Plant Signal Transduction Bulletin
Vol. XII - The Postdoc Issue
January 1999
Greetings to the plant signal transduction aficionados. This volume is
comprised of contributions from several postdocs who describe their current
research projects and share some of the intended future plans. A big THANK
YOU to everyone who sent abstracts of their work.
Braco
________________________________________________________________
Dr. Bratislav Stankovic a.k.a. Braco [Bratso]
Researcher, North Carolina State University
Botany Department, Box 7612 Tel: (919) 515-6043
2214 Gardner Hall Fax: (919) 515-3436
Raleigh, NC 27695 E-mail: braco_stankovic at ncsu.eduhttp://www2.ncsu.edu/ncsu/cals/botany/people/faculty/davies/bs.htm
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1. EXPLOITING PHYSICAL HETEROGENEITY OF PLANT CELLS TO STUDY SIGNAL
TRANSDUCTION DURING GRAVITROPISM
OLEG A. KUZNETSOV (Karl Hasenstein Lab)
USL Biology Department, P.O. Box 42451, Lafayette, LA 70504-2451
oleg at usl.edu; http://www.ucs.usl.edu/~oxk7779/
Plant cell is a highly heterogeneous and complex physical and chemical
system. Cell components differ in their chemical composition and,
therefore, in their physical properties, including density, optical
density, refraction index, viscosity, elasticity, surface electric charge,
magnetic properties, etc. This heterogeneity allows to study different
components of the cell using different physical principles: e.g.,
differences in optical properties are studied by microscopy, differences in
density - by various centrifugation techniques, differences in electric
properties - by electrophoresis, etc.
We are exploiting heterogeneity of magnetic susceptibility (æ) of plant
gravity receptor cells (statocytes) to displace statoliths (dense
organelles, like starch-filled amyloplasts in higher plants and
BaSO4-filled plastids in Chara) inside the cells. Intracellular
sedimentation of statoliths is presumed to be the primary step of gravity
perception in plants. A possibility to displace them without affecting the
rest of the plant by centrifugation or re-orienting in the gravity field is
a potent research tool for studying plant graviperception and signal
transduction. Statoliths are stronger diamagnetics, than the cytoplasm
[1-4]. In a non-uniform magnetic field they are repulsed from stronger
field zones by the ponderomotive magnetic force: Fm= (æp-æcp)Vgrad(H2/2),,
where æp is the magnetic susceptibility of plastids, æcp - that of
cytoplasm, grad(H2/2) is the dynamic factor of the magnetic field. If
magnetic field is very strong and very non-uniform (so-called high gradient
magnetic field [HGMF]), with grad(H2/2)= 109 to 1010 Oe2/cm, this force can
be equivalent to the gravity force. In our experiments with several types
of magnetic systems [1-7] we were able to displace amyloplasts inside
receptor cells in roots [3,4] and shoots [5,6] of higher plants, and inside
protonemata of the moss Ceratodon purpureus [7]. This displacement
(intracellular magnetophoresis) caused positively gravitropic organs (roots
of flax and Arabidopsis, wwr-mutant of Ceratodon) to curve in the direction
of amyloplast displacement (i.e., away from the stronger field), while
negatively gravitropic organs (shoots of tomato and barley, WT of
Ceratodon) curved in the direction opposite to the amyloplast displacement.
This pattern of the physiological response of the plants is consistent
with gravitropism, and the kinetics of the curvature is similar to the
gravitropic curvature [1-7]. Starchless mutant of Arabidopsis did not
curve in HGMF, indicating, that starch-filled bulk organelles are necessary
for the effect, and that other cell components are not significantly
affected by the field [3]. This shows, that magnetic ponderomotive forces
acting on amyloplasts can simulate gravity for plants.
This approach not only can provide information on physical heterogeneity
of the cells and organelles (e.g., determining volumetric shares of starch
grains and the membranous envelope in amyloplasts [4,8] or the volumetric
share of BaSO4 in Chara statoliths by magnetograviphoretic measurements [4]
of the plastids), but also can serve as a research tool for signal
transduction in plants. Such manipulation of amyloplasts is likely to
answer some of the basic questions of the sequence of events in
gravisensing and a possible involvement of the cytoskeleton in
graviperception. We can also expect answers as to whether amyloplast
displacement or the force that amyloplasts exert on the ER system leads to
the cascade of events that results in curvature. Small size of the area of
HGMF with a significant dynamic factor allows to stimulate only a small
portion of receptor cells of a bigger plant organ, allowing to study
relative sensitivity and importance of different regions of the organ for
gravity perception and response [5,6], which is impossible to do by any
other method. Recently we started to work with Chara rhizoid, where
intracellular magnetophoresis of statoliths can be observed in vivo in real
time, and both gravity perception and response take place in the same
single cell. This makes it a perfect system for assessing the possible
roles of microtubules and f-actin in graviperception using magnetic forces
on statoliths in combination with a treatment with cytoskeleton affecting
chemicals (MT-depolymerizer oryzalin, MT-stabilizer Taxol, f-actin
depolymerizers Cytochalasin B or D, and f-actin stabilizer phalloidin).
