA-dependent pathway of oxalate catabolism is important, large gaps in our knowledge of the enzymes catalyzing each step remain. Evidence that an oxalyl-CoA decarboxylase (OXC) catalyzes the second step in this pathway, accelerating the conversion of oxalyl-CoA to formyl-CoA, has been reported. Induction studies revealed that OXC gene expression was upregulated in response to an exogenous oxalate supply. Phylogenetic analysis indicates that OXCs are conserved across plant species. Evolutionarily the plant OXCs can be separated into dicot and monocot classes. Multiple sequence alignments and molecular modeling suggest that OXCs have similar functionality with three conserved domains, the N-terminal PYR domain, the middle R domain, and the C-terminal PP domain. Further study of this CoA-dependent pathway of oxalate degradation would benefit efforts to develop new strategies to improve the nutrition quality of crops.</p

Vacuolar H⁺-ATPase, Ca²⁺/H⁺ and Mn²⁺/H⁺ exchanger activity in CAX mutant plants.

<p>(<b>A</b>) Initial rates of V-ATPase H⁺ transport activity in purified vacuolar-enriched membrane vesicles from Col-0 (wild type) and <i>cax</i> knockout mutant plants determined from the rates of acridine orange fluorescence quenching during the first 60 sec following the addition of Mg-ATP, as shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0047455#pone.0047455.s006" target="_blank">Figure S6</a>. (<b>B</b>) and (<b>C</b>) Initial rates of ΔpH-dependent cation/H⁺ exchange activity in purified vacuolar-enriched membrane vesicles from Col-0 and <i>cax</i> knockout mutant plants determined from the rates of acridine orange fluorescence recovery during the first 60 sec following the establishment of a steady-state pH gradient and the addition of 200 µM CaCl₂ (<b>A</b>) or MnCl₂ (<b>B</b>), as shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0047455#pone.0047455.s006" target="_blank">Figure S6</a>. <i>F</i> indicates relative fluorescence intensity. Membrane vesicles were prepared from 2-week-old plants grown on solid 0.5×MS media and pre-treated with 50 mM CaCl₂ and 1.5 mM MnCl₂ 14 h before harvest. The data represent means±SE from three experiments. ** (<i>P</i><0.01) and * (<i>P</i><0.05) denotes significant difference between CAX mutant lines and Col-0 control as determined by one-way ANOVA.</p

Na sensitivity of CAX mutant plants.

<p>Total chlorophyll content was measured in leaf and shoot tissue of 21 d-old Col-0 (wild type) and <i>cax</i> knockout mutant plants following germination and growth on solid 0.5×MS media (adjusted to pH 5.6) in the absence or presence of 50 mM NaCl. The mean±SE (<i>n</i> = 4–6) chlorophyll content is shown. ** (<i>P</i><0.01) denotes significant difference between control and NaCl treatments as determined by one-way ANOVA.</p

Difference in elemental concentration in dry seeds from CAX mutant plants.

<p>The concentrations of known or putative substrates of CAX transporters: Ca, Mn, Na and Zn (<b>A</b>) and of metals not thought to be transported by CAX: Fe, K, Mg and P (<b>B</b>) are shown as % difference from Col-0 (wild type) seeds. Dry seeds (approximately 15 mg per sample) were obtained from plants grown on soil without additional metal supplementation. Elemental analysis was performed by ICP-AES measurement. Bars indicate the mean±SE of three replicates. * (<i>P</i><0.05) denotes significant difference from Col-0 as determined by one-way ANOVA.</p

Figure 4

<p>Germination profile of CAX mutant seedlings over three time periods. Approximately 100 seeds from Col-0 (wild type) and <i>cax</i> knockout mutant plants were sterilized and sown on 1% agar plates. After a 2-d incubation period at 4°C plates were moved to an environmentally-controlled growth chamber at 22°C. Radicle emergence from the testa was taken as the indicator of germination. Stacked bars indicate the percentage of seeds germinating 0–9 hours, 9–21 hours and 21–37 hours after transfer to 22°C. All values are corrected for seed non-viability, determined by quantifying the absence of seed germination after 10 d.</p

<i>CAX1</i>, <i>CAX2</i> and <i>CAX3</i> expression.

