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Robert J Ferl

Publications and source records attributed to Robert J Ferl.

17 recordsLinked to original sources
PubMed2006

High magnetic field induced changes of gene expression in arabidopsis.

BACKGROUND: High magnetic fields are becoming increasingly prevalent components of non-invasive, biomedical imaging tools (such as MRI), thus, an understanding of the molecular impacts associated with these field strengths in biological systems is of central importance. The biological impact of magnetic field strengths up to 30 Tesla were investigated in this study through the use of transgenic Arabidopsis plants engineered with a stress response gene consisting of the alcohol dehydrogenase (Adh) gene promoter driving the beta-glucuronidase (GUS) gene reporter. METHODS: Magnetic field induced Adh/GUS activity was evaluated with histochemical staining to assess tissue specific expression and distribution, and with quantitative, spectrofluometric assays to measure degree of activation. The evaluation of global changes in the Arabidopsis genome in response to exposure to high magnetic fields was facilitated with Affymetrix Gene Chip microarrays. Quantitative analyses of gene expression were performed with quantitative real-time polymerase-chain-reaction (qRT-PCR). RESULTS: Field strengths in excess of about 15 Tesla induce expression of the Adh/GUS transgene in the roots and leaves. From the microarray analyses that surveyed 8000 genes, 114 genes were differentially expressed to a degree greater than 2.5 fold over the control. These results were quantitatively corroborated by qRT-PCR examination of 4 of the 114 genes. CONCLUSION: The data suggest that magnetic fields in excess of 15 Tesla have far-reaching effect on the genome. The wide-spread induction of stress-related genes and transcription factors, and a depression of genes associated with cell wall metabolism, are prominent examples. The roles of magnetic field orientation of macromolecules and magnetophoretic effects are discussed as possible factors that contribute to the mounting of this response.

Journal Article↗
PubMed2006

Exposed loop domains of complexed 14-3-3 proteins contribute to structural diversity and functional specificity.

The 14-3-3 family of proteins functions through protein:phosphoprotein interactions, the nature of which has been elucidated using x-ray crystallography. However, some key structural features in nonconserved regions have yet to be fully resolved, leaving open questions regarding the functional selectivity of 14-3-3 family members for diverse clients. In an effort to study surface accessible structural features in 14-3-3 containing macromolecular complexes and to illuminate important structure/function variations among the 14-3-3 isoforms, we determined the epitopes for three unique monoclonal antibodies (mAbs) developed against the Arabidopsis (Arabidopsis thaliana) G-box DNA:protein complex. The epitopes mapped to different loops in a phylogenetically important subset of the 13 14-3-3 family members. All three epitopes were on a common exposed face of complexed 14-3-3s. Two of the mAbs recognized linear sequences within loops 5 and 6, while the third mAb recognized 14-3-3 residues surrounding the pivotal medial Gly in the divalent cation-binding domain of loop 8, together with distal residue(s) in the putative dynamic 10th helix that has yet to be determined by crystallography. Gly at this loop 8 position is unique to nonepsilon 14-3-3 isoforms of the plant kingdom, suggesting that this region constitutes a plant-specific key functional 14-3-3 feature and highlighting that the loop 8 region is functionally significant. Mutagenesis of the medial amino acid in the loop 8 domain changed the flexibility of the C terminus and altered client peptide-binding selectivity, demonstrating the functional significance of the surface accessible, evolutionarily distinct loop 8 domain.

14-3-3 Proteins↗
PubMed2006

Proteomic analysis of the 14-3-3 family in Arabidopsis.

In this study, various proteomics-based methods were utilized to examine the 14-3-3 protein family in Arabidopsis thaliana. A protein extract was prepared from an Arabidopsis hypocotyl suspension culture and analyzed by two-dimensional gel electrophoresis and immunoblotting with a 14-3-3 monoclonal antibody that recognizes multiple Arabidopsis isoforms. Protein spots that cross-reacted with the monoclonal antibody as well as the surrounding spots were analyzed by high performance liquid chromatography in conjunction with electrospray-tandem mass spectrometry. Nine separate spots contained 14-3-3s and each spot contained multiple 14-3-3 isoforms. Every isoform observed was verified by the identification of at least one isoform-specific peptide. Further analysis by mass spectrometry revealed that the isoforms Chi, Upsilon, Omega, Phi, and Lambda were acetylated on their N termini and no non-acetylated N termini were recovered. These data, together with the distribution of isoforms and the confirmation that 14-3-3s are not complexed during urea denaturing isoelectric focusing, supports the conclusion that Arabidopsis 14-3-3s are acetylated in vivo and are significantly affected by other post-translational modifications.

