Richard B. Meagher

Phytoremediation of toxic elemental and organic pollutants

Phytoremediation is the use of plants to extract, sequester, and/or detoxify pollutants. Phytoremediation is widely viewed as the ecologically responsible alternative to the environmentally destructive physical remediation methods currently practiced. Plants have many endogenous genetic, biochemical, and physiological properties that make them ideal agents for soil and water remediation. Significant progress has been made in recent years in developing native or genetically modified plants for the remediation of environmental contaminants. Because elements are immutable, phytoremediation strategies for radionuclide and heavy metal pollutants focus on hyperaccumulation above-ground. In contrast, organic pollutants can potentially be completely mineralized by plants.

Engineering tolerance and hyperaccumulation of arsenic in plants by combining arsenate reductase and γ-glutamylcysteine synthetase expression

We have developed a genetics-based phytoremediation strategy for arsenic in which the oxyanion arsenate is transported aboveground, reduced to arsenite, and sequestered in thiol–peptide complexes. The Escherichia coli arsC gene encodes arsenate reductase (ArsC), which catalyzes the glutathione (GSH)-coupled electrochemical reduction of arsenate to the more toxic arsenite. Arabidopsis thaliana plants transformed with the arsC gene expressed from a light-induced soybean rubisco promoter (SRS1p) strongly express ArsC protein in leaves, but not roots, and were consequently hypersensitive to arsenate. Arabidopsis plants expressing the E. coli gene encoding γ-glutamylcysteine synthetase (γ-ECS) from a strong constitutive actin promoter (ACT2p) were moderately tolerant to arsenic compared with wild type. However, plants expressing SRS1p/ArsC and ACT2p/γ-ECS together showed substantially greater arsenic tolerance than γ-ECS or wild-type plants. When grown on arsenic, these plants accumulated 4- to 17-fold greater fresh shoot weight and accumulated 2- to 3-fold more arsenic per gram of tissue than wild type or plants expressing γ-ECS or ArsC alone. This arsenic remediation strategy should be applicable to a wide variety of plant species.

Phytodetoxification of hazardous organomercurials by genetically engineered plants.

Methylmercury is a highly toxic, organic derivative found in mercury-polluted wetlands and coastal sediments worldwide. Though commonly present at low concentrations in the substrate, methylmercury can biomagnify to concentrations that poison predatory animals and humans. In the interest of developing an in situ detoxification strategy, a model plant system was transformed with bacterial genes (merA for mercuric reductase and merB for organomercurial lyase) for an organic mercury detoxification pathway. Arabidopsis thaliana plants expressing both genes grow on 50-fold higher methylmercury concentrations than wild-type plants and up to 10-fold higher concentrations than plants that express merB alone. An in vivo assay demonstrated that both transgenes are required for plants to detoxify organic mercury by converting it to volatile and much less toxic elemental mercury.

Repression of Flowering in Arabidopsis Requires Activation of FLOWERING LOCUS C Expression by the Histone Variant H2A.Z

The histone variant H2A.Z has been implicated in numerous chromatin-mediated processes, including transcriptional activation, euchromatin maintenance, and heterochromatin formation. In yeast and humans, H2A.Z is deposited into chromatin by a conserved protein complex known as SWR1 and SRCAP, respectively. Here, we show that mutations in the Arabidopsis thaliana homologs of two components of this complex, ACTIN-RELATED PROTEIN6 (ARP6) and PHOTOPERIOD-INDEPENDENT EARLY FLOWERING1 (PIE1), produce similar developmental phenotypes and result in the misregulation of a common set of genes. Using H2A.Z-specific antibodies, we demonstrate that ARP6 and PIE1 are required for the deposition of H2A.Z at multiple loci, including the FLOWERING LOCUS C (FLC) gene, a central repressor of the transition to flowering. Loss of H2A.Z from chromatin in arp6 and pie1 mutants results in reduced FLC expression and premature flowering, indicating that this histone variant is required for high-level expression of FLC. In addition to defining a novel mechanism for the regulation of FLC expression, these results support the existence of a SWR1-like complex in Arabidopsis and show that H2A.Z can potentiate transcriptional activation in plants. The finding that H2A.Z remains associated with chromatin throughout mitosis suggests that it may serve an epigenetic memory function by marking active genes and poising silenced genes for reactivation.

