Biomedical subjects

Richard B Meagher

Publications and source records attributed to Richard B Meagher.

28 records · Page 2Linked to original sources

PubMed2003

Arabidopsis actin gene ACT7 plays an essential role in germination and root growth.

Arabidopsis contains eight actin genes. Of these ACT7 is the most strongly expressed in young plant tissues and shows the greatest response to physiological cues. Adult plants homozygous for the act7 mutant alleles show no obvious above-ground phenotypes, which suggests a high degree of functional redundancy among plant actins. However, act7-1 mutant plants are at a strong selective disadvantage when grown in competition with wild-type plants and therefore must have undetected physical defects. The act7-1 and act7-4 alleles contain T-DNA insertions just after the stop codon and within the first intron, respectively. Homozygous mutant seedlings of both alleles showed less than 7% of normal ACT7 protein levels. Mutants displayed delayed and less efficient germination, increased root twisting and waving, and retarded root growth. The act7-4 mutant showed the most dramatic reduction in root growth. The act7-4 root apical cells were not in straight files and contained oblique junctions between cells suggesting a possible role for ACT7 in determining cell polarity. Wild-type root growth was fully restored to the act7-1 mutant by the addition of an exogenous copy of the ACT7 gene. T-DNA insertions just downstream of the major polyadenylation sites (act7-2, act7-3) appeared fully wild type. The act7 mutant phenotypes demonstrate a significant requirement for functional ACT7 protein during root development and explain the strong negative selection component seen for the act7-1 mutant.

Actins↗

PubMed2003

Cell cycle-dependent association of Arabidopsis actin-related proteins AtARP4 and AtARP7 with the nucleus.

Arabidopsis encodes at least eight actin-related proteins (ARPs) most of which have orthologs in other distant organisms. To gain insight into the role of ARPs in plants, we have examined the spatial expression and subcellular distribution of two highly divergent Arabidopsis ARPs, AtARP4 and AtARP7. AtARP4 is a homolog of human BAF53 and yeast Arp4, and AtARP7 is a novel, ancient and plant-specific actin-related protein that is not distinctly related to any known ARPs from other kingdoms. Analysis of both these proteins with AtARP4- and AtARP7-specific antibodies revealed that they were most abundant in young meristematic and floral tissues, but were expressed constitutively in all organs and cell types irrespective of their developmental stage. Immunofluorescence studies showed that both AtARP4 and AtARP7 were localized predominantly to the nucleus during interphase. In mitotic cells lacking a nuclear envelope (e.g. metaphase, anaphase, and early telophase stages), these ARPs were excluded from the condensed chromosomes and dispersed throughout the cytoplasm. In contrast, a putative Arabidopsis histone H2B protein remained associated with the interphase nuclei as well as chromosomes throughout the cell cycle. Based on our results and data on the yeast ortholog of AtARP4, these two nuclear plant ARPs may be involved in the modulation of chromatin structure and transcriptional regulation mainly in interphase cells.

Actins↗

PubMed2003

Expression of mercuric ion reductase in Eastern cottonwood (Populus deltoides) confers mercuric ion reduction and resistance.

Mercury is one of the most hazardous heavy metals and is a particular problem in aquatic ecosystems, where organic mercury is biomagnified in the food chain. Previous studies demonstrated that transgenic model plants expressing a modified mercuric ion reductase gene from bacteria could detoxify mercury by converting the more toxic and reductive ionic form [Hg(II)] to less toxic elemental mercury [Hg(0)]. To further investigate if a genetic engineering approach for mercury phytoremediation can be effective in trees with a greater potential in riparian ecosystems, we generated transgenic Eastern cottonwood (Populus deltoides) trees expressing modified merA9 and merA18 genes. Leaf sections from transgenic plantlets produced adventitious shoots in the presence of 50 microm Hg(II) supplied as HgCl2, which inhibited shoot induction from leaf explants of wild-type plantlets. Transgenic shoots cultured in a medium containing 25 microm Hg(II) showed normal growth and rooted, while wild-type shoots were killed. When the transgenic cottonwood plantlets were exposed to Hg(II), they evolved 2-4-fold the amount of Hg(0) relative to wild-type plantlets. Transgenic merA9 and merA18 plants accumulated significantly higher biomass than control plants on a Georgia Piedmont soil contaminated with 40 p.p.m. Hg(II). Our results indicate that Eastern cottonwood plants expressing the bacterial mercuric ion reductase gene have potential as candidates for in situ remediation of mercury-contaminated soils or wastewater.

