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Biomedical subjects

Richard B Meagher

Publications and source records attributed to Richard B Meagher.

At least 19 recordsLinked to original sources
PubMed2007

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.

Arabidopsis↗
PubMed2006

The shoot-specific expression of gamma-glutamylcysteine synthetase directs the long-distance transport of thiol-peptides to roots conferring tolerance to mercury and arsenic.

Thiol-peptides synthesized as intermediates in phytochelatin (PC) biosynthesis confer cellular tolerance to toxic elements like arsenic, mercury, and cadmium, but little is known about their long-distance transport between plant organs. A modified bacterial gamma-glutamylcysteine synthetase (ECS) gene, S1ptECS, was expressed in the shoots of the ECS-deficient, heavy-metal-sensitive cad2-1 mutant of Arabidopsis (Arabidopsis thaliana). S1ptECS directed strong ECS protein expression in the shoots, but no ECS was detected in the roots of transgenic plant lines. The S1ptECS gene restored full mercury tolerance and partial cadmium tolerance to the mutant and enhanced arsenate tolerance significantly beyond wild-type levels. After arsenic treatment, the root concentrations of gamma-glutamylcysteine (EC), PC2, and PC3 peptides in a S1ptECS-complemented cad2-1 line increased 6- to 100-fold over the mutant levels and were equivalent to wild-type concentrations. The shoot and root levels of glutathione were 2- to 5-fold above those in wild-type plants, with or without treatment with toxicants. Thus, EC and perhaps glutathione are efficiently transported from shoots to roots. The possibility that EC or other PC pathway intermediates may act as carriers for the long-distance phloem transport and subsequent redistribution of thiol-reactive toxins and nutrients in plants is discussed.

Arabidopsis↗
PubMed2006

Hyperaccumulation of arsenic in the shoots of Arabidopsis silenced for arsenate reductase (ACR2).

Endogenous plant arsenate reductase (ACR) activity converts arsenate to arsenite in roots, immobilizing arsenic below ground. By blocking this activity, we hoped to construct plants that would mobilize more arsenate aboveground. We have identified a single gene in the Arabidopsis thaliana genome, ACR2, with moderate sequence homology to yeast arsenate reductase. Expression of ACR2 cDNA in Escherichia coli complemented the arsenate-resistant and arsenate-sensitive phenotypes of various bacterial ars operon mutants. RNA interference reduced ACR2 protein expression in Arabidopsis to as low as 2% of wild-type levels. The various knockdown plant lines were more sensitive to high concentrations of arsenate, but not arsenite, than wild type. The knockdown lines accumulated 10- to 16-fold more arsenic in shoots (350-500 ppm) and retained less arsenic in roots than wild type, when grown on arsenate medium with <8 ppm arsenic. Reducing expression of ACR2 homologs in tree, shrub, and grass species should play a vital role in the phytoremediation of environmental arsenic contamination.

Amino Acid Sequence↗
PubMed2005

The nuclear actin-related protein ARP6 is a pleiotropic developmental regulator required for the maintenance of FLOWERING LOCUS C expression and repression of flowering in Arabidopsis.

Actin-related proteins (ARPs) are found in the nuclei of all eukaryotic cells, but their functions are generally understood only in the context of their presence in various yeast and animal chromatin-modifying complexes. Arabidopsis thaliana ARP6 is a clear homolog of other eukaryotic ARP6s, including Saccharomyces cerevisiae ARP6, which was identified as a component of the SWR1 chromatin remodeling complex. We examined the subcellular localization, expression patterns, and loss-of-function phenotypes for this protein and found that Arabidopsis ARP6 is localized to the nucleus during interphase but dispersed away from the chromosomes during cell division. ARP6 expression was observed in all vegetative tissues as well as in a subset of reproductive tissues. Null mutations in ARP6 caused numerous defects, including altered development of the leaf, inflorescence, and flower as well as reduced female fertility and early flowering in both long- and short-day photoperiods. The early flowering of arp6 mutants was associated with reduced expression of the central floral repressor gene FLOWERING LOCUS C (FLC) as well as MADS AFFECTING FLOWERING 4 (MAF4) and MAF5. In addition, arp6 mutations suppress the FLC-mediated late flowering of a FRIGIDA-expressing line, indicating that ARP6 is required for the activation of FLC expression to levels that inhibit flowering. These results indicate that ARP6 acts in the nucleus to regulate plant development, and we propose that it does so through modulation of chromatin structure and the control of gene expression.

