Element-Specific Distribution in a Plant
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For the first stage of the study of the elements, the distribution of the element within the plant tissue was presented employing neutron activation analysis (NAA). Since NAA allows nondestructive analysis of the elements in the sample, this is the only method to measure the absolute amount of elements in the sample.
The results showed that the element-specific profile varied throughout the whole plant, and this distribution tendency remained similar throughout development. There were many junctions of element-specific concentrations between the tissues, suggesting barriers to the movement of the elements. Generally, heavy elements tended to accumulate in roots, except for Mn and Cr. Of the elements measured, Ca and Mg showed changes in concentration with the circadian rhythm. Since the amount of the element in a plant reflects the features of the soil where the plant grows, multielement analysis of the plant could specify the site of the agricultural products produced.
Before addressing the development of a real-time RI imaging system (RRIS), the production of RIs for essential elements for plant nutrition, 28Mg and 42K, is presented. The reason why concentrating on RIs is because when we examine the history of plant research, physiological research on the elements without available radioisotopes has not been well developed. For example, the boron (B) transporter was recently found and the study of B in plants is far behind compared to the other elements.
Therefore, we developed a preparation method for elements whose available RIs were not previously employed in plant research, 28Mg and 42K. They are the radioisotopes we prepared and a root absorption study using 28Mg as a tracer is presented as an example. It was found that the orientation of Mg transfer was different according to the site of the root where Mg was absorbed. The specific role of Mg has not yet been clarified by florescent imaging because the overwhelming amount of Ca makes it difficult to distinguish Mg and Ca.
KeywordsElement-specific distribution Neutron activation analysis Nondestructive element analysis Barley morning glory 28Mg 28Mg production 42K 42K production Short-day treatment Dark/light cycle
3.1 Nondestructive Element Analysis: Element Profile
Sensitivity of the thermal neutron activation analysis
(3~5) × 10−13
Dy, Eu, In
(1~4) × 10−12
Co, Ag, Rh, V
(6~9) × 10−12
Mn, Br, I
(2~5) × 10−11
Th, Pr, Se, Lu, Nb, Ga, Sm, Cu, Re, Ho, U, Al, Hf
(6~9) × 10−11
Kr, Ba, Au, Ar, Cs
(2~5) × 10−10
Se, Er, Cl, W, Zn, As, La, Na, Pd, Pt, Yb, Gd, Ge
(6~8) × 10−10
Os, Te, Nd
(1~3) × 10−9
TI, Rb, Sb, Sr, Ti, Mo, Xe, Mg, Cr, Hg, Y, Tm, K
(4~7) × 10−9
Ru, Sn, Tb, Ni, Ta, F, Ca
(2~4) × 10−8
Si, Ne, Ce, P, Cd
(4~5) × 10−7
(2~5) × 10−6
Zr, Pb, Fe
(2~6) × 10−4
0.11, 0.33, 19.2
Be, H, C
The measurement of radiation is as follows. After irradiation, the γ-rays emitted from the sample are measured by a γ-counter, such as a pure Ge counter or a Na(Tl)I scintillator, equipped with a pulse height analyzer. The kind of radionuclide produced was identified by the energy of γ-rays detected, and the amount of the element was calculated from the intensity of the γ-rays. However, the half-lives of newly produced radionuclides are different; since some of the radionuclides produced have very short half-lives, they rapidly decay out during the measurement. Therefore, γ-ray measurement should be performed at an appropriate time after irradiation checking the changes in the γ-ray spectrum.
For example, when a small amount of plant tissue is irradiated, a high amount of 28Al is produced from 27Al by the 27Al (n, γ)28Al reaction, and the half-life of 28Al is only 2 min (γ-ray energy: 1.779 MeV). Though 28Al decays out rapidly during the measurement, while a high intensity of radiation from 28Al emits, the background level of γ-ray measurement is high, and this high background prevents the measurement of the γ-rays emitted by other radionuclides, especially those whose γ-ray energies are lower than approximately 1.5 MeV, in this case.
With the decay of 28Al, the γ-rays of other nuclides with longer half-lives than that of 28Al, such as 24Na (1.369 MeV), 27Mg (1.014 MeV), and 42K (1.525 MeV), emerge in the γ-ray spectrum during measurement, revealing γ-ray peaks previously hidden under the overwhelming intensity of the 28A γ-rays. From the counts of each nuclide-specific γ-ray, the absolute amount of the original element can be calculated by comparison to the radiation from a standard sample irradiated at the same time.
