Negative regulation of chitosan-induced stomatal closure by glutathione in Arabidopsis thaliana.

Chitosan (CHT) is a deacylated derivative of chitin and improves growth and yield performance, activates defensive genes, and also induces stomatal closure in plants. Glutathione (GSH) has significant functions in the growth, development, defense systems, signaling, and gene expression. Glutathione negatively regulates abscisic acid (ABA)-, methyl jasmonate (MeJA)-, and salicylic acid (SA)-induced stomatal closure. However, the negative regulation by GSH of CHT-induced stomatal closure is still unknown. Regulation of CHT-induced stomatal closure by GSH in guard cells was investigated using two GSH-deficient mutants, cad2-1 and ch1-1, and a GSH-decreasing chemical, 1-chloro-2,4-dinitrobenzene (CDNB). The cad2-1 and ch1-1 mutations and CDNB treatment enhanced CHT-induced stomatal closure. Treatment with glutathione monoethyl ester (GSHmee) restored the GSH level in the guard cells of cad2-1 and ch1-1 and complemented the stomatal phenotype of the mutants. These results indicate that GSH negatively regulates CHT-induced stomatal closure in A. thaliana.

Enhancing effect of the coexisting alpha-tocopherol on quercetin absorption and metabolism.

The aim of this study is to investigate the modulating effect of coexisting food components on the absorption and metabolism of quercetin and blood plasma antioxidant potentials. The combination of quercetin with α-tocopherol (αT), cellulose, or a commercially available vegetable beverage containing αT and dietary fiber was orally administered to mice. Compared to the single administration of quercetin aglycone, the coadministration of αT with quercetin significantly increased the plasma quercetin concentration at 0.5 h, whereas the combination of quercetin and cellulose decreased it. Interestingly, the administration of quercetin mixed with the vegetable beverage showed no significant change in the quercetin concentration in the mice plasma. The treatment of the cells with the blood plasma after the coadministration of αT with quercetin significantly upregulated the gene expression of the antioxidant enzyme (heme oxygenase-1), whereas the quercetin and cellulose combination did not. In the plasma of the quercetin-administered mice, eight types of quercetin metabolites were detected, and their quantities were affected by the combination with αT. The potentials of the heme oxygenase-1 gene expression by these metabolites were very limited, although several metabolites showed radical scavenging activities comparable to aglycone in the in vitro assays. These results suggested that the combination of αT potentiates the quercetin absorption and metabolism and thus the plasma antioxidant potentials, at least in part, by the quantitative changes in the quercetin metabolites.

An outward-rectifying plant K+ channel SPORK2 exhibits temperature-sensitive ion-transport activity.

Temperature sensing is critical for the survival of living organisms.1,2 Thermosensitive transient receptor-potential (TRP) cation channels function as thermosensors in mammals.2,3,4,5,6 In contrast to animals, land plants lack TRP genes.7,8,9 Previous patch-clamp studies in plant cells suggested the presence of ion channels whose activities are related to temperature, implying the presence of TRP-like channels.10,11,12,13,14 However, the molecular entities of such temperature-sensitive ion channels were still unknown in land plants. In this study, we observed that the unique rainfall-induced leaf-folding movement of the legume tree Samanea saman15 was temperature-sensitive by using a rainfall-mimicking assay. Chilling-induced leaf folding in S. saman was shown to be related to the swelling of the motor cells16,17 at the base of the leaflet. This swelling suggested involvement of temperature-sensitive inactivation of K+ currents, independent of fluctuations in ion channel gene expression in motor cells. These findings led us to examine the temperature sensitivity of an outward-rectifying K+ channel, SPORK2, which was reported as an ion channel responsible for the nyctinastic (circadian-rhythmic) leaf movement of S. saman.18 We also discovered that SPORK2 exhibits temperature-sensitive K+ transport activity in the Xenopus oocyte expression system. Using chimeric channels, we showed that two domains of SPORK2 regulated the temperature sensitivity. Furthermore, heterologously expressed SPORK2 in Arabidopsis guard cells induced temperature-dependent stomatal closure. Therefore, SPORK2 is an ion channel in land plants with temperature-sensitive ion-transport activity that functions similarly to mammalian TRP channels. Our current findings advance the molecular understanding of temperature-sensing mechanisms in plants.

