Cadmium-induced oxalate secretion from root apex is associated with cadmium exclusion and resistance in Lycopersicon esulentum

The mechanisms of heavy metal resistance in plants can be classified into internal tolerance and exclusion mechanisms, but exclusion of heavy metals with the help of organic acids secretion has not been well documented. Here we demonstrated the contribution of oxalate secretion to cadmium (Cd) exclusion and resistance in tomato. Different Cd resistance between two tomato cultivars was evaluated by relative root elongation (RRE) and Cd accumulation. Cultivar 'Micro-Tom' showed better growth and lower Cd content in roots than 'Hezuo903' at different Cd concentrations not only in short-term hydroponic experiment but also in long-term hydroponic and soil experiments, indicating that the genotypic difference in Cd resistance is related to the exclusion of Cd from roots. 'Micro-Tom' had greater ability to secrete oxalate, suggesting that oxalate secretion might contribute to Cd resistance. Cd-induced secretion of oxalate was localized to root apex at which the majority of Cd accumulated. Phenylglyoxal, an anion-channel inhibitor, effectively blocked Cd-induced oxalate secretion and aggravated Cd toxicity while exogenous oxalate supply ameliorated Cd toxicity efficiently. These results indicated that the oxalate secreted from the root apex helps to exclude Cd from entering tomato roots, thus contributes to Cd resistance in the Cd-resistant tomato cultivar.     

Sat1 is dispensable for active oxalate secretion in mouse duodenum

Mice deficient for the apical membrane oxalate transporter SLC26A6 develop hyperoxalemia, hyperoxaluria, and calcium oxalate stones due to a defect in intestinal oxalate secretion. However, the nature of the basolateral membrane oxalate transport process that operates in series with SLC26A6 to mediate active oxalate secretion in the intestine remains unknown. Sulfate anion transporter-1 (Sat1 or SLC26A1) is a basolateral membrane anion exchanger that mediates intestinal oxalate transport. Moreover, Sat1-deficient mice also have a phenotype of hyperoxalemia, hyperoxaluria, and calcium oxalate stones. We, therefore, tested the role of Sat1 in mouse duodenum, a tissue with Sat1 expression and SLC26A6-dependent oxalate secretion. Although the active secretory flux of oxalate across mouse duodenum was strongly inhibited (>90%) by addition of the disulfonic stilbene DIDS to the basolateral solution, secretion was unaffected by changes in medium concentrations of sulfate and bicarbonate, key substrates for Sat1-mediated anion exchange. Inhibition of intracellular bicarbonate production by acetazolamide and complete removal of bicarbonate from the buffer also produced no change in oxalate secretion. Finally, active oxalate secretion was not reduced in Sat1-null mice. We conclude that a DIDS-sensitive basolateral transporter is involved in mediating oxalate secretion across mouse duodenum, but Sat1 itself is dispensable for this process.     

不同氮素形态培养下荞麦叶片中草酸积累的变化

用1/5浓度Hoagland(pH6.0)营养液培养荞麦幼苗3d后,取其中一部分继续用此营养液(硝态氮);另一部分用硫酸氨和氯化钙取代硝态氮(氨态氮)的营养液,均培养至荞麦第一片真叶完全展开。结果表明,以氨态氮为唯一氮源培养荞麦时,植株叶片中草酸含量显著下降。进一步研究表明,氨态氮培养下荞麦根中及根分泌草酸的速率也显著下降,结果排除了叶片中草酸含量的下降是由于叶片中草酸向其根系转运或是因为根分泌草酸速率的差异造成的,而可能与其草酸代谢改变有关。氨态氮培养下叶片中与草酸代谢相关的有机酸含量以及相关酶活性也显著下降,这可能意味着荞麦叶片草酸形成积累可能与相关有机酸代谢有关。     

Expression of heterologous oxalate decarboxylase in HEK293 cells confers protection against oxalate induced oxidative stress as a therapeutic approach for calcium oxalate stone disease