Significant change in kinetics of statoliths movement brought about by one
of these biochemical treatments will indicate involvement of the
corresponding component of the cytoskeleton in the positioning of the
organelles, and a change in the plant's response (curvature) will reveal
its role in the signal transduction.
Heterogeneity of statocytes allows to use other ponderomotive forces for
intracellular displacement of statoliths. For example, if the plant is
subjected to non-symmetric vibrations, amyloplasts experience an inertial
force proportional to the difference between their density and that of
cytoplasm and to the instantaneous acceleration of the cell. This force
causes cyclic motion of statoliths relative to cytoplasm and, depending on
the profile of oscillations, can result in a net displacement of them (due
to a very complex rheology of the cell interior), similar to sedimentation.
This can be described as a so-called "vibrational" ponderomotive force
acting on the statoliths. Arabidopsis seedlings, which were grown
vertically on an agar surface for two days and then subjected to
horizontal, sawtooth shaped oscillations (250 Hz, 1.5 mm amplitude), showed
17±2o root curvature toward and shoot curvature of 11±3o against the
"vibrational" acceleration after 12 h [9,10]. When the polarity of the
oscillations was reversed, the direction of curvature of shoots and roots
was also reversed. Control experiments with starchless mutants (TC7)
produced no net curvature, which indicates that dense starch-filled
amyloplasts are needed for the effect [9,10]. Acoustic ponderomotive
forces, which originate from a transfer of momentum of a sonic beam due to
its scattering and attenuation in a mechanically heterogeneous medium to
the medium, also can displace statoliths. Vertical flax seedlings curved
away from the ultrasonic source (800 kHz, 0.1 W/cm2) presumably as a
reaction to a displacement of amyloplasts by acoustic ponderomotive forces
[2]. Besides investigating the graviperception mechanism, vibrational and
acoustic studies also allow to analyze the mechanical properties of the
cell interior.
These examples show, that physical heterogeneity of plant cells allows to
create a variety of tools to study their properties, processes and signal
transduction in them.
References:
1. Kuznetsov AA, Kuznetsov OA 1989 Simulation of gravity force for plants
by high gradient magnetic field. Biofizika, 35: 835-840.
2. Kuznetsov OA 1993 Non-uniform magnetic field and relaxational
oscillations as instruments for plant cell exploration. PhD thesis, Moscow,
Russia: 132p.
3. Kuznetsov OA, Hasenstein KH 1996 Magnetophoretic Induction of Root
Curvature. Planta 198: 87-94.
4. Kuznetsov OA, Hasenstein KH 1997 Magnetophoretic characterization of
plant gravity receptors. in: Scientific and clinical applications of
magnetic carriers. (U. Hafeli, W. Schutt, J. Teller, M. Zborowski, eds.),
Plenum Press, New York: 429-444.
5. Kuznetsov OA, Hasenstein KH 1997 Magnetophoretic induction of curvature
in coleoptiles. J. of Exp. Botany 48 (316): 1951-1957.
6. Hasenstein KH, Kuznetsov OA 1998 Graviresponse of lazy-2 tomato
seedlings is not affected by curvature-inducing magnetic gradients.
Accepted by Planta
7. Kuznetsov OA, Schwuchow J, Sack FD, Hasenstein KH 1998 Curvature induced
by amyloplast magnetophoresis in protonemata of the moss Ceratodon
purpureus. Accepted by Plant Phys.
8. Kuznetsov OA, Brown CS, Sanwo MM, Hasenstein KH 1997
Magnetograviphoretic studies show differences in starch metabolism of
space-flown soybean. Gravitational and Space Biology Bulletin 11(1): 24.
9. Kuznetsov OA, Kuznetsov AA, Hasenstein KH 1997 Induction of amyloplast
dependent force based on non-symmetrical vibration. 12th Man in Space
Symposium: the Future of Humans in Space, June 8-13, 1997, Washington, DC:
64-65.
10. Kuznetsov OA, Hasenstein KH 1998 Simulation of gravity by vibrational
forces. Plant Physiol. 117: S39
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2. MOLECULAR ANALYSIS OF PLANT GRAVITROPISM: NEW APPROACHES TO AN OLD PROBLEM
SARAH WYATT (Niki Robertson Lab)
North Carolina State University
Transduction of a signal from the point of stimulus perception to the site
of response remains one of the greatest mysteries in science. My research
consists of two complementary approaches to this complex problem: reverse
genetics and mutant analysis.
