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Ultrafast Laser Sampling of a Plant Tissue and ion Conductivity Measurement for Investigation of Light Stress Generation Mechanisms

Description: In this study we applied ultra-short laser pulses on a biological sample (Arabidopsis), in order to cut it precisely in a square pattern and subsequently use it for studying stress generation mechanisms. For this purpose, we utilized femtosecond laser pulses at 100 fs pulse width and 80 MHz repetition rate. We took two processing parameters into consideration such as laser power, laser exposure time which is related to the stage speed. Therefore, we were able to find the laser optimum conditions for ablation of biological tissues. The mutant and wildtype (control) obtained from laser cutting with a size of 500 µm × 500 µm were directly transferred (in-situ with laser cutting) into a microfabricated chamber containing ~500 nanoliters deionized water for measuring ion conductivity. The ion conductivity is a signature of cell-death mechanisms caused by various stresses. A light with intensity of 100 µmol was exposed to the samples for 2 hours and 20 minutes as a source of stress. A quantitative electrical analysis with high accuracy was assured by utilizing a microchamber, which enables a measurement in nanoliter volume. We measured the impedance which is reciprocal of conductivity using a lock-in amplifier and a precise current source at frequency of 130 Hz. Initially high impedance of mutant sample tended to drop within 2 hours and finally approached the constant value which signified that the cell death mechanism was complete. However, the wildtype sample demonstrated approximately constant impedance (conductivity) during the experiment.
Date: August 2010
Creator: Abtahi, Seyed Ali
Partner: UNT Libraries

Expression analysis of the fatty acid desaturase 2-4 and 2-3 genes from Gossypium hirsutum in transformed yeast cells and transgenic Arabidopsis plants.

Description: Fatty acid desaturase 2 (FAD2) enzymes are phosphatidylcholine desaturases occurring as integral membrane proteins in the endoplasmic reticulum membrane and convert monounsaturated oleic acid into polyunsaturated linoleic acid. The major objective of this research was to study the expression and function of two cotton FAD2 genes (the FAD2-3 and FAD2-4 genes) and their possible role in plant sensitivity to environmental stress, since plants may increase the polyunsaturated phospholipids in membranes under environmental stress events, such as low temperature and osmotic stress. Two FAD2 cDNA clones corresponding to the two FAD2 genes have been isolated from a cotton cDNA library, indicating both genes are truly expressed in cotton. Model yeast cells transformed with two cotton FAD2 genes were used to study the chilling sensitivity, ethanol tolerance, and growth rate of yeast cells. The expression patterns of the two FAD2 genes were analyzed by reverse transcription polymerase chain reactions (RT-PCR) and Western blot analyses in cotton plants under different treatment conditions. The coding regions of both FAD2 genes were inserted downstream from the CaMV 35S promoter in the pMDC gateway binary vector system. Five different FAD2/pMDC constructs were transformed into the Arabidopsis fad2 knockout mutant background, and multiple potential transgenic Arabidopsis plant lines harboring the cotton FAD2 genes were generated. The cotton FAD2 genes were amplified by the polymerase chain reaction (PCR) from the genomic DNAs isolated from the transgenic Arabidopsis T1 plant lines. Complementation of the putative transgenic Arabidopsis plants with the two cotton FAD2 genes was demonstrated by gas chromatography analyses of the fatty acid profiles of leaf tissues. The cellular localization of cotton FAD2-4 polypeptides with N-terminal green fluorescence protein (GFP) was visualized by confocal fluorescence microscopy. The phenotype of transgenic Arabidopsis plants transformed with the cotton FAD2-4 gene was compared to Arabidopsis knockout fad2 mutant plants and wild ...
Date: August 2008
Creator: Zhang, Daiyuan
Partner: UNT Libraries

The genome of black cottonwood, Populus trichocarpa (Torr.&Gray)

Description: We report the draft genome of the black cottonwood tree, Populus trichocarpa. Integration of shotgun sequence assembly with genetic mapping enabled chromosome-scale reconstruction of the genome. Over 45,000 putative protein-coding genes were identified. Analysis of the assembled genome revealed a whole-genome duplication event, with approximately 8,000 pairs of duplicated genes from that event surviving in the Populus genome. A second, older duplication event is indistinguishably coincident with the divergence of the Populus and Arabidopsis lineages. Nucleotide substitution, tandem gene duplication and gross chromosomal rearrangement appear to proceed substantially slower in Populus relative to Arabidopsis. Populus has more protein-coding genes than Arabidopsis, ranging on average between 1.4-1.6 putative Populus homologs for each Arabidopsis gene. However, the relative frequency of protein domains in the two genomes is similar. Overrepresented exceptions in Populus include genes associated with disease resistance, meristem development, metabolite transport and lignocellulosic wall biosynthesis.
Date: September 1, 2006
Creator: Tuskan, G. A.; DiFazio, S.; Jansson, S.; Bohlmann, J.; Grigoriev,I.; Hellsten, U. et al.
Item Type: Article
Partner: UNT Libraries Government Documents Department

