The maize WRKY transcription factor ZmWRKY64 confers cadmium tolerance in Arabidopsis and maize (Zea mays L.).

KEY MESSAGE: ZmWRKY64 positively regulates Arabidopsis and maize Cd stress through modulating Cd uptake, translocation, and ROS scavenging genes expression. Cadmium (Cd) is a highly toxic heavy metal with severe impacts on crops growth and development. The WRKY transcription factor is a significant regulator influencing plant stress response. Nevertheless, the function of the WRKY protein in maize Cd stress response remains unclear. Here, we identified a maize WRKY gene, ZmWRKY64, the expression of which was enhanced in maize roots and leaves under Cd stress. ZmWRKY64 was localized in the nucleus and displayed transcriptional activity in yeast. Heterologous expression of ZmWRKY64 in Arabidopsis diminished Cd accumulation in plants by negatively regulating the expression of AtIRT1, AtZIP1, AtHMA2, AtNRAMP3, and AtNRAMP4, which are involved in Cd uptake and transport, resulting in Cd stress tolerance. Knockdown of ZmWRKY64 in maize led to excessive Cd accumulation in leaf cells and in the cytosol of the root cells, resulting in a Cd hypersensitive phenotype. Further analysis confirmed that ZmWRKY64 positively regulated ZmABCC4, ZmHMA3, ZmNRAMP5, ZmPIN2, ZmABCG51, ZmABCB13/32, and ZmABCB10, which may influence Cd translocation and auxin transport, thus mitigating Cd toxicity in maize. Moreover, ZmWRKY64 could directly enhance the transcription of ZmSRG7, a reported key gene regulating reactive oxygen species homeostasis under abiotic stress. Our results indicate that ZmWRKY64 is important in maize Cd stress response. This work provides new insights into the WRKY transcription factor regulatory mechanism under a Cd-polluted environment and may lead to the genetic improvement of Cd tolerance in maize.

Co-application of Parthenium biochar and urea effectively mitigate cadmium toxicity during wheat growth.

Environmental contamination by cadmium (Cd), a highly toxic heavy metal, poses significant health risks to plants and humans. Biochar has been effectively used to promote plant growth and productivity under Cd stress. This study presents an innovative application of biochar derived from the invasive weed Parthenium hysterophorus to promote plant growth and productivity under Cd stress. Our study includes detailed soil and plant analyses, providing a holistic perspective on how biochar and urea amendments influence soil properties, nutrient availability, and plant physiological responses. To address these, we established seven treatments: the control, Cd alone (5 mg kg-1), biochar alone (5 %), urea alone (3 g kg-1), biochar with Cd, urea with Cd, and a combination of biochar and urea with Cd. Cd stress alone significantly reduced plant growth indicators such as shoot and root length, fresh and dry biomass, chlorophyll content, and grain yield. However, the supplementation of biochar, urea, or their combination significantly increased shoot length (by 48%, 34%, and 65%), root length (by 73%, 46%, and 70%), and fresh shoot biomass (by 4%, 31%, and 4%), respectively. This improvement is attributed to enhanced soil properties and improved nutrient absorption. The biochar-urea combination also enhanced Cd tolerance by improving total chlorophyll content by 14 %, 13 %, and 16 % compared to the control, respectively. Similaly, these treatments significantly (p < 0.05) boosted the activity of antioxidant enzymes such as catalase, peroxidase, and superoxide dismutase by 51 %, 30 %, and 51 %, respectively, thereby mitigating oxidative stress as a defensive mechanism. The Cd tolerance was improved by biochar, urea, and their combinations, which reduced Cd content in the shoots (by 60.5 %, 38.9 %, and 51.3 %), roots (by 47.5 %, 23.9 %, and 57.6 %), and grains (by 58.1 %, 30.2 %, and 38.3 %) relative to Cd stress alone, respectively. The synergistic effects of biochar and urea are achieved through improved soil properties, nutrient availability, activating antioxidant defense mechanisms, and minimizing the accumulation of metal ions in plant tissues, thereby enhancing plant defenses against Cd stress. Conclusively, converting invasive Parthenium weed into biochar and combining it with urea offers an environmentally friendly solution to manage its spreading while effectively mitigating Cd stress in crops.

Diversity and functional traits of seed endophytes of Dysphania ambrosioides from heavy metal contaminated and non-contaminated areas.

