Dan Roberts, Ph.D.
Research Statement
I. Membrane water and solute transport processes of plants.
A. Transport processes of legume/rhizobia nitrogen fixing symbioses.
During the formation of legume-rhizobia symbioses, the bacteria infect a specialized plant cell (infected cell) within the core of the root nodule and become enclosed in an organelle called the symbiosome, the major organelle of this cell type. This organelle is delimited by the plant-derived symbiosome membrane which mediates the symbiotic exchange of nutrients (reduced carbon compounds from the plant cytosol in exchange for reduced nitrogen from the bacteroid), and serves to protect the endosymbiont from plant defense responses. Further, as the major organelle in a non vacuolated cell, the symbiosome also is crucial for osmotic and volume regulation. One of the focal points of the laboratory is the characterization of the transporters of this membrane that aid in the establishment and maintenance of the symbiosis. Principal focus is on two areas:
i. Nodulin 26, an aquaglyceroporin protein mediating water and ammonia flux across the symbiosome membrane. Nodulin 26 is a symbiosome membrane-specific protein that is the major protein component constituting 10-15% of the total protein mass of this membrane. Nodulin 26 is a member of the major intrinsic protein (MIP) super family of water and solute channels, and it confers a high water permeability upon the soybean symbiosome membrane and also mediates the rapid movement of uncharged solutes such as fixed ammonia, glycerol and possibly gases. In addition, nodulin 26 is a major phosphorylation target for a calcium-dependent protein kinase (CDPK), which also resides on the symbiosome membrane. These multifunctional protein kinases are now recognized to be principal targets of calcium signals, and catalyze the phosphorylation of multiple proteins including membrane channels, pumps and a number of metabolic enzymes. Phosphorylation of nodulin 26 occurs on one residue, ser262, which resides within the cytosolic carboxyl terminal tail of the protein and phosphorylation modulates an increase in the intrinsic rate of transport. Phosphorylation accompanies maturation of the nodule and is regulated in response to osmotic signals, supporting a role for nodulin 26 in the osmoregulation of the symbiosome membrane.
It is clear that nodulin 26 is a fundamental protein in the symbiosis that may play a multifunctional role in osmoregulation (water and glycerol transport) as well as transport of metabolically relevant solutes (fixed ammonia and possibly gases considering the low O2 tension). In addition, it has recently become clear that nodulin 26 and related proteins represent a unique structural and functional subclass of the larger plant MIP channel family (see below). Presently our interest lies in using a multidisciplinary approach of biophysical, structural, and molecular genetic techniques to investigate: 1. structural characterization of protein from the perspective of pore forming determinants that confer the selectivity and rate of transport; 2. the biological role of calcium-dependent phosphorylation of nodulin 26 and its effect on the function of the protein, and its trafficking between subcellular compartments and possible docking of regulatory proteins; and 3. its importance in stress adaptation in osmotically-challenged nodules.
ii. Ion transporters and channels of the symbiosome membrane Besides our investigation of nodulin 26, we have extended our analysis of the symbiosome membrane to include various other transporters, including ion channels and metabolite transporters. The symbiosome membrane is an energized membrane which generates a proton motive force through an H+-ATPase that drives metabolic exchange between the plant host and symbiont. We have investigated the transport properties of the membrane by using organelle-based patch clamp approaches as well as by the investigation of the properties of symbiosome membrane nodulins by heterologous expression in Xenopus oocytes for two electrode voltage clamp.
B. Plant MIP structure and function, and role in plant water relations.
MIPs represent an ancient family of membrane channel proteins. Over 100 genes encoding these proteins have been identified and they possess a similar structural architecture (tetrameric integral membrane proteins with six transmembrane a helices and an obverse symmetry). Despite this similarity, the proteins are strikingly diverse with respect to their selectivities, rates of transport and regulation. MIPs are thought to exist in all organisms from bacteria to higher eukaryotes, and these proteins are especially abundant in plants, with 35 members in Arabidopsis thaliana. Members of the plant MIP family have been implicated in cell elongation, root tip elongation, changes in hydraulic conductivity in response to environmental cues, and numerous other processes that require rapid transmembrane movements of water.
Recently we have used computational approaches using the recent crystal structures of aquaporin and glyceroporins from animals and microbes to investigate the structural and functional phylogeny of the plant MIP family. A surprising finding is that plant MIP structure transcends the traditional “aquaporin vs. glyceroporin” paradigm, and we identified at least 8 separate “pore subfamilies” some of which have pore forming determinants that are unprecedented in other species, underscoring the diversity and complexity of the MIP family in higher plants. To understand the molecular basis for its unique functional properties, we are investigating the structure of this protein by a variety of approaches including expression in yeast and Xenopus systems for biophysical analyses as well as using molecular genetic approaches to investigate the role of selected proteins in Arabidopsis.
II. Calcium-modulated proteins: targets and plant defense responses.
In higher plants, calcium signaling has been implicated in plant responses to a multitude of environmental stimuli including abiotic stresses such as cold, drought, salinity, and mechanical stimulation/wounding, biotic signals from invading pathogens and elicitors, as well as in numerous developmental and growth processes. The detection and “decoding” of these calcium signals is mediated by a battery of calcium sensor proteins that bind specifically to calcium ions with Kd in the physiological range of the intracellular Ca2+ transients. These sensors undergo a conformational change that results in a change in activity. Typically, proteins with these properties possess specialized calcium binding domains known as EF hands. One of the earliest calcium regulatory proteins detected in plants is calmodulin, a highly conserved, ubiquitous calcium sensor that has been implicated in the regulation of over twenty target proteins in higher plants.
