An Oxalate Transporter Gene, AtOT, Enhances Aluminum Tolerance in Arabidopsis thaliana by Regulating Oxalate Efflux.

Secretion and efflux of oxalic acid from roots is an important aluminum detoxification mechanism for various plants; however, how this process is completed remains unclear. In this study, the candidate oxalate transporter gene AtOT, encoding 287 amino acids, was cloned and identified from Arabidopsis thaliana. AtOT was upregulated in response to aluminum stress at the transcriptional level, which was closely related to aluminum treatment concentration and time. The root growth of Arabidopsis was inhibited after knocking out AtOT, and this effect was amplified by aluminum stress. Yeast cells expressing AtOT enhanced oxalic acid resistance and aluminum tolerance, which was closely correlated with the secretion of oxalic acid by membrane vesicle transport. Collectively, these results underline an external exclusion mechanism of oxalate involving AtOT to enhance oxalic acid resistance and aluminum tolerance.

Cloning, Expression Analysis, and Functional Characterization of Candidate Oxalate Transporter Genes of HbOT1 and HbOT2 from Rubber Tree (Hevea brasiliensis).

Secretion of oxalic acid from roots is an important aluminum detoxification mechanism for many plants such as Hevea brasiliensis (rubber tree). However, the underlying molecular mechanism and oxalate transporter genes in plants have not yet been reported. In this study, the oxalate transporter candidate genes HbOT1 and HbOT2 from the rubber tree were cloned and preliminarily identified. It was found that HbOT1 had a full length of 1163 bp with CDS size of 792 bp, encoding 263 amino acids, and HbOT2 had a full length of 1647 bp with a CDS region length of 840 bp, encoding 279 amino acid residues. HbOT1 and HbOT2 were both stable hydrophobic proteins with transmembrane structure and SNARE_assoc domains, possibly belonging to the SNARE_assoc subfamily proteins of the SNARE superfamily. qRT-PCR assays revealed that HbOT1 and HbOT2 were constitutively expressed in different tissues, with HbOT1 highly expressed in roots, stems, barks, and latex, while HbOT2 was highly expressed in latex. In addition, the expressions of HbOT1 and HbOT2 were up-regulated in response to aluminum stress, and they were inducible by metals, such as copper and manganese. Heterologous expression of HbOT1 and HbOT2 in the yeast mutant AD12345678 enhanced the tolerance to oxalic acid and high concentration aluminum stress, which was closely correlated with the secretion of oxalic acid. This study is the first report on oxalate transporter genes in plants, which provides a theoretical reference for the study on the molecular mechanism of oxalic acid secretion to relieve aluminum toxicity and on aluminum-tolerance genetic engineering breeding.

Estrogen treatment reduced oxalate transporting activity and enhanced migration through the involvement of SLC26A6 in lung cancer cells.

Estrogen therapy has used to prevent bone loss in postmenopausal women. Although therapeutically enhanced estrogen levels have been suggested, patients are exposed to greater risks of nephrolithiasis and cancer. It has been known that oxalate or bicarbonate transporter SLC26A6 is involved in oxalate homeostasis and its deletion results in kidney stone formation and addressed that patients with kidney stones possess higher cancer risk. Thus, the mechanism of the interaction between estrogen and SLC26A6 and the effect of SLC26A6 on cancer cells should be elucidated. In this study, we investigated whether ß-estradiol treatment modulates SLC26A6 expression and its bicarbonate or oxalate transporting activity and affects the proliferative and migratory ability of A549 cells. The ß-estradiol stimulation attenuated oxalate or bicarbonate transporting activities through SLC26A6. Knockdown of SLC26A6 reduced transporter activity whereas enhanced cellular migration. ß-estradiol-mediated cellular migration was independent of SLC26A6 transporter activity, whereas enhanced SLC26A6 expression attenuated cellular migration even in the presence of ß-estradiol treatment. These results indicate ß-estradiol treatment enhances cancer cell migration and dysregulates oxalate transport by inhibiting SLC26A6 activity, suggesting reduced oxalate transporting activity may involve in the oxalate homeostasis.

An optogenetic assay method for electrogenic transporters using Escherichia coli co-expressing light-driven proton pump.

In organisms, nutrients and wastes move across the cellular membrane, in which membrane-embedded transporters facilitate and inhibit the movement. Despite the physiological significances, the currently used assay methods for transporter activities require tedious preparation and analytical processes. In this study, we report the isotope-free and label-free measurement system for the transport activities of electrogenic transporters. In the system, two molecules, a light-driven inward proton pump rhodopsin, xenorhodopsin (XeR), and a representative of an electrogenic transporter, an oxalate transporter (OxlT), were co-expressed in Escherichia coli cells. The light illumination of the cells co-expressing XeR and OxlT showed an increase in the pH of the bulk solution and that the extent of the pH change is significantly enhanced by adding the oxalate, suggesting the light-induced inward proton transport by XeR coupled to the negative electrogenic transport by OxlT. Such a pH increase was dependent on the oxalate concentration, but not on the XeR expression level. Of note, pH increase was not observed for the nonfunctional mutants of OxlT, R272A, and K355Q, supporting the validity of the system. Thus, we successfully developed an optogenetic assay method for electrogenic transporters using E. coli co-expressing light-driven proton pump.

Molecular mechanism of hyperoxalic acid-induced arterial endothelial cell injury / 中华肾脏病杂志

Objective:To investigate the injury effect of hyperoxali acid on human arterial endothelial cells (HAECs) and its mechanism.Methods:HAECs were divided into intervention group and control group according to whether oxalic acid was used for intervention. The cells in the intervention group were stimulated with 30, 100, 200 and 300 μmol/L oxalic for different time. The effect of oxalic acid on the proliferation of HAECs was detected by MTT colorimetry. The change of cell cycle was analyzed by flow cytometry. The content of intracellular calcium was detected by fluorescence detection technology. The protein and mRNA expressions of cell cycle and anion transporter-related proteins were detected by Western blotting and fluorescence quantitative PCR. Besides, JAK2/STAT3 signaling pathway-related proteins were measured by Western blotting.Results:MTT colorimetry results showed that the intervention groups with high concentration of oxalic acid (100, 200, 300 μmol/L) could significantly inhibit the proliferation of HAECs, which was significantly different from the control group (all P<0.05). Fluorescence detection showed that the contents of intracellular calcium of HAECs in the intervention groups with high concentration of oxalic acid (100, 200, 300 μmol/L) were significantly higher than those in the control group after 48 hours ( P<0.05, P<0.001, P<0.001, respectively). Flow cytometry showed that the proportion of S phase of cells in the 200 μmol/L oxalic acid intervention group was significantly higher than that in the control group ( P<0.05). The results of Western blotting and PCR showed that the relative protein and mRNA expressions of anion transporter-related proteins slc26a1, slc26a5, slc26a11 in the intervention groups were higher than those in the control group (all P<0.05). Western blotting showed that the expression of p-JAK2 and p-STAT3 in the intervention groups after 24 hours were significantly higher than those in control group (all P<0.05). Conclusions:Hyperoxalic acid may enter HAECs through transporters slc26a1, slc26a5 and slc26a11 to inhibit cell proliferation and increase the intracellular calcium concentration. The mechanism may be through the activation of JAK2/STAT3 signaling pathway. Therefore, oxalic acid may be one of the uremic toxins leading to atherosclerosis.