Protoplasma (1999) 209:126-131
PROTOPLASMA
9 Springer-Verlag 1999
Printed in Austria
Dynamic studies of phloem and xylem flow in fully differentiated plants
by fast nuclear-magnetic-resonance microimaging
Rapid communication
M. Rokitta 1'*, A. D. Peuke 2, U. Zimmermann 3, and A. Haase ~
Lehrstuhl f/jr Experimentelle Physik V, Physikalisches Institut, 2Julius-von-Sachs-Institut f/Jr Biowissenschaften, and
3Lehrstuhl f/jr Biotechnologie, Biozentrum, Universitfit W/jrzburg, W/Jrzburg
Received February 9, 1999
Accepted May 5, 1999
Summary. A fast nuclear-magnetic-resonance imaging method was
developed in order to measure simultaneously and quantitatively
the water flow velocities in the xylem and the phloem of intact and
transpiring plants. Due to technical improvements a temporal resolution of 7 rain could be reached and flow measurements could be
performed over a time course of 12-30 h. The novel method was
applied to the hypocotyl of 35- to 40-day-old, leafy plants of Ricinus
comrnunis which were subjected to different light-dark regimes. The
results showed that the xylem flow velocities and the xylem volume
flow responded immediately to light on-off changes. Upon illumination the flow velocity and the volume flow increased as expected
in respect to literature. In contrast, the phloem flow velocity did not
change in response to the light-dark regimes. Interestingly, though,
the volume flow in the phloem increased during darkness. These
findings can be explained by assuming that the conducting area
of the phloem becomes enlarged during the dark period due to
opening of sieve pores.
Keywords: Phloem flow; Xylem flow; Water transport; Nuclearmagnetic-resonance imaging; Ricinus communis L.
Introduction
N u c l e a r magnetic r e s o n a n c e ( N M R ) imaging is a noninvasive and nondestructive technique and provides a
great variety of contrast m e c h a n i s m s such as chemical
shift, relaxation times, diffusion or flow velocities.
Recently, flow-weighted N M R imaging was used to
obtain quantitative patterns of the flow velocity in the
xylem of (intact) plants (Xia et al. 1993, K u c h e n b r o d
et al. 1996). D u e to the small d i a m e t e r of the vessels
(up to 50 ~tm) and flow velocities of less than 1 mm/s
sophisticated e q u i p m e n t and pulse sequences are
required to achieve a reasonable signal-to-noise ratio
in the N M R images. P h l o e m flow is even m o r e difficult
to m o n i t o r because the d i a m e t e r of the sieve tubes is
significantly smaller than that of the xylem vessels and,
thus, the total v o l u m e flow in the p h l o e m is m u c h less
than that in the xylem.
Despite these difficulties, K 6 c k e n b e r g e r et al. (1997)
recently r e p o r t e d m e a s u r e m e n t s of the p h l o e m flow
in R i c i n u s c o m m u n i s seedlings by N M R flow-imaging
techniques. H o w e v e r , due to the dimensions of the
m a g n e t bore, only 6-day-old (nontranspiring) seedlings could be investigated. M o r e o v e r , the total time
for m e a s u r i n g a single flow data set was 4.5 h which
m a d e it impossible to detect d y n a m i c flow changes
in response to a light-dark regime.
In this c o m m u n i c a t i o n , we will present m e a s u r e ments of p h l o e m flow t o g e t h e r with xylem flow in
leafy, transpiring R. c o m m u n i s plants. D u e to technical
i m p r o v e m e n t s the t e m p o r a l resolution was only 7 rain
which was sufficient for d y n a m i c (functional) studies.
This could be shown by m e a s u r e m e n t s of the response
of the xylem and p h l o e m flow u p o n light-dark regimes.
Material and methods
*Correspondence and reprints: Physikalisches Institut, EP 5,
Universitfit W/jrzburg, Am Hubland, D-97074 W/jrzburg, Federal
Republic of Germany.
