AtPDR12 contributes to lead resistance in Arabidopsis

Arabidopsis (Arabidopsis thaliana) contains about 130 ATP-binding cassette (ABC) proteins, which are likely to contribute to the transport of diverse materials, including toxic substances. However, the substrates of ABC transporters remain unknown in most cases. We tested which ABC transporter is involved in detoxification of lead [Pb(II)]. Among the many tested, we found that the message level of only AtPDR12 increased in both shoots and roots of Pb(II)-treated Arabidopsis, suggesting that it may be involved in the detoxification of Pb(II). AtPDR12-knockout plants (atpdr12) were used to further test this possibility. In Pb(II)-containing medium, atpdr12 plants grew less well and had higher Pb contents than those of wild-type plants. In contrast, AtPDR12-overexpressing Arabidopsis plants were more resistant to Pb(II) and had lower Pb contents than wild-type plants. The mutant phenotypes and their Pb contents, as well as the localization of the GFP:AtPDR12 fusion protein at the plasma membrane, suggest that AtPDR12 functions as a pump to exclude Pb(II) and/or Pb(II)-containing toxic compounds from the cytoplasm. Inhibition of glutathione synthesis by addition of buthionine sulfoximine to the growth medium exacerbated the Pb(II)-sensitive phenotype of atpdr12 plants, consistent with a glutathione-dependent detoxification mechanism operating in parallel with an AtPDR12-dependent mechanism. Thus, we propose that AtPDR12 is an ABC transporter that contributes to Pb(II) resistance in Arabidopsis.     

Pathogen-responsive expression of a putative ATP-binding cassette transporter gene conferring resistance to the diterpenoid sclareol is regulated by multiple defense signaling pathways in Arabidopsis

The ATP-binding cassette (ABC) transporters are encoded by large gene families in plants. Although these proteins are potentially involved in a number of diverse plant processes, currently, very little is known about their actual functions. In this paper, through a cDNA microarray screening of anonymous cDNA clones from a subtractive library, we identified an Arabidopsis gene (AtPDR12) putatively encoding a member of the pleiotropic drug resistance (PDR) subfamily of ABC transporters. AtPDR12 displayed distinct induction profiles after inoculation of plants with compatible and incompatible fungal pathogens and treatments with salicylic acid, ethylene, or methyl jasmonate. Analysis of AtPDR12 expression in a number of Arabidopsis defense signaling mutants further revealed that salicylic acid accumulation, NPR1 function, and sensitivity to jasmonates and ethylene were all required for pathogen-responsive expression of AtPDR12. Germination assays using seeds from an AtPDR12 insertion line in the presence of sclareol resulted in lower germination rates and much stronger inhibition of root elongation in the AtPDR12 insertion line than in wild-type plants. These results suggest that AtPDR12 may be functionally related to the previously identified ABC transporters SpTUR2 and NpABC1, which transport sclareol. Our data also point to a potential role for terpenoids in the Arabidopsis defensive armory.     

Identification of Natural Diterpenes that Inhibit Bacterial Wilt Disease in Tobacco, Tomato and Arabidopsis

The soil-borne bacterial pathogen Ralstonia solanacearum invades a broad range of plants through their roots, resulting in wilting of the plant, but no effective protection against this disease has been developed. Two bacterial wilt disease-inhibiting compounds were biochemically isolated from tobacco and identified as sclareol and cis-abienol, labdane-type diterpenes. When exogenously applied to their roots, sclareol and cis-abienol inhibited wilt disease in tobacco, tomato and Arabidopsis plants without exhibiting any antibacterial activity. Microarray analysis identified many sclareol-responsive genes in Arabidopsis roots, including genes encoding or with a role in ATP-binding cassette (ABC) transporters, and biosynthesis and signaling of defense-related molecules and mitogen-activated protein kinase (MAPK) cascade components. Inhibition of wilt disease by sclareol was attenuated in Arabidopsis mutants defective in the ABC transporter AtPDR12, the MAPK MPK3, and ethylene and abscisic acid signaling pathways, and also in transgenic tobacco plants with reduced expression of NtPDR1, a tobacco homolog of AtPDR12. These results suggest that multiple host factors are involved in the inhibition of bacterial wilt disease by sclareol-related compounds.     