Actins↗
PubMed2005

Inverted repeat PCR for the rapid assembly of constructs to induce RNA interference.

Expressing stem-loop RNAs in plants, fungi, and animals efficiently silences homologous target gene expression. We devised a novel PCR strategy, called inverted repeat PCR (IR-PCR), which allows rapid assembly and cloning of stem-loop-containing constructs in any vector. IR-PCR relies on differentially tagging antisense and sense copies of the target in one round of PCR and assembling them in a second. We used IR-PCR to assemble constructs targeting profilin, actin, and actin-related protein (ARP) transcripts from Arabidopsis. Immunoblotting of lines expressing a profilin PRF1 3' untranslated region (UTR)-specific construct demonstrated a 77 to 97% reduction in PRF1 protein, but not other profilin isovariants.

Base Sequence↗
PubMed2005

Arabidopsis ARP7 is an essential actin-related protein required for normal embryogenesis, plant architecture, and floral organ abscission.

The actin-related proteins (ARPs) that are localized to the nucleus are present as components of various chromatin-modifying complexes involved in chromatin dynamics and transcriptional regulation. Arabidopsis (Arabidopsis thaliana) ARP7 is a constitutively expressed nuclear protein belonging to a novel plant-specific ARP class. In this study, we demonstrate a vital role for ARP7 protein in embryogenesis and plant development. Knocking out the expression of ARP7 in an arp7-1 T-DNA mutant produced morphologically aberrant, homozygous embryos that were arrested at or before the torpedo stage of development. Hence, the arp7-1 null mutation is homozygous lethal. Knocking down the expression levels of ARP7 protein with RNA interference produced viable Arabidopsis lines affected in multiple developmental pathways and induced dosage-dependent, heritable defects in plant architecture. The transgenic plants containing greatly reduced levels of ARP7 in the nucleus were severely dwarfed with small rosette leaves that are defective in cell expansion and trichome morphology. Moreover, the ARP7-deficient RNA interference plants exhibited retarded root growth, altered flower development, delayed perianth abscission, and reduced fertility. These pleiotropic phenotypic changes suggest a critical role for the Arabidopsis ARP7 protein in the regulation of various phases of plant development through chromatin-mediated, global regulation of gene expression.

Actins↗
PubMed2005

Strategies for the engineered phytoremediation of toxic element pollution: mercury and arsenic.

Plants have many natural properties that make them ideally suited to clean up polluted soil, water, and air, in a process called phytoremediation. We are in the early stages of testing genetic engineering-based phytoremediation strategies for elemental pollutants like mercury and arsenic using the model plant Arabidopsis. The long-term goal is to develop and test vigorous, field-adapted plant species that can prevent elemental pollutants from entering the food-chain by extracting them to aboveground tissues, where they can be managed. To achieve this goal for arsenic and mercury, and pave the way for the remediation of other challenging elemental pollutants like lead or radionucleides, research and development on native hyperaccumulators and engineered model plants needs to proceed in at least eight focus areas: (1) Plant tolerance to toxic elementals is essential if plant roots are to penetrate and extract pollutants efficiently from heterogeneous contaminated soils. Only the roots of mercury- and arsenic-tolerant plants efficiently contact substrates heavily contaminated with these elements. (2) Plants alter their rhizosphere by secreting various enzymes and small molecules, and by adjusting pH in order to enhance extraction of both essential nutrients and toxic elements. Acidification favors greater mobility and uptake of mercury and arsenic. (3) Short distance transport systems for nutrients in roots and root hairs requires numerous endogenous transporters. It is likely that root plasma membrane transporters for iron, copper, zinc, and phosphate take up ionic mercuric ions and arsenate. (4) The electrochemical state and chemical speciation of elemental pollutants can enhance their mobility from roots up to shoots. Initial data suggest that elemental and ionic mercury and the oxyanion arsenate will be the most mobile species of these two toxic elements. (5) The long-distance transport of nutrients requires efficient xylem loading in roots, movement through the xylem up to leaves, and efficient xylem unloading aboveground. These systems can be enhanced for the movement of arsenic and mercury. (6) Aboveground control over the electrochemical state and chemical speciation of elemental pollutants will maximize their storage in leaves, stems, and vascular tissues. Our research suggests ionic Hg(II) and arsenite will be the best chemical species to trap aboveground. (7) Chemical sinks can increase the storage capacity for essential nutrients like iron, zinc, copper, sulfate, and phosphate. Organic acids and thiol-rich chelators are among the important chemical sinks that could trap maximal levels of mercury and arsenic aboveground. (8) Physical sinks such as subcellular vacuoles, epidermal trichome cells, and dead vascular elements have shown the evolutionary capacity to store large quantities of a few toxic pollutants aboveground in various native hyperaccumulators. Specific plant transporters may already recognize gluthione conjugates of Hg(II) or arsenite and pump them into vacuole.

