Functional Imaging of Plants by Magnetic Resonance Experiments Walter Köckenberger

286 Research Update TRENDS in Plant Science Vol.6 No.7 July 2001 Conclusions 3 Bohnert, H.J. et al. (1995) Adaptations to 12 Roncarati, R. et al. (1995) An aldose reductase Preventing oxidative stress or reducing the environmental stresses. Plant Cell 7, homologous gene from barley: regulation and 1099–1111 function. Plant J. 7, 809–822 level of the reactive molecules appears to be a 4 Bray, E.A. (1997) Molecular responses to water 13 Benedetti, A. et al. (1980) Identification of 4- promising approach to obtain plants with deficit. Trends Plant Sci. 2, 48–54 hydroxynonenal as a cytotoxic product originating diverse tolerance to abiotic stress. 5 Ingram, J. and Bartels, D. (1996) The molecular from the peroxidation of liver microsomal lipids. Engineering crop plants that can cope with basis of dehydration tolerance in plants. Annu. Biochem. Biophys. Acta. 620, 281–296 oxidative molecules could have a broad Rev. Plant Physiol. Plant Mol. Biol. 47, 377–403 14 Allen, R.D. (1995) Dissection of oxidative stress 6 Shinozaki, K. and Yamaguchi-Shinozaki, K. tolerance using transgenic plants. Plant Physiol. application in agriculture. The examples (1997) Gene expression and signal transduction in 107, 1049–1054 discussed in this article show that there are water-stress response. Plant Physiol. 115, 15 McKersie, B.D. et al. (1996) Water-deficit several pathways that can be used to obtain 327–334 tolerance and field performance of transgenic stress-tolerant plants. Because there are still 7 Zhu, J.K. (2001) Plant salt tolerance. Trends in alfalfa overexpressing superoxide dismutase. many stress-activated genes with unknown Plant Sci. 6, 66–71 Plant Physiol. 111, 1177–1181 8 Bohren, K.M. et al. (1989) The aldo-keto reductase 16 Sheveleva, E. et al. (1997) Increase salt and functions, future experiments might discover superfamily. cDNAs and deduced amino acid drought tolerance by D-ononitol production in further pathways that lead to reduced levels sequences of human aldehyde and aldose transgenic Nicotiana tabacum L. Plant Physiol. of reactive molecules. Therefore, as yet reductase. J. Biol. Chem. 264, 9547–9551 115, 1211–1219 unidentified stress-induced genes could have 9 Oberschall, A. et al. (2000) A novel 17 Tarczynski, M.C. et al. (1993) Stress protection the potential to engineer plants with aldose/aldehyde reductase protects transgenic in transgenic tobacco producing a putative plants against lipid peroxidation under chemical osmoprotectant, mannitol. Science 259, improved stress tolerance. and drought stresses. Plant J. 24, 437–446 508–510 10 Bartels, D. et al. (1991) An ABA and GA References modulated gene expressed in the barley embryo 1 Boyer, J.S. (1982) Plant productivity and encodes an aldose reductase-related protein. Dorothea Bartels environment. Science 218, 443–448 EMBO J. 10, 1037–1043 University of Bonn, Institute of Botany, 2 McKersie, B.D. and Leshem, Y. (1994) Stress and 11 Mundree, S.G. et al. (2000) An aldose reductase Stress Coping in Cultivated Plants, Kluwer homolog from the resurrection plant Xerophyta Kirschallee 1, D53115 Bonn, Germany. Academic Publishers viscosa Baker. Planta 211, 693–700 e-mail: [email protected] Techniques & Applications Functional imaging of plants by magnetic resonance experiments Walter Köckenberger Microimaging based on magnetic properties such as water diffusion and magnetization, which can be manipulated resonance is an experimental technique relaxation mechanisms in different by irradiating with appropriate radio- that can provide a unique view of a variety cellular compartments. In addition, frequency pulses. The sample of plant physiological processes. electron paramagnetic resonance (EPR) magnetization can then be detected Particularly interesting applications include techniques can be used to detect free through an induction of a weak voltage in investigations of water movement and stable radicals in plant tissue. a coil placed around the sample. The spatially resolved studies of the transport frequency components of the time- and accumulation of labelled molecules in Noninvasive images of virtual transverse dependent signal can be extracted by intact plant tissue. Some of the sections Fourier analysis and represented in a fundamental principles of nuclear and Several nuclides, such as 1H, 13C, 15N and spectrum. electron magnetic resonance microimaging 17O, have angular momentum (nuclear If the sample consists of one type of are explained here and the potential of spin) and a magnetic moment. These two nuclei, such as protons bound in the water these techniques is shown using several nuclear properties are a prerequisite for molecule, and a homogeneous external representative examples. any NMR experiment. In microimaging magnetic field is applied, there