From myewhat at seed.net.tw Fri Jun 10 14:41:28 2005 From: myewhat at seed.net.tw (David Lynch) Date: Sun Jun 12 16:41:29 2005 Subject: [Plant-signal-transduction] Welcome to Jen Sheen's Lab Message-ID: Welcome to Jen Sheen's Lab http://genetics.mgh.harvard.edu/sheenweb/ From myewhat at seed.net.tw Fri Jun 10 14:44:45 2005 From: myewhat at seed.net.tw (David Lynch) Date: Sun Jun 12 16:41:31 2005 Subject: [Plant-signal-transduction] Welcome to Jen Sheen's Lab Message-ID: Welcome to Jen Sheen's Lab http://genetics.mgh.harvard.edu/sheenweb/ From bogus@does.not.exist.com Mon Jun 13 10:09:55 2005 From: bogus@does.not.exist.com () Date: Mon Jun 13 10:09:54 2005 Subject: No subject Message-ID: 11:00am Coffee Break 11:30am James Putney "Coordination of Intracellular Calcium Release and Calcium Entry Across the Plasma Membrane" 12:30pm Lunch (Free to Pre-registered Participants) and Posters 2:30pm Karl Hasenstein "Magnetophoresis and the Cytoskeleton - Studies of Two Aspects of Plant Gravitropism" 3:30pm Jen Sheen "Signaling Pathways Mediated by Calcium-dependent Protein Kinases" Saturday, January 24, 1998 At the North Carolina Biotechnology Center 15 Alexander Drive, Research Triangle Park, North Carolina Registration Forms Are Enclosed Sponsored by the North Carolina State University Nasa Specialized Center of Research and Training in Gravitational Biology >>>>>>>>>>> Registration Form Calcium, Signal Transduction and Gravitational Biology Symposium Saturday, January 24, 1998 Please Return the Forms by January 16, 1998 You must Register to Get a Lunch. Name: Address: Yes Do You Want Lunch? Would You Prefer a Vegetarian Lunch? If You Want to Present a Poster Relating to Calcium, Signal Transduction or Gravity, Please Give the Title, Authors Names and Affiliations Below: Title: Authors: Affiliations: You Can Send this by Mail To: Linda Jenkins, North Carolina State University, Department of Botany, Box 7612, Gardner Hall, Raleigh NC 27695-7612, or E-mail Linda_jenkins@ncsu.edu, or Fax 919-515-3436. From bogus@does.not.exist.com Mon Jun 13 10:09:58 2005 From: bogus@does.not.exist.com () Date: Mon Jun 13 10:09:57 2005 Subject: No subject Message-ID: 11:00am Coffee Break 11:30am James Putney "Coordination of Intracellular Calcium Release and Calcium Entry Across the Plasma Membrane" 12:30pm Lunch (Free to Pre-registered Participants) and Posters 2:30pm Karl Hasenstein "Magnetophoresis and the Cytoskeleton - Studies of Two Aspects of Plant Gravitropism" 3:30pm Jen Sheen "Signaling Pathways Mediated by Calcium-dependent Protein Kinases" Saturday, January 24, 1998 At the North Carolina Biotechnology Center 15 Alexander Drive, Research Triangle Park, North Carolina Registration Forms Are Enclosed Sponsored by the North Carolina State University Nasa Specialized Center of Research and Training in Gravitational Biology >>>>>>>>>>> Registration Form Calcium, Signal Transduction and Gravitational Biology Symposium Saturday, January 24, 1998 Please Return the Forms by January 16, 1998 You must Register to Get a Lunch. Name: Address: Yes Do You Want Lunch? Would You Prefer a Vegetarian Lunch? If You Want to Present a Poster Relating to Calcium, Signal Transduction or Gravity, Please Give the Title, Authors Names and Affiliations Below: Title: Authors: Affiliations: You Can Send this by Mail To: Linda Jenkins, North Carolina State University, Department of Botany, Box 7612, Gardner Hall, Raleigh Nc 27695-7612, or E-mail Linda_jenkins@ncsu.edu, or Fax 919-515-3436. From bogus@does.not.exist.com Mon Jun 13 10:10:06 2005 From: bogus@does.not.exist.com () Date: Mon Jun 13 10:10:06 2005 Subject: No subject Message-ID: Bjorn Drobak myo-Inositol (hexahydroxycyclohexane) is essential for the growth of most bacterial, fungal, plant and mammalian cells. Cells without obvious requirement for exogenous inositol have all been found to be capable of de novo synthesis of inositol. Thus, myo-inositol is now recognized as an essential factor for normal cell growth and function. There are nine possible stereoisomers of inositol and seven of these have been found to occur naturally, the exceptions being epi- and allo-inositol. Myo-, D/L-chiro and scyllo-inositol are the major stereoisomers of inositol in plants although both muco- inositol and neo-inositol have been found to= exist. The presence of inositol in certain bacterial lipids was discovered around 1930 and the presence of inositol in brain phospholipids was reported by Folch and Woolley in 1942. A very wide range functions have in the last decades been proposed for inositol and its derivatives in both plant and animal cells, but it is the discovery that inositol-containing compounds play a key role in the perception and transmission of extracellular signals which has led to the current intense interest in this particular area of science.=20 The basic concepts of the eukaryotic phosphoinositide signal transducing system (PI-system) were delineated in mammalian and insect cells by M.J.Berridge, R.F.Irvine, R.H.Michell and their co- workers in the early 1980's, and PI-systems have since been found to be present in all the eukaryotic cells which so far have been studied.=20 The talk focused on recent advances in the understanding of how the plant PI system participates in the transmission of signals from the plasma membrane to the nucleus. The key topics presented in the talk were : - Generation of phosphoinositides by phosphoinositide kinases - PLC activity and its regulation - Changes in cytosolic Ins(1,4,5)P3 activity in plant cells - Interactions between components of the PI-system and the cytoskeleton - Causes and consequences of an uneven cellular distribution of phosphoinositide metabolising enzymes - Generation of Ca2+ waves and their dependency on Ins(1,4,5)P3 receptor occupancy - Nuclear Ca2+ fluxes and their regulation - 3-phosphoinositides and the regulation nuclear and nucleolar events - An integrated view of PI-signalling from the plasma membrane to the= nucleus Capacitative Calcium Entry=20 by James W. Putney, Jr. Calcium Regulation Section Laboratory of Signal Transduction National Institute of Environmental Health Sciences - NIH Research Triangle Park, NC 27709 In animal cells, a wide variety of hormones, neurotransmitters, growth factors and other physiological and pathological stimuli initiate cellular responses through the process of calcium signalling. Cellular calcium signalling involves a rise in the concentration of ionized calcium in the cytoplasm of cells. This increase in cytoplasmic calcium concentration ([Ca2+]i) can come either from the extracellular space, through channels in the plasma membrane, or from intracellular stores of Ca2+, again through specific channels in the limiting membranes of Ca2+-storing organelles. In the majority of cases, both modes are utilized (For example, see Figure 1). For electrically non-excitable cells (epithelial cells, blood cells, and fibroblasts for example), and in many instances for excitable cells this signalling is initiated by the activation of a polyphosphoinositide-specific phospholipase C which hydrolyzes membrane phospholipids to release the