First, I am collaborating on a reverse genetics approach to dissect the
role of calcium in the signal transduction pathway. Calcium is a second
messenger that controls a wide variety of cellular functions. Because of
its multiple actions, tight regulation of the cytosolic Ca2+ concentrations
is required. This is achieved by a system of Ca2+ -transport and storage
pathways that include Ca2+ buffering proteins in the cytosol and in the
lumen of intracellular storage compartments. In hopes of better
understanding this regulation in plants, I am attempting to alter calcium
stores at the subcellular level by manipulating expression of the
calcium-binding protein, calreticulin (CRT). This will ultimately be
combined with altered calcium transport and altered cellular sensitivity to
calcium.
CRT is an evolutionarily conserved protein found in the lumen of the
endoplasmic reticulum. In animal cells, overexpression of CRT increases
the Ca2+ capacity of IP3-sensitive Ca2+ stores (Bastianutto et al., J. Cell
Biol. 130, 847) and decreases store-operated Ca2+ influx from the
extracellular space (Mery et al. J. Biol. Chem. 271, 9332). In Xenopus
oocytes, overexpression of the CRT P-domain inhibits IP3-induced Ca2+ waves
(Camacho & Lechleiter, Cell 82, 765). To dissect the role of Ca2+ in the
plant signal transduction pathways, we are exploiting the Ca2+ binding
characteristics of CRT. At NCSU, I have generated transgenic material with
altered levels of CRT. I have expressed a maize CRT clone in both sense
and anti-sense orientation under the control of inducible promoters in both
Arabidopsis and tobacco suspension culture (NT1) cells. I have developed
stable transformants with increased and reduced levels of CRT. My
preliminary evidence suggests that our original hypothesis that altered
expression of CRT alters calcium stores in plants is correct. In
Arabidopsis altered CRT alters viability on reduced calcium media and
effects the plant's ability to respond to a gravity stimulus under low
calcium conditions but does not significantly effect growth rates. These
results strongly suggest a role for calcium in the gravity signal
transduction-response pathway. The work to date has focused on gravity as
a stimulus; however, these materials could be used for a variety of other
environmental stresses including light, cold, pathogen attack, or touch.
Goals for future research include 1) characterization of the physiology of
these transgenics with respect to their response to gravity and to broaden
this aspect of the research to include other stimuli, and 2) evaluation of
the changes in Ca 2+ storage and cellular Ca 2+ upon stimulation.
Measurement of cellular and subcellular calcium will be critical both
through 45Ca 2+ loading experiments and imaging of cellular calcium.
Traditionally, cellular calcium has been measured through ratio imaging of
microinjected dextran-linked dyes. However, this allows only cytosolic
free calcium to be measured and is highly time consuming. So in addition
to these dyes, we have begun development of systems utilizing targeted
aequorin and the calmodulin-linked GFP cameleons.
Second, mutational analyses have provided important information about the
regulation of the components of the phototropic signal transduction
pathway. To date, several gravity mutants have also been isolated, but
most are pleiotropic with dramatic effects on growth, and many are also
altered in their response to auxin. My early work to characterize the
graviresponse of the inflorescence stems of Arabidopsis and recent data
published by Fukaki et al (Plant Phys., 1996, 110: 933-943) suggested a
mutant screen that could isolate mutants specifically impaired in the early
events of the signal transduction pathway. The gravitropic response of
Arabidopsis stems is visible within 30 min of stimulation and results in
vertical reorientation of the apical meristem within 2 h. However,
horizontal gravistimulation for 3 h at 4oC does not cause curvature. When
the stems are subsequently placed in the vertical position at 22oC, they
still curve in response to the previous, horizontal gravistimulation.
These results indicate that the gravity perception step can occur at 4oC
but that a part of the response is sensitive to cold. Stems incubated
horizontally at 4oC for 30 min, then vertically at 4oC for up to 60 min,
bend in response to the horizontal gravistimulation when returned to 22oC,
suggesting that the gravistimulation perceived at 4oC was "set," and was
not reversible even at 4oC. Utilizing this cold effect on gravity signal
persistence, I initiated a screen to select for mutants affected in the
signal transduction and/or storage mechanism. Screening, to date, has
identified three classes of mutants defective in their ability to perceive
a gravity stimulus or properly transduce that information to the responding
tissue. For example, the mutant, designated Gravity Persistence Signal 1
(GPS1), is positively gravitropic after presentation at 4oC whereas at 22oC
the stem shows a normal (negative) gravitropic response. Future goals for
this project are the characterization of these mutants and identification
of the genes involved.
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3. SIGNAL TRANSDUCTION IN GRAVITROPISM: STUDIES WITH ROOTS AND RHIZOIDS
ELISON BLANCAFLOR (Simon Gilroy Lab)
Pennsylvania State University
The directional growth and subsequent developmental patterns that plant
organs exhibit in response to gravity, allows for correct anchorage,
nutrient and water acquisition, seedling emergence, and light absorption
for photosynthesis. Gravitropism has been conveniently divided into a
sequence of events namely gravity perception, signal transduction, and the
growth response (i.e. differential organ growth). The mechanisms underlying
the transduction of the gravity signal has remained the most elusive phase
and is the primary focus of my research. We have used two systems for our
research on gravitropism namely primary roots of higher plants and rhizoids
of the green alga Chara.
