PNNL-13501 - Pacific Northwest National Laboratory

selection of seedlings demonstrating no contamination,
and aseptic transfer to sterile nutrient solution (tenthstrength
Hoagland’s) oxygenated with 0.22 µ filtered air.
Growth conditions included a light intensity of 300 µE
m -2 sec -1 , (fluorescent/incandescent mix), 18 to 23°C
day/night temperatures, and 60% R.H. Continual sterility
checks were performed on the roots and nutrient solution.
The microscope for observing siderophore uptake was
assembled along with a newly designed and constructed
plant holder, consisting of a Nikon TE-300 inverted
optical microscope with CFI60 infinity optics, fitted with
Toshiba Tu40A 3-chip color CCD camera, Scion CG7
color frame grabber, imaging software, a Nikon optical
camera, a thermoelectrically cooled Hamamatsu 4220p
photon counting photomultiplier tube, a Stanford
Research SR400 two-channel gated photon counter, and a
Ludl LEP Biopoint computer controlled multi-axis
translation stage. A specially made sample holder was
attached to the sample translation stage permitting direct
observation and laser induced fluorescence imaging of a
live plant root in its growth media.
Results and Accomplishments
Critical to the study was the requirement that there be no
interference from the native fluorescence of plant tissues
or extracellular siderophores on the resolution of
europium in and out of the plant and the confirmation of
spectral changes following uptake and complexation
within the plant. The spectra, shown in Figure 1,
demonstrated that both the oat and grass roots will not
interfere with that of the europium. The characteristic
emissions in the 590 to 620 nm range are plainly evident
in this system. Shifts in the two emission peaks are
characteristic of europium binding. Figure 2 shows that
there is a shift between the uncomplexed supplied Eu(III)
media (both with and without nutrient solution) and that
observed in the root and leaf tissue. Figure 2 also
demonstrates that the Eu is transported to the leaves
(shoot) of the plants. In fact Eu accumulation in the roots
and shoot was nearly linear even over 5 days (data not
shown). The siderophores available during the study,
Desferrol and Ferrichrome, were unable to be resolved in
the system either alone or complexed with Eu(III).
Following several unsuccessful attempts, it was decided
to pursue actual uptake and distribution of the metal itself.
Fluorescence changes within individual tissues and cell
types along developing primary and secondary roots of
oats immersed in nutrient solution containing 10 mM
europium (III) were followed with the confocal scanning
laser microscope. Distribution of the europium was
observed to decrease basipetally along the root axis with
Photon Counts
250x10 3
the highest concentrations consistently found in the
meristematic tissue below the root cap at the tip and up to
300 to 500 µm back (Figure 3). Here, and for up to 2000
to 3000 µm back from the tip, concentations were highest
after 48 to 72 hours after exposure. Transverse scans
were made across the intact roots where a rapid rise in
fluorescence at the epidermis followed by a slight decline
throughout the cortex and another rise through the stele
was observed. Freehand cross sections approximately
300 to 500 µm thick were taken and scanned to precisely
identify the tissue types involved in the fluorescence
patterns. The sections were subsequently stained with
0.1% (w/v) toluidine-o blue dye, rinsed in distilled H2O,
and photographed. The results for individual cell types
are shown in Figure 4. Here the relative fluorescence
shows that the stele contains the highest concentration of
europium and that the xylem parenchyma, known to
control composition of the transpiration stream (Boer and
Wegner 1997), and the sieve element-companion cell
complexes of the phloem were the highest within this
structure.
Relative Intensity
200
150
100
50
0
300x10 3
150
100
50
400
0
560
450
Oat root
Grass root
500
0.05 M EuC3 in
0.01 M EuC3 in growth
580 600 620 640
Wavelength (nm)
Eu(III) in
Eu(III) in
Figure 2. Fluorescence spectra at 394 nm for a 10 mM
europium (III) solution in H 2O, 10 mM europium (III) in
nutrient solution, oat root, and oat leaf following a 48-hour
exposure period
550
600
Wavelength(nm)
l ex =394nm
10 mM Eu (III)
Figure 1. Fluorescence spectra at 394 nm for 10 mM
EuCl 2·6H 2O solution, 21-day-old oat root and 28-day-old
grass root demonstrating that plant root fluorescence does
not conflict with europium signature
650
70 0
Biosciences and Biotechnology 85