calcium signalling molecule, inositol 1,4,5-trisphosphate (IP3). IP3 binds to a receptor molecule located in the endoplasmic reticulum, or a specialized component of it(1,2). The IP3 receptor functions as a ligand-gated channel and its activation by IP3 leads to rapid release of Ca2+ to the cytoplasm. Intuitively, it would seem reasonable that IP3 is also involved in the signalling of Ca2+ entry across the plasma membrane. There are numerous examples in which direct application of IP3 to the cytoplasm of cells, whether by microinjection or by patch-clamp dialysis, activates calcium entry into cells(3). However, the evidence suggests that in the vast majority of cases this action is an indirect one. Rather than directly activating Ca2+ channels in the plasma membrane, it appears that the ability of IP3 to activate Ca2+ entry is secondary to, and dependent upon the mobilization of Ca2+ from intracellular Ca2+ stores. This concept, termed the capacitative model for Ca2+ entry, was first proposed on the basis of experiments examining the kinetics of refilling of intracellular Ca2+ stores following their depletion by a phospholipase C-linked agonist(4). A more direct demonstration of this phenomenon can be seen with the use of specific inhibitors of the intracellular Ca2+ transport ATPases which mediate the active accumulation of Ca2+ in the endoplasmic or sarcoplasmic reticulum (termed SERCA pumps for Sarcoplasmic Endoplasmic Reticulum ATPase pumps). The prototypical reagent in this class is the plant sesquiterpene lactone, thapsigargin(5). Thapsigargin is readily membrane permeant, and its application to cells results in a relatively rapid passive depletion of intracellular Ca2+ stores. Consistent with predictions of the capacitative model, Figure 1B illustrates that thapsigargin activates Ca2+ entry. Following activation of entry by thapsigargin, no further increase in entry is seen on application of a phospholipase C-stimulating agonist. This means that not only does depletion of Ca2+ stores provide a signal for the activation of Ca2+ entry, but also that this mechanism fully activates the available channels with no further potentiation or additional pathways activated by IP3 (or its metabolites). This basic finding, that depletion of intracellular Ca2+ stores provides a signal for activation of calcium entry, has been confirmed in a large number of cell types, and it is now clear that the capacitative model represents a widespread mechanism for control of calcium entry at the plasma membrane. Recent research has focused on the molecular identity of the channels underlying this phenomenon, and the nature of the signal that activates these channels in response to intracellular Ca2+ store= depletion(6). References 1. Rossier, M. F. and Putney, J. W., Jr. (1991). The identity of the calcium storing inositol 1,4,5-trisphosphate-sensitive organelle in non-muscle cells: Calciosome, endoplasmic reticulum, ... or both? Trends Neurosci. 14, 310-314. 2. Pozzan, T., Rizzuto, R., Volpe, P., and Meldolesi, J. (1994). Molecular and cellular physiology of intracellular calcium stores. Physiol.Rev. 74, 595-636. 3. Putney, J. W., Jr. and Bird, G. St. J. (1993). The inositol phosphate-calcium signalling system in non-excitable cells. Endocrine Rev. 14, 610-631. 4. Putney, J. W., Jr. (1986). A model for receptor-regulated calcium entry. Cell Calcium 7, 1-12. 5. Thastrup, O., Dawson, A. P., Scharff, O., Foder, B., Cullen, P. J., Drobak, B. K., Bjerrum, P. J., Christensen, S. B., and Hanley, M. R. (1994). Thapsigargin, a novel molecular probe for studying intracellular calcium release and storage. Agents Actions 43, 187-193. 6. Putney, J. W., Jr. (1997). Capacitative Calcium Entry pp. Landes Biomedical Publishing, Austin, TX. =20 Figure 1: Patterns of Calcium Release and Calcium Entry. Top: A single mouse lacrimal cell containing a fluorescent calcium indicator is activated by methacholine, an agonist for the phospholipase C-coupled muscarinic receptors. Comparison of the [Ca2+]i responses in the presence (green) and absence (red) of extracellular calcium reveals that the signal is comprised of two components, an intracellular release of Ca2+ as well as an activation of Ca2+ entry across the plasma membrane. Bottom: In a single lacrimal cell, the SERCA inhibitor, thapsigargin, activates entry of Ca2+, and this entry is not facilitated by methacholine (MeCh). Regulation of guard cell K+ fluxes by Ca2+-dependent protein kinases Sally Assmann Sally Assmann spoke about ABA-regulated and Ca2+-dependent kinases in guard cell function. Her laboratory has discovered a kinase ("ABA-activated protein kinase" or "AAPK") that is activated when guard cell protoplasts are treated with ABA. The kinase is a 48 kD serine-threonine kinase and is Ca2+ -independent. It is activated by as little as 1 minute of ABA treatment and by ABA concentrations as low as 1 mM. The activity of this kinase was not detected in mesophyll cell protoplasts, epidermal cell protoplasts, or roots, only in guard cells. These results imply that this kinase may play special roles in the complex signal transduction pathways by which ABA regulates stomatal function. Dr. Assmann also discussed data concerning a calcium-dependent protein kinase (CDPK) isolated from guard cells. In vitro, this kinase can phosphorylate the KAT1 K+ channel protein, which is thought to be a major conduit for K+ uptake into guard cells during stomatal opening. These experiments were performed with recombinant KAT1 that was transcribed and translated in vitro. Phosphorylation of KAT1 by CDPK only occurs when KAT1 is translated in the presence of microsomes, suggesting that the correct conformation of KAT1 is essential for the phosphorylation event. It has been known for some time that Ca2+ inhibits the activity of guard cell inward K+ channels; these observations may provide a mechanistic basis for that event. Magnetophoresis and the Cytoskeleton - Studies of two aspects of plant gravitropism Karl H. Hasenstein Plants respond to reorientation in the gravity field by adjusting the differential growth rate along the upper and lower flanks of roots and shoots. Despite longstanding correlative evidence that gravity perception is linked to the presence of starch-filled amyloplasts, manipulating plants by centrifugation or clinostatting always affects the entire plant and not just the presumptive gravisensor. Recent studies employing high gradient magnetic fields (HGMF) have allowed us to induce curvature by manipulating these organelles and not the entire plant. Our studies with HGMFs of different designs and strength showed that a properly arranged and sufficiently strong magnetic gradient can induce intracellular displacement of amyloplasts and results in curvature. In roots the curvature is away from the HGMF. However, in barley coleoptiles, tomato hypocotyls, and the wildtype of the moss Ceratodon the curvature is toward the HGMF. This curvature is thus equivalent to the graviresponse. In all tested examples, the curvature in wrong way or phytochrome dependent mutants was similar to curvature induced by reorienting plants in the gravity field.