3.1.1 Profile of the Elements in Barley
The profile of the elements in barley leaves was obtained by employing NAA as follows. Barley (Hordeum vulgare c.v. Minorimugi) seed was germinated, grown in water culture, and harvested after 12, 19, 23, and 46 days. Then, 2 cm samples of the tip, middle, and bottom sections of each leaf were cut out, and neutron activation was performed. Each sample was sealed doubly in a well-washed polyethylene vinyl bag and then irradiated by a Triga Mark II type atomic reactor at the Institute for Atomic Energy, Rikkyo University in Japan, now decommissioned, with a thermal neutron flux of 1.3 × 1012 n/cm2/s for 3 min. After the sample cooled for 3 min, the γ-rays emitted from each sample were measured by a Ge(Li) detector for 200 s. To determine the P concentration, the samples were placed in a Cd container for irradiation to measure only 28Al produced by the fast neutron reaction of (n, α) from 31P. Since 28Al is also produced by the thermal neutron reaction of (n, γ) from natural 27Al, a Cd container was needed to prevent this thermal neutron reaction. From this short-time activation analysis, 24Na, 27Mg, 28Al, 38Cl, 42K, 40Ca, and 56Mn were produced, and their radioactivity counts corresponded to the amounts of Na, Mg, P, Cl, K, Ca, and Mn, respectively. To determine the element amount, reference materials, namely, JB3 (Geological Survey of Japan) and orchard leaves (National Bureau of Standards, present NIST, USA), were irradiated at the same time.
There was an extremely high accumulation of Na in the first leaf, especially at the bottom part, whose concentration on the 46th day was 20 times higher than those of the other leaves. However, in other leaves, the Na concentration remained rather uniform and low, regardless of the leaf number and the position in the leaf. When the profiles of the 46-day sample of Na were compared to those of K, the tendency was that the Na content in the bottom part of the leaf was the highest in the first leaf and slightly higher in younger leaves, showing the reverse distribution from that of K. Considering this reciprocal tendency of Na concentration to that of K, as well as the high concentration of Na when K was eliminated from the culture medium (data not shown), there might be some compensatory function between K and Na content in the leaf. However, despite the same cation valency number, the distribution patterns of K and Na were drastically different.
The elements Cl and K are the only ones that are not constituents of organic structures but function mainly in osmoregulation. The concentration change within one leaf took place gradually, without any drastic accumulation at one edge of the leaf; therefore, the K and Cl contents in the middle part of the leaf were the average of the amounts in the tip and bottom parts of the leaves. However, it is interesting that Cl, K, and Na, which are known to regulate the osmotic pressure of cells, tended to accumulate in the bottom part of the leaf, which might suggest fast movement to the other tissues.
It is also interesting that K, which is the most abundant cation in the cytoplasm and whose cytoplasmic concentration remains in a relatively narrow range to stabilize the pH concentration, had a large concentration gradient in the leaf.
When the element moves along with the transpiration stream and redistributes to the other tissues smoothly, the element abundance in the middle of the leaf could be an average value of those in the tip and the bottom part. Extremely different concentrations of the element within a leaf might suggest some barriers to movement or fixation of the element at certain sites. A steep gradient in the element concentration within a leaf was shown for P and Mn, where the concentration was especially high at the tip of the leaf. P serves as a constituent of proteins and nucleic acids; therefore, it might not be released readily from the tip. Sometimes phosphate movement is considered to be similar to water movement; therefore, a high concentration of P at the leaf tip might reflect the result of intensive water evaporation. However, although P is known to undergo fast chemical changes in plants, the exact chemical form in which it is fixed is uncertain from this analysis. Regarding the chemical form of P in a plant, although it should be discussed later, we found that 32P was in the form of phosphate, at least for the first 30 min, after being absorbed from the roots, in Lotus japonicus.
Calcium is the only element known to function mainly outside the cytoplasm, and its uptake rate into the cytoplasm is strictly restricted. A concentration of Ca a few times higher at the leaf tip might be explained by the low mobility of Ca from cell to cell. The concentration profile of Ca was similar to that of Mg. Although the behaviors of these elements are expected to be similar because they are in the same group in the periodic table, Mg has its own specific characteristics. One of them is that the Mg concentration is known to be kept constant among different plant species, suggesting a role in maintaining homeostasis in plants.