A. NAD kinases. The first protein to be identified as a target for calmodulin regulation was NAD kinase which catalyzes the following reaction:
ATP + NAD+®ADP + NADP+
NAD+ and NADP+ have become universally recognized as critical carriers of reductive energy for fundamentally different metabolic processes. Besides its well-understood function in reductive biosynthesis in intermediary metabolism, NADPH also serves a role in the generation (via NADPH oxidase) as well as the detoxification (via the glutathione and thioredoxin reductases) of reactive oxygen intermediates. In addition, NADP+ also is a precursor for intracellular signaling molecules, such as the calcium mobilizing agent nicotinic acid adenine dinucleotide phosphate. Hence, the modulation of NADP+/NADPH pools is essential not only for control of metabolism, but also is important in a wide variety of other cellular processes ranging from adaptation to oxidative stress to the production of intracellular signaling molecules.
In previous work, we have investigated the biological role of calmodulin-dependent NAD kinase by ectopic expression of dominant positive derivatives of calmodulin in transgenic tobacco plants. Transgenic plants expressing this calmodulin show an enhanced capacity to produce reactive oxygen species in response to challenge with abiotic or biotic stress signals. Given the central signaling role of calcium and reactive oxygen in mediating plant defense response signal transduction, the findings suggest that NADK activation is part of the metabolic response to challenge by these environmental agents. Our present work is focused on the elucidation of the structural and functional properties of three NAD kinase isoforms found in the Arabidopsis genome, including the analysis of the structural properties of their interaction with calmodulin and other effectors, and the role that each may play in redox signaling in Arabidopsis.
Selected Publications
Vincill ED, Szczyglowski K and Roberts Daniel M (2005) GmN70 and LjN70: Anion transporters of the symbiosome membrane of nodules with a transport preference for nitrate. Plant Physiol. In press (Special Legume Issue)
Wallace IS and Roberts DM (2004) Homology modeling of representative subfamilies of Arabidopsis thaliana major intrinsic proteins: Classification based on the aromatic/arginine selectivity filter. Plant Physiol. 135: 1059-1068. (Special Arabidopsis Issue)
Guenther JF, Chanmanivone N, Galetovic MP, Wallace IS, Cobb JA and Roberts DM (2003) Phosphorylation of Soybean Nodulin 26 on Serine 262 Enhances Water Permeability and is Regulated Developmentally and by Osmotic Signals. Plant Cell 15: 981-991.
Roberts DM and Tyerman Stephen D (2002) Voltage-dependent cation channels permeable to NH4+, K+, and Ca2+ in the symbiosome membrane of the model legume Lotus japonicus. Plant Physiol. 128: 370-378.
Wallace IS, Wills DM, Guenther JF and Roberts DM (2002) Functional selectivity for glycerol of the nodulin 26 subfamily of plant membrane intrinsic proteins. FEBS Lett. 523: 109-112..
Cobb JA and Roberts DM (2000) Structural requirements for N-trimethylation of lysine-115 of calmodulin. J. Biol. Chem. 275: 18969-18975.
Guenther JF and Roberts DM (2000) Water selective and multifunctional aquaporins from nodules of Lotus japonicus. Planta, 210: 741-748.
Cobb JA, Han C -H, Wills DM and Roberts DM (1999) Structural elements within the methylation loop (residues 112-117) and EF hands III and IV of calmodulin are required for lysine-115 trimethylation. Biochem. J. 340: 417-424
Dean RM, Rivers RL, Zeidel ML and Roberts DM (1999) Purification and functional reconstitution of soybean nodulin 26. An aquaporin with water and glycerol transport properties. Biochemistry 38: 347-353.
Harding SA and Roberts DM (1998) Enhanced reactive oxygen production and HR-induced cell death in transgenic tobacco expressing a hyperactive mutant calmodulin. Planta 206: 253-258.
Harding SA, Oh S -H and Roberts DM (1997) Transgenic tobacco expressing a foreign calmodulin gene shows enhanced production of active oxygen species. EMBO J. 16: 1137-1144.
Wallace IS and Roberts DM (2004) Homology modeling of representative subfamilies of Arabidopsis thaliana major intrinsic proteins: Classification based on the aromatic/arginine selectivity filter. Plant Physiol. 135: 1059-1068. (Special Arabidopsis Issue)
Roberts DM, Besl L, Oh S -H, Masterson RM, Schell J and Stacey G (1992) Expression of a calmodulin methylation mutant affects the growth and development of transgenic tobacco plants. Proc. Natl. Acad. Sci. USA 89: 8394-8398.
Contact Information
Office:
Room F-431
Walters Life Sciences
Phone: (865) 974-4070
Lab:
Room D-401
Room D-407
Walters Life Sciences
Phone: (865) 974-2339 (D-401)
Phone: (865) 974-2364 (D-407)
Email: drobert2@utk.edu