Plant Physiol. (1976) 57, 612-616
Effects of 35 C Heat Treatments on Photosensitive Grand Rapids
Lettuce Seed Germination1
Received for publication December 24, 1974 and in revised form December 12, 1975
NICHOLAS C. CARPITA AND MURRAY W. NABORS
Department of Botany and Plant Pathology, Colorado State University, Fort Collins, Colorado 80523
Sylvania 150-w soft white flood lamps underlaid with a 3 mm
of Rohm and Haas 2444 R Plexiglas having a peak
thickness
Grand Rapids lettuce (Lactuca sativa L.) seeds were given 35 C heat transmission at 680 nm. The FR source consisted of four Gentreatments to increase photodormancy in a subsequent 20 C dark period. eral Electric 150-w clear reflector flood lamps underlaid with a 3
Short heat treatments (1-5 hours) induced a significant germination mm thickness of Rohm and Haas V-58015 "black" Plexiglas.
percentage increase of from 16% to over 50% depending on seed lot. Lamps and filters were separated by a water heat trap such that
With longer heat treatments dark germination percentage was gradually the temperature change within the chamber during the treatreduced to zero. If given at the end of 35 C, far red or red followed by far ments did not exceed 1.2 C.
red further increased the amount of dark germination.
Transmission spectra of R and FR sources were obtained by
Thermodormancy also delayed red-stimulated germination by 10 an
ISCO, Model SR, spectroradiometer and calibrated against
hours or more when red was given following a long 35 C treatment. The an ISCO, Model SRC, spectroradiometer calibrator standard
presence of Pfr was required during this time since far red light remained lamp. The FR transmission spectrum of the phytochrome-sensieffective in reversing at least 50% of the red stimulation for up to 16 tive range is given in Figure 1.
hours compared to only 4 hours in nonheat-treated seeds.
Light treatments consisted of unwrapping the dishes of seeds
in total darkness, administering a 5-min R or FR illumination,
and rewrapping the dishes in total darkness. In seeds given a R,
FR treatment, a 5-min R illumination was immediately followed
with a 5-min FR treatment. Seeds were then placed immediately
in the proper growth chamber in darkness.
Grand Rapids lettuce seeds (Lactuca sativa L.) are typically
In the R sensitivity studies, the light source consisted of a
photosensitive and exhibit the classical phytochrome photorev- Durst M 600 75-mm f4.5 enlarger lens with a Sylvania 150-w
ersible response (4, 5, 11). Heat treatments (30-37 C) have enlarger lamp underlaid with the R filter previously described.
been performed on imbibed seeds (7, 9, 10, 14, 16, 19) inducing Light treatments consisted of unwrapping the dishes of seeds in
a thermodormancy in which dark germination percentage is total darkness, administering a 10-sec R illumination of varying
reduced to zero in both photodormant and nonphotodormant intensities, and rewrapping the dishes in total darkness. The
seeds. This phenomenon is typically attributed to a conversion of seeds were then placed in the appropriate growth chamber in
Pfr to Pr (10, 16, 19). Thermodormancy may be completely darkness. The light source was calibrated as described and total
eliminated by R2 at 20 C or, in some cases, by a combination of energies in a band width of 40 nm from 640 nm to 680 nm were
CO2 + ethylene (7, 14).
converted to ujoules/cm2.
Heat treatments are being used routinely to drive dark germiThermodormancy Studies. Imbibing seeds were immediately
nation percentage to zero in many studies of photodormant seed placed into a 35 C growth chamber for 0 to 30 hr, then given an
germination. This report examines the changes in the germina- appropriate light or dark treatment and placed into a 20 C
tion potential during heat treatments, as well as the effective growth chamber for 48 hr to allow maximum possible germinaresponse to R and FR after heat treatments.
tion. In another study, seeds were allowed to imbibe 4 hr at
20 C, given a R treatment, and then placed at 35 C for various
MATERIALS AND METHODS
hours. At the end of the heat treatment seeds were transferred to
20
C for 48 hr to allow maximum possible germination.
Seed Source. Grand Rapids lettuce seeds (Lactuca sativa L.)