Summary of the International Conference on Arabidopsis Research 2011, June 22-25, 2011

Description: This project provided participant support for the gathering of plant biologists at the International Conferences on Arabidopsis Research (ICAR) in 2011. Arabidopsis thaliana, the reference flowering plant, has been intensely studied over the last 20 years and has proven to be an ideal model for studying nearly all aspects of plant biology. The success of this research field has been greatly facilitated by the openness and collegiality of the community fostered through multiple international forums including the ICAR. Advances in basic and applied plant biology are featured at the meeting, which is the primary gathering point for this strongly integrated international community. The ICAR convenes plant researchers, allows discussion and dissemination of the latest research in plant biology, and facilitates dialog among those that may be separated by geography, career stage, and culture. This project focused on facilitating access by early career scientists that have reduced access to attend major meetings.
Date: July 15, 2012
Creator: Meyers, Blake C
Item Type: Report
Partner: UNT Libraries Government Documents Department

Proteolysis in Plastids of Arabidopsis Thaliana: Functional Analysis of ClpS1,2,T and their Physical and Genetic Interactions with the ClpPR Protease Core Complex and Clp Chaperones

Description: Chloroplasts are essential organelles required for plant growth and biomass production. They synthesize many essential secondary metabolites (e.g. hormones, isoprenoids, amino acids, etc.) and house the photosynthetic apparatus needed for conversion of light energy and CO2 into chemical energy [in the form of reduced carbohydrates, ATP and NADPH]. Thus chloroplasts are essential for life on earth and essential for production of bioenergy. Formation and maintenance of a functional chloroplast requires an extensive investment in the biogenesis and homeostasis apparatus. Protease and proteolysis play a critical role in these processes, with the Clp gene family being particularly central. Proteolysis of proteins and protein complexes in plastids is poorly understood, and is not only critical for biogenesis, adaptation and maintenance but is also important for plant development. Several years ago, the vanWijk lab identified a large and relatively abundant ClpP/R/S complex, along with ClpC1,C2 and ClpD chaperones and a putative Clp affinity modulator in plastids. So far, no substrate recognition mechanism has been determined for any Clp complex in plants. The purpose of this grant was to initiate functional analysis of three members of the Clp family.
Date: January 12, 2009
Creator: van Wijk, Klaas
Item Type: Report
Partner: UNT Libraries Government Documents Department

Physiology and Regulation of Calcium Channels in Stomatal Guard Cells

Description: Stomatal pores in the epidermis of leaves regulate the diffusion of CO2 into leaves for photosynthetic carbon fixation and control water loss of plants during drought periods. Guard cells sense CO2, water status, light and other environmental conditions to regulate stomatal apertures for optimization of CO2 intake and plant growth under drought stress. The cytosolic second messenger calcium contributes to stomatal movements by transducing signals and regulating ion channels in guard cells. Studies suggest that both plasma membrane Ca2+ influx channels and vacuolar/organellar Ca2+ release channels contribute to ABA-induced Ca2+ elevations in guard cells. Recent research in the P.I.'s laboratory has led to identification of a novel major cation-selective Ca2+-permeable influx channel (Ica) in the plasma membrane of Arabidopsis guard cells. These advances will allow detailed characterization of Ica plasma membrane Ca2+ influx channels in guard cells. The long term goal of this research project is to gain a first detailed characterization of these novel plasma membrane Ca2+-permeable channel currents in Arabidopsis guard cells. The proposed research will investigate the hypothesis that Ica represents an important Ca2+ influx pathway for ABA and CO2 signal transduction in Arabidopsis guard cells. These studies will lead to elucidation of key signal transduction mechanisms by which plants balance CO2 influx into leaves and transpirational water loss and may contribute to future strategies for manipulating gas exchange for improved growth of crop plants and for biomass production.
Date: May 2, 2007
Creator: Schroeder, Julian I.
Item Type: Report
Partner: UNT Libraries Government Documents Department