The experiments were performed with 35- to 40-day-old R.
communis L. plants, The cultivation of the plants was similar to the
M. Rokitta et al.: Phloem and xylem flow in fully differentiated plants
127
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1. Schematic diagram of the setup used for the high-resolution NMR microimaging of the xylem and ]phloem flow velocities in intact
and transpiring plants under controlled environmental conditions. For detailed explanations, see text
Fig.
procedure described by Peuke et al. (1994), except that the NMR
equipment required some modifications. In order to fit the hypocotyl
to the radio frequency (RF) coil, the seedlings were kept in darkness for a couple of days after germination. Under these conditions
hypocotyls with a length of 15-20 cm were obtained. Then the
seedlings were transferred to plastic pots filled with quartz sand.
(The dimensions of the pots corresponded to the diameter and
length of the magnetic bore.) The plants were watered daily with a
nutrient solution containing 1.33 mol Ca(NO3)a and 1.33 mol K N O 3
per m 3 as N-source and were illuminated artificially [16 h light, 8 h
dark regime; 300-500 gmol photon per (m 2. s)].
For the NMR measurements a leafy plant was installed in a
home-built probe head with the transmitter/receiver RF coil about
8 cm above the roots (see Fig. 1).The probe head was sealed air tight
in order to monitor the changes of the water vapour content and
COz concentration in the air by a commercial gas exchange system
(Walz, Effeltrich, Federal Republic of Germany). Illumination of the
plant was performed with a halogen lamp through the front window
of the probe head. The maximum light intensity (irradiance) was
about 500 gmol photon per (m2. s). The temperature and the relative humidity were kept at about 25 ~ and 50%, respectively.
The probe head was inserted into a Biospec 70/20 horizontal bore
magnet (Bruker, Rheinstetten, Federal Republic of Germany).
Magnetization-prepared NMR imaging (Bourgeois and Decorps
1991, Haase et al. 1993) was used to combine a very sensitive flowweighting sequence with a fast, high-resolution NMR microimaging
technique. A phase contrast method employing stimulated echoes
(Tanner 1970) combined with a suppression of the signals arising
from stationary water (Lafirech et al. 1987, Bourgeois and Decorps
1991) was used for flow weighting. The imaging part of the sequence
was a refocused FLASH (fast low angle shot) experiment (Haase
et al. 1986). Thus, a relatively high spatial resolution (minimum 47 x
47 gm in plane, 3 mm slice thickness) as well as a high temporal resolution was obtained. A complete flow measurement took 7 min
consisting of 16 averages. This resulted in a satisfactory signalto-noise ratio. Eight images were acquired with increasing flowencoding gradients. From this data set the flow velocities and signal
amplitudes were calculated by a pixel-by-pixel fit of an appropriate
function to the signal intensities of the images. In addition to the
velocities, the upward or downward direction of the flows could be
extracted from the above analysis as described in detail by Rokitta
et al. (1999).
The flow velocities given below represent average values of the
conducting areas of the phloem and the xylem, respectively.
However, for the evaluation of the xylem velocity only the areas of
the primary vascular bundles were taken into account because they
appeared uniformly in the NMR images of different plants in contrast to other structural conducting elements (see Fig. 2). It should
also be noted that for the averaging of the data points in the selected
phloem and xylem areas only data points were included which
exhibited nonzero values for the flow velocity. Since the lowest
detection limit for the flow velocity was about 0.2 mm/s (Rokitta
et al. 1999), this procedure leads to a slight overestimation of the
average flow velocity.
For the estimation of the total volume flow from the average flow
velocity values the total geometric conducting area of the xylem and
phloem, respectively, must be known. However, these parameters
are not available. The problem can be solved in the following way.
An image pixel was generally larger than a single vessel as indicated
in Fig. 2c.This means that for the calculation of the conducting area
in this particular pixel the ratio of the regions with flowing and with
nonflowing (stationary) water must be taken into account. A reasonable measure for this ratio is the signal amplitude derived from
the fitting procedure mentioned above. Thus, the total volume flow
can be estimated by multiplication of the average flow velocity value
of each pixel with the corresponding signal amplitude and by subsequent summation.
However, this procedure could lead to an error because regions
of stationary water comprise both vessels with flow velocities below
the detection limit as well as tissue cells. At present, it is not possible to discrimate between these two compartments and thus to
quantify the volume flow in absolute units. Therefore, the values of
the volume flows given below were normalized to the maximum
value of each data set.
For the morphological identification of the conducting elements
in the FLASH images and flow maps a few spin echo images
were made (for conditions, see legend to Fig. 2) as well as microscopical images (Axiophot; Zeiss, Oberkochen, Federal Republic of
Germany).
