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The pollution of soil with heavy metals (HMs) has become an environmental problem of global concern. Phytoremediation, whereby plants extract HMs from soil, can efficiently and substantially reduce HM pollution in soil in an environmentally friendly manner. Cadmium-induced protein AS8 (CIPAS8) is present in many plants and its expression is induced by HMs. In this study, PeCIPAS8 and SlCIPAS8 were transformed into 84K poplar to study their effects on tolerance to, and translocation of, cadmium (Cd) in woody plants. Localization analyses showed that two CIPAS8 proteins were localized at the plasma membrane when transiently expressed in tobacco leaf epidermal cells. Compared with wild-type 84K poplar seedlings, transgenic poplar lines overexpressing PeCIPAS8 or SlCIPAS8 showed increased Cd contents and decreased Cd tolerance. Transgenic poplar lines overexpressing PeCIPAS8 or SlCIPAS8 accumulated more Cd in the roots, stems, and leaves, but the plant height did not differ significantly, compared with wild-type 84K poplar under Cd stress during the vegetative stage. CIPAS8 increased the Cd influx rate of transgenic poplar roots compared with that of the wild type, and affected the transcription levels of other metal transporters. These findings show that CIPAS8 increases Cd flux into plant tissues and demonstrate moderate Cd sensitivity of the plant. Therefore, CIPAS8 is an influx transporter with the potential to increase the uptake of toxic HMs by woody plants growing in HM-contaminated soils., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Masson SAS. All rights reserved.)

Key Message: CIPAS8 is a novel Cd-influx and Co-efflux transporters, and Ser86 and Cys128 might play a decisive role in Co-binding and translocation. Cadmium (Cd) is among the most toxic heavy metals and is a widespread environmental pollutant. Cobalt (Co) is a mineral nutrient that is essential for plant growth and development, but high concentrations may be toxic. Cadmium-induced protein AS8 (CIPAS8) is widely distributed among plant species and might be induced by heavy metals, but its function has not been studied previously. In this study, Populus euphratica PeCIPAS8 and Salix linearistipularis SlCIPAS8 were investigated. The transcription of both genes was significantly enhanced under Cd and Co stresses. PeCIPAS8 and SlCIPAS8 conferred sensitivity to Cd in transgenic yeast, allowing higher quantities of Cd to accumulate within the cells, whereas SlCIPAS8 also conferred tolerance to Co and reduced Co accumulation. The determinants of substrate selectivity of the SlCIPAS8 protein were examined by site mutagenesis, which indicated that the Ser at 86th (S86) substituted for Arg (R) [S86R] and Cys at 128th (C128) substituted for Ser [C128S] mutations limited the protein's capability for Co translocation. These results suggested that PeCIPAS8 and SlCIPAS8 may be involved in Cd uptake into the plant cell. SlCIPAS8 can reduce excess Co accumulation to maintain intracellular Co homeostasis, and the site mutations S86R and C128S were essential for Co transport. These findings provide insight into the function of CIPAS8 and highlight its potential for utilization in phytoremediation applications., (© 2023. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

The OsHMA2, OsLCT1 and OsZIP3 transporters were all involved in zinc (Zn) and cadmium (Cd) transport. So far, only a few researches studied on the co-regulation effect of three transporters related to Zn and Cd transport. The present study showed that rice co-expressing OsLCT1-OsHMA2-OsZIP3 (LHZ) had longer roots and shoots than wild-type (WT) rice after Zn and Cd treatments. The chlorophyll content was significantly higher, and the proline, malondialdehyde and H 2 O 2 contents were significantly lower in co-transgenic lines than in WT under Cd and Zn stress. LHZ in the seedlings of transgenic rice decreased the root-to-shoot translocation of Cd after Cd and Zn treatments. At the filling stage, LHZ line reduced Cd accumulation in grain after Cd treatment. Moreover, LHZ line increased the translocation of Zn to grain and reduced the accumulation of Cd after Zn treatment. These results suggested that LHZ co-expression could effectively decrease the translocation and accumulation of Cd to grains, alleviated the oxidative stress of Cd and Zn, and finally enhanced the quality and safety of rice grains., (Copyright © 2019. Published by Elsevier B.V.)