Arabidopsis↗
PubMed2005

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.

Amino Acid Sequence↗
PubMed2005

The plant MT1 metallothioneins are stabilized by binding cadmiums and are required for cadmium tolerance and accumulation.

The small Arabidopsis genome contains nine metallothionein-like (MT) sequences with classic, cysteine-rich domains separated by spacer sequences, quite unlike the small conserved MT families found vertebrate genomes. Phylogenetic analysis revealed four ancient and divergent classes of plant MTs that predate the monocot-dicot divergence. A distinct cysteine spacing pattern suggested differential metal ion specificity for each class. The in vivo stability of representatives of the four classes of plant MT proteins and a mouse MT2 control expressed in E. coli were enhanced by cadmium (Cd). Particular MTs were also stabilized by arsenic (As), copper (Cu), and or zinc (Zn). To understand why plants have such a diversity of MT sequences, the Arabidopsis MT1 class, comprised of three genes, MT1a, MT1b, and MT1c, was characterized in more detail in plants. MT1 family transcripts were knocked down to less than 5-10% of wild-type levels in Arabidopsis by expression of a RNA interference (RNAi) construct. The MT1 knockdown plant lines were all hypersensitive to Cd and accumulated several fold lower levels of As, Cd, and Zn than wildtype, while Cu and Fe levels were unaffected. The ancient class of MT1 protein sequences may be preserved in plant genomes, because it has distinct metal-binding properties, confers tolerance to cadmium, and can assist with zinc homeostasis.

Animals↗
PubMed2005

Silencing the nuclear actin-related protein AtARP4 in Arabidopsis has multiple effects on plant development, including early flowering and delayed floral senescence.

Actin-related proteins (ARPs) share moderate sequence homology and basal structure with conventional actins and are found in all eukaryotes. While the functions of most of the divergent ARPs are not clear, several of them are localized to the nucleus and have been identified as components of various chromatin-modifying complexes. Using an antibody to Arabidopsis AtARP4, we found this conserved homolog of human BAF53 and yeast Arp4 is concentrated in the nucleoplasm of Arabidopsis, Brassica, and tobacco cells. To gain further insight into the role of ARP4, we have examined Arabidopsis plants that are defective in AtARP4 expression. Phenotypic analysis of the arp4-1 mutant allele, which has a T-DNA insertion in the promoter region and a moderate reduction in the level of AtARP4 protein expression, revealed partial sterility due to defects in anther development. Targeting the distinct, 3' UTR of AtARP4 transcripts with RNA interference caused a drastic reduction in the level of AtARP4 protein expression in several independent transgenic lines, and resulted in strong pleiotropic phenotypes such as altered organization of plant organs, early flowering, delayed flower senescence and high levels of sterility. Western blot analysis and immunolabeling demonstrated a clear correlation between reductions in the level of AtARP4 expression and severity of the phenotypes. Based on our results and data on the orthologs of AtARP4 in yeast and other organisms, we suggest that AtARP4 is likely to exert its effects on plant development through the modulation of chromatin structure and subsequent changes in gene regulation.

Actins↗
PubMed2005

Engineering a root-specific, repressor-operator gene complex.

