Photoresponses in fern gametophytes

masamitsu wada

1.1 Introduction

Fern gametophytes are ideal model systems for study of the mechanisms of photomorphogenesis 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 game-tophytes 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) to study 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

Biology and Evolution of Ferns and Lycophytes, ed. Tom A. Ranker and Christopher H. Haufler. Published by Cambridge University Press. © Cambridge University Press 2008.

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 the nuclear 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) used a long protonemal cell cultured under red light in which cell division occurred at 40-60 |im from 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 photobiologi-cal responses in fern gametophytes, various tools and special techniques have been developed. These include microbeam irradiators to stimulate only a small

Figure 1.1 Photomicrographs of bent protonemata showing the effect of double centrifugation on pre-prophase band formation. (a)-(c) A bent protonema centrifuged parallel with the arrows to sediment cytoplasm, including a nucleus and chloroplasts. Note that a nucleus, indicated by small arrowheads, moved downward. (d), (e) Bent protonemata centrifuged 4 and 8.5 hours (d) and 4 and 12.5 hours (e) after the onset of blue light irradiation and fixed at 14.5 hours. The regions between the bend and the nucleus are shown. One pre-prophase band (marked with a bracket) was found in (d) and two bands were found in (e). The second centrifugation was applied before and during pre-prophase band formation, respectively. (After Murata and Wada, 1991b).

Figure 1.1 Photomicrographs of bent protonemata showing the effect of double centrifugation on pre-prophase band formation. (a)-(c) A bent protonema centrifuged parallel with the arrows to sediment cytoplasm, including a nucleus and chloroplasts. Note that a nucleus, indicated by small arrowheads, moved downward. (d), (e) Bent protonemata centrifuged 4 and 8.5 hours (d) and 4 and 12.5 hours (e) after the onset of blue light irradiation and fixed at 14.5 hours. The regions between the bend and the nucleus are shown. One pre-prophase band (marked with a bracket) was found in (d) and two bands were found in (e). The second centrifugation was applied before and during pre-prophase band formation, respectively. (After Murata and Wada, 1991b).

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). Photoreceptive sites revealed by experiments using microbeam irradiators are summarized in Figure 1.3, and will be explained in the following sections.

C-ob

Figure 1.2 Diagrams showing microbeam irradiators. (a) The first generation irradiator. An ordinal light microscope was remodeled for microbeam irradiation by inserting a diaphragm, and another light source was added for observation. Ph, photographic camera; Oc, ocular lens; Ob, objective lens; S, specimen; Fs, focusing stage; C-ob, condenser objective lens; Sp, silicon photocell; mV, millivolt meter; Bs, beam splitter; D, diaphragm; Sh, shutter; if, interference filter; Pf, plastic filter; Hf, heat filter; Is, irradiation source; St, stabilizer; Cs, CuSO4 solution; Is-ob, irradiation source for observation. (b) A third generation irradiator. Four different wavelength lights can be irradiated simultaneously at one point or two mixed lights can be irradiated side by side. CF, cut-off filter; Dp, depolarizer; Fs, field stop; HM, half-silvered mirror; IF, interference filter; IRV, infrared viewer; LS, light source; M, mirror; Obs, observation point; P, polarizer; PC, photographic camera; Sh, shutter; Sl, slit; St, sample stage; WF, water cell; WP, pump for circulation of cooled water to water cell. See Fig. 6 of Wada (2007) for a diagram of the second generation irraditor. ((a) After Wada and Furuya, 1978. (b) After Iino et al., 1990.)

Light source II

Figure 1.2 Diagrams showing microbeam irradiators. (a) The first generation irradiator. An ordinal light microscope was remodeled for microbeam irradiation by inserting a diaphragm, and another light source was added for observation. Ph, photographic camera; Oc, ocular lens; Ob, objective lens; S, specimen; Fs, focusing stage; C-ob, condenser objective lens; Sp, silicon photocell; mV, millivolt meter; Bs, beam splitter; D, diaphragm; Sh, shutter; if, interference filter; Pf, plastic filter; Hf, heat filter; Is, irradiation source; St, stabilizer; Cs, CuSO4 solution; Is-ob, irradiation source for observation. (b) A third generation irradiator. Four different wavelength lights can be irradiated simultaneously at one point or two mixed lights can be irradiated side by side. CF, cut-off filter; Dp, depolarizer; Fs, field stop; HM, half-silvered mirror; IF, interference filter; IRV, infrared viewer; LS, light source; M, mirror; Obs, observation point; P, polarizer; PC, photographic camera; Sh, shutter; Sl, slit; St, sample stage; WF, water cell; WP, pump for circulation of cooled water to water cell. See Fig. 6 of Wada (2007) for a diagram of the second generation irraditor. ((a) After Wada and Furuya, 1978. (b) After Iino et al., 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 by Dyer (1979b) and Raghavan (1989) and the following reviews: Wada and Kadota (1989), Wada and Sugai (1994), Kanegae and Wada (2006), and Wada (2007).

1.2 Spore germination

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) for more 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

Growth promotion Growth inhibition

G1 shortening G2 elongation

Apical swelling

Photo-

Polarotropism

Chloroplast photoorientation

Figure 1.3 Photoreceptive sites for light-induced phenomena in an Adiantum capillus-veneris protonema. Light grey (blue) and dark grey (red) indicate photoreceptive sites of blue and red light photoreceptors, respectively. (After Wada, 2007.)

species, red-light induced germination is inhibited by far-red light in a red/far-red 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 instantaneously 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

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