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Phytochrome-Mediated Mitotic Activity and Induction of two-Dimensional Growth in Gametophytes of Anemia phyllitidis (L.) Sw.*
J. Plant Physiol. Vol.
141. pp. 457-462 (1993)
Phytochrome-Mediated Mitotic Activity and Induction of twoDimensional Growth in Gametophytes of Anemia phyllitidis (L.) Sw. * RENATE GRILL
and
HELMUT SCHRAUDOLF
Abteilung Allgemeine Botanik, Universitat Ulm, Oberer Eselsberg, W-7900 Ulm/Donau, Germany Received August 20, 1992 . Accepted November 12, 1992
Summary
In non-growing protonemata of Anemia phyllitidis mitotic activity can be induced with a single 5-min red light pulse that is abolished by brief far red light, indicating phytochrome control. After repeatedly raising the PI, to a high level with red light pulses (e.g. one pulse every 4 days) some 10 % of gametophytes respond with transition to 2-D growth, which is first apparent in a drastic reduction of the LIB ratio of apical cells ( < 0.5) followed by spindle reorientation. Lowering the PI, level with brief far red light immediately following red light not only totally prevents a reduction of cell growth but causes a considerable promotion of cell extension growth, which eventually leads to transverse cell divisions. Induction of 2-D growth with red light pulses also occurs in non-growing cells derived after R-pulsed (instead of continuous red light) induction of spore germination; the light treatment of spores must only be so sufficient as to establish a high enough number of protonemata for subsequent experimentation. The slow escape reaction from far red reversibility of red light-induced mitotic activity suggests mediation by a stable form of phytochrome, which appears to be different from that controlling spore germination.
Key words: Anemia phyllitidis, mitotic activity, phytochrome, two-dimensional growth, spore germination. Abbreviations: D = darkness; FR = far red light; R = red light; RF = red-far red; P = phytochrome; PI,
= far red absorbing form of phytochrome; LIB ratio = lengthlbreadth ratio of protonemal cells; 2-D growth
=
two-dimensional growth.
Introduction
The main aim of the present work is predominantly concerned with the question of the photoreceptor involved in mediating two-dimensional (2-D) growth in red light in gametophytes of Anemia phyllitidis. For blue light-induced 2-D growth of fern gametophytes a different photoreceptor is responsible, and differences between red and blue light-induced prothallial growth have been enumerated previously (Grill, 1990).
* Part of this report has been presented as a poster presentation at the Beltsville Symposium XVI, Sept. 22-27,1991. © 1993 by Gustav Fischer Verlag, Stuttgart
Most fern gametophytes develop one-dimensional filaments (protonemata) in response to continuous red light, and gametophytes of Anemia phyllitidis (Schizaeaceae) are no exception to this rule. Phytochrome has been shown to be responsible for several aspects of protonemal development, i.e. filament elongation (Schnarrenberger und Mohr, 1967; Greany and Miller, 1976), apical growth and timing of cell division (Furuya, 1978). Since cell division in Adiantum capillus-veneris after transfer from continuous red light to darkness was delayed by brief far red light given immediately before transfer to darkness, control by phytochrome in the timing of cell division was indicated (Furuya, 1978). Direct evidence that brief red light initiates mitotic activity, which is abolished by subsequent far red light, has to the best of our knowledge never been provided.
458
RENATE
GRILL and HELMUT SCHRAUDOLF
When spores of A. phyllitidis are induced to germinate with 1 day of continuous red light they differentiate one- to three-celled filaments in subsequent darkness. In continued darkness of up to 4 weeks these protonemata do not develop any further without, however, loosing their full viability. Thus, they provide absolutely non-growing cells (Grill, 1987) that are ideally suited to study light effects, since the reference system is equal to zero. In Adiantum capillus·ve· neris (Polypodiacea) growth is also soon arrested upon transfer to darkness, and this system has been used extensively for the study of after-effects of light on apical growth in subsequent darkness (Furuya, 1978) or upon resumption of apical growth of 3 days dark-incubated non-growing cells after a brief red or far red irradiation (Kadota and Furuya, 1981). When, in contrast to Adiantum, non-growing cells of Anemia are incubated in darkness for several weeks, re-irradiation with continuous red light leads to resumption of growth, which in contrast to uninterrupted red light results in a large proportion of gametophytes with biplanar morphology without the requirement for exogenous sugars (Grill, 1987). Whether phytochrome is involved is, however, not yet known with certainty. In the present investigation we therefore studied light effects on mitotic activity and induction of biplanar morphology in more detail in non-growing protonemata of Anemia using brief red or red-far red light pulses.
8.6±5.0%
7
A 2.7±1.7%
6 2.0±2.0%
5
o
2
3
4
Number of R or RF pulses
Fig. 1: Induction of mitotic activity and 2-D growth with red light pulses. Non-growing protonemata were irradiated with one to four 5 min of red (R) or 5 min of red immediately followed by 5 min of far red (RF) light, one R or RF pulse every 4 days. Percentages of 2-D growth and/or transition stages ± S.E. of means are given for each light pulse on the graphs. Measurements were made 16 days after the first light pulse.