The effect of exogenous dihydroxyacetone and methylglyoxal on growth, anthocyanin accumulation, and the glyoxalase system in Arabidopsis.

Dihydroxyacetone (DHA) occurs in wide-ranging organisms, including plants, and can undergo spontaneous conversion to methylglyoxal (MG). While the toxicity of MG to plants is well-known, the toxicity of DHA to plants remains to be elucidated. We investigated the effects of DHA and MG on Arabidopsis. Exogenous DHA at up to 10 mm did not affect the radicle emergence, the expansion of green cotyledons, the seedling growth, or the activity of glyoxalase II, while DHA at 10 mm inhibited the root elongation and increased the activity of glyoxalase I. Exogenous MG at 1.0 mm inhibited these physiological responses and increased both activities. Dihydroxyacetone at 10 mm increased the MG content in the roots. These results indicate that DHA is not so toxic as MG in Arabidopsis seeds and seedlings and suggest that the toxic effect of DHA at high concentrations is attributed to MG accumulation by the conversion to MG.

Cycloartenyl Ferulate Is the Predominant Compound in Brown Rice Conferring Cytoprotective Potential against Oxidative Stress-Induced Cytotoxicity.

Since brown rice extract is a rich source of biologically active compounds, the present study is aimed to quantify the major compounds in brown rice and to compare their cytoprotective potential against oxidative stress. The content of the main hydrophobic compounds in brown rice followed the order of cycloartenyl ferulate (CAF) (89.00 ± 8.07 nmol/g) >> α-tocopherol (αT) (19.73 ± 2.28 nmol/g) > γ-tocotrienol (γT3) (18.24 ± 1.41 nmol/g) > α-tocotrienol (αT3) (16.02 ± 1.29 nmol/g) > γ-tocopherol (γT) (3.81 ± 0.40 nmol/g). However, the percent contribution of CAF to the radical scavenging activity of one gram of whole brown rice was similar to those of αT, αT3, and γT3 because of its weaker antioxidant activity. The CAF pretreatment displayed a significant cytoprotective effect on the hydrogen peroxide-induced cytotoxicity from 10 µM, which is lower than the minimal concentrations of αT and γT required for a significant protection. CAF also enhanced the nuclear factor erythroid 2-related factor 2 (Nrf2) nuclear translocation coincided with the enhancement of the heme oxygenase-1 (HO-1) mRNA level. An HO-1 inhibitor, tin protoporphyrin IX (SnPP), significantly impaired the cytoprotection of CAF. The cytoprotective potential of CAF is attributable to its cycloartenyl moiety besides the ferulyl moiety. These results suggested that CAF is the predominant cytoprotector in brown rice against hydrogen peroxide-induced cytotoxicity.

The microbiota catabolites of quercetin glycosides concertedly enhance the resistance against acetaldehyde-induced oxidative stress.

3,4-Dihydroxyphenylacetic acid (DOPAC) and 3-hydroxyphenylacetic acid (OPAC) are the predominant catabolites of quercetin glycosides, such as quercetin 4'-O-ß-glucoside from the onion, produced by intestinal microbiota. Although each catabolite has been reported to protect the cells from acetaldehyde-induced cytotoxicity, the effect of their combination remains to be clarified. The purpose of this study was to determine whether the combination of DOPAC and OPAC enhances the resistance against the acetaldehyde-induced oxidative stress in the cultured hepatocytes. The pretreatment of the combination of DOPAC (5 µM) and OPAC (5 µM) showed significant protection against the acetaldehyde- and hydrogen peroxide-induced cytotoxicity, even though each compound at the same concentration did not. This combination also significantly inhibited the intracellular dichlorofluorescin diacetate-detectable reactive oxygen species (ROS) level, whereas the solo treatment did slightly, suggesting that reducing mechanisms of ROS or compounds that enhance ROS production are involved in the cytoprotective effect. The combinatory treatment significantly enhanced the gene expression of not only the aldehyde dehydrogenases (ALDHs), but also glutamate-cysteine ligase, catalytic subunit, the first rate-limiting enzyme of glutathione (GSH) synthesis. Accordingly, both the intracellular GSH level and the total ALDH activity were enhanced by DOPAC plus OPAC. Involvement of GSH in the cytoprotection as well as ALDH up-regulation by the combination was confirmed by the experiments using a GSH biosynthesis inhibitor, buthionine sulfoximine. Taken together, the present results suggested that the quercetin microbiota catabolites concertedly protect the cells from acetaldehyde through a pre-enhanced resistance against oxidative stress by the GSH-dependent up-regulation of ALDHs.