Roots offer the unique advantage in that the site of gravity perception
(the columella region in the root cap) and the site of the response (the
elongation zone) occurs in defined, spatially distinct regions. A wealth of
literature supports the idea that starch-filled plastids (amyloplast)
present in the central columella cells of the root cap function as
statoliths. The widely accepted starch-statolith hypothesis proposes that
the sedimentation of amyloplasts upon reorientation of the plant constitute
the initial act of gravity perception. Recent laser ablation studies with
roots of Arabidopsis have offered support for the starch-statolith
hypothesis because these studies show that the columella cells with the
fastest rate of amyloplast sedimentation contribute most to root
gravitropism. Importantly, these laser ablation studies have revealed for
the first time that the cells located in story 2 of the columella region
provides the greatest contribution to gravitropic sensitivity (Blancaflor
et al., 1998).
As a first step in understanding signal transduction events in gravitropism
we have focused on visualizing the activity of putative signal transduction
elements (e.g. calcium and protons) in gravity sensing columella cells
using fluorescent indicator dyes. We have recently shown that the touch
stimulus elicits transient changes in cytoplasmic calcium in roots but no
detectable changes in cytoplasmic calcium were associated with the gravity
response (Legue et al., 1997). However rapid changes in pH occur within
seconds of gravistimulation and could constitute one of the earliest events
in gravitropic signaling in plants. Future research will attempt to map the
precise temporal and spatial patterns of these pH changes as they occur in
the root and explore the possibility of using this as a physiological assay
to identify additional components of early gravity sensing and transduction
events.
To further test the involvement of regulatory molecules in gravitropism,
the rhizoids of the green alga Chara offer a more convenient system for
microscope based studies in gravitropic-signaling. While the statoliths of
roots are thought to be starch-filled amyloplast located in the columella
region of the root cap, the statoliths of Chara rhizoids are the barium
sulfate filled vesicles found at the apical region of the rhizoid. Upon
tilting the rhizoid by 90°, statoliths sediment onto the lower flank of the
cell within minutes and downward bending commences shortly after their
sedimentation. We are interested in characterizing calcium and pH gradients
in tip growing rhizoids and then manipulating these gradients using caged
molecules to link statolith sedimentation, ion gradients, and redirected
growth that leads to gravitropism.
The possibility that the cytoskeleton is involved in gravitropism is an
appealing concept because it has been shown to reorganize in response to a
variety of environmental factors and proposed to be involved in many
cellular processes (e.g. intracellular transport, cell wall synthesis, and
cell division). My previous results have shown that a reorganization of the
microtubule cytoskeleton is correlated with the gravitropic response of
roots (Blancaflor and Hasenstein, 1993). However, the microtubule
reorganization event appears to be a consequence of altered growth patterns
in the root rather than its cause (Blancaflor and Hasenstein, 1995). This
also appears to be the case for the actin component of the cytoskeleton
(Blancaflor and Hasenstein, 1997). Furthermore, microtubule inhibitors do
not affect auxin transport in maize roots (Hasenstein et al., 1999).
Although the cytoskeleton does not appear to be a major factor in the final
growth response during gravitropism, the role of the cytoskeleton in the
perception of the gravity signal has not yet been resolved and therefore
warrants further investigation.
An interesting finding is that the central columella cells in the root cap
are depleted of thick actin filaments (Blancaflor and Hasenstein, 1997). It
has been proposed that this could be one of the adaptations of the gravity
sensing columella cells favoring free sedimentation of amyloplast. We would
like to explore this intriguing observation further by measuring the forces
needed to displace amyloplast in columella cells using laser tweesers and
determine whether there is a correlation between the scarcity of actin
cables and the movement of statoliths. Furthermore, we are interested in
using rapid freezing techniques and immunofluorescence microscopy to
visualize the cytoskeleton in the columella cells of the root cap.
We would also like to characterize further the role of the cytoskeleton in
gravitropic single cells such as the Chara rhizoid. Recent experiments in
our lab have revealed that the microtubule cytoskeleton is important in
directing root hair growth and could potentially interact with the cellular
machinery that maintains a cytoplasmic calcium gradient at the tip
(Bibikova et al. 1998). Similar experiments with Chara rhizoids have also
shown that microtubule disrupting drugs affect the morphology and
directionality of rhizoid growth. Future studies will attempt to clarify
the involvement of the cytoskeleton in rhizoid growth and gravitropism, and
define its interaction with the ion gradients at the tip.