Results Material and Methods
Spores of Anemia phyllitidis (L.) Sw. were collected from plants grown in the greenhouse of the D niversity of DIm and were stored at + 5°C. The culture medium was as described by Mohr in 1956, containing 0.25g MgS0 4 , 1.00g Ca(NO,)z, 0.12g KNO" 0.25g KH 2P04 and a trace of FeCI, in 1 L of double distilled water. After 1 day of dark imbibition, germination was induced with 1 day of red light, unless otherwise stated. After the red preirradiation, cultures were incubated in darkness for at least 2 weeks, providing the nongrowing experimental material. Re-irradiation of non-growing protonemata was performed with brief light pulses, separated by 1-4 days of darkness (see Figure legends); the dark interval between light pulses could be as much as 7 days without change of effect (personal observation). The first visible sign for a transition to 2-D growth was apparent in filaments whose apical cells have their length/ breadth ratio reduced to < 0.5 (see Table 3 and Fig. 2), and such protonemata are termed «transition stages" (see a in Fig. 2). Since one of their short apical cells soon undergoes the first longitudinal cell division (see b in Fig. 2), transition stages are included in the evaluation of the percentage of gametophytes induced to 2-D growth. The temperature was 20°C throughout. The irradiance of the red light field (Philips-TL, 20W/15, two layers of red Plexiglas no. 501 [Rohm & Haas]) was about 0.3 W cm- 2 and that of the far red light field (Osram Wolfram, Linestra, 60 W incandescent lamps, two layers of red Plexiglas no. 501 plus one layer of blue Plexiglas no. 627 [Rohm & Haas]) ca. 0.4 W cm -2. The energy fluence rate was measured with a compensated thermopile (Kipp and Zonen, Holland) equipped with the heat-absorbing filter KP 560 (Zeiss, Germany). Except for the experiment presented in Fig. 2, all experiments were repeated at least two times and in each sample at least 200 gametophytes were measured microscopically after staining with acetocarmine. Vertical bars represent ± S.E. of means.
Non-growing protonemata of A. phyllitidis were irradiated with 5 min of red or 5 min of red immediately followed by a 5-min far red light pulse every 4 days during a total of 12 days in otherwise complete darkness. Figure 1 demonstrates that the first red light pulse induces approximately two cell divisions and each further red light pulse induces ca. one additional cell division in the majority of gametophytes. The first two red-far red light pulses almost completely abolish the red light-induced cell divisions, demonstrating mediation by phytochrome. Only after repeated red-far red exposures is a small increase in mean cell numbers observed. A small proportion of gametophytes (around 10%) responds to repeated red light pulses with the beginning of two-dimensional (2-D) growth. Since brief far red light immediately following red light completely prevents any indication of 2-D growth, phytochrome control is also indicated for this response. The frequency of cell numbers per gametophyte after one to four red (Rl to ~) or red-far red (R1FI to ~F4) pulses, one pulse each day, is shown in one representative experiment in Fig. 2. Clearly, the more red light pulses given, the broader the distribution of cell numbers per gametophyte. The maximal cell number per gametophyte that can be reached after one red pulse (R1) amounts to 7 cells and after four red pulses (~) to 13 cells in < 1 % gametophytes. In contrast, the maximal cell number per gametophyte for all red-far red treatments (R1FI to ~F4; Fig. 2, right) was only five cells, which is one cell more than the maximal cell number of dark controls (Fig. 2, left). After a period of filamentous growth, first transition stages are generally visible in seven-celled gametophytes (a) and appearance of a first longitudinal cell wall can
Phytochrome-mediated mitotic activity and induction of two-dimensional growth in Anemia 60
ocontrol
459
0 _ 12.31 R, 0---<> 13.31 R, lI- · - ·lI 14.71
en w
Fig. 2: Frequency of cell numbers per gametophyte after red or red-far red pulses. Non-growing protonemata received one to four 5 min of red (RI to ~) or 5 min of red immediately followed by 5 min of far red light (RIFt to ~F4)' one light pulse per day . Mean cell numbers are given in parentheses behind the explanation of symbols. Arrows indicate appearance of first transition stages (a) or insertion of the first longitudinal cell wall (b), illustrated also in a schematical drawing.
I-
>
R3 0······016.51 R4 16.81
*.......*
40
:I: 0..
oI-
w :<
« (!)
"*
20
CELLS PER GAMETOPHYTE
Table 1: Effects of continuous far red light on protonemata incubated in darkness for 2 weeks. Mean cell numbers and LIB ratios are given ± S E . . of means. Irradiation
Mean cell number
LIB ratio of cells
0 IdFR+6dD 2dFR+5dD 7 dFR
2.1±O.1 3.7±O.1 5.2±O.2 7.3±O.6
2.7±O.1 7.0±O.6 8.8± 1.2 12.5± 1.7
U
. ~
R Controi
------------ - -------- -----
3.5
.0
E c:
" "c:co
Qi
~
be observed at the ten-cell stage (b). A thirteen-celled gametophyte may contain at least three longitudinal cell walls at its apex. Thus, it is evident that 2-D growth after far red light is prevented because of diminished mitotic activity. Although, in contrast to darkness, irradiation of non-growing protonemata with continuous far red light also leads to the resumption of growth and induction of cell divisions (Table 1), these are exclusively transverse ones. The predominant effect of far red light is to increase cell extension growth, which is reflected in the high length/breadth ratio of cells (LIB ratio). Transition stages never occur in far red light although mean cell numbers are attained at which transition stages would have appeared under R-pulsed conditions. Inasmuch as the red light effect on mitotic activity is largely prevented by subsequent far red light, the question arises of whether phytochrome type I (<
FRContml
J
- ------------------------"1
3.0
-- ------------1J OConhOl
2.5
o
12
24
36
48
60
72
Time
Fig. 3: Escape kinetics of far red reversibility. Non-growing protonemata received either 5 min of red (R control), 5 min of far red (FR control), or remained in darkness (D control). The remaining cultures were given 5 min of red light followed by 5 min of far red light after 0 to 72 h of darkness (R A t FR).
