Visualization of ¹⁴C-labeled Gas Fixation in a Plant

Abstract

We targeted not only the elements we can supply to the nutrient solution but also carbon dioxide gas to visualize the fixation process and the movement of assimilated carbon in a plant. This is another highlight of our study using real-time RI imaging systems (RRIS). The interesting result was that the route of assimilated carbon was different depending on where the fixation took place. In Arabidopsis, most of the metabolites after photosynthesis were transferred to the tip of the main internode and roots when ¹⁴CO₂ gas was fixed and photosynthates were produced at rosette leaves, whereas most of the metabolites moved to the tip of the branch internode and hardly moved down to the roots when ¹⁴CO₂ gas was supplied to the aboveground parts of the plant other than rosette leaves. Interestingly, it was possible to visualize and trace which tissue performed the fixation of ¹⁴CO₂ gas, i.e., carbon could be traced from the fixation site in tissue to tissue formation. However, especially in the case of ¹⁴C imaging, image analysis should be carefully performed because of the self-absorption of the β-rays in tissue. To image ¹⁴CO₂ gas fixation in larger samples, approximately 50 cm in height, a plastic scintillator was introduced, and the assimilation process of the gas was visualized for rice and maize.

1 Performance of RRIS for ¹⁴C imaging

To investigate the performance of RRIS for ¹⁴C imaging, standard sources of ¹⁴C were prepared by spotting ¹⁴C-labeled sucrose on a polystyrene sheet from 1.8 Bq (0.5 Bq/mm²) to 14,800 Bq (4500 Bq/mm²). The spots on the sheet were measured at integration times of 3–60 min. Then, the minimum limit and upper limit of determination were evaluated in the RRIS (Real-time RI Imaging System) as well as the IP using ¹⁴C standard spots, in the same way described for the development of RRIS (Chap. 4). The minimum limit of determination in the RRIS was lower than that in the IP when the accumulation time was relatively short. The linearity between the activity of ¹⁴C and the signal intensity obtained by the RRIS was evaluated using the standards. There was good linearity between the activity count of ¹⁴C and the signal intensity obtained by both the RRIS and an IP (data not shown). The minimum and upper limits as well as the dynamic range of determination measured for both the RRIS and the IP method are listed in Table 5.1.

Table 5.1 Properties of the RRIS and IP for ¹⁴C imaging

The next step was to compare the image of a plant sample, supplied with ¹⁴C-labeled CO₂ gas, taken by RRIS with that taken by an IP. The sample used for imaging was a seedling of Arabidopsis thaliana (Col-0) grown in culture solution for 38 days with flowers and developed pods. ¹⁴C-labeled CO₂ was produced by mixing ¹⁴C-labeled sodium hydrogen carbonate (2–5 MBq) with lactic acid in a 1.5 mL vial with a septum cap equipped with a syringe needle. The plant was placed in a polypropylene bag with the mouth sealed with clay in which a tube was connected. Then, 5 MBq of ¹⁴CO₂ gas, generated in the vial, was introduced to the bag through a syringe for 24 h (Fig. 5.1). To fix the sample to an FOS where Cs(Tl)I scintillator was deposited, a silicone gum sheet was used, and the FOS was covered with polyphenylene sulfide film to prevent contamination with ¹⁴C.

Fig. 5.1

Imaging of the ¹⁴CO₂ gas fixation by the RRIS [1]. (a) ¹⁴CO₂ gas was produced by mixing ¹⁴C-labeled sodium hydrogen carbonate (2–5 MBq) with lactic acid and supplied to a polytropylene bag covering the plant sample. (b) Outline of the RRIS. A plant was prepared on the FOS in a dark box, and the image was monitored by a computer

First, images of the plant supplied with ¹⁴CO₂ gas were taken by both the RRIS and an IP, and the images were compared. The exposure times were equal for the RRIS and IP. The images differed in quality, such as resolution, data depth, and contrast, particularly at sites with complex morphology such as a flower (Fig. 5.2). The main reason for the differences in image quality between the RRIS and IP was derived from the resolution. The resolution of the images captured by the RRIS was approximately 70 dpi, while that of the images captured by the IP was 500 dpi, which was more than seven times higher than that of the RRIS. In addition, the fixation method of the plant also caused differences in the quality of the images. The plant was fixed closely on the IP in a cassette for exposure, whereas in the case of RRIS, the plant was fixed relatively loosely to the FOSs using tape to allow growth during imaging. In total, the quality of the IP image was higher than that of the RRIS image, particularly at sites of complex morphology such as a flower.

