Change of cell structure during tropistic responses

Circular arrays of microtubules and microfilaments, as well as the microfibril pattern at the subapical portion of protonemata, change during phototropism (Wada et al, 1990; Kadota and Wada, 1992a, 1992b). When polarotropism was induced by polarized red light vibrating 45° to the cell axis, the cortical array of microtubules became oblique within 30 minutes after irradiation to the direction of bending, but if the vibration plane was 70°, the microtubule array disappeared. After 1 hour, the tropistic response could be observed using a microscope. By 2 hours after polarotropism induction, the microfibril rearrangement of the innermost layer of the cell wall became oblique to the former growing axis (Wada et al., 1990). During phototropism, reorganization of the microfilament structure precedes that of the microtubule structure (Kadota and Wada, 1992a), suggesting that the microtubule array is influenced by the microfilament array. Interestingly, this hypothesis was confirmed by experiments using cytoskeletal inhibitors (Kadota and Wada, 1992b). Colchicine and amiprophos-methyl disrupted the microtubule array but not the microfilament array. In contrast, cytochalasin B disrupted both arrays, indicating that the microtubule array depends on the array of microfibrils. Taken together, phototropism and polarotropism must occur through sequential changes: the microfilament array controls the direction of the microtubule array, which controls the direction of microfibril arrangement, and finally microfibrils restrict the cell diameter and the direction of cell growth.

1.5 Cell division

When red-light grown protonemata are transferred into darkness or white light, cell division occurs synchronously at the apical portion of the pro-tonemata (Wada and Furuya, 1970, 1972). Structural changes during the cell

Figure 1.9 Timing of cell division induced in red-light grown protonemata of Adiantum capillus-veneris by irradiation with short pulses (10 min) of blue (open circles), red (filled circles) and far-red (triangles) light before transfer to darkness. Blue light shortens the cell cycle but far-red light lengthens it compared with the direct transfer from red light to the dark. L.T, light treatment. (After Wada, 2007, modified from Wada and Furuya, 1972.)

Figure 1.9 Timing of cell division induced in red-light grown protonemata of Adiantum capillus-veneris by irradiation with short pulses (10 min) of blue (open circles), red (filled circles) and far-red (triangles) light before transfer to darkness. Blue light shortens the cell cycle but far-red light lengthens it compared with the direct transfer from red light to the dark. L.T, light treatment. (After Wada, 2007, modified from Wada and Furuya, 1972.)

division were analyzed using thin sections (see the details in Wada and O'Brien, 1975; Wada et al., 1980). The timing of cell division is light dependent. Blue light induces cell division (Wada and Furuya, 1974) (Figure 1.9), whereas red light inhibits cell division (Wada and Furuya, 1970). When protonemata grow under red light, their cell cycle remains at an early G1 phase (Miyata et al., 1979). Altogether, the cell cycle of protonemata is controlled by light. Blue light shortens the period of the G1 phase but far-red light lengthens the G2 phase (Miyata et al., 1979). Within a restricted range, the stronger the intensity of blue light or the longer the light irradiation, the shorter the G1 phase. This light-dependent protonemal cell cycle may be a very useful system for studying the regulation mechanisms of the plant cell cycle precisely from a physiological and cell biological standpoint. In this system, cell division is controlled by light and occurs synchronously in almost all protonemata. The most difficult aspect of this model system is the collection of sufficient protonemal cells if biochemical studies are intended.

The protonemal cell cycle induced by darkness can be reversed by red light irradiation, if the cell cycle is still in the G1 phase (Wada et al., 1984). When

Figure 1.10 ''The point of no return" in cell division in red-light grown Adiantum capillus-veneris protonemata. Under continuous red light, protonemata remain at the beginning of the G1 phase. When the red-light grown protonemata are transferred into the dark, cell division is induced. However, the cell cycle stops and then returns to the beginning of the G1 phase, if the protonemata are irradiated with continuous red light before entering the S phase. Triangles, open circles, and filled circles show the point of no return, timing of cell division, and entry to the S phase, respectively. (After Wada, 2007, modified from Wada et al., 1984.)