Protein expression in E. coli minicells by recombinant plasmids

The polypeptides synthesized in E. coli minicells from recombinant plasmids containing DNA fragments from cauliflower mosaic virus, Drosophila melanogaster, and mouse mitochondria were examined. Molecularly cloned fragments of cauliflower mosaic virus DNA directed the synthesis of high levels of three polypeptides, which were synthesized entirely from within the cloned virus DNA fragments independent of their insertion into the plasmid vehicles. Several fragments of D. melanogaster DNA were capable of initiating polypeptide synthesis; however, termination of these polypeptides was dependent upon the insertion into the plasmid vehicle. The majority of D. melanogaster DNA fragments examined did not direct the detectable synthesis of any polypeptides. Insertion of DNA into the Eco RI site of ColE1 and pSC101 plasmids resulted in the altered expression of plasmid-encoded polypeptides. In the case of ColE1, this site of insertion lies within the colicin E1 structural gene, and insertion of foreign DNA into the site results in the synthesis of an inactive truncated colicin E1 molecule. It is probable that the Eco RI site in pSC101 lies within the structural gene for a polypeptide involved in tetracycline resistance, and insertion of DNA into this site may also result in the synthesis of a truncated or elongated polypeptide.

Actin-organelle interaction: Association with chloroplast in Arabidopsis leaf mesophyll cells

The role of the cytoskeleton in the regulation of chloroplast motility and positioning has been investigated by studying: (1) structural relationship of actin microfilaments, microtubules, and chloroplasts in cryofixed and freeze-substituted leaf cells of Arabidopsis; and (2) the effects of anti-actin (Latrunculin B; LAT-B) and anti-microtubule (Oryzalin) drugs on intracellular distribution of chloroplasts. Immunolabeling of leaf cells with two plant-actin specific antibodies, which react equivalently with all the expressed Arabidopsis actins, revealed two arrangements of actin microfilaments: longitudinal arrays of thick actin bundles and randomly oriented thin actin filaments that extended from the bundles. Chloroplasts were either aligned along the actin bundles or closely associated with the fine filaments. Baskets of actin microfilaments were also observed around the chloroplasts. The leaf cells labeled with an anti-tubulin antibody showed dense transverse arrays of cortical microtubules that exhibited no apparent association with chloroplasts. The application of LAT-B severely disrupted actin filaments and their association with chloroplasts. In addition, LAT-B induced aberrant aggregation of chloroplasts in the mesophyll and bundle sheath cells. Double labeling of LAT-B treated cells with anti-actin and anti-tubulin antibodies revealed that the microtubules in these cells were unaffected. Moreover, depolymerization of microtubules with Oryzalin did not affect the distribution of chloroplasts. These results provide evidence for the involvement of actin, but not tubulin, in the movement and positioning of chloroplasts in leaf cells. We propose that using motor molecules, some chloroplasts migrate along the actin cables directly, while others are pulled along the cables by the fine actin filaments. The baskets of microfilaments may anchor the chloroplasts during streaming and allow control over proper three-dimensional orientation to light.

Comparison of the nucleotide sequence of soybean 18S rRNA with the sequences of other small-subunit rRNAs

SummaryWe present the sequence of the nuclearencoded ribosomal small-subunit RNA from soybean. The soybean 18S rRNA sequence of 1807 nucleotides (nt) is contained in a gene family of approximately 800 closely related members per haploid genome. This sequence is compared with the ribosomal small-subunit RNAs of maize (1805 nt), yeast (1789 nt),Xenopus (1825 nt), rat (1869 nt), andEscherichia coli (1541 nt). Significant sequence homology is observed among the eukaryotic small-subunit rRNAs examined, and some sequence homology is observed between eukaryotic and prokaryotic small-subunit rRNAs. Conserved regions are found to be interspersed among highly diverged sequences. The significance of these comparisons is evaluated using computer simulation of a random sequence model. A tentative model of the secondary structure of soybean 18S rRNA is presented and discussed in the context of the functions of the various conserved regions within the sequence. On the basis of this model, the short basepaired sequences defining the four structural and functional domains of all 18S rRNAs are seen to be well conserved. The potential roles of other conserved soybean 18S rRNA sequences in protein synthesis are discussed.

