Сельскохозяйственная биология, 2010, № 3, с. 118-124.

УДК 581.8:57.086.8:57.088.53

APPLICATION OF NUCLEAR MAGNETIC RESONANCE IMAGING METHOD FOR RESEARCH OF AN INTERNAL STRUCTURE OF PLANTS

I.S. Vinogradova¹, O.V. Falaleev²

Proton nuclear magnetic resonance imaging study of water distribution in plants of haricot and cucurbit was carried out using a Bruker 300 MHz NMR spectrometer. Two-dimensional NMR-images were obtained in seeds with the different contents of water; the germination and freezing phenomena were studied from room temperature to -30 °C for Lima haricot. The applications magnetic resonance imaging were illustrated for study of the roots of haricot plants and conductive system of cucurbit caulis.

Key words: magnetic resonance imaging method (MRI), swelling of seeds, water distribution in pores of seeds, freezing of seeds, imaging of roots and caulis, Lima haricot, plants of cucurbit.

The study was performed under the support of the target program “ The Development of Scientific Potential of Higher School”, the project № 2.1.1/2584.

Most of modern methods for investigation the internal structure of plants are invasive, as they are connected with the destruction of a studied tissue. Firstly, there are standard methods for obtaining light optical images using a microscope. Nuclear magnetic resonance tomography (of NMR imaging) is one of recently developed approaches to obtain images of tissues without their destruction.

The method appeared in mid-1980's (1), and it is based upon the impulse nuclear magnetic resonance (NMR). For the first time, the practical application of NMR was medicine and is still widely used to diagnose bone diseases, tumors detection, routine monitoring of disease treatment, and also for studying functional brain activity. Modern techniques make it possible to obtain images of samples’ internal structure and to study the processes occurring in them with a resolution of up to 100 microns. The main advantage of NMR is that it does not use ionizing radiation, which makes installation of NMR tomography harmless for objects. Among the nuclei involved in NMR, the hydrogen nuclei - protons - are of the particular interest, because protons are the  most prevalent in various objects in water molecules and other hydrogen-containing groups. The sensitivity of proton NMR is relatively higher compared with other nuclei, that’s why the majority of studies using NMR imaging has been performed on protons nuclei.
The most promising use of NMR is studying porous materials containing liquids (2), like plant tissues (3). The method allows imaging fluid distribution in pore space with the amplitude of NMR signal per pixel proportional to water saturation within the corresponding element of a sample. As water makes up 95% of plant biomass and it participates in all physiological processes, therefore, it’s interesting to use NMR imaging as a method of nondestructive testing for plants water regime throughout the life cycle.

In this study, authors obtained NMR images of seeds with different water contents, in a process of swelling, at  temperatures up to -30 °C, and also plant stems and root system.

Methods. The studies were performed upon haricot plants and seeds (cultivars Lima and Saksa) and cucurbit (pumpkin the cultivars Volzhskaya seraya and Stofuntovaya) in the Krasnoyarsk Scientific Center of Siberian Branch of RAS using the microtomography device «Bruker» (Germany) providing the following parameters: a superconducting magnet with a magnetic field induction of 4.7 Tesla, the vertical bore of 89 mm diameter and a set of radio frequency (RF) coils with a maximum diameter of 37 mm.

