PST Bulletin Vol. IX

daemon at net.bio.net daemon at net.bio.net
Tue Oct 8 14:25:30 EST 1996


PLANT SIGNAL TRANSDUCTION BULLETIN

IX

October 1996


The Plant Signal Transduction news bulletin is now in its sixth year of
existence, distributing information related to signals and signaling in
plants.  Starting with this issue, the bulletin will also be posted in the
Bionet.Plants.Signal-Transduction usenet newsgroup.
The next issue (Vol. X) will be coming out in the first half of 1997.
Please submit your PST Bulletin contributions (opinions, perspectives,
meeting notices, funding notices, recent books, technical notes, citations,
positions announcements) and any suggestions to:
Bratislav Stankovic
E-mail:   stankovic.1 at osu.edu
[Ohio State University, Department of Plant Biology, 1735 Neil Avenue,
Columbus, OH 43210, USA]

______________________________________________________________

PERSPECTIVES AND LAB UPDATES
______________________________________________________________


THE RHO GTPASE AND THE DEVELOPMENT AND FUNCTION OF MALE GAMETOPHYTES IN PLAN=
TS

We are interested in the role of the rho family of small GTPases in plant
signal transduction.  These are conserved molecular switches that were
first found to control the organization of actin cytoskeleton in fungi and
mammals.  Recent studies show that they are versatile signal transducers
that also modulate a variety of other processes including gene expression
(by activating MAP kinase cascades), membrane trafficking, and activation
of enzymes such as mammalian NADPH oxidase and fungal glucan synthase.  A
pea homolog of the rho GTPases was identified a few years ago (Yang and
Watson, 1993), we have now shown that plants have a unique subfamily of rho
GTPases, designated as Rop.  Using a polyclonal antibody raised against the
pea Rop, we found that one or more of Rop GTPases are expressed in pollen
and are differentially localized to the apex and the periphery of
generative cells in pea pollen tubes (Lin et al., 1996).  Rop proteins are
also detected in tapetal cells and are localized to network-like
peri-nuclear structures.  Studies are being conducted to determine whether
Rop is localized to ER in tapetal cells.  In situ hybridization and
promoter:GUS fusion analyses suggest that both pollen and tapetum Rop
proteins may be encoded by Rop1At in Arabidopsis.

Zhenbiao Yang, Yakang Lin, and Hai Li, Plant Biotechnology Center, The Ohio
State University, Columbus, OH 43210.  email:yang.147 at osu.edu

Yang, Z. and J.C. Watson.  1993.  Molecular cloning and characterization of
rho, a ras-related small GTP-binding protein from the garden pea. Proc.
Natl. Acad. Sci. USA 90:8732-8736.

Lin, Y., Y. Wang, J.-K. Zhu, and Z. Yang. 1996. Localization of a rho
GTPase implies a role in tip growth and movement of the generative cell in
pollen tubes.  Plant Cell 8: 293-303.


****************************************************************

EVALUATING 3D MICROSCOPY WITH A STANDARDIZED TEST OBJECT

How accurate are confocal images, and how do they compare to non-confocal
images or to images processed by deconvolution?  Based on studies of a
well-defined test object, we have found that a variety of confocal
microscopes often exhibit serious aberrations, and even under the best
circumstances do not accurately reveal the true structure of the test
object.  Aberrations can also arise when non-confocal images of the test
object are processed by some deconvolution methods, but at least one
deconvolution method which we have tested produces a better image of the
test object than that obtained on any confocal microscope thus far.

The test object is a 10um diameter fluorescent bead which we have sectioned
on a microtome to determine its fluorescent distribution.  The microtome
sections show that the bead is composed of a 2um-thick shell of
fluorescence at its periphery surrounding a core that is largely devoid of
fluorescence.  Images on commercial confocal microscopes often show serious
distortions in the bead image.  Distortions include a scalloped xz profile
of the bead image that probably arises from inaccurate focal change, or
irregular jagged edges in an xy or xz view of the bead that probably arise
from improper vibration isolation of the microscope.  In addition, many
confocal instruments exhibit an off-axis tilt of the bead's xz profile
suggesting an off-axis aberration such as astigmatism, which may arise due
to slight errors in confocal alignment.