References
1.Bibikova T.N., Blancaflor E.B., Gilroy S. (1998) Microtubules regulate
tip growth and direction in root hairs of Arabidopsis thaliana (Submitted)
2.Blancaflor E.B., Hasenstein K.H. (1993) Organization of cortical
microtubules in graviresponding maize roots. Planta 191: 231-237.
3.Blancaflor E.B., Hasenstein K.H. (1995) Time course and auxin sensitivity
of cortical microtubule reorientation in maize roots. Protoplasma 185: 72-82.
4.Blancaflor E.B., Hasenstein K.H. (1997) The organization of the actin
cytoskeleton in vertical and graviresponding primary roots of maize. Plant
Physiology 113: 1447-1455.
5.Blancaflor E.B., Fasano, J.M., Gilroy S. (1998). Mapping the functional
roles of cap cells in the response of Arabidopsis primary roots to gravity.
Plant Physiology 116: 213-222
6.Legue V., Blancaflor E.B., Wymer C., Fantin D., Perbal G., Gilroy, S.
(1997) Cytoplasmic free calcium in Arabidopsis roots changes in response
to touch but not gravity. Plant Physiology 114: 789-800.
7.Hasenstein K.H., Blancaflor E.B., Lee J.S. (1999) Does the microtubule
cytoskeleton integrate auxin transport and gravitropism in maize roots?
Physiologia Plantarum (in press)
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4. SIGNAL TRANSDUCTION IN THE BARLEY ALEURONE
SIAN RITCHIE (Simon Gilroy Lab)
Pennsylvania State University
The system Work on the barley aleurone in the laboratory of Simon Gilroy is
centered on identifying components of the signal transduction machinery of
this cell in the response to gibberellic acid (GA) and abscisic acid (ABA).
The cereal aleurone is used as a model system for studying the action of
these plant hormones because it exhibits a well defined set of responses to
GA and ABA and it is possible to work with a single cell type. During
cereal seed germination the aleurone produces hydrolytic enzymes which are
responsible for mobilization of the starchy reserves in the endosperm,
providing energy for seedling development. These processes are activated by
GA supplied by the hydrating embryo, and this GA response is inhibited by
ABA.
GA regulated events The importance of calcium in the response of the
aleurone to GA is well documented (refs 1 & 2). Although the calcium signal
is not required for gene activation, it is necessary for the downstream
events of processing and secretion of the hydrolytic enzymes in response to
GA (ref 1). We were interested in determining if calcium-dependent protein
kinase activity (CDPK) could be involved in these calcium-based events.
Peptide substrates of a range of protein kinases were microinjected or
electroporated into aleurone protoplasts to identify if any could inhibit
the GA response, by competing with the endogenous substrate/s. We found
that one specifically, syntide-2, was able to block the GA response.
Photoaffinity-labeled syntide-2 electroporated into protoplasts bound to
proteins of 33 and 55 kDa. Aleurone extracts contained a 54 kDa CDPK, and
in vitro syntide-2 was phosphorylated in a calcium-dependent manner (ref
1). These data suggest that the target of syntide-2 in vivo was the
activity of this 54 kDa protein kinase which may therefore be involved in
mediating the GA activated, calcium-dependent events in the aleurone.
More recently we have turned to molecular biology techniques to
investigate further the role of CDPK in the aleurone. In collaboration with
Andrew McCubbin (Department of Biochemistry and Molecular Biology at Penn
State) we have isolated a full length CDPK clone from a barley aleurone
cDNA library. The sequences encodes for a protein of 53.3 kDa which is very
similar to the protein identified using biochemical techniques. Initial
characterization of the recombinant protein shows it has very similar
properties to the calcium-dependent syntide-2 phosphorylating activity
identified biochemically. Since the domains of plant CDPKs have been
studied in some detail, we are using this information to design mutated and
truncated forms of the aleurone CDPK which will allow for the introduction
of, among other things, a "dominant-negative" protein into aleurone
protoplasts.
Another project has developed from an interest in the ideas of Bradford
and Trewavas (ref 4) regarding the sensitivity threshold concept as an
explanation for the broad concentration range (10-11 - 10-6 for the
aleurone) through which GA acts. They proposed that this is not due to all
the cells responding in a very gradual way to increasing GA concentrations,
rather each cell has a threshold GA concentration above which it is
switched "on". Within a population of aleurone cells there are
subpopulations which have different thresholds, and increasing the
concentration of GA increases the number of cells recruited to the GA
response. From a detailed study of the GA response of the aleurone layer
and individual protoplasts we have data that supports the hypothesis of
Bradford and Trewavas, and we have also discovered that part of the
difference in sensitivity lies in the spatial organization of the aleurone
(ref 5).
