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This false-color image shows a cell from the
epidermis of an Arabidopsis thaliana plant marked with fluorescent
imaging sensors designed to detect the sugar glucose.
(Image courtesy Sylvie Lalonde and Wolf Frommer)
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Led by Carnegie staff member Wolf Frommer, the
researchers designed genetically-encoded fluorescent tags to monitor
glucose, an important sugar, in leaf and root tissues of the model
plant Arabidopsis thaliana. The technique has allowed the
researchers to track glucose over time and space at unprecedented
detail, in living and undisturbed plant tissues. The work appears in
the September issue of the journal Plant Cell. The group has
also developed a FRET sensor for sucrose, a major transport sugar in
plants. This work will appear in the September issue of the
Journal of Biological Chemistry.
“Until now, we have had few clues regarding how
much sugar is in an individual cell in a multicellular plant,” Frommer
said. “We normally grind up a leaf or a root and average the
information for all cells, but if sugar levels rise in one cell and
drop in another, we would see no change in this average.” Also,
because the cell can distribute sugar among subcellular organelles, it
is nearly impossible to know how much sugar is in any cell compartment
at a given time.
“Time resolution is another problem,” Frommer added.
“We can sample tissue at intervals, but if the sugar changes in waves,
we might miss the right time point. Our new technology addresses all
of these problems by measuring sugar flux in real time in individual
cells, with subcellular resolution.”
Frommer and his colleagues have used similar
imaging tags, called fluorescent resonance energy transfer (FRET)
sensors, to track sugars and neurotransmitters in animal cells. Most
recently, the group used FRET sensors to study glutamate, an important
mammalian neurotransmitter. Frommer has tracked glucose in cultured
mammalian cells, but until now, plant tissues had proven problematic
because of interference from the plants’ virus defense mechanisms, as
well as high background fluorescence in some plants.
To surmount these issues, Frommer’s team
dramatically improved the sensors, while inserting them in mutant
Arabidopsis plants with disabled defense genes. The fluorescent
tags worked well where they had failed before.
“It may not be ideal to use defense-mutant plants -
the ideal would be for the sensors to work in any wild-type genetic
background,” Frommer explained. “But proving that the sensors can work
in plants is an important first step. Now we can begin addressing
important questions about the way plants manage sugar distribution
while we continue to improve the sensors.”
In preliminary experiments, Frommer’s group
compared fluctuations in glucose levels in root tissue and leaf
epidermis—the topmost layer that absorbs sunlight - and found that the
plant maintained glucose at higher levels in leaf tissue than in roots.
In fact, the researchers found that root cells contain sugar at
concentrations at least 100,000 times lower than previous estimates.
FRET sensors are encoded by genes that, in theory,
can be engineered into any cell line or organism. They are made of two
fluorescent proteins that produce different colors of light - one cyan
and one yellow - connected by a third protein that resembles a hinged
clam shell. The two fluorescent proteins are derived from jellyfish,
and the third from a bacterium; the shape of the clam shell protein
determines which sugar or other molecule the sensor can detect. When a
target molecule such as glucose or sucrose binds to the third protein,
the hinge opens, changing the distance and orientation of the
fluorescent proteins. This physical change affects the energy transfer
between the cyan and yellow markers.
When the researchers hit the tags with light of a
specific wavelength, the cyan tag starts to fluoresce. If the yellow
tag is close enough, the cyan tag will transfer its energy to the
yellow tag, causing it to resonate and fluoresce as well. This energy
transfer affects how much cyan and yellow fluorescence can be seen,
and by calculating this ratio, researchers can accurately track
molecules such as glucose and sucrose in both time and space.
“The strength of this technology lies in its
elegant simplicity; with the power of computational design, we can
potentially design FRET tags to detect virtually any small molecule in
living cells,” Frommer said. “Imaging techniques like this are the
next frontier in the study of metabolism, and will help to answer some
of the most pressing questions on plant biologists’ minds, such as the
role of individual genes in the distribution of sugars. This in turn
can help us engineer plants to produce more biomass.” |
