Joint Meeting - Genomics - U.S. Department of Energy

12
Systems Biology for DOE Energy and Environmental Missions
are certain filamentous fungi. One reason that vascular
plant cell walls are so difficult to digest is that their lignin
prevents even the smallest enzymes from penetrating.
Filamentous fungi solve this problem by making lignocellulose
more hydrophilic and porous, with the result
that hydrolytic enzymes can eventually penetrate the
substrate.
To make lignocellulosics permeable to enzymes, filamentous
fungi use a variety of small, diffusible reactive oxygen
species (ROS) such as hydroxyl radicals, peroxyl radicals,
and possibly phenoxy radicals. These radicals diffuse into
the cell walls and initiate biodegradative radical reactions.
When lignin is the target, radical attack results in various
extents of oxidation and depolymerization.
These small diffusible oxidative species are important
tools used by filamentous fungi to make the cell wall
accessible to enzymes. Despite this, we have a poor
knowledge of how these oxidants are spatially distributed
in biodegrading lignocellulose relative to the fungal
hypha that produce them. The goal of this project is to
remedy this deficit through fluorescence microscopy of
newly designed sensors that will serve as in situ reporters
of biodegradative radical production. While developing
these sensors, we will test the specificity of reaction of a
variety of promising fluorophores to increase the arsenal
of ROS specific probes available for work at low pH.
We will also demonstrate how binding these fluorescent
probes to beads improves fluorescent imaging by preventing
dye diffusion, limiting interferences, and allowing the
use of almost all dyes (lipo- or hydrophilic, cell permeant
or not) to be used in extracellular environments. Finally,
we will use these sensors to produce oxidative maps that
will help us to understand how fungi generate ROS and
how they use these ROS to make cell walls more accessible
to enzymes.
Method
We are attaching fluorescent dyes to silica beads. Our
first bead has BODIPY 581/591® on a 3µm porous
HPLC bead. This dye’s emission changes irreversibly
from red to green upon oxidation by ROS. The ratio of
red to green emission provides a quantitative measure
of the cumulative oxidation at that point in space. Dyes
with reactivity to specific ROS, pH, or other metabolites
of interest are envisioned.
* Presenting author
Figure. Fluorescent dye attached to silica bead
There are many advantages gained by fixing the dye to
bead. We design the bead to emit two fluorescent signals,
so that the ratio of the two signal intensities provides
quantitative information. Immobilized dyes are prevented
from moving after reaction, so partitioning is impossible,
they cannot be ingested, and the fluorescence from the
dye is clearly distinguishable from background.
Beads are placed on wood samples and imaged with a
confocal microscope during fungal colonization. Images
can be analyzed to provide the analyte concentration
maps as well as an overlay of the location of fungal
hypha.
12
Dynamic Molecular Imaging of
Lignocellulose Processing in Single
Cells
Amy Hiddessen, Alex Malkin, and Michael Thelen*
(mthelen@llnl.gov)
GTL
Chemistry Directorate, Lawrence Livermore National
Laboratory, Livermore, California
Of central importance to our nation’s energy resources
is the pursuit of alternative fuels directly from plant
material rich in lignin and cellulose. The abundant and
intractable nature of these biopolymers limits their practical
use to produce biofuel precursors, and so presents
an extraordinary scientific challenge. A comprehensive
understanding of the natural or engineered breakdown
of plant cell wall materials must be addressed on several
levels, including the examination of detailed ultrastructural
changes that occur in real time. To accelerate
research on the cellular and molecular details of the cell
wall deconstruction process, we are developing sophisticated
analytical tools specifically to visualize changes
in surfaces, polysaccharides and proteins. Molecular
surface characterization can be directly linked with high