Oxalates stimulate alterations in renal epithelial cells and thereby induce calcium oxalate (CaOx) stone formation. Bacillus subtilis YvrK gene encodes for oxalate decarboxylase (OxdC) which degrades oxalate to formate and CO₂. The present work is aimed to clone the oxdC gene in a mammalian expression vector pcDNA and transfect into Human Embryonic Kidney 293 (HEK293) cells and evaluate the oxdC expression, cell survival rate and oxalate degrading efficiency. The results indicate cell survival rate of HEK293/pcDNAOXDC cells pre-incubated with oxalate was enhanced by 28%. HEK293/pcDNAOXDC cells expressing OxdC treated with oxalate, significantly restored antioxidant activity, mitochondrial membrane potential and intracellular reactive oxygen species (ROS) generation compared with HEK293/pcDNA. Apoptotic marker caspase 3 downregulation illustrates HEK293/pcDNAOXDC cells were able to survive under oxalate-mediated oxidative stress. The findings suggest HEK293 cells expressing oxdC capable of degrading oxalate protect cells from oxidative damage and thus serve as a therapeutic option for prevention of CaOx stone disease.

     

Determination of urinary oxalate with alkylamine glass-bound sorghum oxalate oxidase and horseradish peroxidase

Oxalate in urine was analyzed using sorghum oxalate oxidase and horseradish peroxidase immobilized on alkylamine glass through glutaraldehyde. The minimum detection limit was 0.46 g/0.1 ml urine. The recovery of added oxalate was 97.5%. Within and between assay coefficients of variation were <3.5% and <6.5% respectively. A good correlation (r=0.9234) was found between oxalate values obtained by a commercial kit method and the present method. The method is unaffected by Cl– and NO3– found in urine.     

铝对外生菌根真菌草酸分泌及磷、钾、铝吸收的影响

试验研究了在铝胁迫条件下,6种(株)外生菌根真菌(ECMF)的生长、草酸分泌,以及磷、钾、铝的吸收状况。结果表明,铝对抗(耐)型菌种Pt715、HrSp、CgSIV的生长无抑制作用,但显著抑制敏感型菌株LbS238N、LbS238A和Lb270的生长,说明ECMF对铝胁迫的生长反应可能是筛选抗(耐)铝的指标之一。在铝胁迫条件下,无论是抗(耐)型还是敏感型菌种(株),都会发生一系列有益于抗(耐)铝的生化反应,包括草酸分泌量、菌丝磷和钾含量增加,H+分泌改变等。在培养液中,草酸电离产生的H+仅占H+总浓度的少数,说明溶液中H+的主要来源不是ECMF所分泌的草酸,而是菌丝细胞为保持吸收阳离子的电荷平衡排出的H+或分泌的其它有机酸。     

植物叶片中抗坏血酸含量与草酸积累的关系

不同植物和不同生长期烟草叶片中抗坏血酸含量变化与相应的草酸含量变化之间都无显著的相关性;喂饲外源抗坏血酸后的水稻和荞麦叶中草酸含量提高不明显。据此推测：尽管不能排除抗坏血酸可能是植物草酸合成的前体,但其内源含量高低不一定影响植物中草酸积累。     

A Comparison of Aluminum Resistance among Polygonum Species Originating on Strongly Acidic and Neutral Soils