Time Studies. Seeds were allowed to imbibe at
Germination
were used in all experiments. Lots 670-C, 671-C, and 672-C 20 C or 35 C for
15 hr prior to a R treatment. After the R
were obtained from Rocky Mountain Seed Co., Denver, Colo. treatment, seeds were placed
20 C and samples were taken at
Lot 12310-18639 was obtained from Ferry-Morse Seed Co., Mt. various times from 2 to 30 hr at
to
determine germination percentView, Calif. Seeds were stored at -20 C to maintain light sensi- age. Any visible protrusion of the
radicle through the seed coat
tivity and viability.
was
positive
germination.
considered
Imbibition. All sample sizes were about 200 seeds. Seeds were
FR Reversibility Studies. Seeds were allowed to imbibe for 15
placed in moistened filter paper-lined 9-cm glass Petri dishes, hr at 20
C or 35 C prior to R treatment. After the R treatment,
wrapped in a double thickness of heavy duty aluminum foil, and seeds were
placed at 20 C for 0 to 24 hr after which time a FR
placed in covered metal containers. The containers of seeds were treatment was
given. Seeds were replaced at 20 C for 48 hr to
then put into appropriate 35 C or 20 C growth chambers.
maximum
allow
germination. Germination percentages
Light Sources and Treatments. The R source consisted of four of seeds imbibed possible
at 20 C were corrected for nonphotoreversible,
dark germination.
R Sensitivity Studies. Seeds were imbibed in darkness at 20 C
' This work was supported in part by Colorado State University
or 35 C for 5, 10, or 15 hr and given an appropriate light
Faculty Improvement Grant No. 1834.
2 Abbreviations: R:
intensity treatment. Light-treated seeds plus dark controls were
red light; FR: far red light.
612
ABSTRACT
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Plant
Physiol. Vol. 57,
1976
HEAT TREATMENTS AND SEED GERMINATION
613
i
C4
E
10
400
500
600
WAVELENGTH (nm)
700
20
10
HOURS AT 35C AFTER RED
800
FIG. 1. Transmission spectrum of the FR source in terms of energy
output over the phytochrome-sensitive range.
FIG. 2. Changes in the germination potential of lot 671-C due to
35 C treatment following R stimulation. At the end of 35 C treatments,
seeds were placed at 20 C for 48 hr to express germination potential.
Each point represents the mean of three samples.
placed into a 20 C growth chamber for 48 hr to allow maximum
possible germination. Germination percentages were corrected
for the dark germination corresponding to the imbibition treatment.
RESULTS
Water-imbibed lettuce seeds will not germinate at 35 C in
light or darkness. In the thermodormancy studies, the seeds were 0 K
~
~
~~~~~~~~~~D
given a heat treatment for various hours. At the end of the
treatment a germination potential existing in the seeds will be
expressed only if the seeds are placed at a suitable temperature.
40
10203
A temperature of 20 C allows for an average of 98% germina20
tion of R-stimulated lettuce seeds. Thus, heat-treated seeds were
placed at 20 C to allow for the expression of the germination
potential.
HOURS AT 35C
Imbibed seeds given a R stimulation and placed at 35 C for
3.
in
FIG.
the
germination potential of lot 671-C due to
Changes
germiover
time
in
the
a
decrease
varying lengths of time show
nation percentage determined at 20 C after 48 hr (Fig. 2). Once imbibition time at 35 C prior to dark; FR; R, FR; or R treatment and
the germination has fallen to zero, however, the seeds may be placement at 20 C. Each point represents mean +SE of five samples.
restimulated by R and express 98% germination at 20 C.
In all four lots of seeds investigated, the heat treatment ultimately resulted in complete thermodormancy in seeds which
35C * 20C, 48hr
were kept in darkness throughout the experiment. The germination percentage, however, did not decrease from the initial dark
germination, but rose during the first 5 hr to over 50% before
y
the expected decline in the dark germination percentage was
noted (Figs. 3 and 4). When FR was given after heat treatment,
the germination percentage was initially lower than the dark
germination, as expected, but also quickly rose to over 70% in z
FE
61C
30RRYthree of the lots used. The characteristic decline to complete
thermodormancy was then observed (Figs. 3 and 5).
672-C
When R immediately preceded the FR treatment, two seed
lots showed no FR enhancement of dark germination, whereas
the two remaining lots rose to over 70% before the expected
decline as the seeds became thermodormant (Fig. 6). In one
particular lot, 671-C, the FR and the R-FR enhancement of the
germination percentage was maintained for 15 to 20 hr before
falling to below 15% (Figs. 3, 5, and 6). Red light alone was 000
20
10
HOURS AT 35 C
effective in stimulating germination irrespective of the time imbibed at 35 C.