=20 Despite the excellent correlation between intracellular displacement and curvature, the ponderomotive force generated by the magnetic field or the magnetic field itself may affect cellular structures other than bulk diamagnetics (amyloplasts), for example the assembly of dynamically rearranged proteins such as the microtubule (MT) or actin cytoskeleton. Corresponding experiments showed that the structure and presumably assembly of MTs was not affected by the HGMF.=20 Other experiments concerning the organization of the cytoskeleton revealed that neither MTs nor microfilaments contribute to the growth reaction during graviresponse. This evidence is based on the effect of MT disrupting and stabilizing drugs such as oryzalin and taxol. Despite locking the dynamic assembly of MTs, the graviresponse was not affected. Alternatively, drugs that inhibit auxin transport and graviresponse such as naphthylphthalamic acid (NPA) had no effect on the organization of the MTs. Similarly, experiments designed to depolymerize the actin mirofilaments through the activity of the cytochalasins B or D succeeded in depolymerizing the microfilaments but had no effect on the graviresponse. Analogous to the effect on MTs, NPA inhibited the graviresponse but showed no effect on the actin cytoskeleton. Since the actin cytoskeleton may depend on co-factors that facilitate turn over or polymerization of g-actin, such as profilin, this cytoskeletal protein was also examined. Preliminary data suggest that a high proportion of this protein exists in the cell wall region of discrete tissues within the stele of roots but shows no apparent redistribution in graviresponding roots.=20 (Supported by NASA grant NAGW-3565 and NAG10-0190). Dr. Karl H. Hasenstein Biology - Univ. SW Louisiana Lafayette, LA 70504-2451 Phone: 318.482.6750 Fax: 318.482.5834 email: hasenstein@usl.edu http://www.usl.edu/Departments/Biology/hasenstein.html ********************************************************************* Announcements: Postdoctoral position A postdoctoral position is available in my laboratory starting any time soon, and I am particularly looking for somebody with experience in protein chemistry. An abstract of our current research follows. SIGNAL TRANSDUCTION IN PHOTOTROPISM: IN PURSUIT OF AN ELUSIVE PHOTORECEPTOR Eva Huala, John Christie, Paul Oeller, Emmanuel Liscum, In-Seob Han, Elise Larsen, and Winslow R. Briggs=20 Department of Plant Biology, Carnegie Institution of Washington, 260 Panama St., Stanford, CA 94305, USA The NPH1 gene was previously hypothesized to encode the photoreceptor for phototropism in Arabidopsis thaliana. On irradiation with blue light it carries out autophosphorylation both in vivo and in vitro. We have now sequenced both genomic and cDNA clones for A. thaliana, cDNA clones of two NPH1 homologues from Avena sativa and a cDNA clone from Zea mays. The A. thaliana genomic sequence is unusual in that it is broken into 20 exons. The A. thaliana gene encodes a protein of 996 amino acids, the A. sativa genes proteins of 926 and 933 amino acids respectively. The deduced protein sequence for all three genes has all eleven of the conserved motifs found in serine/threonine kinases, as we predicted earlier from biochemical experiments. These are located near the C-terminal end of the protein. The sequence also contains two repeated domains of 107 amino acids (that we are currently designating LOV1 and LOV2) that share significant homology with a heterogeneous group of gene products from several non-plant organisms: bat from Halobacterium halobium (Archaea); nifL from Klebsiella pneumoniae, Azotobacter vinelandii, and Enterobacter agglomerans (Eubacteria), recently shown to be a flavoprotein in A. vinelandii; aer from Escherichia coli (Eubacteria), also thought to be a flavoprotein; wc-1 from Neurospora crassa (Fungi); and members of the eag family from Drosophila, rat and man. The activities of all of these proteins are regulated either by light, oxygen, or voltage (hence LOV). The deduced NPH1 protein is also highly homologous with three plant gene fragments (ice plant, pea, and spinach) in GenBank that were obtained by using PCR on kinase signature sequences. They have all of the kinase signature domains found in the A. thaliana gene, all have at least the A1 domain, and spinach, the longest fragment, clearly has the A2 domain as well. The overall protein sequence similarity is extremely high. We are investigating whether the LOV1 and LOV2 domains of NPH1 may function as redox sensors, and whether they function as flavin-binding domains. Autophosphorylation of NPH1 protein produced heterologously by the Baculovirus/insect cell system is induced by blue light, suggesting that the protein is acquiring a blue light-absorbing chromophore, likely a flavin, from the insect cells, and that the holoprotein is the photoreceptor for phototropism. We have named the photoreceptor photokinase. =20 (Note new street number, new area code!) Winslow R. Briggs,=20 Dept. Plant Biology,=20 Carnegie Institution of Washington,=20 260 Panama St., Stanford, California 94305 Phone (650) 325-1521 Ex 207 Fax (650) 325-6857 email briggs@andrew.stanford.edu ***************************************************************** Recent Publications Muschietti,J.P., Eyal,Y. and McCormick,S. 1998. Pollen tube localization implies a role in pollen-pistil interactions for the tomato receptor-like protein kinases LePRK1 and LePRK2. Plant Cell 10: 319-330.=20 Abstract: We screened for pollen-specific kinase genes, which are potential signal transduction components of pollenpistil interactions, and isolated two structurally related receptor-like kinases (RLKs) from tomato, LePRK1 and LePRK2. These kinases are similar to a pollen-expressed RLK from petunia, but they are expressed later during pollen development than is the petunia RLK. The abundance of LePRK2 increases when pollen germinates, but LePRK1 remains constant. Both LePRK1 and LePRK2 are localized to the plasma membrane/cell wall of growing pollen tubes. Both kinase domains have kinase activity when expressed in Escherichia coli. In phosphorylation assays with pollen membrane preparations, LePRK2, but not LePRK1, is phosphorylated, and the addition of tomato style, but not leaf, extracts to these membrane preparations results at least partially in specific dephosphorylation of LePRK2. Taken together, these results suggest that LePRK1 and LePRK2 play different roles in postpollination events and that at least LePRK2 may mediate some pistil response.