Cadmium (Cd) is a widespread soil contaminant that readily accumulates in wheat, and posing a potential threat to human health. Our aim is to investigate Cd toxicity effect and molecular mechanisms for wheat. In this study, the physiological indexes, morphology, and gene expression patterns of diploid wheat (Triticum urartu) seedlings were evaluated after 2 and 5 d of a Cd treatment (10 μM CdSO 4 ). The Cd treatment resulted in increased proline and glutathione contents in shoots and roots, slight damage to leaf tips, severe damage to root tips, and increased root secretions. Transcriptome analysis showed that there were significantly more differentially expressed genes (DEGs) in shoots and roots after 5 d of Cd stress than after 2 d of Cd stress, and the DEGs of the shoots were more different than the roots. A Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis indicated that the pathways enriched under Cd treatment were "DNA replication" and "phenylpropanoid biosynthesis". These findings provide information about the responses to Cd stress in wheat, and provide a theoretical basis for reducing Cd toxicity and protecting food safety., (Copyright © 2019. Published by Elsevier B.V.)

Background: HMA4 transporters are involved in the transport and binding of divalent heavy metals (Cd, Zn, Pb [lead] and Co [cobalt]). In general, as efflux pumps, HMA4 transporters can increase the heavy metal tolerance of yeast and Escherichia coli. Additional research has shown that the C-terminus of HMA4 contains a heavy metal-binding domain and that heterologous expression of a portion of peptides from this C-terminal domain in yeast provides a high level of Cd tolerance and Cd hyperaccumulation., Results: We cloned BjHMA4 from Brassica juncea, and quantitative real-time PCR analysis revealed that BjHMA4 was upregulated by Zn and Cd in the roots, stems and leaves. Overexpression of BjHMA4 dramatically affects Zn/Cd distribution in rice and wheat seedlings. Interestingly, BjHMA4 contains a repeat region named BjHMA4R within the C-terminal region; this repeat region is not far from the last transmembrane domain. We further characterized the detailed function of BjHMA4R via yeast and E. coli experiments. Notably, BjHMA4R greatly and specifically improved Cd tolerance, and BjHMA4R transformants both grew on solid media that contained 500 μM CdCl 2 and presented improved Cd accumulation (approximately twice that of wild-type [WT] strains). Additionally, visualization via fluorescence microscopy indicated that BjHMA4R clearly localizes in the cytosol of yeast. Overall, these findings suggest that BjHMA4R specifically improves Cd tolerance and Cd accumulation in yeast by specifically binding Cd 2+ in the cytosol under low heavy metal concentrations. Moreover, similar results in E. coli experiments corroborate this postulation., Conclusion: BjHMA4R can specifically bind Cd 2+ in the cytosol, thereby substantially and specifically improving Cd tolerance and accumulation under low heavy metal concentrations.

Soil microelement deficiency and heavy metal contamination affects plant growth and development, but improving trace element uptake and reducing heavy metal accumulation by genetic breeding can help alleviate this. Cell number regulator 2 (TaCNR2) from common wheat (Triticum aestivum) are similar to plant cadmium resistance proteins, involved with regulating heavy metal translocation. Our aim was to understand the effect of TaCNR2 on heavy metal tolerance and translocation. In this study, real-time quantitative PCR indicated TaCNR2 expression in the wheat seedlings increased under Cd, Zn and Mn treatment. Overexpression of TaCNR2 in Arabidopsis and rice enhanced its stress tolerance to Cd, Zn and Mn, and overexpression in rice improved Cd, Zn and Mn translocation from roots to shoots. The grain husks in overexpressed rice had higher Cd, Zn and Mn concentrations, but the brown rice accumulated less Cd but higher Mn than wild rice. The results showed that TaCNR2 can transport heavy metal ions. Thus, this study provides a novel gene resource for increasing nutrition uptake and reducing toxic metal accumulation in crops.