FIG. 4. Changes in the germination potential of 4 lots of seeds due to
When the sensitivity to low intensity irradiation was examined imbibition time at 35 C prior to placement at 20 C. Each point repreas a function of time and temperature of imbibition (Fig. 7), it sents the mean of three samples (671-C, five samples).
was found that seeds imbibed at 35 C expressed over 60%
Seeds imbibed at 20 C for 15 hr achieved 50% germination
germination with less than 10 ujoules/cm2 after only 5 hr imbibition. Comparable sensitivity was not achieved in the 20 C im- between 11 and 12 hr after the R stimulation, whereas a 15-hr
heat treatment delayed subsequent germination approximately
bibed seeds until 15 hr.
0
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Copyright © 1976 American Society of Plant Biologists. All rights reserved.
CARPITA AND NABORS
614
Plant Physiol. Vol. 57, 1976
10 hr, as 50% germination was not achieved until 21 hr (Fig. 8).
From the FR reversal curves (Fig. 9) we see 50% of the seeds
escaped FR reversibility of the R stimulation by 4 hr after the R
treatment when imbibed at 20 C. Heat-treated seeds were FR
reversible up to 16 hr after the R stimulation.
DISCUSSION
The dark germination percentage of the seeds fell completely
.67j-C-;
MORSE
X
to zero after 20 hr incubation at 35 C (Fig. 2), but initially rose
20
670-C671-C"
to almost twice the normal dark germination. We know of no
.'~~~~~~~~~~~~~~~~~~%
precedent for such a response. This response is not inherent in
one particular seed lot, but existed in all four lots investigated
0(Fig. 3). Ikuma and Thimann (12) showed that pretreatment at
35 C did not promote germination but decreased it steadily after
4 hr imbibition. Their variance in the dark germination was high
20
10
0
(±+10%), however, and they did not observe changes in the
HOURS AT 35C
germination potential between 0 and 4 hr.
FIG. 5. Changes in the germination potential of four lots of seeds due
The rise and fall in the germination potential could be exto imbibition time at 35 C prior to FR treatment and placement at 20 C.
on the basis of a rise and fall in the amount of active Pfr
plained
Each point represents the mean of three samples (671-C, five samples). alone. The Pfr increase during short heat treatments could come
from Pr or from another form of phytochrome, as much recent
work suggests that more than the two classical forms of phytochrome exist in plant tissue (1, 3, 13, 17). These results cannot
be fully explained in terms of the classical Pfr to Pr conversion
due to heat proposed by several papers (10, 16, 19), even though
35C
FR
48hr
20C,
R,
AC "
*
*
Schafer and Schmidt (15) present direct spectrophotometric
evidence for an increased rate of decay of Pfr to Pr in squash
seedlings at 35 C. The phytochrome components may greatly
differ during the early stages of seed germination, and the apparent conversion of Pfr to Pr may have little importance during this
0~~~~~~.
stage of development. Results from Figure 2 also demonstrate
7240 ;O...
that no net loss in the active Pfr needed to stimulate germination
occurred before 5 hr. Boisard and his colleagues (2) present
evidence for an apparent Pr to Pfr "inverse" conversion in
imbibing lettuce seeds which could result in an increased germination potential. Although this may conveniently explain the rise
in the germination potential during the heat treatment there is a
30
20
0
10
problem with this proposal in that there is no means of explainHOURS AT 35C
ing why a Pr to Pfr conversion is at first favorable and then after
FIG. 6. Changes in germination potential of 4 lots of seeds due to the 5th hr unfavorable. Furthermore, Briggs and Rice (6) point
imbibition time at 35 C prior to R, FR treatment, and placement at out that the inverse conversion may be only apparent and ac20 C. Each point represents the mean of three samples (671-C, five
tually results from hydration of the seed. Tobin and Briggs (18)
samples).
found that a low level of spectrophotometrically measurable
phytochrome could be tripled in Pinus embryos in less than 2
min by simply adding water to the chopped sample. Briggs and
Rice (6) propose that if the Pfr in the unhydrated dormant
80 -35C,15hr
embryo is a weakly absorbing form while the Pr is a normally
absorbing form, then hydration of both phytochrome compo35C,10hr
_____"
9.
nents would appear as an apparent Pr to Pfr conversion.
°~
25h
Germination of photosensitive lettuce seeds will usually be
induced by R within 15 min from the start of imbibition in our
lots of seeds. Although these seeds quickly become photosensitive,
complete water uptake by the seed may take as long as 4 to
40_rr____
8 hr, and complete hydration of all the components of the seed
takes considerably longer (10, 18). If a rise in Pfr is responsible
for the increase in the germination potential while the Pr to Pfr
conversion is artifactual, as Briggs and Rice suggest, then Pfr
must come from another phytochrome pool and be stable in the
unhydrated form during the heat treatment.