=20 E. Davies, S. Abe, B.A. Larkins, A.M. Clore, R.S. Quatrano and S. Weidner (1998) The role of the cytoskeleton in plant protein synthesis. In: A Look Beyond Transcription: Mechanisms determining mRNA Stability and Translation in Plants. J. Bailey-Serres, D.R. Gallie, eds. ASPP pp. 115-124. B. Stankovic, E. Davies (1998) The wound response in tomato involves rapid growth and electrical responses, systemically up-regulated transcription of proteinase inhibitor and calmodulin and down-regulated translation. Plant & Cell Physiology 39: 268-274 ******************************************************************** Abstracts submitted to ASPP for this summer's meeting: The Arabidopsis thaliana photoreceptor mutants phyb and hy4 have conditional phenotypes. Dale E. Blum and Elizabeth Van Volkenburgh, Dept. of Botany, University of Washington, Seattle, WA 98195-1330 Two different null photoreceptor mutants, phyb, which lacks the apoprotein for phytochrome B and hy4, which lacks cryptochrome 1, fail to show light-induced hypocotyl inhibition and have reduced cotyledon expansion when grown in the light quality they cannot perceive. On the other hand, it has been reported that leaf area of hy4 is greater than wild-type when grown in blue light while leaf expansion of phyb is reduced, unaltered, or enhanced when grown in white or red light. The opposite response of cotyledon and leaf expansion to light could be due either to the age of the plants or growth conditions as well as differences in light quality. Cotyledon areas have mostly been measured on young seedlings grown on agar while leaf data are commonly collected from older plants grown on soil. Here we show that both cotyledons and leaves of phyb and hy4 have reduced expansion compared to wild-type when grown on agar in blue (hy4) or red (phyb) light. When grown in soil, however, cotyledons and leaves of hy4 and phyb have equal or even enhanced growth compared to wild-type. In either growth condition the plants maintain their mutant phenotypes of longer hypocotyls and an increase in the length/width ratio of the leaf. These results show that cotyledons and leaves of Arabidopsis respond to light in a similar manner and that the mutant phenotype of reduced light-induced leaf and cotyledon expansion is conditional. While phytochrome B and cryptochrome 1 enhance light-induced expansion of cotyledons and leaves, under certain growth conditions they are not necessary for full expansion. --- Kinetics of light-stimulated growth, proton pumping, and hyperpolarization of the mesophyll cells of growing pea leaves (Pisum sativum argenteum)=20 Rainer Stahlberg and Elizabeth Van Volkenburgh Botany Department, University of Washington, Seattle, WA 98195-1330 The association of light-stimulated leaf growth with proton pump activation has been shown before, but it is not understood how these processes related kinetically to each other and to the rapid electric photoresponses of the membrane potential observed in mesophyll cells or protoplasts. To connect these three processes causally we tested the effect of several chemical inhibitors on the light-induced changes in the electric photoresponse, extracellular pH, and growth. The photosynthetic inhibitor DCMU completely abolished the early steps of the photoelectric response without abolishing light-induced proton extrusion, hyperpolarization or growth acceleration. The PM proton pump inhibitor ortho-vanadate did not affect these early steps in the electric photoresponse but suppressed (1) a light-induced hyperpolarization occurring in untreated cells after a lag of 5-10 min, (2) a proton net efflux normally starting after a lag of 15 min, and (3) the light-induced growth acceleration in leaf strips. These three vanadate-sensitive processes have similar kinetics and lag phases suggesting a common photoregulatory mechanism. Exposures ranging from 1 to 5 min of white light show the need for continuous light exposure during the entire lag phase. If illumination is stopped after the onset of growth stimulation, however, leaf expansion continues in the dark with a higher rate than before the light treatment. Only light treatments exceeding the duration of the lag period result in an elevated growth rate of the leaf tissue as well as a lowered extracellular pH and a hyperpolarized membrane potential of the mesophyll cells. Elizabeth Van Volkenburgh telephone: 206-543-6286 Associate Professor FAX: 206-543-3262 Botany Department 351330 University of Washington Seattle, WA 98195-1330 Electrical signals and gene expression in tomato=20 Bratislav Stankovic, Alain Vian, Chantal Henry-Vian, Justine Wilson, Hallema Mitchell , Flora Shabani and Eric Davies North Carolina State University, Botany Department, Box 7612, Raleigh NC= 27695 There is increasing evidence to suggest that electrical signals, including variation potentials (VP) and action potentials (AP) are important signaling systems in an array of plant responses to environmental stimuli. We are using tomato as a model system to understand the role of the AP and VP in gene expression. We have heat-wounded the 3rd leaf of young tomato plants, analyzed the electrical signals traveling to the youngest (4th) leaf, excised this leaf and use the extracted mRNA to make "heat-wounded" cDNA. We have isolated mRNA from the 4th leaf of untreated plants and used this to make "control" cDNA. We then constructed a subtractive (heat-wounded minus control) cDNA library and have isolated 800 putatively up-regulated clones. We have used these clones to probe northern blots of mRNA at different times after heat-wounding, and have found that 53 of the 55 clones so far analyzed are up-regulated after wounding. Many of these clones become maximally abundant within 5-15 minutes in tissue 5 cm from the region wounded (phase I), decrease to basal levels by 15-30 minutes (phase II), undergo a slower and more sustained increase (phase III) before being recruited into polysomes (phase IV). We are trying to determine whether the same or different signals evoke each of these responses. To visualize the role of Ca2+ in these responses, we have used Arabidopsis plants transgenic for aequorin, applied a short heat pulse close to one leaf and examined changes in light emission using a Hamamatsu photon counting camera. Within 3 seconds of the heat pulse, Ca2+ levels were higher throughout the plant, becoming maximal by 6 s before declining to basal levels by 15 s. We are now trying to evoke an AP in these plants to see whether Ca2+ changes can be visualized after this treatment. We will then apply various Ca2+ antagonists to see whether the Ca2+ increases can be lessened or even prevented and whether the up-regulation of specific genes is decreased or not. --- Gravity-induced increases in polysomes in the maize pulvinus Eric Davies, Thomas J. Whitlock, Heike Winter and Bratislav Stankovic North Carolina State University, Botany Department, Box 7612, Raleigh NC= 27695 When maize plants are put horizontally, they respond by differential growth at previously non-growing regions (pulvini) immediately above the node. Differential growth becomes evident within about 10 hours and the plant regains its upright position within 2-3 days. In younger (4-week old) plants, the major growth response is in pulvini 1 and 2, whereas in older plants (6-8 week old), the response is primarily in pulvini 4-6. The total amount of ribosomes and the proportion of ribosomes in polysomes (indicators of protein synthesis in vivo) are much higher in pulvini capable of responding to gravi-stimulation. In pulvinus #2, the most rapidly responding pulvinus in 4 week old plants, increases in polysomes in response to gravity occur