Strong, tissue-specific and genetically regulated expression systems are essential tools in plant biotechnology. An expression system tool called a 'repressor-operator gene complex' (ROC) has diverse applications in plant biotechnology fields including phytoremediation, disease resistance, plant nutrition, food safety, and hybrid seed production. To test this concept, we assembled a root-specific ROC using a strategy that could be used to construct almost any gene expression pattern. When a modified E. coli lac repressor with a nuclear localization signal was expressed from a rubisco small subunit expression vector, S1pt::lacIn, LacIn protein was localized to the nuclei of leaf and stem cells, but not to root cells. A LacIn repressible Arabidopsis actin expression vector A2pot was assembled containing upstream bacterial lacO operator sequences, and it was tested for organ and tissue specificity using beta-glucuronidase (GUS) and mercuric ion reductase (merA) gene reporters. Strong GUS enzyme expression was restricted to root tissues of A2pot::GUS/S1pt::lacIn ROC plants, while GUS activity was high in all vegetative tissues of plants lacking the repressor. Repression of shoot GUS expression exceeded 99.9% with no evidence of root repression, among a large percentage of doubly transformed plants. Similarly, MerA was strongly expressed in the roots, but not the shoots of A2pot::merA/S1pt::lacIn plants, while MerA levels remained high in both shoots and roots of plants lacking repressor. Plants with MerA expression restricted to roots were approximately as tolerant to ionic mercury as plants constitutively expressing MerA in roots and shoots. The superiority of this ROC over the previously described root-specific tobacco RB7 promoter is demonstrated.

Journal Article↗
PubMed2005

Arsenic and mercury tolerance and cadmium sensitivity in Arabidopsis plants expressing bacterial gamma-glutamylcysteine synthetase.

Cysteine sulfhydryl-rich peptide thiols are believed to play important roles in the detoxification of many heavy metals and metalloids such as arsenic, mercury, and cadmium in plants. The gamma-glutamylcysteine synthetase (gamma-ECS) catalyzes the synthesis of the dipeptidethiol gamma-glu-cys (gamma-EC), the first step in the biosynthesis of phytochelatins (PCs). Arabidopsis thaliana, engineered to express the bacterial gamma-ECS gene under control of a strong constitutive actin regulatory sequence (A2), expressed gamma-ECS at levels approaching 0.1% of total protein. In response to arsenic, mercury, and cadmium stresses, the levels of gamma-EC and its derivatives, glutathione (GSH) and PCs, were increased in the A2::ECS transgenic plants to three- to 20-fold higher concentrations than the increases that occurred in wild-type (WT). Compared to cadmium and mercury treatments, arsenic treatment most significantly increased levels of gamma-EC and PCs in both the A2::ECS transgenic and WT plants. The A2::ECS transgenic plants were highly resistant to arsenic and weakly resistant to mercury. Although exposure to cadmium produced three- to fivefold increases in levels of gamma-EC-related peptides in the A2::ECS lines, these plants were significantly more sensitive to Cd(II) than WT and trace levels of Cd(II) blocked resistance to arsenic and mercury. A few possible mechanisms for gamma-ECS-enhanced arsenic and mercury resistance and cadmium hypersensitivity are discussed.

Arabidopsis↗
PubMed2004

Plant actin-related proteins.

Actin-related proteins (ARPs) constitute a family of divergent and evolutionarily ancient eukaryotic proteins whose primary sequences display homology to conventional actins. Whereas actins play well-characterized cytoskeletal roles, the ARPs are implicated in various cellular functions in both the cytoplasm and in the nucleus. Cytoplasmic ARPs, for example, are known to participate in the assembly of branched actin filaments and dynein-mediated movement of vesicles in many eukaryotes. Nuclear ARPs, by contrast, are enigmatic components of various chromatin-modifying complexes involved in transcriptional regulation. Here, we review homologs to several known classes of ARPs and two distinct ARP classes in plants, and summarize recent work elucidating the biological functions of ARPs in eukaryotes.

Actins↗
PubMed2004

Overexpression of phytochelatin synthase in Arabidopsis leads to enhanced arsenic tolerance and cadmium hypersensitivity.

