Change of cell structure during tropistic responses

1.4.1 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 thecell 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.,themicrotubule 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 amiprophosmethyl 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.

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Change of cell structure during tropistic responses

1.4.1 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 thecell 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.,themicrotubule 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 amiprophosmethyl 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.

1.4 Phototropism and polarotropism

1.4 Phototropism and polarotropism
The direction of protonemal growth is controlled by light. Protonemal cells grow toward a red light source (phototropism) or perpendicular toavibration plane of polarized red light, following a phenomenon known as ‘‘polarotropism” (B¨and 1958; Etzold, 1965). Phototropism  and polarotropism are phenomenologically different responses because in phototropism a protonema grows toward a light source whereas in polarotropism a
protonema grows perpendicular to the polarized incident light and to its vibration plane, regardless of the direction of incident light . Thus, the protonema grows toward the side that absorbs more light. Polarotropism occurs because of the orderly intracellular arrangement of phytochrome molecules attached to the plasma membrane. However the two light-induced tropisms may be considered equivalent if the direction of growth is determined by the highest
concentration of the far-red light absorbing form of phytochrome (Pfr) in the protonemal apical dome (explained in the following section). Hence, here I treat these two responses as one physiological phenomenon controlled by the same phytochrome molecular species and the same mechanism. When polarotropism is induced in protonemata growing on the surface of an agar medium, growth is not only perpendicular to the vibration plane but also toward the light source of the polarized light. In order to avoid phototropism under polarized light, to
irradiate cells evenly without reflection and refraction, and to eliminate the lens effect caused when cylindrical protonemal cells are elevated above the growing surface, all experiments were performed using protonemata cultured on an agar medium covered with a cover slip, or under similar submerged conditions.

When the direction of incident red light is changed, a change in the direction of protonemal growth toward the new light source can be detected about 1 hour after the light treatment. However, intracellular events show that the cells respond instantaneously to the new light by modifying the cytoskeletal pattern and subsequently the pattern of microfibrils (Wada et al., 1990). Analytical studies of the response have focused on either polarotropism or microbeam-induced phototropism, because the responses induced by these methods can be
controlled more accurately. The tropistic curvature of protonemata is very sharp when induced through whole cell irradiation by polarized light or by partial cell irradiation with a microbeam , but is more rounded when the whole cell is irradiated with ordinary light. It is likely that in the former instances light absorption by phytochrome molecules is restricted to a very small area compared to that in the latter.

Etzold (1965)proposed a hypothesis to explain the polarotropic response in Dryopteris protonemata. According to this hypothesis the red light absorbing form of phytochrome (Pr) is localized at the cell periphery (close to the plasma membrane) and has a transition moment parallel to the plasma membrane. Growth occurs in the portion of the cell where the highest concentration of phytochrome is transformed to the far-red light absorbing form (Pfr) by red light absorption . Based on this hypothesis, when polarized red light vibrating perpendicular to the cell axis is applied to the apical dome of protonemata, theprotonemata grow straight as before at their tip, because the phytochromes with transition moments parallel to the vibration plane of the polarized light are localized only at the tip of the apical dome. When the vibration plane is twisted (no longer perpendicular to the cell axis), a portion of the apical dome whose plasma membrane becomes parallel to the direction of the new vibration
plane can absorb more polarized light, and can then become a new growing cell tip.