Even the best confocal images that we have obtained so far exhibit a
significant blurring of the bead's fluorescent shell into the bead's
non-fluorescent core.  In the confocal images, fluorescent intensity
gradually decays from the bead's outer perimeter to its center point, even
though the actual distribution from microtome sections shows a very sharp
boundary between the fluorescent outer shell and the non-fluorescent inner
core.  This blur is much greater than would be predicted in theory,
suggesting that all of the instruments we have tested are operating well
below theoretical resolution limits.  Our current efforts are aimed at
identifying why this might be the case.

At least one deconvolution method applied to non-confocal images produces a
better image of the bead than any of the confocal images we have obtained
so far.  The method, (the maximum-likelihood estimation-maximization
algorithm - available gratis at
http://wuibc.wustl.edu/bcl/xcosm/xcosm.html), resolves a much sharper
boundary between the outer shell and inner core of the bead.  Other
deconvolution methods (some of them commercially available) also reveal a
sharper boundary than seen in confocal images, but these deconvolution
methods leave the bead image elongated in xz profiles.  We are currently
applying these deconvolution methods to the confocal images of the bead.

In sum, we have found that a test object is a useful tool for identifying
distortions and limitations in 3D microscope images.  In addition to
aberrations that may be specific to certain installations or brands of
confocal microscope,  we have found that all confocal images of this object
suffer more blurring than expected.

Jim McNally
E-mail:  mcnally at tyrone.wustl.edu

*******************************************************

ROLE IDENTIFIED FOR 14-3-3 PROTEINS IN REGULATION OF NITRATE REDUCTASE

Control of nitrate reductase (NR) in higher plants involves regulation of
enzyme protein level and reversible protein phosphorylation.  The
inactivation of spinach leaf NR by phosphorylation is a two-stage process:
first, NR is phosphorylated on Ser543 and second, the phospho-NR binds a
60-kDa inhibitor protein in the presence of divalent cations to form an
inactive complex.  The inhibitor protein has recently been identified in
two labs (1.,2) as a mixture of 14-3-3 proteins.  The 14-3-3 proteins are
ubiquitous among plants and animals and are thought to function as binding
proteins modulating interactions between proteins involved in signal
transduction pathways.  Identification of the phospho-NR inhibitor protein
as one or more 14-3-3 proteins provides additional insight about the
control of this important enzyme of nitrate assimilation. Some of these
aspects will be covered in a forthcoming review (3).

1. Bachman M, Huber JL, Liao PC, Gage DA and Huber SC (1996) The inhibitor
protein of phosphorylated nitrate reductase from spinach leaves is a 14-3-3
protein. FEBS Lett 387: 127-131

2. Moorhead G, Douglas P, Morrice N, Scarabel M, Aitken A, MacKintosh C
(1996) Phosphorylated nitrate reductase from spinach leaves is inhibited by
14-3-3 proteins and activated by fusicoccin. Current Biology 6, 1104-1113.

3. Huber SC , Bachmann M, Huber JL (1996) Post-translational regulation of
nitrate reductase activity: a role for Ca2+ and 14-3-3 proteins. Trends
Plant Sci., in press.


Steven C. Huber, PhD
USDA Professor of Crop Sci & Bot
North Carolina State University
Raleigh, NC 27695-7631
(919) 515-3906
(919) 856-4598 FAX

********************************************************

______________________________________________________________

ANNOUNCEMENTS:
______________________________________________________________


Second Annual Meeting of the NASA/NSF Joint Program in Plant Biology
Network for Research on Plant Sensory Systems

NASA and the NSF are cosponsors of a five-year program to support a network
of researchers studying mechanisms of plant responses to environmental
signals such as light, temperature, and mechanical stimulation (gravity,
wind, touch).  The program was designed by NASA and NSF to foster
collaborations between laboratories with different perspectives and
research strengths, with the expectation that this will quicken the pace of
discovery.  Information on the activities of the network can be obtained
from the world wide web by visiting http://trna.chem.yale.edu/pss/.