Phospholipase D mediated ABA signaling We have identified phospholipase D
(PLD) as an element in the early signal transduction pathway/s of ABA in
this cell type. The evidence for this link comes from biochemical studies
of aleurone protoplasts, primarily in vitro phospholipase D assays, and in
vivo measurement of phospholipids. We found that the application of ABA to
protoplasts increased the activity of PLD 10 min after application. In vivo
levels of PtOH (the product of PLD activity) also increased transiently at
this time. The application of PtOH to protoplasts led to an ABA-like
inhibition of the GA response and induction of an ABA upregulated protein
Rab (Responsive to ABA). Finally, inhibition of the production of PtOH by
PLD by the application of 0.1% 1-butanol during the initial 20 min after
ABA treatment resulted in inhibiton of ABA-regulated processes. This
inhibiton coincided with the timing of PLD activation by ABA and was
overcome by simultaneous application of PtOH (ref 6). More recently we have
developed an in vitro assay in which the stimulation of PLD by ABA mimicks
that seen in vivo. This is allowing us to characterize further the nature
of the stimulation including cellular localization and involvement of other
signaling proteins.
1. Gilroy S (1996) Signal transduction in barley aleurone protoplasts in
calcium dependent and independent. Plant Cell 8: 2193-2209.
2. Ritchie S, Gilroy S (1998) Gibberellins: regulating genes and
germination. The New Phytologist in press.
3. Ritchie S, Gilroy S (1998) Calcium-dependent protein phosphorylation may
mediate the gibberellic acid response in barley aleurone. Plant Physiol
116: 765-776.
4. Bradford KJ, Trewavas AJ (1994) Sensitivity thresholds and variable time
scales in plant hormone action. Plant Physiol 105: 1029-1036
5. Ritchie S, McCubbin A, Ambrose G, Kao T-h, Gilroy S. The sensitivity of
barley aleurone cells to GA in spatially determined. submitted to Plant
Physiol
6. Ritchie S, Gilroy S (1998) Phospholipase-D mediates ABA signaling in
barley aleurone cells. PNAS USA 95: 2697-2702.
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5. PHOSPHORYLATION OF SUCROSE SYNTHASE: MEMBRANE ASSOCIATION AND ACTIN BINDING
HEIKE WINTER, SARAH E. WYATT, AND JOANNE C. LONG (Joan L. Huber, Steven C.
Huber, and Dominique Robertson Labs)
North Carolina State University
Sucrose Synthase (SuSy), which catalyzes the reversible conversion of
sucrose and UDP into UDP-glucose and fructose, occurs both as a soluble and
membrane-associated enzyme in higher plants. Recently, it was found that
some of the SuSy protein is associated with the plasma membrane and it was
postulated that it may exsist in a complex with b-glucan synthases to
channel UDP-glucose directly into cellulose synthesis (Armor et al, 1995).
We have been studying the mechanism underlying the membrane-association of
SuSy in graviresponding maize pulvini and elongating leaves. SuSy can be
phoshorylated in vivo on a single serine residue (Ser-15) in maize leaves
(Huber et al., 1996). Experiments were conducted to determine whether
phosphorylation could directly affect SuSy localization in vitro. A
clarified crude pulvinus extract (containing the microsomal membrane
fraction and soluble proteins) was preincubated at 25°C in the absence
(endogenous protein phosphatase (PP)) or presence of protein phosphatase
inhibitors (NaF and microcystin-LR; "Control"), or with addition of
alkaline phosphatase to facilitate dephosphorylation of proteins including
SuSy. After preincubation, the distribution of SuSy protein between the
soluble and membrane fraction was assessed by immunoblotting and activity
assay. We found that pretreatment conditions that promoted protein
dephosphorylation (endogenous and alkaline PP) reduced SuSy protein in the
soluble fraction while increasing the protein associated with the membrane
fraction. When phosphorylation was promoted by addition of ATP+PKA
(catalytic subunit of cAMP-activated protein kinase) in the presence of
phosphatase inhibitors, the membrane-associated SuSy protein was reduced
while SuSy protein in the soluble fraction was increased. These reciprocal
changes in SuSy protein and activity distribution are consistent with the
notion that protein dephosphorylation may mediate the translocation of SuSy
to the membrane, and that protein phosphorylation of SuSy is part of the
mechanism that releases SuSy from the membrane. In order to test whether
phosphorylation of SuSy itself could be involved in its intracellular
localisation, the microsomal membrane fraction was treated under
phosphorylating conditions in the presence of [g-32P]ATP and Ca2+.
Phosphorylation was terminated with EGTA and the soluble and membrane
fraction separated. An autoradiograph of the immunoprecipitated SuSy showed
that SuSy released from the membrane was phosphorylated (Winter et al,
1997). Phosphorylation and release of phosphorylated SuSy protein from the
membrane was greatest in the presence of PKA catalytic subunit. In the
absence of PKA, less SuSy was released into the soluble fraction, and at
least part of the phosphorylated SuSy remained associated with the membrane.
This suggests that either the activity of endogenous SuSy-kinase
(membrane-associated) is limiting and/or other membrane protein(s) are
phosphorylated in order to release SuSy protein into the soluble fraction.