Phytochelatin synthase (PCS) catalyzes the final step in the biosynthesis of phytochelatins, which are a family of cysteine-rich thiol-reactive peptides believed to play important roles in processing many thiol-reactive toxicants. A modified Arabidopsis thaliana PCS sequence (AtPCS1) was active in Escherichia coli. When AtPCS1 was overexpressed in Arabidopsis from a strong constitutive Arabidopsis actin regulatory sequence (A2), the A2::AtPCS1 plants were highly resistant to arsenic, accumulating 20-100 times more biomass on 250 and 300 microM arsenate than wild type (WT); however, they were hypersensitive to Cd(II). After exposure to cadmium and arsenic, the overall accumulation of thiol-peptides increased to 10-fold higher levels in the A2::AtPCS1 plants compared with WT, as determined by fluorescent HPLC. Whereas cadmium induced greater increases in traditional PCs (PC2, PC3, PC4), arsenic exposure resulted in the expression of many unknown thiol products. Unexpectedly, after arsenate or cadmium exposure, levels of the dipeptide substrate for PC synthesis, gamma-glutamyl cysteine (gamma-EC), were also dramatically increased. Despite these high thiol-peptide concentrations, there were no significant increases in concentrations of arsenic and cadmium in above-ground tissues in the AtPCS1 plants relative to WT plants. The potential for AtPCS1 overexpression to be useful in strategies for phytoremediating arsenic and to compound the negative effects of cadmium are discussed.

Aminoacyltransferases↗
PubMed2003

Detection of deleterious genotypes in multigenerational studies. III. Estimation of selection components in highly selfing populations.

New paradigms in genetics have increased the chance of finding genes that appear redundant but in fact may have been preserved due to a small level of positive selection potential acting during each generation. Monitoring changes in genotypic frequencies within and between generations allows the dissection of the fertility, viability and meiotic drive selection components acting on such genes in natural and experimental populations. Here, a formal maximum likelihood procedure is developed to identify and estimate these selection components in highly selfing populations by fitting the time-dependent solutions for genotypic frequencies to observed multigenerational counts. With adult census alone, we can not simultaneously estimate all three selection components considered. In such cases, we instead consider a hierarchy of 11 models with either fewer selection components, complete dominance, or multiplicative meiotic drive with a single parameter. We identify the best-fitting of these models by applying likelihood ratio tests to nested models and Akaike's Information Criterion (AIC) and the Bayesian Information Criterion (BIC) to non-nested models. With seed census, fertility and viability selection are not distinguishable and thus can only be estimated jointly. A combination of joint seed and adult census data allows us to estimate all three selection components simultaneously. Simulated data validate the estimation procedure and provide some practical guidelines for experimental design. An application to Arabidopsis data establishes that viability selection is the major selective force acting on the ACT2 actin gene in laboratory-grown Arabidopsis populations.

Arabidopsis↗
PubMed2003

Multiple conserved 5' elements are required for high-level pollen expression of the Arabidopsis reproductive actin ACT1.

The Arabidopsis thaliana actin gene family comprises eight genes, which are divided into two ancient classes, vegetative and reproductive. We dissected various 5' elements and the conserved expression pattern of the reproductive actin gene ACT1, which is the most strongly expressed pollen actin gene. A basal construct containing only 310 bp of sequence upstream of the major transcriptional start site showed essentially full promoter activity in pollen and ovules. Further truncations of the 5'-flanking region and two different 10 bp replacements within a 55 bp conserved domain each caused a several-fold reduction in mature pollen expression. Intron L, located in the 5'-untranslated region (5'-UTR) was also required for high-level expression of pollen and organ primordia, and it had the properties of an enhancer. Pollen expression was not preserved when intron L was precisely replaced by intron L2 from the vegetatively expressed actin gene ACT2. ACT1 reporter gene constructs were strongly expressed in both pollen and ovules of tobacco and in the pollen of rice. Promoter-reporter fusions of the most distantly related Arabidopsis reproductive actin gene ACT1 showed strong expression in tobacco pollen and ovules indistinguishable from that directed by ACT1. Thus, multiple conserved cis-sequence elements within the 5'-flanking region and 5'-UTR of ACT1 direct high levels of reproductive tissue-specific expression.

5' Flanking Region↗