with only pulses of red light. In the first instance, therefore, the effect of red light pulses on induction of germination was established. From Table 2 it is obvious that germination of Anemia spores is under phytochrome control since continuous red light can be replaced by brief red light pulses, which are far red reversible. From Table 2 it is also apparent that a single red light pulse of 5 min does not suffice to induce full germination but only induces about 9 %. Two red light pulses separated by 8 h of darkness are more effective than one R pulse, and nine R pulses given at hourly intervals approach the effect of 8 h continuous red light. A brief far red light pulse applied immediately after 8 h of continuous red light reduced germination by about 50 %. However large the percentage of germination resulting from the various light treatments of spores, all germinated spores developed into
460
RENATE GRILL
and HELMUT SCHRAUDOLF
Table 2: Induction of spore germination of A. phyllitidis. After 1 day of dark imbibition spores were treated for an 8 h period with continuous light, with various red light pulses, or with red light pulses followed immediately by 5 min far red light. Percent germination was evaluated 9 days after sowing and is given ± S.E. of means. % Germination
Irradiation ShR ShR+5minFR 5minR 5minR + 5minFR 5minR+ShD+5minR 5 min R + 5 min FR+S h D+5 min R+5 min FR 9 x (5 min R + 55 min D) 9 x (5 min R + 5 min FR + 50 min D)
86.4± 1.5 41.S± 10.8 S.9± 2.0 0.6± 0.1 27.S± 6.9
0.2± 0.2 6S.7± 3.5 14.7± 3.3
9 15.6 ±5.3%
R
8 /
/
/
//9.5:':3.7%
7
6
/
/
/
/
/
/
/
/
0%
Q; ..c
RF
15 5
//0%
c:
Qi u c:
4
til
Q)
~
3
,-
,y ,-
/
/
/
/
/
/
/
/
0%
2
o
4
8
Table 3: Effect of repeated red or red-far red light pulses on cell extension growth. From cultures in Fig. 4, twenty randomly chosen cells of gametophytes after irradiation of non-growing protonemata with eight light pulses were used for measurements of cell length, cell breadth and determination of the L/B ratio. For culture conditions see legend to Fig. 4. Light treatments
Cell length
Cell breadth LIB ratio
!lm ± S.D.
o
98.0±11.0 S x (5 min R + 2 d D) Filamentous cells 105.4±27.7 Apical cells of transition stages 14.2± 5.9 8 x (5 min R + 5 min FR + 2 d D) 202.0±44.5
22.6±1.7
4.33
28.8±2.5
3.66
30.8±2.S 24.2±2.4
0.46 8.37
growth were induced with four or eight red light pulses, one pulse every 2 days. The results show that there is no significant difference between the two pretreatments on re-irradiation-induced mean cell numbers or on percentages of gametophytes induced to 2-D growth. Thus 2-D growth and! or transition stages can also be induced in gametophytes derived after R-pulsed induction of germination. After red-far red (RF) light pulses mean cell numbers of protonemata rise gradually. However, the effect on 2-D growth development after four or eight red light pulses is entirely abolished with red-far red exposures, although eight RF pulses result in mean cell numbers that approach those obtained after four red light pulses. Cell extension growth and lengthlbreadth ratio resulting after the eight red or red-far red light pulses given in Fig. 4, is shown in Table 3. Obviously, repeated red-far red light pulses lead to a doubling in cell length as compared with that after red light pulses only. As there is no gross effect on cell breadth, a more than 2-fold increase in the LIB ratio of cells results. In apical cells of red light-induced transition stages cell lengthening is drastically decreased as compared with that of more basal cells, and the LIB ratio is reduced to <0.5.
Number of R or RF pulses
Fig. 4: Comparison of red preirradiation applied continuously or intermittently on subsequent induction of cell division and 2-D growth. After 1 day of dark imbibition, spores received either 8 h continuous red light (solid lines) or five 5-min red light pulses, each separated by 2h of darkness (broken lines). After incubation in darkness for 2 weeks non-growing protonemata were re-irradiated with four or eight 5-min red light pulses (R: e--e, 0- - - - -0), each separated by 2 days of darkness, or received 5 min of far red light immediately following each red light pulse (RF: A--A, i':,.- - - - -i':,.). Percentages of 2-D growth and/or transition stages ± S.E. of means are indicated on the graphs.
one- to three-celled protonemata in subsequent darkness. The light treatment of spores must only be so sufficient as to induce the formation of enough protonemata for further experimentation. In Figure 4 an 8 h pretreatment was given either with continuous red light or with five brief red light pulses each separated by 2 h of darkness. After an ensuing dark period of 3 weeks, induction of cell divisions and initiation of 2-D
Discussion
In the present study three distinct phytochrome-mediated morphogenic responses are demonstrated in A. phyllitidis: i} induction of mitotic activity and reduction of the LIB ratio of apical cells with subsequent spindle reorientation, mediated by the high Pf/P ratio established in red light; ii} increase in cell extension growth gradually followed by transverse cell divisions, mediated by a low Pf/P ratio as established in far red light; and iii} necessity for repeated brief red light exposures for full germination. The approach to the first two responses was possible only because of the absolute light requirement of resumption of growth of non-growing protonemata. There are some known fern species in which growth is also soon arrested after transfer from light to darkness, but studies of phytochrome effects have mainly concentrated on apical growth (Furuya, 1978). R-pulsed induction of mitotic activity of non-growing protonemata of Anemia can also lead to the beginning of 2-D growth in apical cells in a small proportion of
Phytochrome-mediated mitotic activity and induction of two-dimensional growth in Anemia