Seed endophytes played a crucial role on host plants stress tolerance and heavy metal (HM) accumulation. Dysphania ambrosioides is a hyperaccumulator and showed strong tolerance and extraordinary accumulation capacities of multiple HMs. However, little is known about its seed endophytes response to field HM-contamination, and its role on host plants HM tolerance and accumulation. In this study, the seed endophytic community of D. ambrosioides from HM-contaminated area (H) and non-contaminated area (N) were investigated by both culture-dependent and independent methods. Moreover, Cd tolerance and the plant growth promoting (PGP) traits of dominant endophytes from site H and N were evaluated. The results showed that in both studies, HM-contamination reduced the diversity and richness of endophytic community and changed the most dominant endophyte, but increased resistant species abundance. By functional trait assessments, a great number of dominant endophytes displayed multiple PGP traits and Cd tolerance. Interestingly, soil HM-contamination significantly increased the percentage of Cd tolerance isolates of Agrobacterium and Epicoccum, but significantly decreased the ration of Agrobacterium with the siderophore production ability. However, the other PGP traits of isolates from site H and N showed no significant difference. Therefore, it was suggested that D. ambrosioides might improve its HM tolerance and accumulation through harboring more HM-resistant endophytes rather than PGP endophytes, but to prove this, more work need to be conducted in the future.

Cadmium tolerance and hyperaccumulation in plants - A proteomic perspective of phytoremediation.

Cadmium (Cd) is a major environmental pollutant and poses a risk of transfer into the food chain through contaminated plants. Mechanisms underlying Cd tolerance and hyperaccumulation in plants are not fully understood. Proteomics-based approaches facilitate an in-depth understanding of plant responses to Cd stress at the systemic level by identifying Cd-inducible differentially abundant proteins (DAPs). In this review, we summarize studies related to proteomic changes associated with Cd-tolerance mechanisms in Cd-tolerant crops and Cd-hyperaccumulating plants, especially the similarities and differences across plant species. The enhanced DAPs identified through proteomic studies can be potential targets for developing Cd-hyperaccumulators to remediate Cd-contaminated environments and Cd-tolerant crops with low Cd content in the edible organs. This is of great significance for ensuring the food security of an exponentially growing global population. Finally, we discuss the methodological drawbacks in current proteomic studies and propose that better protocols and advanced techniques should be utilized to further strengthen the reliability and applicability of future Cd-stress-related studies in plants. This review provides insights into the improvement of phytoremediation efficiency and an in-depth study of the molecular mechanisms of Cd enrichment in plants.

Ectopic Expression of PvHMA2.1 Enhances Cadmium Tolerance in Arabidopsis thaliana.

Cadmium (Cd) in soil inhibits plant growth and development and even harms human health through food chain transmission. Switchgrass (Panicum virgatum L.), a perennial C4 biofuel crop, is considered an ideal plant for phytoremediation due to its high efficiency in removing Cd and other heavy metals from contaminated soil. The key to understanding the mechanisms of switchgrass Cd tolerance is to identify the genes involved in Cd transport. Heavy-metal ATPases (HMAs) play pivotal roles in heavy metal transport, including Cd, in Arabidopsis thaliana and Oryza sativa, but little is known about the functions of their orthologs in switchgrass. Therefore, we identified 22 HMAs in switchgrass, which were distributed on 12 chromosomes and divided into 4 groups using a phylogenetic analysis. Then, we focused on PvHMA2.1, which is one of the orthologs of the rice Cd transporter OsHMA2. We found that PvHMA2.1 was widely expressed in roots, internodes, leaves, spikelets, and inflorescences, and was significantly induced in the shoots of switchgrass under Cd treatment. Moreover, PvHMA2.1 was found to have seven transmembrane domains and localized at the cell plasma membrane, indicating that it is a potential transporter. The ectopic expression of PvHMA2.1 alleviated the reduction in primary root length and the loss of fresh weight of Arabidopsis seedlings under Cd treatment, suggesting that PvHMA2.1 enhanced Cd tolerance in Arabidopsis. The higher levels of relative water content and chlorophyll content of the transgenic lines under Cd treatment reflected that PvHMA2.1 maintained water retention capacity and alleviated photosynthesis inhibition under Cd stress in Arabidopsis. The roots of the PvHMA2.1 ectopically expressed lines accumulated less Cd compared to the WT, while no significant differences were found in the Cd contents of the shoots between the transgenic lines and the WT under Cd treatment, suggesting that PvHMA2.1 reduced Cd absorption from the environment through the roots in Arabidopsis. Taken together, our results showed that PvHMA2.1 enhanced Cd tolerance in Arabidopsis, providing a promising target that could be engineered in switchgrass to repair Cd-contaminated soil.