Fig. 5.2

¹⁴C images of an Arabidopsis acquired by an IP and RRIS [1]. Five MBq of ¹⁴CO₂ gas was supplied to the plant for 24 h after 38 days of germination. A and B are the images by an IP and RRIS with resolution of approximately 500 and 70 dpi, respectively. One of the reasons for the high resolution of the IP image was that the plant could be tightly fixed on the IP in a cassette for exposure

The β-ray energy emitted from ¹⁴C is low; hence, the effect of self-absorption cannot be ignored. Since the main factor in self-absorption is the thickness of the sample, the effect of self-absorption on the efficiency of signal detection by the RRIS was investigated before the analysis of the image. The radioactivity of the different tissues was measured, and the β-ray counts were compared with the intensity of the images. After 24 h of ¹⁴CO₂ supply to the plant, plant tissues such as the leaves, siliques, stems, and flowers were separated, and the radioactivity of ¹⁴C in each tissue was counted for 2 min by a liquid scintillation counter (ALOKA LSC-6100). For the quenching effect in the counting, a known activity of ¹⁴C was added to each sample as an internal standard, and the counts were corrected.

Figure 5.3 shows the relationship between the signal intensity acquired by the RRIS and the actual radioactivity measured. The result shown in the figure indicates that the ¹⁴C signal intensity found in some tissues cannot be linear. Thick tissues, such as mature leaves, flowers, siliques, and stems, showed no correlation between ¹⁴C activity and the intensity of the image. These results indicated that it was difficult to quantify the ¹⁴C activity in a thick sample using the autoradiographic technique in both imaging methods, IP and RRIS, because of self-absorption. On the other hand, there was potential for quantifying the ¹⁴C activity in young and mature leaves using the RRIS. When we compared the intensity of the RRIS with the PSL value of the IP, linearity was maintained in most of the tissues: mature leaves, flowers, siliques, and stems. The results showed that although self-absorption was confirmed, the images taken by RRIS and IP seemed to show the same images of the ¹⁴C profile, indicating that the IP image could compensate for the RRIS image.

Fig. 5.3

Calibration of the ¹⁴C activity in the tissue images of Arabidopsis acquired by RRIS [1]. (a) ¹⁴C intensity versus radioactivity counts; (b) Magnification of the lower counts in a; c: relation between PSL value obtained by an IP and the intensity of the image obtained by RRIS

As an example of ¹⁴C imaging other than ¹⁴CO₂ gas fixation, ¹⁴C images of Arabidopsis are briefly introduced. In this case, ¹⁴C-labeled sucrose, an initial metabolic product of photosynthesis, was supplied from the leaves as a kind of foliar fertilization. Figure 5.4 shows successive RRIS images of ¹⁴C in a 30-day-old Arabidopsis plant supplied with ¹⁴C-labeled sucrose from the rosette of the plant (1.85 MBq/500 μL) during the measurement. Although self-absorption had to be taken into account, the tendency of ¹⁴C accumulation over time could be observed, since when the imaging area in the tissue was fixed, the change in the ¹⁴C signal in the area indicated the relative change in the sucrose amount. Then, IP images of the whole plant at this stage and further aged samples were taken. Figure 5.5 is an IP image of the Arabidopsis plant at different stages of growth after 30 and 60 days of culture. Both figures showed the tendency of ¹⁴C accumulation, such as that the sucrose assimilation products accumulated in maturing tissue and in joint parts. Since it was difficult to discern the detailed distribution of the silique, the pods were harvested, and the distribution of ¹⁴C was investigated. The young pods, after 2–3 days of flowering, showed uniform accumulation of ¹⁴C in the longitudinal direction by IP. However, a higher accumulation of ¹⁴C was observed by IP at a higher position in the pod after 7 days of flowering (data not shown).

Fig. 5.4

An example of ¹⁴C-sucrose images of Arabidopsis (1) [2]. 1.85 MBq/500 μL of ¹⁴C-sucrose solution was applied to a rosette leaf of Arabidopsis after 30 days of germination. Successive ¹⁴C images of the plant were acquired by RRIS. The integration time was 15 min every hour. The light was turned off during the image acquisition

Fig. 5.5

Example of ¹⁴C-sucrose images of Arabidopsis (2) [2]. 1.85 MBq/500 μL of ¹⁴C-sucrose solution was supplied to rosette leaves of Arabidopsis after 30 (A) and 60 days (B) of germination

2 Imaging the ¹⁴CO₂ Gas fixation

After ¹⁴CO₂ gas is fixed by photosynthesis, the metabolites move to other tissues by phloem flow. This means that when the tissue to apply ¹⁴CO₂ gas for fixation is selected and the movement of photosynthate is traced, the sink-source relationship between the tissues can be analyzed. To study this relationship, ¹⁴CO₂ gas fixation imaging was performed.