The extremely low concentrations in the senescent leaves, namely, the 46-day-old samples, suggested that the elements were recycled or transferred within the plant and moved toward another tissue where these elements were needed. Actually, in the case of a rice plant, it is known that N is transferred from senescent leaves to other tissues and used again, which might be regarded as an element recycling activity.
3.1.2 Profile of the Elements in Morning Glory During Growth
The morning glory bears the flower bulb, flower, and seeds after 56, 61, and 78 days of germination, respectively. The plant developmental stage from the juvenile phase to the adult phase occurs between 23 days and 56 days. As shown in the figures, systematic barriers appeared according to each element profile. The first barrier was found between the root and the upper part of the plant.
Among the elements, the profile of Mn was representative, showing a high concentration in senescent tissue, especially in the elderly leaves, suggesting rapid movement of the element to the senescent tissue with growth. Mn seemed to move with the water in xylem tissue and then accumulate at the leaf tip (Fig. 3.3).
The concentration profiles of Mg and Ca (Fig. 3.4) showed similar distribution patterns, where the highest concentration was found below the cotyledon before flowering was induced. With the development of the plant, there appeared to be a distinct difference in element distribution between the juvenile phase and adult phase. When the flower bulb was developed, on day 56, the stored Mg and Ca in the lower tissue were moved to the upper part of the cotyledon.
Although both alkaline elements Na and K produce monovalent cations, the profiles were drastically different (Fig. 3.5). Most Na accumulated in roots, and the concentration of Na in the aboveground part was very low, whereas most of the K absorbed was transferred to the aboveground part. The reciprocal distribution of Na and K was also observed in barley (Fig. 3.2).
A barrier around the cotyledon was also shown for Cl and Br (Fig. 3.6) but did not disappear completely until the seed ripening stage. The halogen elements Cl and Br are volatile and easily lost from the plants during the developmental stage; however, higher concentrations of Cl and Br were still found in the petiole of the cotyledon and the first leaf at the senescent stage.
In the case of K, the concentration at the leaf petiole was always high during development, indicating that the high concentration of K at the leaf petiole always exerted pressure on the element to move toward the connecting leaf. This ratio tended to increase in the younger leaves, suggesting the regulation of the movement of the elements to accumulate in younger leaves. In addition to the leaf, the concentration of K in the petiole was also higher than that in the connecting internode, especially during flower formation. Therefore, it was suggested that the regulation of K movement in both directions, toward the connecting leaf and the internode, indicated some role in the petiole (Fig. 3.5). This tendency toward higher concentrations at the petiole than at the connecting leaf and internode was also observed for the elements Ca, Cl, and Br (Figs. 3.4 and 3.6).
In the case of the seed maturing process, there was a clear element partition among the seed tissues, calyx, seed coat, seed wall, or endosperm. In the mature seed, where the seed wall was well developed, it was interesting that the element selectively accumulated in the seed wall, and only a small amount was partitioned into the embryo or endosperm (data not shown).
Juvenile phase and adult phase
During the juvenile phase, Ca and Mg accumulated below the cotyledon, and then, when the plant reached the adult phase, they moved upward. In the internode, these element concentrations increased during the juvenile phase and decreased during the adult phase.
Element barriers in the tissue
The barrier between roots and the aboveground part of the plant was high, especially for Na, Al, and heavy elements other than Cr and Mn. The concentrations of K, Ca, Mg, Cl and Cl in the leaf petiole were higher than those in the connecting internodes and leaves.
Seed maturing process
During the maturing process, the element concentration ratio of seed to seed stem increased. When the seed was mature, most of the elements occurred predominantly in the seed coat or seed wall, not in the embryo or endosperm.
3.1.3 Profile of the Elements in Young Seedlings of Morning Glory
The overall element profile during 78 days of development is shown. The next analysis presented is the change in the elemental profile within a few days, with a connection to circadian rhythm. The circadian rhythm plays an important role in plant development, such as stem elongation and leaf movement according to light conditions. A Japanese morning glory (Pharbitis nil. cv. violet) is known to be very sensitive to light conditions, and a single treatment of seedlings with an increased dark period, from 8 to 16 h, can induce flowering. Although it is known that the circadian rhythm of light acts to promote floral induction in the shoot apical meristem, through the FT signaling system (expression of flowering locus T (FT) gene), the elemental profile at flower induction in the meristem is not well known.