This research group held its second annual meeting at Washington University
in St. Louis on October 2-4.  The main purposes of the meeting were to
present progress reports on collaborative projects between the laboratories
of the network and to assess initial progress on a recently chosen central
project to serve as a research focus for the group.  The focus of the
central project will be on the sensory properties of cells in the distal
elongation zone (DEZ) of roots.  The long term goal of the project is to
determine the primary mechanism(s) by which growth rate adjustments occur
in the DEZ in response to environmental stimuli, especially gravity.  The
distal elongation zone of roots is a group of cells positioned between the
main portion of the root apical meristem and the zone of rapid elongation.
Recent research has shown that the DEZ plays a central role in the response
of roots to a wide variety of stimuli including gravity, touch, mechanical
impedance, electric field imposition, water stress, cation gradients, and
responses to a variety of hormones.

Incentive for choosing the DEZ for the focus of the central project
includes the observations that: 1) The response of cells of the DEZ to
environmental stimuli is dramatically different from that of adjacent cells
in the main elongation zone, 2) Screening of T-DNA mutagenized seedlings by
workers outside the network has revealed genes expressed only within the
DEZ and conversely, genes expressed in tissues on either side of the DEZ
but not within the DEZ itself, 3) the DEZ is strategically located between
the root cap (thought to be the main region of graviperception) and the
main elongation zone, 4) The DEZ shows rapid and strong changes in membrane
potential within a matter of seconds following gravistimulation indicating
that these cells will be amenable to electrophysiological studies designed
to identify some of the primary responses linked to stimulus-induced growth
rate changes, and 5) the cells of the DEZ should be relatively easy to work
with in that they do not have large vacuoles and they lack chloroplasts.

One of the main approaches to examining the DEZ will be to compare patterns
of gene expression in the DEZ of Arabidopsis with patterns in bordering
tissue regions.  This will be pursued  utilizing both differential gene
expression studies as well as the DNA chip technology developed in the
laboratory of Ron Davis at Stanford University.  Toward that end, we have
collected 1000 samples each of isolated DEZ tissue from Arabidopsis as well
as samples of the apically adjacent cap/meristem tissue and samples of the
basally adjacent elongation zone tissue for comparisons of patterns of gene
expression.  We have also begun characterizing changes in physiological
properties of the DEZ associated with the changes in hormone sensitivity
observed in a variety of Arabidopsis mutants.  Parallel approaches to
characterizing the properties of the DEZ will include analysis of ion
channel activity in these cells as well as patterns of macromolecule
distribution using antibodies to plasma membrane proteins from Arabidopsis.

=46or a brief review of the significance of the DEZ to studies of sensory
physiology in plant cells, see Ishikawa and Evans (1995) Plant Physiology
109:725-727.

Mike Evans
E-mail: evans.20 at osu.edu



*******************************************************************

I am writing a review for Ann. Rev. Cell & Devel. Biol. which we will send
off in January 1997.  The topic will be centered around the area of plant
membrane/cytoskeleton/cell wall interactions.  I am writing to all of you
to ask a favor.  Would it be possible for you to send me your relevant
reprints and preprints as well as recent references on this topic ?
Obviously I am trying to canvass the field of researchers and publications
on this topic but we will undoubtedly miss some.  I hope with this request,
that I will miss fewer !

