Plant Physiol. (1972) 49, 531-534
Two Effects of Prolonged Far Red Light on the Response of
Lettuce Seeds to Exogenous Gibberellin'
Received for publication August 27, 1971
A. N. BURDETT
Photobiology Group, Department of Biology, Simon Fraser University, Burnaby 2, British Columbia, Canada
ABSTRACT
Prolonged far red irradiation of imbibed lettuce seeds
(Lactuca sativa L. cv. Grand Rapids) makes them unresponsive
to subsequent treatment with gibberellin. It has been found
that this effect is overcome by supplying gibberellin buffered
at a low pH. On the basis of this and other evidence it is suggested that an extended far-red exposure causes a loss of gibberellin sensitivity through an effect on the permeability of
the endosperm. In seeds treated simultaneously with gibberellin and far red light, the hormone is taken up at the beginning
of the irradiation, but its action is suspended until the seeds
are placed in the dark.
with maximal energy at 718 nm (half peak height bandwidth:
10 nm) was used. All other light treatments consisted of a 1min exposure to red or far red filtered light sources with maximal energy at 660 and 731 nm, respectively (half peak height
bandwidth: 10 nm; irradiance: 6 X 10-' J cm-' sec-1). Seeds,
referred to below as receiving a preimbibition light treatment,
were irradiated for 1 min after their water content had been
raised to 20% by holding them in a water-saturated atmosphere for 2 hr at 20 C. They were then dried in the dark before being reimbibed for assay of germination.
From four to eight replicates of 50 seeds were used for each
treatment. Each lot was placed on two Whatman No. 1 filter
100,
\_
so.
%%
Prolonged far red light treatment prevents, or retards, GAinduced germination in lettuce seeds (5, 8). Negbi et al. (8)
found that the response to GA was antagonized by an extended tar red exposure before, as well as atter, treatment with
the hormone. On the assumption that the mechanism is the
same in each case, they proposed a single model to account
for these effects.
In the present study it has been found that prolonged far
red light affects the response to exogenous GA in different
ways, depending on whether the irradiation precedes, or occurs
at the same time as, the hormone treatment. Far red pretreatment causes a permanent loss of sensitivity to GA, and this
appears to be the result of its effect on the permeability of the
endosperm. In seeds treated simultaneously with GA and far
red light, the action of GA taken up at the beginning of the
irradiation is suspended but not prevented during a subsequent
dark incubation.
As will be discussed, these results cannot be accommodated
by the model noted above.
~ ~
~
~
~
-Red
60.G
AE
{:>
C on trolI
%
40.
\
s
\
84 hr do rk
---36 hr dark
20
'S'S
0
24
12
For-red Pretreatment (hr)
48
FIG. 1. The effect of different periods of far red (718 nm) irradiation on the germination of seeds subsequently treated with
GA (1 mM) or red light. Where no far red pretreatment is indicated the seeds received a preimbibition irradiation with red or far
red (control and GA treated) light. Each point represents the mean
germination percentage of four replicates.
papers in a 5-cm Petri dish and moistened with 1.8 ml of
water or GA, solution. When seeds were transferred from a
solution of GA to water, they were first placed, with the filter
papers, in a Buchner funnel to which a light vacuum was applied, and rinsed for 10 sec under a stream of distilled water.
They were then placed in a dish with fresh filter papers and remoistened. The same method, without the rinse, was followed
when seeds were transferred from water to a solution of GA.
Procedures with imbibed seeds were carried out under a dim
green lamp in a room maintained close to 20 C. All incubations were at 20 C, either in the dark or under far red light.
Emergence of the radicle, determined by inspection with the
naked eye, was the criterion of germination adopted.
MATERIALS AND METHODS
Seeds (Lactuca sativa L. cv. Grand Rapids) harvested in
1970 were purchased in December of that year and stored
in airtight containers at -20 C. Those used in the experiments
described were selected for freedom from damage and uniformity of size and color. For prolonged low intensity (2.5 X
10-5 J cni -2 sec-') far red irradiations a filtered light source
'The research was supported in part by Canadian National Research Council Grant A2908.
531
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Plant
BURDETT
532
GA
e
(84hr)
c
c
E
/Control
0
0
30 36
Incubation
20
10
r) a rk
10
Hcurs
20
30 36
FIG. 2. The effect of a prolonged far red (718 nm) pretreatment
on the response of punctured seeds to GA (10 /AM) or red light. a:
No prolonged far red pretreatment. Seeds imbibed in water after a
red (E-) or far red (A-A) preimbibition light treatment.
