Zn Tolerance Research Articles

PtHSFA4a gene play critical roles in the adaptation of Arabidopsis thaliana plants to high-Zinc stress

ABSTRACTHeat shock transcription factors (HSFs) play pivotal roles in various abiotic stresses. However, only one of the studies on HSFs that participated in excess zinc (Zn) stress in our previous study in Populus ussuriensis. Here, overexpression of P. trichocarpa PtHSFA4a gene in Arabidopsis thaliana significantly improved excess Zn tolerance. It was found that PtHSFA4a-OE lines have higher seed germination rate than wild type (WT) when exposed to excess Zn. Also, PtHSFA4a-OE lines exhibit high viability and stronger root growth than WT in soil. PtHSFA4a reduced the intracellular concentration of free zinc ion of roots when overexpressed in A. thaliana. Our data indicate PtHSFA4a is the candidate gene to act as positive regulators in the resistance to excess Zn, extending our knowledge of excess Zn tolerance transcription factors

Nicotianamine Synthase Gene 1 from the hyperaccumulator Sedum alfredii Hance is associated with Cd/Zn tolerance and accumulation in plants

Sedum alfredii Hance is a hyperaccumulator of cadmium (Cd) and zinc (Zn) that exhibits extraordinary accumulation of these metals in various tissues. The Nicotianamine synthase (NAS) genes play key roles in regulating the production of nicotianamine, a non-protein amino acid that facilitates metal homeostasis in plants, but the functions of these genes in S. alfredii remain unknown. The aims of this study were to identify and characterize the NAS gene from S. alfredii (SaNAS1) and explore NAS roles in Cd or Zn tolerance and accumulation. One Cd-induced NAS gene from S. alfredii (SaNAS1) was cloned. We determined the subcellular localization of the encoded protein and evaluated SaNAS1 expression. We also assessed SaNAS1 function by conducting a complementary assay with yeast mutants Δzrc1 exposed to Cd or Zn. Finally, transgenic Arabidopsis plants expressing SaNAS1 were produced, and SaNAS1 function was further examined in these plants. SaNAS1 was highly expressed in response to Cd or Zn exposure, and the encoded protein was distributed throughout the cytoplasm and nucleus. Furthermore, yeast expressing SaNAS1 exhibited increased tolerance to Cd or Zn. Finally, expression of SaNAS1 in Arabidopsis increased nicotianamine production and promoted Cd or Zn accumulation in roots and shoots. SaNAS1-expressing transgenic Arabidopsis lines showed improved seedling growth under Cd or Zn stress compared with wild type, indicating that SaNAS1 enhances tolerance to both Cd and Zn. SaNAS1 may play a critical role in Cd or Zn tolerance and hyperaccumulation by regulating nicotianamine level in S. alfredii.

Study of Zn accumulation and tolerance of HMA4 TILLING mutants of Brassica rapa grown under Zn deficiency and Zn toxicity

Nowadays, Zinc (Zn) deficiency is the most widespread micronutrient deficiency but simultaneously Zn toxicity is produced due to environmental pollution. A potential method to alleviate Zn deficiency and to reduce Zn concentration in soils is through the generation of plants with enhanced capacity for Zn accumulation and higher tolerance. This could be achieved through the modification of HMA4 transporter. BraA.hma4a-3 is a TILLING mutant plant that presents one modification in HMA4 transporter. Thus, in this study we analyzed the potential of BraA.hma4a-3 for Zn accumulation and Zn deficiency and toxicity tolerance. BraA.hma4a-3 and parental R-o-18 plants were grown with different Zn doses: 1 μM ZnSO4 (Control), 0.01 μM ZnSO4 (Zn deficiency) and 100 μM ZnSO4 (Zn toxicity). Parameters of biomass, Zn concentration, photosynthesis, oxidative stress, N metabolism and amino acids (AAs) were measured. BraA.hma4a-3 did not affect plant biomass but did increase Zn accumulation in leaves under an adequate Zn supply and Fe under control and Zn deficiency doses. Regarding stress tolerance parameters and N metabolism, BraA.hma4a did not produce alterations under control conditions. In addition, under Zn toxicity, parameters suggest a greater tolerance. Briefly, the obtained results point to BraA.hma4a-3 as a useful mutant to increase Zn accumulation.