Two kinds of Polygonum species (Polygonum aviculare L. and Polygonum lapathifolium L.) grown in tea garden soils at pH around 3.5 and one Polygonum bungeanum Turcz grown in neutral soils were collected to investigate the mechanisms involved in their high Al resistance. Hydroponic experiments showed that the root elongation was only inhibited by 15% in P. aviculare and 35% in P. lapathifolium after exposure to 50 μM Al for 24 h. Their Al resistance was respectively higher than and similar to that in an Al resistant buckwheat (Fagopyrum esculentum Moench) cultivar. In contrast, P. bungeanum was much more Al sensitive since the root elongation was inhibited by 80% under the same condition. The difference in Al resistance among Polygonum species was confirmed in a 10-d intermittent Al treatment experiment, the root biomass of the first two species were unaltered and decreased by 50% in the latter species. However, high Al accumulation was not found in the leaves, indicating these species were not Al accumulators. Oxalate efflux was detected in root exudates of both Al resistant species, efflux initiated within 30 min treatment of 50 μM Al. No organic acid anions were detected in the root exudates of the Al sensitive species. The anion channel inhibitor phenylglyoxal (PG) inhibited the oxalate efflux greatly. Inhibition of root elongation was greater in the presence of PG, confirming that oxalate efflux was associated with the Al resistance. However, since the efflux rate was much lower than their related species buckwheat, other mechanisms must be involved in Al resistance and these need to be studied further.     

Protecting Cell Walls from Binding Aluminum by Organic Acids Contributes to Aluminum Resistance

Aluminum-induced secretion of organic acids from the root apex has been demonstrated to be one major AI resistance mechanism in plants. However, whether the organic acid concentration is high enough to detoxify AI in the growth medium is frequently questioned. The genotypes of Al-resistant wheat, Cassia tora L. and buckwheat secrete malate, citrate and oxalate, respectively. In the present study we found that at a 35% inhibition of root elongation, the AI activities in the solution were 10, 20, and 50 μM with the corresponding malate, citrate, and oxalate exudation at the rates of 15, 20 and 21 nmol/cm2 per 12 h, respectively, for the above three plant species. When exogenous organic acids were added to ameliorate Al toxicity, twofold and eightfold higher oxalate and malate concentrations were required to produce the equal effect by citrate. After the root apical cell walls were isolated and preincubated in 1 mM malate, oxalate or citrate solution overnight, the total amount of AI adsorbed to the cell walls all decreased significantly to a similar level, implying that these organic acids own an equal ability to protect the cell walls from binding AI. These findings suggest that protection of cell walls from binding Al by organic acids may contribute significantly to AI resistance.     

大豆耐铝性品种差异及其与有机酸的关系

从 1 0个大豆品种中筛选出两个耐铝性差异显著的品种 ,研究了其耐铝性与有机酸的关系。经铝处理后 ,吴川品种的相对根长为 1 3 3 .5 % ,化州只有 68.9% ,表明吴川相对耐铝 ,化州对铝较敏感。将不同浓度的AlCl3 加入营养液中处理大豆 1 0d,化州较吴川根长受到较大影响 ,进一步证实吴川相对耐铝毒 ,而化州对酸铝敏感。机理研究发现大豆在铝胁迫下根系可分泌两种有机酸 (草酸、柠檬酸 ) ,其中吴川根系草酸分泌速率提高了 74% ,化州几乎没有提高 ,表明耐铝性大豆品种的根系草酸分泌速率明显提高 ,可增强其缓解酸铝毒性的能力。而二者分泌柠檬酸的速率虽然均有显著提高 ,但处理后感抗品种之间差异不大 ,表明柠檬酸在缓解铝毒性中的作用不大。铝处理下大豆根系虽然分泌两种有机酸 ,但草酸在大豆耐酸铝机制中的作用可能更为重要。     

Characterisation of nuclear pore complex oxalate binding protein from human kidney

Both rat and human kidney nuclei exhibited time and pH dependent oxalate or histone-oxalate uptake which was inhibited by anion transport inhibitor, 4,4-dithiocyanostilbene-2,2-disulphonic acid. Sodium chloride had no effect. Nuclear membrane had oxalate binding at pH 7.4. Extraction of nuclear membrane by Triton–high salt mixture showed maximal oxalate binding activity with nuclear pore complex while nuclear lamin had no oxalate binding. The rat and human kidney nuclear pore complex showed oxalate binding of 144 and 220 pmoles/mg protein respectively. Subsequent purification of the protein on diethyl amino ethyl–Sephadex A 50 column and Sephadex G-200 column yielded 4-fold purification. The protein revealed a molecular weight of 205 kDa on SDS-PAGE. The protein was found to be saturable at 2 M oxalate and had a Kd of 2.98 pM and a Bmax of 197 pmoles. Antibody for 205 kD was separated from primary biliary cirrhosis serum containing auto antibody against 205 kDa using affinity column chromatography. The oxalate binding activity as well as the nuclear uptake of oxalate or histone-oxalate were inhibited by its antibody.     