Journal Article↗

PubMed2003

Subcellular targeting of methylmercury lyase enhances its specific activity for organic mercury detoxification in plants.

Methylmercury is an environmental pollutant that biomagnifies in the aquatic food chain with severe consequences for humans and other animals. In an effort to remove this toxin in situ, we have been engineering plants that express the bacterial mercury resistance enzymes organomercurial lyase MerB and mercuric ion reductase MerA. In vivo kinetics experiments suggest that the diffusion of hydrophobic organic mercury to MerB limits the rate of the coupled reaction with MerA (Bizily et al., 2000). To optimize reaction kinetics for organic mercury compounds, the merB gene was engineered to target MerB for accumulation in the endoplasmic reticulum and for secretion to the cell wall. Plants expressing the targeted MerB proteins and cytoplasmic MerA are highly resistant to organic mercury and degrade organic mercury at 10 to 70 times higher specific activity than plants with the cytoplasmically distributed wild-type MerB enzyme. MerB protein in endoplasmic reticulum-targeted plants appears to accumulate in large vesicular structures that can be visualized in immunolabeled plant cells. These results suggest that the toxic effects of organic mercury are focused in microenvironments of the secretory pathway, that these hydrophobic compartments provide more favorable reaction conditions for MerB activity, and that moderate increases in targeted MerB expression will lead to significant gains in detoxification. In summary, to maximize phytoremediation efficiency of hydrophobic pollutants in plants, it may be beneficial to target enzymes to specific subcellular environments.

Adaptation, Physiological↗

PubMed2003

Toward detoxifying mercury-polluted aquatic sediments with rice genetically engineered for mercury resistance.

Mercury contamination of soil and water is a serious problem at many sites in the United States and throughout the world. Plant species expressing the bacterial mercuric reductase gene, merA, convert ionic mercury, Hg(II), from growth substrates to the less toxic metallic mercury, Hg(0). This activity confers mercury resistance to plants and removes mercury from the plant and substrates through volatilization. Our goal is to develop plants that intercept and remove Hg(II) from polluted aquatic systems before it can undergo bacterially mediated methylation to the neurotoxic methylmercury. Therefore, the merA gene under the control of a monocot promoter was introduced into Oryza sativa L. (rice) by particle gun bombardment. This is the first monocot and first wetland-adapted species to express the gene. The merA-expressing rice germinated and grew on semisolid growth medium spiked with sufficient Hg(II) to kill the nonengineered (wild-type) controls. To confirm that the resistance mechanism was the conversion of Hg(II) to Hg(0), seedlings of merA-expressing O. sativa were grown in Hg(II)-spiked liquid medium or water-saturated soil media and were shown to volatilize significantly more Hg(0) than wild-type counterparts. Further genetic manipulation could yield plants with increased efficiency to extract soil Hg(II) and volatilize it as Hg(0) or with the novel ability to directly convert methylmercury to Hg(0).

Bacteria↗

PubMed2002

Engineering tolerance and hyperaccumulation of arsenic in plants by combining arsenate reductase and gamma-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 gamma-glutamylcysteine synthetase (gamma-ECS) from a strong constitutive actin promoter (ACT2p) were moderately tolerant to arsenic compared with wild type. However, plants expressing SRS1p/ArsC and ACT2p/gamma-ECS together showed substantially greater arsenic tolerance than gamma-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 gamma-ECS or ArsC alone. This arsenic remediation strategy should be applicable to a wide variety of plant species.

Arabidopsis↗

PubMed2002

Plant profilin isovariants are distinctly regulated in vegetative and reproductive tissues.

Profilin is a low-molecular weight, actin monomer-binding protein that regulates the organization of actin cytoskeleton in eukaryotes, including higher plants. Unlike the simple human or yeast systems, the model plant Arabidopsis has an ancient and highly divergent multi-gene family encoding five distinct profilin isovariants. Here we compare and characterize the regulation of these profilins in different organs and during microspore development using isovariant-specific monoclonal antibodies. We show that PRF1, PRF2, and PRF3 are constitutive, being strongly expressed in all vegetative tissues at various stages of development. These profilin isovariants are also predominant in ovules and microspores at the early stages of microsporogenesis. In contrast, PRF4 and PRF5 are late pollen-specific and are not detectable in other cell types of the plant body including microspores and root hairs. Immunocytochemical studies at the subcellular level reveal that both the constitutive and pollen-specific profilins are abundant in the cytoplasm. In vegetative cell types, such as root apical cells, profilins showed localization to nuclei in addition to the cytoplasmic staining. The functional diversity of profilin isovariants is discussed in light of their spatio-temporal regulation during vegetative development, pollen maturation, and pollen tube growth.