Figure 1.10 ''The point of no return" in cell division in red-light grown Adiantum capillus-veneris protonemata. Under continuous red light, protonemata remain at the beginning of the G1 phase. When the red-light grown protonemata are transferred into the dark, cell division is induced. However, the cell cycle stops and then returns to the beginning of the G1 phase, if the protonemata are irradiated with continuous red light before entering the S phase. Triangles, open circles, and filled circles show the point of no return, timing of cell division, and entry to the S phase, respectively. (After Wada, 2007, modified from Wada et al., 1984.)

red-light grown, single-celled protonemata are transferred into darkness, the cell cycle starts and cell growth slows. However, the protonemata begin to elongate again and the cell division trigger is cancelled if the protonemata are returned to red light after the transfer to darkness. But if the timing of the transfer to red light is delayed to beyond the entry to the S phase, cell division occurs and the cell cycle cannot return to the G1 phase even under continuous red light. The switch between ''returnable" and ''unreturnable" is called a ''point of no return" (Wada et al., 1984) (Figure 1.10). The point of no return in blue light induced cell division is different from that of cell division induced in the dark. In this case the point of no return by red light irradiation is in the middle of the G1 phase, much earlier than the entry to the S phase (Wada, unpublished data). The mechanisms and pathways of cell cycle progress may be different for blue light induced cell division and dark induced cell division.

Intracellular localization of the blue light receptor for the induction of cell division was studied by microbeam irradiation (Wada and Furuya, 1978). Various portions of the tip of red-light grown, single-celled protonemata were irradiated with a short pulse of a blue light microbeam through a slit (30 ^m in width). The time at which cell division occurred after microbeam irradiation was calculated. The portion that induced the highest frequency of cell division was located about 60 ^m from the tip, where a spindle-shaped nucleus is usually found (Wada and Furuya, 1978). To confirm that the photoreceptive site is on or in the nucleus or near the nuclear region, protonemal cells were centrifuged to move the nuclei basipetally. The region now containing a nucleus or the former region now lacking a nucleus was irradiated with blue light through a narrow slit (Kadota et al., 1986). The results clearly showed that the new nuclear region, rather than the former region, was responsible for initiating cell division, indicating that photoreceptors were localized within the cytoplasm mass near the nucleus. We are not yet sure what receives the blue light, nor do we know whether the photoreceptors are within the nucleus or outside. One or more cryptochrome(s) of five (AcCRY 1 to 5) already cloned in A. capillus-veneris (Kanegae and Wada, 1998; Imaizumi et al., 2000) may be a photoreceptor candidate for this response. Depending on the distribution of GUS-AcCRY nucleocytoplasma (Imaizumi et al., 2000), AcCRY3/4 are plausible candidates for this response (Kanegae and Wada, 2006).

1.6 Apical cell bulging

When red-light grown protonemata are irradiated with blue light continuously, the apical part of the protonemata starts to swell and becomes bulbous prior to cell division (Wada et al., 1978). The cell plate occurs usually near the neck of the swollen part (Wada and Furuya, 1970; Wada and O'Brien, 1975), that is, at the junction between the bulbous and filamentous parts of the protonemata. Apical bulging has been recognized as the event that initiates two-dimensional growth (Mohr, 1956b). Using microbeam irradiation, the apical dome of protonemal cells was identified as the location of blue light reception for this phenomenon, but not in or around the nucleus. Polarized blue light irradiation shows a dichroic effect: polarized light vibrating parallel to the plasma membrane is more effective than that vibrating perpendicular to the plasma membrane. These data indicate that the blue light receptor should be localized on the plasma membrane and should be arranged dichroically parallel to the plasma membrane (Wada et al., 1978). The evidence appears to indicate that both cell division and apical cell bulging can be induced by a single application of blue light, and that these responses are sequential phenomena along one signal transduction pathway, starting with apical bulging leading to cell division. However, by careful analyses of intracellular localization of photoreceptor(s) by microbeam experiments, it was discovered that the two phenomena are completely independent from each other (Wada and Furuya, 1978; Wada et al., 1978).

The photoreceptor for cell division is localized in the nuclear region (Wada and Furuya, 1978) but that for apical bulging is on the plasma membrane (Wada et al., 1978). Presumably the photoreceptors for apical bulging are phototropins, although that remains to be determined through further studies.

Typical apical cell bulging is not observed when cell division is induced in the dark (Wada et al., 1980) or even under weak white light, although there is a tendency for the apical part of the protonemal cell to swell slightly when cell division occurs under any light conditions, in the dark or under red light.