S-Nitrosothiol Signaling Is involved in Regulating Hydrogen Peroxide Metabolism of Zinc-Stressed Arabidopsis.

Accumulation of heavy metals such as zinc (Zn) disturbs the metabolism of reactive oxygen (e.g. hydrogen peroxide, H2O2) and nitrogen species (e.g. nitric oxide, NO; S-nitrosoglutathione, GSNO) in plant cells; however, their signal interactions are not well understood. Therefore, this study examines the interplay between H2O2 metabolism and GSNO signaling in Arabidopsis. Comparing the Zn tolerance of the wild type (WT), GSNO reductase (GSNOR) overexpressor 35S::FLAG-GSNOR1 and GSNOR-deficient gsnor1-3, we observed relative Zn tolerance of gsnor1-3, which was not accompanied by altered Zn accumulation capacity. Moreover, in gsnor1-3 plants Zn did not induce NO/S-nitrosothiol (SNO) signaling, possibly due to the enhanced activity of NADPH-dependent thioredoxin reductase. In WT and 35S::FLAG-GSNOR1, GSNOR was inactivated by Zn, and Zn-induced H2O2 is directly involved in the GSNOR activity loss. In WT seedlings, Zn resulted in a slight intensification of protein nitration detected by Western blot and protein S-nitrosation observed by resin-assisted capture of SNO proteins (RSNO-RAC). LC-MS/MS analyses indicate that Zn induces the S-nitrosation of ascorbate peroxidase 1. Our data collectively show that Zn-induced H2O2 may influence its own level, which involves GSNOR inactivation-triggered SNO signaling. These data provide new evidence for the interplay between H2O2 and SNO signaling in Arabidopsis plants affected by metal stress.

PuHSFA4a Enhances Tolerance To Excess Zinc by Regulating Reactive Oxygen Species Production and Root Development in Populus.

Zinc (Zn) is an essential micronutrient but in excess is highly toxic to plants. Plants regulate Zn homeostasis and withstand excess Zn through various pathways; these pathways are generally tightly regulated by a specific set of genes. However, the transcription factors involved in excess Zn tolerance have yet to be identified. Here, we characterized a Populus ussuriensis heat shock transcription factor A4a (PuHSFA4a) that acts as a positive regulator of excess Zn tolerance in P ussuriensis We used overexpression (PuHSFA4a-OE) and chimeric dominant repressor (PuHSFA4a-SRDX) lines to identify the targets of PuHSFA4a PuHSFA4a transcription is specifically induced in roots by high Zn. Overexpression of PuHSFA4a conferred excess Zn tolerance and a dominant repressor version of PuHSFA4a increased excess Zn sensitivity in P ussuriensis by regulating the antioxidant system in roots. PuHSFA4a coordinately activates genes related to abiotic stress responses and root development and directly binds to the promoter regions of glutathione-s-transferase U17 (PuGSTU17) and phospholipase A2 (PuPLA2 ). PuGSTU17 overexpression significantly increased GST activity and reduced reactive oxygen species levels in roots while PuGSTU17-RNA interference lines exhibited the opposite phenotype. Furthermore, PuPLA2 overexpression promoted root growth under high Zn stress. Taken together, we provide evidence that PuHSFA4a coordinately activates the antioxidant system and root development-related genes and directly targets PuGSTU17 and PuPLA, thereby promoting excess Zn tolerance in P ussuriensis roots.

