Plant Physiol. (1986) 82, 1158-11600032-0889/86/82/1158/03/$01.00/0

Communication

In Vivo Magnetic Resonance Microscopy of Changing WaterContent in Pelargonium hortorum Roots'

Received for publication August 4, 1986

JOHN M. BROWN, G. ALLAN JOHNSON*, AND PAUL J. KRAMERDepartment ofHorticultural Science, North Carolina State University, Raleigh, North Carolina (J.M.B.),Box 3302, Department ofRadiology (G.A.J.), and Department ofBotany (P.J.K.), Duke University,Durham, North Carolina 27710

ABSTRACT

Magnetic resonance imaging (MRI) was used to nondestructivelyobserve changes in water content in roots of Pelargonium hortorum xBailey during a period of relatively rapid transpiration. Anatomicalregions of the root could be differentiated with a spatial resolution of 0.1x 0.1 mm. MRI shows great potential for study of plant-water relations.

Nondestructive measurements of changes in the water contentof plant tissue and of the rooting medium would be very usefulin studies of water absorption and transport and of changes inwater content of various tissues during the development ofwaterstress. The development of MRI,2 also known as NMR, equip-ment for medical diagnosis and research provides the possibilityof making such measurements on plants. The method uses astrong static magnetic field and weak radio frequency radiationwhich readily penetrate plant tissues and measure the concentra-tion of protons, chiefly those associated with water (2, 6). Theprocedure is nondestructive and repeated observations can bemade over time on the same tissue without causing injury. Omasaet al. (7) used MRI with a resolution of about 2 mm to imageroots of seedlings and to show changes in water content of theseedlings and the soil in which they were growing. Bottomley etal. (1) used a resolution of 0.6 mm with no slice definition toobserve movement ofwater doped with Cu2SO4 into and throughroots of Viciafaba seedlings.

Recent developments in MRI technology have increased thespatial resolution to 0.05 mm with a slice definition of 1.25 mm(5), making it possible to observe differences in the water contentof various tissues of plant organs. The purpose of this study wasto investigate the utility ofMRI technology for nondestructivelyobserving changes in water content in roots during a period ofrelatively rapid transpiration.

MATERIALS AND METHODS

Study plants ofPelargonium hortorum x Bailey were producedfrom stem cuttings rooted in synthetic phenolic foam medium

'Supported by the National Institute ofEnvironmental Health ScienceContract No. 273-84-I-0025.

2Abbreviations: MRI, magnetic resonance imaging; rf, radio fre-quency.

(Rootcube,3 Smithers Oasis, Kent, Ohio) under intermittent mistand grown under greenhouse conditions. The cuttings wererooted in foam cylinders 5 cm diameter x 8 cm long which werewrapped in polyethylene film and aluminum foil to preventevaporation from the medium and root exposure to sunlight.Synthetic foam medium was used to avoid soil diamagetismand ferromagnetism which may cause spatial distortion andsignal loss in MRI data acquisition (1), and to provide a uniform,rigid medium for quantifying water content adjacent to the root.In approximately 5 weeks, after the cuttings were well rooted,the root systems of the plants were imaged.MRI images were obtained at 63.8 MHz on a prototype 1.5

Tesla (T) General Electric medical research system housed atDuke Medical Center, Durham, NC. A 5 cm diameter "birdcage"rf transceiver coil (3), mounted within 28 cm gradient coils (ableto generate field gradients of 4.7 mT/cm) (5), was placed insidea 1.0 m diameter whole-body superconducting magnet. A plantwas placed horizontally within the 5 cm receiver coil such thatthe area to be observed was in the center of the coil. Threedimensional Fourier transform (3DFT) spin warp imaging wasused for all images. Images were reconstructed on a 256 x 256x 16 array with a resulting spatial resolution Of 0.1 mm x 0.1mm in the imaging plane (transverse plane of root) and a 1.2mm slice thickness (longitudinal plane). A more complete de-scription of the MRI techniques is available elsewhere (5).The signal intensity in anMR image is dependent on a number

of intrinsic parameters:a. Spin density, i.e. the number of hydrogen protons per cc.