Arabidopsis↗

PubMed2002

Functional nonequivalency of actin isovariants in Arabidopsis.

Plants encode at least two ancient and divergent classes of actin, reproductive and vegetative, and each class produces several subclasses of actin isovariants. To gain insight into the functional significance of the actin isovariants, we generated transgenic Arabidopsis lines that expressed a reproductive actin, ACT1, under the control of the regulatory sequences of a vegetative actin gene, ACT2. In the wild-type plants, ACT1 is predominantly expressed in the mature pollen, growing pollen tubes, and ovules, whereas ACT2 is constitutively and strongly expressed in all vegetative tissues and organs, but not in pollen. Misexpression of ACT1 in vegetative tissues causes dwarfing of plants and altered morphology of most organs, and the effects are in direct proportion to protein expression levels. Similar overexpression of ACT2 has little effect. Immunolocalization of actin in leaf cells from transgenic plants with highest levels of ACT1 protein revealed massive polymerization, bundling, and reorganization of actin filaments. This phenomenon suggests that misexpression of ACT1 isovariant in vegetative tissues affects the dynamics of actin and actin-associated proteins, in turn disrupting the organization of actin cytoskeleton and normal development of plants.

Actin Cytoskeleton↗

PubMed2002

Arabidopsis contains ancient classes of differentially expressed actin-related protein genes.

Actin-related proteins (ARPs) share less than 60% amino acid sequence homology with conventional actins and have roles in diverse cytoskeletal processes in the cytoplasm and nucleus. The genome of Arabidopsis was explored for possible ARP gene family members. Eight potential ARP gene sequences were found dispersed on three of the five Arabidopsis chromosomes. AtARP2 and AtARP3 are protein orthologs of their similarly named counterparts in other kingdoms. AtARP4, AtARP5, and AtARP6 are orthologs of two classes of nuclear ARPs previously characterized in animals and fungi, BAF53s and ARP6s. AtARP7 and AtARP8 appear to be novel proteins that are not closely related to any known animal or fungal ARPs, and may be plant specific. The complex Arabidopsis ARP gene structures each contain from five to 20 exons. Expressed transcripts were identified and characterized for AtARP2 through AtARP8, but not for AtARP9, and transcripts representing two splice variants were found for AtARP8. The seven expressed genes are predicted to encode proteins ranging from 146 to 471 amino acids in length. Relative to conventional actin and the other ARPs, AtARP2 and AtARP3 transcripts are expressed at very low levels in all organs. AtARP5, AtARP6, and AtARP8 each have distinct transcript expression patterns in seedlings, roots, leaves, flowers, and siliques. Using isovariant-specific monoclonal antibodies, AtARP4 and AtARP7 proteins were shown to be most highly expressed in flowers. The likely involvement of plant ARPs in actin nucleation, branching of actin filaments, chromatin restructuring, and transcription are briefly discussed.

Actin-Related Protein 2↗

PubMed2002

Both vegetative and reproductive actin isovariants complement the stunted root hair phenotype of the Arabidopsis act2-1 mutation.

The ACT2 gene, encoding one of eight actin isovariants in Arabidopsis, is the most strongly expressed actin gene in vegetative tissues. A search was conducted for physical defects in act2-1 mutant plants to account for their reduced fitness compared with wild type in population studies. The act2-1 insertion fully disrupted expression of ACT2 RNA and significantly lowered the level of total actin protein in vegetative organs. The root hairs of the act2-1 mutants were 10% to 70% the length of wild-type root hairs, and they bulged severely at the base. The length of the mutant root hairs and degree of bulging at the base were affected by adjusting the osmolarity and gelling agent of the growth medium. The act2-1 mutant phenotypes were fully rescued by an ACT2 genomic transgene. When the act2-1 mutation was combined with another vegetative actin mutation, act7-1, the resulting double mutant exhibited extensive synergistic phenotypes ranging from developmental lethality to severe dwarfism. Transgenic overexpression of the ACT7 vegetative isovariant and ectopic expression of the ACT1 reproductive actin isovariant also rescued the root hair elongation defects of the act2-1 mutant. These results suggest normal ACT2 gene regulation is essential to proper root hair elongation and that even minor differences may cause root defects. However, differences in the actin protein isovariant are not significant to root hair elongation, in sharp contrast to recent reports on the functional nonequivalency of plant actin isovariants. Impairment of root hair functions such as nutrient mining, water uptake, and physical anchoring are the likely cause of the reduced fitness seen for act2-1 mutants in multigenerational studies.

Actins↗