14-3-3 Proteins↗
PubMed2006

Exposure of Arabidopsis thaliana to hypobaric environments: implications for low-pressure bioregenerative life support systems for human exploration missions and terraforming on Mars.

Understanding how hypobaria can affect net photosynthetic (P (net)) and net evapotranspiration rates of plants is important for the Mars Exploration Program because low-pressured environments may be used to reduce the equivalent system mass of near-term plant biology experiments on landers or future bioregenerative advanced life support systems. Furthermore, introductions of plants to the surface of a partially terraformed Mars will be constrained by the limits of sustainable growth and reproduction of plants to hypobaric conditions. To explore the effects of hypobaria on plant physiology, a low-pressure growth chamber (LPGC) was constructed that maintained hypobaric environments capable of supporting short-term plant physiological studies. Experiments were conducted on Arabidopsis thaliana maintained in the LPGC with total atmospheric pressures set at 101 (Earth sea-level control), 75, 50, 25 or 10 kPa. Plants were grown in a separate incubator at 101 kPa for 6 weeks, transferred to the LPGC, and acclimated to low-pressure atmospheres for either 1 or 16 h. After 1 or 16 h of acclimation, CO(2) levels were allowed to drawdown from 0.1 kPa to CO(2) compensation points to assess P (net) rates under different hypobaric conditions. Results showed that P (net) increased as the pressures decreased from 101 to 10 kPa when CO(2) partial pressure (pp) values were below 0.04 kPa (i.e., when ppCO2 was considered limiting). In contrast, when ppCO(2) was in the nonlimiting range from 0.10 to 0.07 kPa, the P (net) rates were insensitive to decreasing pressures. Thus, if CO(2 )concentrations can be kept elevated in hypobaric plant growth modules or on the surface of a partially terraformed Mars, P (net) rates may be relatively unaffected by hypobaria. Results support the conclusions that (i) hypobaric plant growth modules might be operated around 10 kPa without undue inhibition of photosynthesis and (ii) terraforming efforts on Mars might require a surface pressure of at least 10 kPa (100 mb) for normal growth of deployed plant species.

Arabidopsis↗
PubMed2005

Isoform-specific subcellular localization among 14-3-3 proteins in Arabidopsis seems to be driven by client interactions.

In most higher eukaryotes, the predominantly phosphoprotein-binding 14-3-3 proteins are the products of a multigene family, with many organisms having 10 or more family members. However, current models for 14-3-3/phosphopeptide interactions suggest that there is little specificity among 14-3-3s for diverse phosphopeptide clients. Therefore, the existence of sequence diversity among 14-3-3s within a single organism begs questions regarding the in vivo specificities of the interactions between the various 14-3-3s and their clients. Chief among those questions is, Do the different 14-3-3 isoforms interact with different clients within the same cell? Although the members of the Arabidopsis 14-3-3 family of proteins typically contain highly conserved regions of sequence, they also display distinctive variability with deep evolutionary roots. In the current study, a survey of several Arabidopsis 14-3-3/GFP fusions revealed that 14-3-3s demonstrate distinct and differential patterns of subcellular distribution, by using trichomes and stomate guard cells as in vivo experimental cellular contexts. The effects of client interaction on 14-3-3 localization were further analyzed by disrupting the partnering with peptide and chemical agents. Results indicate that 14-3-3 localization is both isoform specific and highly dependent upon interaction with cellular clients.

14-3-3 Proteins↗
PubMed2005

Plant molecular biology in the space station era: utilization of KSC fixation tubes with RNAlater.