For this work we assume that the majority of protons are waterprotons.

b. Spin-lattice relaxation time (T1); rf pulses are used in MRIto excite the protons. The excited protons remain in their higherenergy state for some time, decaying with a time constant TIthat is dependent on the ability of these protons to release theirenergy to their surroundings (lattice). Thus a proton in a watermolecule hydrogen bonded to a membrane relaxes much morequickly than one in free water. This makes it possible to distin-guish between free and bound water.

c. Spin-spin relaxation (T2); the signal intensity is a result ofmany protons acting coherently. As the proton population losescoherence the signal decays. For example two water protons inslightly different tissue environments may be subjected to suffi-ciently different magnetic fields to precess at slightly differentfiequencies. Thus after a few milliseconds they will no longer act

'The use of trade names in this publication does not imply endorse-ment by Duke University of the products named nor criticism of similarones not mentioned.

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MAGNETIC RESONANCE MICROSCOPY OF ROOT WATER CONTENT

FIG. I a, Images of cross-sections of adventitious roots showing apparent changes in distribution of water after a period of transpiration.Variations in brightness of the image represent differences in concentration of mobile protons usually associated with differences in concentrationof water molecules and 1 1, the spin lattice relaxation time. Roots A and B represent the plant imaged in the dark, and A' and B' represent the rootsimaged after the plant was exposed to light and allowed to transpire for 8 h. b, Graphs superimposed on the corresponding image represent therelative MR signal intensity for each pixel on its respective transacting line. These graphs better illustrate changes in signal intensity caused bychanges in proton concentration within the root during transpiration.

synchronously, causing a loss of signal.Finally, one can choose the relative weighting of these three

variables in an image by adjusting the timing used for theacquisition. In this work we have used a partial saturationsequence which employs multiple rf excitations for spatial en-coding. The time between the individual phase encodings (TR)was 400 ms. The time between the 90° rf pulse and 180° echo-forming pulse of each phase encoding (TE) was 20 ms. Thisyields an image in which signal intensity is dependent primarilyon T1 and spin density. The reader interested in more details isdirected to several general descriptions of the signal dependencein MRI images (8).The plant was allowed to equilibrate in the MRI unit overnight

and a set of axial images was acquired prior to exposing it tolight. The shoot was then exposed to light and two sagittal(longitudinal) scans and one axial (transverse) scan were acquiredat approximately 2.5 h intervals such that the last set of imagesrepresented plant water status after 8 h ofmoderate transpiration.Only images of cross sections of roots are presented in this paper.

Light was supplied to the plant by two 650 W, 120 VAC quartz-iodide lamps mounted in flood light housings on adjustablestands and focused on the leaf surface. Lights had to be placedapproximately 4 m from the magnetic core's center, becausestrong magnetic fields rapidly destroy the metallic filaments inlamps. This gave an incident photon flux density of approxi-mately 450 uE m 2s-' at the leaf surface. During imaging, theair temperature in the coil ranged from ambient (220C) with theflood lights off to 32C with the flood lights on. RH was approx-imately 36% in the room during the experiment. However, itcould not be measured at the leaf surface because the strongmagnetic field prevents the operation ofmotorized or mechanicalpsychrometers.

After imaging, samples of the plant tissue were preserved forsubsequent microscopic evaluation. The fresh plant material wassectioned into 1 cm portions then placed in vials containing FAEsolution (formalin 200 ml, acetic acid 100 ml, ethanol 1000 ml,water 700 ml) (4). A freehand section of the preserved tissue,located approximately 3 cm back from the root tip, was stained

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Plant Physiol. Vol. 82, 1986

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FIG. 2. Photomicrograph of a cross-section of the type of root imagedin Figure 1, a and b, with a diameter of about 0.9 mm.

with iodine/potassium iodide for examination under a lightmicroscope.