gametophytes after a period of filamentous growth. It should, however, be mentioned that this is a rather variable response. In some cultures first transition stages can already be observed after a single red light pulse whereas in others under identical conditions no transition stages occur after repeated red light pulses. The reason for this is, however, not clear. It also remains a challenge why Anemia protonemata require a long dark period interruption of red light to respond to red re-irradiation with biplanar morphology (Grill, 1987) instead of continuing the one-dimensional mode of growth occurring in uninterrupted red light. For other fern species it has been shown that a dark period interruption of red light is not required to elicit 2-D growth (Grill, 1990). Demonstration of the effect of a low PI/P ratio is evident under far red light conditions. In contrast to darkness, already the low Plr level established in far red light leads to resumption of growth of non-growing protonemata, indicating that in darkness phytochrome is entirely in the inactive Pr from. However, in contrast to red re-irradiation, elongation of developing cells is greatly promoted, resulting in quite considerable increases in LIB ratios, but cell divisions are exclusively transverse ones. Thus, concerning cell divisions, a low Plr level acts in the same direction as a high Plr level. In contrast, cell lengthening is greatly promoted by a low Plr level but is unaffected or inhibited by a high Plr level. This is an unsolved paradox that cannot yet be explained. Filament elongation of several fern species is also more promoted by far red than by red light (reviewed by Furuya, 1983). A dual action of phytochrome, viz. high Plr and low Plr reactions, has also been postulated for photoperiodic induction of flowering in higher plants (Vince-Prue, 1975). The phytochrome involved in inducing mitoses appears to be a type II phytochrome (<
461
must be different from that controlling mitotic activity, unless conditions exist within the spore-cell environment that favour rapid reversion. At least increasing knowledge on phytochromes within single plant species, for example Arabidopsis thaliana, has pointed to the existence of three or possibly more phytochromes (Quail et aI., 1991). Thus, the above assumption appears justified although caution is indicated since phytochromes from higher and lower plants are different at least at the molecular level (Thiimmler et aI., 1990). Interestingly, the entire development to initial stages of 2-D growth in red light can occur with only pulses of red light right from the induction of spore germination, indicating phytochrome as the only photoreceptor required. Thus, for induction of prothallial growth in red light, in a proportion of gametophytes, involvement of photosynthesis appears not to be required, and gametophytes may rely entirely on mobilizing reserve materials. This supposition has, however, still to be tested. Acknowledgements
We are extremely grateful to Dr. J. Oliver for carefully checking the English.
References FRANKLAND, B. and R. TAYLORSON: Light control of seed germination. In: SHROPSHIRE, W., Jr. and H. MOHR (eds.): Photo morphogenesis, Encyclopedia of Plant Physiology, N.S., vol. 16 A, 428-456. Springer, Berlin, Heidelberg, New York, Tokyo (1983). FURUYA, M.: Photocontrol of developmental processes in fern gametophytes. Bot. Mag. Tokyo Special Issue 1, 219-242 (1978). - Photo morphogenesis in ferns. In: SHROPSHIRE, W., Jr. and H. MOHR (eds.): Photomorphogenesis, Encyclopedia of Plant Physiology, N.S., vol. 16 B, 569-600. Springer, Berlin, Heidelberg, New York, Tokyo (1983). GREANY, R. H. and J. H. MILLER: An interaction of dose-response curves for light-induced cell elongation in fern protonemata. Am. J. Bot. 63, 1031-1037 (1976). GRILL, R .: Induction of two-dimensional growth by red and green light in the fern Anemia phyllitidis L. Sw. J. Plant Physiol. 131, 363 -371 (1987). - Red light-mediated two-dimensional growth in gametophytes of the ferns Adiantum capillus-veneris L. and Dryopteris filix·mas (L.) Schott. J. Plant Physiol. 136, 765 -768 (1990). HAAs, C. J., H. Kopp, and R. SCHEUERLEIN: Nitrate effect on Plrmediated and GAl-induced germination in spores of Anemia phyllitidis L. Sw. Photochem. Photobiol. 56, 633 - 640 (1992). JABBEN, M. and M. G. HOLMES: Phytochrome in light-grown plants. In: SHROPSHIRE, W., Jr. and H. MOHR (eds.): Photomorphogenesis, Encyclopedia of Plant Physiology, N.S., vol. 16 B, 704 -722. Springer, Berlin, Heidelberg, New York, Tokyo (1983). KAnOTA, A. and M. FURUYA: Apical growth of protonemata in Adiantum capillus-veneris. N . Phytochrome-mediated induction in non-growing cells. Plant Cell Physiol. 22, 629-638 (1981). MOHR, H.: Die Abhangigkeit des Protonemawachstums und der Protonemapolaritat bei Farnen vom Licht. Plant a 47, 127 -158 (1956). NESTER, J. E. and R. C. COOLBAUGH: Factors influencing germination and early gametophyte development in Anemia
462
RENATE GRILL
and HELMUT SCHRAUDOLF
mexicarut and Anemia phyllitidis. Plant Physiol. 82, 230-235 (1986). QUAIL, P. H., H. R. HERSHEY, K. B. IDLER, R. A. SHAlUI.OCK, A. H .. CHIUSTENSEN, B. M. PARKS, D. SOMERS, J. TEPPERMAN, W. B. BRUCE, and K. DEBESH: Phy-gene structure, evolution, and expression. In: THOMAS, B. and C. B. JOHNSON (eds.): Phytochrome properties and biological action, 13-38. Springer, Berlin, Heidelberg, New York, London, Paris, Tokyo, Hongkong, Barcelona, Budapest (1991). SCHNARRENBERGER, C. and H. MOHR: Die Wechselwirkung von Hellrot, Dunkelrot und Blaulicht bei der Photomorphogenese
von Farngametophyten [Dryopteris jilix-mas (L) Schott]. Planta (Berl.) 75, 114-124 (1967). THOMMLER, F., A. BEETZ, and W. RUDIGER: Phytochrome in lower plants. Detection and partial sequence of a phytochrome gene in the moss Ceratodon purpureus using the polymerase chain reaction. FEBS Letters 275, 125-129 (1990). VINCE-PRUE, D.: Photoperiodism in plants. McGraw-Hill, London, New York, St. Louis, San Francisco, Dusseldorf, Johannesburg, Kuala Lumpur, Mexico, Montreal, New Delhi, Panama, Paris, Sao Paulo, Singapore, Sydney, Toronto (1975).