Spaceflight experiments involving biological specimens face unique challenges with regard to the on orbit harvest and preservation of material for later ground-based analyses. Preserving plant material for gene expression analyses requires that the tissue be prepared and stored in a manner that maintains the integrity of RNA. The liquid preservative RNAlater (Ambion) provides an effective alternative to conventional freezing strategies, which are limited or unavailable in current spaceflight experiment scenarios. The spaceflight use of RNAlater is enabled by the Kennedy space center fixation tube (KFT), hardware designed to provide the necessary containment of fixatives during the harvest and stowage of biological samples in space. Pairing RNAlater with the KFT system provides a safe and effective strategy for preserving plant material for subsequent molecular analyses, a strategy that has proven effective in several spaceflight experiments. Possible spaceflight scenarios for the use of RNAlater and KFTs are explored and discussed.

Arabidopsis↗
PubMed2005

Identification and characterization of GIP1, an Arabidopsis thaliana protein that enhances the DNA binding affinity and reduces the oligomeric state of G-box binding factors.

Environmental control of the alcohol dehydrogenase (Adh) and other stress response genes in plants is in part brought about by transcriptional regulation involving the G-box cis-acting DNA element and bZIP G-box Binding Factors (GBFs). The mechanisms of GBF regulation and requirements for additional factors in this control process are not well understood. In an effort to identify potential GBF binding and control partners, maize GBF1 was used as bait in a yeast two-hybrid screen of an A. thaliana cDNA library. GBF Interacting Protein 1 (GIP1) arose from the screen as a 496 amino acid protein with a predicted molecular weight of 53,748 kDa that strongly interacts with GBFs. Northern analysis of A. thaliana tissue suggests a 1.8-1.9 kb GIP1 transcript, predominantly in roots. Immunolocalization studies indicate that GIP1 protein is mainly localized to the nucleus. In vitro electrophoretic mobility shift assays using an Adh G-box DNA probe and recombinant A. thaliana GBF3 or maize GBF1, showed that the presence of GIP1 resulted in a tenfold increase in GBF DNA binding activity without altering the migration, suggesting a transient association between GIP1 and GBF. Addition of GIP1 to intentionally aggregated GBF converted GBF to lower molecular weight macromolecular complexes and GIP1 also refolded denatured rhodanese in the absence of ATP. These data suggest GIP1 functions to enhance GBF DNA binding activity by acting as a potent nuclear chaperone or crowbar, and potentially regulates the multimeric state of GBFs, thereby contributing to bZIP-mediated gene regulation.

Arabidopsis↗
PubMed2004

Plasma membrane H(+)-ATPase and 14-3-3 isoforms of Arabidopsis leaves: evidence for isoform specificity in the 14-3-3/H(+)-ATPase interaction.

The plasma membrane H(+)-ATPase is activated by binding of 14-3-3 protein to the phosphorylated C terminus. Considering the large number of 14-3-3 and H(+)-ATPase isoforms in Arabidopsis (13 and 11 expressed genes, respectively), specificity in binding may exist between 14-3-3 and H(+)-ATPase isoforms. We now show that the H(+)-ATPase is the main target for 14-3-3 binding at the plasma membrane, and that all twelve 14-3-3 isoforms tested bind to the H(+)-ATPase in vitro. Using specific antibodies for nine of the 14-3-3 isoforms, we show that GF14epsilon, mu, lambda, omega, chi, phi, nu, and upsilon are present in leaves, but that isolated plasma membranes lack GF14chi, phi and upsilon. Northern blots using isoform-specific probes for all 14-3-3 and H(+)-ATPase isoforms showed that transcripts were present for most of the isoforms. Based on mRNA levels, GF14epsilon, mu, lambda and chi are highly expressed 14-3-3 isoforms, and AHA1, 3, and 11 highly expressed H(+)-ATPase isoforms in leaves. However, mass peptide fingerprinting identified AHA1 and 2 with the highest score, and their presence could be confirmed by MS/MS. It may be calculated that under 'unstressed' conditions less than one percent of total 14-3-3 is attached to the H(+)-ATPase. However, during a condition requiring full activation of H+ pumping, as induced here by the presence of the fungal toxin fusicoccin, several percent of total 14-3-3 may be engaged in activation of the H(+)-ATPase.