Genotypic variation in the tolerance to moderate cadmium toxicity among 20 maize genotypes with contrasting root systems.

BACKGROUND: Cadmium (Cd) contamination in farmland is a serious environmental and safety issue affecting plant growth, crop productivity, and human health. This study aimed to investigate genotypic variation in root morphology and Cd accumulations under moderate Cd stress among diverse maize genotypes. Twenty maize genotypes with contrasting root systems were assessed for Cd tolerance 39 days after transplanting (V6, six-leaf stage) under 20 µmol L-1 CdCl2 using a semi-hydroponic phenotyping platform in a glasshouse. RESULTS: Cadmium stress significantly inhibited plant growth across all genotypes. Genotypic variation in response to Cd toxicity was apparent: shoot dry weight varied from 0.13 (genotype NS2020) to 0.35 g plant-1 (Dongke301) with deductions up to 63% compared with non-Cd treatment (CK). Root dry weight of 20 genotypes ranged from 0.06 (NS2020) to 0.18 g plant-1 (Dongke301) with a deduction up to 56%. Root length ranged from 2.21 (NS590b) to 9.22 m (Dongke301) with a maximal decline of 76%. Cadmium-treated genotypes generally had thicker roots and average diameter increased by 34% compared with CK. Genotypes had up to 3.25 and 3.50 times differences in shoot and root Cd concentrations, respectively. Principal component and cluster analyses assigned the 20 genotypes into Cd-tolerant (five genotypes) and Cd-sensitive (15 genotypes) groups. CONCLUSIONS: Maize genotypes varied significantly in response to moderate Cd stress. Cadmium-tolerant genotypes optimized root morphology and Cd accumulation and distribution. This study could assist in the selection and breeding of new cultivars with improved adaptation to Cd-contaminated soil for food and feed or land remediation purposes. © 2022 The Authors. Journal of The Science of Food and Agriculture published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Application of exogenous salicylic acid reduces Cd toxicity and Cd accumulation in rice.

Cd pollution in farmland is becoming a serious problem because it affects the safety of rice production and human health. Salicylic acid (SA) plays crucial roles in plant development and mediates plant responses to biotic and abiotic stress. This study assessed the molecular and physiological mechanisms of SA spraying effects on Cd tolerance and Cd accumulation in rice. Spraying of 0.1 mM SA had no great effect on the agronomic traits of rice, but significantly decreased Cd accumulation in rice grains, and SA spraying increased the Cd contents in leaves (only at the mature stage) and decreased the Cd contents in panicles (only at the filling and mature stage), but had no evident impact on the Cd content of other tissues and other growth stages. SA spraying reduced Cd accumulation in rice grains by promoting the deposition and fixation of Cd in the cell wall of leaves, thus preventing Cd being transferred from leaves to rice grains at the filling stage. SA spraying also decreased Cd toxicity by reducing H2O2 and MDA accumulation and increasing the chlorophyll content in rice leaves. Furthermore, SA spraying remarkably decreased Cd accumulation in rice grains by modulating the expression level of the genes associated with Cd translocation and accumulation to control the Cd accumulation in rice. Hence, SA spraying reduced the inhibition of Cd on the plant height caused by Cd and increased the dry weight of shoots in the vegetative growth period of rice seedlings, and it reduced Cd transport from leaves to grains, thus reducing Cd content in rice. These findings provide a novel perspective and a new method for reducing Cd accumulation in rice.

1,8-dihydroxy naphthalene (DHN) - melanin confers tolerance to cadmium in isolates of melanised dark septate endophytes.

