Calcium signal transduction Calcium plays an integral role as a messenger molecule in most eukaryotic cells and certainly has been implicated as a pleiotropic regulator in higher plant cell responses. We are interested in understanding the structure, function and mechanism of action of plant calcium modulatory proteins with a focus on two: calmodulin and the calmodulin-like domain protein kinase (CDPK). 2. Structure and function of Aquaporins and plant MIPs Based on the finding that calcium and CDPK appear to regulate various stress phenomena in higher plants, a more recent focus has been the investigation of a family of membrane proteins known as the major intrinsic protein family. This family includes proteins that play a critical role in water relations by facilitating rapid water flow through biological membranes. Several of these are regulated by calcium-dependent phosphorylation, including members of the nodulin 26 sub family. Our goal is to understand from a structural and functional perspective, the selectivity of these channels and the mechanism through which phosphorylation gates their activities.
Description of Research
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.
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.
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.
Li, T., Choi, W. G., Wallace, I. S., Baudry, J., and Roberts, D. M. (2011) Biochemistry, in press. "Arabidopsis thalianaNIP7;1: An Anther-Specific Boric Acid Transporter of the Aquaporin Superfamily Regulated by an Unusual Tyrosine in Helix 2 of the Transport Pore"
Hwang, J. H., Ellingson, S. R., and Roberts, D. M. (2010) FEBS Lett 584, 4339-4343. "Ammonia permeability of the soybean nodulin 26 channel"
Masalkar, P., Wallace, I. S., Hwang, J. H., and Roberts, D. M. (2010) J Biol Chem 285, 23880-23888. "Interaction of cytosolic glutamine synthetase of soybean root nodules with the C-terminal domain of the symbiosome membrane nodulin 26 aquaglyceroporin"
Roberts, D. M., Hwang JH and Choi WG (2010) Strategies for adaptation to waterlogging and hypoxia in nitrogen fixing nodules of legumes. in Waterlogging Signalling and Tolerance in Plants. (S. Mancuso and S. Shabala, eds.). Springer-Verlag Berlin Heidelberg, pp. 37-59.
Shakesby, A. J., Wallace, I. S., Isaacs, H. V., Pritchard, J., Roberts, D. M., and Douglas, A. E. (2009) Insect Biochem Mol Biol 39, 1-10. "A water-specific aquaporin involved in aphid osmoregulation"
Tanaka M, Wallace IS, Takano J, Roberts, D. M. and Fujiwara T (2008) NIP6;1 is a boric acid channel for preferential transport of boron to growing shoot tissues in Arabidopsis. Plant Cell. 20:2860-75.
Choi WG and Roberts, D. M. (2007) Arabidopsis NIP2;1, a major intrinsic protein transporter of lactic acid induced by anoxic stress. J Biol Chem. 282:24209-18.
Wallace IS, Choi WG and Roberts, D. M. (2006) The structure, function and regulation of the nodulin 26-like intrinsic protein family of plant aquaglyceroporins. Biochim Biophys Acta. 1758:1165-75.
Guenther JF, Seki S, Kleinhans FW, Edashige K, Roberts, D. M. and Mazur P. Extra- and intra-cellular ice formation in Stage I and II Xenopus laevis oocytes. Cryobiology. 52:401-16.
Kleinhans FW, Guenther JF, Roberts, D. M. and Mazur P (2006) Analysis of intracellular ice nucleation in Xenopus oocytes by differential scanning calorimetry. Cryobiology. 52:128-38.