Tandem oligomeric expression of metallothionein enhance heavy metal tolerance and bioaccumulation in Escherichia coli

Metallothioneins (MTs) are a family of low molecular weight, cysteine-rich, metal-binding proteins, which play important roles in metal homeostasis and heavy metal detoxification. In our previous study, a novel full length MT cDNA was successfully cloned from the freshwater crab (Sinopotamon henanense). In the present study, tandem repeats of two and three copies of the crab MT gene were integrated by overlap extension PCR (SOE-PCR) and expressed in Escherichia coli. The SUMO fusion expression system was adopted to increase the stability and solubility of the recombinant MT proteins. The recombinant proteins were purified and their metal-binding abilities were further analyzed by the ultraviolet absorption spectral scan. Furthermore, the metal tolerance and bioaccumulation of E. coli cells expressing oligomeric MTs were determined. Results showed that the recombinant plasmids pET28a-SUMO-2MT and pET28a-SUMO-3MT were successfully constructed. SDS-PAGE analysis showed that the SUMO-2MT and SUMO-3MT were expressed mainly in the soluble forms. Oligomeric MTs expression significantly enhanced Cu, Cd or Zn tolerance and accumulation in E. coli in the order: SUMO-3MT˃SUMO-2MT˃SUMO-MT˃control. Cells harboring pET28a-SUMO -3MT exhibited the highest Cu, Cd or Zn bioaccumulation at 5.8-fold, 3.1-fold or 6.7-fold higher than that of the control cells. Our research could lay a foundation for large-scale preparation of MTs and provide a scientific basis for bioremediation of heavy metal pollution by oligomeric MTs.

Zinc Excess Induces a Hypoxia-Like Response by Inhibiting Cysteine Oxidases in Poplar Roots.

Poplar (Populus spp.) is a tree species considered for the remediation of soil contaminated by metals, including zinc (Zn). To improve poplar's capacity for Zn assimilation and compartmentalization, it is necessary to understand the physiological and biochemical mechanisms that enable these features as well as their regulation at the molecular level. We observed that the molecular response of poplar roots to Zn excess overlapped with that activated by hypoxia. Therefore, we tested the effect of Zn excess on hypoxia-sensing components and investigated the consequence of root hypoxia on poplar fitness and Zn accumulation capacity. Our results suggest that high intracellular Zn concentrations mimic iron deficiency and inhibit the activity of the oxygen sensors Plant Cysteine Oxidases, leading to the stabilization and activation of ERF-VII transcription factors, which are key regulators of the molecular response to hypoxia. Remarkably, excess Zn and waterlogging similarly decreased poplar growth and development. Simultaneous excess Zn and waterlogging did not exacerbate these parameters, although Zn uptake was limited. This study unveils the contribution of the oxygen-sensing machinery to the Zn excess response in poplar, which may be exploited to improve Zn tolerance and increase Zn accumulation capacity in plants.

Improved Cd, Zn and Mn tolerance and reduced Cd accumulation in grains with wheat-based cell number regulator TaCNR2

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.

Different cadmium tolerance of two isolates of Hebeloma mesophaeum showing different basal expression levels of metallothionein (HmMT3) gene

Hebeloma mesophaeum is an ectomycorrhizal fungus frequently associated with metal disturbed environments. In this work, we examined Ag, Cd, and Zn tolerance of H. mesophaeum isolates from heavy metal-polluted (isolate Prib) and clean (isolate Rez) sites. Both mycelia showed essentially the same level of Ag and Zn tolerance, but Prib was more Cd tolerant. In short-term exposures, Prib accumulated slightly less Cd than Rez. Size exclusion chromatography of cell-free extracts and fluorescence microscopy of hyphae with a Cd-specific fluorescent tracer revealed that substantial proportion of Cd was contained in the vacuoles in both isolates. Considering that the proportion of Cd associated with fractions attributable to Cd complexes with cytosolic, metallothionein (MT) peptides was higher in Prib, we examined the copy number and basal levels of HmMTs genes in Rez and Prib. While no difference between the isolates was observed in the gene copy numbers and basal levels of HmMT1 transcripts, the basal transcription of HmMT3 was 3-fold higher in Prib. These observations suggest that MTs provide in Prib better protection against Cd. Furthermore, the higher Cd tolerance in Prib can be to some extent also supported by the efflux or reduced uptake of Cd in the hyphae.

Acclimatization of Rhizophagus irregularis Enhances Zn Tolerance of the Fungus and the Mycorrhizal Plant Partner.