The contribution of 1,8-dihydroxy naphthalene (DHN) melanin to cadmium (Cd) tolerance in two dark septate endophytes (DSE) of the genus Cadophora with different melanin content was investigated in vitro. The DSE isolate Cad#148 with higher melanin content showed higher tolerance to Cd than the less melanised Cad#149. Melanin synthesis was significantly reduced by Cd in both isolates with uninhibited melanin synthesis, in a dose-dependent manner. Inhibition of melanin synthesis by tricyclazole reduced the relative growth of Cad#148 exposed to Cd and did not affect Cad#149. Cd accumulation was not altered by tricyclazole in the two isolates, but it increased catalase and reduced glutathione reductase activity in more melanised Cad#148, indicating higher stress levels. In contrast, in Cad#149 the enzyme activity was less affected by tricyclazole, indicating a more pronounced role of melanin-independent Cd tolerance mechanisms. Cd ligand environment in fungal mycelia was analysed by extended EXAFS (X-ray absorption fine structure). It revealed that Cd was mainly bound to O- and S-ligands, including hydroxyl, carboxyl, phosphate and thiol groups. A similar proportion of S- and O- ligands (~35% and ~65%) were found in both isolates with uninhibited melanin synthesis. Among O-ligands two types with Cd-O-C- and Cd-O-P- coordination were identified. Tricyclazole altered Cd-O- ligand environment in both fungal isolates by reducing the proportion of Cd-O-C- and increasing the proportion of Cd-O-P coordination. DHN-melanin, among other tolerance mechanisms, significantly contributes to Cd tolerance in more melanised DSE fungi by immobilising Cd to hydroxyl groups and maintaining the integrity of the fungal cell wall.

Foliar application of anthocyanin extract regulates cadmium accumulation and distribution in rice (Oryza sativa L.) at tillering and booting stages.

Anthocyanin extract has been applied in agricultural production and enhanced tolerance of plants to adverse effects of Cd stress. Rice was subjected to different concentration of Cd and blueberry anthocyanin, and the effects on rice growth, antioxidative defense, Cd distribution in rice tissues, FTIR and TEM characterization of rice leaves were examined to explain the Cd reductions in rice grains and the protective mechanisms by blueberry anthocyanin. Foliar spray of blueberry anthocyanin at tillering and booting stages was effective for reducing Cd concentration in rice grains and increasing the rice yield, anthocyanin and Cd concentration of rice leaves under 1.0 and 10.0 mg/kg Cd stress. The Cd concentration in rice grains was less than the China national standard of Cd for rice grains (0.2 mg/kg) after surface spraying by 5.0-12.5 g/L blueberry anthocyanin under 1.0 mg/kg Cd stress, while 7.5 g/L spray concentration was the best choice. Blueberry anthocyanin spraying prevents Cd from being transferred from leaves to rice grains mainly by fixation of Cd in soluble and organelle fractions at the tillering and booting stages, and reduces H2O2 and MDA accumulation in rice leaves to decrease Cd toxicity. Combined with FTIR and TEM characterization of rice leaves, the results indicated that surface spraying of 7.5 g/L blueberry anthocyanin under 1.0 mg/kg Cd stress could effectively relieve Cd oxidative damage, and form chelates with Cd ions to immobilize Cd in rice leaves. Hence, blueberry anthocyanin could be used as a foliar resistance control agent to reduce Cd accumulation in rice grains through chelate compound synthesis and decrease Cd toxicity by preventing membrane lipid peroxidation and H2O2 accumulation.

Serratia sp. CP-13 alleviates Cd toxicity by morpho-physio-biochemical improvements, antioxidative potential and diminished Cd uptake in Zea mays L. cultivars differing in Cd tolerance.