Seeds imbibed in GA after a preimbibition far red irradiation
(0
0). b: Far red-pretreated (48 hr) seeds receiving: no further
0), or red light (E-). Each
treatment (A
A), GA (0
point represents the mean germination percentage of four replicates.
100.
n
Table I. Effects of GA anzd a Low pH Buiffer oti Germiniationz after a
Preimbibitiont Irradiationi with Red or Far Red Light
The seeds were imbibed either in water or potassium biphthalate
(1 mM):HCI buffer (pH 2.5), with or without GA (1 mM), and their
germination was recorded 14 hr after the beginning of imbibition.
Eight replicates were used for each treatment.
Irradiation
Buffer
GA
Far red
Far red
Far red
Far red
Red
Red
Red
Red
+
+
+
+
+
+
+
+
14 hr
I
60.
c
w
Germination
Treatment
Punctured
tact
1972
irradiation (cf. "Materials and Methods") germinate as rapidly
as red light-treated seeds imbibed in water. From the shape of
the curves it is evident that this effect is primarily due to the
greater sensitivity of the punctured seeds to low GA concentrations.
A similar increase in GA sensitivity was observed when a
solution of the hormone was injected underneath the endosperm, rather than supplied externally (4). In view of this effect, it has been suggested that puncturing the seeds increases
their sensitivity to GA by eliminating the endosperm as an
effective barrier to its uptake (9). According to this interpretation, the absence of an effect of prolonged far red pretreatment
on the GA sensitivity of punctured seeds indicates that the irradiation impairs the response of intact seeds by reducing the
permeability of the endosperm to GA.
This conclusion is supported by other evidence reported be-
80.
-
Physiol. Vol. 49,
40.
0\0
0
7
47
12
48
64
75
63
76
20.
LSD p < 0.05
13
0.
0
-9
-8
-7
-6
-5
Log
-4
10
-3
GA
0 - 9 -8 -7 -6
Concentration (M)
-5
-4
3
FIG. 3. Dose response curves of GA-promoted germination in
(a) intact and (b) punctured seeds. All seeds received a preimbibition far red irradiation except for one control which was red light
treated (broken line). Germination was recorded after 18 hr imbibition. Each point represents the mean germination percentage
of eight replicates, and the vertical bar is four times the standard
error.
Table II. Effects of Red Light, GA, anzd a Low pH Buffer oni the
Germinationz of Far Red-pretreated Seeds
The seeds were imbibed in water and irradiated with far red
(718 nm) light for 48 hr. After giving some of the seeds a red light
treatment, they were incubated in the dark, either in water or
potassium biphthalate (1 mM):HCl buffer (pH 2.5), with or without GA (1 mM). Germination was recorded at the indicated times
during the dark incubation. Eight replicates were used for each
treatment.
Unlike the response to red light, it has been reported that
GA-induced germination in lettuce seeds is prevented by a
prolonged far red pretreatment (8). A similar effect has been
observed in experiments with the seeds used in this investigation. Their response to GA or red light after different periods
of far red irradiation is shown in Figure 1.
In contrast with that of intact seeds, Figure 2 shows that
the GA sensitivity of seeds punctured through the middle of
the cotyledons with a fine needle is not affected by a prior far
red exposure. The dose response curves illustrated in Figure
3 show that puncturing the seeds also has the effect of reducing, by several orders of magnitude, the concentration of
GA necessary to make seeds receiving a preimbibition far red
Germination
Treatment
RESULTS
Red light
Buffer
-
_
+
+
+
+
I
GA
15 hr
24 hr
36 hr
-
-
1
1
1
+
+
+
+
+
+
+
1
0
6
14
19
7
17
55
3
1
68
91
96
93
98
10
10
+
LSD p <
0.05
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Copyright © 1972 American Society of Plant Biologists. All rights reserved.
2
99
99
100
99
100
3
Plant Physiol. Vol. 49, 1972
FAR RED LIGHT, GA EFFECTS ON GERMINATION
100_
533
GA
0
0
Unpret reoted
24hr
Fa
r
Re d
-r ed
80._
60.
Red
40c
'i
20.
u
Cont rol
c
O_
I
I
0
*-
100.
a
12hr Far - red
48hr Fo r
r ed
A
80.
E
o
60
40.
20.
