A heavy metal-associated protein (AcHMA1) from the halophyte, Atriplex canescens (Pursh) Nutt., confers tolerance to iron and other abiotic stresses when expressed in Saccharomyces cerevisiae.
ABSTRACT: Many heavy metals are essential for metabolic processes, but are toxic at elevated levels. Metal tolerance proteins provide resistance to this toxicity. In this study, we identified and characterized a heavy metal-associated protein, AcHMA1, from the halophyte, Atriplex canescens. Sequence analysis has revealed that AcHMA1 contains two heavy metal binding domains. Treatments with metals (Fe, Cu, Ni, Cd or Pb), PEG6000 and NaHCO3 highly induced AcHMA1 expression in A. canescens, whereas NaCl and low temperature decreased its expression. The role of AcHMA1 in metal stress tolerance was examined using a yeast expression system. Expression of the AcHMA1 gene significantly increased the ability of yeast cells to adapt to and recover from exposure to excess iron. AcHMA1 expression also provided salt, alkaline, osmotic and oxidant stress tolerance in yeast cells. Finally, subcellular localization of an AcHMA1/GFP fusion protein expressed in tobacco cells showed that AcHMA1 was localized in the plasma membrane. Thus, our results suggest that AcHMA1 encodes a membrane-localized metal tolerance protein that mediates the detoxification of iron in eukaryotes. Furthermore, AcHMA1 also participates in the response to abiotic stress.
Project description:Plant productivity is limited by salinity stress, both in natural and agricultural systems. Identification of salt stress-related genes from halophyte can provide insights into mechanisms of salt stress tolerance in plants. Atriplex canescens is a xero-halophyte that exhibits optimum growth in the presence of 400 mM NaCl. A cDNA library derived from highly salt-treated A. canescens plants was constructed based on a yeast expression system. A total of 53 transgenic yeast clones expressing enhanced salt tolerance were selected from 10? transformants. Their plasmids were sequenced and the gene characteristics were annotated using a BLASTX search. Retransformation of yeast cells with the selected plasmids conferred salt tolerance to the resulting transformants. The expression patterns of 28 of these stress-related genes were further investigated in A. canescens leaves by quantitative reverse transcription-PCR. In this study, we provided a rapid and robust assay system for large-scale screening of genes for varied abiotic stress tolerance with high efficiency in A. canescens.
Project description:BACKGROUND:Atriplex canescens is a typical C4 secretohalophyte with salt bladders on the leaves. Accumulating excessive Na+ in tissues and salt bladders, maintaining intracellular K+ homeostasis and increasing leaf organic solutes are crucial for A. canescens survival in harsh saline environments, and enhanced photosynthetic activity and water balance promote its adaptation to salt. However, the molecular basis for these physiological mechanisms is poorly understood. Four-week-old A. canescens seedlings were treated with 100 mM NaCl for 6 and 24 h, and differentially expressed genes in leaves and roots were identified, respectively, with Illumina sequencing. RESULTS:In A. canescens treated with 100 mM NaCl, the transcripts of genes encoding transporters/channels for important nutrient elements, which affect growth under salinity, significantly increased, and genes involved in exclusion, uptake and vacuolar compartmentalization of Na+ in leaves might play vital roles in Na+ accumulation in salt bladders. Moreover, NaCl treatment upregulated the transcripts of key genes related to leaf organic osmolytes synthesis, which are conducive to osmotic adjustment. Correspondingly, aquaporin-encoding genes in leaves showed increased transcripts under NaCl treatment, which might facilitate water balance maintenance of A. canescens seedlings in a low water potential condition. Additionally, the transcripts of many genes involved in photosynthetic electron transport and the C4 pathway was rapidly induced, while other genes related to chlorophyll biosynthesis, electron transport and C3 carbon fixation were later upregulated by 100 mM NaCl. CONCLUSIONS:We identified many important candidate genes involved in the primary physiological mechanisms of A. canescens salt tolerance. This study provides excellent gene resources for genetic improvement of salt tolerance of important crops and forages.
Project description:Little information is available on gene expression profiling of halophyte A. canescens. To elucidate the molecular mechanism for stress tolerance in A. canescens, a full-length complementary DNA library was generated from A. canescens exposed to 400 mM NaCl, and provided 343 high-quality ESTs. In an evaluation of 343 valid EST sequences in the cDNA library, 197 unigenes were assembled, among which 190 unigenes (83.1% ESTs) were identified according to their significant similarities with proteins of known functions. All the 343 EST sequences have been deposited in the dbEST GenBank under accession numbers JZ535802 to JZ536144. According to Arabidopsis MIPS functional category and GO classifications, we identified 193 unigenes of the 311 annotations EST, representing 72 non-redundant unigenes sharing similarities with genes related to the defense response. The sets of ESTs obtained provide a rich genetic resource and 17 up-regulated genes related to salt stress resistance were identified by qRT-PCR. Six of these genes may contribute crucially to earlier and later stage salt stress resistance. Additionally, among the 343 unigenes sequences, 22 simple sequence repeats (SSRs) were also identified contributing to the study of A. canescens resources.