Using the same kind of morning glory, the circadian rhythm of elemental concentration was studied. First, the concentration profile in the seedling was analyzed after growth for approximately a week after germination in water culture under 12 h L/12 h D light/dark conditions. The seedlings were periodically harvested and separated into 9 tissues: cotyledon, petiole, shoot apex, and three parts each of the stem and roots, upper, middle, and bottom. To determine the amount of the elements, neutron activation analysis with γ-ray spectroscopy was performed using the research reactor JRR3M installed at the Japan Atomic Energy Agency (JAEA). Each sample was sealed in an ultrapure polyethylene bag and irradiated for 10 s. The total thermal neutron dose was 1.9 × 1014 n/cm2. After irradiation, the sample was cooled for 2 min, and the gamma-rays from the sample were measured for 150 s by a pure Ge counter.
3.1.4 Ca and Mg Concentrations
Each element showed its specific distribution pattern within a seeding, as shown above. Although the macroscopic concentration pattern of the element did not change during growth, on a finer level, the concentration within each tissue was found to change with hours. To study the change in elemental concentration with respect to the light conditions, the seedlings were harvested periodically during the water culture, and the elements in each tissue were measured in the same way by neutron activation analysis (NAA).
Since the Ca concentrations in the shoot apex, stem 3 connected to the shoot apex, and stem 2 and stem 1 showed similar levels (data omitted), approximately 2 times higher than that in root 3, it was suggested that the Ca concentration was maintained at the same level among the organs above the ground.
Compared to the Ca distribution, there was a distinctive gap in Mg concentration between the shoot apex and stem 3, although these tissues were connected to each other. The magnesium concentration in stem 3 was only approximately 60% of that in the shoot apex. Considering the different positions of the concentration gaps of Ca and Mg, between roots and stems, and between stems and the shoot apex, respectively, it was suggested that the Mg concentration was more severely regulated than that of Ca.
In the case of Ca, the increase in Ca concentration stopped at the beginning of the short-day treatment, plateaued during the first dark period, and then decreased during the light period of day 6 in the shoot apex as well as stem 1, suggesting that the Ca concentration was maintained at the same level in both tissues. After day 7, the diurnal rhythm in the concentration appeared again. The large concentration gap between stem 1 and root 3 indicates that there is some mechanism that controls the Ca distribution between the bottom of the stem and the root.
Then, what could be suggested by the different changes in the concentrations of Mg and Ca? The changes in Ca and Mg concentrations showed similar tendencies during ordinary light–dark conditions throughout the whole plant, especially in the shoot apex. However, a clear difference between the two elements was found under short-day treatment. After the first longer period of darkness, 16 h, during the 8 h light period on day 6, the concentration of Ca in the shoot apex decreased, while that of Mg increased, which might indicate a compensatory role of Mg for decreased Ca, for example, to maintain the proper pH value and avoid the accumulation of organic acids. The increase in concentration during the light period could be well explained by transpiration, the major driving force of mass flow in the xylem. The increase during the dark period, on the other hand, could be understood only when endogenous “circadian rhythms” were taken into consideration. That is, diurnal concentration change was lost during the response to the short-day treatment and then “reset” again to the circadian rhythms even during the dark period.
During growth in the vegetative phase, cells in the center of the top layers accumulated high amounts of Mg2+. Exposure to a single short-day treatment induced the flowering process and dramatically reduced the fluorescence associated with Mg2+ accumulation in the top layers, suggesting that Mg2+ contributed to the flower induction process. The fluorescence associated with Ca2+ did not show this distribution difference before and after the short-day treatment. A night break treatment also showed a similar Mg fluorescence pattern. Since Mg2+ might play an important role in flower induction (Fig. 3.17), this Mg study was further developed to produce 28Mg tracers and to develop a real-time RI imaging system (see next Sect. 3.2).
The flowering process is crucial for plant development. Plants begin life in the vegetative phase and shift to a reproductive phase depending on environmental signals or aging. In the case of morning glory, one hundred percent of flowering was attained when removal of the cotyledon took place 2 h after the end of the short-day treatment, indicating that the flowering signal(s) reached the shoot apical meristem at this time (data not shown).
Although the flowering process is not well understood, flowering is reported to be controlled by endogenous diurnal rhythms mediated by phytochrome and pH, which show oscillating patterns, as well as abscisic acid (ABA), which affects photoperiodic flowering in relation to a dark period. The time course and the movement of some components are key to understanding the mechanism of flowering. Our fluorescent staining study showed that there was a specific accumulation of Mg in the shoot apex, and this accumulation disappeared during flowering induction.