植物中草酸积累与光呼吸乙醇酸代谢的关系

对几种C3 和C4 植物中草酸含量及相应的乙醇酸氧化酶活性测定结果表明 :叶片光呼吸强度及其关键酶活性大小与草酸积累量没有相关性 ;植物根中均能积累草酸 ,但未测出乙醇酸氧化酶活性。烟草根、叶中的草酸含量在不同生长时期差异明显 ,且二者呈极显著正相关 (y =2 .5 6 5lnx 2 .137,r =0 .749,P <0 .0 0 1) ,说明根中草酸可能来自叶片。氧化乙醇酸的酶的活性与氧化乙醛酸的酶的活性呈极显著线性正相关 (y =0 .2 41x 0 .0 0 6 ,r=0 .96 7,P <0 .0 0 0 1) ,进一步证实是乙醇酸氧化酶催化了两种底物的反应。烟草在不同生长期叶片中草酸总含量变化与相应的乙醇酸氧化酶活性变化亦没有相关性 ;低磷胁迫可显著诱导烟草根叶中的草酸形成和分泌 ,但并未影响乙醇酸氧化酶活性 ,进一步证明草酸积累与该酶活性大小无关     

Adsorption of ethylenediaminetetraacetic dianhydride modified oxalate decarboxylase on calcium oxalate

We used ethylenediaminetetraacetic acid dianhydride (EDTAD) to modify oxalate decarboxylase (OXDC) to improve its adsorption on calcium oxalate stones. The modified sites were identified by Ultra performance liquid chromatography-mass spectrometry (UPLC-MS) and the adsorption mechanism of the EDTAD-modified OXDC on calcium oxalate (CaOx) was investigated. We investigated adsorption time, initial enzyme concentration, temperature and solution pH on the adsorption process. Data were analyzed using kinetics, thermodynamics and isotherm adsorption models. UPLC-MS showed that EDTAD was attached to OXDC covalently and suggested that the chemical modification occurred at both the free amino of the side chain and the α-NH₂ of the peptide. The adsorption capacity of the EDTAD-OXDC on calcium oxalate was 53.37% greater than that of OXDC at the initial enzyme concentration of 5 mg/ml, pH = 7.0, at 37° C. The modified enzyme (EDTAD-OXDC) demonstrated improved oxalate degradation activity at pH 4.5?6.0. Kinetic data fitting analysis suggested a pseudo second order kinetic model. Estimates of the thermodynamic parameters including ΔG⁰, ΔH⁰ and ΔS⁰ of the adsorption process showed it to be feasible, spontaneous and endothermic. Isotherm data fitting analysis indicated that the adsorption process is reduced to monolayer adsorption at a low enzyme concentration and to multilayer adsorption at a high enzyme concentration. It may be possible to apply OXDC to degradation of calcium oxalate stones.     

Aluminum-induced cell death of barley-root border cells is correlated with peroxidase- and oxalate oxidase-mediated hydrogen peroxide production

The function of root border cells (RBC) during aluminum (Al) stress and the involvement of oxalate oxidase, peroxidase and H₂O₂ generation in Al toxicity were studied in barley roots. Our results suggest that RBC effectively protect the barley root tip from Al relative to the situation in roots cultivated in hydroponics where RBC are not sustained in the area surrounding the root tip. The removal of RBC from Al-treated roots increased root growth inhibition, Al and Evans blue uptake, inhibition of RBC production, the level of dead RBC, peroxidase and oxalate oxidase activity and the production of H₂O₂. Our results suggest that even though RBC actively produce active oxygen species during Al stress, their role in the protection of root tips against Al toxicity is to chelate Al in their dead cell body.     