Arbuscular mycorrhizal (AM) fungi confer heavy metal tolerance to plants, but this characteristic differs between different AM fungal strains. We tested the hypotheses if acclimatization of an AM fungus to Zn stress is possible and if this leads also to higher Zn tolerance of mycorrhizal plants. The AM fungus Rhizophagus irregularis was acclimatized in root organ cultures (Daucus carota L.) to Zn resulting in an acclimatized (Acc+) strain. The non-acclimatized (Acc-) strain remained untreated. Fungal development and RNA accumulation of a set of stress-related genes were analyzed in root organ cultures and the capacity of conferring Zn tolerance to maize plants was investigated in pot cultures. Development of Acc+ strain was significantly higher than Acc- strain, when strains were grown in Zn-enriched root organ cultures, whereas the growth of the Acc+ strain was reduced on normal medium probably due to a higher Zn demand compared to the Acc- strain. RNA accumulation analyses revealed different expression patterns of genes encoding glutathione S-transferase (RiGST), superoxide dismutase (RiSOD) and glutaredoxin (RiGRX) between the two strains. Plants inoculated with the Acc+ strain showed higher biomass and lower Zn content than those inoculated with the Acc- strain. The results showed that R. irregularis can be acclimatized to increased amounts of Zn. This acclimatization leads not only to improved fungal development in Zn-stress conditions, but also to an increase of mycorrhiza-induced Zn tolerance of colonized plants.

Wheat Cell Number Regulator CNR10 Enhances the Tolerance, Translocation, and Accumulation of Heavy Metals in Plants.

Heavy metal contamination affects crop growth and development and can indirectly threaten human health. Therefore, improving the content of microelements and reducing the accumulation of toxic metals by genetic breeding in crops is an effective strategy to solve this environmental problem. Previous reports show plant cadmium resistance (PCR) protein can transport zinc (Zn) and cadmium (Cd). The cell number regulator (CNR) protein, which functions to regulate organ size, has high similarity to, and shares conserved motifs with, PCR. Therefore, CNR may be involved in regulating heavy metal translocation. We isolated TuCNR10 from diploid wheat, Triticum urartu. Real-time quantitative PCR showed TuCNR10 expression increased in the shoots and roots of seedlings under Cd, Zn, and manganese (Mn) stresses. Confocal imaging indicated TuCNR10 was localized at the plasma membrane. Overexpression of TuCNR10 in Arabidopsis and rice enhanced Cd, Zn, and Mn tolerance and improved Cd, Zn, and Mn translocation from roots to shoots. Compared with wild-type rice, rice overexpressing TuCNR10 had lower Cd and higher Zn and Mn contents in grains. These results indicated that TuCNR10 may be a transporter of Cd, Zn, and Mn. TuCNR10 may be a useful genetic resource for microelement fortification and reducing toxic metal accumulation in crops.

AQUA1 is a mercury sensitive poplar aquaporin regulated at transcriptional and post-translational levels by Zn stress

Aquaporins are water channel proteins that regulate plant development, growth, and response to environmental stresses. Populus trichocarpa is one of the plants with the highest number of aquaporins in its genome, but only few of them have been characterized at the whole plant functional level. Here we analyzed a putative aquaporin gene, aqua1, a gene that encodes for a protein of 257 amino acid with the typical NPA (Asp-Pro-Ala) signature motif of the aquaporin gene family. aqua1 was down-regulated of ∼10 fold under excess Zn in both leaves and roots, and conferred Zn tolerance when expressed in yeast Zn hypersensitive strain. In vivo localization of AQUA1-GFP in Arabidopsis protoplast showed a heterogeneous distribution of this protein on different membranes destined to form aggregates related to autophagic multivesicular bodies. Zn-dependent AQUA1-GFP re-localization was perturbed by phosphatases' and kinases' inhibitors that could affect both intracellular trafficking and aquaporins' activity. Exposed to high concentration of Zn, AQUA1 also co-localized with AtTIP1;1, a well-known Arabidopsis vacuolar marker, probably in pro-vacuolar multivesicular bodies. These findings suggest that high concentration of Zn down-regulates aqua1 and causes its re-localization in new forming pro-vacuoles. This Zn-dependent re-localization appears to be mediated by mechanisms regulating intracellular trafficking and aquaporins' post-translational modifications. This functional characterization of a poplar aquaporin in response to excess Zn will be a useful reference for understanding aquaporins' roles and regulation in response to high concentration of Zn in poplar.