2.1 Imaging of 43-Day-Old Plant

To visualize the flow of photosynthate, ¹⁴CO₂ gas was supplied to the rosette leaves of Arabidopsis after 43 days of growth. The rosette leaves were covered with polyethylene bags 1.2 μm in thickness. ¹⁴CO₂ gas was introduced to the bag for 24 h under light irradiation in a phytotron. Then, the upward movement of ¹⁴C-labeled photosynthate was visualized by RRIS.

Figure 5.6 shows the change in the signal intensity during 24 h of ¹⁴CO₂ gas supply. The amount of ¹⁴C-labeled metabolite in the main stem was very low, and hardly any signal appeared in the upper part of the main stem. In contrast, at the tip of the lateral stem, the amount of ¹⁴C-labeled metabolite continued to accumulate, suggesting that the rosette leaves were the source organs supplying photosynthates primarily to the lateral stems. This result was surprising: despite the sink tissue present in the main stem, such as developed flowers and siliques, the main part of the sink was in the secondary stem. However, in these images, the phloem flow along the sieve tube connecting the basal shoot and the tip region around the main stem was not observed.

Fig. 5.6

Successive images of ¹⁴C photosynthates after fixation of ¹⁴CO₂ gas in Arabidopsis [3]. (Photograph of the plant and successive images of the ¹⁴C-labeled metabolite movement. ¹⁴CO₂ was supplied to rosette leaves of 43-day-old plants, and the upward movement of ¹⁴C-phtosynthates was visualized by RRIS. The accumulation time of the imaging was set as 15 min. Scale bar: 2 cm)

The plant sample tested was well developed, with mature siliques in the main stem and the lateral stem developing younger tissue than the main stem. Since the primary photosynthates preferentially accumulated at the lateral stem, it was hypothesized that the rosette leaves are the source organs when the stem is young, but after flowering, the necessary carbon source in the stem is supplied by photosynthates produced in the siliques, stems, and cauline leaves. To test this hypothesis, the same experiment was performed using a younger plant.

2.2 Younger Sample Imaging

To test the hypothesis mentioned above, the same experiment was performed using a younger plant, 30 days after germination. Accordingly, the movement of the photosynthates was different from that in the 43-day-old plants. When ¹⁴CO₂ gas was introduced to the rosette leaves, the photosynthate produced in the rosette leaves was preferentially transferred to the main stem tip (Fig. 5.7). The direction of phloem flow from the rosette leaves towards each stem was changed in the basal shoot region and was influenced by the age of the stem.

Fig. 5.7

¹⁴C-photosynthate movement in Arabidopsis when ¹⁴CO₂ gas was supplied to different tissues [3]. Successive images of ¹⁴C photosynthate are shown after/while supplying ¹⁴CO₂ to rosette leaves, whole plant or inflorescence. Serial images were acquired by RRIS. ¹⁴CO₂ gas was supplied as pulses (A) or continuously (B). The imaging time for each frame was 15 min. Scale bar: 20 mm

In contrast, there was no difference in the amounts of ¹⁴C detected at the tips of the main and lateral stems when ¹⁴CO₂ gas was supplied to the whole shoots. However, when ¹⁴CO₂ gas was supplied to the aboveground part of the plant, except for the rosette leaves, the ¹⁴C intensity was higher at the lateral stem than at the main stem, an opposite movement from that supplied from the rosette (Fig. 5.7). This observation suggested the existence of a source organ other than the rosette leaves that supplied photosynthates to the lateral stems.

To determine whether the potential source organ to the lateral stem was the inflorescence, ¹⁴CO₂ gas was supplied in a pulse of 1 h to the inflorescence only. The result was that the ¹⁴C signal intensity in the lateral stem tip was much higher than that in the main stem tip, and this high intensity of the ¹⁴C signal in the lateral tips continued up to 24 h, suggesting that the ¹⁴C metabolites produced in the main stem are continuously transported towards the lateral stems. In the case of the photosynthates produced in the rosette leaves, preferential transfer to the main stem tip was observed after a pulse supply of ¹⁴CO₂ gas, similar to that resulting from the continuous supply.

2.3 Photosynthate Transfer Route by Image Analysis

To analyze the photosynthate transfer route, the changes in the ¹⁴C signal in the main stem tip and lateral stem tip were plotted. During the continuous ¹⁴CO₂ gas supply, the orientation of the photosynthate movement showed that there was a difference in the route as well as in the photosynthate accumulating tissue according to the gas fixation site, the rosette or the aboveground part without the rosette.

As shown in Fig. 5.8, the preferential transfer of photosynthate to the main stem tip was observed when ¹⁴CO₂ gas was supplied from the rosette for 24 h. However, the ¹⁴C accumulation at the lateral stem tip linearly increased during 24 h of continuous ¹⁴CO₂ supply from the inflorescences, whereas the rate of increase at the main stem tip was lower than that of the lateral stem after approximately 12 h. In the case of ¹⁴CO₂ gas supplied from the whole plant, the increase in the ¹⁴C signal was similar between the main stem tip and lateral stem tip.