According to Etzold’s hypothesis the transition moment of Pfr should be perpendicular to the plasma membrane (Etzold, 1965). The photo-conversion of the transition moment between Pr parallel and Pfr perpendicular to the plasma membrane by red and far-red light irradiation respectively, was confirmed through chloroplast movement in Mougeotia by Haupt and his colleagues (Haupt et al., 1969). In A. capillus-veneris we determined the actual photoreceptive
site in a protonema by microbeam irradiation with polarized red light at various portions of the cell and found that the cell margin, especially at the basal part of the apical dome, is the most effective site for polarized light absorption (Wada et al., 1981). The dichroic orientation of phytochrome was also confirmed in the polarotropism of A. capillus-veneris protonemata by very precise analyses (Kadota et al., 1982, 1985;Wada et al., 1983). Recently neochrome1
(neo1) (formerly called phytochrome 3 (phy3)) was discovered in A. capillus-veneris


Phototropism and polarotropism can be induced by blue light also, although, because blue light inhibits protonemal cell growth, it is better to irradiate with red light simultaneously to stimulate cell growth (Kadota et al., 1979, 1989). In these experiments, blue light was applied unilaterally or as polarized light, but red light was applied vertically in the former case and as non-polarized light in the latter case to avoid the directional influence of red light. The blue
light receptor of this phenomenon is not yet known, although phototropins are plausible candidates.

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1.3.2 Resumption of cell growth

1.3.2 Resumption of cell growth

When red-light grown protonemata are transferred into the dark, cell division occurs in the apical region of the linear protonemata (Wada and Furuya, 1972). If protonemal cells are kept in the dark after cell division for several days, almost all cytoplasm in the apical cells, including chloroplasts and a nucleus, moves toward the cell plate at the basal end, so that the cells are occupied by a large vacuole and become transparent. These cells appear dormant and neither grow nor divide until light is provided. It is not known how long the cells can survive without light. When protonemata are irradiated with red light continuously, the nucleus moves toward the cell tip, the cytoplasm disperses over the entire cell, and then elongation resumes at the apex of the cell (Kadota and Furuya, 1981). When red-light grown protonemata kept in the dark for 3 days were used, 24 hours of irradiation was required for all protonemata to recover and grow normally (Kadota and Furuya, 1981). Resumption of cell growth is controlled in a phytochrome red/far-red light reversible manner. However, full reversibility by far-red light is lost when cells are irradiated by red light for longer than 4 s at 4.6 W m-2,although it is not dependent on an escape reaction (Kadota and Furuya, 1981). This is an unusually rapid and sensitive response compared to other phytochrome-dependent phenomena in fern gametophytes,
e.g., the timing of cell division, which is induced by transferring protonemata from red light to darkness and is still reversible by far-red light after irradiation by red light for 10 minutes (Wada and Furuya, 1972).

Red light controlled protonemal growth, cessation, and resumption of growth discussed in this section are mediated by phytochrome, but it is not yet known which of the three phytochrome genes cloned in A. capillus-veneris mediates these phenomena.

Cell growth in ferns

In most homosporous ferns, after spore germination under red light, a filamentous protonemal cell grows at the apical dome towards a red light source without (or at least with a low frequency of) cell division. The cell is about 15–20 µmindiameter, although this varies with species and culture conditions. The nucleus is always located about 60 µmfrom the tip during cell growth in A. capillus-veneris,indicating that the nucleus migrates in the cell toward the tip, maintaining a constant distance (Figure 1.4)(Wada and O’Brien, 1975;Wada et al.,
1980). In a growing protonema, microtubule and microfilament strands connect thenucleus to the cortex of the apical and basal parts of the cell (Kadota and Wada, 1995), although how these cytoskeletal strands control nuclear migration is not yet known. The growth rate varies with species and also with environmental conditions in the same species. In the case of A. capillus-veneris under continuous red light (0.5 W m-2s-1)at25 .Ctheprotonemata grew at an average rate of about 200 µm/day (Wada, 1988a).

In some species (including those of Anemia, Osmunda, and Lygodium), even under red light conditions, gametophytes germinate as two-dimensional prothallia, and no protonemal stage is observed (Raghavan, 1989). In Ceratopteris, when spore germination was induced by white light irradiation for 1 day after imbibition and then the spores were kept in the dark, gametophytes germinated as a two-dimensional, strap-shaped prothallium in four cell-columns. A cell mass proliferated at the apical part of the gametophyte and each cell at the basal part of the cell mass grew in the dark parallel to the cell polarity (Murata et al., 1997). In this species, cells can grow in the dark, similar to protonemal cells of A. capillus-veneris grownunder red light, but the cells are not protonemata.

