Ol

Fig. 5 SAR study of the leaf-closing factor of Albizzia plants

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Stained by F1TC-labeled natural-type probe 15 Stained by FITC-labeled enantiorner-type probe 1 & Fig. 6 Enantiodifferential fluorescence staining of pulvinus

Stained by F1TC-labeled natural-type probe 15 Stained by FITC-labeled enantiorner-type probe 1 & Fig. 6 Enantiodifferential fluorescence staining of pulvinus

Fig. 7 Enantiodifferential fluorescence staining of pulvinus, stem and root

specific binding, which was affected by the natural stereochemistry. In addition, the strong fluorescence observed in the xylem for both enantiomers was attributed to nonspecific binding of the probes.

Moreover, no other part of A. saman bound probe 15 stereospecifically (Fig. 7) (Nakamura et al. 2008). Thus, the actual target cell for the leaf-closing factor was confirmed to be the motor cell. These results strongly suggested the involvement of some specific target protein in the motor cell.

3.3.2 Photoaffinity Labeling of the Target Protein for the Leaf-Closing Factor

We designed and synthesized photoaffinity probe 17 with a benzophenone and a biotinyl group in the sugar moiety (Nakamura et al. 2008) . An enantiodifferential photoaffinity labeling experiment was carried out using 17 and 18 against protoplasts of motor cells (Nakamura et al. 2008) that were prepared from A. saman leaves according to Satter's method (Fig. 8) (Gorton and Satter 1984) . Protoplasts were prepared from the ca. 200 leaflet pulvini collected, and photocrosslinking was carried out on the cell surface using probe 17 or 18 . After treatment with streptavidin-FITC conjugate, labeled protoplasts with the biologically active probe 17 gave green fluorescence due to fluorescein on the plasma membrane of protoplasts (Fig. 9). This result strongly suggested that the target protein that recognizes the stereochemistry of the aglycone in probe 17 is associated with the plasma membrane of motor cells.

Fig. 8 Photoaffinity probes based on the leaf-closing factor of Albizzia plants
Fig. 9 Enantiodifferential photoaffinity labeling of Albizzia motor cells

lane 1 lane 2 lane 3 lane 4

Fig. 10 Enantiodifferential photoaffinity labeling of membrane proteins of Albizzia motor cells lane 1 lane 2 lane 3 lane 4

Fig. 10 Enantiodifferential photoaffinity labeling of membrane proteins of Albizzia motor cells

SDS-PAGE analysis and chemiluminescence analysis of photocrosslinked membrane proteins of protoplasts were carried out (Fig. 10) (Nakamura et al. 2008). In Fig. 10 , lane 2 contained the crude membrane fraction without any probe incubation, lane 3 contained the membrane fraction incubated with probe 18, and lane 4 contained the membrane fraction incubated with probe 17. Several bands below 30 kDa were observed in lanes 3 and 4, indicating nonspecific binding of the probe. However, one difference between probe 17 and 18 was evident around 38kDa, indicating that this protein showed stereospecific recognition of the aglycone of the probe. Additionally, the binding of probe 7 with this protein was competitively inhibited by the photoaffinity labeling experiment when an excess amount (1*10-3M) of 1 was present. Our enantiodifferential approach clearly discriminated specific from nonspecific binding of the probe. The observation that only the biologically active stereoisomer was recognized by this protein strongly suggested that this membrane protein is the true target protein of 1 involved in the control of nyctinasty in A. saman.