in both the lower and upper sides, even though growth occurs predominantly in the lower side. The increase in polysomes can be detected within 15 minutes and is greater on the upper side, although by 3 hours (before growth becomes evident) the increase in polysomes is greater on the lower side. These increases in polysomes are sustained for at least 48 hours. We are currently trying to determine whether a) changes in polysome metabolism occur in pulvini explants (where precursors and inhibitors can be easily introduced), b) whether the tissue is responding directly to gravity or to compression-tension, and c) whether electrical changes can be monitored prior to changes in polysomes or in growth. Eric Davies Tel. 919-515-2727 North Carolina State University Fax: 919-515-3436 Botany Department, Box 7612 Raleigh NC 27695 From bogus@does.not.exist.com Mon Jun 13 10:10:06 2005 From: bogus@does.not.exist.com () Date: Mon Jun 13 10:10:06 2005 Subject: No subject Message-ID: Bjorn Drobak myo-Inositol (hexahydroxycyclohexane) is essential for the growth of most bacterial, fungal, plant and mammalian cells. Cells without obvious requirement for exogenous inositol have all been found to be capable of de novo synthesis of inositol. Thus, myo-inositol is now recognized as an essential factor for normal cell growth and function. There are nine possible stereoisomers of inositol and seven of these have been found to occur naturally, the exceptions being epi- and allo-inositol. Myo-, D/L-chiro and scyllo-inositol are the major stereoisomers of inositol in plants although both muco- inositol and neo-inositol have been found to exist. The presence of inositol in certain bacterial lipids was discovered around 1930 and the presence of inositol in brain phospholipids was reported by Folch and Woolley in 1942. A very wide range functions have in the last decades been proposed for inositol and its derivatives in both plant and animal cells, but it is the discovery that inositol-containing compounds play a key role in the perception and transmission of extracellular signals which has led to the current intense interest in this particular area of science. The basic concepts of the eukaryotic phosphoinositide signal transducing system (PI-system) were delineated in mammalian and insect cells by M.J.Berridge, R.F.Irvine, R.H.Michell and their co- workers in the early 1980's, and PI-systems have since been found to be present in all the eukaryotic cells which so far have been studied. The talk focused on recent advances in the understanding of how the plant PI system participates in the transmission of signals from the plasma membrane to the nucleus. The key topics presented in the talk were : - Generation of phosphoinositides by phosphoinositide kinases - PLC activity and its regulation - Changes in cytosolic Ins(1,4,5)P3 activity in plant cells - Interactions between components of the PI-system and the cytoskeleton - Causes and consequences of an uneven cellular distribution of phosphoinositide metabolising enzymes - Generation of Ca2+ waves and their dependency on Ins(1,4,5)P3 receptor occupancy - Nuclear Ca2+ fluxes and their regulation - 3-phosphoinositides and the regulation nuclear and nucleolar events - An integrated view of PI-signalling from the plasma membrane to the nucleus Capacitative Calcium Entry by James W. Putney, Jr. Calcium Regulation Section Laboratory of Signal Transduction National Institute of Environmental Health Sciences - NIH Research Triangle Park, NC 27709 In animal cells, a wide variety of hormones, neurotransmitters, growth factors and other physiological and pathological stimuli initiate cellular responses through the process of calcium signalling. Cellular calcium signalling involves a rise in the concentration of ionized calcium in the cytoplasm of cells. This increase in cytoplasmic calcium concentration ([Ca2+]i) can come either from the extracellular space, through channels in the plasma membrane, or from intracellular stores of Ca2+, again through specific channels in the limiting membranes of Ca2+-storing organelles. In the majority of cases, both modes are utilized (For example, see Figure 1). For electrically non-excitable cells (epithelial cells, blood cells, and fibroblasts for example), and in many instances for excitable cells this signalling is initiated by the activation of a polyphosphoinositide-specific phospholipase C which hydrolyzes membrane phospholipids to release the calcium signalling molecule, inositol 1,4,5-trisphosphate (IP3). IP3 binds to a receptor molecule located in the endoplasmic reticulum, or a specialized component of it(1,2). The IP3 receptor functions as a ligand-gated channel and its activation by IP3 leads to rapid release of Ca2+ to the cytoplasm. Intuitively, it would seem reasonable that IP3 is also involved in the signalling of Ca2+ entry across the plasma membrane. There are numerous examples in which direct application of IP3 to the cytoplasm of cells, whether by microinjection or by patch-clamp dialysis, activates calcium entry into cells(3). However, the evidence suggests that in the vast majority of cases this action is an indirect one. Rather than directly activating Ca2+ channels in the plasma membrane, it appears that the ability of IP3 to activate Ca2+ entry is secondary to, and dependent upon the mobilization of Ca2+ from intracellular Ca2+ stores. This concept, termed the capacitative model for Ca2+ entry, was first proposed on the basis of experiments examining the kinetics of refilling of intracellular Ca2+ stores following their depletion by a phospholipase C-linked agonist(4). A more direct demonstration of this phenomenon can be seen with the use of specific inhibitors of the intracellular Ca2+ transport ATPases which mediate the active accumulation of Ca2+ in the endoplasmic or sarcoplasmic reticulum (termed SERCA pumps for Sarcoplasmic Endoplasmic Reticulum ATPase pumps). The prototypical reagent in this class is the plant sesquiterpene lactone, thapsigargin(5). Thapsigargin is readily membrane permeant, and its application to cells results in a relatively rapid passive depletion of intracellular Ca2+ stores. Consistent with predictions of the capacitative model, Figure 1B illustrates that thapsigargin activates Ca2+ entry. Following activation of entry by thapsigargin, no further increase in entry is seen on application of a phospholipase C-stimulating agonist. This means that not only does depletion of Ca2+ stores provide a signal for the activation of Ca2+ entry, but also that this mechanism fully activates the available channels with no further potentiation or additional pathways activated by IP3 (or its metabolites). This basic finding, that depletion of intracellular Ca2+ stores provides a signal for activation of calcium entry, has been confirmed in a large number of cell types, and it is now clear that the capacitative model represents a widespread mechanism for control of calcium entry at the plasma membrane. Recent research has focused on the molecular identity of the channels underlying this phenomenon, and the nature of the signal that activates these channels in response to intracellular Ca2+ store depletion(6). References 1. Rossier, M. F. and Putney, J. W., Jr. (1991). The identity of the calcium storing inositol 1,4,5-trisphosphate-sensitive organelle in non-muscle cells: Calciosome, endoplasmic reticulum, ... or both? Trends Neurosci. 