Cadmium (Cd) is highly toxic for plant metabolic processes even in low concentration due to higher retention rates, longer half-life and non-biodegradable nature. The current study was designed to assess the bioremediation potential of Cd tolerant PGPR, Serratia sp. CP-13 together with two differentially Cd tolerant maize cultivars (MMRI-Yellow, Sahiwal-2002) selected amongst ten cultivars after screening. The maize cultivars were grown under different Cd treatments (0, 6, 12, 18, 24, 30 µM) in Petri plates both with and without Serratia sp. CP-13 inoculation. Treated plants were analyzed for their biomass accumulation, chlorophylls, carotenoids, proline, anthocyanin, protein, malondialdehyde (MDA), H2O2 as well as for antioxidants (POD, SOD, CAT) and mineral elements (Ca, Mg, Zn, K, Fe, Na, Cd). The maize cultivar MMRI-Yellow (tolerant) and Sahiwal-2002 (sensitive) exhibited significant reduction in leaf area, nutrient contents, plant biomass, activity of antioxidants, total proteins, photosynthetic pigments as well as flavonoids with increased production of H2O2, proline, MDA and relative membrane permeability (RMP) under Cd stress. However, this reduction was cultivar specific and recorded higher in cv. Sahiwal-2002 as compared to MMRI-Yellow. Application of Serratia sp. CP-13 significantly augmented plant biomass, photosynthetic pigments, antioxidative machinery, as well as flavonoids and proline while diminishing H2O2, RMP MDA production even under Cd stress in studied cultivars. Furthermore, CP-13 inoculation assisted the Cd stressed plants to sustain an optimal level of essential nutrients (Ca, Mg, Zn, K, Fe) except for Na and Cd which responded antagonistically. It was inferred that both inoculated maize cultivars exhibited better health and metabolism but substantial Cd tolerance was acquired by the sensitive cv. Sahiwal-2002 than the tolerant cv. MMRI-Yellow under applied Cd regimes. Furthermore, studied maize cultivars depicted maximum Cd tolerance in order of 30 < 24 < 18 < 12 < 6 < 0 µM Cd treatments under Serratia sp. CP-13 inoculation. Findings of current work highlighted the importance of Serratia sp. CP-13 and its inoculation impact on morpho-physio-biochemical attributes of maize growth under Cd dominant environment, which is likely an addition towards efficient approaches for bacterially-assisted Cd bioremediation and minimal Cd retention in edible plant parts.

Elucidating Cd-mediated distinct rhizospheric and in planta ionomic and physio-biochemical responses of two contrasting Zea mays L. cultivars.

Cadmium (Cd) in soil-plant system can abridge plant growth by initiating alterations in root zones. Hydroponics and rhizoboxes are useful techniques to monitor plant responses against various natural and/or induced metal stresses. However, soil based studies are considered more appropriate in order to devise efficient food safety and remediation strategies. The present research evaluated the Cd-mediated variations in elemental dynamics of rhizospheric soil together with in planta ionomics and morpho-physio-biochemical traits of two differentially Cd responsive maize cultivars. Cd-sensitive (31P41) and Cd-tolerant (3062) cultivars were grown in pots filled with 0, 20, 40, 60 and 80 µg/kg CdCl2 supplemented soil. The results depicted that the maize cultivars significantly influenced the elemental dynamics of rhizosphere as well as in planta mineral accumulation under applied Cd stress. The uptake and translocation of N, P, K, Ca, Mg, Zn and Fe from rhizosphere and root cell sap was significantly higher in Cd stressed cv. 3062 as compared to cv. 31P41. In sensitive cultivar (31P41), Cd toxicity resulted in significantly prominent reduction of biomass, leaf area, chlorophyll, carotenoids, protein contents as well as catalase activity in comparison to tolerant one (3062). Analysis of tolerance indexes (TIs) validated that cv. 3062 exhibited advantageous growth and efficient Cd tolerance due to elevated proline, phenolics and activity of antioxidative machinery as compared to cv. 31P41. The cv. 3062 exhibited 54% and 37% less Cd bio-concentration (BCF) and translocation factors (TF), respectively in comparison to cv. 31P41 under highest Cd stress regime. Lower BCF and TF designated a higher Cd stabilization by tolerant cultivar (3062) in rhizospheric zone and its potential use in future remediation plans.

Mutation in OsCADT1 enhances cadmium tolerance and enriches selenium in rice grain.

How cadmium (Cd) tolerance in rice is regulated remains poorly understood. We used a forward genetic approach to investigate Cd tolerance in rice. Using a root elongation assay, we isolated a rice mutant with enhanced Cd tolerance, cadt1, from an ethyl methanesulphonate (EMS)-mutagenized population of a widely grown Indica cultivar. The mutant accumulated more Cd in roots but not in shoots and grains. Using genomic resequencing and complementation, we identified OsCADT1 as the causal gene for the mutant phenotype, which encodes a putative serine hydroxymethyltransferase. OsCADT1 protein was localized to the nucleus and the OsCADT1 gene was expressed in both roots and shoots. OsCADT1 mutation resulted in higher sulphur and selenium accumulation in the shoots and grains. Selenate influx in cadt1 was 2.4 times that of the wild-type. The mutant showed higher expression of the sulphate/selenate transporter gene OsSULTR1;1 and the sulphur-deficiency-inducible gene OsSDI1. Thiol compounds including cysteine, glutathione and phytochelatins were significantly increased in the mutant, underlying its increased Cd tolerance. Growth and grain biomass were little affected. The results suggest that OsCADT1 acts as a negative regulator of sulphate/selenate uptake and assimilation. OsCADT1 mutation increases Cd tolerance and enriches selenium in rice grains, providing a novel solution for selenium biofortification.