Fig. 5.8

Preferential route of the ¹⁴C-photosynthate movement in the main or lateral stem in Arabidopsis [3]. ¹⁴CO₂ gas was supplied under both pulsed (A) and continuous (B) conditions to rosette leaves, whole plants or inflorescences. ROIs were set as the main (blue) and lateral (red) stem tips for A and B. Two ROIs were set on the cauline leaves (C1 and C2) for C, and three ROIs were set for the lateral stem. D: Two cauline leaves and the lateral stem tip. The preferential transfer of ¹⁴C-photosynthate to the main stem tip was shown when ¹⁴CO₂ gas was supplied to the rosette leaves, whereas the preferential move to branch tip was shown when the gas was supplied inflorescence

When ¹⁴CO₂ gas was supplied for 1 h as a pulse, the ¹⁴C signal increase was similar to that when ¹⁴CO₂ gas was supplied continuously, except for the supply from the inflorescences. The photosynthate supply clearly moved towards the lateral stem tip, where the accumulated amount of photosynthates was more than two times higher than that in the main stem tip. The high ¹⁴C signal intensity kept in the lateral stems by the pulse supply and the steady increase during the continuous supply from the inflorescences suggested that ¹⁴C-labeled photosynthates generated in the main stem, other than the rosette leaves, are continuously transported to the lateral stems. Although the route of photosynthate movement was different depending on the production site of ¹⁴CO₂ gas fixation, in total, there was no significant difference in the accumulation amount among plant tissues in a younger plant, as shown when the gas was supplied to the whole plant.

To analyze the route of photosynthates after production in the aboveground part of the plant except for the rosette leaves in more detail, cauline leaves and tips in the lateral stem were plotted. The ¹⁴C signal in the cauline leaves of the lateral stems was decreased, although the total signal intensity of ¹⁴C in the cauline leaves and lateral stem tips was maintained (Fig. 5.8c). This observation might indicate that cauline leaves also act as a carbon source for lateral stem tips, although the amount of ¹⁴C-photosynthate in the lateral stem tip area, including the tips and cauline leaves, was kept constant.

The next analysis was to determine which tissue producing photosynthate provided a carbon source to the silique. The region of interest (ROI) was set as shown in Fig. 5.9, and siliques were numbered Si1 to Si4, from the lower to the upper part of the plant. When ¹⁴CO₂ gas was supplied to the rosette leaves as a pulse for 1 h, the ¹⁴C amounts in all the siliques increased. The ¹⁴C signal also increased according to the position of the silique from low to high, i.e. the younger siliques accumulated higher amounts. This result indicated that the C source in the siliques was derived from rosette leaves. On the other hand, when ¹⁴CO₂ gas was supplied to the inflorescence as a pulse, the ¹⁴C amount decreased with time in all the siliques, and there was hardly any difference in amount among the siliques. This result, that the ¹⁴C amount in the siliques was slightly decreased when the photosynthate from rosette leaves contained ¹⁴C, suggested that there was hardly any movement of the photosynthate from the siliques to the other tissues and that the loss of ¹⁴C at the silique might be due to respiration. A pulse supply of ¹⁴CO₂ gas to the whole plant showed that even when photosynthate was supplied from the rosette leaves, the amount of ¹⁴C decreased in older siliques. With a continuous supply of ¹⁴CO₂ gas to the inflorescences, the ¹⁴C amount increased in all the siliques measured. This result indicated that photosynthate from the main stem, including the cauline leaves or siliques, could be supplied to younger siliques.

Fig. 5.9

Preferential route of the ¹⁴C-photosynthate movement to silique in Arabidopsis [4]. ¹⁴CO₂ gas was supplied as a pulse (1 h) to rosette leaves, whole plants or shoots (inflorescences). In the case of shoots, ¹⁴CO₂ gas was continuously supplied. ROIs were chronologically set at siliquis in the main stem from Si1 to Si4. There was a preference for the ¹⁴C-photosynthate transfer route when supplied from rosettes

The method of photosynthate transfer could be discussed in comparison with rape seed plants, whose rosette leaves fall down after flowering with increasing photosynthesis activities of cauline leaves. Although the rosette leaves of Arabidopsis do not fall down after flowering, the photosynthesis activity gradually decreases after maximum expansion of rosette leaf development, and even after senescence of the rosette leaves, the emergence of siliques continues for a while suggesting the important role of photosynthate produced in the cauline leaves or siliques in Arabidopsis, similar to that in rape seed plants.