Key Message: Cysteine in the N-terminal metal-binding domain (N-MBD) of TaHMA2 participates in Zn 2+ /Cd 2+ binding and translocation in Arabidopsis. Wheat heavy metal ATPase 2 (TaHMA2) can transport Zn 2+ and Cd 2+ across membranes. A previous study showed that cysteine (Cys) and glutamate residues in the N-terminal metal-binding domain (N-MBD) were necessary for metal-binding and translocation of TaHMA2 in yeast. However, the function of TaHMA2 in plants was not fully revealed. In this study, we investigated the roles of the CCxxE and CPC motifs in the N-MBD and the N/C-terminal regions of TaHMA2 in Zn 2+ /Cd 2+ translocation in root and shoot of Arabidopsis. Compared with the wild type, overexpression of TaHMA2 and the TaHMA2 derivative (glutamic substituted for alanine from CCxxE) in Arabidopsis increased root length, fresh weight and enhanced Zn 2+ /Cd 2+ root-to-shoot translocation. The plants with a truncated N/C-terminal of TaHMA2 were impaired in Zn 2+ /Cd 2+ tolerance and translocation, while mutagenesis of Cys in the N-MBD reduced the tolerance and transport activity of TaHMA2, suggesting the involvement of Cys in Zn 2+ /Cd 2+ binding and translocation in Arabidopsis. This study therefore provides a theoretical possibility for the application of TaHMA2 in transgenic breeding to regulate metal element balance in crop plants.

Key Message: SnYSL3 encodes a plasma-localized transporter delivering various metal-nicotianamine complexes. The expression of SnYSL3 is up-regulated by excess Cd, suggesting an important role for SnYSL3 in response to Cd stress. The Yellow Stripe-Like (YSL) transporters have been proposed to participate in metal uptake and long-range transport in model plants. In this study, we isolated and characterized a novel member of the YSL gene family, SnYSL3, from the cadmium hyperaccumulator Solanum nigrum. SnYSL3 was constitutively expressed and encodes a plasma membrane-localized protein. In situ RNA hybridization localized the SnYSL3 transcripts predominantly in vascular tissues and epidermal cells of the roots and stems, while in leaves, the mRNA levels were high in the vasculature. The SnYSL3 expression level was up-regulated by excess Cd, excess Fe and Cu deficiency. Heterologous expression of SnYSL3 in yeast revealed that SnYSL3 transports nicotianamine complexes containing Fe(II), Cu, Zn and Cd. SnYSL3 overexpression in Arabidopsis thaliana decreased Fe and Mn concentrations in the roots and increased the root-to-shoot translocation ratios of Fe and Mn. Under Cd exposure, the transgenic plants showed increased translocation ratios of Fe and Cd, but no difference was observed in Mn translocation from roots to shoots between the transgenic and wild-type lines. Although the accurate function of SnYSL3 remains to be confirmed, these results suggest that SnYSL3 is a transporter delivering a broad range of metal-nicotianamine complexes and is potentially important for the response to heavy metal stress, especially due to Cd and Fe.