is only one experiments with plants, images are frequency component in the spectrum Only a few techniques make it possible to frequently formed from the dominant (Fig. 1a). The signal can be spatially map physical and chemical parameters in signal of the protons bound in the water encoded by exploiting one of the most intact, living plants. Imaging methods molecule. However, it is also possible to fundamental principles of magnetic based on magnetic resonance are among detect protons in metabolites with much resonance, which states that the the most versatile techniques within this lower concentrations or to use 13C nuclei resonance frequency is proportional to the group. The information that is available in an imaging experiment. The principles interacting local magnetic field. from the use of nuclear magnetic underlying the detection of nuclei by Therefore, if a magnetic field gradient is resonance (NMR) techniques includes the NMR are summarized in Box 1. In applied across the sample, the local in vivo distribution of metabolites, water essence, the application of a strong magnetic field becomes spatially flow in the vascular conduits and physical magnetic field creates a weak sample dependent and the detected signal http://plants.trends.com 1360-1385/01/$ – see front matter © 2001 Elsevier Science Ltd. All rights reserved. PII: S1360-1385(01)01984-7 Research Update TRENDS in Plant Science Vol.6 No.7 July 2001 287 Box 1. Nuclear magnetic resonance An externally applied magnetic field Bo, created with either a radio-frequency pulses. This signal is amplified superconductive magnet or an electromagnet, interacts with the and digitized. After the application of a radio frequency pulse, nuclear magnetic moments. As a result, the nuclei split into different the sample magnetization returns to the initial equilibrium populations, which occupy different energy levels. In the classical by relaxation (c). Although there are several different description of nuclear magnetic resonance, the magnetic dipoles are processes contributing to relaxation, frequently the time depicted as vectors that precess (rotate) with a typical frequency ω dependence of the sample magnetization can be described by two around the axis of the applied magnetic field Bo (a). In Fig. I, the phenomenological first-order equations. The rate of return of the nuclear dipoles are represented by just eight vectors. They are either sample magnetization after excitation to the initial equilibrium state aligned parallel (five) or antiparallel (three) to the applied magnetic is mediated by dissipative processes and is characterized by the time field. A weak sample magnetization (Mequilibrium), depicted by a red constant T1. This process is called longitudinal relaxation. Note that vector in the diagram, arises from the unequal distribution between the initial distribution between parallel and antiparallel magnetic the two energy levels. The initial distribution can be pushed away dipoles is re-established after the completion of longitudinal from the equilibrium state through the application of radio- relaxation and therefore Mequilibrium has returned to its initial value. frequency pulses that induce transitions between the energy levels The decrease of detectable sample magnetization by a loss of phase and synchronize the precession of the magnetic dipoles (b). They coherence is described by the time constant T2 and the process is precess now in phase and a detectable magnetization (Mdetectable) is called transverse relaxation. In Fig. I, this loss of phase coherence is created perpendicular to the axis of the applied magnetic field. In represented by the difference in the orientation of the magnetic Fig. I, Mequilibrium is rotated into the plane perpendicular to the axis of dipoles (black vectors) compared with their orientation after the the applied field by a ‘90o-radio-frequency pulse’ The precessing radio-frequency pulse (red vectors). Note that owing to the loss of sample magnetization creates a small electromotive force in a phase coherence between the individual magnetic dipoles, the detection coil after its manipulation by an appropriate sequence of resulting magnetization Mdetectable is attenuated. ω Bo Mequilibrium 90o Radio frequency Longitudinal relaxation (T1) pulse Mequilibrium ω Mdetectable ω Transverse relaxation (T2) Mdetectable (a) (b) (c) TRENDS in Plant Science Fig. I consists of a range of frequencies. From a few hundred µm (for a comprehensive and morphological NMR microscopy this signal, a one-dimensional projection introduction, see Ref. 1). NMR studies have been carried out with of the sample can be derived by Fourier microimages of plant organs with such plants1–5. Recent applications include the transformation (Fig. 1b). High magnetic

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