Investigations of cytoskeletal changes during apical bulging revealed that the diameter of protonemal cells was regulated by actin filaments, microtubules, and microfibrils. Grown under red light, microtubules (Murata et al., 1987) and microfilaments (Kadota and Wada, 1989a) at the subapical part of protonemata showed a circular arrangement. When these red-light grown protonemata were transferred to blue light, the circular array of microtubules and microfilaments disappeared prior to apical bulging. Both structures disappeared at about the same time (Kadota and Wada, 1992a). Under red light, the arrangement of microfibrils in the innermost layer of the cell wall at the subapical part of the protonemata was perpendicular to the cell axis. Thus, the microfibrils were parallel to the circular arrays of microtubules and microfilaments. When transferred to blue light, however, the microfibrils changed to a random arrangement (Murata and Wada, 1989a) (Figure 1.11). The circular microtubule array disappeared before the microfibril pattern became random, prior to the detection of apical bulging, indicating that the cortical microtubule array regulates the microfibril arrangement, which, in turn, controls the cell diameter. Apical cell bulging is also induced by disruption of the microtubule array by colchicine and amiprophos-methyl (Murata and Wada, 1989c), confirming the regulation of the cell diameter by microtubules.

1.7 Chloroplast movement

Chloroplast photorelocation has been well known since the nineteenth century in groups ranging from algae to seed plants (Wada et al., 1993, 2003). Under weak light, chloroplasts move toward a light source or to a brighter part of a cell (accumulation response) for efficient light absorption. Under excess light, they move away from the light (avoidance response), preventing photo-damage of chloroplasts (Figure 1.12). When Arabidopsis mutants deficient in avoidance response were kept under strong light (1400 |imol m-2 s-1) for more than 10 hours, the leaves became necrotic because the chloroplasts were destroyed and mesophyll cells became seriously damaged (Kasahara et al., 2002). Hence, the avoidance response is very important for plant survival. Augustynowicz and

Figure 1.11 Diagrams showing changes in the arrangement of cortical microtubules (MTs) and microfibrils (MFs). The pattern of cortical microtubules at the apical part of the protonemata grown under red light is modified by blue light irradiation. The transverse arrangement of microtubules disappears and a random arrangement becomes dominant. The pattern of microfibril arrangement follows the change in microtubule pattern, suggesting that the microfibril pattern is controlled by microtubules. The numbers indicate hours after blue light irradiation. (After Murata and Wada, 1989a.)

Gabrys (1999) reported the ecological significance of chloroplast movement in fern sporophytes. They reported that A. capillus-veneris and Pteris cretica showed clear photorelocation movement under both strong and weak light. However, Adiantum caudatum, found in high light environments, does not show photorelocation movement and Adiantum diaphanum, living in shady environments, shows only weak photorelocation movement. Augustynowicz and Gabrys (1999) concluded that chloroplast photorelocation is only found in plants living in environments with fluctuating light intensities. Using a microscope, we detected chloro-plast photorelocation movement only in very young leaves of A. capillus-veneris (Kawai et al., 2003). I am not certain that the chloroplast avoidance response occurs under strong light in adult fern leaves, even if they live under fluctuating light conditions, but it is clear that almost all fern gametophytes so far tested show both the accumulation response and the avoidance response. In general, blue light stimulates both accumulation and avoidance responses in many fern species, except in the genus Pteris (Kadota et al., 1989; Kagawa and Wada, 2002).

Figure 1.11 Diagrams showing changes in the arrangement of cortical microtubules (MTs) and microfibrils (MFs). The pattern of cortical microtubules at the apical part of the protonemata grown under red light is modified by blue light irradiation. The transverse arrangement of microtubules disappears and a random arrangement becomes dominant. The pattern of microfibril arrangement follows the change in microtubule pattern, suggesting that the microfibril pattern is controlled by microtubules. The numbers indicate hours after blue light irradiation. (After Murata and Wada, 1989a.)

Figure 1.12 Chloroplast photorelocation movement in a prothallium of Adiantum capillus-veneris. The portions outside the letters "FERN" were irradiated with strong light and chloroplasts moved away from the area, showing an avoidance response. The portions within the letters were irradiated with weak light to induce an accumulation response.

Figure 1.12 Chloroplast photorelocation movement in a prothallium of Adiantum capillus-veneris. The portions outside the letters "FERN" were irradiated with strong light and chloroplasts moved away from the area, showing an avoidance response. The portions within the letters were irradiated with weak light to induce an accumulation response.

In lower plants, such as Mougeotia (Haupt et al., 1969), Mesotaenium (Kraml et al., 1988), and Physcomitrella (Kadota et al., 2000), red light is also effective. We focus here on chloroplast movement in fern gametophytes. For other plant species refer to Haupt, 1999; Wada et al., 1993; or Wada et al., 2003.

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