If FR is administered at the end of the heat treatment a normal
inhibition of dark germination is evident only for the 1st hr (Figs.
3 and 5). This can be expected since extremely short heat
30
20
treatments would probably have little effect on the seeds. After 1
ENERGY lpjouls/cm2I
hr, the per cent germination rose rapidly to even greater than the
FIG. 7. Low intensity R sensitivity curve for heat-treated and non- dark germination. In lot 671-C, the high rate of germination
remained over 70% for almost 15 hr before falling to below 15 %
heat-treated seeds. Each point represents the mean of three samples.
FERRY-
t
"
20
670- C
*O..
z
° X
FERRY-
MORSE
~~~~%
-----
Z
/
20C,lOhr
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Copyright © 1976 American Society of Plant Biologists. All rights reserved.
Plant
Physiol. Vol. 57,
1976
HEAT TREATMENTS AND SEED GERMINATION
615
zso
0
E
z
f
i 40LU
0
R*20820C
,
s,
t
35C*
~~~~~~0
15 hr
~~~~~~~~~~~~~20C
0,R 0,20C
0
~~~~~~~35Cl15hr
20
0
10
20
~~~~~~~~~~~~~~48hr
30
HOURS AFTER RED TREATMENT
FIG. 8. Time required for germination of heat-treated and nonheattreated seeds. Each point represents one sample.
(Fig. 3). The high germination potential also was seen in the
three other lots (Fig. 5). If R immediately preceded the FR
treatment, FR remained equally effective in enhancing the dark
germination (Fig. 2). In two other lots, however, the FR was
much more effective in inhibiting germination if R preceded the
FR treatment. These results may only reflect differences in seed
age, storage conditions, or pre-harvest light conditions. Scheibe
and Lang (16) also found enhancement of the germination
potential if FR followed short heat treatments. They suggested
that the promotion of germination could arise from an acceleration of metabolic processes preceding germination or an admittedly less likely possibility that Pfr action could be accelerated
during the short heat treatments. The enhancement of the dark
germination by FR is puzzling. Eisenstadt and Mancinelli (8)
found an apparent escape from phytochrome control by cucumber seeds at temperatures above 20 C. Inhibition could be reimposed by a narrow band 730 nm source. Contamination of the
FR source by shorter wavelengths could alter the Pfr-P ratios
such that an enhanced germination potential results. When the
FR transmission spectrum was analyzed over the phytochromesensitive range no energies below 685 nm were detected and
only 0.7% of the total energy from the FR source between 685
and 800 nm existed below 700 nm (Fig. 1). Although this would
appear insufficient to increase the germination potential of FRtreated seeds, the sensitivity to low intensity irradiation after
various imbibition times at 35 C increased markedly after only 5
hr imbibition compared to seeds imbibed at 20 C (Fig. 7). This
increased sensitivity may then modify the dark germination potentials.
Not only are germination potential changes occurring during
the heat treatments, but also after full thermodormancy has been
imposed; the germination time at 20 C following R stimulation is
nearly doubled (Fig. 8). In addition to the delayed germination
time, the Pfr must remain active for an additional 12 hr in the
heat-treated seeds (Fig. 9). This would not be expected if the
35 C was merely enhancing a Pfr to Pr conversion since R would
restore high Pfr levels and a normal germination time would
result.
In terms of formulating possible modes of action to explain
these results, one must consider whether phytochrome alone is
involved in the rise and fall of germination potential or whether
nonphytochrome components are involved. Only the FR and R,
FR data must involve phytochrome. A multitude of other models
may be proposed to explain the data. Several which we have
considered are: (a) a hormone precursor or hormone-inhibitor
complex which first is activated by mild heat and then ultimately
turned over; (b) an enzyme precursor or enzyme-inhibitor com-
10
20
HOURS BETWEEN R AND FR
FIG. 9. Far red reversal curve for heat treated and nonheat-treated
seeds. Data indicate hours that FR remains effective in reversing the R
stimulation of germination. Each point represents one sample.
plex which would act similarly; (c) an activator molecule stimulated by mild heat treatment, coupled with a turnover of the
activated substrate; (d) inactive gene stimulated to transcription
by the heat treatment; (e) inactive mRNA stimulated to translation. In all cases a turnover process is involved to explain the rise
and fall in germination potential. As suggested by the FR reversibility and germination time curves (Figs. 8 and 9), the delay
caused by the heat treatments is due to the resynthesis necessary
to replace the turned-over component which would normally be
present.