The spin-echo technique with subjecting a sample to periodically repeated sequence of RF impulses (90 and 180°), was used.  The obtained images (tomograms) contain light and dark areas; the lighter in color regions correspond to the higher local content of mobile water molecules. Measurements were performed in several projections with slice thickness 1.0 mm. When imaging, the seeds can be located in the spectrometer RF circuit so that their largest axis is parallel or perpendicular to the external magnetic field (respectively, vertical or horizontal position), and tomographic slices would be made in three mutually perpendicular planes (in this work, the transverse and longitudinal sections were used, respectively, perpendicular and parallel to an embryonic axis).
In Lima haricot, the air-dry seeds were analyzed, and also the dynamics of water income and distribution in swelling seeds. For this purpose, a glass ampoule with 2-3 samples of air-dry seeds filled with distilled water were placed inside the RF circuit of the spectrometer NMR 300 MHz («Bruker», Germany). At certain intervals, tomograms for the longitudinal and transverse sections were recorded (most of the measurements were performed on cross sections, as they were found to be the most informative in this study). To illustrate the possibility of obtaining tomographic images at different temperatures, authors investigated freezing of water in Lima haricot swollen seeds by analyzing the cross sections of the sample placed in a RF frequency circuit vertically to the largest dimension, with cooling by nitrogen vapor (25, 0, -15 and -30 °C; the temperature was monitored with a thermocouple). Besides, the possibility of tomographic studies of a root system without extraction of plants from soil was evaluated in Lima haricot. For this purpose, the pre-soaked seeds starting to germinate were planted in a test tube 30 mm diameter filled with ordinary commercial ground. In about 1 week, the shoots appeared and then plants were grown until the first leaves. Then the tube was placed in the RF circuit of the spectrometer. Lima haricot cultivar has hypogeal seed germination, ie its seed leaves, or cotyledons, remain in soil. In our experiments, they occupied most of the useful volume, preventing root system to develop normally. That’s why there were additionally recorded tomograms of the same plants’ root system removed from soil.

In Saksa haricot, authors studied the seeds passing the stage from swelling to the first root appearance.
In the cucurbit Volzhskaya seraya, the seeds derived from a fruit immediately before the experiment were analyzed.

In the curcubit Stofuntovaya, the mikrotomograms of stems transverse sections were obtained to be compared with the results of light microscopy performed by standard methods (4) on thin (10-15 micron) sections with safranin or other dyes staining.

Results. In contrast to the conventional impulse NMR, the applied imaging uses a gradient external field (in this case, the resonant frequency changes in proportion to coordinate in the chosen direction). To study the voluminous bodies, the gradient field is applied in three mutually perpendicular directions providing three-dimensional anatomical slices of the body. To do this, at the time of sample excitation, the external field gradient is formed perpendicular to the plane of the expected cut. As a result, at a given frequency of atomic nuclei excitation the excited state will be reached only by the nuclei for which this frequency is resonant, that is, those that lie in the selected planar cross-section of the body. The method allows obtaining images of individual sections, whose depth and orientation are specified by the operator. The spatial resolution of the method is 0,5 mm. The signals are increased by their accumulation. The image of the object is created using a computer, which performs two-dimensional Fourier transform (FT). Usually, up to 30 sections and more can be simultaneously recorded. To our point of view, the study of NMR images of root system of plants growing in soil, monitoring the dynamics of its development, the effects of high and low temperatures, heavy metals, as well as other important factors are very promising for the theory and practice of agriculture and plant breeding, while other methods can’t implement these tasks.
NMR research of plant seeds with different water content. Water plays a particular role in seeds and it determines their status at all stages of maturation before germination. At the same time, the water supply of seeds is the least studied issue. It has been reliably established, that the seed of milk ripeness keep moisture content equal to 65-70%; then, by the phase of full ripeness, it reduces to 30-40%, and finally, in the third phase of a life cycle, the seeds quickly dehydrate and pass into the air-dry state with water content ranging from 3-5 to 15-18% in different types of seeds (5). The dry seeds can long exist as an independent individual until a new contact with water initiates them to the stage of swelling with subsequent germination.

Different stages of seed interaction with water were clearly reproduced on the obtained tomographic images. On the  NMR image of air-dry seeds of Lima haricot (Fig. 1 a), the three samples were placed vertically in the RF circuit with distilled water filling, which created the white background contrasting with the seeds. The image shows the cross section approximately in the middle of the seeds - two cotyledons and the air gap between them (it looks darker). When seeds go swelling, water flows inward and gradually brightens areas in which  it comes.

On cross sections of one of the imbibed seeds starting to germinate (Saks haricot, see Fig. 1, b), the protruding radicle is seen. The samples were completely saturated with water, with an air filling the space between cotyledons. The seeds of this cultivar perform a fairly uniform imbibing.

