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Inhibition of Onoclea sensibilis Spore Germination by Far-red Light and Cis-4-Cyclohexene-1,2-dicarboximide
Short Communication
Inhibition of Onoclea sensibilis Spore Germination by Far-red Light and Cis-4-Cyclohexene-t,2-dicarboximide CLARK
s. HUCKABY, KAMBIZ KALANTARI and JOHN H. MILLER
Department of Biology, Syracuse University, Syracuse, New York 13210, USA Received September 25,1981 . Accepted November 2,1981
Summary Red light-induction of Onoclea sensibilis spore germination has been reversed by far-red light, revealing the photoreceptive role of phytochrome. Cis-4-cyclohexene-1,2-dicarboximide, which inhibits processes involved with phytochrome-mediated seed germination, appears mainly to inhibit non-phytochrome-related processes in Onoclea spore germination.
Key words: Onoclea sensibilis, cis-4-cyclohexene-1,2-dicarboximide, fern spore germination, phytochrome.
Introduction Germination in spores of the fern Onoclea sensibilis is promoted by red light, and in darkness by high (30°C) incubation temperatures (Hartt, 1925; Towill and Ikuma, 1973; Towill, 1978; Chen and Ikuma, 1979). High temperature- and light-induced germination are believed to occur by different mechanisms (Towill, 1978). Towill and Ikuma (1973) did not observe far-red photo reversibility of red light-induced germination, and thus only tentatively suggested that phytochrome was the photoreceptor involved in light-induced germination. However, we have observed far-red photoreversibility of germination in some of our Onoclea spore isolates. In this report, we describe this photoreversibility along with the effects of CHDC on germination. This compound is an endogenous inhibitor of seed germination isolated from sugar beet seeds (Michell and Tolbert, 1968). It is thought to inhibit the action of Pfr in photo blastic seeds (Bewley and Oaks, 1980).
Materials and Methods Fertile fronds of Onoclea sensibilis L. were collected in the spring of 1980 near Jericho, Vermont. Spores from a single frond were isolated, stripped of their perines in dilute sodium hypo-
Abbreviations: CHDC form of phytochrome.
=
cis-4-cyclohexene-1,2-dicarboximide; Pfr
=
far-red light absorbing
z. Pjlanzenphysiol. Bd. 105. S. 375 -378. 1982.
376
CLARK S. HUCKABY, KAMBIZ KALANTARI and JOHN H. MILLER
chlorite bleach, and cultured on liquid Knop's solution using previously described methods (Miller and Greany, 1974: Vogelmann and Miller, 1981). Knop's solution was supplemented in some cases with synthetic CHDC, which was generously provided by Dr. N. E. Tolbert of Michigan State University. The cultures were held in darkness for 2 h, given light treatments, then returned to darkness for the remaining 72 h duration of an experiment. Dark incubations were at 25 or 30°C, depending on the experiment, and all light treatments were at 27 - 28°C. Red light of 68.4 ~W . cm- 2 was provided by two Champion F15T12 15 W red fluorescent tubes wrapped with one layer each of Roscolene no. 821 and no. 807 cellulose acetate. Far-red light of 32.4 ~W . cm- 2 was obtained from the light of alSO W tungsten reflector lamp transmitted through a Corning Glass no. 2600 filter. All experiments were repeated at least three times, and at least 300 spores from each culture were scored for germination using the acetocarmine/chloral hydrate method (Edwards and Miller, 1972).
Results and Discussion While maximal photoinduced germination (ca. 95 %) in our spores required at least 8 h of red light irradiation, 2 h of red light significantly enhanced germination at both 25 and 30°C; dark germination was greater at 30 °C than at 25°C, as shown previously by Towill (1978). Figure 1 shows that the red light effect was reversed by 2 h 80
~
60
c·
.2
g
'e ~
~
40 20
OL-LL-l no ~~ FR
R R·FI!