141. pp. 457-462 (1993)
Phytochrome-Mediated Mitotic Activity and Induction of twoDimensional Growth in Gametophytes of Anemia phyllitidis (L.) Sw. * RENATE GRILL
and
HELMUT SCHRAUDOLF
Abteilung Allgemeine Botanik, Universitat Ulm, Oberer Eselsberg, W-7900 Ulm/Donau, Germany Received August 20, 1992 . Accepted November 12, 1992
Summary
In non-growing protonemata of Anemia phyllitidis mitotic activity can be induced with a single 5-min red light pulse that is abolished by brief far red light, indicating phytochrome control. After repeatedly raising the PI, to a high level with red light pulses (e.g. one pulse every 4 days) some 10 % of gametophytes respond with transition to 2-D growth, which is first apparent in a drastic reduction of the LIB ratio of apical cells ( < 0.5) followed by spindle reorientation. Lowering the PI, level with brief far red light immediately following red light not only totally prevents a reduction of cell growth but causes a considerable promotion of cell extension growth, which eventually leads to transverse cell divisions. Induction of 2-D growth with red light pulses also occurs in non-growing cells derived after R-pulsed (instead of continuous red light) induction of spore germination; the light treatment of spores must only be so sufficient as to establish a high enough number of protonemata for subsequent experimentation. The slow escape reaction from far red reversibility of red light-induced mitotic activity suggests mediation by a stable form of phytochrome, which appears to be different from that controlling spore germination.
Key words: Anemia phyllitidis, mitotic activity, phytochrome, two-dimensional growth, spore germination. Abbreviations: D = darkness; FR = far red light; R = red light; RF = red-far red; P = phytochrome; PI,
= far red absorbing form of phytochrome; LIB ratio = lengthlbreadth ratio of protonemal cells; 2-D growth
=
two-dimensional growth.
Introduction
The main aim of the present work is predominantly concerned with the question of the photoreceptor involved in mediating two-dimensional (2-D) growth in red light in gametophytes of Anemia phyllitidis. For blue light-induced 2-D growth of fern gametophytes a different photoreceptor is responsible, and differences between red and blue light-induced prothallial growth have been enumerated previously (Grill, 1990).
* Part of this report has been presented as a poster presentation at the Beltsville Symposium XVI, Sept. 22-27,1991. © 1993 by Gustav Fischer Verlag, Stuttgart
Most fern gametophytes develop one-dimensional filaments (protonemata) in response to continuous red light, and gametophytes of Anemia phyllitidis (Schizaeaceae) are no exception to this rule. Phytochrome has been shown to be responsible for several aspects of protonemal development, i.e. filament elongation (Schnarrenberger und Mohr, 1967; Greany and Miller, 1976), apical growth and timing of cell division (Furuya, 1978). Since cell division in Adiantum capillus-veneris after transfer from continuous red light to darkness was delayed by brief far red light given immediately before transfer to darkness, control by phytochrome in the timing of cell division was indicated (Furuya, 1978). Direct evidence that brief red light initiates mitotic activity, which is abolished by subsequent far red light, has to the best of our knowledge never been provided.
458
RENATE
GRILL and HELMUT SCHRAUDOLF
When spores of A. phyllitidis are induced to germinate with 1 day of continuous red light they differentiate one- to three-celled filaments in subsequent darkness. In continued darkness of up to 4 weeks these protonemata do not develop any further without, however, loosing their full viability. Thus, they provide absolutely non-growing cells (Grill, 1987) that are ideally suited to study light effects, since the reference system is equal to zero. In Adiantum capillus·ve· neris (Polypodiacea) growth is also soon arrested upon transfer to darkness, and this system has been used extensively for the study of after-effects of light on apical growth in subsequent darkness (Furuya, 1978) or upon resumption of apical growth of 3 days dark-incubated non-growing cells after a brief red or far red irradiation (Kadota and Furuya, 1981). When, in contrast to Adiantum, non-growing cells of Anemia are incubated in darkness for several weeks, re-irradiation with continuous red light leads to resumption of growth, which in contrast to uninterrupted red light results in a large proportion of gametophytes with biplanar morphology without the requirement for exogenous sugars (Grill, 1987). Whether phytochrome is involved is, however, not yet known with certainty. In the present investigation we therefore studied light effects on mitotic activity and induction of biplanar morphology in more detail in non-growing protonemata of Anemia using brief red or red-far red light pulses.
8.6±5.0%
7
A 2.7±1.7%
6 2.0±2.0%
5
o
2
3
4
Number of R or RF pulses
Fig. 1: Induction of mitotic activity and 2-D growth with red light pulses. Non-growing protonemata were irradiated with one to four 5 min of red (R) or 5 min of red immediately followed by 5 min of far red (RF) light, one R or RF pulse every 4 days. Percentages of 2-D growth and/or transition stages ± S.E. of means are given for each light pulse on the graphs. Measurements were made 16 days after the first light pulse.