<p>Changes in <i>CAX</i> expression in <i>cax1</i>, <i>cax2</i> and <i>cax3</i> single and double knockout lines relative to Col-0 (wild type) were determined by real-time PCR using actin and ubiquitin as constitutive control primers. RNA was extracted from whole seedling tissue grown on 0.5×MS plates without additional metal supplementation for 21 d. Expression relative to actin is shown and expression relative to ubiquitin was equivalent. Relative fold changes in gene expression were calculated using the 2^−ΔΔCt method. Bars indicate the mean log expression±SE from three samples each replicated twice. ‘n’ denotes no increase in expression detected.</p

Mg and Mn sensitivity of CAX mutant plants.

<p>Fresh weight of Col-0 (wild type) and <i>cax</i> knockout mutant plants following germination and growth on solid 0.5×MS media (adjusted to pH 5.6) in the absence (<b>A</b>) or presence of 25 mM MgCl₂ (<b>B</b>) or 1.5 mM MnCl₂ (<b>C</b>). Bars indicate the mean±SE (<i>n</i> = 12–15) fresh weight measured in 21 d-old plants. ** (<i>P</i><0.01) and * (<i>P</i><0.05) denotes significant difference between CAX mutant lines and Col-0 control as determined by one-way ANOVA.</p

Germination time and maximum seed viability in CAX mutants.

<p>Seeds from Col-0 (wild type) and <i>cax</i> knockout mutant plants (approximately 70 of each line) were sterilized and sown on 1% agar before stratification for 2 d at 4°C in the dark. Plates were incubated (22°C, 16 h light/8 h dark) for 9 h then seeds were observed every 2 h for 12 h, then every 12 h until 10 d after which any seeds not germinated were considered non-viable. All values are the mean of three replicate experiments.</p

Figure 5

<p>Germination of CAX mutant seedlings in response to abscisic acid (ABA). The germination of seeds from Col-0 (wild type) and <i>cax</i> knockout mutant plants was quantified after 24 h on 0.1 µM ABA. Approximately 100 seeds of each line were sterilized and sown on 1% agar plates containing ABA. After a 2-d incubation period at 4°C plates were moved to an environmentally-controlled growth chamber at 22°C. Radicle emergence from the testa was taken as the indicator of germination. Bars indicate the mean±SE percentage of germination. ** (<i>P</i><0.01) denotes significant difference between CAX mutant lines and Col-0 control as determined by one-way ANOVA.</p

Quantitative Imaging of Glutathione in Live Cells Using a Reversible Reaction-Based Ratiometric Fluorescent Probe

Glutathione (GSH) plays an important role in maintaining redox homeostasis inside cells. Currently, there are no methods available to quantitatively assess the GSH concentration in live cells. Live cell fluorescence imaging revolutionized the field of cell biology and has become an indispensable tool in current biological studies. In order to minimize the disturbance to the biological system in live cell imaging, the probe concentration needs to be significantly lower than the analyte concentration. Because of this, any irreversible reaction-based GSH probe can only provide qualitative results within a short reaction time and will exhibit maximum response regardless of the GSH concentration if the reaction is completed. A reversible reaction-based probe with an appropriate equilibrium constant allows measurement of an analyte at much higher concentrations and, thus, is a prerequisite for GSH quantification inside cells. In this contribution, we report the first fluorescent probeThiolQuant Green (TQ Green)for quantitative imaging of GSH in live cells. Due to the reversible nature of the reaction between the probe and GSH, we are able to quantify mM concentrations of GSH with TQ Green concentrations as low as 20 nM. Furthermore, the GSH concentrations measured using TQ Green in 3T3-L1, HeLa, HepG2, PANC-1, and PANC-28 cells are reproducible and well correlated with the values obtained from cell lysates. TQ Green imaging can also resolve the changes in GSH concentration in PANC-1 cells upon diethylmaleate (DEM) treatment. In addition, TQ Green can be conveniently applied in fluorescence activated cell sorting (FACS) to measure GSH level changes. Through this study, we not only demonstrate the importance of reaction reversibility in designing quantitative reaction-based fluorescent probes but also provide a practical tool to facilitate redox biology studies