One mechanism by which phosphorylation could affect membrane association of
SuSy would involve conformational changes that affect exposure of
hydrophobic residues. This notion is supported by experiments with the
hydrophobic flourescent probe bis-ANS. In aqueous solution, the
flourescence of bis-ANS is very low, but in apolar solvent or when
associated with hydrophobic sites of a protein, flourescence intensity is
enhanced markedly [Rosen and Weber, 1969; Musci et al., 1985]. When
affinity-purified SuSy was incubated with bis-ANS, there was a
time-dependent increase in fluorescence, reflecting binding of the probe to
the protein (Winter et al, 1997). After about 30 min, the fluorescence
emission was constant, but an additional increase in flourescence occurred
after addition of alkaline phosphatase. Appropriate controls indicated that
dephosphorylation of SuSy by the alkaline phosphatase was responsible for
the flourescence increase. These results suggest that dephosphorylation
promotes a conformational change, in which hydrophobic residues are more
exposed to the solvent. To further test this postulate, we attempted to
increase the phosphorylation status of SuSy to a higher level by incubation
with PKA + ATP. There was a time-dependent decrease in bis-ANS flourescence
when SuSy was mixed with ATP, PKA and sucrose. We also found that all three
components were required and that there was no decrease in flourescence in
the absence of SuSy, the presumed target of the flourescent probe. These
results support the postulate that phosphorylation caused a conformational
change resulting in decreased surface hydrophobicity, presumably by burial
of hydrophobic side chains. The requirement for sucrose suggests that
substrate binding may be necessary for the presumed conformational change
of phospho-SuSy to occur. Quantifying the distribution of SuSy between
soluble and membrane fractions, we found that a substantial amount of SuSy
protein partitioned into the detergent-insoluble pellet. This fraction is
known to be enriched in cytoskeletal polymers (microfilament and
microtubules). The amount of F-actin in this fraction can be increased by
using cytoskeleton-stabilizing buffer (Abe and Davies, 1991;Tan and Boss,
1992). Under conditions stabilizing F-Actin, we found increased amounts of
SuSy in the detergent-insoluble pellet. Immunoprecipitation of SuSy from
the soluble (100.000 x g supernatant) and the detergent-resolubilized
microsomal membrane fraction with monoclonal anti-maize-SuSy antibodies
resulted in co-immunoprecipitation of actin from the soluble fraction only
(Winter et al, 1998).
In order to determine whether SuSy could bind directly to actin, and
whether this binding is specific or not, we polymerized rabbit muscle actin
in vitro in the presence and absence of purified SuSy. As controls for
specificity of actin binding, we added BSA (not actin binding) or rabbit
aldolase (actin binding). Binding of proteins by F-actin was then monitored
by sedimentation. We found that the F-actin pellet contained approximately
25% of the total SuSy protein, but less than 5 % of BSA, and that aldolase
competes with SuSy for F-actin binding (Winter et al, 1998), suggesting
that SuSy and aldolase may share a common binding site on actin. Increasing
the total concentration of SuSy in an in-vitro polymerization assay with a
constant amount of actin resulted in an increase in the amount of bound
SuSy with saturation of binding at approximately 0.2 nmol SuSy monomer per
nmol of actin (monomer). Assuming that SuSy binds as a tetramer, then one
native SuSy molecule may be associated with F-actin equivalent to 20
monomers. Binding of purified SuSy to actin in vitro requires the presence
of di- or tri-saccharides (sucrose, trehalose, raffinose). We found that
monosaccharides or glycerol at the same concentration did not induce SuSy
binding to actin. If sucrose binding to the catalytic site regulates actin
binding, then trehalose or raffinose should compete for this binding site
-affecting the catalytic activity of sucrose cleavage. Raffinose and
trehalose are not substrates for SuSy and did not affect the velocity of
sucrose cleavage when present in equimolar concentration to sucrose. This
suggests that SuSy might have a binding site for sucrose (and raffinose or
trehalose) that is distinct from the catalytic site. The existence of a
regulatory sucrose-binding site on SuSy is also indicated by the
differences in sucrose concentrations required for cleavage activity versus
actin binding. We are currently attempting to visualize SuSy localization
in vivo using a SuSy green fluorescent protein (GFP) fusion protein.
Initially we are using particle bombardment to transiently express SuSy-GFP
in onion bulb epidermal cells.
In conclusion, phosphorylation of SuSy may be at least part of the
mechanism controlling SuSy localization, and is supported by three lines of
evidence: i) dephosphorylation of SuSy causes increased association with
the membrane fraction; ii) phosphorylation of membrane-associated SuSy
caused its release from the membrane, and iii) in vivo phosphorylation
studies indicate that the membrane-associated enzyme contains less 32P than
the soluble enzyme (SuSy protein basis). At least part of the underlying
mechanism of reversible protein translocation may be
phoshorylation-dependent conformation changes that affect surface
hydrophobicity. That surface properties are altered is supported by studies
with the hydrophobic probe bis-ANS and observations relating to differences
in protein solubility.