Best regards,
Ralph

Ralph Quatrano
Department of Biology
Coker Hall, South Road
Chapel Hill, NC 27599-3280
Phone: (919)-962-2098
=46ax: (919)-962-6840
=46ax: (919)-962-3690
Email: rsq at unc.edu

***************************************************************

A book that should be of interest to some is:
Hoch, H. C., L. W. Jelinski, and H. Craighead (Editors).  1996.
Nanofabrication and Biosystems: Integrating Materials Science, Engineering
and Biology.  Cambridge University Press, 423 pp. ISBN 0-521-46264-9

The contents are:

Preface

Table of Contents

Contributors

Introduction --- The Frontiers and Challenges
L. W. Jelinski, H. C. Hoch, and H. G. Craighead

Microfabrication: contemporary and emerging techniques

Chapter 1       A. Forchel, P. Ils, R. Steffen, M. Bayer
High Resolution Lithographic Techniques for Semiconductor Nanofabrication

Chapter 2       R. Germann
Principles of Materials Etching

Chapter 3       K. D. Wise
The Development and Application of Micromechanical Devices in Biosystems

Chapter 4       M. Esashi
Microsensors and Microactuators for Biomedical Applications

Chapter 5       M. Washizu
The Use of Micromachined Structures for the Manipulation of Biological Objec=
ts

Chapter 6       H. M. McConnell
Light Addressable Potentiometric Sensor: Applications to Drug Discovery

Chapter 7       M. Malmqvist
A Surface Plasmon Resonance Biosensor for Characterization of Biospecific
Interactions

Chapter 8       R. Oldenbourg, S. Inou=E9, R. Tiberio, A. Stemmer, G. Mei,
and M. Skvarla
Standard Test Targets for High Resolution Light Microscopy

Chapter 9       P. J. Beyer, R. A. Lee, M. R. Wood, N. Winograd, A. G. Ewing
Cell Constituent Analysis: Single Cell Sensitivity

Chapter 10      C. Bustamante, D. A. Erie, G. Yang
Scanning Force Microscopy of Biological Macromolecules: Present and Future

Chapter 11      D. L. Allara
Nanoscale Structures Engineered by Molecular Self-Assembly of
=46unctionalized Monolayers

Chapter 12      R. Tamp=E9, C. Dietrich, S. Gritsch, G. Elender, L. Schmitt
Biofunctionalized Membranes on Solid Surfaces

Chapter 13      M. Aizawa, K. Nishiguchi, M. Imamura, E. Kobatake, T.
Haruyama, Y. Ikariyama
Molecular Assembly Technology for Biosensors

Chapter 14      K. Ploog
Self-Organized Ordered Growth of III-V Semiconductor Quantum Wires

Chapter 15      A. Kawana
=46ormation of a Simple Model Brain on Microfabricated Electrode Arrays

Chapter 16      P. E.  Hockberger, B. Lom, A. Soekarno, K. E. Healy
Cellular Engineering: Control of Cell-Substrate Interactions

Chapter 17      H. M. Buettner
Micro-control of Neuronal Outgrowth

Chapter 18      H. C. Hoch, R. J. Bojko, G. L. Comeau, D. A. Lilienfeld
Microfabricated Surfaces in Signaling for Cell Growth and Differentiation
in Fungi

Chapter 19      D. M. Brunette
Effects of Surface Topography of Implant Materials on Cell Behavior in
vitro and in vivo

Chapter 20      P. Clark
Cell and Growth Cone Behavior on Micropatterned Surfaces

Chapter 21      F. Gittes, E. Meyh=F6fer, S. Baek, D. Coy, B. Mickey, J. How=
ard
=46orce Generation by the Microtubule-Based Motor Protein Kinesin

Chapter 22      G. H. Pollack
Contemporary Problems in Biology: Contractile Materials