0
FR
R R·FR
light Treatment
Fig. 1: Germination of Onoclea spores in darkness (D), after 2 h of red light (R), after 2 h of farred light (FR), and after 2 h of red light followed immediately by 2 h of far-red light (R-FR). Dark incubations were at 25°C (unshaded bars) or 30°C (shaded bars). Error bars represent doubled standard errors in cases where the standard error was greater than 1 %. of far-red light. These results clearly implicate phytochrome mediation in the photoinduction of Onoclea spore germination. Variability between collections of spores, or differences in sources of far-red light may be responsible for photoreversibility not being observed by Towill and Ikuma (1973); these authors discussed several possible explanations for lack of photoreversibility in their collection of spores. CHDC has been characterized as an inhibitor of phytochrome-dependant seed germination; the compound does not affect the germination of non-light-requiring seeds (Bewley and Oaks, 1980). As a way of comparing seed and fern spore germination, we investigated the effects of CHDC on dark and photoinduced germination in Onoclea spores. Figure 2 a shows that the most severe inhibition of germination at CHDC Z. Pjlanzenphysiol. Bd. 105. S. 375-378.1982.
Onoclea spore germination
377
o
<¥-
J60 '0c:
.~
.,
o
40
20
b
o
o I' o .001
,
.01
1
1 10 0
~HDC]. mM
40
80
120
Time in far·red li9ht,
160
2h
se<;
Fig. 2: Inhibition of Onoclea spore germination by (a) CHDC and (b) far-red light alone and in combination with CHDC. Results of a single representative experiment are shown in each case; all dark incubations were at 30°C. (a) Spores cultured on various concentrations of CHDC were given 8 h (0) or 4 h (0) of red light irradiation, or held in darkness (e). (b) Spores cultured on Knop's solution (0) or Knop's solution + 1.0 mM CHDC (0) were given 4 h of red light followed immedediately by various exposures to far-red light. concentrations between 0.001 and 1.0 mM occurred in spores held in d;trkness. For example, germination at 1.0 mM CHDC was only 42 % of the O-CHDC control level in spores held in darkness, but was 92 % of the control level in spores given 8 h of red light. This suggested that the non-light-requiring, thermoinduced processes leading to spore germination were more sensitive to inhibition by CHDC than the photoinduced processes. To further characterize the inhibition of Onoclea spore germination by CHDC, combined submaximal treatments of CHDC and far-red light were given to red lighttreated spores. Brief far-red light irradiations interacted additively with 1.0 mM CHDC in inhibiting germination (Fig. 2 b). The simplest interpretation of this result is that inhibition of spore germination by CHDC is independent of the phytochrome state. This interpretation seems consistent with the conclusion of Towill (1978) that photoinduced and thermoinduced germination involve different processes, and our data indicate that the latter is the more sensitive to inhibition by CHDC. Phytochrome-related germination processes are strongly inhibited by CHDC in seeds (Bewley and Oaks, 1980), but not in Onoclea spores. This suggests that (1) phytochrome-initiated events leading to Onoclea spore germination are not identical to these events in seed germination, and (2) CHDC is not an inhibitor of all phytochrome-mediated germination processes.
References BEWLEY,}. D. and A. OAKS: Proc. Nat!. Acad. Sci. U.S.A. 77, 3408-3411 (1980). CHEN, c.-Y. and H. IKUMA: Plant Physiol. 63, 704-708 (1979).
Z. Pjlanzenphysiol. Bd. 105. S. 375-378.1982.
378
CLARK S. HUCKABY, KAMBIZ KALANTARI and JOHN H. MILLER
EDWARDS, M. E. and]. H. MILLER: Amer.]. Bot. 59, 458-465 (1972). HARTT, C. E.: Bot. Gaz. 79,427-441 (1925). MITCHELL, E. D. Jr. and N. E. TOLBERT: Biochemistry 7, 1019-1025 (1968). MILLER,]. H. and R. H. GREANY: Amer.]. Bot. 61, 296-302 (1974). TOWILL, L. R.: Plant Physiol. 62,116-119 (1978). TOWILL, L. R. and H. IKUMA: Plant Physiol. 51, 973-978 (1973). VOGELMANN, T. C. and]. H. MILLER: Amer.]. Bot. 68, 1177 -1183 (1981).