Benzyl isothiocyanate and its metabolites inhibit cell proliferation through protein modification in mouse preosteoclast RAW264.7 cells.

Benzyl isothiocyanate (BITC), derived from cruciferous vegetables, is an organosulfur compound exerting antiproliferative effects in several human cancer cells. In this study, we assessed BITC as a potential osteoclastogenesis inhibitor and investigated its underlying mechanism. BITC at 5 µM significantly decreased the viability of the osteoclast-like differentiating RAW264.7 cells, coinciding with the downregulation of the primary biomarkers for osteoclast differentiation, such as the tartrate-resistant acid phosphatase activity and nuclear factor of activated T-cells gene expression. Not only BITC but also its metabolites, inhibited cell proliferation in the normal RAW264.7 cells, suggesting that BITC shows an anti-osteoclastogenesis effect in vivo after its ingestion and metabolism, possibly through an antiproliferative action. Both BITC and its metabolites also enhanced the DNA fragmentation and the caspase-3 activity, whereas their higher concentrations tended to suppress these effects. BITC was intracellularly accumulated when the cells were treated with its metabolites via their degradation into the free form. A quantitative experiment using the proteolysis/high performance liquid chromatography technique showed that the amount of BITC-lysine thiourea in the cells was also increased in a time-dependent manner, suggesting that lysine modification of the cellular proteins actually took place in the cells treated by BITC. Among the cellular proteins, the cleaved caspase-3 was identified as a potential target for lysine modification by BITC. Taken together, BITC dissociated from its metabolites as well as its free form might modulate osteoclastogenesis, possibly through inhibition of cell proliferation by protein modification.

Extracellular malate induces stomatal closure via direct activation of guard-cell anion channel SLAC1 and stimulation of Ca2+ signalling.

Plants secrete malate from guard cells to apoplast under stress conditions and exogenous malate induces stomatal closure. Malate is considered an extracellular chemical signal of stomatal closure. However, the molecular mechanism of malate-induced stomatal closure is not fully elucidated. We investigated responses of stomatal aperture, ion channels, and cytosolic Ca2+ to malate. A treatment with malate induced stomatal closure in Arabidopsis thaliana wild-type plants, but not in the mutants deficient in the slow (S-type) anion channel gene SLOW ANION CHANNEL-ASSOCIATED 1 (SLAC1). The treatment with malate increased S-type anion currents in guard-cell protoplasts of wild-type plants but not in the slac1 mutant. In addition, extracellular rather than intracellular application of malate increased the S-type currents of constitutively active mutants of SLAC1, which have kinase-independent activities, in a heterologous expression system using Xenopus oocytes. The treatment with malate transiently increased cytosolic Ca2+ concentration in the wild-type Arabidopsis guard cells and the malate-induced stomatal closure was inhibited by the Ca2+ channel blocker and the Ca2+ chelator. These results indicate that extracellular malate directly activates SLAC1 and simultaneously stimulates Ca2+ signalling in guard cells, resulting in steady and solid activation of SLAC1 for stomatal closure.

Malate induces stomatal closure via a receptor-like kinase GHR1- and reactive oxygen species-dependent pathway in Arabidopsis thaliana.

A primary metabolite malate is secreted from guard cells in response to the phytohormone abscisic acid (ABA) and elevated CO2. The secreted malate subsequently facilitates stomatal closure in plants. Here, we investigated the molecular mechanism of malate-induced stomatal closure using inhibitors and ABA signaling component mutants of Arabidopsis thaliana. Malate-induced stomatal closure was impaired by a protein kinase inhibitor, K252a, and also by the disruption of a receptor-like kinase GHR1, which mediates activation of calcium ion (Ca2+) channel by reactive oxygen species (ROS) in guard cells. Malate induced ROS production in guard cells while the malate-induced stomatal closure was impaired by a peroxidase inhibitor, salicylhydroxamic acid, but not by the disruption of Nicotinamide adenine dinucleotide (phosphate) (NAD(P)H) oxidases, RBOHD and RBOHF. The malate-induced stomatal closure was impaired by Ca2+ channel blockers, verapamil, and niflumic acid. These results demonstrate that the malate signaling is mediated by GHR1 and ROS in Arabidopsis guard cells.