2.4 Whole Plant Image of Photosynthate by an IP

To obtain ¹⁴C images of the whole plant, including roots, the plants were harvested and placed on an IP after imaging by RRIS. Figure 5.10 shows the autoradiograph of the plant after ¹⁴CO₂ gas was supplied for 1 h. As shown in the figure, the profile of the ¹⁴C signal in the aboveground part of the plant showed the same profile as that obtained by RRIS. The root image acquired by the IP, which was not obtained in the RRIS, also indicated the difference in the ¹⁴C signal with respect to the difference in the ¹⁴CO₂ gas supply site, from rosette leaves or inflorescences. The red and blue arrows in Fig. 5.10 indicate the root part in the picture and in the radiograph, respectively. It was noted that no ¹⁴C image of the roots was observed when the ¹⁴CO₂ gas was supplied only to the aboveground part of the plant other than the rosette leaves, as shown by the blue arrow in the root autograph. However, when ¹⁴CO₂ gas was supplied to the rosette leaves, a higher amount of ¹⁴C was shown in the root than when the gas was supplied to the whole plant. The results indicated that rosettes are the primary carbon source for roots. Thus, the photosynthate fixed in the inflorescence seemed to be transported, metabolized, and accumulated only within the inflorescence itself.

Fig. 5.10

Preferential route of the ¹⁴C-labeled metabolite transfer to the root in Arabidopsis [3]. The ¹⁴C-labeled metabolite distribution images by IP were acquired after imaging by RRIS, where ¹⁴CO₂ gas was supplied as pulses (1 h) to rosette, whole plant or inflorescences. In each ¹⁴CO₂ gas supply method, the grayscale image by an IP (left) and photograph (right) are shown. Scale bar: 20 mm. The arrows indicate the roots. When ¹⁴CO₂ gas was supplied to the rosette, ¹⁴C-labeled metabolites were preferentially transferred to the root, whereas ¹⁴C-labeled metabolites were not observed in the root (blue arrow) when the gas was supplied from inflorescences

3 Photosynthate Movement in Soybean Plants When ¹⁴CO₂ Was Supplied

Using the RRIS, the carbon dioxide gas fixation process, as well as the orientation of the photosynthate in Arabidopsis, was visualized by applying the developed method of ¹⁴C-labeled gas supply. The next experiment was to visualize photosynthate movement by employing soybean plants. Since soybean is much larger than Arabidopsis, it was easier to supply ¹⁴CO₂ gas to specific tissues and to study how the photosynthate moved from one tissue to other tissues. For this purpose, soybean plants (Glycine max. cv, Enrei) were grown in culture solution under 16 h L/8 h D, light/dark conditions in a phytotron. First, ¹⁴CO₂ gas was supplied as a pulse for 30 min to the whole plant 40 days after germination. The ¹⁴C signal was distributed uniformly among the developed leaves, suggesting that a similar amount of photosynthate was produced by 30 min of photosynthesis in each leaf. Figure 5.11 is the image of the plant acquired by an IP. There was no ¹⁴C signal observed in the roots, suggesting that most of the photosynthate produced in the leaves remained at the site where photosynthesis occurred; therefore, it was not transferred to the roots.

Fig. 5.11

¹⁴C-photosynthate distribution in a soybean plant [5]. ¹⁴CO₂ gas was prepared and supplied to a whole soybean plant for 30 min after 40 days of germination. Then, the plant was exposed to an IP (imaging plate) for 30 min. a: Picture of the plant; b: Radiograph of the image in the IP. The ¹⁴C photosynthate was uniformly distributed throughout the expanded leaves; however, the ¹⁴C signal was not observed in the roots

Since the amount of photosynthate is similar among the expanded trifoliate leaves, the first trifoliate leaves of 20-day-old plants were chosen, and ¹⁴CO₂ gas was supplied only to these leaves for 30 min. Then, the photosynthate movement to other tissues was analyzed from the decrease in the ¹⁴C signal from these leaves. Figure 5.12 shows the change in the ¹⁴C signal during 8 h after the gas was supplied. It was shown that the photosynthate produced in the original trifoliate leaves gradually decreased with time and plateaued after approximately 4 h. The decreasing curve of the relative intensity in the treated leaves showed that within 8 h, most of the transfer movement of the photosynthate in the phloem seemed to cease.