Z. Pjlanzenphysiol. Bd. 105. S. 375-378.1982.
Inhibition of Onoclea sensibilis Spore Germination by Far-red Light and Cis-4-Cyclohexene-t,2-dicarboximide CLARK
s. HUCKABY, KAMBIZ KALANTARI and JOHN H. MILLER
Department of Biology, Syracuse University, Syracuse, New York 13210, USA Received September 25,1981 . Accepted November 2,1981
Summary Red light-induction of Onoclea sensibilis spore germination has been reversed by far-red light, revealing the photoreceptive role of phytochrome. Cis-4-cyclohexene-1,2-dicarboximide, which inhibits processes involved with phytochrome-mediated seed germination, appears mainly to inhibit non-phytochrome-related processes in Onoclea spore germination.
Key words: Onoclea sensibilis, cis-4-cyclohexene-1,2-dicarboximide, fern spore germination, phytochrome.
Introduction Germination in spores of the fern Onoclea sensibilis is promoted by red light, and in darkness by high (30°C) incubation temperatures (Hartt, 1925; Towill and Ikuma, 1973; Towill, 1978; Chen and Ikuma, 1979). High temperature- and light-induced germination are believed to occur by different mechanisms (Towill, 1978). Towill and Ikuma (1973) did not observe far-red photo reversibility of red light-induced germination, and thus only tentatively suggested that phytochrome was the photoreceptor involved in light-induced germination. However, we have observed far-red photoreversibility of germination in some of our Onoclea spore isolates. In this report, we describe this photoreversibility along with the effects of CHDC on germination. This compound is an endogenous inhibitor of seed germination isolated from sugar beet seeds (Michell and Tolbert, 1968). It is thought to inhibit the action of Pfr in photo blastic seeds (Bewley and Oaks, 1980).
Materials and Methods Fertile fronds of Onoclea sensibilis L. were collected in the spring of 1980 near Jericho, Vermont. Spores from a single frond were isolated, stripped of their perines in dilute sodium hypo-
Abbreviations: CHDC form of phytochrome.
=
cis-4-cyclohexene-1,2-dicarboximide; Pfr
=
far-red light absorbing
z. Pjlanzenphysiol. Bd. 105. S. 375 -378. 1982.
376
CLARK S. HUCKABY, KAMBIZ KALANTARI and JOHN H. MILLER
chlorite bleach, and cultured on liquid Knop's solution using previously described methods (Miller and Greany, 1974: Vogelmann and Miller, 1981). Knop's solution was supplemented in some cases with synthetic CHDC, which was generously provided by Dr. N. E. Tolbert of Michigan State University. The cultures were held in darkness for 2 h, given light treatments, then returned to darkness for the remaining 72 h duration of an experiment. Dark incubations were at 25 or 30°C, depending on the experiment, and all light treatments were at 27 - 28°C. Red light of 68.4 ~W . cm- 2 was provided by two Champion F15T12 15 W red fluorescent tubes wrapped with one layer each of Roscolene no. 821 and no. 807 cellulose acetate. Far-red light of 32.4 ~W . cm- 2 was obtained from the light of alSO W tungsten reflector lamp transmitted through a Corning Glass no. 2600 filter. All experiments were repeated at least three times, and at least 300 spores from each culture were scored for germination using the acetocarmine/chloral hydrate method (Edwards and Miller, 1972).
Results and Discussion While maximal photoinduced germination (ca. 95 %) in our spores required at least 8 h of red light irradiation, 2 h of red light significantly enhanced germination at both 25 and 30°C; dark germination was greater at 30 °C than at 25°C, as shown previously by Towill (1978). Figure 1 shows that the red light effect was reversed by 2 h 80
~
60
c·
.2
g
'e ~
~
40 20
OL-LL-l no ~~ FR
R R·FI!