14, 310-314. 2. Pozzan, T., Rizzuto, R., Volpe, P., and Meldolesi, J. (1994). Molecular and cellular physiology of intracellular calcium stores. Physiol.Rev. 74, 595-636. 3. Putney, J. W., Jr. and Bird, G. St. J. (1993). The inositol phosphate-calcium signalling system in non-excitable cells. Endocrine Rev. 14, 610-631. 4. Putney, J. W., Jr. (1986). A model for receptor-regulated calcium entry. Cell Calcium 7, 1-12. 5. Thastrup, O., Dawson, A. P., Scharff, O., Foder, B., Cullen, P. J., Drobak, B. K., Bjerrum, P. J., Christensen, S. B., and Hanley, M. R. (1994). Thapsigargin, a novel molecular probe for studying intracellular calcium release and storage. Agents Actions 43, 187-193. 6. Putney, J. W., Jr. (1997). Capacitative Calcium Entry pp. Landes Biomedical Publishing, Austin, TX. Figure 1: Patterns of Calcium Release and Calcium Entry. Top: A single mouse lacrimal cell containing a fluorescent calcium indicator is activated by methacholine, an agonist for the phospholipase C-coupled muscarinic receptors. Comparison of the [Ca2+]i responses in the presence (green) and absence (red) of extracellular calcium reveals that the signal is comprised of two components, an intracellular release of Ca2+ as well as an activation of Ca2+ entry across the plasma membrane. Bottom: In a single lacrimal cell, the SERCA inhibitor, thapsigargin, activates entry of Ca2+, and this entry is not facilitated by methacholine (MeCh). Regulation of guard cell K+ fluxes by Ca2+-dependent protein kinases Sally Assmann Sally Assmann spoke about ABA-regulated and Ca2+-dependent kinases in guard cell function. Her laboratory has discovered a kinase ("ABA-activated protein kinase" or "AAPK") that is activated when guard cell protoplasts are treated with ABA. The kinase is a 48 kD serine-threonine kinase and is Ca2+ -independent. It is activated by as little as 1 minute of ABA treatment and by ABA concentrations as low as 1 mM. The activity of this kinase was not detected in mesophyll cell protoplasts, epidermal cell protoplasts, or roots, only in guard cells. These results imply that this kinase may play special roles in the complex signal transduction pathways by which ABA regulates stomatal function. Dr. Assmann also discussed data concerning a calcium-dependent protein kinase (CDPK) isolated from guard cells. In vitro, this kinase can phosphorylate the KAT1 K+ channel protein, which is thought to be a major conduit for K+ uptake into guard cells during stomatal opening. These experiments were performed with recombinant KAT1 that was transcribed and translated in vitro. Phosphorylation of KAT1 by CDPK only occurs when KAT1 is translated in the presence of microsomes, suggesting that the correct conformation of KAT1 is essential for the phosphorylation event. It has been known for some time that Ca2+ inhibits the activity of guard cell inward K+ channels; these observations may provide a mechanistic basis for that event. Magnetophoresis and the Cytoskeleton - Studies of two aspects of plant gravitropism Karl H. Hasenstein Plants respond to reorientation in the gravity field by adjusting the differential growth rate along the upper and lower flanks of roots and shoots. Despite longstanding correlative evidence that gravity perception is linked to the presence of starch-filled amyloplasts, manipulating plants by centrifugation or clinostatting always affects the entire plant and not just the presumptive gravisensor. Recent studies employing high gradient magnetic fields (HGMF) have allowed us to induce curvature by manipulating these organelles and not the entire plant. Our studies with HGMFs of different designs and strength showed that a properly arranged and sufficiently strong magnetic gradient can induce intracellular displacement of amyloplasts and results in curvature. In roots the curvature is away from the HGMF. However, in barley coleoptiles, tomato hypocotyls, and the wildtype of the moss Ceratodon the curvature is toward the HGMF. This curvature is thus equivalent to the graviresponse. In all tested examples, the curvature in wrong way or phytochrome dependent mutants was similar to curvature induced by reorienting plants in the gravity field. Despite the excellent correlation between intracellular displacement and curvature, the ponderomotive force generated by the magnetic field or the magnetic field itself may affect cellular structures other than bulk diamagnetics (amyloplasts), for example the assembly of dynamically rearranged proteins such as the microtubule (MT) or actin cytoskeleton. Corresponding experiments showed that the structure and presumably assembly of MTs was not affected by the HGMF. Other experiments concerning the organization of the cytoskeleton revealed that neither MTs nor microfilaments contribute to the growth reaction during graviresponse. This evidence is based on the effect of MT disrupting and stabilizing drugs such as oryzalin and taxol. Despite locking the dynamic assembly of MTs, the graviresponse was not affected. Alternatively, drugs that inhibit auxin transport and graviresponse such as naphthylphthalamic acid (NPA) had no effect on the organization of the MTs. Similarly, experiments designed to depolymerize the actin mirofilaments through the activity of the cytochalasins B or D succeeded in depolymerizing the microfilaments but had no effect on the graviresponse. Analogous to the effect on MTs, NPA inhibited the graviresponse but showed no effect on the actin cytoskeleton. Since the actin cytoskeleton may depend on co-factors that facilitate turn over or polymerization of g-actin, such as profilin, this cytoskeletal protein was also examined. Preliminary data suggest that a high proportion of this protein exists in the cell wall region of discrete tissues within the stele of roots but shows no apparent redistribution in graviresponding roots. (Supported by NASA grant NAGW-3565 and NAG10-0190). Dr. Karl H. Hasenstein Biology - Univ. SW Louisiana Lafayette, LA 70504-2451 Phone: 318.482.6750 Fax: 318.482.5834 email: hasenstein@usl.edu http://www.usl.edu/Departments/Biology/hasenstein.html ********************************************************************* Announcements: Postdoctoral position A postdoctoral position is available in my laboratory starting any time soon, and I am particularly looking for somebody with experience in protein chemistry. An abstract of our current research follows. SIGNAL TRANSDUCTION IN PHOTOTROPISM: IN PURSUIT OF AN ELUSIVE PHOTORECEPTOR Eva Huala, John Christie, Paul Oeller, Emmanuel Liscum, In-Seob Han, Elise Larsen, and Winslow R. Briggs Department of Plant Biology, Carnegie Institution of Washington, 260 Panama St., Stanford, CA 94305, USA The NPH1 gene was previously hypothesized to encode the photoreceptor for phototropism in Arabidopsis thaliana. On irradiation with blue light it carries