Arabidopsis desaturase 2 gene is involved in the regulation of cadmium and lead resistance

Aims

It was shown previously that Arabidopsis (Arabidopsis thaliana) desaturase 2 (ADS2) cDNA was isolated and it was shown that the expression of ADS2 was organ-dependent and up-regulated by low temperature. However, little is known about the role of ADS2 gene in heavy metal resistance in plants. In this study, we showed that ADS2 gene is involved in the regulation of cadmium (Cd) and lead (Pb) resistance.

Methods

For heavy metal resistance tests, seeds were germinated and grown on 1/2 MS media supplemented with the indicated concentrations of metal ions. To quantify root length, plants were grown vertically in plates. For heavy metal treatments, two-week old wild-type seedlings grown on MS media were treated with cadmium (Cd) or lead (Pb) for 24 h, and then sampled for metal content measurement and qPCR analysis.

Results

ADS2 was strongly repressed by Cd(II), and ads2-1 mutant plants showed increased Cd(II) resistance. A lower Cd content was detected in ads2-1 plants than in wild-type plants subjected to Cd(II) treatment, which was associated with activation in expression of AtPDR8 gene, a pump excluding Cd(II) and/or Cd(II)-containing toxic compounds from the cytoplasm, suggesting that ADS2-mediated Cd(II) resistance is AtPDR8 dependent. We also found that ads2-1 plants showed increased Pb(II) sensitivity, and ADS2 was strongly repressed by hydrogen peroxide (H₂O₂) but not by Pb(II). The ads2-1 mutant showed increased sensitivity to oxidative stresses mediated by H₂O₂ and paraquat, and higher levels of H₂O₂ accumulation were observed in leaves of ads2-1 plants than those of wild-type plants when subjected to Pb(II) and H₂O₂, indicating that ADS2 mediates Pb(II) resistance indirectly by impaired ROS scavenging.

Conclusions

ADS2 gene mediates Cd(II) and Pb(II) resistance, at least in part, through two distinct mechanisms, an AtPDR8-dependent mechanism and a ROS detoxification system-mediated mechanism, respectively.     

Expression of a truncated tobacco NtCBP4 channel in transgenic plants and disruption of the homologous Arabidopsis CNGC1 gene confer Pb2+ tolerance

Recently we reported on a plasma membrane tobacco protein (designated NtCBP4) that binds calmodulin. When overexpressed in transgenic plants, NtCBP4 confers Pb2+ hypersensitivity associated with enhanced accumulation of this toxic metal. To further investigate possible modulation of Pb2+ tolerance in plants, we prepared transgenic plants that express a truncated version of this protein (designated NtCBP4DeltaC) from which its C-terminal, with the calmodulin-binding domain and part of the putative cyclic nucleotide-binding domain, was removed. In contrast to the phenotype of transgenic plants expressing the full-length gene, transgenic plants expressing the truncated gene showed improved tolerance to Pb2+, in addition to attenuated accumulation of this metal. Furthermore, disruption by T-DNA insertion mutagenesis of the Arabidopsis CNGC1 gene, which encodes a homologous protein, also conferred Pb2+ tolerance. We suggest that NtCBP4 and AtCNGC1 are components of a transport pathway responsible for Pb2+ entry into plant cells.     