3.1.5 Al Concentration
Finally, the change in the Al concentration in the root tip is presented. Aluminum is a toxic element to plants, and most studies focus on the negative effect of Al on root tips when the Al content in soil increases. However, the Al concentration naturally contained in roots and the changes in Al content have attracted less attention. Since the detection limit of Al in neutron activation analysis is extremely high, the Al concentration in the root of morning glory was analyzed under normal conditions without the addition of any Al chemicals in the culture solution.
3.1.6 Summary of NAA
By applying neutron activation analysis, the absolute amounts of the elements were determined nondestructively in both barley and morning glory. Each element has its own distribution pattern, suggesting a specific physiological role of each element in different tissues of the plant. It seemed that there are many concentration junctions for each element to pass through, and these junctions exist at every connection between tissues in the whole plant. This distribution pattern was maintained throughout the developmental stages. The element-specific distribution pattern of barley was similar to that of morning glory, suggesting that the roles of each element in different plants were similar. Heavy elements, except for Cr and Mn, accumulated in roots and hardly moved to the aboveground part of the plant. Although the macroscopic pattern of the element distribution in tissues showed a similar tendency during growth, the element profiles changed in the course of hours, especially under different light conditions. Among the elements studied, Ca and Mg showed circadian rhythms in their shoot concentrations, increasing during the day and decreasing during the night. There was also a rhythmic change in the Al concentration in the root tip according to the dark/light conditions.
Since plant nutrients are mainly inorganic ions, elemental movement is expected to provide some clues to analyze the physiological development of plants. These findings triggered the further development of a real-time element-specific imaging system using RI (See Part II, Chap. 4).
3.2 Radioactive Nuclide Production for Mg and K
Although plants require 17 elements, the plant physiology of the elements for which radioisotopes are not available, such as B or Si, an essential element and a useful element, respectively, has not been well studied.
Because of the lack of radioactive nuclides available for tracer work, the physiological study of some elements, such as B or Si, is far behind that of other elements. For example, the physiological study of B has begun to develop only after the recent identification of the transporter of B, BOR1 in Arabidopsis, owing to the development of an analytical method for determining trace amounts of B. Another difficulty in studying B was the difficulty out of determining the chemical reactions of B in plants. Although B reacts with the chemical configuration of cis –OH groups, such as marine plants, the sugars of higher plants do not have cis –OH groups. Therefore, the role of B in higher plants was difficult to pursue. In the case of Si, a highly sensitive analytical method has not yet been developed. However, the abundance of Si in the surface of the Earth is 25.8%, which is extremely high, second only to O, which has an elemental abundance of 49.5%, according to the Clarke number. Although the amounts of both elements, B and Si, can now be measured by inductively coupled plasma-mass spectrometry (ICP-MS) or optical emission spectrometry (ICP-OES), it is still difficult to analyze and trace the behavior of these elements at the level of radioactive tracer work.
There are still three other essential elements, O, Mg, and K, for which radioactive nuclides are not commercially available, because of the short half-lives of the corresponding radioactive nuclides, 15O, 28Mg, and 38K or 42K. In the case of 15O, we produced and employed 15O-labeled water to analyze water movement as described in Part I, Chap. 2, Sect. 2.3. Because of the extremely short half-life and rapid decay of 15O, 2 min, the nuclide had to be prepared immediately prior to the experiment, and a large amount of 15O was employed to conduct the experiment. The difficulty of preparing 15O-labeled compounds within a short time before starting the experiment is another reason the physiology of 15O-labeled compounds in a plant has been little studied. In addition to 15O, we prepared 28Mg and 42K ourselves and were able to perform radioactive tracer work before the nuclides decayed out. In the following sections, the preparation of Mg, with a short introduction to tracer work using 28Mg, and the production of 42K tracers are presented.
3.2.1 Production of 28Mg
First, we briefly introduce the features of Mg before describing the preparation of 28Mg. Although Mg is an essential element for plant growth, since Mg is a component of chlorophyll to carry out photosynthesis, the radioactive tracer was not applied to study plant physiology. Magnesium is the second most abundant cation in living cells, and over 300 enzymes are known to be Mg dependent. Mg concentration changes significantly affect the membrane potential. The important feature of Mg is that the concentration of Mg in plants is constant, within a small range, among different plant species; therefore, Mg is estimated to have a role in maintaining the homeostasis of plant physiological activity.