out autophosphorylation both in vivo and in vitro. We have now sequenced both genomic and cDNA clones for A. thaliana, cDNA clones of two NPH1 homologues from Avena sativa and a cDNA clone from Zea mays. The A. thaliana genomic sequence is unusual in that it is broken into 20 exons. The A. thaliana gene encodes a protein of 996 amino acids, the A. sativa genes proteins of 926 and 933 amino acids respectively. The deduced protein sequence for all three genes has all eleven of the conserved motifs found in serine/threonine kinases, as we predicted earlier from biochemical experiments. These are located near the C-terminal end of the protein. The sequence also contains two repeated domains of 107 amino acids (that we are currently designating LOV1 and LOV2) that share significant homology with a heterogeneous group of gene products from several non-plant organisms: bat from Halobacterium halobium (Archaea); nifL from Klebsiella pneumoniae, Azotobacter vinelandii, and Enterobacter agglomerans (Eubacteria), recently shown to be a flavoprotein in A. vinelandii; aer from Escherichia coli (Eubacteria), also thought to be a flavoprotein; wc-1 from Neurospora crassa (Fungi); and members of the eag family from Drosophila, rat and man. The activities of all of these proteins are regulated either by light, oxygen, or voltage (hence LOV). The deduced NPH1 protein is also highly homologous with three plant gene fragments (ice plant, pea, and spinach) in GenBank that were obtained by using PCR on kinase signature sequences. They have all of the kinase signature domains found in the A. thaliana gene, all have at least the A1 domain, and spinach, the longest fragment, clearly has the A2 domain as well. The overall protein sequence similarity is extremely high. We are investigating whether the LOV1 and LOV2 domains of NPH1 may function as redox sensors, and whether they function as flavin-binding domains. Autophosphorylation of NPH1 protein produced heterologously by the Baculovirus/insect cell system is induced by blue light, suggesting that the protein is acquiring a blue light-absorbing chromophore, likely a flavin, from the insect cells, and that the holoprotein is the photoreceptor for phototropism. We have named the photoreceptor photokinase. (Note new street number, new area code!) Winslow R. Briggs, Dept. Plant Biology, Carnegie Institution of Washington, 260 Panama St., Stanford, California 94305 Phone (650) 325-1521 Ex 207 Fax (650) 325-6857 email briggs@andrew.stanford.edu ***************************************************************** Recent Publications Muschietti,J.P., Eyal,Y. and McCormick,S. 1998. Pollen tube localization implies a role in pollen-pistil interactions for the tomato receptor-like protein kinases LePRK1 and LePRK2. Plant Cell 10: 319-330. Abstract: We screened for pollen-specific kinase genes, which are potential signal transduction components of pollenpistil interactions, and isolated two structurally related receptor-like kinases (RLKs) from tomato, LePRK1 and LePRK2. These kinases are similar to a pollen-expressed RLK from petunia, but they are expressed later during pollen development than is the petunia RLK. The abundance of LePRK2 increases when pollen germinates, but LePRK1 remains constant. Both LePRK1 and LePRK2 are localized to the plasma membrane/cell wall of growing pollen tubes. Both kinase domains have kinase activity when expressed in Escherichia coli. In phosphorylation assays with pollen membrane preparations, LePRK2, but not LePRK1, is phosphorylated, and the addition of tomato style, but not leaf, extracts to these membrane preparations results at least partially in specific dephosphorylation of LePRK2. Taken together, these results suggest that LePRK1 and LePRK2 play different roles in postpollination events and that at least LePRK2 may mediate some pistil response. E. Davies, S. Abe, B.A. Larkins, A.M. Clore, R.S. Quatrano and S. Weidner (1998) The role of the cytoskeleton in plant protein synthesis. In: A Look Beyond Transcription: Mechanisms determining mRNA Stability and Translation in Plants. J. Bailey-Serres, D.R. Gallie, eds. ASPP pp. 115-124. B. Stankovic, E. Davies (1998) The wound response in tomato involves rapid growth and electrical responses, systemically up-regulated transcription of proteinase inhibitor and calmodulin and down-regulated translation. Plant & Cell Physiology 39: 268-274 ******************************************************************** Abstracts submitted to ASPP for this summer's meeting: The Arabidopsis thaliana photoreceptor mutants phyb and hy4 have conditional phenotypes. Dale E. Blum and Elizabeth Van Volkenburgh, Dept. of Botany, University of Washington, Seattle, WA 98195-1330 Two different null photoreceptor mutants, phyb, which lacks the apoprotein for phytochrome B and hy4, which lacks cryptochrome 1, fail to show light-induced hypocotyl inhibition and have reduced cotyledon expansion when grown in the light quality they cannot perceive. On the other hand, it has been reported that leaf area of hy4 is greater than wild-type when grown in blue light while leaf expansion of phyb is reduced, unaltered, or enhanced when grown in white or red light. The opposite response of cotyledon and leaf expansion to light could be due either to the age of the plants or growth conditions as well as differences in light quality. Cotyledon areas have mostly been measured on young seedlings grown on agar while leaf data are commonly collected from older plants grown on soil. Here we show that both cotyledons and leaves of phyb and hy4 have reduced expansion compared to wild-type when grown on agar in blue (hy4) or red (phyb) light. When grown in soil, however, cotyledons and leaves of hy4 and phyb have equal or even enhanced growth compared to wild-type. In either growth condition the plants maintain their mutant phenotypes of longer hypocotyls and an increase in the length/width ratio of the leaf. These results show that cotyledons and leaves of Arabidopsis respond to light in a similar manner and that the mutant phenotype of reduced light-induced leaf and cotyledon expansion is conditional. While phytochrome B and cryptochrome 1 enhance light-induced expansion of cotyledons and leaves, under certain growth conditions they are not necessary for full expansion. --- Kinetics of light-stimulated growth, proton pumping, and hyperpolarization of the mesophyll cells of growing pea leaves (Pisum sativum argenteum) Rainer Stahlberg and Elizabeth Van Volkenburgh Botany Department, University of Washington, Seattle, WA 98195-1330 The association of light-stimulated leaf growth with proton pump activation has been shown before, but it is not understood how these processes related kinetically to each other and to the rapid electric photoresponses of the membrane potential observed in mesophyll cells or protoplasts. To connect these three processes causally we tested the effect of several chemical inhibitors on the light-induced changes in the electric photoresponse, extracellular pH, and growth. The photosynthetic inhibitor DCMU completely abolished the early steps of the