Acid treated biochar enhances cadmium tolerance by restricting its uptake and improving physio-chemical attributes in quinoa (Chenopodium quinoa Willd.).

Heavy metals contamination of soil especially with cadmium (Cd) is a serious environmental concern in the current industrial era. Biochar serves as an excellent ameliorating agent depending upon its properties and application rates. In the pot scale study, effect of acid treated (AWSB) and untreated wheat straw biochar (WSB) was studied on physiology, grain yield, Cd accumulation, and tolerance of quinoa with possible health risks. Different levels of Cd (0, 25, 50 and 75 mg kg-1), AWSB and WSB (1% and 2% (w/w)) were applied in soil. Accumulation of Cd in control plant tissues led to oxidative stress which was shown in terms of increased lipid peroxidation. While biochar application relieved the oxidative damage as confirmed by the low production of H2O2 and TBARS contents. Application of AWSB improved plant growth, pigment contents and gas exchange attributes by limiting the accumulation of Cd in root, shoot and grain of quinoa. Results revealed a significant improvement in the activity of superoxide (SOD), catalase (CAT), ascorbate peroxidase (APX) and peroxidase (POD) with biochar at elevated levels of Cd in soil. Target Hazard Quotient (THQ) remained < 1 in the quinoa grains with WSB and AWSB under Cd stress. These results revealed that AWSB most effectively alleviated Cd toxicity in quinoa thereby decreasing Cd accumulation and regulation of Cd induced oxidative stress triggered by the antioxidant enzymatic system.

The influence of humic and fulvic acids on Cd bioavailability to wheat cultivars grown on sewage irrigated Cd-contaminated soils.

The soil cadmium (Cd) contamination is a ubiquitous environmental problem that has resulted from intense irrigation with wastewater. This pot trial was conducted with aim to produce safe food with Cd tolerant wheat cultivar in wastewater irrigated soils. For this purpose, two wheat cultivars NARC-2011 (Cd tolerant) and Shafaq-2006 (Cd sensitive) were screened out and selected, after conducting a pilot trial of twelve local wheat cultivars against Cd stress. Both cultivars were grown in naturally contaminated soils with Cd concentrations (4.18, 3.23, 2.29 and 1.25 mg kg-1). After harvesting, NARC-2011 showed significant photosynthetic attributes, grain biochemical parameters and yield. Additionally, Cd concentrations in edible grains of NARC-2011 cultivars were found within standard limits (200 mg kg-1), in all contaminated soils. Furthermore, a marked decrease in Cd bioavailability was noted with cultivar NARC-2011, where contribution of mobile Cd fractions (exchangeable and reducible) percentage was decreased, while immobile Cd fractions percentage increased (oxidizable and residual). Fourier transform infrared (FTIR) spectroscopy reflects the maturity and stability of humic and fulvic like acid fractions and revealed that humification of these compounds after prolonged sludge enriched wastewater irrigation lowered the Cd availability. The wheat cultivar NARC-2011 (Cd tolerant) could be opted to grow on soils irrigated with wastewater for a long time, as Cd bioavailability decreased with ageing due to stabilized humic substances and varietal tolerance.

Comparison of cadmium uptake and transcriptional responses in roots reveal key transcripts from high and low-cadmium tolerance ryegrass cultivars.