When Mg is deficient in soil, such as at low pH and high concentrations of K+ or NH4+, chlorosis occurs, especially in young mature leaves, changing the green color to yellow. Mg deficiency is also liable to appear during the ripening process of the fruit and decrease the yield. The mechanism is estimated to be closely related to sugar transport. The accumulation of Mg in the fruit drives Mg deficiency in leaves, which induces the deactivation of sugar transport in the phloem and inhibits translocation.
There are two strategies by which plants address element deficiency. One is the retranslocation of the element to maintain growth, which requires mostly phloem transport of the element from mature tissues. The other is to increase the uptake of deficient elements by the roots, such as by inducing the expression of root transporters of the element or secreting chemicals to produce compounds of the element.
In the case of Mg, the early response to Mg deficiency is now an important issue, i.e. what occurs before the accumulation of sugars and subsequent reactions to reduce photosynthesis or to increase the heavy element concentration, which induces reactive oxygen species and chlorosis, etc. Because of the lack of available Mg tracers, Co or Ni is sometimes used to replace Mg, but the physical properties of Mg cannot be not well determined by using these elements.
To perform Mg tracer work, the useful radioactive nuclides for Mg should be taken into account. There are two candidates for Mg tracers, 27Mg and 28Mg, whose half-lives are 9.46 min and 21.1 h, respectively. There are several ways to produce 27Mg, one of which is by the nuclear reaction of 26Mg(n, γ)27Mg. However, the target nuclide and the produced radioactive nuclide are the same element, which means that when 27Mg is produced from 26Mg, 27Mg cannot be separated from 26Mg, a stable nuclide. This means that the produced nuclide, 27Mg, cannot be a carrier-free nuclide and always occurs together with an overwhelming amount of stable Mg elements. That is, when 27Mg was employed as a tracer for the Mg experiment, because of the high Mg ratio to 27Mg, a low concentration of Mg solution could not be prepared for the work: the radioactivity of 27Mg in Mg solution is too low to be detected. Therefore, the element of the irradiating target and the element of the nuclide produced should be different to be able to prepare any concentration of Mg solution for the experiment.
Together with the consideration of the half-life, 28Mg was chosen, and the reaction 27Al(α, 3p)28Mg was employed for production. The key was the chemical purification of the trace amount of 28Mg produced in the target, which means the separation of 28Mg from the macroscopic amount of Al. The chemistry of Al is rather difficult, and the brief preparation is as follows.
Ten pieces of pure aluminum foil (99.999%, 10 × 10 × 0.1 mm, each) were irradiated with a 50–75 MeV He(α) beam for 4–6 h by an AVF cyclotron installed at Tohoku University or QST (National Institutes for Quantum and Radiological Science and Technology) in Japan. Then, the irradiated Al foils in the vessel were removed and dissolved with 3 M HCl. After drying, the residue was dissolved with 2 M NH4SCN and passed through a Sep-Pak Plus tC18 column (Environment, Waters). The eluted solution was applied to a column filled with AG50W-X4 resin (H+ form, 100–200 mesh, Bio-Rad), and 28Mg was retained by the resin. After washing with 0.5 M oxalic acid and 0.01 N HCl, 28Mg was eluted with 2 M HCl. Then, the eluted solution was dried, and 28Mg was dissolved in pure water. In the present study, the 28Mg acquired through this chemical procedure was approximately 4–5 MBq per preparation.
3.2.2 Mg Uptake Activity Using 28Mg as a Tracer
Using 28Mg as a tracer, the Mg uptake activity of the rice root was studied. The Mg uptake activity changed rapidly with Mg concentration. In the case of an Arabidopsis seedling, high- and low-affinity transport systems were revealed by a kinetic study, and the high-affinity transport system was upregulated by Mg-deficiency treatment, similar to that applied to a rice seedling. The increased absorption activity at lower Mg concentrations and the rapid response to Mg concentrations suggested an active Mg absorption mechanism in roots. The Mg2+ uptake system in roots was upregulated within 1 h in response to the low Mg2+ condition. However, the Mg deficiency-induced Mg2+ uptake system was shut down within 5 min when Mg2+ was resupplied to the environment (data not shown).
This result that a relatively large amount of Mg was transported downward from R-C was of particular interest. The pulse chase experiment clearly demonstrated that Mg absorbed from R-C was transported toward the root tip area without going through the upper root part or shoot (data not shown). The results suggested that phloem loading of Mg occurred vigorously in the lateral root developing zone within minutes after uptake from the culture solution.