Control
O.i
IL
O
0I
20
316
30
D
a
r
k
0
I
n c
20
10
u
b
a
t i
o n
30
40
50
60
( H o u r s )
FIG. 4. The germination of intact seeds treated with GA (1 mM) during and after a prolonged far red (718 nm) exposure, compared with tha
of seeds imbibed in water and receiving a red light treatment at the end of the far red irradiation. a: No far red pretreatment. Seeds imbibed in
water after a red (u--u) or far red (A--A) preimbibition irradiation; seeds imbibed in GA after a far red preimbibition light treatment
(0- *). b, c, and d: Twelve, 24, and 48 hr far-red light, respectively. GA present throughout the incubation (
0): red light after the
far red exposure (--E): controls receiving no additional treatment (A
A). Each point represents the mean germination percentage of
four replicates.
low. However, it must also be considered possible that puncturing the seeds influences their responsiveness to GA supplied
after a far red irradiation in some other way, perhaps through
its effect on their gas exchange properties or by inducing a
wound response. The capacity of punctured seeds, unlike intact ones, to germinate slowly after prolonged far red irradiation without an inductive treatment must be explained by such
an alternative mechanism (cf. Figs. 2b and 4d-controls).
Cathey et al. (3) reported that the response of lettuce seeds
to exogenous GA is enhanced, if the substance is supplied in a
solution buffered at a low pH. The effect was found to be independent of the ionic composition of the buffer, and they
suggested it was due to the pH dependence of GA uptake.
Since only the endosperm is in contact with the buffered medium, this implies that pH influences endosperm permeability
to GA. If far red light reduces the permeability of the endosperm, it would, therefore, be expected that low pH will cause
a greater increase in the GA response of seeds which have received a far red pretreatment than of those which have not.
Such a difference has been observed. With the seeds used in
this study, no effect of a buffer at pH 2.5 (potassium biphthal-
ate [1 mM]: HCl) was observed on their germination after a
preimbibition exposure to red or far red light, either in the
presence or absence of GA (cf. Table I). However, the GA
response after a prolonged far red pretreatment was found to
be almost completely dependent on the presence of the buffer.
This was also true of red light-treated seeds although they
eventually germinated completely even in the absence of GA
(cf. Table II).
Seeds given a preimbibition far red irradiation and then
imbibed in 1 mm GA germinated as rapidly as red light-treated
seeds held in water, the process being virtually complete within
24 hr (cf. Fig. 4a). Seeds held in a solution of GA and irradiated with far red light for 12 hr before being placed in the
dark did not germinate at all during the first 24 hr of imbibition. This is in agreement with a similar experiment reported
by Negbi et al. (8). However, these seeds germinated rapidly
during the next 12 hr, the time course being closely comparable with that of seeds imbibed in water and irradiated with red
light after a 12-hr far red exposure (cf. Fig. 4b). Germination
was delayed by longer far red treatments, by the duration of
the irradiation, but began soon after the seeds were placed in
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Plant
BURDETT
534
Table Ill. The Dark Germiniationi of Seeds Treated wit/i GA for a
Limited Time at the Beginniinlg of a Far Red Irradiationi
Seeds imbibed in water or GA (1 mM) and irradiated with far
red (718 nm) light for 12 hr were washed and subsequently incubated in water. At the end of the GA treatment they were placed in
the dark, either immediately or after an additional far red exposure. Germination was recorded at the indicated times during the
dark incubation. Five replicates were used for each treatment.
Treatment
Germination
Total duration
GA
far red
light
of
hrc
12
12
24
36
48
+
+
+
36 hr
24 hr
12 hr
84 hr
cI
0
0
1
29
64
0
47
65
47
60
44
60
0
2
4
the dark (cf. Fig. 4, c and d). After 24 and 48 hr far red, the
germination of the GA-treated seeds started before that of the
red light-treated seeds.
If prolonged far red light inhibits GA uptake, the dark germination of seeds treated with this substance and far red light
at the same time implies that they take up GA during the
first few hours of the irradiation. Since the GA-treated seeds
do not germinate as long as they are exposed to far red light,
it must also be inferred that far-red light inhibits GA action, the effect being dark reversible. It is, therefore, probable
that GA taken up during the first part of an extended far red
treatment,
or a
product of its action
necessary
for its effect
on
germination, is not appreciably degraded during the subsequent irradiation period.
Data confirming these inferences are given in Table III.
They show that seeds treated with GA during the first 12 hr
of a far red exposure germinate when incubated in the dark
without GA. This indicates that seeds under the far red light
can take up GA at the beginning of the irradiation. The results demonstrate that far red light prevents the germination
of seeds which have taken up enough GA to germinate in the
dark. This verifies an inhibitory effect of far red light on GA
action. The data also show that the dark germination of seeds
treated with GA during the first 12 hr of imbibition is not diminished in rate or final percentage by a far red exposure between the end of the GA treatment and the beginning of the
dark incubation. This is in accord with the inferred stability
of GA,, or a product of its action necessary for its effect on
germination, in far red irradiated seeds.