Negative regulation of salicylic acid-induced stomatal closure by glutathione in Arabidopsis thaliana.

Salicylic acid (SA) is a ubiquitous phenolic phytohormone that induces stomatal closure. Glutathione (GSH) negatively regulates stomatal closure induced by other plant hormones such as abscisic acid (ABA) and methyl jasmonate (MeJA). However, the involvement of GSH in SA-induced stomatal closure is still unknown. We investigated the regulation of SA signaling by GSH in guard cells using an Arabidopsis thaliana mutant, cad2-1, which is deficient in the first GSH biosynthesis enzyme, γ-glutamylcysteine synthetase. Application of SA decreased stomatal apertures with decreasing intracellular GSH level in guard cells. Decreasing GSH by the cad2-1 mutation and by a GSH-decreasing chemical, 1-chloro-2,4-dinitrobenzene, enhanced the SA-induced stomatal closure. Treatment with glutathione monoethyl ester restored the GSH level in the cad2-1 guard cells and complemented the stomatal phenotype of the mutant. These results indicate that GSH negatively modulates SA-induced stomatal closure in A. thaliana.

Phytocytokine signalling reopens stomata in plant immunity and water loss.

Stomata exert considerable effects on global carbon and water cycles by mediating gas exchange and water vapour1,2. Stomatal closure prevents water loss in response to dehydration and limits pathogen entry3,4. However, prolonged stomatal closure reduces photosynthesis and transpiration and creates aqueous apoplasts that promote colonization by pathogens. How plants dynamically regulate stomatal reopening in a changing climate is unclear. Here we show that the secreted peptides SMALL PHYTOCYTOKINES REGULATING DEFENSE AND WATER LOSS (SCREWs) and the cognate receptor kinase PLANT SCREW UNRESPONSIVE RECEPTOR (NUT) counter-regulate phytohormone abscisic acid (ABA)- and microbe-associated molecular pattern (MAMP)-induced stomatal closure. SCREWs sensed by NUT function as immunomodulatory phytocytokines and recruit SOMATIC EMBRYOGENESIS RECEPTOR-LIKE KINASE (SERK) co-receptors to relay immune signalling. SCREWs trigger the NUT-dependent phosphorylation of ABA INSENSITIVE 1 (ABI1) and ABI2, which leads to an increase in the activity of ABI phosphatases towards OPEN STOMATA 1 (OST1)-a key kinase that mediates ABA- and MAMP-induced stomatal closure5,6-and a reduction in the activity of S-type anion channels. After induction by dehydration and pathogen infection, SCREW-NUT signalling promotes apoplastic water loss and disrupts microorganism-rich aqueous habitats to limit pathogen colonization. The SCREW-NUT system is widely distributed across land plants, which suggests that it has an important role in preventing uncontrolled stomatal closure caused by abiotic and biotic stresses to optimize plant fitness.

Plant hormone regulation of abiotic stress responses.

Plant hormones are signalling compounds that regulate crucial aspects of growth, development and environmental stress responses. Abiotic stresses, such as drought, salinity, heat, cold and flooding, have profound effects on plant growth and survival. Adaptation and tolerance to such stresses require sophisticated sensing, signalling and stress response mechanisms. In this Review, we discuss recent advances in understanding how diverse plant hormones control abiotic stress responses in plants and highlight points of hormonal crosstalk during abiotic stress signalling. Control mechanisms and stress responses mediated by plant hormones including abscisic acid, auxin, brassinosteroids, cytokinins, ethylene and gibberellins are discussed. We discuss new insights into osmotic stress sensing and signalling mechanisms, hormonal control of gene regulation and plant development during stress, hormone-regulated submergence tolerance and stomatal movements. We further explore how innovative imaging approaches are providing insights into single-cell and tissue hormone dynamics. Understanding stress tolerance mechanisms opens new opportunities for agricultural applications.

Green Tea Catechins, (-)-Catechin Gallate, and (-)-Gallocatechin Gallate are Potent Inhibitors of ABA-Induced Stomatal Closure.