This characteristic movement of Mg according to the absorption site of the root was not observed for 32P-phosphate or 45Ca, as the upper part of the root did not show high transport activity, and R-C and R-B showed similar transport behavior (data not shown). These results indicated that the long-distance transport of Mg was controlled by a different mechanism from that of phosphate and Ca.
These findings led to our performing a study to determine the predominant response to Mg deficiency in rice seedlings. Each leaf was analyzed in terms of chlorophyll, starch, anthocyanin, and carbohydrate metabolites, and among several mineral deficiencies, only the absence of Mg was found to cause irreversible senescence of the fifth leaf (L5). The results suggested that the predominant response to Mg deficiency is a defect in transpiration flow. Furthermore, changes in myo-inositol and citrate concentrations were detected only in L5 when transpiration decreased, suggesting that they may constitute new biological markers of Mg deficiency (data not shown).
The movement of an element results from the combination or balance between xylem and phloem flow and is very complicated, similar to water flow . Therefore, the only method to solve these problems in the dynamic movement of elements is the application of radioisotopes for tracer work.
3.2.3 Radioactive Tracer Production of K
Potassium (K) is one of the major essential elements in plants, and its physiological role and the role of transporters mediating transmembrane K transport within plant tissue have been intensively studied. Despite these studies, the behavior of long-distance K transport and the factors affecting K, as well as the distribution and function of the transporters, have not yet been clarified. To understand K behavior in a living plant, it is necessary to develop a new technique using a radioactive nuclide of K. There are two radioactive nuclides as candidates to trace K behavior, 38K and 42K, with half-lives of 7.6 min and 12.4 h, respectively. Because of their short half-lives, neither of the nuclides is commercially available.
38K can be produced by the 38Ar(p, n)38K reaction using a small cyclotron. Although we once employed 38K as a tracer to study K uptake in a rice root (See previous section), only a short experiment, within approximately an hour, was possible because of the extremely short half-life. Therefore, the other radioactive nuclide, 42K, is preferable to trace a longer time of K movement. The device to produce 42K is as follows.
The greatest advantage of using the 42Ar-42K generator is that 42K can be prepared repeatedly in the laboratory. Using the 42K tracer, real-time imaging of the element movement in a plant was performed, and the results are shown in Chap. 4.
3.3 Other Elements
Finally, when considering the elements in plants, it should be noted that naturally grown plants have different concentrations of elements according to the site where they grow. Plants adapted to the nature of the soil have been selected or acquired specific physiological features to survive through the long history of evolution. Sometimes, at high concentrations of heavy elements, plants adapt to live without showing any effect from the poisonous element. For example, some Astragalus sp., a grass that grows in meadows, has adapted to grow at high soil concentrations of Se and accumulates high amounts of Se, sometimes1000 times higher than those in other plants, thereby becoming a poisonous plant. Se toxicity can kill a mouse that is fed wheat grown in soil containing 1 mg of Se per 1 kg. When animals eat this grass, S in the chemical structure of two essential amino acids is replaced with Se, causing serious disease in animals. The deaths of sheep or horses caused by this poisonous plant were described in Marco Polo’s diary in the thirteenth century. This grass favors Se and acquired a new metabolic pathway to escape the poisonous effect of Se. In addition to this example, the profiles of elements in naturally grown plants could provide information about the features of the area and what elements are present in high quantities. Considering the adaptation of plants to areas with different kinds and concentrations of the elements in the soil, the elements in a plant could be described as another DNA, reflecting the environmental history of the growth site.
3.3.1 Production Districts of Onion and Beef
Since plant growth is highly dependent on the elemental concentrations in soil, the elemental profile of a plant is expected to be correlated well with that of the soil. This means that the amount or profile of the elements contained in a plant could be an indicator of where the plant was grown.
Recently, consumers have paid close attention to the quality of agricultural products, especially the production districts, to verify the safety of the products. For the same cultivar, there is no difference in the DNA sequence of plants growing in different districts. However, the same kind of plant must have different elemental profiles when produced in different districts.