RESULTS AND DISCUSSION

Figure la shows images of cross-sections of two adventitiousroots acquired approximately 3 cm back from the root tips. Thediameters of roots A and B were 1.0 and 0.8 mm, respectively,and they are typical of the many adventitious roots formed ongeranium cuttings. The images labeled A and B were made whilethe cuttings were in darkness, those labeled A' and B' were madeafter the plant had been exposed to light and relatively warm,

dry air, transpiring, for about 8 h. Variations in brightness of theimages represent differences in concentration of mobile protons,presumably associated with differences in concentration of watermolecules and T 1, the spin lattice relaxation time.The images made in darkness show a higher concentration

and more even distribution of water in the stele than in thecortex. Regions distinctly higher than average in water content,roughly resembling spokes of a wheel extending out from thestele to the periphery of the root are visible in the corticalparenchyma of these and other roots that were imaged. Theseareas are puzzling because no specialized cells are seen under a

light microscope (Fig. 2). Images made after exposure to light for8 h show a decrease in brightness in the peripheral region and inthe radial regions connecting the stele and outer cortex (A' and

B'), suggesting that these regions have lost water.The differences in water content are shown graphically in

Figure lb. Regions of interest, indicated by the horizontal lines,were analyzed for signal intensity and the results are plottedacross each root. The region studied consists of picture elements(pixels) 0.1 x 0.1 mm in the transverse direction, by 1.2 mm inthe longitudinal direction of the root. These graphs better illus-trate changes in signal intensity within the root during transpi-ration.The presence of radial regions high in water in the cortex is

puzzling because, as mentioned earlier, the cortex is uniform inappearance under a light microscope. This difference emphasizesthe fact that MRI of living tissue is likely to reveal situations thatcannot be detected by other methods. At present one can onlyspeculate concerning the reasons for the local areas high in watercontent. It is unlikely that they lead to xylem points in the stelebecause the stele of these adventitious roots are diarch and thelocalized regions appear to randomly intersect the stele. Anotherpossibility is that these areas are simply the most direct pathwayto the xylem from points on the root surface in contact with theroot medium that supplies water. In any event, these observationssuggest that water movement across roots may not involve allcells of the cortex equally. This possibility raises questions con-cerning the current water and mineral transport models whichassume that all of the cortical cells are equally involved. Furtherstudy, including the use of labeled water and improved MRItechnology may provide interesting information concerning thepathway of radial salt and water movement in roots.

CONCLUSIONSThese observations show that MRI can successfully differen-

tiate between regions of the root (epidermal region, cortex andvascular cylinder) with a spatial resolution of100 Mm. The imagesshow changes in pixel signal intensities which apparently indicatechanges in water content during the 8 h of transpiration. How-ever, at this time, quantitative analysis of individual pixel signalintensities is not fully understood. As noted earlier, signal inten-sity is dependent on Tl and spin density. One cannot rule outthe possibility that the changes in signal intensity are due tochanges in local Tl instead of spin density. Further experimen-tation is being conducted to provide a more precise understand-ing of the nature of MR signal intensity as it relates to the waterstatus of plant tissue.

Acknowledgments-The authors are grateful to William A. Jackson, Judith F.Thomas, and William C. Fonteno at North Carolina State University for manyhelpful discussions. We would also like to thank Jim Boodley of Smithers OasisCompany for supplying the foam medium. We thank Gary Cofer and John Karisof Duke Medical Center for their technical expertise.

LITERATURE CITED

1. BOTTOMLEY, PA, HH ROGERS, TH FOSTER 1986 NMR imaging shows waterdistribution and transport in plant root systems in situ. Proc Natl Acad SciUSA 83: 87-89

2. EDELSTEIN WA, JMS HUTCHISON, G JOHNSON, T REDPATH 1980 Spin warpNMR imaging and applications to whole body imaging. Phys Med Biol 25:751

3. HAYES, CE, WA EDELSTEIN, JF SCHENCK 1985 An efficient, highly homoge-nous radio frequency coil for whole body imaging at 1.5T. J Magnet Reson63: 622-628

4. JOHANSEN, DA 1940 Plant Microtechnique. McGraw-Hill, New York5. JOHNSON, GA, MB THOMPSON, SL GEWALT, CE HAYES 1986 Nuclear magnetic

resonance imaging at microscopic resolution. J Magnet Reson 68: 129-1376. LAUTERBUR, PC 1973 Image formation by induced local interactions-examples

employing nuclear magnetic resonance. Nature 242: 190-1917. OMASA, K, M ONOE, H YAMADA 1985 NMR imaging for measuring root

systems and soil water content. Environ Control Biol 23: 99-1028. WEHRLI,FW, JR MAcFALL, GH GLOVER 1985 The dependence of nuclear

magnetic resonance (NMR) image contrast on intrinsic and operator select-able parameters. Appl Opt Instrum Med SPIE 419:256-265

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