14-3-3 Proteins↗
PubMed2003

Hypobaric biology: Arabidopsis gene expression at low atmospheric pressure.

As a step in developing an understanding of plant adaptation to low atmospheric pressures, we have identified genes central to the initial response of Arabidopsis to hypobaria. Exposure of plants to an atmosphere of 10 kPa compared with the sea-level pressure of 101 kPa resulted in the significant differential expression of more than 200 genes between the two treatments. Less than one-half of the genes induced by hypobaria are similarly affected by hypoxia, suggesting that response to hypobaria is unique and is more complex than an adaptation to the reduced partial pressure of oxygen inherent to hypobaric environments. In addition, the suites of genes induced by hypobaria confirm that water movement is a paramount issue at low atmospheric pressures, because many of gene products intersect abscisic acid-related, drought-induced pathways. A motivational constituent of these experiments is the need to address the National Aeronautics and Space Administration's plans to include plants as integral components of advanced life support systems. The design of bioregenerative life support systems seeks to maximize productivity within structures engineered to minimize mass and resource consumption. Currently, there are severe limitations to producing Earth-orbital, lunar, or Martian plant growth facilities that contain Earth-normal atmospheric pressures within light, transparent structures. However, some engineering limitations can be offset by growing plants in reduced atmospheric pressures. Characterization of the hypobaric response can therefore provide data to guide systems engineering development for bioregenerative life support, as well as lead to fundamental insights into aspects of desiccation metabolism and the means by which plants monitor water relations.

Adaptation, Physiological↗
PubMed2002

The 14-3-3s.

Multiple members of the 14-3-3 protein family have been found in all eukaryotes so far investigated, yet they are apparently absent from prokaryotes. The major native forms of 14-3-3s are homo- and hetero-dimers, the biological functions of which are to interact physically with specific client proteins and thereby effect a change in the client. As a result, 14-3-3s are involved in a vast array of processes such as the response to stress, cell-cycle control, and apoptosis, serving as adapters, activators, and repressors. There are currently 133 full-length sequences available in GenBank for this highly conserved protein family. A phylogenetic tree based on the conserved middle core region of the protein sequences shows that, in plants, the 14-3-3 family can be divided into two clearly defined groups. The core region encodes an amphipathic groove that binds the multitude of client proteins that have conserved 14-3-3-recognition sequences. The amino and carboxyl termini of 14-3-3 proteins are much more divergent than the core region and may interact with isoform-specific client proteins and/or confer specialized subcellular and tissue localization.

14-3-3 Proteins↗
PubMed2002

Molecular aspects of stress-gene regulation during spaceflight.

Spaceflight-associated stress has been the topic of investigation since the first terrestrial organisms were exposed to this unique environment. Organisms that evolved under the selection pressures of earth-normal environments can perceive spaceflight as a stress, either directly because gravity influences an intrinsic biological process, or indirectly because of secondary effects imparted by spaceflight upon environmental conditions. Different organisms and even different organs within an organism adapt to a spaceflight environment with a diversity of tactics. Plants are keenly sensitive to gravity for directed development, and are also sensitive to other stresses associated with closed-system spaceflight environments. Within the past decade, the tools of molecular biology have begun to provide a sophisticated evaluation of spaceflight-associated stress and the genetic responses that accompany metabolic adaptation to spaceflight.

Adaptation, Physiological↗
PubMed2002

Evolution and isoform specificity of plant 14-3-3 proteins.

The 14-3-3 proteins, once thought of as obscure mammalian brain proteins, are fast becoming recognized as major regulators of plant primary metabolism and of other cellular processes. Their presence as large gene families in plants underscores their essential role in plant physiology. We have examined the Arabidopsis thaliana 14-3-3 gene family, which currently is the largest and most complete 14-3-3 family with at least 12 expressed members and 15 genes from the now completed Arabidopsis thaliana genome project. The phylogenetic branching of this family serves as the prototypical model for comparison with other large plant 14-3-3 families and as such may serve to rationalize clustering in a biological context. Equally important for ascribing common functions for the various 14-3-3 isoforms is determining an isoform-specific correlation with localization and target partnering. A summary of localization information available in the literature is presented. In an effort to identify specific 14-3-3 isoform location and participation in cellular processes, we have produced a panel of isoform-specific antibodies to Arabidopsis thaliana 14-3-3s and present initial immunolocalization studies that suggest biologically relevant, discriminative partnering of 14-3-3 isoforms.