Results
Figure 2 shows a typical FLASH
i m a g e (a; 47 x 4 7 g m
in-plane resolution, 3 mm slice thickness) and the corresponding flow velocity map (b; 78 x 313 gm in-plane
resolution, 4 mm slice thickness) of a cross section of
128
M. Rokitta et al.: Phloem and xylem flow in fully differentiated plants
Fig. 2. Cross sections of the shoot of 35- to 40-day-old intact and transpiring plant of R. communis made by NMR microimaging (a, b, and
d) and light microscopy (c). a FLASH image (echo time, TE=4.3 ms; repetition time, TR= 11.5ms; field of view, FOV=
6 x 6 mm2; matrix, 128 x 128 zerofilled to 256 x 256; slice thickness, 3 ram; measuring time, 2 s; nominal in-plane resolution, 47 x
47 ~tm2).b Flow velocity map (matrix, 128 x 32 zerofilled to 128 x 128; slice thickness, 4 mm; FOV = 10 x 10 mm2reduced to 6 x 6 ram2;measuring time, 7 rain; nominal in-plane resolution, 78 x 313 gmZ). c Light microscopy; the size of one pixel in NMR flow imaging (b) is indicated in the upper right corner of the picture, d Spin echo image (TE = 15 ms; TR = 0.5 s; slice thickness, 6 mm; FOV = 8 x 8 mm2 reduced
to 6 x 6 ram2; measuring time, 4.8 h; 64 averages; nominal in-plane resolution, 16 x 16 gm2). For further explanations, see text
a R. c o m r n u n i s plant. The F L A S H image reflects the
water distribution and thus the a r r a n g e m e n t of the
structural elements in the cross section. Since b o t h
images were m a d e at the same position of the shoot
the flow image was projected on the F L A S H image in
Fig. 2 b in o r d e r to facilitate the correlation b e t w e e n
the flow p a t t e r n and the structural architecture. For
unequivocal assignment of the structural and flow elements revealed by the N M R images to the m o r p h o logical elements seen usually in the light m i c r o s c o p e a
typical microscopical cross section of the shoot of a
plant is given additionally in Fig. 2c. C o m p a r i s o n of
the F L A S H image with the light m i c r o s c o p y and the
spin echo image shows that it is possible to identify the
xylem and p h l o e m regions in the F L A S H image, but
that the resolution of the F L A S H image was not as
high as that of a spin echo image (Fig. 2 d).
H o w e v e r , in contrast to the acquisition time of a
F L A S H image ( a b o u t 2 s) a spin echo image of very
high resolution (as shown in Fig. 2 d ) needs several
hours. This period was not tolerable because this time
would have b e e n missing for the m e a s u r e m e n t s of the
kinetics of the xylem and p h l o e m flow velocities as a
function of a light-dark regime. Therefore, flow images
were m a d e in the following experiments only in combination with a F L A S H image. This was obviously also
M. Rokitta et al.: Phloem and xylem flow in fully differentiated plants
justified because the FLASH image apparently contained the necessary structural information. Both
images show that the eight vascular bundles (initially
separated from each other in younger plants) formed
a closed ring due to secondary thickening. This ring
appeared dark in the FLASH image because of the
reduced free-water concentration per volume in the
xylem area. However, the large vessels which can be
seen very clearly in the spin echo image (Fig. 2 d) are
also visible in the FLASH image (Fig. 2 a). The resolution of the FLASH image, particularly in combination with the flow map (Fig. 2 b), was also high enough
to reveal the separation of the xylem from the phloem
by the cambium (about 100-150 gm in thickness; see
also Fig. 2 c).
The flow image (Fig. 2b) shows that the xylem flow
was directed upwardly. In order to demonstrate this,
this flow direction is indicated by blue pixels in the
image and the flow velocity values are provided with
a positive sign. The phloem flow velocities were negative, i.e., opposite to the xylem flow velocities. This is
indicated in Fig. 2b by the red pixels. Flow datasets as
shown in Fig. 2b were performed regularly in 7 rain
time intervals on five different plants subjected to
various light-dark regimes. The corresponding average
flow velocities and the calculated (normalized) volume
flows in the xylem and in the phloem of two plants (A
and B) are given in Fig. 3 a and b, respectively. Figure
3 c represents the transpiration and the assimilation
rates measured simultaneously.