Stomatal movement is indispensable for plant growth and survival in response to environmental stimuli. Cytosolic Ca2+ elevation plays a crucial role in ABA-induced stomatal closure during drought stress; however, to what extent the Ca2+ movement across the plasma membrane from the apoplast to the cytosol contributes to this process still needs clarification. Here the authors identify (-)-catechin gallate (CG) and (-)-gallocatechin gallate (GCG), components of green tea, as inhibitors of voltage-dependent K+ channels which regulate K+ fluxes in Arabidopsis thaliana guard cells. In Arabidopsis guard cells CG/GCG prevent ABA-induced: i) membrane depolarization; ii) activation of Ca2+ permeable cation (ICa ) channels; and iii) cytosolic Ca2+ transients. In whole Arabidopsis plants co-treatment with CG/GCG and ABA suppressed ABA-induced stomatal closure and surface temperature increase. Similar to ABA, CG/GCG inhibited stomatal closure is elicited by the elicitor peptide, flg22 but has no impact on dark-induced stomatal closure or light- and fusicoccin-induced stomatal opening, suggesting that the inhibitory effect of CG/GCG is associated with Ca2+ -related signaling pathways. This study further supports the crucial role of ICa channels of the plasma membrane in ABA-induced stomatal closure. Moreover, CG and GCG represent a new tool for the study of abiotic or biotic stress-induced signal transduction pathways.

A Major Intestinal Catabolite of Quercetin Glycosides, 3-Hydroxyphenylacetic Acid, Protects the Hepatocytes from the Acetaldehyde-Induced Cytotoxicity through the Enhancement of the Total Aldehyde Dehydrogenase Activity.

Aldehyde dehydrogenases (ALDHs) are the major enzyme superfamily for the aldehyde metabolism. Since the ALDH polymorphism leads to the accumulation of acetaldehyde, we considered that the enhancement of the liver ALDH activity by certain food ingredients could help prevent alcohol-induced chronic diseases. Here, we evaluated the modulating effects of 3-hydroxyphenylacetic acid (OPAC), the major metabolite of quercetin glycosides, on the ALDH activity and acetaldehyde-induced cytotoxicity in the cultured cell models. OPAC significantly enhanced the total ALDH activity not only in mouse hepatoma Hepa1c1c7 cells, but also in human hepatoma HepG2 cells. OPAC significantly increased not only the nuclear level of aryl hydrocarbon receptor (AhR), but also the AhR-dependent reporter gene expression, though not the nuclear factor erythroid-2-related factor 2 (Nrf2)-dependent one. The pretreatment of OPAC at the concentration required for the ALDH upregulation completely inhibited the acetaldehyde-induced cytotoxicity. Silencing AhR impaired the resistant effect of OPAC against acetaldehyde. These results strongly suggested that OPAC protects the cells from the acetaldehyde-induced cytotoxicity, mainly through the AhR-dependent and Nrf2-independent enhancement of the total ALDH activity. Our findings suggest that OPAC has a protective potential in hepatocyte models and could offer a new preventive possibility of quercetin glycosides for targeting alcohol-induced chronic diseases.

Neither glutamate nor alanine but arginine sensitizes BY-2 cells to arsenate.

Arsenic is toxic for plants. Our previous results showed that the application of proline enhanced the sensitivity of tobacco BY-2 cells to arsenate. In order to clarify the enhancement mechanism, we investigated the effects of other amino acids on the arsenate-stressed BY-2 cells. Glutamate at up to 10 m m did not affect the cell growth in the absence or presence of arsenate. Arginine at up to 10 m m did not affect the growth in the absence of arsenate but arginine at 10 m m enhanced the inhibition of the cell growth by arsenate. Alanine at up to 10 m m did not affect the cell growth under nonstressed condition but alanine at 10 m m significantly improved the cell growth under arsenate stress. These results suggest that alanine mitigates arsenate stress in BY-2 cells and that arginine like proline enhances the sensitivity of BY-2 cells to arsenate.

White rice ethanol extract is qualitatively, but not quantitatively, equivalent to that of brown rice as an antioxidant source.