Fig. 1. Two-dimensional MRI images (MRI - magnetic resonance imaging) of seeds of haricot and cucurbit at various stages of a life cycle (upper row) and the imbibed seeds of Lima haricot at different temperatures (lower row). Top row: а, c - Lima haricot (respectively, cross sections of air-dry seeds and longitudinal sections of imbibing seeds), b – Saks haricot (transverse sections of seeds imbibed up to the radicle emergence); d – seeds of Volzhskaya seraya cucurbit (transverse sections of seeds extracted from a fruit before the experiment). On the right side of scans, the specified scale (cm) is shown which persists for all the plotted tomograms.

The studied longitudinal sections were also very informative. Thus, the image of two imbibed seeds of Lima haricot (see Fig., 1) shows a visible air gap (dark area) in the central part of one seed and cotyledons being filled with water (most intense - in the bottom).

In the top, there’s the embryonic axis with an ovule and a radicle. Seeds of this type tend to inhomogeneous hydration: even the completely imbibed samples have dark areas. The greatest content of water was concentrated in the radicle, which corresponds to the results obtained previously by other methods (5). The image of seed on the right corresponds to initial stage of imbibing: water starts to enter the areas located under seed coat, and an ovule is visible in the lower left side of the seed. The peculiarity of Lima haricot - a relatively large size and weight of seeds (may exceed 2 g).

The tomograms of seeds of the cucurbit Volzhskaya seraya (see Fig. 1, d) show sufficiently high content of water in them, as these seeds were extracted from a fruit before the experiment and had not yet become air-dry. The fresh seeds are maximally and uniformly saturated with water. The image shows transverse sections of the seeds horizontally placed in the RF circuit (the air between them provides a dark background).

Thus, the tomographic experiment allows to evaluate water content in seeds and can be used in agronomic practice for solving problems of grain preservation.

Investigation of seeds subjected to low temperatures. Plant death under low temperature is caused by the formation of ice crystals, which makes necessary studying the conditions, localization, and mechanism of this process. NMR is a promising and informative method allowing to solve these problems.

The results of the experiment with seeds of Lima haricot are presented in the bottom row of Figure 1. The image of the seed had imbibed  at room temperature (25 °C) in air environment (dark background) shows two cotyledons completely saturated with water and separated by an air gap, which remains in the right side of the seed. When temperature was decreased to 0 oC, almost no changes appeared; then (-15 °C) water starts to crystallize, which is described as the appearance of dark spots inside the seed. When -30° C, seed image completely disappears, which corresponds to complete freezing. When seed is defrosting, the temperature  hysteresis is  observed, and at 0°C not all of the water passes to liquid state.

Dynamics of seed imbibing. The most interesting subject here is discovering the dynamics and ways of water transport in seeds when they are imbibing. Most researchers believe that the water penetrates through certain parts of seed surface, in haricot seeds - probably via hilum (6) or micropyle (7) tissues. However, this issue has been the most controversial, since previously the direct "noninvasive" observation was impossible. Using NMR-tomography allows such monitoring and analyzing the stages of water penetration, the content of water in different parts of a seed, and the rate of water flow into particular parts of a seed, as well as other peculiarities of the process.

Fig. 2. Two-dimensional MRI images (MRI - magnetic resonance imaging) of transverse sections of two seeds the Lima haricot, reflecting the dynamics of imbibing, starting form the air-dry state (0 h). The upper and lower rows are distinct in height where the sections were performed (the lower row - the scans of sections passing through embryonic axis and including a cross section of radicle).

These experiments were carried out in several repetitions upon different samples of Lima haricot seeds. The effective volume of the RF circuit allows placing in it several samples. In this study, usually two or three samples were placed, which were vertically  located in the circuit and filled with distilled water. The required amount of water was determined earlier - the weight of imbibed seeds depending on time of their contact with water was measured in several replications. It has been found, that most of the seeds complete the first phase of swelling within 12-15 hours, but the process was considerably longer in some samples due to  specificity of their quality. The weight of imbibed seeds increased by an average of 100% from the initial air-dry weight.