photoelectric response without abolishing light-induced proton extrusion, hyperpolarization or growth acceleration. The PM proton pump inhibitor ortho-vanadate did not affect these early steps in the electric photoresponse but suppressed (1) a light-induced hyperpolarization occurring in untreated cells after a lag of 5-10 min, (2) a proton net efflux normally starting after a lag of 15 min, and (3) the light-induced growth acceleration in leaf strips. These three vanadate-sensitive processes have similar kinetics and lag phases suggesting a common photoregulatory mechanism. Exposures ranging from 1 to 5 min of white light show the need for continuous light exposure during the entire lag phase. If illumination is stopped after the onset of growth stimulation, however, leaf expansion continues in the dark with a higher rate than before the light treatment. Only light treatments exceeding the duration of the lag period result in an elevated growth rate of the leaf tissue as well as a lowered extracellular pH and a hyperpolarized membrane potential of the mesophyll cells. Elizabeth Van Volkenburgh telephone: 206-543-6286 Associate Professor FAX: 206-543-3262 Botany Department 351330 University of Washington Seattle, WA 98195-1330 Electrical signals and gene expression in tomato Bratislav Stankovic, Alain Vian, Chantal Henry-Vian, Justine Wilson, Hallema Mitchell , Flora Shabani and Eric Davies North Carolina State University, Botany Department, Box 7612, Raleigh NC 27695 There is increasing evidence to suggest that electrical signals, including variation potentials (VP) and action potentials (AP) are important signaling systems in an array of plant responses to environmental stimuli. We are using tomato as a model system to understand the role of the AP and VP in gene expression. We have heat-wounded the 3rd leaf of young tomato plants, analyzed the electrical signals traveling to the youngest (4th) leaf, excised this leaf and use the extracted mRNA to make "heat-wounded" cDNA. We have isolated mRNA from the 4th leaf of untreated plants and used this to make "control" cDNA. We then constructed a subtractive (heat-wounded minus control) cDNA library and have isolated 800 putatively up-regulated clones. We have used these clones to probe northern blots of mRNA at different times after heat-wounding, and have found that 53 of the 55 clones so far analyzed are up-regulated after wounding. Many of these clones become maximally abundant within 5-15 minutes in tissue 5 cm from the region wounded (phase I), decrease to basal levels by 15-30 minutes (phase II), undergo a slower and more sustained increase (phase III) before being recruited into polysomes (phase IV). We are trying to determine whether the same or different signals evoke each of these responses. To visualize the role of Ca2+ in these responses, we have used Arabidopsis plants transgenic for aequorin, applied a short heat pulse close to one leaf and examined changes in light emission using a Hamamatsu photon counting camera. Within 3 seconds of the heat pulse, Ca2+ levels were higher throughout the plant, becoming maximal by 6 s before declining to basal levels by 15 s. We are now trying to evoke an AP in these plants to see whether Ca2+ changes can be visualized after this treatment. We will then apply various Ca2+ antagonists to see whether the Ca2+ increases can be lessened or even prevented and whether the up-regulation of specific genes is decreased or not. --- Gravity-induced increases in polysomes in the maize pulvinus Eric Davies, Thomas J. Whitlock, Heike Winter and Bratislav Stankovic North Carolina State University, Botany Department, Box 7612, Raleigh NC 27695 When maize plants are put horizontally, they respond by differential growth at previously non-growing regions (pulvini) immediately above the node. Differential growth becomes evident within about 10 hours and the plant regains its upright position within 2-3 days. In younger (4-week old) plants, the major growth response is in pulvini 1 and 2, whereas in older plants (6-8 week old), the response is primarily in pulvini 4-6. The total amount of ribosomes and the proportion of ribosomes in polysomes (indicators of protein synthesis in vivo) are much higher in pulvini capable of responding to gravi-stimulation. In pulvinus #2, the most rapidly responding pulvinus in 4 week old plants, increases in polysomes in response to gravity occur in both the lower and upper sides, even though growth occurs predominantly in the lower side. The increase in polysomes can be detected within 15 minutes and is greater on the upper side, although by 3 hours (before growth becomes evident) the increase in polysomes is greater on the lower side. These increases in polysomes are sustained for at least 48 hours. We are currently trying to determine whether a) changes in polysome metabolism occur in pulvini explants (where precursors and inhibitors can be easily introduced), b) whether the tissue is responding directly to gravity or to compression-tension, and c) whether electrical changes can be monitored prior to changes in polysomes or in growth. Eric Davies Tel. 919-515-2727 North Carolina State University Fax: 919-515-3436 Botany Department, Box 7612 Raleigh NC 27695 From bogus@does.not.exist.com Mon Jun 13 10:10:12 2005 From: bogus@does.not.exist.com () Date: Mon Jun 13 10:10:12 2005 Subject: No subject Message-ID: filling phase durations may be considered, especially in children, a first sign of rejection. I would like to know if somebody is interested to collaborate with me to provide me with data to be analysed with this methodic. Thank you. I wait also for your ideas in this matter. From biosci at net.bio.net Sat Jun 18 13:58:07 2005 From: biosci at net.bio.net (BIOSCI Administrator) Date: Sat Jun 18 15:07:32 2005 Subject: [Plant-signal-transduction] BIOSCI/Bionet has a new home Message-ID: <200506181858.j5IIw7c20282@net.bio.net> Dear PLANT-SIGNAL-TRANSDUCTION readers, BIOSCI/Bionet is the long-running Biology news and discussion groups. The management of these groups has been handled ably at MRC/Rosalind Franklin Centre for several years now. They are turning over management to me, Don Gilbert, at Indiana University Biology department. The MRC/RFC folks deserve our thanks for their devotion to maintaining this useful and unique biology news service. Web access continues at http://www.bio.net/ Find there PLANT-SIGNAL-TRANSDUCTION links to Read/Subscribe/Post messages. E-mail postings continue to be through plsignal @ net.bio.net The new home is at IUBio Archive, iubio.bio.indiana.edu, which I've maintained for over 15 years. The names net.bio.net and www.bio.net, and their related BIOSCI roles continue to work. Please keep using these bio.net addresses, they will continue when the host computer changes. There may be some hiccups over the coming weeks as the new BIOSCI home gets a work out. Please bear with us, and let us know if problems persist longer. Regards, Don Gilbert Biology Dept., Indiana University Bloomington, Indiana, USA 47405 BIOSCI help mail: biosci-help @ net.bio.net ----------------------------------------------