Fig. 5.12

¹⁴C-photosynthate transfer manner in the leaves of a soybean plant [5]. ¹⁴CO₂ gas was prepared and supplied to the trifoliate leaves for 30 min; then, real-time imaging (RRIS) was performed. The radioactivity intensity of the trifoliate leaves was monitored from the intensity of the sequential images taken by RRIS. a: Schematic illustration of the ¹⁴CO₂ gas supply to the trifoliate leaves; b Relative radioactivity intensity curve of the trifoliate leaves with time

Using plants at the same growth stage, ¹⁴CO₂ gas was supplied to the selected tissue for 30 min, and the movement of the ¹⁴C image was observed by the RRIS until 8 h after the treatment. From the trifoliate leaves, the metabolites were transferred preferentially to the youngest tissues and accumulated there. However, when ¹⁴CO₂ gas was supplied only to the youngest leaves, including the meristem, the photosynthate produced in this tissue remained at this site and hardly moved to the other tissues (Fig. 5.13). This preferential movement of photosynthate from trifoliate leaves to the youngest tissue was shown, in different to the position where the trifoliate leaves developed (data not shown). There are many kinds of tissue at different developmental stages in one plant; therefore, it was suggested that premier importance was placed on promoting the growth of the youngest tissue by transferring photosynthate, which was the source to produce the structure of the plant.

Fig. 5.13

¹⁴C-photosynthate movement in a soybean plant [5]. The ¹⁴CO₂ gas was supplied to the trifoliate leaves of the plant after 20 days of germination for 30 min, and the movement of the photosynthate was monitored by RRIS. Sequential images are shown after ¹⁴CO₂ gas was supplied. ¹⁴CO₂ gas was supplied to the first (a, b) and third (c) trifoliate leaves. The accumulation time for each imaging frame was set to 15 min. The signal intensity is assigned using pseudocolor (white represents the highest intensity)

The movement of photosynthate from trifoliate leaves to the younger tissue could be observed at an earlier time when ¹⁴CO₂ gas was supplied for 30 min. However, the movement of photosynthate from the aboveground part to the root was much slower than that to the younger tissue, as shown above. The ¹⁴C signal was not shown in roots by a 30-minute supply of ¹⁴CO₂ gas (Fig. 5.11). Since it took a longer time to move and accumulate the photosynthate in roots, Fig. 5.14 shows an image of the whole plant taken by an IP after 24 h of continuous supply of ¹⁴CO₂ gas. Since the leaves supplied with ¹⁴CO₂ gas emit higher radiation than the other tissues due to the remaining ¹⁴C-labeled compounds, the background level of the image was increased when the whole plant was exposed to the same IP. Therefore, the treated leaves were disconnected from the plant, and the images of the cut off leaves and the rest of the plant were taken by different IPs. The original growing site of these trifoliate leaves is indicated by an arrow in the picture. The IP images showed that when ¹⁴CO₂ gas was supplied to the expanded trifoliate leaves, most of the photosynthate was preferentially moved to the youngest tissue, and only a small amount of the photosynthate was moved to the roots or to other tissues, regardless of the position of the trifoliate leaves. The route preference was the same as that shown by RRIS. However, when ¹⁴CO₂ gas was supplied to the youngest tissue, all of the photosynthate produced at this site remained at the site, and movement to other tissues, including roots, was not observed. Photosynthate transfer from the youngest tissue was hardly detected.

Fig. 5.14

Photosynthate profile in a soybean plant [5]. After 24 h of ¹⁴CO₂ gas supply to the trifoliate leaves and the youngest tissue, the plant was exposed to an IP. The upper figures in a, b, and c are pictures of the plant corresponding to each IP image below (d, e, and f). ¹⁴CO₂ gas was supplied to the expanded trifoliate leaves (a, b, d, and e) and youngest tissue (c and f). To reduce the background noise of the IP images, the treated leaves were disconnected from the plant and exposed to different IPs (d–f), and the positions where the leaves were connected are shown by arrows

Another question was from what source photosynthate is supplied to the pods. To determine the orientation of the photosynthate movement from leaves to developing pods, plants after 55 days of germination were selected. Then, ¹⁴CO₂ gas was supplied for 30 min to the trifoliate leaves grown at the site close to the pod. The accumulation images obtained from the RRIS and the IP are shown in Fig. 5.15. In the case of the pods, carbon fixed from the ¹⁴CO₂ gas in the matured trifoliate leaves was preferentially transferred to the closest pod, and the accumulated amount was still increasing even after 8 h of ¹⁴CO₂ gas supply. Although several pods developed on the plant, the accumulation of photosynthate was not observed in those developed at higher positions than the treated trifoliate leaves, although they were younger than the closest pod. However, a small amount of photosynthate accumulation was shown in the pods grown below the tissue where the ¹⁴CO₂ gas was supplied, in leaves as well as in roots.

Fig. 5.15

Photosynthate movement from leaves to a pod of a soybean plant [5]. ¹⁴CO₂ gas was supplied to the expanded trifoliate leaves of a soybean plant after 55 days of germination, and images of the ¹⁴C-photosynthate movement with time were acquired by RRIS. After sequential images were taken, the plant was exposed to an IP. To decrease the background noise, the treated tissues were disconnected and exposed to different IPs. The disconnected site is shown by arrows (b and c)

It was interesting to note that in the growth stage of pod development, the main carbon source, photosynthate, produced in expanded trifoliate leaves was not supplied primarily to the youngest tissues with meristems but to the closest pod. It was suggested that most photosynthate was not transferred for a long distance when pods were developing.