Manganese (Mn) can interact with cadmium (Cd) in environments and influence the toxic effect of Cd on plants. However, few studies have investigated the relationship between the Mn/Cd ratio and plant Cd-toxicity along Cd concentrations. In this paper, we studied the effects of external Mn/Cd molar ratios (0, 10, 30, 50 and 60) on Cd toxicity in the Mn hyperaccumulator and Cd tolerant plant, Phytolacca acinosa Roxb., at three Cd levels (50, 100 and 200 μM) under hydroponic conditions. Our result showed that seedling growth (y) under Cd stress was strongly positively related to the solution Mn/Cd molar ratio (SMCR). The relationship between the two variables under solution Cd concentrations was well explained by the linear regression model y=a+b1 (SMCR)+b2 (Solution-Cd). Increasing SMCR significantly reduced the Cd concentration and increased the Mn concentration in plant tissues. However, seedling growth was consistent with the shoot Mn/Cd molar ratio rather than with the Mn or Cd concentrations in plant tissues. At low levels of SMCR (e.g. 0 and 10), elevation of Mn distribution in shoot tissues might be a mechanism in P. acinosa seedlings to defend against Cd-toxicity. In comparison with low levels of SMCR, high levels of SMCR (e.g. 50 and 60) greatly alleviated lipid peroxidation and plant water-loss, and enhanced photosynthesis. However, the alleviated lipid peroxidation in the Mn-mitigation of Cd toxicity was likely to be the secondary effect resulting from the antagonism between Mn and Cd in the plant., (Copyright © 2013 Elsevier Masson SAS. All rights reserved.)

Solanum nigrum L. is considered to be a potential plant for restoring Cd-contaminated soils. Si could enhance plants tolerance to heavy metal; however, the mechanism of Si-mediated alleviation of Cd toxicity in S. nigrum was not clear. Three-week-old S. nigrum seedlings were grown in Hoagland solution containing 0 or 100 μM Cd with or without 1 mM Si for 4 days. The results showed that the Cd concentration both in roots and shoots of Si-supplied plant was significantly reduced, especially in expanding and old leaves. The relative proportion of ethanol-extractable Cd, water-extractable Cd and NaCl-extractable Cd in roots was increased by adding Si, while the root-to-shoot Cd translocation was not decreased. Furthermore, in comparison with single Cd treatment, supplying Si could reduce H₂O₂ accumulation and cell death in roots, and the electrolyte leakage and H₂O₂ concentration in functional leaves. Moreover, the activity of superoxide dismutase (SOD, EC 1.15.1.1), catalase (CAT, EC 1.11.1.6), peroxidase (POD, EC 1.11.1.7) and ascorbate peroxidase (APX, EC 1.11.1.11) in functional leaves was markedly increased by Cd exposure, while the antioxidative enzyme activities in Cd plus Si treatment seedlings were significantly lower than that in Cd treatment alone, this decrease might be attributed to the reduction of Cd concentration and Cd-induced oxidative damages. These results demonstrate that Si-enhanced Cd tolerance in S. nigrum is mainly due to the decrease of Cd uptake in roots and Cd distribution in expanding and old leaves, as well as lowering oxidative stress induced by Cd in plants., (Copyright © 2013 Elsevier Masson SAS. All rights reserved.)

Brassica juncea L. is a Zn/Cd accumulator. To determine the physiological basis of its metal accumulation phenotype, the functional properties and role of the metal efflux transporter BjCET2 were investigated using transgenic technology. Heterologous expression of BjCET2 in the double mutant yeast strain Deltazrc1Deltacot1 enhanced the metal tolerance of the yeast strain and led to decrease in Zn or Cd accumulation. Detection of green fluorescence from green fluorescent protein (GFP) in the root tip of transgenic tobacco further revealed that BjCET2::GFP is localized at the plasma membrane. Semi-quantitative RT-PCR analysis showed that BjCET2 was most abundant in the root and was weakly expressed in the stem and leaves. The expression of BjCET2 was up-regulated by heavy metals. However, exposure to low temperature, salt and drought did not affect the expression of BjCET2. Overexpression of BjCET2 in transgenic B. juncea plants conferred heavy metal tolerance and increased Cd/Zn accumulation in the leaves. BjCET2-deficient B. juncea mediated by antisense RNA resulted in hypersensitivity to heavy metals and decreased Zn/Cd accumulation in the plants. These results suggest that the heavy metal efflux of BjCET2 plays important roles in the metal tolerance of B. juncea and in Zn/Cd accumulation in B. juncea.