Cadmium (Cd), which seriously affects plant growth and crop production, is harmful to humans. Previous studies revealed ryegrass (Lolium multiflorum Lam.) exhibits Cd tolerance, and may be useful as a potential hyperaccumulator because of its wide distribution. In this study, the physiological and transcriptional responses of two ryegrass cultivars [i.e., high (LmHC) and low (LmLC) Cd tolerance] to Cd stress were investigated and compared. The Cd tolerance of LmHC was greater than that of LmLC at various Cd concentrations. The uptake of Evans blue dye revealed that Cd-induced root cell mortality was higher in LmLC than in LmHC after a 12-h Cd treatment. Furthermore, the content and influx rate of Cd in LmLC roots were greater than in LmHC roots under Cd stress conditions. The RNA sequencing and quantitative real-time PCR data indicated that the Cd transport regulatory genes (ABCG37, ABCB4, NRAMP4, and HMA5) were differentially expressed between the LmLC and LmHC roots. This expression-level diversity may contribute to the differences in the Cd accumulation and translocation between LmLC and LmHC. These findings may help clarify the physiological and molecular mechanisms underlying ryegrass responses to Cd toxicity. Additionally, ryegrass may be able to hyperaccumulate toxic heavy metals during the phytoremediation of contaminated soil.

Phosphorus deficiency induces root proliferation and Cd absorption but inhibits Cd tolerance and Cd translocation in roots of Populus × euramericana.

To disclose how phosphorus deficiency influence phytoremediation of Cd contamination using poplars, root architecture, Cd absorption, Cd translocation and antioxidant defense in poplar roots were investigated using a clone of Populus × euramericana. Root growth was unaltered by Cd exposure regardless of P conditions, while the degree of root proliferation upon P deficiency was changed by high level of Cd exposure. The concentration and content of Cd accumulation in roots were increased by P deficiency. This can be partially explained by the increased expression of genes encoding PM H + -ATPase under the combined conditions of P deficiency and high Cd exposure, which enhanced Cd2+-H+ exchanges and led to an increment of Cd uptake under P deficiency. Despite of the increasing Cd accumulation in roots, the translocation of Cd from roots to aerial tissues sharply decreased upon P deficiency. The relative expression of genes responsible for Cd translocation (HMA4) decreased upon P deficiency and thus inhibited Cd translocation via xylem. GR activity was decreased by P deficiency, which can inhibit the form of GSH and GSH-Cd complexes and decrease Cd translocation via GSH-Cd complexes. The transportation of PC-Cd complexes into vacuole decreased under P deficiency as a result of the low expression of PCS and ABCC1, and thus suppressed Cd tolerance and Cd detoxification in roots. Moreover, P deficiency decreased the levels of antioxidase (GR and CAT) and phytohormones including JA, ABA and GA3, which synchronously reduced antioxidant capacity in roots.

Responses of glutathione and phytochelatins biosysthesis in a cadmium accumulator of Perilla frutescens (L.) Britt. under cadmium contaminated conditions.

Screening new accumulators of heavy metal and identifying their tolerance, enrichment capacity of heavy metals are currently hot issues in phytoremediation research. A series of hydroponic experiments were conducted to analyze the effects of glutathione and phytochelatins in roots, stems, and leaves of Perilla frutescens under cadmium stress. The results showed that the non-protein thiols in roots and stems mainly existed in the form of GSH, PC2, PC3, and PC4 under Cd stress condition, while in leaves they existed in the form of GSH, PC2, and PC3. Furthermore, the contents of GSH and PCs positively correlated with Cd, but negatively correlated with root vigor and chlorophyll content under Cd stress conditions. After 21 days of treatments, the contents of Cd in different parts of the plant were 1465.2-3092.9 mg· kg-1 in the roots, 199.6-478.4 mg·kg-1 in the stems and 61.3-96.9 mg· kg-1 in the leaves at 2, 5, 10 mg·L-1 Cd levels respectively, and the amount of Cd uptakes were up to 3547.7-5701.7 µg·plant-1. Therefore, P. frutescens performed high capacity in Cd accumulation, and PCs played a key role in Cd tolerance. The application prospect of the plant in phytoremediation Cd polluted soil was also discussed.

Physiological and molecular mechanism of cadmium (Cd) tolerance at initial growth stage in rapeseed (Brassica napus L.).