The cell diameter under red light is reasonably constant. How do cells know the diameter and how do they maintain it? At the basal part of the apical dome of protonemata, a circular array of microtubules and microfilaments is observed (Murata et al., 1987;Kadota and Wada, 1992b). Because this will be discussed in detail in Section 1.6, it is sufficient to note that these cytoskeletal structures play a key role in maintaining a constant diameter, as has been well established in higher plant cells (Shibaoka, 1994).

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Spore germination in ferns

There are two kinds of fern spores based on their color: one is green (chlorophyllous) and the other is brown (non-chlorophyllous). Green spores have chloroplasts even before water imbibition and, unless refrigerated, their germination ability (spore viability) does not persist long after harvest. See Raghavan (1989)formore information.

Most fern spore germination is light dependent. In a tetrahedral, non-chlorophyllous, dormant spore, the nucleus sits in one corner surrounded by three furrows. When spores are irradiated with red light after imbibition in the dark, they become round, and the nucleus, still in its corner position, divides, followed by cell division to produce large protonemal and small rhizoidal mother cells (Furuya et al., 1997). In A. capillus-veneris, Pteris vittata, and probably other species, red-light induced germination is inhibited by far-red light in a red/farred reversible manner, indicating the involvement of phytochrome (Sugai and Furuya, 1967;Furuya et al., 1997). The red light effect is inhibited by blue light, on exposure before or after the red light treatment (Sugai and Furuya, 1967; Furuya et al., 1997). The blue light inhibition effect, however, cannot be reversed nstantaneously by subsequent exposure to a pulse of red light, suggesting the involvement of a blue light receptor, but not a phytochrome system. Inhibition
can be prevented when the spores are kept in the dark for about a week (Sugai and Furuya, 1968;Furuya et al., 1997). The time period required for prevention of blue light inhibition is very much reduced if the spores are irradiated with red light. The red light effect can be reversed by far-red light, indicating phytochrome dependence (Sugai and Furuya, 1968;Furuya et al., 1997). The inhibitory effects of far-red and blue light could not be observed after the first mitosis in spores, suggesting that cell division is a crucial step for spore germination
(Furuya et al., 1997). Partial spore irradiation with red or blue microbeam lights showed that the blue light receptor is located in the nucleus, but the location of the red light hotoreceptor could not be identified (Furuya et al., 1997). The photoreceptors mediating spore germination (both phytochrome and blue light receptors) have not yet been identified, although several candidate genes have beenclonedandsequenced.

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Photoresponses in fern gametophytes

1.1 Introduction
Fern gametophytes are ideal model systems for study of the mechanisms of  hotomorphogenesis from the standpoint of physiology, photobiology, and cell biology (Wada, 2003, 2007;Kanegae and Wada, 2006). Positive aspects of the fern system include the following.

(1) Spores can be preserved at room temperature and they germinate under appropriate conditions within about a week in many species, becoming gametophytes that grow rapidly, at least in their critical early stages.
(2) Gametophytes are nutritionally autonomous, facilitating ease of cultivation.
 (3) Gametophytes are not enclosed by other tissue, so that observation, light irradiation, and experimental manipulation are readily performed.
(4) Each developmental step can be controlled synchronously because gametophytes are highly sensitive to light. Each step in development is completely dependent on light; indeed, without light, development does not proceed.