Figure 2 shows tomograms for the two haricot seeds placed vertically in the RF circuit and filled with distilled water (totally 25 slices in 1 mm were made in water penetration dynamics; the figure shows some of the obtained images). Initially, the air-dry (0 h) seeds were dark on a white background of the distilled water. Water penetration in the seeds was recorded as white areas appearing in the image. The observations has suggested that water actually penetrates into a seed via certain parts of a surface (micropyle, hilum and strophiolar cleft tissues). Water appears under seed cover and causes wrinkles on it, which can be observed both visually and on the tomograms (especially, the lower row images). Inside the seed, water penetrates first into embryonic axes – to the base of embryonic root, which is located near the micropyle (it started to light first and remains the brightest point indicating high content of water in this area) and can be seen as the bright lighting round on the sample in the lower row to the right (see Fig. 2). These results agree well with published data: for example, it has been reported that water content in maize corn during the period of contact with water increases by 800 times in embryo, and in the cotyledons - only 5-50 times (5). Then water saturates the thin layer of cotyledons at their perimeter and fills the air gap between the cotyledons. Only after that, water starts to penetrate into the cotyledons, but not as a continuous front, as was previously assumed (7). Seed is inhomogeneous inside, so the assertions proposed by J.D. Bewley and V. Black about a uniform transport of water can’t be performed. This heterogeneity was quite visible on the image of the right sample (the last one in the upper row, see Fig. 2).

Considering the diversity in seed quality,  presence of internal micro-and macroscopic defects, structure and properties of seed shell, the model obtained in authors’ experiments can be possible. The particular details can differ in various groups of seeds.

Fig. 3. Two-dimensional MRI images (MRI - magnetic resonance imaging) of the root system of Lima haricot (а - in soil, b - after extraction from the soil;1 and 2 - respectively, cotyledons and the main root) (top row), and the schematic structure of a stem of the cucurbit  Stofuntovaya according to microscopic (c) and tomography (d) researches (bottom row).  The scale (mm) is shown in the right of images (scans).

While water was penetrating into the seeds, its amount in the space between seeds decreased, which was shown on the tomogram as the appearance of a dark area filled with air (water was refilled in the circuit after 8 h).

Investigation of a root system and stems. The seeds of Lima haricot happened to be very suitable for studying the processes of imbibitions; however, this cultivar couldn’t be the subject for investigation of a root system, because its cotyledons remain in soil and occupy most of tube space. Because of this, the main root bended upward while the lateral roots and a few shoots grew from the cotyledons. Most of the image area was occupied by swollen cotyledons, the main root was held under the cotyledons and curved upward (Fig. 3). The selected slice of Lima haricot sample removed from soil shows that most of its area is occupied by cotyledons, which structure is inhomogeneous due to changes in growth process. Between the cotyledons, there is a cross section of hypocotil - the interjacent region between root and stem. Its structure and varying degrees of water content in individual parts are clearly visible.
According to light microscopy, the stem of the Stofuntovaya cucurbit was found to be rounded or rounded-pentagonal with internal five-way air cavity. Between the protrusions of air cavity, there are five major vascular bundles. In opposite of the protrusions closer to stem surface, there is a second ring of five similar smaller beams. The image of the stem reproduces well the details of this structure (see Fig. 3) - the areas filled with water look darker. In the center of the tomogram, a white five-way air cavity and black vascular bundles can be observed (the content of liquid water in them was much greater than in other tissues of the stem).

Thus, nuclear magnetic resonance microtomography imaging (NMR imaging, or MRI) can be successfully applied  for studying the ontogeny of plants and many issues related to water regime monitoring. The described method is especially promising for investigation of plant seeds, because their water status when imbibing and germination can affect the subsequent development and growth of plants. Although the data on water absorption by seeds are  numerous, little is known about its spatial and temporal distribution. Knowing the structural changes caused by water penetration in dry seeds is important for understanding of activation processes in plant life.  It is possible that the discovered spatial heterogeneity of water distribution in cotyledons correlates with gradients of reserved substances accumulation. The obtained data has shown that activation of micropyle and hilum tissues as water penetration channels, water gradient to a radicle within seed cover, cotyledons swelling and a further increase of water content are physiologically different processes which can be distinguished at MRI by localization of water in different tissues of a seed. The biological role of water distribution can be important for both germination and the assessment of seed quality.

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