3.3.3 Double Fluorescence Labeling of Plant Pulvini Using

Fluorescence-Labeled Leaf-Closing and Leaf-Opening Factors

There are two types of motor cells in pulvini of nyctinastic plants: extensors and flexors. Since leaflets move upward during closure and downward during opening, extensors are located on the upper (adaxial) side of the leaf and flexors on the lower (abaxial) side. To examine whether closing and opening factors differentially target

Fig. 11 Double fluorescence labeling of Albizzia pulvinus using fluorescence-labeled leaf-closing and -opening factors

extensors and flexors, we performed a double fluorescence labeling study using FITC-labeled leaf-closing factor 15 and rhodamine-labeled leaf-opening factor 19 (Fig. 11) (Nakamura et al. 2006b). Figure11 shows a photograph of the fluorescence image of a plant section that was cut perpendicular to the vessel. Somewhat unexpectedly, both of the probes bound to the extensor cells but not the flexor cells in the pulvini. Therefore, the motor cell with a set of target proteins for leaf-movement factors is located in the extensor side of the pulvini in A. saman. As extensor cells are defined as cells that increase turgor during opening and decrease turgor during closing, the leaf-movement factors may regulate potassium channels, which in turn change potassium salt levels and thus turgor pressure.

As described, leaf-closing and -opening factors act in a genus-specific manner. Therefore, we investigated whether the labeled factors bind to the target cells in a genus-specific manner. As expected, the fluorescence-labeled probes 15 and 19 bound to motor cells of A. saman and A. juribrissin, whereas they did not bind to the cells of Cassia mimosoides L., Phyllanthus urinaria, and Leucaena leucocephara (Nakamura et al. 2006a; Nagano et al. 2003).

4 The Chemical Mechanism of Rhythm in Nyctinasty

If a pair of leaf-movement factors regulate nyctinasty, there should be some relationship between their levels in plants and the circadian clock. The changes in the contents of leaf-closing and -opening factors in the plant P. urinaria over time are highlighted in Fig. 12 (Ueda et al. 1999c). HPLC was used to determine the levels of these factors

aglycon 20

Fig. 12 Changes in the concentrations of leaf-opening and leaf-closing factors in Phyllanthus urinaria over time aglycon 20

Fig. 12 Changes in the concentrations of leaf-opening and leaf-closing factors in Phyllanthus urinaria over time

Leaf-closing factor (const.) Leaf-opening factor (charge)

Fig. 13 Chemical mechanism of nyctinasty in Lespedeza cuniata

Leaf-closing factor (const.) Leaf-opening factor (charge)

Fig. 13 Chemical mechanism of nyctinasty in Lespedeza cuniata every 4h over a daily cycle. It was found that the content of the leaf-opening factor 8 remains nearly constant during the day, whereas that of the leaf-closing factor 3 changes by as much as 20-fold. This behavior could be accounted for by the conversion of the leaf-closing factor to its corresponding aglycon 20 in a hydrolytic reaction. It follows from this type of analysis that significant changes in the ratio of the concentrations of the leaf-closing and leaf-opening factors in the plant are responsible for leaf movement.

In Lespedeza cuneata, the concentration of potassium lespedezate 10 (a glucoside-type leaf-opening factor, Shigemori et al. 1989, 1990) decreases in the evening, whereas the concentration of the leaf-closing factor 9 remains constant during the day (Ohnuki et al. 1998). Leaf-opening factor 10 is metabolized to the biologically inactive aglycon 21 in the evening (Fig. 13). These findings are consistent with the changes in b-glucosidase activity in the plant body that occur during the day, where significant activity is only observed in plants collected in the evening. This suggests that there is a temporal mechanism that regulates b-glucosidase activity and influences these factors during the diurnal cycle. Recently, the b-glucosidase associated with the hydrolysis of 10 was purified and named LOFG (leaf-opening factor b-glucosi-dase), and was revealed to be a family III type glucosidase from partial sequence analysis (Kato et al. 2008).

In all of the five pairs of leaf-closing and -opening factors 1-10 from the five nyctinastic plants discovered so far, one from each pair of factors is a glycoside, and in all cases the concentrations of these glycoside-type leaf-movement factors change during the day in a similar manner to that described for L. cuneata.

This suggests that all nyctinastic leaf movement can be explained by a single mechanism involving two leaf movement factors, of which one is a glucoside. b-Glucosidase activity is then regulated by some mechanism that deactivates the glucoside and controls the relative concentrations of leaf-closing and -opening factors. Thus, nyctinastic leaf movement is controlled by regulated b-glucosidase activity with a daily cycle.

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