Membrane Targeting of P-type ATPases in Plant Cells

Description: How membrane proteins are targeted to specific subcellular locations is a very complex and poorly understood area of research. Our long-term goal is to use P-type ATPases (ion pumps), in a model plant system Arabidopsis, as a paradigm to understand how members of a family of closely related membrane proteins can be targeted to different subcellular locations. The research is divided into two specific aims. The first aim is focused on determining the targeting destination of all 10 ACA-type calcium pumps (Arabidopsis Calcium ATPase) in Arabidopsis. ACAs represent a plant specific-subfamily of plasma membrane-type calcium pumps. In contrast to animals, the plant homologs have been found in multiple membrane systems, including the ER (ACA2), tonoplast (ACA4) and plasma membrane (ACA8). Their high degree of similarity provides a unique opportunity to use a comparative approach to delineate the membrane specific targeting information for each pump. One hypothesis to be tested is that an endomembrane located ACA can be re-directed to the plasma membrane by including targeting information from a plasma membrane isoform, ACA8. Our approach is to engineer domain swaps between pumps and monitor the targeting of chimeric proteins in plant cells using a Green Fluorescence Protein (GFP) as a tag. The second aim is to test the hypothesis that heterologous transporters can be engineered into plants and targeted to the plasma membrane by fusing them to a plasma membrane proton pump. As a test case we are evaluating the targeting properties of fusions made between a yeast sodium/proton exchanger (Sod2) and a proton pump (AHA2). This fusion may potentially lead to a new strategy for engineering salt resistant plants. Together these aims are designed to provide fundamental insights into the biogenesis and function of plant cell membrane systems.
Date: June 30, 2004
Creator: Jeffrey F. Harper, Ph.D.
Item Type: Report
Partner: UNT Libraries Government Documents Department

Characterization of a New Family of Metal Transporters

Description: Metal ions are critical nutrients, yet overaccumulation of these same metals can also be toxic. To maintain appropriate intracellular levels, cells require specific metal uptake systems that are subject to precise homeostatic regulation. The long-range goal of our research is to define the molecular mechanism(s) and regulation of metal ion uptake in eukaryotic cells. Integrating genetic, molecular biological and biochemical approaches, we have examined these processes in the yeast Saccharomyces cerevisiae and the plant Arabidopsis thaliana. Both are proven model systems for studying fundamental cellular processes. Our work has focused on the ZIP family of metal transporters which we identified; this family has representatives in bacteria, fungi, plants and animals. IRT, one of the founding members of the ZIP family, is an essential cation transporter that is expressed in the epidermal cells of iron deficient plant roots and is responsible for uptake of iron from the soil. We now know that there are 15 ZIP genes in the Arabidopsis and the similarities among their encoded gene products. The ZIP family members display different substrate specificities for metals and different tissue distributions in Arabidopsis. Moreover, the family members respond differentially to metal deficiencies. For example, IRT1, ZIP6 and ZIP9 mRNA are expressed mainly in the roots of iron deficient plants whereas ZIP4 responds to both iron and zinc deficiency. Work in both yeast and Arabidopsis has addressed substrate specificity as well as how these transporters are regulated in response to metal availability
Date: April 29, 2002
Creator: Geurinot, Mary Lou & Eide, David
Item Type: Report
Partner: UNT Libraries Government Documents Department

Final Technical Report: Genetic and Molecular Analysis of a new control pathway in assimilate partitioning.

Description: Assimilate partitioning refers to the systemic distribution of photoassimilate from sites of primary assimilation (source tissue) to import-dependent tissues and organs (sinks). One of the defining questions in this area is how plants balance source productivity with sink demand. We discovered a sucrose-sensing signal transduction pathway that controls the activity of BvSUT1, a proton-sucrose symporter in sugar beet leaf tissue. Sucrose symporters are responsible for sucrose accumulation in the phloem of many plants and, therefore, they mediate the pivotal step in the long-distance transport of photoassimilate to non-photosynthetic tissues, such as roots and seed. We previously showed that sucrose transport activity is directly proportional to the transcription rate of BvSUT1 and that symporter mRNA and protein have high rates of turnover with half-lives on the order of 2 h. We further demonstrated that symporter transcription is regulated by sucrose levels in the leaf and that sucrose-dependent regulation of BvSUT1 transcription is mediated, at least in part, by a protein phosphorylation relay pathway. The goal of the experiments during this current grant were to use genetic and molecular approaches to identify essential components of this vital regulatory system. The initial objectives were to: (1) to characterize Arabidopsis mutants we've isolated that are resistant to growth inhibition by sucrose analogues that are recognized by the sucrose-sensor, (2) to screen for loss of function mutants in BvSUT1-promoter:luciferase transgenic plants that no longer respond to sucrose accumulation in the leaf using non-destructive visualization of luciferase activity, (3) to use gel mobility-shift assays and nuclease protection experiments to identify cis elements in the symporter promoter and DNA-binding proteins that are involved in sucrose regulation of symporter expression.
Date: March 10, 2009
Creator: Bush, Daniel, R.
Item Type: Report
Partner: UNT Libraries Government Documents Department