Results Material and Methods
Spores of Anemia phyllitidis (L.) Sw. were collected from plants grown in the greenhouse of the D niversity of DIm and were stored at + 5°C. The culture medium was as described by Mohr in 1956, containing 0.25g MgS0 4 , 1.00g Ca(NO,)z, 0.12g KNO" 0.25g KH 2P04 and a trace of FeCI, in 1 L of double distilled water. After 1 day of dark imbibition, germination was induced with 1 day of red light, unless otherwise stated. After the red preirradiation, cultures were incubated in darkness for at least 2 weeks, providing the nongrowing experimental material. Re-irradiation of non-growing protonemata was performed with brief light pulses, separated by 1-4 days of darkness (see Figure legends); the dark interval between light pulses could be as much as 7 days without change of effect (personal observation). The first visible sign for a transition to 2-D growth was apparent in filaments whose apical cells have their length/ breadth ratio reduced to < 0.5 (see Table 3 and Fig. 2), and such protonemata are termed «transition stages" (see a in Fig. 2). Since one of their short apical cells soon undergoes the first longitudinal cell division (see b in Fig. 2), transition stages are included in the evaluation of the percentage of gametophytes induced to 2-D growth. The temperature was 20°C throughout. The irradiance of the red light field (Philips-TL, 20W/15, two layers of red Plexiglas no. 501 [Rohm & Haas]) was about 0.3 W cm- 2 and that of the far red light field (Osram Wolfram, Linestra, 60 W incandescent lamps, two layers of red Plexiglas no. 501 plus one layer of blue Plexiglas no. 627 [Rohm & Haas]) ca. 0.4 W cm -2. The energy fluence rate was measured with a compensated thermopile (Kipp and Zonen, Holland) equipped with the heat-absorbing filter KP 560 (Zeiss, Germany). Except for the experiment presented in Fig. 2, all experiments were repeated at least two times and in each sample at least 200 gametophytes were measured microscopically after staining with acetocarmine. Vertical bars represent ± S.E. of means.
Non-growing protonemata of A. phyllitidis were irradiated with 5 min of red or 5 min of red immediately followed by a 5-min far red light pulse every 4 days during a total of 12 days in otherwise complete darkness. Figure 1 demonstrates that the first red light pulse induces approximately two cell divisions and each further red light pulse induces ca. one additional cell division in the majority of gametophytes. The first two red-far red light pulses almost completely abolish the red light-induced cell divisions, demonstrating mediation by phytochrome. Only after repeated red-far red exposures is a small increase in mean cell numbers observed. A small proportion of gametophytes (around 10%) responds to repeated red light pulses with the beginning of two-dimensional (2-D) growth. Since brief far red light immediately following red light completely prevents any indication of 2-D growth, phytochrome control is also indicated for this response. The frequency of cell numbers per gametophyte after one to four red (Rl to ~) or red-far red (R1FI to ~F4) pulses, one pulse each day, is shown in one representative experiment in Fig. 2. Clearly, the more red light pulses given, the broader the distribution of cell numbers per gametophyte. The maximal cell number per gametophyte that can be reached after one red pulse (R1) amounts to 7 cells and after four red pulses (~) to 13 cells in < 1 % gametophytes. In contrast, the maximal cell number per gametophyte for all red-far red treatments (R1FI to ~F4; Fig. 2, right) was only five cells, which is one cell more than the maximal cell number of dark controls (Fig. 2, left). After a period of filamentous growth, first transition stages are generally visible in seven-celled gametophytes (a) and appearance of a first longitudinal cell wall can
Phytochrome-mediated mitotic activity and induction of two-dimensional growth in Anemia 60
ocontrol
459
0 _ 12.31 R, 0---<> 13.31 R, lI- · - ·lI 14.71
en w
Fig. 2: Frequency of cell numbers per gametophyte after red or red-far red pulses. Non-growing protonemata received one to four 5 min of red (RI to ~) or 5 min of red immediately followed by 5 min of far red light (RIFt to ~F4)' one light pulse per day . Mean cell numbers are given in parentheses behind the explanation of symbols. Arrows indicate appearance of first transition stages (a) or insertion of the first longitudinal cell wall (b), illustrated also in a schematical drawing.
I-
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R3 0······016.51 R4 16.81
*.......*
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20
CELLS PER GAMETOPHYTE
Table 1: Effects of continuous far red light on protonemata incubated in darkness for 2 weeks. Mean cell numbers and LIB ratios are given ± S E . . of means. Irradiation
Mean cell number
LIB ratio of cells
0 IdFR+6dD 2dFR+5dD 7 dFR
2.1±O.1 3.7±O.1 5.2±O.2 7.3±O.6
2.7±O.1 7.0±O.6 8.8± 1.2 12.5± 1.7
U
. ~
R Controi
------------ - -------- -----
3.5
.0
E c:
" "c:co
Qi
~
be observed at the ten-cell stage (b). A thirteen-celled gametophyte may contain at least three longitudinal cell walls at its apex. Thus, it is evident that 2-D growth after far red light is prevented because of diminished mitotic activity. Although, in contrast to darkness, irradiation of non-growing protonemata with continuous far red light also leads to the resumption of growth and induction of cell divisions (Table 1), these are exclusively transverse ones. The predominant effect of far red light is to increase cell extension growth, which is reflected in the high length/breadth ratio of cells (LIB ratio). Transition stages never occur in far red light although mean cell numbers are attained at which transition stages would have appeared under R-pulsed conditions. Inasmuch as the red light effect on mitotic activity is largely prevented by subsequent far red light, the question arises of whether phytochrome type I (<
FRContml
J
- ------------------------"1
3.0
-- ------------1J OConhOl
2.5
o
12
24
36
48
60
72
Time
Fig. 3: Escape kinetics of far red reversibility. Non-growing protonemata received either 5 min of red (R control), 5 min of far red (FR control), or remained in darkness (D control). The remaining cultures were given 5 min of red light followed by 5 min of far red light after 0 to 72 h of darkness (R A t FR).