4 Downward Movement of Photosynthate to Roots

When ¹⁴CO₂ gas was supplied to the rosette leaves, ¹⁴C-labeled photosynthates were found to be transported to the root (Fig. 5.10). To visualize the sink tissues within the roots, imaging of the downward movement of ¹⁴C-labeled photosynthates was performed after ¹⁴CO₂ gas was supplied only to the aboveground part of 14-day-old Arabidopsis seedlings, which are juvenile plants before flowering. Plants were grown in a 0.4% gellan gum and full-nutrient culture solution using a dish prepared with several vent holes. Plant roots were then placed on gellan gum on a polyethylene sheet (thickness: 10 μm) for imaging. The plants on the FOS were placed vertically, and images were acquired for 15 min at intervals of 1 h. The plants were irradiated by light-emitting diode light (100 μmol/m²/s) for 45 min between the image acquisition periods.

The RRIS images of ¹⁴C in roots visualized the arrival of ¹⁴C-labeled photosynthates at the root tip areas, including developing lateral roots, as early as 3 h after ¹⁴CO₂ was supplied. Thereafter, the accumulation of ¹⁴C-labeled photosynthates in the root tips increased for 12 h. Then, the accumulation of ¹⁴C-labeled photosynthates at the main root tip was observed under micro-RRIS. The root elongation rate in 2-week-old Arabidopsis plants was 5.1 ± 0.4 (SD) mm over 12 h. Therefore, the root tip segments shown in Fig. 5.16 under micro-RRIS were inferred to be newly developed tissues constructed with the ¹⁴C-labeled photosynthate and thus were the sink of the photosynthates.

Fig. 5.16

Visualization of the downward movement of ¹⁴C-labeled metabolites from leaves to roots [3]. (a) Schematic illustration of the sample covered with a polyethylene bag in preparation for the ¹⁴CO₂ gas supply. (b) Serial images of the ¹⁴C-labeled metabolite movement taken by macro-RRIS. (c) After taking successive images by macro-RRIS, we obtained micro-RRIS images of ¹⁴C-labeled metabolites in the root (root tip, maturation area and lateral root). (1): Grayscale image; (2): pseudocolor image; (3): distribution profile of ¹⁴C-labeled metabolites in a root. Scale bar = 100 mm. The 14C signal was high at approximately 200 and 800 μm to the main root tip

The result that ¹⁴ C-labeled photosynthates preferentially accumulated in the tip area is in agreement with the findings in Brassica napus seedlings, in which the photosynthate produced in leaves was translocated to the meristematic root regions (Dennis et al. 2010). The phloem unloading activity around the root tip of Arabidopsis has been previously visualized using carboxyfluorescein (CF) dye applied to a single cotyledon (Oparka et al. 1994). Based on sequential CF images taken by confocal laser scanning microscopy, the protophloem located 200–700 μm behind the root tip was suggested by the authors to function in phloem unloading and subsequent lateral transport. Consistent with this suggestion, a high ¹⁴C signal intensity was detected approximately 200 and 800 μm distal to the main root tip using the micro-RRIS (Fig. 5.16). This region, now suggested to be the major sink tissue in roots, can be considered the part extending from the middle part of the apical meristem to the start of the elongation zone.

5 ¹⁴CO₂ Fixation in a Large-Scale Plant

The RRIS was developed using a fiber optic plate (FOS), on which a CsI (Tl) scintillator was deposited to convert radiation into light. However, the scintillator size was fixed at 10 × 10 cm, which was too small to observe the entire plant. Therefore, a plastic scintillator, Lumineard-C, was used to image the ¹⁴C-labeled photosynthate movement in a plant (see Chap. 4, Sect. 4.2.6). The performance of the plastic scintillator was studied, and it was demonstrated that a plastic scintillator was applicable for ¹⁴C imaging in a plant. To study the long-distance transportation of photosynthate, this system can visualize ¹⁴C-labeled photosynthate movement between shoots and roots when the plant length is long.

A 40-day-old rice seedling (Oryza sativa L. cv. Nipponbare) and a 70-day-old maize seedling (Zea mays L.), a commercial hybrid sweet corn, were employed to analyze photosynthate movement in plants. They were grown in culture solution under 16 h L/8 h D, light/dark conditions. The aboveground heights of the rice and maize plants were approximately 550 mm and 400 mm, respectively. ¹⁴CO₂ gas was produced by mixing ¹⁴C-labeled sodium hydrogen carbonate and lactic acid in a 1.5 mL vial with a septum cap. Each plant was sealed with a polyethylene bag. Then, the vial and bag were connected using a tube to introduce ¹⁴CO₂ gas into the bag. Doses of 4 MBq and 8 MBq were applied to rice and maize seedlings for 90 and 120 min, respectively. After the ¹⁴CO₂ gas was supplied, the plant was removed from the bag, fixed to Lumineard-C (170 × 750 mm) covered with an Al sheet (2 μm in thickness), and placed in a large dark box. In the box, light was kept off for 15 min after a 15 min light period, and imaging was performed during the dark period. The imaging was continued until 24 h after the treatment.