Down regulation of a heavy metal transporter gene influences several domestication traits and grain Fe-Zn content in rice

Biofortification of rice (Oryza sativa L.) would alleviate iron and zinc deficiencies in the target populations. We identified two alleles 261 and 284 of a Gramineae-specific heavy metal transporter gene OsHMA7 by analyzing expression patterns and sequences of genes within QTLs for high Fe & Zn, in Madhukar x Swarna recombinant inbred lines (RILs) with high (HL) or low (LL) grain Fe & Zn. Overexpression of 261 allele increased grain Fe and Zn but most of the transgenic plants either did not survive or did not yield enough seeds and could not be further characterized. Knocking down expression of OsHMA7 by RNAi silencing of endogenous gene resulted in plants with altered domestication traits such as plant height, tiller number, panicle size and architecture, grain color, shape, size, grain shattering, heading date and increased sensitivity to Fe and Zn deficiency. However, overexpression of 284 allele resulted in transgenic lines with either high grain Fe & Zn content (HL-ox) and tolerance to Fe and Zn deficiency or low grain Fe & Zn content (LL-ox) and phenotype similar to RNAi-lines. OsHMA7 transcript levels were five-fold higher in the HL-ox plants whereas LL-ox and RNAi plants showed 2–3 fold reduced levels compared to Kitaake control. Spraying LL-ox and RNAi lines with Fe & Zn at grain filling stage resulted in increased grain yield, significant increase in Fe & Zn content and brown pericarp. Altered expression of OsHMA7 influenced transcript levels of iron-responsive genes indicating cellular Fe-Zn homeostasis and also several domestication-related genes in rice. Our study shows that a novel heavy metal transporter gene influences yield and grain Fe & Zn content and has potential to improve rice production and biofortification.

Systemic Upregulation of MTP2- and HMA2-Mediated Zn Partitioning to the Shoot Supplements Local Zn Deficiency Responses.

Low bioavailable concentrations of the micronutrient zinc (Zn) limit agricultural production on 40% of cultivated land. Here, we demonstrate that plant acclimation to Zn deficiency involves systemic regulation. Physiological Zn deficiency of Arabidopsis thaliana shoots results in increased root transcript levels of the membrane transport protein-encoding genes METAL TRANSPORT PROTEIN2 (MTP2) and HEAVY METAL ATPASE2 (HMA2), which are unresponsive to the local Zn status of roots. MTP2 and HMA2 act additively in the partitioning of Zn from roots to shoots. Chimeric GFP fusion proteins of MTP2 complement an mtp2 mutant and localize in the endoplasmic reticulum (ER) membrane of the outer cell layers from elongation to root hair zone of lateral roots. MTP2 restores Zn tolerance in a hypersensitive yeast mutant. These results are consistent with cell-to-cell movement of Zn toward the root vasculature inside the ER-luminal continuum through the desmotubules of plasmodesmata, under Zn deficiency. The previously described Zn deficiency response comprises transcriptional activation of target genes, including ZINC-REGULATED TRANSPORTER IRON-REGULATED TRANSPORTER PROTEIN genes ZIP4 and ZIP9, by the F-group bZIP transcription factors bZIP19 and bZIP23. We show that ZIP4 and ZIP9 respond to the local Zn status in both roots and shoots, in contrast to the systemic regulation identified here. Our findings are relevant for crop management and improvement toward combating human nutritional Zn deficiency that affects 30 to 50% of the world's population.

Salinity alleviates zinc toxicity in the saltmarsh zinc-accumulator Juncus acutus