with only pulses of red light. In the first instance, therefore, the effect of red light pulses on induction of germination was established. From Table 2 it is obvious that germination of Anemia spores is under phytochrome control since continuous red light can be replaced by brief red light pulses, which are far red reversible. From Table 2 it is also apparent that a single red light pulse of 5 min does not suffice to induce full germination but only induces about 9 %. Two red light pulses separated by 8 h of darkness are more effective than one R pulse, and nine R pulses given at hourly intervals approach the effect of 8 h continuous red light. A brief far red light pulse applied immediately after 8 h of continuous red light reduced germination by about 50 %. However large the percentage of germination resulting from the various light treatments of spores, all germinated spores developed into
460
RENATE GRILL
and HELMUT SCHRAUDOLF
Table 2: Induction of spore germination of A. phyllitidis. After 1 day of dark imbibition spores were treated for an 8 h period with continuous light, with various red light pulses, or with red light pulses followed immediately by 5 min far red light. Percent germination was evaluated 9 days after sowing and is given ± S.E. of means. % Germination
Irradiation ShR ShR+5minFR 5minR 5minR + 5minFR 5minR+ShD+5minR 5 min R + 5 min FR+S h D+5 min R+5 min FR 9 x (5 min R + 55 min D) 9 x (5 min R + 5 min FR + 50 min D)
86.4± 1.5 41.S± 10.8 S.9± 2.0 0.6± 0.1 27.S± 6.9
0.2± 0.2 6S.7± 3.5 14.7± 3.3
9 15.6 ±5.3%
R
8 /
/
/
//9.5:':3.7%
7
6
/
/
/
/
/
/
/
/
0%
Q; ..c
RF
15 5
//0%
c:
Qi u c:
4
til
Q)
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3
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/
/
/
/
/
/
/
/
0%
2
o
4
8
Table 3: Effect of repeated red or red-far red light pulses on cell extension growth. From cultures in Fig. 4, twenty randomly chosen cells of gametophytes after irradiation of non-growing protonemata with eight light pulses were used for measurements of cell length, cell breadth and determination of the L/B ratio. For culture conditions see legend to Fig. 4. Light treatments
Cell length
Cell breadth LIB ratio
!lm ± S.D.
o
98.0±11.0 S x (5 min R + 2 d D) Filamentous cells 105.4±27.7 Apical cells of transition stages 14.2± 5.9 8 x (5 min R + 5 min FR + 2 d D) 202.0±44.5
22.6±1.7
4.33
28.8±2.5
3.66
30.8±2.S 24.2±2.4
0.46 8.37
growth were induced with four or eight red light pulses, one pulse every 2 days. The results show that there is no significant difference between the two pretreatments on re-irradiation-induced mean cell numbers or on percentages of gametophytes induced to 2-D growth. Thus 2-D growth and! or transition stages can also be induced in gametophytes derived after R-pulsed induction of germination. After red-far red (RF) light pulses mean cell numbers of protonemata rise gradually. However, the effect on 2-D growth development after four or eight red light pulses is entirely abolished with red-far red exposures, although eight RF pulses result in mean cell numbers that approach those obtained after four red light pulses. Cell extension growth and lengthlbreadth ratio resulting after the eight red or red-far red light pulses given in Fig. 4, is shown in Table 3. Obviously, repeated red-far red light pulses lead to a doubling in cell length as compared with that after red light pulses only. As there is no gross effect on cell breadth, a more than 2-fold increase in the LIB ratio of cells results. In apical cells of red light-induced transition stages cell lengthening is drastically decreased as compared with that of more basal cells, and the LIB ratio is reduced to <0.5.
Number of R or RF pulses
Fig. 4: Comparison of red preirradiation applied continuously or intermittently on subsequent induction of cell division and 2-D growth. After 1 day of dark imbibition, spores received either 8 h continuous red light (solid lines) or five 5-min red light pulses, each separated by 2h of darkness (broken lines). After incubation in darkness for 2 weeks non-growing protonemata were re-irradiated with four or eight 5-min red light pulses (R: e--e, 0- - - - -0), each separated by 2 days of darkness, or received 5 min of far red light immediately following each red light pulse (RF: A--A, i':,.- - - - -i':,.). Percentages of 2-D growth and/or transition stages ± S.E. of means are indicated on the graphs.
one- to three-celled protonemata in subsequent darkness. The light treatment of spores must only be so sufficient as to induce the formation of enough protonemata for further experimentation. In Figure 4 an 8 h pretreatment was given either with continuous red light or with five brief red light pulses each separated by 2 h of darkness. After an ensuing dark period of 3 weeks, induction of cell divisions and initiation of 2-D
Discussion
In the present study three distinct phytochrome-mediated morphogenic responses are demonstrated in A. phyllitidis: i} induction of mitotic activity and reduction of the LIB ratio of apical cells with subsequent spindle reorientation, mediated by the high Pf/P ratio established in red light; ii} increase in cell extension growth gradually followed by transverse cell divisions, mediated by a low Pf/P ratio as established in far red light; and iii} necessity for repeated brief red light exposures for full germination. The approach to the first two responses was possible only because of the absolute light requirement of resumption of growth of non-growing protonemata. There are some known fern species in which growth is also soon arrested after transfer from light to darkness, but studies of phytochrome effects have mainly concentrated on apical growth (Furuya, 1978). R-pulsed induction of mitotic activity of non-growing protonemata of Anemia can also lead to the beginning of 2-D growth in apical cells in a small proportion of
Phytochrome-mediated mitotic activity and induction of two-dimensional growth in Anemia