Occurrence of histone-related oxalate binding in rat liver nucleus

The rat liver nuclear oxalate binding protein was isolated, purified by anion and cation exchange column chromatography using Diethyl Amino Ethyl Sephadex, Carboxy Methyl Cellulose and Carboxy Methyl Sephadex C-50 ion exchangers. The purified oxalate binding protein was found to be H1B of H1 fraction of histories. Kinetic analysis of oxalate binding showed the presence of two affinity sites, one with Kd of 133.5 nM and Bmax of 40 pmoles and another with Kd of 262.5 nM and Bmax of 210 pmoles. The optimal oxalate binding was at pH 4.2 and at 28°C. The oxalate binding was specific and reversible and not due to ionic charge interaction. The IC₅₀ of other dicarboxylates was higher than that of oxalate. EGTA had no effect on oxalate binding but di- and tri-carboxylate carrier inhibitors and thiol modifying agents significantly lowered the binding activity. Oxalate binding to histones was significantly reduced in the presence of DNA or nucleotides, but RNA had no effect. ATP completely inhibited the oxalate binding activity at 1 mM concentration. Different tissues exhibited oxalate binding showing ubiquitous nature. Calf thymus H1 showed maximal binding similar to liver histones.Abbreviations ADP Adenosine diphosphate - ATP Adenosine triphosphate - DNA Deoxyribonucleic acid - RNA Ribonucleic acid     

Conductive pathways for chloride and oxalate in rabbit ileal brush-border membrane vesicles

To evaluate thepossibility that an apical membrane conductive pathway for oxalate ispresent in the rabbit distal ileum, we studied oxalate([¹⁴C]oxalate) andchloride (³⁶Cl) uptake intobrush-border membrane vesicles enriched 15- to 18-fold in sucraseactivity. Voltage-sensitive pathways for oxalate and chloride wereidentified by the stimulation of uptake provided by an inwardlydirected potassium diffusion potential in the presence of valinomycin.Additionally, outwardly directed oxalate (or chloride) gradientsstimulated[¹⁴C]oxalate (or³⁶Cl) uptake to a greater degreein the absence of valinomycin (when intracellular and extracellularpotassium are equal) than in the presence of valinomycin.Voltage-dependent anion uptake was poorly saturable: apparent affinityconstants were 141 ± 17 and 126 ± 8 mM for chlorideand oxalate, respectively. Activation energies for thevoltage-dependent uptake processes were low: 4.7 and 6.3 kcal/mol forchloride and oxalate, respectively. Sensitivity profiles ofvoltage-dependent chloride and oxalate uptake to anion transport inhibitors were similar. We conclude that an anion conductance ispresent in the apical membranes of ileal enterocytes and that thisconductance is a candidate pathway for oxalate efflux from theenterocyte during transepithelial oxalate secretion.

     

Comparative studies on the effect of a protein-synthesis inhibitor on aluminium-induced secretion of organic acids from Fagopyrum esculentum Moench and Cassia tora L. roots