14-3-3 Proteins↗
PubMed2002

Remote sensing of gene expression in Planta: transgenic plants as monitors of exogenous stress perception in extraterrestrial environments.

Transgenic arabidopsis plants containing the alcohol dehydrogenase (Adh) gene promoter fused to the green fluorescent protein (GFP) reporter gene were developed as biological sensors for monitoring physiological responses to unique environments. Plants were monitored in vivo during exposure to hypoxia, high salt, cold, and abcissic acid in experiments designed to characterize the utility and responses of the Adh/GFP biosensors. Plants in the presence of environmental stimuli that induced the Adh promoter responded by expressing GFP, which in turn generated a detectable fluorescent signal. The GFP signal degraded when the inducing stimulus was removed. Digital imaging of the Adh/GFP plants exposed to each of the exogenous stresses demonstrated that the stress-induced gene expression could be followed in real time. The experimental results established the feasibility of using a digital monitoring system for collecting gene expression data in real time from Transgenic Arabidopsis Gene Expression System (TAGES) biosensor plants during space exploration experiments.

Alcohol Dehydrogenase↗
PubMed2002

Plant adaptation to low atmospheric pressures: potential molecular responses.

There is an increasing realization that it may be impossible to attain Earth normal atmospheric pressures in orbital, lunar, or Martian greenhouses, simply because the construction materials do not exist to meet the extraordinary constraints imposed by balancing high engineering requirements against high lift costs. This equation essentially dictates that NASA have in place the capability to grow plants at reduced atmospheric pressure. Yet current understanding of plant growth at low pressures is limited to just a few experiments and relatively rudimentary assessments of plant vigor and growth. The tools now exist, however, to make rapid progress toward understanding the fundamental nature of plant responses and adaptations to low pressures, and to develop strategies for mitigating detrimental effects by engineering the growth conditions or by engineering the plants themselves. The genomes of rice and the model plant Arabidopsis thaliana have recently been sequenced in their entirety, and public sector and commercial DNA chips are becoming available such that thousands of genes can be assayed at once. A fundamental understanding of plant responses and adaptation to low pressures can now be approached and translated into procedures and engineering considerations to enhance plant growth at low atmospheric pressures. In anticipation of such studies, we present here the background arguments supporting these contentions, as well as informed speculation about the kinds of molecular physiological responses that might be expected of plants in low-pressure environments.

Acclimatization↗
PubMed2002

Near-term lander experiments for growing plants on Mars: requirements for information on chemical and physical properties of Mars regolith.

In order to support humans for long-duration missions to Mars, bioregenerative Advanced Life Support (ALS) systems have been proposed that would use higher plants as the primary candidates for photosynthesis. Hydroponic technologies have been suggested as the primary method of plant production in ALS systems, but the use of Mars regolith as a plant growth medium may have several advantages over hydroponic systems. The advantages for using Mars regolith include the likely bioavailability of plant-essential ions, mechanical support for plants, and easy access of the material once on the surface. We propose that plant biology experiments must be included in near-term Mars lander missions in order to begin defining the optimum approach for growing plants on Mars. Second, we discuss a range of soil chemistry and soil physics tests that must be conducted prior to, or in concert with, a plant biology experiment in order to properly interpret the results of plant growth studies in Mars regolith. The recommended chemical tests include measurements on soil pH, electrical conductivity and soluble salts, redox potential, bioavailability of essential plant nutrients, and bioavailability of phytotoxic elements. In addition, a future plant growth experiment should include procedures for determining the buffering and leaching requirements of Mars regolith prior to planting. Soil physical tests useful for plant biology studies in Mars regolith include bulk density, particle size distribution, porosity, water retention, and hydraulic conductivity.

Biological Availability↗