The potential importance of Juncus acutus for remediation of Zn-contaminated lands has been recognized, because of its Zn tolerance and capacity to accumulate Zn. Since it is also a halophyte, the extent to which salinity influences its Zn tolerance requires investigation. A factorial greenhouse experiment was designed to assess the effect of NaCl supply (0 and 85 mM NaCl) on the growth, photosynthetic physiology and tissue ions concentrations of plants exposed to 0, 30 and 100 mM Zn. Our results indicated that NaCl supplementation alleviated the effects of Zn toxicity on growth, as Zn at 100 mM reduced relative growth rate (RGR) by 60% in the absence of NaCl but by only 34% in plants treated also with NaCl. This effect was linked to a reduction in Zn tissue concentrations, as well as to overall protective effects on various stages in the photosynthetic pathway. Thus, at 85 mM NaCl plants were able to maintain higher net photosynthesis (AN) than in the absence of added NaCl, although there were no differences in stomatal conductance (gs). This contributed to preserving the trade-off between CO2 acquisition and water loss, as indicated by higher intrinsic water use efficiency (iWUE). Hence, AN differences were ascribed to limitation in the RuBisCO carboxylation, manifested as higher intercellular CO2 concentration (Ci), together with dysfunction of PSII photochemistry (in term of light harvest and energy excess dissipation), as indicated by higher chronic photoinhibition percentages and variations in the photosynthetic pigment profiles in presence of Zn under non-saline conditions.

Tolerance, toxicity and transport of Cd and Zn in Populus trichocarpa

Metal inputs to terrestrial ecosystems are of great concern due their toxicity to biota, especially for elements with no biological function such as cadmium. Fast-growing trees such as poplars may have potential in phytoremediation schemes. We assessed accumulation, metal partitioning, gene expression (Pt-HMA4) and overall tolerance to, and interaction between, cadmium (Cd) and zinc (Zn) in Populus trichocarpa ‘Trichobel’. We predicted that Zn would have an antagonistic effect in Cd accumulation and anticipated some level of tolerance to these metals. Poplars were grown in sandy substrate under different metal applications, ranging from 1 to 243 mg kg−1 Cd; or 30 to 7290 mg kg−1 Zn; and also two combined treatments: 27 mg kg−1 Cd with 90 or 270 mg kg−1 Zn. Growth parameters and metal contents in shoots and roots were determined. Transcriptional levels of the Pt-HMA4 gene were assessed in roots and leaves. P. trichocarpa showed a surprisingly high tolerance to Cd, with root biomass being affected only at the highest doses applied. Metals accumulated mainly in roots (up to 6537 mg kg−1 Cd and 21,500 mg kg-1 Zn), root-to-shoot translocation peaked at the 9 mg kg−1 dose for Cd (41%) and 90 mg kg−1 for Zn (40%). At high Cd/Zn applications, expression of Pt-HMA4 in roots decreased significantly. Contrary to the initial presumption, Zn addition increased Cd uptake, reaching hyperaccumulator-like concentrations in shoots (≥100 mg kg−1 Cd). Differential root-to-shoot partitioning has a major role in Cd tolerance in P. trichocarpa; partly by down-regulating the Pt-HMA4 gene in roots. Zn addition promoted high Cd uptake without any detriment to plant growth. P. trichocarpa was tolerant to extreme Cd concentrations, offering a great potential to be used in phytoremediation techniques for stabilization/extraction of Cd from soils contaminated by both Cd and Zn.

Integrative Conjugative Element ICEHs1 Encodes for Antimicrobial Resistance and Metal Tolerance in Histophilus somni.

The objectives of this study were to determine antimicrobial resistance and metal tolerance, and identify associated genes and mobile genetic elements in clinical strains of Histophilus somni isolated from feedlot cattle in Alberta during years 2012–2016 (contemporary isolates, n = 63) and years 1980–1990 (historical isolates, n = 31). Comparison of antimicrobial resistance (AMR) showed a significant increase in resistance among contemporary isolates compared to historical isolates (P < 0.001). Tolerance to copper (Cu) and zinc (Zn) concentrations above 1 mM was observed in 68 and 52% of the contemporary isolates, respectively. The tet(H) gene associated with oxytetracycline resistance and multicopper oxidase (mco) and cation efflux (czcD) genes associated with Cu and Zn tolerance were identified. An integrative conjugative element; ICEHs1, was identified in whole genome sequences of strains resistant to oxytetracycline, which had Cu and Zn minimum inhibitory concentrations (MIC) >1 mM. The length of ICEHs1 was 64,932 bp and it contained 83 genes, including tetracycline resistance gene tetH, a multidrug efflux pump gene ebrB, and metal tolerance genes mco, czcD, and acr3. Comparative genomics of ICEs revealed that ICEHs1 shares high homology with previously described ICEs of Histophilus somni, Pasteurella multocida, and Mannheimia haemolytica. The ICEHs1 is an active element capable of intra- and inter-genus transfer as demonstrated by successful transfer to H. somni and P. multocida recipients. All isolates carrying ICEHs1 were resistant to tetracycline, a commonly used antibiotic in feedlots, and had Cu and Zn MIC higher than 1 mM. Since Cu and Zn are routinely used in feedlots, there is the possibility of co-selection of AMR in H. somni due to selection pressure created by Cu and Zn. Based on results of in-vitro conjugation experiments, ICEHs1 mediated transmission of antimicrobial and metal resistance genes is possible between BRD pathogens in the respiratory tract, potentially undermining treatment options available for histophilosis and BRD.

