No subject


Mon Jun 13 10:10:06 EST 2005


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 at 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 at 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




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