Catalase (CAT), an important enzyme of antioxidant system, was investigated the role in preventing the plant from Cd-induced oxidative stress caused by reactive oxygen species. A CAT gene from Brassica juncea was cloned and up-regulated in response to Cd/Zn. The CAT cDNA (BjCAT3) under the control of CaMV35S promoter was introduced into tobacco via Agrobacterium-mediated transformation. Northern blot analysis verified the BjCAT3 was expressed at high level in different transgenic lines. In morphological observation, we found that seedlings from transgenic tobacco plants grew better and showed longer root length in the presence of Cd versus wild-type (WT) seedlings. Under 100 microM Cd stress, WT plants became chlorotic and almost dead while transgenic tobacco plants still remained green and phenotypically normal. The CAT activity of transgenic T(1) generations was approximately two-fold higher than that of WT plants. In WT, endogenous CAT activity is rapidly reduced as a result of 200 microM CdCl2 exposure. Compared with WT plants, lower level of Cd-induced H2O2 accumulation and cell death were detected in roots of transgenic plants with high level of CAT activity. All our findings strongly support that overexpressing BjCAT3 in tobacco could enhance the tolerance under Cd stress.

Mineral nutrients, such as manganese (Mn) and iron (Fe), play essential roles in many biological processes in plants but their over-enrichment is harmful for the metabolism. Metal tolerance proteins (MTPs) are involved in cellular Mn and Fe homeostasis. However, the transporter responsible for the transport of Mn in wheat is unknown. In our study, TuMTP8, a Mn-CDF transporter from diploid wheat (Triticum urartu), was identified. Expression of TuMTP8 in yeast strains of Δccc1 and Δsmf1 and Arabidopsis conferred tolerance to elevated Mn and Fe, but not to other metals (zinc, cobalt, copper, nickel, or cadmium). Compared with TuVIT1 (vacuole Fe transporter), TuMTP8 shows a significantly higher proportion in Mn transport and a smaller proportion in Fe transport. The transient analysis in tobacco epidermal cells revealed that TuMTP8 localizes to vacuolar membrane. The highest transcript levels of TuMTP8 were in the sheath of the oldest leaf and the awn, suggesting that TuMTP8 sequesters excess Mn into the vacuole in these organs, away from more sensitive tissues. These findings indicate that TuMTP8, a tonoplast-localized Mn/Fe transporter, functions as a primary balancer to regulate Mn distribution in T. urartu under elevated Mn conditions and participates in the intracellular transport and storage of excess Mn as a detoxification mechanism, thereby conferring Mn tolerance. [ABSTRACT FROM AUTHOR]

Currently, there are few studies concerning the function of heavy metal ATPase 2 ( HMA2), particularly in monocotyledons, and the potential application of this protein in biofortification and phytoremediation. Thus, we isolated and characterized the Ta HMA2 gene from wheat ( Triticum aestivum L.). Our results indicate that Ta HMA2 is localized to the plasma membrane and stably expressed, except in the nodes, which showed relatively high expression. Zinc/cadmium ( Zn/ Cd) resistance was observed in Ta HMA2-transformed yeast. The over-expression of Ta HMA2 increased the elongation and decreased the seed-setting rate in rice ( Oryza sativa L.), but not Arabidopsis thaliana, tobacco ( Nicotiana tabacum L.) or wheat. Ta HMA2 over-expression also improved root-shoot Zn/ Cd translocation, especially in rice. The seeds of transgenic rice and wheat, not tobacco, showed decreased Zn concentrations. The Zn concentration was decreased in all parts of the transgenic rice seeds, but was decreased only in the ventral endosperm of wheat, which showed an increased Zn concentration in the embryo and aleurone. The over-expression of Ta HMA2 improved plant tolerance under moderate Zn stress and Zn deficiency, but Zn and Cd resistance decreased under high levels of Zn and Cd stress, respectively. The Cd concentration in transgenic rice seedlings was dramatically increased under Zn deficiency. Thus, over-expression of Ta HMA2 showed a more obvious phenotype in monocotyledons than in dicotyledons. These findings provide important information for Ta HMA2, and more efforts should be made in the future to characterize the reduced Zn concentration in Ta HMA2 transgenic grains and the diversity of Ta HMA2 substrate specificity. [ABSTRACT FROM AUTHOR]