17th International Conference on Arabidopsis Research

Description: The 17th International Conference on Arabidopsis Research was held at the University of Madison, Wisconsin from June 27- July 2, 2006. ICAR-2006 included approximately 625 scientists from across the world. The scientific program was of excellent quality featuring 73 talks, including 30 from invited speakers. There were also 6 community-organized workshops (facilitated by conference staff) featuring additional talks on topics including ‘Submitting data to long-term repositories,’ ‘TAIR introductory workshop,’ ‘Web services and demonstration,’ ‘Public engagement: broadening the impact of your research,’ ‘Systems biology approaches to analysis of metabolic and regulatory networks of Arabidopsis,’ and ‘Mechanotransduction in Arabidopsis.’ Approximately 440 posters were presented in general topic areas including, among others, Development, Modeling/Other Systems, Energy, Environment, and Genetic/Epigenetic mechanisms. Graduate students, postdoctoral researchers, junior faculty, and underrepresented minorities made up a significant portion of the oral presentations thereby promoting the training of young scientists and facilitating important career development opportunities for speakers. Several poster sessions provided an opportunity for younger participants to freely meet with more established scientists. The North American Arabidopsis Steering Committee (NAASC) continued its outreach effort and again sponsored two special luncheons to encourage personal and professional development of young scientists and also underrepresented minorities. The ‘Emerging Scientists Luncheon’ featured 10 graduate students selected on the basis of scientific excellence of their submitted research abstracts. The ‘Minority Funding Luncheon,’ featured 8 awardees selected by the NAASC through a widely-publicized application process. This luncheon was established specifically to provide an opportunity for underrepresented minorities, and/or scientists from Minority-Serving Institutions/Historically Black Colleges and Universities to network with NAASC members and representatives from federal funding agencies in an informal and intimate setting. This luncheon included introductions of each award recipient and discussion of outreach efforts and informal research and career discussions. Outreach Activities: The conference is the largest annual international Arabidopsis venue ...
Date: July 1, 2006
Creator: Bender, Judith
Item Type: Article
Partner: UNT Libraries Government Documents Department

Final Progress Report for FG02-89ER14030

Description: Intracellular Dynamics of Energy-Transducing Organelles. The location and interaction of intracellular organelles is important for exchange of substrate and product between compartments for optimum functioning of biochemical pathways and energy transduction. Plastids and stromules, tubular plastid extensions, are highly dynamic in many plant tissues. Stromules can connect two or more plastids and proteins and macromolecular complexes can be transferred between them. Stromules have been observed to form close contacts with other organelles, the plasma membrane, and can pass through channels in the nucleus. Chloroplasts move in response to light and mechanical stimulus. Especially in non-green cells, plastids change shape and position, and stromules extend and retract. Stromules appear to be involved in recycling of chloroplast proteins when photosynthesis is limited, through an autophagic process that results in degradation of portions of the stromal contents without complete destruction of the chloroplast. Mutations in several genes known to mediate chloroplast division result in altered stromule morphology in some cells. Plastid and stromule motility is mediated by the actin cytoskeleton. The possible role of myosins in chloroplast movement was investigated by labeling the cargo-binding tails of six Arabidopsis myosin XI proteins with yellow fluorescent protein (YFP). The fluorescent proteins were found to localize to vesicles and peroxisomes. The portion of the myosin tail domain fused to YFP affected whether specific or non-specific localization was observed. In contrast to experiments in animal cells, movement of labeled organelles was not entirely inhibited by expression of defective myosin in which the motor domain was replaced with a fluorescent protein. None of the six myosin proteins tested labeled plastids or chloroplasts. However, the Arabidopsis myosin XI gene family expresses an additional seven myosins that await further examination. These experiments have provided more information on stromule formation and function and the actin-myosin machinery that mediates the intracellular trafficking ...
Date: October 26, 2011
Creator: Hanson, Maureen R
Item Type: Report
Partner: UNT Libraries Government Documents Department