The Aspergillus fumigatus transcription factor AceA is involved not only in Cu but also in Zn detoxification through regulating transporters CrpA and ZrcA.

Copper (Cu) and zinc (Zn) are essential nutrients for both pathogens and hosts; however, their excess accumulation is toxic for all cells. The Aspergillus transcription factor AceA has been identified having function for Cu detoxification through up-regulation of the expression of the P-type ATPase, CrpA. Here, we demonstrate that Aspergillus fumigatus CrpA is involved in both Cu and Zn detoxification and a putative metallothionein AfCrdA plays a major function in Cu detoxification, and a putative transporter AfZrcA has a dominant role in Zn detoxification, but all three members are transcriptionally dependent on AfAceA. Moreover, the Cys, RGHR, and KGRP motifs in the conserved N-terminus of AfAceA are essential, but not sufficient for AfAceA-mediated Cu and Zn tolerance. Our findings suggest that fungal pathogens have developed very precise systems with overlapping machinery to respond to the two different metal stressors. Meanwhile, there is separate specific machinery for Zn detoxification in response to high environmental Zn. Importantly, virulence testing demonstrated that the conserved Cu and Zn detoxification-related Cys residues in AfAceA have key roles in pathogenesis. Therefore, these findings will broaden the current understanding of the adaption of saprophytic fungi to Cu and Zn stress and their survival in hosts and other environmental niches.

Hemin Through the Heme Oxygenase 1/Ferrous Iron, Carbon Monoxide System Involved in Zinc Tolerance in Oryza Sativa L.

Several pharmacological experiments were performed to investigate the possible roles of heme oxygenase 1 (HO1) and its catalytic products on hemin-induced attenuation of excess zinc toxicity. The results showed that hemin markedly reduced concentrations of zinc in rice seedlings (including roots and shoots); this effect may be achieved by down-regulating the relative expression of OsZIP1, OsZIP3, OsZIP7, and OsZIP8. However, zinc protoporphyrin IX (Znpp, an HO1 inhibitor) application could reverse hemin-induced decreases in zinc accumulation and upregulate the relative expression of the above four genes. These results showed that HO1 is indeed involved in the mitigation of zinc stress. The following experiments were conducted to illustrate the contributive effect of different hemin by-products in the hemin-mediated attenuating effect. The results showed that bilirubin (BR) does not have any alleviative effect, whereas carbon monoxide (CO) has a similar effect as hemin; the alleviative effect of ferrous ion (Fe2+) is even better than that of hemin. Altogether, these findings suggested that hemin, through the HO1/Fe2+, CO system, may reduce zinc accumulation in rice seedlings and ultimately strengthen Zn tolerance of rice seedlings.

Draft Genome Sequence of Bacillus subtilis 2C-9B, a Strain with Biocontrol Potential against Chili Pepper Root Pathogens and Tolerance to Pb and Zn.

ABSTRACTBacillus subtilis 2C-9B, obtained from the rhizosphere of wild grass, exhibits inhibition against root rot causal pathogens in Capsicum annuum, Pb and Zn tolerance, and plant growth promotion in medium supplemented with Pb. The genome of B. subtilis 2C-9B was sequenced and the draft genome assembled, with a length of 4,215,855 bp and 4,723 coding genes.