A new aspect of SuSy is its potential to associate with the actin
cytoskeleton. Although much remains to be done, our data strongly indicate
that at least part of "soluble" SuSy might not be so soluble in vivo but
associated with actin. The association of "soluble" and presumably
phosphorlyated SuSy with actin appears to be under metabolic control. High
concentrations of sucrose (and other polysaccharides) are required for SuSy
association with actin. This suggests a regulatory sucrose binding site on
SuSy responsible for its actin association.
References
Abe, S., Davies, E. (1991) Protoplasma 163, 51-61.
Amor, Y., Haigler, C.H., Johnson, S., Wainscott, M., Delmer, D.P. (1995)
Proc. Natl. Acad. Sci. USA 92,9353.
Carlson, S.J., Chourey, P.S. (1996) Mol, Gen. Genet. 252, 303-310.
Huber, S.C., Huber, J.L., Liao, P.C., Gage, D.A., McMicheal, Jr., Chourey
P.S., Hannah, L.C., Koch, K. 1996) Plant Phys. 112, 793
Kaufman, P.B., Brock, T.G., Song, I., Rho, Y.B., Ghosheh, N.S. (1987) Amer.
J. Bot 74 (9), 1446-1457.
Musci, G., Metz, G.D., Tunematsu, H., Berliner, L.J. (1985), Biochemistry
24, 2034-2039.
Rosen, C.G, Weber, G. (1969) Biochemistry 8 (10), 3915-3920.
Tan ,Z., Boss, W.F. (1992) Plant Physiol. 100,2116-2120.
Winter, H., Huber, J.L. , Huber, S.C., (1997) FEBS letters 420,151-155.
Winter, H., Huber, J.L., Huber, S.C., (1998) FEBS letters 430, 205-208.
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6. INVESTIGATING THE ROLE OF CALMODULIN IN GRAVITROPISM OF ARABIDOPSIS
THALIANA
JOHN LOVE (Bill Thompson Lab)
North Carolina State University
Evidence suggests that calmodulin, a small, acidic protein (±150 AA) and
the main regulator of cytosolic Ca2+ in plant cells, is involved in the
transduction of gravitropic signaling. Whether calmodulin is involved
directly during signaling, or has a more subtle role by modulating cellular
sensitivity to Ca2+, is unknown.
My research aims to further elucidate the role of calmodulin in plant cell
signaling, with particular reference to root gravitropism. These questions
are being addressed in the model plant Arabidopsis thaliana, using
recombinant DNA technology. I am currently developing an organ targeted,
inducible promoter system in A. thaliana that will enable me to alter
intracellular calmodulin levels and activity in both space and time. In a
first instance, I have targetted the root cap and the distal elongation
zone. The expression of calmodulin in transgenic plants will be correlated
with various subcellular and physiological effects following
gravistimulation, for instance the intensity of Ca2+ signals, modulations
of other secondary messengers such as IP3, variations in protein
phosphorylation, and root curvature. The hypothesis that calmodulin might
alter a root's sensitivity to gravity will be addressed using magnetic
ponderomotive forces to simulate gravitational fields of varying intensity.
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7. ACTIN IN THE GRAVITY-SENSING CELLS OF THE MAIZE ROOT CAP
DAVID COLLINGS (Nina Allen Lab)
North Carolina State University
We have re-investigated the actin cytoskeleton in the central columella
cells of the root cap and the stem pulvinus of maize, because in a recent
review, Baluska and Hasenstein [Planta (1997) vol 203 s69-s78] stated that
the inability to visualise actin within the columella cells of root caps
showed that these gravity-sensing cells are substantially depleted of
F-actin bundles, presumably because this would allow plastid sedimentation
to occur more freely.
We have shown, using immunofluorescence microscopy, that maize stem
pulvinal cells contain an extensive actin cytoskeleton of randomly oriented
bundles that is indistinguishable from that in the neighbouring,
non-graviperceptive cells (Collings et al. (1998) [Planta vol 207
246-258]). We could not observe any changes in this actin cytoskeleton
during gravity-perception, nor during the bending response, although we are
now pursuing rhodamine-phalloidin injections of these cells to determine
the finer nature of this actin cytoskeleton, and possible changes following
gravistimulation. However, that the pulvinus contained actin suggested that
the reduced-actin hypothesis of Baluska and Hasenstein is either incorrect
in root cap cells, or non-tenable in the pulvinus. Our re-investigation of
actin within the root used an identical methodology to that which gave
successful actin labelling in the pulvinus. A polyclonal antibody raised
against maize actin gave extensive labelling throughout the root. Initial
experiments indicate randomly-oriented actin within the central columella
cells of the root cap, although high levels of autofluorescence and
fixation-induced fluorescence have to date limited observations to date.