Cadmium (Cd) contaminated soil has threatened plant growth and human health. Rapeseed (Brassica napus L.), an ideal plant for phytoremediation, is an important source of edible vegetable oil, vegetable, animal fodder, green manure and biodiesel. For safe utilization of Cd polluted soil, physiological, biochemical, and molecular techniques have been used to understand mechanisms of Cd tolerance in B. napus. However, most of these researches have concentrated on vegetative and adult stages, just a few reports focus on the initial growth stage. Here, the partitioning of cadmium, gene expression level and activity of enzymatic antioxidants of H18 (tolerant genotype) and P9 (sensitive genotype) were investigated under 0 and 30 mg/L Cd stress at seedling establishment stage. Results shown that the radicle length of H18 and P9 under Cd stress were decreased by 30.33 (0.01 < P < 0.05) and 88.89% (P < 0.01) respectively. Cd concentration at cotyledon not radicle and hypocotyl in P9 was significantly higher than that in H18. The expression level of BnaHMA4c, which plays a key role in root-to-shoot translocation of Cd, was extremely higher in P9 than in H18 under both normal and Cd stress conditions. We also found that SOD, CAT and POD were more active in responding to Cd stress after 48 h, and the activity of SOD and CAT in H18 were higher than that in P9 at all observed time points. In conclusion, high activity of enzymatic antioxidants at initial Cd stress stage is the main detoxification mechanism in Cd-tolerant rapeseed, while the higher Cd transfer coefficient, driven by higher expression level of BnaHMA4c is the main mechanism for surviving radicle from initial Cd toxicity in Cd-sensitive rapeseed.

Characterization of the Heavy-Metal-Associated Isoprenylated Plant Protein (HIPP) Gene Family from Triticeae Species.

Heavy-metal-associated (HMA) isoprenylated plant proteins (HIPPs) only exist in vascular plants. They play important roles in responses to biotic/abiotic stresses, heavy-metal homeostasis, and detoxification. However, research on the distribution, diversification, and function of HIPPs in Triticeae species is limited. In this study, a total of 278 HIPPs were identified from a database from five Triticeae species, and 13 were cloned from Haynaldia villosa. These genes were classified into five groups by phylogenetic analysis. Most HIPPs had one HMA domain, while 51 from Clade I had two, and all HIPPs had good collinear relationships between species or subgenomes. In silico expression profiling revealed that 44 of the 114 wheat HIPPs were dominantly expressed in roots, 43 were upregulated under biotic stresses, and 29 were upregulated upon drought or heat treatment. Subcellular localization analysis of the cloned HIPPs from H. villosa showed that they were expressed on the plasma membrane. HIPP1-V was upregulated in H. villosa after Cd treatment, and transgenic wheat plants overexpressing HIPP1-V showed enhanced Cd tolerance, as shown by the recovery of seed-germination and root-growth inhibition by supplementary Cd. This research provides a genome-wide overview of the Triticeae HIPP genes and proved that HIPP1-V positively regulates Cd tolerance in common wheat.

WRKY12 represses GSH1 expression to negatively regulate cadmium tolerance in Arabidopsis.

KEY MESSAGE: The WRKY transcription factor WRKY12 negatively regulates Cd tolerance in Arabidopsis via the glutathione-dependent phytochelatin synthesis pathway by directly targeting GSH1 and indirectly repressing phytochelatin synthesis-related gene expression. Cadmium (Cd) is a widespread pollutant toxic to plants. The glutathione (GSH)-dependent phytochelatin (PC) synthesis pathway plays key roles in Cd detoxification. However, its regulatory mechanism remains largely unknown. Here, we showed a previously unknown function of the WRKY transcription factor WRKY12 in the regulation of Cd tolerance by repressing the expression of PC synthesis-related genes. The expression of WRKY12 was inhibited by Cd stress. Enhanced Cd tolerance was observed in the WRKY12 loss-of-function mutants, whereas increased Cd sensitivity was found in the WRKY12-overexpressing plants. Overexpression and loss-of-function of WRKY12 were associated respectively with increased and decreased Cd accumulation by repressing or releasing the expression of the genes involved in the PC synthesis pathway. Transient expression assay showed that WRKY12 repressed the expression of GSH1, GSH2, PCS1, and PCS2. Further analysis indicated that WRKY12 could directly bind to the W-box of the promoter in GSH1 but not in GSH2, PCS1, and PCS2 in vivo. Together, our results suggest that WRKY12 directly targets GSH1 and indirectly represses PC synthesis-related gene expression to negatively regulate Cd accumulation and tolerance in Arabidopsis.