Metabolic regulation of the plant hormone indole-3-acetic acid

Description: The phytohormone indole-3-acetic acid (IAA, auxin) is important for many aspects of plant growth, development and responses to the environment yet the routes to is biosynthesis and mechanisms for regulation of IAA levels remain important research questions. A critical issue concerning the biosynthesis if IAA in plants is that redundant pathways for IAA biosynthesis exist in plants. We showed that these redundant pathways and their relative contribution to net IAA production are under both developmental and environmental control. We worked on three fundamental problems related to how plants get their IAA: 1) An in vitro biochemical approach was used to define the tryptophan dependent pathway to IAA using maize endosperm, where relatively large amounts of IAA are produced over a short developmental period. Both a stable isotope dilution and a protein MS approach were used to identify intermediates and enzymes in the reactions. 2) We developed an in vitro system for analysis of tryptophan-independent IAA biosynthesis in maize seedlings and we used a metabolite profiling approach to isolate intermediates in this reaction. 3) Arabidopsis contains a small family of genes that encode potential indolepyruvate decarboxylase enzymes. We cloned these genes and studied plants that are mutant in these genes and that over-express each member in the family in terms of the level and route of IAA biosynthesis. Together, these allowed further development of a comprehensive picture of the pathways and regulatory components that are involved in IAA homeostasis in higher plants.
Date: November 1, 2009
Creator: Cohen, Jerry D.
Item Type: Report
Partner: UNT Libraries Government Documents Department

The Role of a Host Protein (TIP) in the Resistance Response of Arabidopsis to Turnip Crinkle Virus Infection.

Description: Our research on Turnip crinkle virus (TCV) has shown that the viral capsid protein (CP) is both a virulence factor as well as the elicitor of a hypersensitive resistance response (HR) to the virus in Arabidopsis. Initially, we identified a protein from Arabidopsis that specifically interacted with the viral CP using a yeast two-hybrid screen. This protein, designated TIP for TCV-Interacting Protein, is a member of the NAC family of plant transcription factors implicated in the regulation of development and senescence. When TCV CP was mutated to eliminate its ability to interact with TIP, the corresponding virus mutants broke the HR-mediated resistance conferred by the HRT resistance (R) gene in Arabidopsis ecotype Dijon (Di)-17. This result suggested that TIP is a component of the signal transduction pathway that leads to the genetically specified TCV resistance. We next confirmed that TIP and the viral CP interact in plant cells and that this interaction prevents nuclear localization of this transcription factor. We demonstrated that TCV CP suppresses post-transcriptional gene silencing (PTGS), a newly discovered RNA-mediated defense system in plants. Together these results suggest that the CP is a virulence factor that could well be functioning through its interaction with TIP. We have proposed a model involving the role of TIP and CP in triggering HR mediated plant defense that fits with the current thinking about how gene-for-gene resistance may function. A unique component of our system is the opportunity to link R-gene function with the newly discovered RNA silencing pathway that is not only a potent defense against viral pathogens, but also regulates early development in plants. In the current funding period we made several significant findings: First, we completed an array analysis comparing gene expression in Arabidopsis infected with TCV and a mutant virus unable to bind TIP. Second, we produced ...
Date: October 20, 2008
Creator: Morris, T. Jack
Item Type: Report
Partner: UNT Libraries Government Documents Department

Functional analysis of U1-70K interacting SR proteins in pre-mRNA splicing in Arabidopsis

Description: Proteins of a serine/arginine-rich (SR) family are part of the spliceosome and are implicated in both constitutive and alternative splicing of pre-mRNAs. With the funding from DOE we have been studying alternative of splicing of genes encoding serine/arginine-rich (SR) proteins and the roles of SR proteins that interact with U1-70K in regulating basic and alternative splicing. Alternative splicing of pre-mRNAs of Arabidopsis serine/arginine-rich proteins and its regulation by hormones and stresses: We analyzed the splicing of all 19 Arabidopsis genes in different tissues, during different seedling stages and in response to various hormonal and stress treatments. Remarkably, about 90 different transcripts are produced from 15 SR genes, thereby increasing the transcriptome complexity of SR genes by about five fold. Using the RNA isolated from polysomes we have shown that most of the splice variants are recruited for translation. Alternative splicing of some SR genes is controlled in a developmental and tissue-specific manner (Palusa et al., 2007). Interestingly, among the various hormones and abiotic stresses tested, temperature stress (cold and heat) and ultraviolet light dramatically altered alternative splicing of pre-mRNAs of several SR genes whereas hormones altered the splicing of only two SR genes (Palusa et al., 2007). Localization and dynamics of a novel serine/arginine-rich protein that interacts with U1-70K: We analyzed the intranuclear movement of SR45 fused to GFP by fluorescence recovery after photobleaching (FRAP) and fluorescence loss in photobleaching (FLIP). We demonstrate that the movement of GFP-SR45 is ATP-dependent. Interestingly, inhibition of transcription or phosphorylation slowed the mobility of GFP-SR45 (Ali et al., 2006). Our studies have revealed that the nuclear localization signals are located in arg/ser-rich domains (RS) 1 and 2, whereas the speckle targeting signals are exclusively present in RS2 (Ali et al., 2006). The regulation of SR45 mobility by ATP and a transcriptional inhibitor is in ...
Date: November 25, 2008
Creator: Reddy, A.S.N.
Item Type: Report
Partner: UNT Libraries Government Documents Department