Phytoremediation of Mercury- and Methylmercury-Polluted Soils Using Genetically Engineered Plants

Inorganic mercury in contaminated soils and sediments is relatively immobile, though biological and chemical processes can transform it to more toxic and bioavailable methylmercury. Methylmercury is neurotoxic to vertebrates and is biomagnified in animal tissues as it is passed from prey to predator. Traditional remediation strategies for mercury contaminated soils are expensive and site-destructive. As an alternative we propose the use of transgenic aquatic, salt marsh, and upland plants to remove available inorganic mercury and methylmercury from contaminated soils and sediments. Plants engineered with a modified bacterial mercuric reductase gene, merA, are capable of converting Hg(II) taken up by roots to the much less toxic Hg(0), which is volatilized from the plant. Plants engineered to express the bacterial organo-mercurial lyase gene, merB, are capable of converting methylmercury taken up by plant roots into sulfhydryl-bound Hg(II). Plants expressing both genes are capable of converting ionic mercu...

Gene structure and molecular analysis of the laccase-like multicopper oxidase (LMCO) gene family in Arabidopsis thaliana

Completed genome sequences have made it clear that multicopper oxidases related to laccase are widely distributed as multigene families in higher plants. Laccase-like multicopper oxidase (LMCO) sequences culled from GenBank and the Arabidopsis thaliana genome, as well as those from several newly cloned genes, were used to construct a gene phylogeny that clearly divided plant LMCOs into six distinct classes, at least three of which predate the evolutionary divergence of angiosperms and gymnosperms. Alignments of the predicted amino acid sequences highlighted regions of variable sequence flanked by the highly conserved copper-binding domains that characterize members of this enzyme family. All of the predicted proteins contained apparent signal sequences. The expression of 13 of the 17 LMCO genes in A. thaliana was assessed in different tissues at various stages of development using RT-PCR. A diversity of expression patterns was demonstrated with some genes being expressed in a constitutive fashion, while others were only expressed in specific tissues at a particular stage of development. Only a few of the LMCO genes were expressed in a pattern that could be considered consistent with a major role for these enzymes in lignin deposition. These results are discussed in the context of other potential physiological functions for plant LMCOs, such as iron metabolism and wound healing.

The Arabidopsis ACT7 Actin Gene Is Expressed in Rapidly Developing Tissues and Responds to Several External Stimuli

ACT7 encodes one of the six distinct and ancient subclasses of actin protein in the complex Arabidopsis actin gene family. We determined the sequence and structure of the Arabidopsis thaliana ACT7 actin gene and investigated its tissue-specific expression and regulation. The ACT7 mRNA levels varied by 128-fold among several different tissues and organs. The highest levels of ACT7 mRNA were found in rapidly expanding vegetative organs, the lowest in pollen. A translational fusion with the 5[prime] end of ACT7 (1.9 kb) joined to the [beta]-glucoronidase reporter gene was strongly and preferentially expressed in all young, developing vegetative tissues of transgenic Arabidopsis plants. ACT7 was the only Arabidopsis actin gene strongly expressed in the hypocotyl and seed coat. Although no [beta]-glucoronidase expression was seen in developing ovules or immature seeds, strong expression was seen in dry seeds and immediately after imbibition in the entire seedling. ACT7 was the only Arabidopsis actin gene to respond strongly to auxin, other hormone treatments, light regime, and wounding, and may be the primary actin gene responding to external stimuli. The ACT7 promoter sequence contains a remarkable number of motifs with sequence similarity to putative phytohormone response elements.