Since ¹⁴CO₂ gas was supplied for a limited time, the imaging showed how the ¹⁴C metabolites moved after fixation. As expected, the amount of ¹⁴C continuously decreased in both rice and maize leaves (Fig. 5.17). The pattern of decrease in the rice plant showed that the photosynthetic ability per unit area and the decreasing speed of photosynthate were approximately the same among the leaves. In the case of maize, older leaves showed a more rapid decrease in ¹⁴C-labeled photosynthate, which seemed to be caused by the translocation of metabolites from leaves to other tissues, including roots, as well as loss from the tissue as ¹⁴CO₂ gas by respiration. Since the photosynthate flow changes with the developmental stage, this result does not provide a reason to discuss the differences in the pattern of decrease in the photosynthate between the plants. However, the similar decrease in photosynthate among the leaves in the rice plant suggested that the rice plant itself was at a younger developmental stage than the maize plant.

Fig. 5.17

Large-scale imaging of the ¹⁴C-photosynthate movement in a plant [6]. (Photograph and sequential images of ¹⁴C photosynthate in (a) 40-day-old rice and (b) 70-day-old maize using a Lumineard-C scintillator (170 × 750 mm). Scale bar: 100 mm. Time course of ¹⁴C movement in (c) rice and (d) maize. The number of ROIs was set from new to old leaves)

6 Summary and Further Discussion

In the study on the performance of the RRIS imaging system, it was found to be possible to trace the movement of ¹⁴C in plant tissue. This means that it was possible to trace the process of photosynthesis, carbon fixation, and photosynthate movement after ¹⁴CO₂ gas was fixed in plant tissue. However, the imaging result had to be carefully analyzed because the effect of self-absorption of the β-rays emitted from ¹⁴C could not be ignored. Considering all these conditions, the findings were as follows.

Visualizing the route of photosynthate movement in Arabidopsis after the assimilation of ¹⁴CO₂ gas in plant tissue revealed a new finding about the flow. The images showed that the transfer route of the metabolites was dependent on the original tissue where the photosynthate was produced. Since the photosynthate was moving via the phloem flow, it was also possible to trace the phloem flow by tracing the signal of ¹⁴C. In addition to how the photosynthate was transferred, it was possible to analyze the phloem partition site and timing.

Notably, these source-sink relationships changed over time with the development of each tissue in the plant. In the case of Arabidopsis, it was shown, as we expected, that rosette leaves were the source organs when the stem was young, but after flowering, the necessary carbon source in the stem was supplied by photosynthates produced in the siliques, stems, and cauline leaves. This means that the role of the expanded leaves in supporting the other tissue changes with the development of the whole tissue of the plant.

The result presented above is one of the examples of analyzing photosynthate movement, but there is also much information in successive images; therefore, by setting a suitable ROI (region of interest) in the image, it was possible to analyze the movement of the photosynthate in more detail. It was amazing for us to be able to trace the photosynthate and define which tissue was created in the meristems from the fixed carbon visualized. However, to further analyze the newly created tissue and the dynamics of phloem unloading of photosynthates, micro-RRIS needs to be improved to supply the labeled gas under light conditions, especially for real-time imaging.

In the case of a soybean plant, high accumulation of photosynthate in the youngest tissue was visualized, similar to that of the other plants. When the photosynthate flow to the pod was visualized, although the amount of photosynthate production was at the same level among the leaves, the assimilated carbon was preferentially transferred from the trifoliate leaves to the closest pod. It is known that the pod is producing the optimal conditions for the growth of seeds, such as a high concentration of CO₂ gas and high photosynthesis activity within the pod. Though the self-absorption of the pod is high, with further development of the devices for the imaging system, the visualization of the CO₂ gas in a pod might be possible.

To visualize a larger sample, a plastic scintillator, Lumineard, was employed, and it was possible to visualize photosynthate movement in the leaves. As described earlier, although the self-absorption of ¹⁴C, especially in the internode, is high, when the analyzed tissue was carefully chosen and the change in ¹⁴C was carefully taken into account, it was possible to trace the photosynthate movement within the plant, especially plants growing in soil. In summary, it was shown that the detection of a gaseous radionuclide in macro- and micro-RRIS could drastically enhance the versatility of RRIS.