Since the nineteenth century, especially in Germany, fern gametophytes have been used (see Dyer, 1979a)tostudy photo-physiological phenomena, such as light dependent spore germination (Mohr, 1956a), differentiation from one-dimensional protonemata to two-dimensional prothalli (Mohr, 1956b), and intracellular dichroic orientation of phytochrome (Etzold, 1965). Even though fern  gametophytes are very good materials for the study of both photobiology and  cell biology, only a few laboratories use them presently, probably for the following reasons.
(1) Although mutants can be obtained easily by phenomenological screening (gametophytes are haplophase), making crosses for genetic studies is difficult and time consuming.
(2) The biochemistry is also challenging because collecting enough gametophyte tissue for biochemical analyses is difficult.
(3) Molecular biological techniques are not yet established (e.g., stable transformation
is not available, although transient gene expression is possible).
(4) Most ferns are not commercially valuable plants, although some species, such as Osmunda japonica, Pteridium aquilinum,and Matteuccia struthiopteris,are edible and obtainable commercially in eastern Asia, or are used as ornamental plants, or for cleaning soil polluted by heavy metals including arsenic (Ma et al., 2001).

Nevertheless, fern gametophytes have structural and physiological characteristics that seed plants do not have, making them more tractable systems for studying many phenomena that are common to ferns and seed plants. For example, we have analyzed factors controlling the pre-prophase band (PPB) formation and its disruption (Murata and Wada, 1989b, 1991a, 1991b, 1992)(Figure 1.1). The PPB is recognized as a factor controlling the attachment site of newly synthesized cell plates to mother cell walls (Mineyuki, 1999). It appears before prophase of
thenuclear division cycle at the future site of cell plate fusion to the mother cell wall, but disappears before cell plate formation. The kind of information remaining at the PPB region has long been a mystery, as have the factors that determine the future cell plate attachment site and disrupt the PPB. To study this issue physiologically, Murata and Wada (1989b, 1991b, 1992)useda long protonemal cell cultured under red light in which cell division occurred at
40–60 µmfrom the tip where the division site is pre-determined by the PPB. During the period when the PPB was polymerizing, protonemal cells with a premature PPB were centrifuged to reposition the nucleus. A new PPB formed at the new nuclear site, distant from the original position, and then cell division occurred, suggesting that the nucleus must be close to the PPB polymerization site. In these cells the first PPB at the apical part did not de-polymerize even after cell division occurred, but if a dividing nucleus was returned to the former PPB
site, the PPB de-polymerized. This result indicates that PPB de-polymerization requires a nucleus and/or surrounding cytoplasm. Experiments such as these could not be done using seed plant cells because long cells like protonemal cells are not found in seed plants, except in some special cases such as cambium cells, where cell division occurs periclinally, making them inappropriate for the experiment. Experiments using long protonemal cells were also performed to study the recovery of a nucleus elongated by cell centrifugation (Wunsch and
Wada, 1989; Wunsch et al.,1989).

To analyze the physiological characteristics at each step of the developmental process or during transitions from one step to another of photobiological responses in fern gametophytes, various tools and special techniques have been developed. These include microbeam irradiators to stimulate only a small part of a cell and identify the photoreceptive site, i.e. the localization of photoreceptor molecules mediating a target phenomenon. The first machine was constructed in 1978 (Wada and Furuya, 1978)(Figure 1.2). Current microbeam projectors are now in their fourth or fifth generation, and are equipped with
various accessories depending on their purpose (Iino et al., 1990;Yatsuhashi and Wada, 1990).


This chapter will focus on recent analyses performed mostly by my laboratory  group using Adiantum capillus-veneris.I also include some results that have not been published but are based on a synthesis of nearly 40 years of my experience with fern gametophytes. Our knowledge, mostly obtained from A. capillus-veneris, assumes that this species follows a pattern of development that is typical of most ferns. However, because of the large diversity in species and gametophytes, numerical data such as the growth rate of protonemata mentioned here may  or may not be applicable to other fern species. For more information refer to  books byDyer(1979b)and Raghavan (1989)andthefollowing reviews: Wada and Kadota (1989), Wada and Sugai (1994), Kanegae and Wada (2006), and Wada (2007).

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