Project description:Microbial diversity associated with micropropagated Atriplex species was assessed using microscopy, isolate culturing, and sequencing. Light, electron, and confocal microscopy revealed microbial cells in aseptically regenerated leaves and roots. Clone libraries and tag-encoded FLX amplicon pyrosequencing (TEFAP) analysis amplified sequences from callus homologous to diverse fungal and bacterial taxa. Culturing isolated some seed borne endophyte taxa which could be readily propagated apart from the host. Microbial cells were observed within biofilm-like residues associated with plant cell surfaces and intercellular spaces. Various universal primers amplified both plant and microbial sequences, with different primers revealing different patterns of fungal diversity. Bacterial and fungal TEFAP followed by alignment with sequences from curated databases revealed 7 bacterial and 17 ascomycete taxa in A. canescens, and 5 bacterial taxa in A. torreyi. Additional diversity was observed among isolates and clone libraries. Micropropagated Atriplex retains a complex, intimately associated microbiome which includes diverse strains well poised to interact in manners that influence host physiology. Microbiome analysis was facilitated by high throughput sequencing methods, but primer biases continue to limit recovery of diverse sequences from even moderately complex communities.
Project description: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??M 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.
Project description:Metal tolerance proteins (MTPs) are plant divalent cation transporters that play important roles in plant metal tolerance and homeostasis. Poplar is an ideal candidate for the phytoremediation of heavy metals because of its numerous beneficial attributes. However, the definitive phylogeny and heavy metal transport mechanisms of the MTP family in poplar remain unknown. Here, 22 MTP genes in P. trichocarpa were identified and classified into three major clusters and seven groups according to phylogenetic relationships. An evolutionary analysis suggested that PtrMTP genes had undergone gene expansion through tandem or segmental duplication events. Moreover, all PtrMTPs were predicted to localize in the vacuole and/or cell membrane, and contained typical structural features of the MTP family, cation efflux domain. The temporal and spatial expression pattern analysis results indicated the involvement of PtrMTP genes in poplar developmental control. Under heavy metal stress, most of PtrMTP genes were induced by at least two metal ions in roots, stems or leaves. In addition, PtrMTP8.1, PtrMTP9 and PtrMTP10.4 displayed the ability of Mn transport in yeast cells, and PtrMTP6 could transport Co, Fe and Mn. These findings will provide an important foundation to elucidate the biological functions of PtrMTP genes, and especially their role in regulating heavy metal tolerance in poplar.
Project description:For environmental safety, the high concentration of heavy metals in the soil should be removed. Cadmium (Cd), one of the heavy metals polluting the soil while its concentration exceeds 3.4 mg/kg in soil. Potential use of cotton for remediating heavy Cd-polluted soils is available while its molecular mechanisms of Cd tolerance remains unclear in cotton. In this study, transcriptome analysis was used to identify the Cd tolerance genes and their potential mechanism in cotton. Finally 4,627 differentially expressed genes (DEGs) in the root, 3,022 DEGs in the stem and 3,854 DEGs in leaves were identified through RNA-Seq analysis, respectively. These genes contained heavy metal transporter genes (ABC, CDF, HMA, etc.), annexin genes, heat shock genes (HSP) amongst others. Gene ontology (GO) analysis showed that the DEGs were mainly involved in the oxidation-reduction process and metal ion binding. The DEGs mainly enriched in two pathways, the influenza A and the pyruvate pathway. GhHMAD5 protein, containing a heavy-metal domain, was identified in the pathway to transport or to detoxify the heavy ion. GhHMAD5-overexpressed plants of Arabidopsis thaliana showed the longer roots compared with the control. Meanwhile, GhHMAD5-silenced cotton plants showed more sensitive to Cd stress compared with the control. The results indicated that GhHMAD5 gene is remarkably involved in Cd tolerance, which gives us a preliminary understanding of Cd tolerance mechanisms in upland cotton. Overall, this study provides valuable information for the use of cotton to remediate the soil polluted with heavy metals. Overall design: leaf, stem and whole root of three leaf-heart cotton seedlings were collected for 9h under 4 mM CdCl2 solution.