To determine the elemental concentration in plant samples, inductively coupled plasma-mass spectrometry (ICP-MS) is now widely performed, which requires acid digestion of the sample. However, some volatile elements, such as I and Cl, are lost during the sample preparation process. To avoid this problem, it is preferable to apply a nondestructive analytical method that does not require acid digestion of the sample. Therefore, neutron activation analysis (NAA) and prompt gamma-ray analysis (PGA) methods were employed to determine the amounts of different elements, including elements that cannot be determined from the method using acid-digested samples. These methods are the best tools for this kind of analysis since they allow nondestructive multielement determination.
Considering the agricultural products from different districts, not only plants but also animals could be candidates to analyze the growth site. For example, cows feed on haylage, fermented local pasture that might contain the specific elemental profile of the site. As an example of using this kind of analysis to search for differences in production district among the agricultural products, two cases are presented below, onion and beef.
Beef samples produced in different districts in Japan, Australia, and the USA were analyzed by NAA and PGA. Freeze-dried samples of chuck, sirloin, fillet, round and other parts were prepared for PGA analysis, and the elements were analyzed in the same way as those in onions. NAA was used to determine Na, Na, Mg, Cl, K, Br, and Sm, and PGA was used to determine H, C, N, and S by γ-ray spectroscopy. With the same data processing used for onions, PCA, it was possible to group Japanese black cattle beef separately from beef from the USA.
However, Holstein beef from Japan and from Australia were not sufficiently grouped by PCA modeling with the elemental data set. It was also found that there was no difference among the parts of the beef, round, sirloin, chuck, and fillet in the grouping of production districts by PCA modeling. These are the first examples of identifying the production site of onions and the provenance of beef through elemental analysis.
3.3.2 Other Elements
Since plants grown in different districts could reflect the mineral composition or concentration of the soil, the author tried to analyze gold (Au) in plants to search for gold mines. The sensitivity of NAA to Au is extremely high, and trace amounts of 198Au can be produced in a reactor by the (n, γ) reaction (Table 3.1) and measured by γ-spectroscopy. This high sensitivity is beyond what people normally estimate; as cited above, when gold accessories or wristwatches are worn during the sample preparation, the gold vapor from the gold materials contaminates the sample and can be detected in the measurement. Many kinds of plants and the corresponding soils were collected at an interval of a certain distance from the gold mine. Then, the plant samples were washed well, and the Au amount in both plants and soils was measured by NAA. The Au amount in the plant increased at a greater distance from the mine than the amount in the soil. Analysis of the plants grown around the mine showed that Callicarpa mollis, beauty-berry, accumulated high amounts of gold and was promising as an indicator for the gold mine; however, the gold amount in the plant was on the order of 10−9 g.
3.4 Summary and Further Discussion
NAA is the only method to determine the absolute amounts of elements in both solid and liquid samples.
The sensitivity of NAA is extremely high, especially the sensitivity to heavy elements, although the sensitivity differs among elements.
It allows multielement analysis at one time, and a time-dependent measurement is needed to determine each element.
The multielement analysis of plants provided much information. Within a plant, element-specific concentrations were found to spread throughout the whole plant tissue. Each element formed a specific gradient or illustrated barriers between the tissues, which were suggested to regulate the movement of the elements. The barrier between roots and aboveground parts is well known and is a common feature of heavy elements. Element barriers were found even in the same root or in the same tissue of the aboveground part, showing imbalance of the element profile. Although the element profile of the elements showed a similar tendency during the developmental stage, changes in the profile were observed between the juvenile and senescent stages.
When there is a change in the element profile, the need for element-specific movement can be estimated, and there is a specific requirement time for each element. Then, why do the elements move? They might respond to growth and environmental conditions, especially light, which could feature plant activity. Then, what about the roots? There were complicated routes for element absorption and movement within a root in the dark, depending on the position. The root tip showed its specific feature for element accumulation and movement. It was interesting to know the Al movement at the root tip. Though the individual tissues showed different concentration profiles of the elements, the multielement movement in a whole plant should always be taken into account because every tissue is connected with each other.
In addition to the element profiles in the plant, the concentration of the elements in a plant reflected the mineral composition or concentration of the soil, and the amount of the elements contained in a plant could be one of the indicators of the site where the plant was grown.
Radioactive nuclides are indispensable tools for tracer work in plants. However, when a suitable RI is not commercially available, the method of production should be considered. In this sense, the production of 28Mg and 42K was presented. Since 28Mg was not previously used for plant research, the physiological function of Mg could be shown in more detail using 28Mg as a tracer. Although further experiments using 28Mg were omitted in this book, some experiments are presented to show that the physiological study of the plant is highly dependent on radioactive tracers.
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