0
FR
R R·FR
light Treatment
Fig. 1: Germination of Onoclea spores in darkness (D), after 2 h of red light (R), after 2 h of farred light (FR), and after 2 h of red light followed immediately by 2 h of far-red light (R-FR). Dark incubations were at 25°C (unshaded bars) or 30°C (shaded bars). Error bars represent doubled standard errors in cases where the standard error was greater than 1 %. of far-red light. These results clearly implicate phytochrome mediation in the photoinduction of Onoclea spore germination. Variability between collections of spores, or differences in sources of far-red light may be responsible for photoreversibility not being observed by Towill and Ikuma (1973); these authors discussed several possible explanations for lack of photoreversibility in their collection of spores. CHDC has been characterized as an inhibitor of phytochrome-dependant seed germination; the compound does not affect the germination of non-light-requiring seeds (Bewley and Oaks, 1980). As a way of comparing seed and fern spore germination, we investigated the effects of CHDC on dark and photoinduced germination in Onoclea spores. Figure 2 a shows that the most severe inhibition of germination at CHDC Z. Pjlanzenphysiol. Bd. 105. S. 375-378.1982.
Onoclea spore germination
377
o
<¥-
J60 '0c:
.~
.,
o
40
20
b
o
o I' o .001
,
.01
1
1 10 0
~HDC]. mM
40
80
120
Time in far·red li9ht,
160
2h
se<;
Fig. 2: Inhibition of Onoclea spore germination by (a) CHDC and (b) far-red light alone and in combination with CHDC. Results of a single representative experiment are shown in each case; all dark incubations were at 30°C. (a) Spores cultured on various concentrations of CHDC were given 8 h (0) or 4 h (0) of red light irradiation, or held in darkness (e). (b) Spores cultured on Knop's solution (0) or Knop's solution + 1.0 mM CHDC (0) were given 4 h of red light followed immedediately by various exposures to far-red light. concentrations between 0.001 and 1.0 mM occurred in spores held in d;trkness. For example, germination at 1.0 mM CHDC was only 42 % of the O-CHDC control level in spores held in darkness, but was 92 % of the control level in spores given 8 h of red light. This suggested that the non-light-requiring, thermoinduced processes leading to spore germination were more sensitive to inhibition by CHDC than the photoinduced processes. To further characterize the inhibition of Onoclea spore germination by CHDC, combined submaximal treatments of CHDC and far-red light were given to red lighttreated spores. Brief far-red light irradiations interacted additively with 1.0 mM CHDC in inhibiting germination (Fig. 2 b). The simplest interpretation of this result is that inhibition of spore germination by CHDC is independent of the phytochrome state. This interpretation seems consistent with the conclusion of Towill (1978) that photoinduced and thermoinduced germination involve different processes, and our data indicate that the latter is the more sensitive to inhibition by CHDC. Phytochrome-related germination processes are strongly inhibited by CHDC in seeds (Bewley and Oaks, 1980), but not in Onoclea spores. This suggests that (1) phytochrome-initiated events leading to Onoclea spore germination are not identical to these events in seed germination, and (2) CHDC is not an inhibitor of all phytochrome-mediated germination processes.
References BEWLEY,}. D. and A. OAKS: Proc. Nat!. Acad. Sci. U.S.A. 77, 3408-3411 (1980). CHEN, c.-Y. and H. IKUMA: Plant Physiol. 63, 704-708 (1979).
Z. Pjlanzenphysiol. Bd. 105. S. 375-378.1982.
378
CLARK S. HUCKABY, KAMBIZ KALANTARI and JOHN H. MILLER
EDWARDS, M. E. and]. H. MILLER: Amer.]. Bot. 59, 458-465 (1972). HARTT, C. E.: Bot. Gaz. 79,427-441 (1925). MITCHELL, E. D. Jr. and N. E. TOLBERT: Biochemistry 7, 1019-1025 (1968). MILLER,]. H. and R. H. GREANY: Amer.]. Bot. 61, 296-302 (1974). TOWILL, L. R.: Plant Physiol. 62,116-119 (1978). TOWILL, L. R. and H. IKUMA: Plant Physiol. 51, 973-978 (1973). VOGELMANN, T. C. and]. H. MILLER: Amer.]. Bot. 68, 1177 -1183 (1981).
Z. Pjlanzenphysiol. Bd. 105. S. 375-378.1982.