Characterization of Laccase-like Multicopper Oxidases (LMCOs) in Arabidopsis thaliana

Description: Laccase-like multicopper oxidases (LMCOs) have repeatedly been associated with the process of lignification in plants, and previous work suggested that these enzymes might be acting as specific marker for highly localized, small-scale lignification events in tissues not typically thought of as lignified. However, plant LMCOs typically occur as members of gene families and different family members can display disparate enzyme activities and overlapping patterns of expression in bulk tissues. This study used reporter genes and knockout mutants to document the involvement of a specific Arabidopsis thaliana LMCO family member (At2g30210 ) in early root development, specifically with development of endodermal tissues. Expression of the gene product was found to be under the control of sucrose levels, but the gene also responded to fluctuations in salt concentrations. The expression patterns of this gene were consistent with its involvement in the formation of suberin in the Casparian strip of root endodermis. An additional LMCO (At5g58910) displayed a more generalized expression in the radicles emergent seedlings. Additional members of the Arabidopsis LMCO family (At2g29130, At5g01190, and At5g05390) were also investigated with reporter gene constructs and knockout mutants. Expression of these LMCOs was associated with lignifying xylem, and the genes had over-lapping expression. Single knockout mutants did not display obvious phenotypes, suggesting that the gene products might have degenerate functionality that could compensate for loss of a single LMCO function.
Date: June 9, 2008
Creator: Dean, Jeffrey F.D.
Item Type: Report
Partner: UNT Libraries Government Documents Department

Regulating Intracellular Calcium in Plants: From Molecular Genetics to Physiology

Description: To grow, develop, adapt, and reproduce, plants have evolved mechanisms to regulate the uptake, translocation and sorting of calcium ions into different cells and subcellular compartments. Yet how plants accomplish this remarkable feat is still poorly understood. The spatial and temporal changes in intracellular [Ca2+] during growth and during responses to hormonal and environmental stimuli indicate that Ca2+ influx and efflux transporters are diverse and tightly regulated in plants. The specific goals were to determine the biological roles of multiple Ca pumps (ECAs) in the model plant Arabidopsis thaliana. We had pioneered the use of K616 yeast strain to functionally express plant Ca pumps, and demonstrated two distinct types of Ca pumps in plants (Sze et al., 2000. Annu Rev Plant Biol. 51,433). ACA2 represented one type that was auto-inhibited by the N-terminal region and stimulated by calmodulin. ECA1 represented another type that was not sensitive to calmodulin and phylogenetically distinct from ACAs. The goal to determine the biological roles of multiple ECA-type Ca pumps in Arabidopsis has been accomplished. Although we demonstrated ECA1 was a Ca pump by functional expression in yeast, the in vivo roles of ECAs was unclear. A few highlights are described. ECA1 and/or ECA4 are Ca/Mn pumps localized to the ER and are highly expressed in all cell types. Using homozygous T-DNA insertional mutants of eca1, we demonstrated that the ER-bound ECA1 supports growth and confers tolerance of plants growing on medium low in Ca or containing toxic levels of Mn. This is the first genetic study to determine the in vivo function of a Ca pump in plants. A phylogenetically distinct ECA3 is also a Ca/Mn pump that is localized to endosome, such as post-Golgi compartments. Although it is expressed at lower levels than ECA1, eca3 mutants are impaired in Ca-dependent root growth and ...
Date: June 22, 2008
Creator: Sze, Heven
Item Type: Report
Partner: UNT Libraries Government Documents Department