gametophytes after a period of filamentous growth. It should, however, be mentioned that this is a rather variable response. In some cultures first transition stages can already be observed after a single red light pulse whereas in others under identical conditions no transition stages occur after repeated red light pulses. The reason for this is, however, not clear. It also remains a challenge why Anemia protonemata require a long dark period interruption of red light to respond to red re-irradiation with biplanar morphology (Grill, 1987) instead of continuing the one-dimensional mode of growth occurring in uninterrupted red light. For other fern species it has been shown that a dark period interruption of red light is not required to elicit 2-D growth (Grill, 1990). Demonstration of the effect of a low PI/P ratio is evident under far red light conditions. In contrast to darkness, already the low Plr level established in far red light leads to resumption of growth of non-growing protonemata, indicating that in darkness phytochrome is entirely in the inactive Pr from. However, in contrast to red re-irradiation, elongation of developing cells is greatly promoted, resulting in quite considerable increases in LIB ratios, but cell divisions are exclusively transverse ones. Thus, concerning cell divisions, a low Plr level acts in the same direction as a high Plr level. In contrast, cell lengthening is greatly promoted by a low Plr level but is unaffected or inhibited by a high Plr level. This is an unsolved paradox that cannot yet be explained. Filament elongation of several fern species is also more promoted by far red than by red light (reviewed by Furuya, 1983). A dual action of phytochrome, viz. high Plr and low Plr reactions, has also been postulated for photoperiodic induction of flowering in higher plants (Vince-Prue, 1975). The phytochrome involved in inducing mitoses appears to be a type II phytochrome (<
461
must be different from that controlling mitotic activity, unless conditions exist within the spore-cell environment that favour rapid reversion. At least increasing knowledge on phytochromes within single plant species, for example Arabidopsis thaliana, has pointed to the existence of three or possibly more phytochromes (Quail et aI., 1991). Thus, the above assumption appears justified although caution is indicated since phytochromes from higher and lower plants are different at least at the molecular level (Thiimmler et aI., 1990). Interestingly, the entire development to initial stages of 2-D growth in red light can occur with only pulses of red light right from the induction of spore germination, indicating phytochrome as the only photoreceptor required. Thus, for induction of prothallial growth in red light, in a proportion of gametophytes, involvement of photosynthesis appears not to be required, and gametophytes may rely entirely on mobilizing reserve materials. This supposition has, however, still to be tested. Acknowledgements
We are extremely grateful to Dr. J. Oliver for carefully checking the English.
References FRANKLAND, B. and R. TAYLORSON: Light control of seed germination. In: SHROPSHIRE, W., Jr. and H. MOHR (eds.): Photo morphogenesis, Encyclopedia of Plant Physiology, N.S., vol. 16 A, 428-456. Springer, Berlin, Heidelberg, New York, Tokyo (1983). FURUYA, M.: Photocontrol of developmental processes in fern gametophytes. Bot. Mag. Tokyo Special Issue 1, 219-242 (1978). - Photo morphogenesis in ferns. In: SHROPSHIRE, W., Jr. and H. MOHR (eds.): Photomorphogenesis, Encyclopedia of Plant Physiology, N.S., vol. 16 B, 569-600. Springer, Berlin, Heidelberg, New York, Tokyo (1983). GREANY, R. H. and J. H. MILLER: An interaction of dose-response curves for light-induced cell elongation in fern protonemata. Am. J. Bot. 63, 1031-1037 (1976). GRILL, R .: Induction of two-dimensional growth by red and green light in the fern Anemia phyllitidis L. Sw. J. Plant Physiol. 131, 363 -371 (1987). - Red light-mediated two-dimensional growth in gametophytes of the ferns Adiantum capillus-veneris L. and Dryopteris filix·mas (L.) Schott. J. Plant Physiol. 136, 765 -768 (1990). HAAs, C. J., H. Kopp, and R. SCHEUERLEIN: Nitrate effect on Plrmediated and GAl-induced germination in spores of Anemia phyllitidis L. Sw. Photochem. Photobiol. 56, 633 - 640 (1992). JABBEN, M. and M. G. HOLMES: Phytochrome in light-grown plants. In: SHROPSHIRE, W., Jr. and H. MOHR (eds.): Photomorphogenesis, Encyclopedia of Plant Physiology, N.S., vol. 16 B, 704 -722. Springer, Berlin, Heidelberg, New York, Tokyo (1983). KAnOTA, A. and M. FURUYA: Apical growth of protonemata in Adiantum capillus-veneris. N . Phytochrome-mediated induction in non-growing cells. Plant Cell Physiol. 22, 629-638 (1981). MOHR, H.: Die Abhangigkeit des Protonemawachstums und der Protonemapolaritat bei Farnen vom Licht. Plant a 47, 127 -158 (1956). NESTER, J. E. and R. C. COOLBAUGH: Factors influencing germination and early gametophyte development in Anemia
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and HELMUT SCHRAUDOLF
mexicarut and Anemia phyllitidis. Plant Physiol. 82, 230-235 (1986). QUAIL, P. H., H. R. HERSHEY, K. B. IDLER, R. A. SHAlUI.OCK, A. H .. CHIUSTENSEN, B. M. PARKS, D. SOMERS, J. TEPPERMAN, W. B. BRUCE, and K. DEBESH: Phy-gene structure, evolution, and expression. In: THOMAS, B. and C. B. JOHNSON (eds.): Phytochrome properties and biological action, 13-38. Springer, Berlin, Heidelberg, New York, London, Paris, Tokyo, Hongkong, Barcelona, Budapest (1991). SCHNARRENBERGER, C. and H. MOHR: Die Wechselwirkung von Hellrot, Dunkelrot und Blaulicht bei der Photomorphogenese
von Farngametophyten [Dryopteris jilix-mas (L) Schott]. Planta (Berl.) 75, 114-124 (1967). THOMMLER, F., A. BEETZ, and W. RUDIGER: Phytochrome in lower plants. Detection and partial sequence of a phytochrome gene in the moss Ceratodon purpureus using the polymerase chain reaction. FEBS Letters 275, 125-129 (1990). VINCE-PRUE, D.: Photoperiodism in plants. McGraw-Hill, London, New York, St. Louis, San Francisco, Dusseldorf, Johannesburg, Kuala Lumpur, Mexico, Montreal, New Delhi, Panama, Paris, Sao Paulo, Singapore, Sydney, Toronto (1975).