Aluminium (Al)-induced secretion of organic acids from plant roots is considered a mechanism of Al resistance, but the processes leading to the secretion of organic acids are still unknown. In the present study, a protein-synthesis inhibitor, cycloheximide (CHM), was used to investigate its effect on Al-induced organic acid secretion in a pattern I (rapid exudation of organic acids under Al stress) plant buckwheat (Fagopyrum esculentum Moench) and a pattern II (exudation of organic acids was delayed by several hours under Al stress) plant Cassia tora L. A dose-response experiment showed that the secretion of oxalate by buckwheat roots was not affected by CHM when added in the range from 0 to 50 microM, with or without exposure to 100 microm Al, but the secretion of citrate was completely inhibited by 30 microM CHM in C. tora. A time-course experiment showed that even prolonged exposure to 20 microM CHM did not affect oxalate secretion in buckwheat, but significantly inhibited citrate secretion in C. tora. However, citrate synthase (CS) activity in C. tora was not affected during 12 h exposure to 100 microM Al when compared with that in control roots, although CHM can inhibit CS activity effectively. These results indicated that CS activity was not related to Al-regulated citrate efflux in C. tora. The total protein was decreased by 14.0% and 32.3% in C. tora and buckwheat root tip, respectively, after 3-h treatment with 20 microM CHM. A 3-h pulse with 20 microM CHM completely inhibited citrate efflux in C. tora during the next 6-h exposure to Al, although a small amount of citrate was exuded after 9-h exposure. However, oxalate efflux in buckwheat was not influenced by a similar treatment. In buckwheat, a 3-h pulse with 100 microM Al maintained oxalate secretion at a high level during the next 9 h, with or without CHM treatment. Conversely, in C. tora a 6-h pulse with 100 microM Al induced significant secretion of citrate which was inhibited by the CHM. Taken together, these findings suggest that both de novo synthesis and activation of an anion channel are needed for Al-induced secretion of citrate in C. tora, but in buckwheat the plasma membrane protein responsible for oxalate secretion pre-exists.     

Ileal oxalate absorption and urinary oxalate excretion are enhanced in Slc26a6 null mice

Intestinal oxalate transport, mediated by anion exchange proteins, is important to oxalate homeostasis and consequently to calcium oxalate stone diseases. To assess the contribution of the putative anion transporter (PAT)1 (Slc26a6) to transepithelial oxalate transport, we compared the unidirectional and net fluxes of oxalate across isolated, short-circuited segments of the distal ileum of wild-type (WT) mice and Slc26a6 null mice [knockout (KO)]. Additionally, urinary oxalate excretion was measured in both groups. In WT mouse ileum, there was a small net secretion of oxalate (J(net)(Ox) = -5.0 +/-5.0 pmol.cm(-2).h(-1)), whereas in KO mice J(net)(Ox) was significantly absorptive (75 +/- 10 pmol.cm(-2)h.h(-1)), which was the result of a smaller serosal-to-mucosal oxalate flux (J(sm)(Ox)) and a larger mucosal-to-serosal oxalate flux (J(ms)(Ox)). Mucosal DIDS (200 microM) reduced J(sm)(Ox) in WT mice, leading to reversal of the direction of net oxalate transport from secretion to absorption (J(net)(Ox) = 15.0 +/- 5.0 pmol.cm(-2).h(-1)) , but DIDS had no significant effect on KO ileum. In WT mice in the absence of mucosal Cl(-), there were small increases in J(ms)(Ox) and decreases in J(sm)(Ox) that led to a small net oxalate absorption. In KO mice, J(net)(Ox) was 1.5-fold greater in the absence of mucosal Cl(-), due solely to an increase in J(ms)(Ox). Urinary oxalate excretion was about fourfold greater in KO mice compared with WT littermates. We conclude that PAT1 is DIDS sensitive and mediates a significant fraction of oxalate efflux across the apical membrane in exchange for Cl(-); as such, PAT1 represents a major apical membrane pathway mediating J(sm)(Ox).     

Colorimetric detection of oxalate in aqueous solution by a pyrogallol red‐based Cu2+ complex

The pyrogallol red (PR)‐based Cu²⁺ complex was proven to be an effective and selective colorimetric chemosensing ensemble for recognition of oxalate over other anions in a perfect aqueous solution. The addition of oxalate to the PR–Cu²⁺ complex resulted in a colour change from purple to orange colour due to the regeneration of PR by the chelation of oxalate with Cu²⁺, while other anions did not induce any significant colour change. Moreover, it was revealed that no obvious interference was observed during the titrations with oxalate into each other anion. Therefore, the PR–Cu²⁺ complex can be used as a simple and practical colorimetric chemosensor for detecting oxalate.