Background: HMA4 transporters are involved in the transport and binding of divalent heavy metals (Cd, Zn, Pb [lead] and Co [cobalt]). In general, as efflux pumps, HMA4 transporters can increase the heavy metal tolerance of yeast and Escherichia coli. Additional research has shown that the C-terminus of HMA4 contains a heavy metal-binding domain and that heterologous expression of a portion of peptides from this C-terminal domain in yeast provides a high level of Cd tolerance and Cd hyperaccumulation. Results: We cloned BjHMA4 from Brassica juncea, and quantitative real-time PCR analysis revealed that BjHMA4 was upregulated by Zn and Cd in the roots, stems and leaves. Overexpression of BjHMA4 dramatically affects Zn/Cd distribution in rice and wheat seedlings. Interestingly, BjHMA4 contains a repeat region named BjHMA4R within the C-terminal region; this repeat region is not far from the last transmembrane domain. We further characterized the detailed function of BjHMA4R via yeast and E. coli experiments. Notably, BjHMA4R greatly and specifically improved Cd tolerance, and BjHMA4R transformants both grew on solid media that contained 500 μM CdCl2 and presented improved Cd accumulation (approximately twice that of wild-type [WT] strains). Additionally, visualization via fluorescence microscopy indicated that BjHMA4R clearly localizes in the cytosol of yeast. Overall, these findings suggest that BjHMA4R specifically improves Cd tolerance and Cd accumulation in yeast by specifically binding Cd2+ in the cytosol under low heavy metal concentrations. Moreover, similar results in E. coli experiments corroborate this postulation. Conclusion: BjHMA4R can specifically bind Cd2+ in the cytosol, thereby substantially and specifically improving Cd tolerance and accumulation under low heavy metal concentrations. [ABSTRACT FROM AUTHOR]

Toxic heavy metals originating from human activities have caused irreversible harm to the environment. Toxic heavy metal ions absorbed by crop plants can seriously threaten human health. Therefore, decreasing heavy metal contents in crop plants is an urgent need. The plant cadmium resistance protein (PCR) is a heavy metal ion transporter. In this study, PePCR10 was cloned from Populus euphratica. Bioinformatics analyses revealed its transmembrane structure and gene sequence motifs. The transcript profile of PePCR10 was analyzed by RT-qPCR, and its transcript levels increased under toxic heavy metal (cadmium, lead, aluminum) treatments. Subcellular localization analyses in tobacco cells revealed that PePCR10 localizes at the plasma membrane. Compared with wild type (WT), PePCR10 -overexpressing lines showed significantly higher values for plant height, root length, fresh weight, and dry weight under heavy metal stress. Electrolyte leakage, nitroblue tetrazolium staining, and chlorophyll fluorescence analyses indicated that Cd/Al tolerance in PePCR10 -overexpressing lines was stronger than that in WT. The Cd/Al contents were lower in the PePCR10 -overexpressing lines than in WT under Cd/Al stress. Our results show that PePCR10 can reduce the heavy metal content in poplar and enhance its Cd/Al tolerance. Hence, PePCR10 is a candidate genetic resource for effectively reducing heavy metal accumulation in crops. [Display omitted] • PePCR10 was confirmed to be a plasma membrane-localized protein. • PePCR10 enhanced Cd/Al tolerance by promoting Cd/Al efflux in poplar. • PePCR10 can be used to reduce the risks of heavy metals to humans. [ABSTRACT FROM AUTHOR]