Chapter 23      David T. Burke
Technology Needs for the Human Genome Project



______________________________________________________________

GRADUATE STUDENT POSITIONS
______________________________________________________________

NCSU-NSCORT Graduate Research Fellowships

North Carolina State University-NASA Specialized Center of Research and
Training (NCSU-NSCORT) in Gravitational Biology has three graduate research
fellowships available.  The NCSU-NSCORT is a consortium of 12 faculty from
North Carolina State University, Kennedy Space Center, Wake Forest
University and the City University of New York (Baruch College).  The
program offers training in the areas of plant molecular and cellular
biology, biochemistry and physiology.  Trainees will interact directly with
multiple project leaders and potdoctoral research associates using
state-of-the-art techniques to study the effects of altered calcium
homeostasis on graviperception, signal transduction and response.  Specific
areas of training, with Project Leaders listed parenthetically, include 1)
creating genetic constructs for imaging and manipulating the expression
level, tissue specificity and organelle targeting of calcium-modulating
proteins/peptides in transgenic plants (William Thompson, Dominique
Robertson, Ron Sederoff and Ross Whetten), 2) imaging calcium, protons,
calcium-binding proteins cytoskeletal proteins during gravistimulation and
response (Nina Stromgren Allen, Wendy Boss, Gloria Muday and Edward
Tucker), 3) using vibrating probe and patch clamp techniques to study the
movements of ions in response to gravity (Nina Stromgren Allen and Eric
Davies), 4) biochemical studies of inositol lipid metabolism (Wendy Boss)
and auxin transport (Gloria Muday), 5) regulation of carbon and nitrogen
metabolism in gravity-stimulated or microgravity-grown plants (Steven
Huber, Joan Huber and Christopher Brown), and 6) molecular and biochemical
studies of cell wall biosynthesis in gravistimulated pine xylem (Ron
Sederoff and Ross Whetten).  For more information contact Dr. Christopher
Brown, Associate Director NCSU-NSCORT, Department of Botany, Box 7612,
North Carolina State University, Raleigh, NC 27695-7612; tel (919)
515-2727, fax (919) 515-3436, internet christopher_brown at ncsu.edu.  Please
visit the NCSU-NSCORT website at http://www2.ncsu.edu/ncsu/cals/nscort
NCSU is an equal opportunity employer.


______________________________________________________________

MEETING ANNOUNCEMENTS
______________________________________________________________


CALCIUM AND GRAVITATIONAL BIOLOGY SYMPOSIUM

Sponsored by the North Carolina State University NASA Specialized Center of
Research and Training in Gravitational Biology

Date:        Saturday November 2, 1996

Location: The North Carolina Biotechnology Center
        15 Alexander Drive, Research Triangle Park, NC, USA

Welcome at 8:50. Lectures begin at 9 AM.  Lunch will be provided for all
registered participants.

Speakers:

Peter Hepler     "Pollen Tube Growth: Calcium and the Cytoskeleton"

Tony Trewavas    "Targeting and Imaging of Transgenic Plants for Cell
Calcium Measurement"

Janet Braam      "Regulation of Expression and Functions of XET and
Calmodulin-related TCH Genes of Arabidopsis"

Lunch and Posters

Roger Hangarter   "Phytochrome Regulation of Gravitropism: Integration of
Environmental Sensory Systems"

Jim Haseloff   "Imaging Living Cells with Mutant Green Fluorescent Protein"

To register email: nscort at ncsu.edu
=46or more information contact Wendy Boss (email:wendy_boss at ncsu.edu or phon=
e
919-515-3496)

**********************************************************


=46EBS ADVANCED COURSE    "SINGLE CELL TECHNIQUES IN SIGNAL
SIGNAL TRANSDUCTION RESEARCH"

LEIDEN / AMSTERDAM, THE NETHERLANDS
MAY 3-10, 1997

 The Course 'Single cell techniques in signal transduction research'
comprises a rapidly expanding field with an increasing number of
possibilities and technical aspects. The course provides insight in a
variety of powerful single cell techniques and attempts to give an overview
of new developments in the field. In doing so, the course is suitable for
both specialist 'single cell researchers' and for researchers from outside
the 'single cell field'. In signal transduction research, combination of
e.g. biochemistry/molecular biology with single cell techniques provides a
powerful way to study mechanisms and explore new directions.
The course consists of  lectures and practical/theoretical workshops in
small groups:
        1. Lectures, explaining how a technique works, what the problems
and limitations are etc., illustrated with research examples.
        2. Theoretical workshops discussing aspects of  different
techniques in detail.
        3. Practical workshops with hands on experiments.