A comparative proteomics approach to detect unintended effects in transgenic Arabidopsis

Recently, as part of biosafety assessments, unintended effects have been given much attention. In this study, we applied a proteomics approach to elucidate the unintended effects of random T-DNA insertion in transgenic plants. Separated proteins extracted from 12 transgenic Arabidopsis thaliana with different T-DNA insertion sites and from wild-type (ecotype Col-o) were analyzed. In the transgenic plants, 102 significantly altered protein spots were detected, in which 59 were up-regulated and 43 down-regulated. MALDI-TOF MS analysis showed that most of these expression level-altered proteins were involved in energy transfer, oxidative respiration and photosynthesis. However, none of these proteins was a toxic protein or allergen. Using plants with or without cold treatment, a natural environmental stress, as controls, we found that the number of the altered proteins was even less in those transgenic plants than those triggered by the cold treatment, suggesting that the transgenic events had a weaker impact on the plants than the environmental stresses. Interestingly,the phosphinothricin acetyl transferase (PAT), the BAR-encoded protein, was detected in nine out of twelve different T-DNA insertion lines at five different insertion sites. These data suggest that the most significant impact of transgenic events on the host plants is from the transgene itself, i.e., from the predictable intended effects, rather than unintended effects. This study also suggests that the proteomics approach has the potential to detect the unintended effects in transgenic plants.     

A comparative proteomics approach to detect unintended effects in transgenic Arabidopsis

Recently, as part of biosafety assessments, unintended effects have been given much attention. In this study, we applied a proteomics approach to elucidate the unintended effects of random T-DNA insertion in transgenic plants. Separated proteins extracted from 12 transgenic Arabidopsis thaliana with different T-DNA insertion sites and from wild-type （ecotype Col-o） were analyzed. In the transgenic plants, 102 significantly altered protein spots were detected, in which 59 were up-regulated and 43 down-regulated. MALDI-TOF MS analysis showed that most of these expression level-altered proteins were involved in energy transfer, oxidative respiration and photosynthesis. However, none of these proteins was a toxic protein or allergen. Using plants with or without cold treatment, a natural environmental stress, as controls, we found that the number of the altered proteins was even less in those transgenic plants than those triggered by the cold treatment, suggesting that the transgenic events had a weaker impact on the plants than the environmental stresses. Interestingly, the phosphinothricin acetyl transferase （PAT）, the BAR-encoded protein, was detected in nine out of twelve different T-DNA insertion lines at five different insertion sites. These data suggest that the most significant impact of transgenic events on the host plants is from the transgene itself, i.e., from the predictable intended effects, rather than unintended effects. This study also suggests that the proteomics approach has the potential to detect the unintended effects in transgenic plants.     

Engineering tolerance and accumulation of lead and cadmium in transgenic plants

We have studied the utility of the yeast protein YCF1, which detoxifies cadmium by transporting it into vacuoles, for the remediation of lead and cadmium contamination. We found that the yeast YCF1-deletion mutant DTY167 was hypersensitive to Pb(II) as compared with wild-type yeast. DTY167 cells overexpressing YCF1 were more resistant to Pb(II) and Cd(II) than were wild-type cells, and accumulated more lead and cadmium. Analysis of transgenic Arabidopsis thaliana plants overexpressing YCF1 showed that YCF1 is functionally active and that the plants have enhanced tolerance of Pb(II) and Cd(II) and accumulated greater amounts of these metals. These results suggest that transgenic plants expressing YCF1 may be useful for phytoremediation of lead and cadmium.     

Characterization of an Arabidopsis cadmium-resistant mutant <Emphasis Type="Italic">cdr3</Emphasis>-<Emphasis Type="Italic">1D</Emphasis> reveals a link between heavy metal resistance as well as seed development and flowering

A lot of studies have identified many key genes involved in heavy metal detoxification and tolerance in plants; however, our understanding of its molecular mechanisms is far from complete. To gain insight into the regulatory mechanisms for heavy metal detoxification and tolerance, we performed a mutant screen for identifying Arabidopsis (Arabidopsis thaliana) cadmium (Cd)-resistant mutants. A Cd-resistant mutant cdr3-1D (c a d mium-r esistant) was isolated because of its increased root growth and fresh weight in Cd stress, and genetic analysis showed that cdr3-1D is a single dominant nuclear mutation. Compared with the wild type, the cdr3-1D mutant was more resistant to heavy metals Cd, Pb, and copper as well as hydrogen peroxide. Moreover, we also observed that seeds of the cdr3-1D mutant were larger than those of wild type, and that cdr3-1D displayed early flowering compared with wild type. A lower Cd/Pb content was detected in cdr3-1D plants than in wild-type plants when subjected to Cd/Pb treatment, which was associated, at least in part, with increase of expression of AtPDR8/AtPDR12, a pump excluding Cd/Pb and/or Cd/Pb-containing toxic compounds from the cytoplasm, respectively. In addition, enhanced Cd/Pb resistance of the cdr3-1D mutant was partially glutathione (GSH) dependent, which was related to increase of expression of GSH1 gene involved in GSH synthesis and consequently increased GSH content. Taken together, our results provide genetic evidence indicating that CDR3 is involved in the regulation of heavy metal resistance as well as seed development and flowering.     