As can be seen from Fig. 3, an increase in the transpiration and assimilation rate upon illumination is
immediately reflected in an increase of the xylem flow
velocity and volume flow. As also expected, when the
light was switched off, the transpiration rate and the
assimilation rate decreased (whereby the assimilation
rate assumed negative values because of CO2 release
from the plant due to respiration). Correspondingly,
the flow velocity and the volume flow in the xylem
decreased. In contrast to the xylem flow, the phloem
velocity was completely independent of the light-dark
regime, at least within the limits of accuracy. However,
the volume flow apparently increased during the dark
period in order to decrease again after the light was
switched on.
Discussion
The objective of the present work was to demonstrate
that the noninvasive method of NMR microimaging
129
allows the time-resolved observation of phloem and
xylem flow simultaneously provided that appropriate
modifications of the hardware and the pulse sequences
were made. Up to now, this technique could only be
applied to seedlings without transpiring leaves. The
temporal resolution was with 4.5 h also much too
long to resolve the kinetics both of phloem and xylem
flow (K6ckenberger et al. 1997). In this study, the time
resolution could be reduced to 7 min. This was fast
enough to monitor the immediate effect of illumination on the kinetics of both flows which were opposed
to each other as expected.
The improved N M R imaging technique yielded
values for the flow velocity in the xylem and the
phloem which were in the ranges reported by Kuchenbrod et al. (1996) and by K6ckenberger et al. (1997),
respectively. We could further demonstrate that the
flow velocity and the volume flow in the xylem
revealed a pattern in response to a light-dark regime
which was consistent with the current belief of the
effect of transpiration on xylem tension and flow
(Schneider et al. 1997, Kuchenbrod et al. 1996). The
most remarkable result was, however, that the flow
velocity in the phloem was not changed by the lightdark regime despite an apparent increase in the
phloem volume flow during darkness. These findings
have two implications. First, the light independence
of the phloem velocity is not necessarily in contrast
to the pressure-flow hypothesis of Mtinch (1930)
provided that a steady-state pressure gradient is
maintained diurnally and nocturnally by osmoregulatory processes between the phloem and the accessory
parenchyma cells or the xylem vessels (Zimmermann
1978; Van Bel 1990, 1993; Ziegler 1977). Second, in the
light of the Hagen-Poiseuille law, the finding of an
increase in volume flow during the dark period at constant velocity can only be explained by the assumption
that the conducting area of phloem flow is increased
during this period. This could mean that part of the
sieve pores are continuously open, but that additional
sieve pores which may be plugged by protein threads
during the light period become temporarily open
during darkness. This conclusion is in agreement with
recent work of Knobtauch and Van Bel (1998).
Van Bel and co-workers pointed out that the structure and mode of the sieve elements can only be elucidated when they are intact and actually translocating
because of their extreme sensitivity to any kind of
manipulation (cutting, chemical fixation, etc.). The
noninvasive N M R flow-imaging technique presented
130
M. Rokitta et al.: Phloem and xylem flow in fully differentiated plants
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3. Xylem flow velocity and volume flow (a) and phloem flow velocity and volume flow (b) in dependence of a light-dark regime measured on two different plants (A and B). Simultaneous measurements of the transpiration and assimilation are given in e. The light and
dark periods are indicated by white and black horizontal bars. Note that the assimilation rate assumes negative values during darkness
because of respiration of the plants. For detailed discussion, see text
Fig.
here may, therefore, c o n t r i b u t e in f u t u r e to the elucid a t i o n of the local c o u p l i n g b e t w e e n x y l e m a n d
p h l o e m flow and, in turn, to the l o n g - d i s t a n c e transport in the p h l o e m in r e s p o n s e to t i m e - v a r y i n g envir o n m e n t a l conditions.
Acknowledgments
The authors thank Ralf Deichmann for improvements in the software used for evaluation of the flow datasets. This work was sup-
ported by two grants from the Deutsche Forschungsgemeinschaft:
Schwerpunkt Apoplast Zi 99/9-2 to U.Z. and Graduiertenkolleg
GRK 64/2 to A.H. and U.Z.
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