Light responses in Photoperiodism in Arabidopsis thaliana

Description: ADO1: An Arabidopsis blue light photoreceptor We have reported the characterization of an Arabidopsis gene encoding the ADAGIO 1 (ADO1) protein (Jarillo et al., 2001a). ADO1 contains a LOV domain, similar to WHITE COLLAR 1 (WC1), a photoreceptor for entrainment of Neurospora circadian rhythms (Froehlich et al., 2002), as well as PHOT1 and PHOT2, the blue light photoreceptors for phototropism (Briggs et al., 2001; Christie et al., 1998; Jarillo et al., 2001b; Kinoshita et al., 2001). Loss of function ado1 mutants show an unusually long periodicity for their free running circadian rhythm (Jarillo et al., 2001a). This observation holds for plants grown under white light as well as blue light and surprisingly, plants grown under red light also show altered circadian properties. The similarity of the LOV domain of ADO1 to those of PHOT1, PHOT2 and WC1 (known flavoprotein photoreceptors) as well as the genetic and molecular properties of ADO1, indicate that ADO1 is likely a new class of blue light photoreceptor. Indeed, the LOV domain of the related FKF1/ADO3 has been shown to bind FMN, and exhibit the in vitro photochemistry characteristic of PHOT1 (Imaizumi et al., 2003). Furthermore, ZTL/ADO1 has been shown to participate in the circadian and proteasome mediated degradation of the Arabidopsis clock protein, TOC1 (Mas et al., 2003). We also showed that the ado1 mutation selectively confers hypersensitivity to red light — when grown under red light (but not blue light) the ado1 mutant possesses an unusually short hypocotyl. This red light hypersensivity is even more severe in a triple ado1 ado2 ado3 mutant — ADO2 and ADO3 being the two other members of this ADAGIO gene family. This finding of a mutant phenotype under red light is somewhat unexpected for a protein thought to function as a photoreceptor for blue light. We have pursued ...
Date: August 1, 2006
Creator: Cashmore, Anthony R.
Item Type: Report
Partner: UNT Libraries Government Documents Department

Phytoremediation of Ionic and Methyl Mercury Pollution

Description: Phytoremediation is defined as the use of plants to extract, resist, detoxify, and/or sequester toxic environmental pollutants. The long-term goal of the proposed research is to develop and test highly productive, field-adapted plant species that have been engineered for the phytoremediation of mercury. A variety of different genes, which should enable plants to clean mercury polluted sites are being tested as tools for mercury phytoremediation, first in model laboratory plants and then in potential field species. Several of these genes have already been shown to enhance mercury phytoremediation. Mercury pollution is a serious, world-wide problem affecting the health of human and wildlife populations. Environmentally, the most serious mercury threat is the production of methylmercury (CH3Hg+) by native bacteria at mercury contaminated wetland sites. Methylmercury is inherently more toxic than metallic (Hg(0)) or ionic (Hg(II)) mercury, and because methylmercury is prolifically biomagnified up the food chain, it poses the most immediate danger to animal populations. We have successfully engineered two model plants, Arabidopsis and tobacco, to use the bacterial merB gene to convert methylmercury to less toxic ionic mercury and to use the bacterial merA gene to further detoxify ionic mercury to the least toxic form of mercury, metallic mercury. Plants expressing both MerA and MerB proteins detoxify methylmercury in two steps to the metallic form. These plants germinate, grow, and set seed at normal growth rates on levels of methylmercury or ionic mercury that are lethal to normal plants. Our newest efforts involve engineering plants with several additional bacterial and plant genes that allow for higher levels of mercury resistance and mercury hyperaccumulation. The potential for these plants to hyperaccumulate mercury was further advanced by developing constitutive, aboveground, and root-specific gene expression systems. Our current strategy is to engineer plants to control the chemical speciation, electrochemical state, transport, and aboveground ...
Date: June 1, 2005
Creator: Meagher, Richard B.
Item Type: Report
Partner: UNT Libraries Government Documents Department