        Looking forward to seeing you in the Netherlands!

The organizing committee

  Topics and Lecturers

=46luorescence
W.F. Boron (USA):          Regulation and measurement of cytoplasmic pH.
P.H.G.M. Willems (NL):  Regulation of cytoplasmic calcium.
M. Fricker (UK):              Introduction to ion imaging. Confocal
imaging: from cells to tissue.
R.W. Horobin (UK):        Fluorescent dyes: where do they go and why?

Electrophysiology
E.L. Boulpaep (USA):           Introduction to ion transport and patch-clamp=
.
A.M.J. van Dongen (USA):   Ion channels and their regulation.
D. Colquhoun (UK):             Analysis of ion channel recordings.
M. Mazzanti (IT):                  Ion channels in the nucleus and special
voltage clamp protocols.
S.M. Assmann (USA):           Regulation of ion channels in plant cells.

Other Techniques
W.J. Wadman (NL):               Introduction to single cell research
C.J. Weijer (UK):                  Cell tracking.
S. Damjanovich (HUN):        Atomic force microscopy.
K.O. Greulich (GER):           Application of lasers in single cell research=
.
H.C. Gerritsen (NL):             Fluorescence life-time imaging measurements=
.
A.J. Trewavas (UK):            Molecular biology and calcium measurements.
R.Y. Tsien (USA):                Looking into the future of single cell
research.

Theoretical Workshops

Recording of electrophysiological signals
Advanced patch-clamp
Analysis of single channel data/stochastic channel behaviour
Molecular biology of ion channels
Patch clamp on plant cell protoplasts
Photomultipliers and cameras for ion imaging measurements
Calibration and measurement of pH signals
Energy transfer measurements
Application of lasers


Practical Workshops

Patch-clamp circuitry, recording of signals
Patch clamp measurements
Patch-clamp analysis
=46luorescence measurements/imaging
Confocal microscopy
Analysis of imaging data/ calibration
=46luorescent dyes; where do they go
Cell tracking
Atomic force microscopy
Laser micro surgery

Location  and Time Schedule

The course will be held from May 3 - May 10, 1997 in the magnificent
seventeenth century estate "Oud Poelgeest" near Leiden. Some of the
practical workshops will be held on different locations in Leiden and
Amsterdam.

Course Fee and Grants

The course fee is DM 1150,-. The fee includes registration, all meals, tea
and coffee, excursions, accommodation on basis of a double room, course
book. A limited number of FEBS Youth Travel Grants covering fee and (part
of ) travel expenses are available for members of FEBS constituent
societies under the age of 31.   Some other grants may be available through
the Single Cell Research Foundation (see application form).

Abstracts and Posters

Participants are asked to submit an abstract on their work and to present a
poster during the course. Some of the abstracts will be selected for short
oral presentations.

 Social program

An attractive social program including visits to the flower fields and
Dutch lakes will be organized.

Application

64 participants will be admitted.
The organisers select participants on basis of their application.
=46or application please send as soon as possible to the address below:
      The completed application form
      A short curriculum vitae, current research interest description and
motivation to join this course (maximal 1 page)
      A letter of recommendation by supervisor or head of department for
students

The deadline for application is February 15, 1997
Applicants will be notified about their admission before March 1, 1997.

 Organising committee
        A. Wiltink, M.T. Flikweert, A.H. de Boer, S. Damjanovich,
        S. Heimovaara-Dijkstra, B. Van Duijn

 Further information and application
        Dr. Bert Van Duijn,
        Center for Phytotechnology RUL/TNO, Wassenaarseweg 64,
        2333 AL Leiden,  The Netherlands.
        Tel. +31-71-5274923,  Fax +31-71-5274863
        e-mail: FEBS at AMC.UVA.NL
        Webpage http://wwwbio.leidenuniv.nl/~imp/febs1.html
 -------------------------------





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