The data presented only allow us to speculate on the activities
of the seed during heat treatment. It is clearly evident that many
processes are affected by the 35 C treatment which must be
taken into consideration in future studies of thermodormancy
and photosensitive seeds.
Acknowledgments -The authors wish to thank C. Ross for his help in the preparation of the
manuscript and J. J. Hanan for the use of the spectroradiometric equipment.
LITERATURE CITED
1. ANDERSON. G. R., E. L. JENNER, AND F. E. MUMFORD. 1969. Temperature and pH studies
on phytochrome in vitro. Biochemistry 8: 1182-1187.
2. BOISARD. J.. C. J. P. SPRUIT, AND P. ROLLIN. 1968. Phytochrome in seeds and an apparent
dark reversion of Pr to Pfr. Mededelingen Landouwhogeschool Wageningen 68: 1-5.
3. BOISARD, J., D. MARME, AND E. SCHAFER. 1971. The demonstration in vivo of more than
one form of Pfr. Planta 99: 302-310.
4. BORTHWICK, H. A., S. B. HENDRICKS, M. W. PARKER, E. H. TOOLE, AND V. K. TOOLE. 1952.
A reversible photoreaction controlling seed germination. Proc. Nat. Acad. Sci. U.S.A. 38:
662-666.
5. BoRTHwicK, H. A., S. B. HENDRICKS, E. H. TOOLE, AND V. K. TOOLE. 1954. Action of light
on lettuce-seed germination. Bot. Gaz. 115: 205-225.
6. BiuGGs, W. R. AND H. V. RICE. 1972. Phytochrome: chemical and physical properties and
mechanism of action. Annu. Rev. Plant Physiol. 23: 293-334.
7. BURDETr, A. N. 1972. Antagonistic effects on high and low temperature pretreatments on
the germination and pregermination ethylene synthesis of lettuce seeds. Plant Physiol. 50:
201-204.
8. EISENSTADT, F. A. AND A. L. MANCINELLI. 1974. Phytochrome and seed germination. VI.
Phytochrome and temperature interaction in the control of cucumber seed germination.
Plant Physiol. 53: 114-117.
9. EVENARI, M. 1952. The germination of lettuce seed. 1. Light, temperature, and coumarin as
germination factors. Pales. J. Bot. Jerus. Ser. 5: 138-160.
10. EVENARI, M. 1972. Phytochrome and temperature relations in Lactuca sativa L. Grand
Rapids seed germination after thermodormancy. Nat. New Biol. 235: 144-145.
11. HENDRICKS, S. B., H. A. BORTHWICK, AND R. J. DOWNS. 1956. Pigment conversion in the
formative responses of plants to radiation. Proc. Nat. Acad. Sci. U.S.A. 42: 19-26.
12. IKUMA, H. AND K. V. THIMANN. 1964. Analysis of germination processes of lettuce seed by
means of temperature and anaerobiosis. Plant Physiol. 39: 756-767.
13. KENDRICK, R. E. AND C. J. P. SPurr. 1973. Phytochrome intermediates in vivo. 1. Effects of
temperature, light intensity, wavelength, and oxygen on intermediate accumulation. Photochem. Photobiol. 18: 139-144.
14. NEGM, F. B., 0. E. SMTm, AND J. KUMAMOTO. 1973. The role of phytochrome in an
Downloaded from on July 31, 2017 - Published by www.plantphysiol.org
Copyright © 1976 American Society of Plant Biologists. All rights reserved.
616
CARPITA AND NABORS
interaction with ethylene and carbon dioxide in overcoming lettuce seed thermodormancy.
Plant. Physiol. 51: 1089-1094.
15. SCHAFER, E. AND W. SCHMIDT. 1974. Temperature dependence of phytochrome dark
reactions. Planta 116: 257-266.
16. SCHEIBE, J. AND A. LANG. 1965. Lettuce seed germination: evidence for a reversible lightinduced increase in growth potential and for phytochrome mediation of the low temperature effect. Plant Physiol. 40: 485-492.
Plant Physiol. Vol. 57, 1976
17. SPRUrr, C. J. P. AND R. E. KENDRICK. 1973. Phytochrome intermediates in vivo. II.
Characterisation of intermediates by difference spectrophotometry. Photochem. Photobiol. 18: 145-152.
18. TOBIN, E. M. AND W. R. BRIGGs. 1969. Phytochrome in embryos of Pinus palustris. Plant
Physiol. 44: 148-150.
19. TOOLE, E. H. 1961. The effect of light and other variables on the control of seed germination.
Proc. Int. Seed Test. Assoc. 26: 659-673.
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