A Phytoremediation Strategy for Arsenic

Description: A Phytoremediation Strategy for Arsenic Progress Report May, 2005 Richard B. Meagher Principal Investigator Arsenic pollution affects the health of several hundred millions of people world wide, and an estimated 10 million Americans have unsafe levels of arsenic in their drinking water. However, few environmentally sound remedies for cleaning up arsenic contaminated soil and water have been proposed. Phytoremediation, the use of plants to extract and sequester environmental pollutants, is one new technology that offers an ecologically sound solution to a devastating problem. We propose that it is less disruptive to the environment to harvest and dispose of several thousand pounds per acre of contaminated aboveground plant material, than to excavate and dispose of 1 to 5 million pounds of contaminated soil per acre (assumes contamination runs 3 ft deep). Our objective is to develop a genetics-based phytoremediation strategy for arsenic removal that can be used in any plant species. This strategy requires the enhanced expression of several transgenes from diverse sources. Our working hypothesis is that organ-specific expression of several genes controlling the transport, electrochemical state, and binding of arsenic will result in the efficient extraction and hyperaccumulation of arsenic into aboveground plant tissues. This hypothesis is supported by theoretical arguments and strong preliminary data. We proposed six Specific Aims focused on testing and developing this arsenic phytoremediation strategy. During the first 18 months of the grant we made significant progress on five Specific Aims and began work on the sixth as summarized below. Specific Aim 1: Enhance plant arsenic resistance and greatly expand sinks for arsenite by expressing elevated levels of thiol-rich, arsenic-binding peptides. Hyperaccumulation of arsenic depends upon making plants that are both highly tolerant to arsenic and that have the capacity to store large amounts of arsenic aboveground. Phytochelatins bind diverse thiol-reactive elements like As(III) and ...
Date: June 1, 2005
Creator: Meagher, Richard B.
Item Type: Report
Partner: UNT Libraries Government Documents Department

Phytoremediation of Ionic and Methyl Mercury Pollution

Description: Phytoremediation is defined as the use of plants to extract, resist, detoxify, and/or sequester toxic environmental pollutants. The long-term goal of the proposed research is to develop and test highly productive, field-adapted plant species that have been engineered for the phytoremediation of mercury. A variety of different genes, which should enable plants to clean mercury polluted sites are being tested as tools for mercury phytoremediation, first in model laboratory plants and then in potential field species. Several of these genes have already been shown to enhance mercury phytoremediation. Mercury pollution is a serious, world-wide problem affecting the health of human and wildlife populations. Environmentally, the most serious mercury threat is the production of methylmercury (CH3Hg+) by native bacteria at mercury contaminated wetland sites. Methylmercury is inherently more toxic than metallic (Hg(0)) or ionic (Hg(II)) mercury, and because methylmercury is prolifically biomagnified up the food chain, it poses the most immediate danger to animal populations. We have successfully engineered two model plants, Arabidopsis and tobacco, to use the bacterial merB gene to convert methylmercury to less toxic ionic mercury and to use the bacterial merA gene to further detoxify ionic mercury to the least toxic form of mercury, metallic mercury. Plants expressing both MerA and MerB proteins detoxify methylmercury in two steps to the metallic form. These plants germinate, grow, and set seed at normal growth rates on levels of methylmercury or ionic mercury that are lethal to normal plants. Our newest efforts involve engineering plants with several additional bacterial and plant genes that allow for higher levels of mercury resistance and mercury hyperaccumulation. The potential for these plants to hyperaccumulate mercury was further advanced by developing constitutive, aboveground, and root-specific gene expression systems.
Date: December 1, 2004
Creator: Meagher, Richard B.
Item Type: Report
Partner: UNT Libraries Government Documents Department

Molecular and Genetic Analysis of Hormone-Regulated Differential Cell Elongation in Arabidopsis

Description: The authors have utilized the response of Arabidopsis seedlings to the plant hormone ethylene to identify new genes involved in the regulation of ethylene biosynthesis, perception, signal transduction and differential cell growth. In building a genetic framework for the action of these genes, they developed a molecular model that has facilitated the understanding of the molecular requirements of ethylene for cell elongation processes. The ethylene response pathway in Arabidopsis appears to be primarily linear and is defined by the genes: ETR1, ETR2, ERS1, ERS2, EIN4, CTR1, EIN2, EIN3, EIN5 EIN6, and EIN. Downstream branches identified by the HLS1, EIR1, and AUX1 genes involve interactions with other hormonal (auxin) signals in the process of differential cell elongation in the hypocotyl hook. Cloning and characterization of HLS1 and three HLS1-LIKE genes in the laboratory has been supported under this award. HLS1 is required for differential elongation of cells in the hypocotyl and may act in the establishment of hormone gradients. Also during the award period, they have identified and begun preliminary characterization of two genes that genetically act upstream of the ethylene receptors. ETO1 and RAN1 encode negative regulators of ethylene biosynthesis and signaling respectively. Progress on the analysis of these genes along with HOOKLESS1 is described.
Date: December 3, 2002
Creator: Ecker, Joseph R.
Item Type: Report
Partner: UNT Libraries Government Documents Department