The purpose of this study is to compare the potentials to exhibit biologically active antioxidant actions between white rice (WR) and brown rice (BR) in in vitro assays and a cellular model. The Trolox equivalent (TE) per 1 mg ethanol extract of WR for the 1,1-diphenyl-2-picrylhydrazyl assay was slightly higher than that of BR, whereas the TE per 1 g whole WR was much lower than that for BR. This tendency was very comparable to those for the oxygen radical absorbance capacity and total polyphenol content. Both of the ethanol extracts also similarly suppressed the hydrogen peroxide-induced cytotoxicity and enhanced the gene expression of drug-metabolizing enzymes. Based on the α-tocopherol quantity, its contribution to the cytoprotective effect of the rice extracts is very limited. Taken together, the ethanol extract of WR might be a qualitatively, but not quantitatively, equivalent source of antioxidative phytochemicals to that of BR.

Modulation of frequency and height of cytosolic calcium spikes by plasma membrane anion channels in guard cells.

Cytosolic calcium ([Ca2+]cyt) elevation activates plasma membrane anion channels in guard cells, which is required for stomatal closure. However, involvement of the anion channels in the [Ca2+]cyt elevation remains unclear. We investigated the involvement using Arabidopsis thaliana anion channel mutants, slac1-4 slah3-3 and slac1-4 almt12-1. Extracellular calcium induced stomatal closure in the wild-type plants but not in the anion channel mutant plants whereas extracellular calcium induced [Ca2+]cyt elevation both in the wild-type guard cells and in the mutant guard cells. The peak height and the number of the [Ca2+]cyt spike were lower and larger in the slac1-4 slah3-3 than in the wild type and the height and the number in the slac1-4 almt12-1 were much lower and much larger than in the wild type. These results suggest that the anion channels are involved in the regulation of [Ca2+]cyt elevation in guard cells.

A multidrug resistance-associated protein inhibitor is a potential enhancer of the benzyl isothiocyanate-induced apoptosis induction in human colorectal cancer cells.

The increasing drug efflux through the ATP-binding cassette (ABC) transporters is the most plausible mechanism that mediates resistance to the anticancer phytochemicals, such as benzyl isothiocyanate (BITC), as well as chemotherapy drugs. To identify a potential component to overcome this resistance by combinatory utilization, we focused on multidrug resistance-associated proteins (MRPs) pumping various drug metabolites with glutathione as well as the organic anions. The pharmacological treatment of an MRP inhibitor, MK571, significantly potentiated the BITC-induced antiproliferation, coincided with the enhanced accumulation of BITC and glutathione in human colorectal cancer HCT-116 cells. MK571 also enhanced the apoptosis induction as well as activation of the mitogen-activated protein kinases and caspase-3, whereas it did not affect their basal levels. These results suggested that, since MRPs might play a pivotal role in the BITC efflux, MK571 potentiates the BITC-induced antiproliferation in human colorectal cancer cells through inhibition of the glutathione-dependent BITC efflux.

Calcium and ethylene glycol tetraacetic acid mitigate toxicity and alteration of gene expression associated with cadmium stress in chickpea (Cicer arietinum L.) shoots.

In the aim to estimate the protective role of calcium (Ca) and ethylene glycol tetraacetic acid (EGTA) against cadmium (Cd)-induced damage, chickpea (Cicer arietinum L.) seeds were exposed to 200 µM Cd stress for 6 days or 3 days then subjected to co-treatment of the metal with either 100 mM CaCl2 or 100 µM EGTA for 3 additional days. The addition of Ca and EGTA improved seedling growth. This protecting effect was correlated to the alleviation of the metal-induced oxidative stress, exemplified by the reduction of hydrogen peroxide (H2O2) contents. Besides, Ca and EGTA stimulated thioredoxin (Trx) and thioredoxin reductase (NTR) activities (2.75- and 1.75-fold increase when compared to Cd-stressed, respectively) protecting, thereby, protein -SH groups from the Cd-mediated oxidation, and modulated ferredoxin (Fdx) activity to a control level. Moreover, Ca and EGTA reinstated the glutathione redox steady state, mainly via preserving a high level of glutathione reduced form (GSH). This effect coincided with the maintaining of the Cd-stimulated glutathione reductase (GR) activity and the decline of glutathione peroxidase (GPX, 43% lower than Cd-stressed shoots) activity. Ca and EGTA counteracted the inhibitory effect of Cd on the activity and gene expression of Cu/Zn-superoxide dismutase (Cu/Zn-SOD) isoenzyme and modulated the activities of catalase (CAT) and ascorbate peroxidase (APX). Overall, our results provided evidence that Ca and EGTA supplement could be a promising approach in the remediation of Cd-contaminated environment.