Project description:In response to heavy metal stress, plants and certain fungi, such as the fission yeast Schizosaccharomyces pombe, synthesize small metal-binding peptides known as phytochelatins. We have identified a cadmium sensitive S. pombe mutant deficient in the accumulation of a sulfide-containing phytochelatin-cadmium complex, and have isolated the gene, designated hmt1, that complements this mutant. The deduced protein sequence of the hmt1 gene product shares sequence identity with the family of ABC (ATP-binding cassette)-type transport proteins which includes the mammalian P-glycoproteins and CFTR, suggesting that the encoded product is an integral membrane protein. Analysis of fractionated fission yeast cell components indicates that the HMT1 polypeptide is associated with the vacuolar membrane. Additionally, fission yeast strains harboring an hmt1-expressing multicopy plasmid exhibit enhanced metal tolerance along with a higher intracellular level of cadmium, implying a relationship between HMT1 mediated transport and compartmentalization of heavy metals. This suggests that tissue-specific overproduction of a functional hmt1 product in transgenic plants might be a means to alter the tissue localization of these elements, such as for sequestering heavy metals away from consumable parts of crop plants.
Project description:Metal tolerance proteins (MTPs) are a gene family of cation efflux transporters that occur widely in plants and might serve an essential role in metal homeostasis and tolerance. Our research describes the identification, characterization, and localization of OsMTP11, a member of the MTP family from rice. OsMTP11 was expressed constitutively and universally in different tissues in rice plant. Heterologous expression in yeast showed that OsMTP11 complemented the hypersensitivity of mutant strains to Mn, and also complemented yeast mutants to other metals, including Co and Ni. Real time RT-PCR analysis demonstrated OsMTP11 expression was substantially enhanced following 4 h under Cd, Zn, Ni, and Mn treatments, suggesting possible roles of OsMTP11 involvement in heavy metal stress responses. Promoter analysis by transgenic assays with GUS as a reporter gene and mRNA in situ hybridization experiments showed that OsMTP11 was expressed specifically in conducting tissues in rice. DNA methylation assays of genomic DNA in rice treated with Cd, Zn, Ni, and Mn revealed that decreased DNA methylation levels were present in the OsMTP11 promoter region, which was consistent with OsMTP11 induced-expression patterns resulting from heavy metal stress. This result suggested that DNA methylation is one of major factors regulating expression of OsMTP11 through epigenetic mechanisms. OsMTP11 fused to green fluorescent protein (GFP) localized to the entire onion epidermal cell cytoplasm, while vacuolar membrane exhibited increased GFP signals, consistent with an OsMTP11 function in cation sequestration. Our results indicated that OsMTP11 might play vital roles in Mn and other heavy metal transportation in rice.
Project description:This paper unravels the occurrence of plasmid-mediated antibiotic resistance in association with tolerance to heavy metals among clinically relevant bacteria isolated from sewage wastewater. The bacteria isolated were identified following conventional phenotypic and/or molecular methods, and were subjected to multiple-antibiotic resistance (MAR) profiling. The isolates were tested against the heavy metals Hg2+, Cd2+, Cr2+ and Cu2+. SDS-PAGE and agarose gel electrophoretic analyses were performed, respectively, for the characterization of heavy metal stress protein and R-plasmid among the isolated bacteria. Principal component analysis was applied in determining bacterial resistance to antibiotics and heavy metals. Both lactose-fermenting ( Escherichia coli ) and non-fermenting ( Acinetobacter baumannii and Pseudomonas putida ) Gram-negative bacterial strains were procured, and showed MAR phenotypes with respect to three or more antibiotics, along with resistance to the heavy metals Hg2+, Cd2+, Cr2+ and Cu2+. The Gram-positive bacteria, Enterococcus faecalis , isolated had 'ampicillin-kanamycin-nalidixic acid' resistance. The bacterial isolates had MAR indices of 0.3-0.9, indicating their ( E. faecalis , E. coli , A. baumannii and P. putida ) origin from niches with high antibiotic pollution and human faecal contamination. The Gram-negative bacteria isolated contained a single plasmid (?54?kb) conferring multiple antibiotic resistance, which was linked to heavy metal tolerance; the SDS-PAGE analysis demonstrated the expression of heavy metal stress proteins (?59 and ?10?kDa) in wastewater bacteria with a Cd2+ stressor. The study results grant an insight into the co-occurrence of antibiotic resistance and heavy metal tolerance among clinically relevant bacteria in sewage wastewater, prompting an intense health impact over antibiotic usage.