DISCUSSION
The present study indicates that prolonged far red light has
two effects on the germination of seeds treated with exogenous
GA. The first suspends the action of GA already taken up by
the seeds, but does not prevent them from responding to it
during a subsequent dark incubation. The second, evident
when seeds are pretreated with far red light, causes a persistent
loss of GA sensitivity. Results discussed above provide rea-
Physiol. Vol. 49,
1972
sonable grounds for the view that this is due to an effect of
the irradiation on the permeability of the endosperm to GA.
To account for its effect on the germination of GA-treated
seeds, Negbi et al. (8) proposed that prolonged far red light
inactivates a pigment, other than phytochrome, which is necessary for, but formed independently of, GA action. This model
cannot account for both of the effects reported here.
If it accounts for the inhibition of germination in seeds
treated simultaneously with GA and far red light, their germination during a subsequent dark period indicates that the far
red-inactivated pigment is formed over an extended period of
time (at least 48 hr; cf. Fig. 4d). If this is the case, the GA
insensitivity of far red-pretreated seeds must be due to another
mechanism.
It could be maintained that this model accounts for one or
other of the effects of far red light reported here. However,
recent studies implicating phytochrome as the photoreceptor
in most, if not all, prolonged far red effects do not support
this possibility (1, 2, 7, 10). They also indicate a number of
other mechanisms which might account for the action of far
red light on GA-induced germination. One of particular interest may be derived from Boisard's (1) conclusion that prolonged far red light inhibits dark germination in lettuce seeds
by preventing the accumulation of Pfr formed from Pr by a
nonphotochemical process.
If it is postulated that GA stimulates a process capable of
transforming Pr to Pfr, an explanation of GA-induced germination and its dark-reversible inhibition by far red light is provided. Since it has been reported (6) that red light promotes
the formation of GA in light-sensitive lettuce seeds, this hypothesis also suggests a possible role of GA in light-stimulated
germination. By initiating a process capable of transforming
Pr to Pfr, GA formed as a result of a brief red light treatment
would maintain a high level of Pfr in seed subsequently incubated in the darkness, even if reversion to Pr occurs. Evidence of such a positive feedback mechanism has been reported. Boisard (1) was able to detect absorbance changes in
dark-dormant lettuce seeds beginning 8 hr after they received
a red light exDosure, which fact he considered to be indicative
of the dark formation of Pfr.
LITERATURE CITED
aklnes
1. BoISARD, J. 1969. Role du ph-tochrome clans la photosensihilite des
de Laittue variWti "Reine de 'Mai." Physiol. Veg. 7: 119-133.
2. BORTHNWICK, H. A., S. B. HEN-DRICKS, 'M. J. SCHNEIDER. R. B. TAYI,ORSON,
AND V. K. TOOLE. 1969. The high-energy light action controlling plant responses and development. Proc. 'Nat. Acad. Sci. U. S. A. 64: 479-486.
3. CATHEY, H. M., N. W. STUART, V. K. TOOLE, AND S. ASENN. 1961. Enlhancement of gibberellin-induced phenomena. Advan. Chem. Ser. 28: 135-141.
4. IKUMA, H. AND K. V. THINIAN-N. 1960. Action of gibberellic acid on lettuce
seed germination. Plant Physiol. 35: 557-566.
5. KAHN, A. 1960. Promotion of lettuce seed germination by gibberellin. Plant
Phvsiol. 35: 333-339.
6. KOHLER, D. 1966. Varindlerungen des Gibberellingehaltes von Salatsamen nach
Belichtung. Planta 70: 42-45.
7. 'MOHR, H., I. BEINGER, AN-D H. LANGE. 1971. Primary reaction of phytoctirome.
Nature 230: 56-58.
8. NEGBI, M., NI. BLACK, AND J. D. BEWLEY. 1968. Far-red sensitive dark process
essential for light- and gibberellin-induced germination of letttuce seed.
Plant Physiol. 43: 35-40.
9. SCHEIME, J. AND A. LANG. 1965. Lettuce seed germination: evidence for a reversible light-induced increase in growth potential and for phytochrome
mediation of the low temperature effect. Plant Physiol. 40: 485-492.
10. SPR-IT, C. J. P. AND A. L. MIANCINELLI. 1969. Phytochrome in cuctimber
seedls. Planta 88: 303-310.
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