Loss of the vacuolar cation channel, AtTPC1, does not impair Ca2+ signals induced by abiotic and biotic stresses.

The putative two-pore Ca(2+) channel TPC1 has been suggested to be involved in responses to abiotic and biotic stresses. We show that AtTPC1 co-localizes with the K(+)-selective channel AtTPK1 in the vacuolar membrane. Loss of AtTPC1 abolished Ca(2+)-activated slow vacuolar (SV) currents, which were increased in AtTPC1-over-expressing Arabidopsis compared to the wild-type. A Ca(2+)-insensitive vacuolar cation channel, as yet uncharacterized, could be resolved in tpc1-2 knockout plants. The kinetics of ABA- and CO(2)-induced stomatal closure were similar in wild-type and tpc1-2 knockout plants, excluding a role of SV channels in guard-cell signalling in response to these physiological stimuli. ABA-, K(+)-, and Ca(2+)-dependent root growth phenotypes were not changed in tpc1-2 compared to wild-type plants. Given the permeability of SV channels to mono- and divalent cations, the question arises as to whether TPC1 in vivo represents a pathway for Ca(2+) entry into the cytosol. Ca(2+) responses as measured in aequorin-expressing wild-type, tpc1-2 knockout and TPC1-over-expressing plants disprove a contribution of TPC1 to any of the stimulus-induced Ca(2+) signals tested, including abiotic stresses (cold, hyperosmotic, salt and oxidative), elevation in extracellular Ca(2+) concentration and biotic factors (elf18, flg22). In good agreement, stimulus- and Ca(2+)-dependent gene activation was not affected by alterations in TPC1 expression. Together with our finding that the loss of TPC1 did not change the activity of hyperpolarization-activated Ca(2+)-permeable channels in the plasma membrane, we conclude that TPC1, under physiological conditions, functions as a vacuolar cation channel without a major impact on cytosolic Ca(2+) homeostasis.     

Functional expression of a bacterial heavy metal transporter in Arabidopsis enhances resistance to and decreases uptake of heavy metals

Large parts of agricultural soil are contaminated with lead (Pb) and cadmium (Cd). Although most environments are not heavily contaminated, the low levels observed nonetheless pose a high risk of heavy metal accumulation in the food chain. Therefore, approaches to develop plants with reduced heavy metal uptake are important. Recently, many transgenic plants with increased heavy metal resistance and uptake of heavy metals were developed for the purpose of phytoremediation. However, to reduce heavy metal in the food chain, plants that transfer less heavy metals to the shoot are required. We tested whether an Escherichia coli gene, ZntA, which encodes a Pb(II)/Cd(II)/Zn(II) pump, could be useful for developing plants with reduced heavy metal content. Yeast cells transformed with this gene had improved resistance to Pb(II) and Cd(II). In Arabidopsis plants transformed with ZntA, ZntA was localized at the plasma membrane and improved the resistance of the plants to Pb(II) and Cd(II). The shoots of the transgenic plants had decreased Pb and Cd content. Moreover, the transgenic protoplasts showed lower accumulation of Cd and faster release of preloaded Cd than wild-type protoplasts. These results show that a bacterial transporter gene, ZntA, can be functionally expressed in plant cells, and that that it may be useful for the development of crop plants that are safe from heavy metal contamination.